Handbook of Bioseparations (Separation Science and Technology, Volume 2)

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HANDBOOK OF BIOSEPARATIONS

This Is Volume 2 of SEPARATION SCIENCE A N D T E C H N O L O G Y A reference series edited by Satinder Ahuja

HANDBOOK OF BIOSEPARATIONS Edited by

Satinder Ahuja Ahuja Consulting Calabash, North Carolina

ACADEMIC PRESS A Harcourt Science and Technology Company

San Diego San Francisco New York Boston London Sydney Tokyo

Cover photo credit: Molecular image courtesy of Molecular Simulations, Inc. using WebLab. http://www.msi.com. This book is printed on acid-free paper, fe) Copyright © 2000 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt, Inc., 6277 Sea Harbor Drive, Orlando, Florida 32887-6777

Academic Press A Harcourt Science and Technology Company 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http ://www. academicpress. com

Academic Press Harcourt Place, 32 Jamestown Road, London NWl 7BY, UK http://www.hbuk.co.uk/ap/ Library of Congress Catalog Card Number: 00-102786 International Standard Book Number: 0-12-045540-4 PRINTED IN THE UNITED STATES OF AMERICA 00 01 02 03 04 05 QW 9 8 7 6

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3 2 1

CONTENTS

PREFACE

xiii

I Bioseparations: An Overview S. AHUJA

I. II. III. IV.

Introduction 1 Analytical Methodologies 3 Separation and Purification Methods Other Important Considerations 18 Reference 21

2 Analysis of Protein Impurities in Pharmaceuticals Derived from Recombinant DNA DONALD O. O'KEEFE

I. Introduction 23 II. Protein Impurity Analysis 28 III. Experimental Summary 57

VI

CONTENTS

IV. Case Studies 58 References 64

3 Physicochemical Factors in Polypeptide and Protein Purification and Analysis by High-Performance Liquid Chromatographic Techniques: Current Status and Challenges for the Future MILTON T. W. HEARN

I. II. III. IV. V. VI. VII. VIII. IX. X. XI.

Introduction 72 Basic Chromatographic Terms and Concepts Id The Chemical Structure of Polypeptides and Proteins 79 Physicochemical Factors That Underpin Ligate Interactions with Polypeptides and Proteins in HPLC Separation Systems 84 Strategic Considerations behind the HPLC Separations 107 Specific Physicochemical Considerations on the Individual Chromatographic Modes 117 The Effect of Temperature and the Thermodynamics of Polypeptide- or Protein-Ligate Interactions 135 Factors That Control Performance and Efficiency 156 Scaling-Up Possibilities: Heuristic Approaches and Productivity Considerations 172 Effect of Mass Transfer Resistances in Preparative HPLC of Polypeptides and Proteins 178 Summary 218 References 222

4 Capillary Electrophoresis of Compounds of Biological Interest S. AHUJA

I. II. III. IV. V. VI. VII. VIII. IX. X.

Introduction 238 Capillary Zone Electrophoresis 239 Migration Behavior of Peptides and Proteins 243 Modifications of Fused Silica Capillaries 247 Effect of Temperature on Separations 252 Strategy for Protein Separations 252 Capillary Gel Electrophoresis 253 Micellar Electrokinetic Chromatography 255 Capillary Electrochromatography 255 Applications 256 References 262

CONTENTS

VII

5 Isoelectric Focusing DAVID E. GARFIN

I. II. III. IV. V. VI. VII. VIII.

Introduction 263 The Principles of Isoelectric Focusing 264 Analytical Isoelectric Focusing 276 Two-Dimensional Gel Electrophoresis 285 Preparative Isoelectric Focusing 287 Capillary Isoelectric Focusing 291 Summary 292 Appendix 292 References 295

6 Mass Spectrometry of Biomolecules DAN GIBSON AND CATHERINE E. COSTELLO

I. Introduction 299 II. Examples of Applications of Mass Spectrometry to Biological Research 309 III. Conclusions 325 References 325

7 Liquid-Liquid Partitioning Methods for Bioseparations TINGYUE GU

I. II. III. IV.

Introduction 329 Solvent Extraction for Bioseparations 330 Aqueous Two-Phase Partitioning for Bioseparations Summary 360 References 361

8 Separation of Nucleic Acids and Proteins ROHIT HARVE AND RAKESH BAJPAI

I. II. III. IV. V. VI.

Introduction 365 Precipitation of Nucleic Acids 368 Nuclease Treatment 370 Aqueous Two-Phase Extraction 370 A Case Study 372 Conclusions 376 References 376

348

VIII

CONTENTS

9 Bioseparations by Displacement Chromatography ABHINAV A. SHUKLA AND STEVEN M. CRAMER

I. Introduction 380 II. Purification of Amino Acids and Peptides by Displacement Chromatography 382 III. Purification of Proteins by Displacement Chromatography 383 IV. Alternative Modes of Displacement Chromatography 390 V. Methods Development for Displacement Chromatography 393 VI. Displacement Chromatography for the Purification of Biomolecules: Industrial Case Studies 400 VII. Design of Lov^ Molecular Weight Displacers 406 VIII. Conclusions 410 References 412

10 Physicochemical Basis of Expanded-Bed Adsorption for Protein Purification B. MATTIASSON AND M. P. NANDAKUMAR

I. Introduction 417 II. Typical Procedure to Operate an Expanded-Bed Chromatographic System 418 III. Ligand Selection 423 IV. Applications 424 V. Conclusion 427 References 428

I I Expanded-Bed Adsorption Process for Protein Capture JOSEPH SHILOACH AND ROBERT M. KENNEDY

I. II. III. IV. V. VI. VII.

Introduction 431 Principles of Expanded-Bed Operation Experimental Strategy 435 Instrumentation 437 Matrices 438 Applications 438 Discussion and Conclusions 449 References 450

433

12 Adsorptive Membranes for Bioseparations RANJIT R. DESHMUKH AND TIMOTHY N. WARNER

I. Introduction 454 II. Comparison of Membrane Chromatography to Traditional Chromatography 458

CONTENTS

IX

III. Scale-Up of Chromatography Membranes 460 IV. AppUcations of MA to Preparative Bioseparations V. Conclusions 470 References 471

464

13 Simulated Moving-Bed Chromatography for Biomolecules R. M. NICOUD

I. II. III. IV. V. VI.

Introduction 475 Basic Principle 476 Operating Conditions 482 Main Applications and Developments 490 Practical Application: Separation of Sugars 499 Conclusion 506 References 508

14 Large-Scale Chromatographic Purification of Oligonucleotides RANJIT R. DESHMUKH. WILLIAM E. LEITCH II, YOGESH S. SANGHVI, AND DOUGLAS L COLE

I. Introduction 512 II. General Purification Strategies for Oligonucleotides 516 III. Large-Scale Purification of Therapeutic Oligonucleotides 519 IV. Purification of Related Molecules—DNA Fragments, Plasmids, Ribozymes, and RNA 529 V. Economics of Oligonucleotide Purification 530 VI. Summary 531 References 531

I 5 Separation of Antibodies by Liquid Chromatography EGISTO BOSCHETTI AND ALOIS JUNGBAUER

I. II. III. IV. V. VI. VII.

Introduction 536 Antibodies: An Overview 538 Biological Starting Material 546 Prepurification 551 Purification of Antibodies by Liquid Chromatography 556 Regulatory Considerations 612 General Conclusion on Antibody Separation Technologies 620 References 621

CONTENTS

16 Processing Plants and Equipment p. BOWLES

I. II. III. IV. V.

Introduction 633 Industries Using Bioseparations 634 Process-Scale Bioseparations 636 Process-Scale Considerations 653 Summary 656 References 657

17 Engineering Process Control of Bioseparation Processes RANDEL M. PRICE AND AJIT SADANA

I. Need for Process Control in Bioseparations 660 II. Brief Overview of Current Control Methods 661 III. Application Examples 662 IV. Opportunities for Continuing Development 664 References 664

18 Economics of Bioseparation Processes ANAND RAMAKRISHNAN AND AJIT SADANA

I. Introduction 667 II. Drugs Market and Sales 670 III. Applications of Models and Flow Sheets in Bioseparation Economics 673 References 684

19 Future Developments S. AHUJA

I. II. III. IV. V. VI. VII.

Introduction 687 The Partnership of Proteins and Nucleic Acids Biotech Drugs 690 Assuring Production and Purity 694 Genomics 698 Lab on Chip 699 Recovery of Biological Products 700 References 710

INDEX

713

690

CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors' contributions begin.

Satinder Ahuja (1, 237, 687) Ahuja Consulting, 330 S. Middleton Drive, Suite 803, Calabash, North Carolina 28467 Rakesh Bajpai (365) Department of Chemical Engineering, Department of Biological and Agricultural Engineering, University of Missouri-Columbia, Columbia, Missouri 65211 Egisto Boschetti (535) Life Technologies-BioSepra, 95804 Cergy Saint Christophe, France P. Bowles (633) Kvaerner Process (UK) Ltd., Whiteley, Hants, United Kingdom Douglas L. Cole (511) Development Chemistry and Pharmaceutical Development, Isis Pharmaceuticals, Inc., Carlsbad, California 92008 Catherine E. Costello (299) Mass Spectrometry Resource, Boston University School of Medicine, Boston, Massachusetts 02118 Steven M. Cramer (379) Department of Chemical Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180 Ranjit R. Deshmukh (453, 511) Manufacturing Process Development, Isis Pharmaceuticals, Inc., Carlsbad, California 92008 XI

XII

CONTRIBUTORS

David E. Garfin (263) Life Science Group, Bio-Rad Laboratories, Hercules, California 94547 Dan Gibson (299) Mass Spectrometry Resource, Boston University School of Medicine, Boston, Massachusetts 02118; and Department of Pharmaceutical Chemistry, School of Pharmacy, Hebrew University of Jerusalem, Jerusalem, Israel Tingyue Gu (329) Department of Chemical Engineering, Ohio University, Athens, Ohio 45701 Rohit Harve (365) Wyeth Ayerst Research, Marietta, Pennsylvania 17547 Milton T. W. Hearn (71) Centre for Bioprocess Technology, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia Alois Jungbauer (535) Institute of Applied Microbiology, University of Agriculture, A-1190 Vienna, Austria Robert M. Kennedy (431) Separations Group, Amersham Pharmacia Biotech, Piscataway, New Jersey 08855 William E. Leitch, II (511) Argyll Associates, Palm Desert, Cahfornia 92210 B. Mattiasson (417) Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, Lund, Sweden M. P. Nandakumar (417) Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, Lund, Sweden R. M. Nicoud (475) NovaSep, Vandoeuvre-les-Nancy, France Donald O. O'Keefe (23) Macromolecular Structure and Biopharmaceuticals, Bristol-Myers Squibb, Princeton, New Jersey 08543 Randel M. Price (659) Department of Chemical Engineering, University of Mississippi, University, Mississippi 38677 Anand Ramakrishnan (667) Department of Chemical Engineering, University of Mississippi, University, Mississippi 38677 Ajit Sadana (659, 667) Department of Chemical Engineering, University of Mississippi, University, Mississippi 38677 Yogesh S. Sanghvi (511) Manufacturing Process Development, Isis Pharmaceuticals, Inc., Carlsbad, California 92008 Joseph Shiloach (431) Biotechnology Unit, National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, Bethesda, Maryland 20892 Abhinav A. Shukla (379) ICOS Corporation, Bothell, Washington 98021 Timothy N. Warner (453) Sartorius Corporation, Edgewood, New York 11717

PREFACE

The commercial success of biotechnology products is highly dependent on the successful development and application of reliable and sensitive bioseparation methods. Bioseparations entail separations of proteins and other materials from biological matrices. This book is planned to serve as a handbook v^ith the primary focus on separations of proteins; how^ever, separations of other materials of interest, such as nucleic acids and oligonucleotides, are also covered to assist the readers in tackling their particular bioseparation problems. Included in this text is a chapter on the separation of monoclonal antibodies. Monoclonal antibodies and recombinant antibodies have become one of the largest classes of proteins that have received FDA approval as therapeutics and diagnostics. Antisense drugs have been covered because of their unique ability to bind to targeted messenger RNA (mRNA) while avoiding attachment to other proteins. Bioseparations are also helping w^ith the development of a large number of drugs for the treatment of a variety of diseases such as cancer, AIDS, rheumatoid arthritis, and Alzheimer's disease. The regulatory considerations applying to bioseparations are discussed in various sections of this book. It is important to remember that the FDA requires a thorough validation program, quality assurance oversight, statistically sound sampling methods, rigorous training, and a comprehensive documentation trail. The guidelines recommended by the International Conference on Harmonization addressing quality, safety, and efficacy have been covered to provide additional insight into this area. XIII

XIV

PREFACE

This book has been broadly divided into three sections: The analytical methodology section covers a variety of methods that are commonly used in bioseparations. Analytical methodology includes an interesting montage of chromatographic methods, capillary electrophoresis, isoelectric focusing, and mass spectrometry. Separation and purification methods provide detailed information on Hquid-liquid distribution, displacement chromatography, expanded-bed adsorption, membrane chromatography, and simulated moving-bed chromatography. This section also provides significant information for process-scale separations. Plant and process equipment, engineering process control of bioseparation processes, economic considerations, and future developments are discussed under the heading of Other Important Considerations—those elements that are sometimes forgotten but should never be ignored when one is dealing with bioseparations. The chapter on future developments provides some insight into what is coming down the road in the field of bioseparations; to this end, short summaries of various oral presentations made at the Ninth Conference on Recovery of Biological Products (held on May 2 3 - 2 8 , 1999, in Whistler, Canada) have also been included since this conference has become the preeminent meeting in the field of bioseparations. The excellent contributions to the Handbook of Bioseparations are likely to make it an essential reference and guidebook for separation scientists working in the pharmaceutical and biotechnology industries, academia, and government laboratories. February 2000

Satinder Ahuja Calabash, North Carolina

EDITORIAL ADVISORY BOARD

Steven M. Cramer Rensselaer Polytechnic Institute Troy, New York

William S. Hancock Hewlett Packard Palo Alto, California

Milton T. W. Hearn Monash University Clayton, Victoria Australia

Brian Hubbard Genetics Institute Andover, Massachusetts

This Page Intentionally Left Blank

I

BIOSEPARATIONS: AN OVERVIEW S. AHUJA Ahuja Consulting, Calabash, North Carolina 28467

I. INTRODUCTION A. Regulatory Considerations II. ANALYTICAL METHODOLOGIES A. HPLC B. Capillary Electrophoresis C. Isoelectric Focusing D. Mass Spectrometry E. Methodology Montage III. SEPARATION A N D PURIFICATION METHODS A. Liquid-Liquid Distribution B. Separation of Proteins and Nucleic Acids C. Displacement Chromatography D. Expanded-Bed Adsorption E. Membrane Chromatography F. Simulated Moving Bed Chromatography G. Purification of Oligonucleotides H. Monoclonal Antibodies IV. OTHER IMPORTANT CONSIDERATIONS A. Processing Plant and Equipment B. Engineering Process Control C. Economics of Separations D. Future Developments REFERENCE

I. INTRODUCTION The biotechnology industry has evolved significantly since the introduction in 1982 of human insulin synthesized in Escherichia coli—the first Food and Drug Administration (FDA)-approved recombinant therapeutic agent in the United States. Since then, over 75 other recombinant proteins have been introduced. The list is comprised of cytokines, hormones, monoclonal antibodies, and vaccines. There are more than 1100 companies competing for this market, and the current sale of these products comprises approximately 10% of the sales of all therapeutic products sold in the United States. One such product, erythropoietin, an erythropoiesis-stimulating factor also known Separation Science and Technology, Volume 2 Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

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as Epogen, is a circulating glycoprotein that stimulates red blood cell formation in higher organisms and has worldwide sales in excess of 1 billion U.S. dollars. The financial potential of these products is indeed great. This is apparent from the fact that over 500 biotechnology-related drugs are currently in clinical trials. Bioseparations, or separations of biological interest, have played a significant role in the development and growth of the biotechnology industry. These separations have to be performed on both analytical and industrial scales—and everything in between. Bioseparations frequently entail separations of proteins and related materials from biological matrices.^ This book is planned to serve as a handbook of bioseparations, where the primary focus is separations of proteins; however, separations of other materials of interest such as nucleic acids and oligonucleotides are also covered to assist the reader in tackling their particular bioseparation problems. Included in this text is a chapter on the separation of monoclonal antibodies, as these materials have found numerous uses in the biopharmaceutical industry. As a matter of fact, in the last few decades, monoclonal antibodies and recombinant antibodies have become one of the largest classes of proteins that have received FDA approval as therapeutics and diagnostics.

A. Regulatory Considerations The regulatory considerations applying to bioseparations are covered in various sections of this book. It is important to assure that separation and purification methods, when operating within the established limits, produce a product of appropriate and consistent quality. The method and process validations provide assurance that product quality is derived from a careful consideration of various factors such as process design, selection, and control of the process through appropriate in-process and end-process testing.^ Validation studies should be performed through each of the three phases of a product's life span: development, pilot scale, and end-process testing. In addition to validated testing methods and standards, the FDA requires a thorough validation program, quality assurance (QA) oversight, statistically sound sampling methods, rigorous training, and a comprehensive documentation trail. Undeniably, biopharmaceuticals should be safe and effective. This must be demonstrated by effectively planned studies as well as documentation to the satisfaction of regulatory agencies. The young age of this industry is demonstrated by the fact that in 1985, the FDA issued a document entitled "Points to Consider in the Production and Testing of New Drugs and Biologicals Produced by Recombinant DNA Technology." In 1997 a similar document was issued for monoclonal antibodies. Also in 1997, the Center for Biologies Evaluation and Research (CBER) issued guidance on the preparation of a Biologies License Application (BLA). For the first time, manufacturers can file a BLA instead of an Establishment License Application (ELA) and a Product License Application (PLA). The BLA brings the drug and biotechnology therapeutics registration process closer together.

BIOSEPARATIONS: AN OVERVIEW

The CBER was established in 1987 as a spin-off of the FDA's Center for Drugs and Biologies in response to a growing number of applications for new biotechnology products. "Guidelines," "Guidance," "Points to Consider," and other documents are available from CBER (Office of Training and Manufacturers Assistance, HFM-40, Rockville, MD, 20852. Information can be obtained by telephone at 800-835-4709 or by fax at 301-827-3844). It is important to keep current with the latest regulations. Generally, this information can be obtained from the FDA Web site, www.fda.gov/cber/publications.htm. A joint regulatory-industry initiative was taken to provide international harmonization of the drug approval process. The guidelines recommended by the International Conference on Harmonization (ICH) address quality, safety, and efficacy. The ICH issued draft guidelines on analytical validation procedures in 1996 and a document entitled "Draft Consensus Guidelines and Specifications: Test Procedures and Acceptance Criteria for Biotechnological/ Biological Products" in 1998. Further information relating to ICH can be found at the Web site www.ifpma.org of the International Federation of Pharmaceutical Manufacturers Association. The contents of this book have been broadly classified into three sections: • Analytical methodologies • Separation and purification methods • Other important considerations The analytical methodology section covers a variety of methodologies that are commonly used in bioseparations. The section on separation and purification methods covers a broad range of methods, including process-scale separations. Plant and process equipment, engineering process control of bioseparation processes, economic considerations, and future developments are discussed under the heading of other important considerations—those elements that are sometimes forgotten but should never be ignored when one is dealing with bioseparations. Processing plants and equipment are discussed in this book to assist the scientist or engineer in selecting a method of bioseparation that will be suited to the particular requirements of the process and the product at a commercial scale of operation. A chapter on economics of bioseparations has been included to help evaluate cost considerations prior to the initiation of any project. Finally, the chapter on future developments attempts to provide some insight into what is coming down the pike in the field of bioseparations, a field that is continually evolving and thus defies any fixed descriptive definitions.

II. ANALYTICAL METHODOLOGIES The purity analysis of a recombinant produced product is difficult because the accuracy of protein purity is method-dependent and is influenced by the shortcomings of the analytical procedures (Chapter 2). Proteins are highly complex molecules; therefore, it is generally very desirable to utilize more than one method to define a given protein's purity. To assure the purity of

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the desired product, it is important to evaluate process-related and productrelated impurities (for details, see Chapter 2). Protein purity in excess of 99% is often expected of therapeutic products. Significant impurities, such as host-cell proteins, are expected to be present at no more than trace levels (parts per million). Most proteins can be analyzed by high-pressure, or high-performance, liquid chromatography (HPLC) and electrophoretic methods. These methods are discussed in great detail in this book. A number of analytical methods are discussed at length throughout this book; Chapters 2 - 6 , 10, 14, and 15 offer fairly extensive coverage of analytical methodologies. Chapter 2 provides an excellent coverage of methods primarily used for protein impurities in pharmaceuticals derived from recombinant DNA. Protocols for selected examples are included to assist the reader in carrying out analyses of interest to them. It should be readily recognized that these methods are also useful for purity analysis of proteins as well. Because of the relative importance of analytical methodology, special chapters are devoted to HPLC (Chapter 3), capillary electrophoresis (Chapter 4), isoelectric focusing (Chapter 5), and mass spectrometry (Chapter 6). Chapter 10 covers analytical aspects of expanded-bed chromatography. The variety of methodologies used for the analysis of oligonucleotides and antibodies are covered extensively in Chapters 14 and 15, respectively.

A. HPLC Chapter 3 provides an overview of physicochemical factors that impact analysis and purification of polypeptides and proteins by HPLC techniques. The current status and some of the future challenges facing this major field of separation sciences are considered from both didactic and practical perspectives (Chapter 3). This chapter attempts to provide an overview of terms, concepts, principles, practical aspects, and primary references that underpin the recent developments in this field. Where appropriate, key relationships and dependencies that describe the interactive behavior of polypeptides and proteins with chemically immobilized ligands are discussed. This understanding is central to any subsequent exploration of alternative avenues now available for further research and development into the field of polypeptide or protein purification and analysis. HPLC techniques have occupied a dominant position for over two decades in peptide and protein chemistry, in molecular chemistry, and in biotechnology. These techniques with their various selectivity modes (listed later) can be considered the bridges that link cellular and molecular biology (viz., structural proteomics and atomic biology) and industrial process development associated with the recovery and purification technologies that turn these opportunities into realities. Different dominant interactive modes of HPLC are as follows: • Normal phase • Ion exchange • Reversed phase

BIOSEPARATIONS: AN OVERVIEW

• Hydrophobic interaction • Biospecific and biomimetic affinity This chapter considers the specific physicochemical considerations of various chromatographic modes and provides strategic considerations in HPLC separations as v^ell as heuristic approaches and productivity considerations in scale-up operations. B. Capillary Electrophoresis Electrophoresis is defined as transport of electrically charged particles in a direct-current electric field. The particles may be simple ions or complex macromolecules including proteins, colloids, or particulate matter such as living cells (bacteria or erythrocytes). Electrophoretic separation is based on differential rate migration in the bulk of the liquid phase and is not concerned with any reactions occurring at the electrodes. The highest resolution is obtained when an element of discontinuity is introduced in the liquid phase, such as a pH gradient or the sieving effect of high-density gels. Membrane barriers may also be introduced into the path of migrating particles. Electrophoresis can be classified on the basis of whether it is carried out as a free solution or on the support media. When support media are used, the technique is called zone electrophoresis. Capillary electrophoresis (CE), which is commonly used today, fits into the latter category, and at one time was called capillary zone electrophoresis. Strictly speaking, CE without any of the modifications mentioned below is not a chromatography technique because two phases are not involved in the separation process (Chapter 4). Recall that the two phases in chromatography are designated as the stationary phase and the mobile phase, based on their role in the separation process. Technically, there is no stationary phase in capillary electrophoresis unless the capillary walls are assigned that role. Some chromatographers promote this concept, but it is not entirely correct. In any event, most chromatographers are comfortable using CE because it enjoys a number of similarities to chromatography in that some of the manipulations used to optimize chromatographic separations are also suitable for CE. And symposia on CE are often included in the major chromatographic meetings. In Chapter 4, the following approaches to peptides and proteins separations with CE are discussed: Capillary zone electrophoresis (CZE) Micellar electrokinetic capillary chromatography (MECC) Capillary gel electrophoresis (CGE) Capillary electrochromatography (CEC) Capillary isoelectric focusing, another form of capillary electrophoresis, is covered in Chapter 5 and discussed briefly in Section II.C. Capillary electrophoresis has been found to be quite useful for resolving a very large number of compounds including peptides and proteins. The primary advantage of capillary electrophoresis is that it can offer rapid, high

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resolution of water-soluble components present in small volumes. The separations are based in general on the principles of the electrically driven flow of ions in solution. Selectivity is accomplished by alternation of electrolyte properties, such as pH, ionic strength, and electrolyte composition, or by the incorporation of electrolyte additives. Some of the typical additives include organic solvents, surfactants, and complexing agents. Biomolecules such as proteins, nucleic acids, and polysaccharides are often present in small quantities, and sample sizes are often limited, requiring highly selective and sensitive techniques. Since samples of biological origin are often complex, two or more different yet complementary techniques are often used to perform qualitative or quantitative analysis. The use of complementary techniques provides greater confidence in the analytical results. HPLC and CE that represent chromatography and electrophoresis fulfill this requirement. For example, in reversed-phase HPLC (see Chapter 3), the species are separated on the basis of hydrophobicity; in CE, charge-to-mass ratios play a key role. The difference in separation mechanism is helpful in the characterization or elucidation of the structure of complex molecules of biological origin. Furthermore, these techniques provide fully automated, microprocessor-controlled quantitative assays, as well as high resolution with short analysis time.

C. Isoelectric Focusing Chapter 5 covers isoelectric focusing (IFF), which is one of the commonly used techniques for the separation of proteins. It is a high-resolution method that is well suited for both analytical and preparative applications. IFF fractionations are based on the pH dependence of the electrophoretic mobilities of the protein molecules. Isoelectric focusing, as the name implies, makes use of the electrical charge properties of molecules to focus them in defined zones in the separation medium. It is the focusing mechanism that distinguishes IFF from the other separation processes and makes it unique among the separation methods. In most other separation methods, diffusion and interactions with the medium act to disperse the bands of separated materials. In contrast, the basic mechanism of isoelectric focusing imposes forces on molecules that directly counteract the dispersive effects of diffusion. During the separation process, the molecules in the sample accumulate in specific and predictable locations in the medium, regardless of their initial distribution. The focusing mechanism distinguishes IFF from the other modes of electrophoresis as well. With the other modes of electrophoresis, the applied electrical field moves molecules through the separation media at fixed rates, whereas the applied field in IFF establishes and maintains steady-state distributions of sample molecules. These distributions collapse once the field is discontinued. The basis of the electrofocusing mechanism lies in the properties of the charge-bearing constituents of proteins. The information thus provided by IFF is very useful and complements information obtained for other physical parameters. In comparison to some other separation methods, IFF is easy to

BIOSEPARATIONS: AN OVERVIEW

/

understand and relatively easy to use. The methodologies are straightforw^ard and the results can be readily interpreted. The separations are carried out under nondenaturing conditions in that proteins maintain most of their physical and chemical characteristics. During an lEF separation, proteins are subjected to the simultaneous influences of an electric field and a pH gradient. As proteins migrate electrophoretically through the pH gradient, they gain or lose protons, depending on the local pH. Their net charges assume positive, negative, or zero values according to their positions in the gradient. For every protein, there is a particular pH at which its net charge, and hence, its electrophoretic mobility are zero. This pH is called the isoelectric point (pi). Once a protein migrates to its pi, the net migration of that protein is reduced to zero. The differences in pis account for separation of proteins in lEF. Proteins are positively charged at a pH below^ their pi and negatively charged above their isoelectric points. The net charge on a protein determines its electrophoretic mobility. The key to understanding IFF is the recognition that the net charges carried by proteins are pH-dependent. Furthermore, it is important to note that net charge on a protein is the algebraic sum of all its positive and negative charges. Chapter 5 includes sufficient details on IFF to provide a better understanding of this technique. It also includes a number of applications of various proteins. Isoelectric focusing is applicable only to the fractionation of amphoteric species, such as proteins and peptides, that can act both as acids and bases. Nonamphoteric species, nucleic acids in particular, cannot be resolved by IFF. Both analytical and preparative modes of IFF, included in this chapter, have been developed as valuable tools for studying proteins. D. Mass Spectrometry The advances in technology in the last decade have transformed mass spectrometry from an analytical tool for the study of small and relatively stable molecules to a virtually indispensable technique for studying biomolecules (Chapter 6). The newrly developed ionization methods such as electrospray ionization (FSI) and matrix-assisted laser desorption-ionization (MALDI), coupled w^ith advances in instrumentation, laser and computer technologies, and data processing algorithms, enable routine detection and structural analysis of biomolecules. In addition to molecular mass determination of biomolecules, it is nov^ possible to sequence peptides, proteins, oligonucleotides, and oligosaccharides; probe protein folding; and study inter- and intramolecular noncovalent interactions. The new^ commercial mass spectrometers have a large accessible mass range equal to or greater than 300,000 Da; high sensitivity in the lov^-femtomole range; high accuracy, and mass resolution of 1 in 100,000. In contrast w^ith some of the older instruments, v^hich required an experienced mass spectroscopist to operate, the new^-generation instruments are user-friendly and can be successfully operated by various researchers in the scientific and medical communities. Unlike other spectroscopic techniques, mass spectrometry (MS) does not require the analytes to possess any special physical properties such as charge, electric or magnetic moments, radioactivity, etc. Furthermore, the short

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measurement times make this technique unique for answering a broad range of questions in a multitude of biological and medical research areas. In addition to its traditional role as an analytical tool used to solve a specific research problem, MS has become an enabling technique in the emerging field of proteomics. Mass spectrometry has played a central role in the attempts to isolate and characterize over 100,000 human proteins. It is increasingly used by biotechnology companies in conjunction with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The goal of Chapter 6 is to familiarize investigators in biological and medical research with mass spectrometry and its potential applications in these fields, with the anticipation that it will encourage and enable them to utilize MS in their research. An attempt has been made to provide a clear basic description of the modern mass spectrometers and of the most relevant types of experiments that they can perform. Discussed also are the advantages and drawbacks of the different methods in the context of biological research with examples of the ability of mass spectrometry to solve problems in this field of research. To benefit general readers, the discussion has been limited to methodologies that are accessible to nonspecialists and that can be carried out on commercially available spectrometers without special modifications. The chapter illustrates the principles of mass spectrometry by demonstrating how various techniques [MALDI, ESI, Fourier transform ion cyclotron resonance (FT-ICR), ion traps, and tandem mass spectrometry (MS-MS)] work. It also provides examples of utilizing mass spectrometry to solve biological and biochemical problems in the field of protein analysis, protein folding, and noncovalent interactions of protein-DNA complexes.

E. Methodology Montage Chapter 2 describes a number of methods that can be useful for analysis of proteins. These methods can be broadly classified as follows: • Gel electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Other modes of gel electrophoresis • High-performance liquid chromatography Reversed-phase HPLC Hydrophobic interaction chromatography Size-exclusion chromatography Ion-exchange chromatography • Capillary electrophoresis Capillary zone electrophoresis Capillary isoelectric focusing Capillary gel electrophoresis Micellar electrokinetic capillary chromatography

BIOSEPARATIONS: AN OVERVIEW

• Immunoassays Enzyme-linked immunosorbant assay (ELISA) Western blot analysis Immunoligand assay As mentioned before, a number of these methods are discussed at length in this book (see Chapters 2-6). The methodologies for separation of nucleic acids, oligonucleotides, and monoclonal antibodies are covered in Chapters 8, 14, and 15. The follow^ing chromatographic methods, as they relate to separations of monoclonal antibodies, are discussed in Chapter 15: • • • • • • • • • •

Ion-exchange chromatography Hydrophobic interaction chromatography Hydroxyapatite chromatography Protein affinity chromatography Thiophilic chromatography Hydrophobic charge induction chromatography Immobilized boronic acid ligand chromatography Dye interaction chromatography Metal chelate affinity chromatography Immunoaffinity chromatography

III. SEPARATION AND PURIFICATION METHODS As mentioned earlier, the biopharmaceutical industry is grow^ing rapidly, vv^ith over IS biotechnology drugs approved for sale in the United States alone and over 500 biopharmaceutical candidates in various phases of clinical trials. In contrast to most of the earlier biotechnology therapeutics that w^ere produced on a relatively small scale (kilograms per year), many of the recent products are expected to have production scales on the order of hundreds of kilograms per year. In addition, many biopharmaceuticals are making the transition to generic drugs, with more than one manufacturer competing for market share. Thus, there is an urgent need for the development of efficient, large-scale purification processes in the biotechnology industry. Discussed in this section are various processes used for separation and purification of proteins and other materials of biological interest, such as oligonucleotides and monoclonal antibodies. A. Liquid-Liquid Distribution The International Union of Pure and Applied Chemistry (lUPAC) recommends the use of liquid-liquid distribution rather than the traditional term, solvent extraction. However, solvent extraction is still used commonly in the literature, and that is why it is also being used here interchangeably (Chapter 7). Solvent extraction utilizes the partition of a solute between two practically immiscible liquid phases—one a solvent phase and the other an aqueous phase. Liquid-liquid partitioning methods are important separation tools in modern biotechnology. They have become increasingly popular as part of a

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downstream process for the recovery and purification of biomolecules including alcohols, aliphatic car boxy lie acids, antibiotics, amino acids, and proteins. Solvent extraction has long been established as a basic unit operation for chemical separations. Chapter 7 summarizes the effects of temperature, pH, ion pairs, and solvent selection on solvent extraction for biomolecules. Solvent extraction of fermentation products such as alcohols, aliphatic carboxylic acids, amino acids, and antibiotics are discussed. Enhanced solvent extraction using reversed micelles and electrical fields are also discussed. Solvent-extraction equipment and operational considerations are adequately covered in this chapter. Aqueous tv^o-phase partitioning using w^ater-soluble polymers and salts has proven to be an effective method in the purification of various biomolecules, especially proteins, which can be denatured by solvents in conventional solvent extraction. The effects of polymer weight and concentration, temperature, salt, and affinity ligands on aqueous two-phase partitioning have been studied. Equipment and operational considerations and large-scale aqueous two-phase partitioning of biomolecules have also been investigated. This chapter also points to sources in the existing literature for both solvent extraction and aqueous two-phase partitioning of biomolecules. Various unit operations are used in the downstream processing of biomolecules. These recovery and purification methods include cell disruption, centrifugation, micro- and ultrafiltration, precipitation, liquid-liquid partitioning, and various forms of liquid chromatography. Among them, liquid-liquid partitioning methods are well established, often inexpensive, and suitable for steady-state large-scale operations. There are two main categories in liquid-liquid partitioning. One is the conventional solvent extraction, which is used for the separations of many metabolites from fermentation, such as alcohols, carboxylic acids, amino acids, and antibiotics. The other is the aqueous two-phase partitioning using water-soluble polymers such as polyethylene glycol (PEG) and dextran, and salts such as potassium phosphate. The latter method is very attractive for the separation of biomolecules, such as proteins and peptides, and including many enzymes that may be denatured by solvents. As the scale of bioseparation processes goes up, liquid-liquid partitioning becomes more and more competitive because it is easy to scale up and it enables continuous steady-state operation. The cost for liquid-liquid partitioning is much lower than that for other more sophisticated bioseparation methods, such as liquid chromatography. This chapter provides a detailed coverage of solvent extraction for bioseparations as well as aqueous two-phase partitioning for bioseparations. B. Separation of Proteins and Nucleic Acids A large number of biologically active molecules are obtained from naturally occurring plants and animal resources. The advances in biotechnology in the past several decades enable the production of many desired compounds under controlled conditions using engineered microorganisms and cells from animals and plants. The recovery of desired products from various sources

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involves sequences of operations with the final aim of obtaining desired products at a prespecified level of purity. The steps involved in the recovery of biological products from their natural environment can be divided into four categories: separation of solubles from insolubles, isolation, purification, and pohshing. A classification of different separation methods based on physicochemical properties is given in Table 1 of Chapter 8. This chapter focuses primarily on unique problems encountered during recovery of intracellularly produced proteins. In a typical recovery and purification process for an intracellular protein, nucleic acids are first removed by precipitation. The isolation and fractionation of undesirable proteins generally follow^ this step. It is v^ell known that precipitation of nucleic acids and removal of contaminating proteins can incur large losses of desirable proteins. In this chapter, various methods of removing nucleic acid are reviewed. Also included is a case study that evaluates several precipitation methods and aqueous two-phase extraction for removal of nucleic acids from a cell homogenate of tartrate dehydrogenase (TDH)-producing strain of Pseudomonas putida, C. Displacement Chromatography Displacement chromatography is an efficient mode of preparative chromatography (Chapter 9). Operationally, displacement chromatography is performed in a manner similar to step-gradient chromatography in which the column is subjected to sequential step changes in the inlet conditions (see Chapter 9). The column is initially equilibrated with a carrier buffer in which the feed solutes exhibit a relatively high retention on the chromatographic stationary phase (e.g., low ionic strength in ion exchange, high salt concentrations in hydrophobic interaction chromatography, and low mobile-phase modifier concentrations in reversed-phase chromatography). Following the equilibration step, the feed mixture is introduced into the column, which is then followed by a constant infusion of the displacer solution. The displacer is selected on the basis of the fact that it has a higher affinity for the stationary phase than any of the feed components. Under appropriate conditions, the displacer induces the feed components to develop into adjacent "square-wave" zones of highly concentrated pure material. After the breakthrough of the displacer, the column is regenerated and is reequilibrated with the carrier buffer. The displacer, having a higher affinity than any of the feed components, competes effectively, under nonlinear conditions, for the adsorption sites on the stationary phase. An important distinction between displacement and gradient chromatography is that the displacer front always remains behind the adjacent feed zones in the displacement train, whereas desorbents, e.g., organic modifiers in reversed-phase HPLC, move through the feed zones. It is important to note that displacement chromatography takes advantage of the thermodynamic characteristics of the chromatographic system to overcome many of the shortcomings of preparative elution chromatography. Since preparative chromatography is the single most widely used unit operation for process-scale purification of biologicals, the development of

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more efficient chromatographic operations is assuming increasing importance. This chapter describes the state of the art of displacement chromatography for the downstream processing of biomolecules for purification of products from complex industrial mixtures including selective displacement and the use of retained pH gradients to displace proteins. It also provides a valuable listing, along with suitable references, of high and low molecular weight displacers employed for proteins in the ion-exchange displacement chromatography of proteins. The object of this chapter is to summarize the recent developments in this field and to place in perspective the role that displacement chromatography could play in preparative separations in the years to come. D. Expanded-Bed Adsorption When purifying biomolecules from a complex mixture, a sequence of unit operations is normally needed. Each of these steps involves losses of substance, adding to the overall cost of the process. One way to simplify the situation has been through process integration. This may be done by combining a bioconversion step with one or two steps involving separation. However, it is possible to combine two or more steps in downstream processing and thus reduce the number of different processing steps that are needed. Still another way may be to design processing steps that eliminate the need for a certain treatment. This is the case when the concept of affinity-mediated separation is used (Chapter 10). The use of biospecificity in the interaction replaces several different steps that previously had to be used. A key problem that does not involve difficult theoretical challenges, but rather technical and economical ones, is the need to remove particulate matter before any substantial chromatography can be appfied (Chapter 10). The problem is that particulate matter can clog the column and thereby destroy the separation power. Another option is to use a batch procedure in which an adsorbent is added directly to the feedstock in a stirred tank. The advantage of this method is that the product is captured directly from the unclarified feedstock; however, the disadvantage is that the stirred tank acts as one theoretical plate in a separation process, leading to a long process time because of poor contacting efficiency. One way to circumvent such problems would be to use fluidized-bed adsorption instead of a packed-bed mode of operation. If just the adsorption-desorption of one single entity is wanted, then the fluidized-bed technique may be sufficient. However, there is a constant mixing, and thus extreme band broadening upon passage of a pulse of hquid through such a reactor. The concept of expanded-bed chromatography combines the advantages of good distances between the chromatographic beads when operated in the expanded mode and the adsorption power of the adsorbent particles without severe back mixing. The particles tend to be stored spontaneously with regard to size and density, so that the smaller and lighter particles will be found in the top fraction of an expanded bed that has been operating until equilibrium is established. The particles tend to be sorted spontaneously with regard to size and density, so that the smaller and lighter particles will be found in the top fraction of an expanded bed that has been

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operating until equilibrium is established. The larger and denser particles are found in the bottom section of the bed. It has furthermore been proven that a fraction from the top will be found in the top the next time the column is in operation. It is therefore realistic to state that an expanded-bed column is stable with regard to particle size-particle density distribution. Therefore, back mixing must be less than that found in a fluidized bed. It has, in fact, been shown that expanded beds show a relatively low dispersion, and thus such beds would be useful for separation purposes. The complete process for obtaining pure proteins can be divided into three main steps: capture, purification, and polishing (Chapter 11). The first step is the immobilization of the target protein onto some adsorptive surface, and it can be viewed as a combination of clarification, concentration, stabilization, and initial purification. Because the starting protein solution (feed stock) is usually crude, it is essential to clarify the solution. The traditional or conventional approach involves centrifugation, microfiltration, ultrafiltration, or diafiltration before the target protein solution can be loaded on an adsorbing material, utilizing packed-bed chromatography. The clarification step is a demanding operation and is particularly difficult when processing large quantities of microorganisms, especially disrupted microorganisms. High-speed, large-scale centrifugation and microfiltration are the most common processes used to obtain protein solutions that are suitable for packedbed chromatography; therefore, it is obvious that an approach that eliminates the clarification step can significantly simplify and improve the overall purification process. Direct adsorption of the protein not only eliminates the clarification step, but also produces a concentrated and partially purified product ready for the next purification step (see Fig. 1 in Chapter 11). Several protein capture procedures, such as batch adsorption, solvent extraction, and expanded-bed adsorption, do not require centrifugation and filtration. This chapter describes the expanded-bed adsorption approach for capturing target proteins. In the expanded-bed mode, the starting protein solution is pumped through a bed of adsorbent beads that are constrained by a flow adapter. As a result of the upward flow and the properties of the beads, the bed expands as spaces open up between the beads. If the physical properties of the beads are significantly different from those of the particles in the feed stock, the particles can pass through the bed without being trapped. An effective process depends on parameters such as viscosity, ionic strength, sofid content, and pH of the feed stock as well as the linear flow rate. A number of applications are given in this chapter that detail the capture and recovery of intracellular proteins including recombinant proteins. The initial protein recovery steps, regardless of the source, are usually associated with large volumes and crude solutions, requiring removal of particles and reduction of volume before their purification can take place. Centrifugation, filtration, precipitation, solvent extraction, and batch adsorption are common unit operations involved in the preliminary steps of protein recovery. Expanded-bed adsorption, as described here, is an approach for the initial protein recovery that eliminates the need for clarification and volume reduction. In this process, a crude starting solution is pumped directly onto an

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adsorbent matrix, which is in an expanded state in a special column. This expanded state provides enough space for contaminating particles to move through, and at the same time, it enables the interaction between the targeted protein and the matrix. Following this capturing step, the protein can be eluted from the matrix, which at this stage is in an unexpanded state. The result of this process is volume reduction and partial purification. The chapter lays out the principles of expanded-bed adsorption, it describes the columns and the matrices that are used, and it provides examples for recovering various proteins from various sources. As mentioned before, the use of chromatographic particles in fluidized beds often gives poor separation because of back mixing. However, when a heterogeneous population of chromatographic particles (with regard to size and density) is used, an ordered arrangement is detected, where the denser and larger particles are found at the bottom and the smaller and lighter particles at the top of the bed. Such beds are called expanded beds. When expanding the bed, distances are introduced between the individual particles in the bed. This fact forms the basis for the ability of expanded beds to be used in connection with particle-containing material, e.g., fermentation broths. When applied in downstream processing, expanded-bed chromatography offers possibilities to recover products without previous separation of cell mass or cell debris. This new concept is often mentioned as capturing. E. Membrane Chromatography Membrane chromatography is gaining wider interest and acceptance in the process bioseparation industry (Chapter 12). Better understanding of membrane materials, large-scale availability, and identification of niche applications have promoted this new technology. The focus of Chapter 12 is on aspects of membrane adsorbers (MAs) that have the greatest impact on large-scale preparative chromatography applications in the bioindustry. It also addresses a few novel technologies, such as thin columns, monolithic matrices, and innovative media, which seem to cross the classical definition of chromatography media. The general technology is fairly well known. This chapter attempts to provide a state-of-the-art look at the various MA separation modes, discusses commercially available technology, and provides guidelines to develop large-scale applications based on this technology. MAs are membranes with chemically functionalized surface sites for chromatography. The appeal of the membrane-based chromatographic surface stems from the fact that it is an ideal monolithic support, i.e., it is a uniformly distributed chromatographic surface with convection-enhanced separation. On the practical side, membrane absorbers can be in modular form, leading to easy and convenient use. The scale-up for MA builds on the knowledge base gained in scale-up of technologies of both chromatography and membrane filtration. To rival traditional chromatography, MA technology must address the age-old chromatography problems. The ligand chemistry should allow high dynamic binding capacity, as well as very low nonspecific binding. The development of membrane housings should consider appropriate fluid distribution to take advantage of the high resolution. The

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ultimate challenge is to develop usable membrane devices capable of good chromatography. A viable alternative to bead-based chromatographic supports should be able to deliver consistent results after repeated cleaning cycles and should be able to fulfill all FDA regulatory guidelines. Table 1 in Chapter 12 lists the commercially manufactured modules and their available formats and chemistries. The geometries reflect the v^idely used formats in membrane filtration: primarily, flat sheets in filter holders, flat sheets wound in spiral or cylindrical configuration, and hoUow^-fiber membranes. Syringe filters are popular housings containing flat sheet MAs. These enable easy lov^-pressure chromatography v^ith low^ cost, disposability, and easy connectivity of units for series operation. These may also be connected to chromatographic v^orkstations. These syringe-type filters can function in place of small chromatographic columns and are w^ell suited for quick method development. The hallmark of membrane separations is its speed; membranes are capable of flow^ rates 10- to 100-fold faster than classical chromatography. Furthermore, they offer good resolution and capacity. The rate-limiting step in the mass transfer is the diffusion of solutes due to the functional groups. Discussion covers these topics as well as scale-up and a variety of interesting applications that include purification of a recombinant vaccine protein, reduction of viral DNA and endotoxin under good manufacturing practice (GMP) conditions, monoclonal antibody purification, and purification of oligonucleotides. F. Simulated Moving Bed Chromatography A number of different products are now purified by chromatographic processes, from the laboratory scale (gram quantities) up to the industrial pharmaceutical scale (a few tons per year). Among the possible technologies, elution HPLC technology (sometimes with recycle) has taken a very important part of the small-scale (10 tons/year) market during the previous decade. And simulated moving bed (SMB) technology has been extensively used for very large scale fractionation of sugars and xylenes for the last 30 years (Chapter 13). Presently, there is considerable interest in the preparative applications of liquid chromatography, even though it is often considered expensive. To make the chromatographic process more attractive, attention is focused on the choice of the operating mode in an effort to minimize eluent consumption and to maximize productivity, which is of key importance when expensive packings are used. Among the alternatives to the classical process (elution chromatography), much attention is paid to SMB. Although SMB is well known as a process that is able to maximize productivity and minimize eluent consumption in some industries, it has been ignored in the pharmaceutical and fine chemical industries during the last 30 years. The reasons may be the patent situation and the complexity of the concept. Recently, separations of pharmaceutical compounds have been performed using SMB technology. It is now considered a real production tool (for instance, the Belgian pharmaceutical company U.C.B. Pharma recently announced the use of SMB for performing multiton-scale separation of

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optical isomers). Small plants are now commercially available for fine chemical industry, the pharmaceutical industry, and biotechnologies. The basic idea of a moving bed system is to promote a countercurrent contact between the solid and the hquid phases. The concept and principles of SMB are discussed at length in Chapter 13, and applications of protein purifications and other complex molecules are given. G. Purification of Oligonucleotides There is a great interest in nucleic acids and oligonucleotides because of recent developments in biochemistry, genetic engineering, genomics, and antisense therapeutics. Oligonucleotides are primarily used as diagnostic tools in biochemical research, and they are being developed as therapeutic agents (Chapter 14). The purification problem in these cases is different in the amount of oligonucleotides required. For example, in one case a large amount of a fewer number of compounds is required in stringent therapeutic quality, whereas, in the second case, a large number of compounds is required with very high throughput in small quantities. However, the general application of separation techniques is similar in both cases. There are presently more than 12 antisense oligonucleotides in human clinical trials. Recently, Fomivirsen (Isis Pharmaceuticals) became the first antisense drug approved by the U.S. FDA. Further success of such compounds is likely to spur greater innovation and development in all facets of oligonucleotide manufacturing and purification. Chapter 14 focuses on two areas: • Application of various modes of separations for purifications of these compounds • State-of-the-art large-scale technologies for purification of therapeutic antisense oligonucleotides In general, antisense oligonucleotides are short single-strand DNA or RNA analogues. The current therapeutic candidates in clinical trials are mostly within 30 nucleotides in length. Many of these compounds are phosphorothioate analogues, where the nonbridging oxygen of the DNA backbone is replaced by a sulfur atom. This chemical modification improves the stability of the oligonucleotide from degradation by cellular nucleases. There are many other chemical modifications of DNA molecules in the literature, under development, and in human clinical trials. Chapter 14 deals primarily with phosphorothioate-modified oligonucleotides because of their rapid progress in clinical trials and the possibilities of NDA (New Drug Application) submission to the FDA for approval as drugs. In recent years, DNA synthesis technology has advanced significantly because of the need for large-scale oligonucleotides for human clinical trials. Gene machines that barely made 1 mg of oligonucleotide now have been scaled up with advanced technology to synthesize almost a kilogram of oligonucleotide per synthesis campaign. Solid-phase oligonucleotide synthesis technology has progressed further up to now compared with solution-phase synthesis. Various column geometries and fluid contact mechanisms have been tested, but packed axial flow columns have been most successful and are

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commonly used at the largest scales. The reagents are pumped in through automated process computer-controlled protocols. The use of a packed column affords optimal solvent efficiency and better process control. Purification strategies and large-scale purification of therapeutic oligonucleotides are also discussed in Chapter 14. H. Monoclonal Antibodies The FDA has approved monoclonal antibodies and recombinant antibodies as therapeutics and diagnostics relatively recently (Chapter 15). Monoclonal antibodies have now become one of the largest classes of proteins currently in clinical trials. This success has resulted from the large intellectual and capital investment that has been made in them. As a result, significant progress has been made in understanding antibody function, host-defense mechanisms, the role of antibodies in cancer, and substantial improvement of production and purification technology. The development of protein-free culture media, continuous production of animal cells in perfusion culture, genetically engineered "humanized" antibodies, single-chain antibodies, phage display, and cellsurface display libraries have been important steps in this dynamic discipline. The design of antibodies according to the special needs for therapy, diagnosis, and purification technology is now^ possible. Specific properties for in vivo behavior such as defined pharmacokinetics and tumor targeting are simply achieved by combining various fragments with desired properties. These represent a few examples of recent progress. Antibodies are expressed by hybridoma cells formed by cell fusion of sensitized animal or human B lymphocytes with myeloma cells, or they are generated by EBV (Epstein-Barr virus) transformation of sensitized B lymphocytes. Other heterologous expression systems such as bacteria, yeast, insect cells, and mammaHan cells have also been used for expression of antibodies and their fragments. However, because of renaturation problems, glycosylation, and expression levels, mammalian cells are mostly used for the expression of monoclonal antibodies. More recently, technologies have been extensively developed for the expression of antibodies in transgenic animals and transgenic plants. Intact antibodies with biologically active glycosylation profiles, crucial for the effector functions, require eukaryotic expression {in vitro or in vivo). These circumstances have inspired many scientists to find effective methods for production, as well as methods for the selection of the best extractionpurification methods. Purity, safety, potency, and cost-effectiveness are some of the main factors that should be considered when designing an expression method and, more importantly, when defining the purification processes. The purification of antibodies was most likely initiated with the separation of proteins, mainly paraproteins, several decades ago. A plethora of protocols have now been described involving precipitation with a variety of chemical agents, electrophoretic separation, membrane methodologies, and liquid chromatography. The latter probably represents the most popular technique because of the ease of implementation, the capability to play on the selectivity, and the

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level of purity that can be achieved. Specific liquid chromatography methodologies and resins have been especially developed for this purpose. To date, monoclonal antibodies and immunoglobulins v^ith all their derivatives represent by far the largest class of produced and purified proteins in numbers and mass. Chapter 15 focuses mainly on antibody purification by chromatographic means. Numerous sorbents have been developed for protein separation, and they are based on a variety of adsorption-desorption principles. Selection of suitable materials and principles depends on the properties of the particular immunoglobulins to be separated and on the composition of the impurities that constitute the feedstock. Antibodies are very diverse in molecular properties, chemical characteristics, and biological activity. Purification strategies are therefore also diverse, since they are based on a large variety of molecular interactions. Antibodies have several common properties that are frequently exploited from the initial feedstock. Knov^ledge about the nature and concentration of impurities is the key to success. Therefore, the initial composition of feedstock is important when designing the separation process. The expression system can be selected to simplify the extraction-purification procedures. Chapter 15 provides an in-depth coverage of various chromatographic methods, such as ion exchange, hydrophobic interaction, affinity, ligand, immunoaffinity, gel filtration, etc., that can be used for separations of antibodies by liquid chromatography.

lY. OTHER IMPORTANT CONSIDERATIONS The selection of appropriate processing plants and equipment, economics, and future developments are important considerations that are discussed in this section. A. Processing Plant and Equipment The diversity of industries that involve bioseparations has led to the development of a w^ide range of techniques and unit operations for the efficient processing of biological materials. Chapter 16 is planned to aid the scientist or engineer in selecting a method of bioseparation that w^ill be suited to the particular requirements of the process and the product at a commercial scale of operation. The complexity of biological processes generally requires many stages to produce a final, purified product from a particular composition of rav^ materials. Although a typical bioprocess consists of tw^o main parts, upstream fermentation and downstream product recovery, it is not unusual to have betv^een 10 and 20 steps in the overall process. This reflects the complex nature of a typical fermentation broth, v^hich w^ill consist of an aqueous mixture of cells, intracellular or extracellular products, unreacted substrates, and by-products of the fermentation process. From this mixture, the desired

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product must be isolated at a given purity and specification, and all of the unw^anted contaminating materials must be removed. The choice of a bioseparation technique w^ill depend on a number of factors, including the initial location of the product inside or outside the cell, as w^ell as the product size, charge, solubility, chemical or physical affinity to other materials, and so on. Economic factors also come into play, including the value of the product, the regulatory environment in w^hich the product is manufactured, and the balance betw^een the capital cost of the bioseparation equipment and the operating cost of running it. In moving from laboratory- or pilot-scale processing to full-scale manufacturing, it can be difficult to scale up certain types of bioseparation equipment easily; for example, high " g " centrifuges are available as benchmounted units (using test tubes), but an equivalent industrial machine v^ith a similar g force is unlikely to be a cost-effective solution, even if it were possible to build a suitable unit. It w^ould not be realistic to consider 10 or 100 identical units as a realistic alternative. Compromises are therefore required as a process is commercialized, to ensure that the process remains technically and economically feasible. Chapter 16 provides guidance relating to the choice of industrial bioseparation equipment that is available and the issues that must be taken into account when selecting a suitable system to meet both technical and economic objectives. B. Engineering Process Control Chapter 17 deals briefly with the engineering process control, which primarily involves measurement of a product property and comparison to a desired value. The process operation can be thus immediately adjusted to reduce deviation from the specifications. This feedback procedure can be used to adjust the process whenever the product deviates from the set point and can be used to change operating points and to reject the effect of outside disturbances. C. Economics of Separations Valuable products are being produced increasingly by biotechnological methods. By the year 2000, the worldwide sales of these biotechnological products will be around $100 billion (Chapter 18). The Western hemisphere countries, particularly the United States, Germany, France, and England, are the leading players. For example, diabetic and thrombolytic drugs have a very large market throughout the world; hence it would be desirable to review the sales of the above-mentioned classes of drugs. In the United States, the estimated market for diabetes drugs is $1.8 biUion, including $800 million for insulin. The estimated market for thrombolytic drugs is $355 million. Biotechnology products include not only pharmaceutical drugs but also other biological macromolecules of interest. One must separate the biological macromolecule of interest; and herein lies a very significant cost of the entire manufacturing process. For different processes, the fraction of the entire cost of the process

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required for bioseparation will vary. The purification and recovery costs may be as high as 80% of the total manufacturing costs. These costs may be higher if ultrahigh purity DNA-involved products are manufactured. Recognize that during processing, one may have to purify products at 99.9% levels with virtually complete removal of DNA, viruses, and endotoxins. The key to cutting production costs is to emphasize improvements in downstream processing. Traditionally, all the steps occurring in the fermenter that result in the production of the desired biological macromolecule can be considered as upstream processes. All the other processes occurring after the fermentation and which result in the separation, purification, concentration, and conversion of the biomolecule to a form suitable for its intended final use can be classified as downstream processes. Thus, it is helpful to better analyze and understand the different facets involved during downstream processing. Better physical insights are required and are continuously being obtained in downstream processes, and these will eventually lead to a more efficient and economical process. Note that upstream processes are already well understood. Even though further improvements in upstream processes are possible, they do not have the potential of making as significant an impact on production costs as improvements in downstream processes. Also, one should not treat the upstream and downstream processes separately, but should integrate the downstream processes with the upstream processes. For instance, any change or improvement envisaged in an upstream process should also consider the possible effect of this change on the downstream process. Minor changes in upstream conditions may have a significant impact on downstream processes. Thus, early in the design of processes, one must consider the impact of upstream processes on downstream processing. One technique where different possible "what-if" scenarios may be analyzed is with the development of an appropriate model for the process. The importance of computer-aided process simulation, and the early necessity of providing a workable process flow sheet can not be overemphasized. These activities should be carried out during the early stages of process development and can serve as an important tool to help optimize the process expeditiously. Considering the high stakes that are involved in getting a drug to the market and the fierce competition involved, it seems appropriate to get as much useful information on a process as early as possible during the development process. This also explains the extreme secrecy involved in the research and development of key steps in the bioprocessing of a highly marketable and valuable product. Chapter 18 emphasizes that drug manufacturing is a high-risk, high-gain business that requires economic analysis at each stage of the developmental process to minimize costs. Several examples are provided to assist the reader in evaluating the economics of bioseparation of a given process.

D. Future Developments The field of bioseparations is very dynamic. As a result, new developments are constantly being made in the techniques discussed here. At the same time.

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new techniques are also evolving that w^ill have an impact on this field in the future. Chapter 19 attempts to address this topic. The important point to recognize is that all future developments are targeting larger separations in the shortest possible time, v^ith the objective of low^ering costs so that these processes become economically more feasible. REFERENCE 1. Sadana, A. (1998). In "Bioseparation of Proteins" (S. Ahuja, ed.). Academic Press, San Diego, CA.

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ANALYSIS OF PROTEIN IMPURITIES IN PHARMACEUTICALS DERIVED FROM RECOMBINANT DNA« DONALD O. O'KEEFE Bristol-Myers Squibb, Macromolecular Structure and Biopharmaceuticals, Princeton, New Jersey 08543 I. INTRODUCTION A. The Regulatory Environment B. Purity Analysis of Recombinant Pharmaceuticals C. Sources and Types of Impurities D. Levels and Identification of Impurities II. PROTEIN IMPURITY ANALYSIS A. Gel Electrophoresis B. High-Performance Liquid Chromatography C. Capillary Electrophoresis D. Immunoassays E. Identification of Host-Cell Protein Impurities III. SUMMARY IV. CASE STUDIES A. Identification of a Host-Cell Protein Impurity in Recombinant Acidic Fibroblast Growth Factor B. Selective Resolution of a Protein Impurity Using RP-HPLC and Fluorescence Derivatization C. Detection of N-terminal Variants Using Peptide Mapping and Fluorescence Detection REFERENCES

INTRODUCTION Recombinant DNA methodology has come of age. It has spawned a growing industry that seeks to commerciahze products derived from this technology. Most notable among these biotechnology products are those for human therapeutic use, and of these there are two categories: proteins and nucleic acids. The research, development, and commercialization of therapeutic proteins derived from biotechnology is far more advanced than that of nucleic *To reproduce or otherwise use this article in whole or in part, permission must be obtained from Bristol-Myers Squibb, the author, and Academic Press.

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Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

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acids and therefore the former will be the focus of this chapter. In 1982 the Food and Drug Administration (FDA) approved human insulin synthesized in Escherichia coli as the first recombinant therapeutic in the United States. Since then over 75 other recombinant proteins have achieved this same status.^'^ Among these are hormones, cytokines, vaccines, and monoclonal antibodies. Simultaneous v^ith this success has been the proliferation of biotechnology companies. In the United States alone there are more than 1100 such companies,^ and the economic impact of this grov^th is substantial. Current annual revenues of biotechnology drugs in the United States alone total $8 billion and account for nearly 10% of all therapeutic sales in the United States.^ Significant among these sales is that of recombinant erythropoietin, which in 1997 had worldwide sales in excess of $1 billion. This financial potential partially explains the approximately 500 biotechnology drugs currently in clinical trials.^ The increased onslaught of biotechnology drugs in the last two decades has challenged both the regulatory agencies responsible for approving new drugs and the biotechnology industry, which must consistently produce a definable and safe product. A. The Regulatory Environment As with traditional drugs, biopharmaceuticals must be safe and effective to be approved by national regulatory agencies. From the very beginning, it was intuitive that a high degree of purity is elemental to the safety of recombinant therapeutics. But what level of purity is sufficient? Initially there were no predetermined guidelines. This led to extensive and in-depth discussions between regulatory agencies and the first companies attempting to develop recombinant proteins as human therapeutics.^'^ Thereafter, the FDA issued working draft guidelines to industry for producing and testing these biologicals in the form of "Points to Consider" documents.*''^"^ These guidelines are not binding requirements but instead are recommendations on how to direct the clinical development of a biopharmaceutical. Likewise, other national and international agencies have done the same.^~^ Fundamentally, all these agencies have the same responsibility, i.e., assuring the quality (purity), safety, and efficacy of biopharmaceuticals. Over time, however, the technical and clinical standards for biopharmaceuticals under development have diverged among the national regulatory agencies to the point that independent studies and licensing applications must be made in the individual countries. The drawbacks here are obvious, and they have led to a regulatory renaissance at the start of the 1990s. In 1990 a joint regulatory-industry initiative was conceived to provide international harmonization of the drug approval process. The result has been the ongoing International Conference on Flarmonization (ICH). The ICH is charged with developing harmonized guidelines on technical issues relating to drug development. The ICH is attempting to codify consensus guidelines for obtaining market approval of drugs on a worldwide scale. The * These documents, as well as others produced by the FDA, can be obtained through the Internet at the website of the FDA at www.fda.gov.

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25

guidelines recommended by the ICH are listed under the four separate topics of quality, safety, efficacy, and multidisciplinary.* Within the quality topic are specifications for testing biotechnology products.^^ This guideline and others proposed by the ICH appear to be the future for international drug development. Therefore, these documents will provide the boundaries for any discussions regarding purity, impurities, and contaminants.

B. Purity Analysis of Recombinant Pharmaceuticals An important fact inherent in the purity analysis of a recombinant pharmaceutical is that the absolute purity of any protein is an elusive, if not an unobtainable, measurement. For biopharmaceuticals, purity is a relative term. Protein purity is method-dependent and is defined by the shortcomings of the analytical procedure. Also, unlike small traditional drugs, proteins are highly complex molecules. For these two reasons, more than one method must be utilized to define a protein's purity. The greater the number of methods used in the purity analysis, the greater the assurance is that the product is pure. Furthermore, the purity determined by an analytical method can only be properly interpreted based on the method's validation. Analytical methods vaUdation is critical to and inseparable from purity determinations. A detailed discussion on analytical methods validation is beyond the scope of this chapter but other sources of information are available for the interested reader.ii-i^ Purity analysis of therapeutic recombinant proteins generally occurs at two stages of the production process. The first material tested is the drug substance, or bulk material, which is the final purified active product prior to formulation. Upon dilution to the final dose, the addition of excipients, and possibly lyophilization, the protein preparation is referred to as the drug product or finished product. Any constituent within these two preparations that is not the active product or an excipient, excluding contaminants, is an impurity. ^^ At these two stages, the purity assays of the bioanalyst are selected based on the potential impurities that are found in the drug substance or the drug product.

C. Sources and Types of Impurities There are two categories of impurities: process-related impurities and product-related impurities. Process-related impurities are components derived from the manufacturing process. Included here are fermentation ingredients, host organism components, and process additives to Hst a few. Product-related impurities are variants of the desired protein product that do not have the desired biological activity, safety, or efficacy. Examples of these impurities might be aggregates, degradates, or misfolded isomers of the protein. Product-related impurities can also arise during storage and are an indication of * ICH topics and guidelines can be obtained at the Internet website of the International Federation of Pharmaceutical Manufacturers Association (IFPMA) at www.ifpma.org.

26

DONALD O. O'KEEFE

the instability of the drug substance or the drug product.* Product-related impurities are distinguishable from product-related substances. The later are molecular variants that have activity, safety, and efficacy indistinguishable from the desired product.^^ An example of product-related substances might be different glycosylated forms of a glycoprotein, for example, recombinant tissue plasminogen activator.^ The analysis of product-related substances will not be presented in this chapter, but many of the analytical techniques for their testing are the same as those used for impurity analysis. A compilation of important process- and product-related impurities is given in Table 1 along v^ith some techniques commonly used for their analysis. Little attention in this chapter will be given to contaminants, which are distinct from impurities. A contaminant is any entity that adventitiously enters the production process, drug substance, or drug product. This includes viruses, mycoplasma, bacteria, fungi, and their products. The control of contamination relates to process validation issues and will not be discussed here. * Protein stability, and the techniques for its analysis, is a related but separate topic from this chapter and will not be covered in depth, but references are available for the interested reader.i'^'i^

TABLE 1 Important Impurities in Recombinant Pharmaceuticals impurity Product-related impurities Aggregates (including dimers) Denatured forms Degradates Deamidations Oxidations (methionine sulfoxides) Amino acid substitutions Misfolded conformers (S-S isomers) N-terminal variants Fragmented products Process-related impurities Culture derived Culture media proteins Amino acids Inducers Antibiotics Downstream derived Solvents Protein denaturants Reducing agents Column leachables (antibodies, protein A) Trace metals Enzymes (nucleases) Host cell derived Host proteins DNA (genomic and vector) Endotoxin and other pyrogens

Common methods of analysis

SEC; PAGE; CGE HIC lEF; CIEF; lEX; peptide mapping; chromatofocusing Peptide mapping Peptide mapping HIC; peptide mapping RP-HPLC; peptide mapping SDS-PAGE; RP-HPLC; HIC

Immunoassay RP-HPLC HPLC HPLC Gas chromatography HPLC HPLC Immunoassay Atomic absorption spectroscopy Immunoassay SDS-PAGE; HPLC; CE; immunoassay Hybridization LAL; rabbit test for pyrogens

PROTEIN IMPURITY ANALYSIS

27

The analytical measurement of process-related impurities is not always routine. Some of these impurities are well defined and therefore detecting and quantitating these are straightforward. Examples here include reducing agents, chaotropes, detergents, eluent components used in chromatography, or media additives to benefit the growth of the host organism. The same is generally true for product-related impurities. Conversely, other process-related impurities are not well defined, and therefore their detection and quantitation can be more difficult. Herein Hes the challenge facing the bioanalyst. Included in this category are nucleic acids and host-cell proteins. Of these two types of potential impurities, host-cell proteins are the most difficult to address because of the large diversity of proteins that exists. In E. coli, the vanguard of recombinant organisms, the sequenced genome suggests there may be greater than 4200 different proteins in this organism, each one a potential impurity. ^^ Moreover, other recombinant hosts, such as the yeast Saccharomyces cervisiae^ mammalian cell lines, and transgenic animals, are eucaryotes and their greater complexity leads to ever more potential protein impurities. D. Levels and Identification of Impurities Well-documented guidelines do exist for the analysis of impurities in traditional small molecular weight drugs.^^ This analysis includes the identification, quantitation, and qualification of all impurities. Once the analysis is complete, initial limits are set for each impurity based on its known toxicity profile. For biopharmaceuticals, on the other hand, the allowable levels and the identification of impurities are less standardized, despite the progress of the ICH. The required purity level for a recombinant pharmaceutical is dependent on several factors. Possible factors considered by regulatory agencies include the size and the frequency of the dose, the route of administration [e.g., topical versus intra muscular (IM)], the duration of the administration (chronic or short term), the intended use of the drug (therapeutic versus prophylactic), the seriousness of the disease (risk versus benefit assessment), the patient population (elderly versus young), and the results of preclinical studies. Impurities in recombinant pharmaceuticals have been commonly categorized by quantity. Those in excess of 0.5% are considered major impurities and, if possible, their toxicity, immunogenicity, and pharmacology should be evaluated if they cannot be eliminated. Impurities less than 0.5% are minor but still should be identified.^^ Major impurities are often product-related impurities, while minor impurities are generally process-related impurities. It is the process-related impurities that are the most worrisome from a potential health hazard perspective and their levels should be reduced to parts per million (ppm, nanogram impurity per milligram recombinant product) or less. Potential hazards due to impurities can include oncogenicity (both protein and DNA), unwanted immunological responses that create anaphylactic or allergic reactions, different pharmacology or antigenicity of product-related impurities, and general or specific toxicity. Furthermore, impurities might adversely affect the protein pharmaceutical by altering either its activity or its stability prior to administration. With these potentially

28

DONALD O. O'KEEFE

deleterious effects, proteins derived from recombinant DNA are expected to be of high purity. Protein purities in excess of 99% are not uncommon, and are often expected for therapeutics. A complete purity analysis, however, not only reports the purity as a percentage (generally weight-to-v^eight) but also reports the level of impurities. Significant impurities such as host-cell proteins are reduced to ppm levels v^ith today's sophisticated purification techniques. Yet even at 99.99% purity (100 ppm of impurities) a 0.1 m g / k g dose for a 70 kg patient exposes the subject to 0.7 fig of impurities. With repeated dosing over a long time, the cumulative health effect of these impurities might be significant. Therefore, it might be necessary to identify these minor impurities to assure greater safety of the drug product. This might appear to be a daunting task for the bioanalyst, but as the sophistication and the sensitivity of both analytical instruments and procedures continues to increase this challenge will be met. When is a recombinant therapeutic pure enough? What levels of impurities are acceptable? The foregoing discussion makes it apparent that such questions are not easily answered. The regulatory agencies have generally agreed with this assessment and therefore have adopted a policy of considering each therapeutic protein on a case-by-case basis. In this regard, information on the potential patient population and the proposed therapy, results of preclinical and clinical studies, and a complete analytical package are indispensable. The latter, of course, should include a thorough and complete purity and impurity analysis, both qualitative and quantitative. It is apparent that impurity analysis in recombinant pharmaceuticals is a broad topic. This chapter is not intended to be either an all inclusive or comprehensive treatise on the subject. Such a chapter would be impossible, given the diversity of both recombinant products and the processes used for their production. Hence, there is no generic protocol or blueprint for performing impurity analysis. Therefore, this chapter will focus on presenting and discussing the methodology most commonly used to analyze process- and product-related protein impurities in therapeutic proteins derived from recombinant DNA. It is hoped that this chapter will provide a starting point for these analyses and expose the reader to many available options. Further in-depth pursuits can be satisfied by consulting the list of references at the end of the chapter. II. PROTEIN IMPURITY ANALYSIS Protein impurities are either process- or product-related impurities. Processrelated impurities include proteins added to the culture medium, proteins used during purification, such as nucleases and chromatography ligands, and proteins from the host organism. Product-related impurities include degradates, aggregates, and conformational isomers of the recombinant drug product. Eliminating all protein impurities in a recombinant pharmaceutical is realistically impossible. In fact, proteins are the most common impurity in

PROTEIN IMPURITY ANALYSIS

29

recombinant drugs and they also may be potentially the most deleterious. Furthermore, protein impurities, as a class, are more complex compared to other potential impurities. This complexity makes the use of a single method for impurity analysis unsuitable. Methods that separate based on different physicochemical properties need to be utilized jointly. All these factors explain both the effort and the number of techniques for protein impurity analysis. The major techniques to analyze protein impurities are gel and capillary electrophoresis, high-performance liquid chromatography, and immunoassays.

A. Gel Electrophoresis i. Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis

Polyacrylamide gel electrophoresis (PAGE) of proteins is a high-resolution separation technique for purity analysis. Proteins, which are multicharged macromolecules, migrate in an electric field. When proteins in a porous polyacrylamide gel are subjected to an electrical current, they migrate based on their total charge and molecular size, i.e., their charge-to-mass ratio. When the anionic detergent sodium dodecyl sulfate (SDS) is added to the gel, the detergent binds uniformly to proteins at a ratio of 1.4 g SDS per 1.0 g of protein.^^ SDS imparts an overall negative charge to proteins, giving each one an identical charge-to-mass ratio. Hence, SDS-PAGE separates proteins based solely on their mass. Although protein electrophoresis exists in many forms,^^ the best methods utilize a discontinuous system. In these methods different components comprise the buffers for the gel, the sample, and the reservoir chambers. Upon application of an electric current, a steep potential gradient is created that causes the proteins to undergo the stacking that is responsible for the high resolution of discontinuous systems. SDS-PAGE is the most common electrophoretic method used for impurity analysis and the protocol adopted from Laemmli is the standard.^^ This method is often the first step employed to analyze a protein's impurity profile because of its ease of use, and it requires little development time. The ICH recommends that SDS-PAGE impurity analysis be done under both reducing and nonreducing conditions with increasing amounts of purified protein.^^ The actual amounts of protein analyzed will depend on the staining technique used after electrophoresis (see later). The protocol given next has been found suitable for a broad range of recombinant proteins. Protocol I: SDS-PAGE

Suitable glass plates are assembled to produce a resolving gel of 14 cm X 15 cm X 1.0 mm. The Protean II electrophoresis apparatus from BioRad has worked well in the author's laboratory but other equipment can be used. The resolving gel is prepared according to the following recipe:

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DONALD O. O'KEEFE

SDS'PAGE Resolving Gel Percentage polyacrylamide

4%

8%

10%

3.0 M Tris-HCl,pH 8.8 7.5 7.5 7.5 Deionized water 17.9 13.9 11.9 30% acrylamide 0.8% bis-acrylamide 4.0 8.0 10.0 Degas under vacuum for at least 5 min 10%SDS 0.6 0.6 0.6 10% ammonium persulfate 0.2 0.2 0.2 TEMED 0.02 0.02 0.02

12%

7.5 9.9 2.0 0.6 0.2 0.02

14%

7.S 7.9 14.0 0.6 0.2 0.02

16%

7.5 5.9 16.0 0.6 0.2 0.02

Recipe notes: All volumes are in milliliters. Ammonium persulfate is made fresh before use. TEMED is N, N, N\ N'-tetramethylethylenediamine. Once the resolving gel is poured between the sealed glass plates, it is overlaid with water-saturated isobutanol and allowed to polymerize for approximately 30 min. Afterward, the water-saturated isobutanol is removed and the 4% polyacrylamide stacking gel mixture is poured on top of the resolving gel and an appropriate sample comb is inserted. SDS-^PAGE Stacking Gel

2.5 mL 0.5 M Tris-HCl, pH 6.8 5.8 mL deionized water 1.3 mL 30% acrylamide-0.8% bis-acrylamide Degas under vacuum for at least 5 min 0.2 mL 10% SDS 75 fiL 10% ammonium persulfate 75 ^tL 1% bromophenol blue 10 fiL TEMED After the stacking gel has polymerized for at least 30 min, the sample comb is removed and the upper and lower reservoir chambers are filled with Electrode Buffer (25 m M Tris-HCl, 192 m M glycine, 0.1% SDS, pH 8.3). The samples are prepared by mixing four parts of sample with one part of 5X Sample Buffer (175 m M Tris-HCl, pH 6.8, 1 1 % SDS, 0.14% bromophenol blue, 55% glycerol, ±2 M DTT). The samples are heated at 95°100° C for 2 - 5 min. Each sample well is rinsed with Electrode Buffer before applying the samples to individual wells. The gel's electrodes are then connected to the power supply and the gels are run at a constant current of 40 mA per gel until the tracking dye reaches the bottom of the gel ( ^ 4 hr). An alternative to Protocol 1 is to use a resolving gel containing a polyacrylamide gradient. Such a gel allows for the analysis of a wider range of molecular weight impurities and yields higher resolution. A 4 - 1 6 % polyacrylamide gel is prepared by placing 15 mL of the 4% polyacrylamide resolving gel solution in the forward chamber of a gradient mixer and an equal volume of the 16% polyacrylamide resolving gel solution into the rear chamber. A small amount of bromophenol blue is added to the rear chamber.

PROTEIN IMPURITY ANALYSIS

31

enabling the analyst to check the gradient after it is formed. The gradient is formed using a peristaltic pump to drive the solutions through a long needle fully inserted between the assembled gel plates. After the solutions are completely poured, the needle is withdrawn, the gel is overlaid with watersaturated isobutanol, and then it is allowed to polymerize for a minimum of 30 min prior to adding the stacking gel as already outlined. ii. Other Modes of Gel Electrophoresis

Additional electrophoretic methods of impurity analysis exist besides that of SDS-PAGE. The resolution of proteins smaller than 20 kDa is better when tricine is used as the trailing ion instead of glycine. The method of Schagger and von Jagow describes a discontinuous denaturing PAGE system utilizing tricine.^^ Native PAGE does not incorporate a denaturing step during sample preparation and SDS is not included in any of the gel solutions or sample buffers. The same procedure outlined in Protocol 1 can be used except SDS is removed from all the solutions and replaced with an equimolar amount of sodium sulfate. Also, the samples are not heated to 95°-100° C but instead are applied directly to the gel in the modified sample buffer. This method suffers from an inability to detect the more basic proteins because their charge does not allow them to migrate under these conditions. Two-dimensional SDS-PAGE has the ability to separate proteins based on their size and charge. The method of O'Farrell is often used.*'^^'^"* Methods of discontinuous urea PAGE have the ability to separate proteins based on charge, size, and conformation.^^ Electrophoretic separations based on several physicochemical properties might resolve impurities that are not detected by one-dimensional SDS-PAGE, i.e., those that comigrate with the recombinant protein, but their general application for impurity analysis has not been widespread. Isoelectric focusing gel electrophoresis (lEF) separates proteins based entirely on their charge.^ A large-pore polyacrylamide gel is prepared containing small charged molecules called ampholytes. When an electric current is applied to the gel, the charged ampholytes migrate rapidly and create a pH gradient in the gel. Simultaneously, proteins will migrate in the gel, although more slowly, to their isoelectric points, where the protein's total charge is zero and migration ceases. lEF gels are stained with Coomassie Blue as described in the next subsection but they do not provide quantitative results. lEF provides the analyst with a qualitative assessment of product-related impurities for highly pure proteins such as deamidated variants of the product, i.e., the amide side chain of glutamine or asparagine residues is converted to a carboxyl side chain. However, this analysis is complicated if the recombinant protein contains covalent carbohydrates with terminal sialic acid residues. Isoelectric focusing in capillary electrophoresis is starting to replace lEF, and this will be discussed later in the chapter. * A detailed protocol for analytical two-dimensional gel electrophoresis is found on the Internet at http://expasy.hcuge.ch/ch2d/technical.info.html. Detailed protocols for lEF are given in Ref. 24 and 26.

32

DONALD O. O'KEEFE

iii. Detection in Gel Electrophoresis

Proteins separated electrophoretically in gels are detected by staining. Most commonly, Coomassie Brilliant Blue R-250 and silver stains are used. The mechanism of protein staining by either remains uncertain. The anionic form of Coomassie Blue is believed to interact electrostatically with arginine, lysine, and histidine residues.^^'^^ Furthermore, van der Waals forces and hydrophobic contacts with aromatic amino acids might enhance dye binding.^^'^^ Despite this uncertainty, the advantage of this stain is the nearly universal quantitative binding of Coomassie Blue to proteins. Davis showed that the staining of 26 proteins in solution with the dye varied by less than twofold.^^ A simple staining solution of Coomassie Brilliant Blue R-250 consists of the dye dissolved in 50% methanol-10% acetic acid at 1 mg/mL. Gels are placed in this solution for > 1 hr and then destained using washes consisting of 50% methanol/10% acetic acid. In recent years, more sensitive methods of Coomassie Blue staining have evolved and these take advantage of the colloidal properties of Coomassie Brilliant Blue G-250 (also known as Xylene Cyanine Brilliant G). One such procedure the author has found useful is the GelCode® Blue Stain Reagent made by Pierce Chemical Company. The procedure is fast, the background staining is minimal, and the sensitivity can be as low as 10 ng. SDS-PAGE gels stained with Coomassie Blue are both a qualitative and a quantitative method of impurity analysis. Coomassie Bluestained gels are made quantitative after image analysis such as with a scanning densitometer. In these instances, the author has found the assay with GelCode® Blue to be linear from 25 ng to 1 ^tg. However, the dynamic range of the assay is greater when larger protein loads are analyzed. For example, an impurity detected at 25 ng, as interpolated from a standard curve, with a total protein load of 2 fig yields a single impurity level of 1.2% (12,000 ppm). Staining proteins with silver often yields a method 100-fold more sensitive than that of Coomassie Blue, reportedly detecting proteins at the subnanogram levels.^^'^^ The argument has been, however, that silver staining methods are significantly more variable than Coomassie Blue staining meth^jg 32,33 ^j^g mechanism of protein silver staining is understood less than that of Coomassie Blue. Lysine residues, negatively charged residues, and the sulfur-containing residues of cysteine and methionine have all been suggested to bind silver cations.^"^ Paradoxically, however, some proteins do not stain at all with silver.^^'^^ Reports have also appeared suggesting that silver staining of proteins is method-dependent.^"^ Acidic-based methods stain acidic proteins more intensely, while basic proteins are detected better in an alkaline environment. Despite all these shortcomings, regulatory agencies recommend silver staining of SDS-PAGE gels as a method of purity analysis'^. In comparing numerous silver-staining protocols, the author has found the method of Wray et al. to be suitable in many circumstances and it is presented in Protocol 2?"^ Note, however, that silver staining of SDS-PAGE gels can at best be considered a qualitative form of impurity analysis. The ICH also acknowledges that silver staining methods are less quantitative than

PROTEIN IMPURITY ANALYSIS

33

Coomassie methods.^"^ Silver staining also suffers from its potential ability to detect nonprotein impurities including nucleic acids, carbohydrates, and lipids."^ Protocol 2: Silver Staining of Polyacrylamide Gels

• Gloves should be worn throughout this procedure to prevent fingerprints from appearing on the developed gel. The gel is agitated moderately during the entire protocol. • The electrophoresed gel is fixed for 1 hr in 50% methanol with solution changes after 20 and 40 min. • Prepare solution A by dissolving 1.6 g of silver nitrate in 8 mL of deionized water. • Prepare solution B by mixing 42 mL of 0.36% (w/v) NaOH with 2.8 mL of 14.8 M NH4OH. • Prepare solution C by adding solution A dropwise to solution B while stirring vigorously. If a brown precipitate persists the solutions should be discarded and prepared again. Bring the final volume to 200 mL with deionized water. Solution C must be used immediately. Dispose of any remaining solution C properly because the decomposition product is explosive. • Place the gel in solution C for 15 min. • Rinse the gel with deionized water. Wash the gel for 5 min in deionized water with one change after 3 min. • Prepare solution D by dissolving 25 mg of citric acid in 2.5 mL of deionized water. Add 250 /xL of 38% formaldehyde and mix. Bring the final volume to 500 mL with deionized water. Prepare this solution fresh. • Place the gel in solution D. Bands should appear within 10 min. Develop the gel until the appropriate sensitivity is achieved. If the color is developing too quickly then the addition of methanol to 5-10% can slow its progress. If solution D begins to turn brown then remove it, rinse the gel with deionized water, and add fresh solution D. • Stop development by removing solution D, rinsing with deionized water, and placing the gel in 4 5 % methanol/10% acetic acid. • After 15 min, equilibrate the gel in deionized water. Staining methods other than silver and Coomassie Blue do exist for detecting proteins in polyacrylamide gels. Often these methods involve labeling the proteins with fluorescent probes or radioisotopes.^^ In both cases, the labeling is based on definable properties, i.e., the labels target specific protein functional groups. The most notable targets for fluorescent probes are protein thiols and amine groups. The advantages and disadvantages of these methods have been discussed previously.^^ In general, these methods are not universal detection methods for protein impurities but they might have application under defined circumstances.^^ Recently though, a new series of fluorescent dyes, SYPRO® Red and SYPRO® Orange (Molecular Probes), has become available for labeling proteins in SDS-PAGE gels.^^'^^ These dyes label protein-SDS complexes noncovalently and preferentially. Since all proteins bind equivalent amounts of SDS based on their mass, it is beheved that these

34

DONALD O. O'KEEFE

dyes will bind to protein-SDS complexes equally, and therefore should be quantitative. Their sensitivity is reportedly comparable to that of silver staining with as little as 1 ng of protein detectable.^^ This sensitivity, along with the reported universal quantitative binding of these dyes, might lead to their replacing silver staining as a standard method for protein impurity analysis. B. High-Performance Liquid Chromatography High-performance liquid chromatography (HPLC) is the primary quantitative technique for purity and impurity analysis of recombinant proteins. Recommended by regulatory agencies, this high-resolution technique routinely detects impurities at the level of 0.1% (1000 ppm).^^ Reversed-phase has been the HPLC method of choice ever since the efficiency of protein separations increased with the development of small particle (3-5 /im), wide pore (300 A) silica bonded to C4 through C^g ligates. Additional modes of HPLC are receiving increased usage for impurity analysis, but in general, their application is more specific and limiting. These include ion-exchange chromatography, hydrophobic interaction chromatography, and size-exclusion chromatography. Each one will be discussed briefly. i. Reversed-Phase High-Performance Liquid Chromatography In reversed-phase high-performance liquid chromatography (RP-HPLC), protein hydrophobic regions interact with the alkyl or aromatic ligates of the stationary phase until a critical concentration of organic solvent in the mobile phase comes along and releases the protein from the column."^^ These hydrophobic regions are surface exposed or internal to the protein. The latter are rendered accessible if the protein is denatured by contact with either the stationary phase or the mobile phase. In either case, the hydrophobic region that dictates retention on the column is the contact region. It is differences in the contact region among the many proteins that affects separation in RP-HPLC. There are three types of organic modifiers commonly used in the mobile phase of RP-HPLC methods. They are isopropanol, acetonitrile, and methanol listed in their decreasing ability to elute proteins. These solvents are generally combined with water in a gradient of increasing organic. Other aqueous solutions, such as acetate or phosphate buffers, have replaced water on occasion but these are generally for more specific applications. The mobile phase might also include a small amount of an ion-pairing agent, generally 0.1% trifluoroacetic acid (TFA) is the most common. Other acids used include phosphoric, hydrochloric, perchloric, and heptafluorobutyric acid. A larger amount of a weaker acid, such as acetate or formate, can also be used. These acids maintain the pH of the mobile phase below 3.5, where uncapped silanol residues on silica columns are prevented from interacting with the proteins and giving rise to mixed mode separations. These unwanted interactions can also be minimized by increasing the ionic strength of the mobile phase or by adding a competing cation such as triethylammonium phosphate. The pH of the mobile phase can be adjusted to achieve the desired separation

PROTEIN IMPURITY ANALYSIS

35

and to ensure protein solubility in the organic phase but pH values above 8 are not compatible with columns composed of silica particles. The use of polymeric supports eliminates this problem and enables the analyst to perform RP-HPLC at alkaline pH. However, RP-HPLC with a wateracetonitrile gradient in 0.1% TFA is the most widely used mobile phase system for protein analysis. Protocol 3 is a general method for analyzing the purity and impurity profile of a protein by RP-HPLC. Based on initial results, the analyst should modify this method to achieve optimal separation. Protocol 3: Reversed-Phase HPLC

• Column Vydac 214 TP (C4 ligate)* 300 A pore size 150 X 4.6 mm i.d. 5 jum particle size • Mobile phases A: 0.1% TFA in water B: 0.05% TFA in acetonitrile • Detection UV at 210 nm • Linear gradient 0 to 100% B over 100 min • Flow Rate 1.0 mL/min • Injection 50 /jiL

The lower concentration of TFA in mobile phase B partly counteracts the increased background absorbance normally seen when using a wateracetonitrile gradient containing TFA. Protocol 3 should be considered as a starting point for protein impurity analysis by RP-HPLC. Modifications will have to be made based on the particular sample undergoing analysis. For example, a more hydrophobic protein might be difficult to elute and require a stationary phase that absorbs the protein less strongly, such as one containing a phenyl ligate."*^ A stationary phase intended for hydrophobic interaction chromatography can be operated under re versed-phase conditions for extremely hydrophobic proteins. Alternatively, a more hydrophobic mobile phase might be employed to elute the strongly adsorbed protein, for instance, one containing isopropanol or a mixture of isopropanol and acetonitrile. An important advance in the RP-HPLC impurity analysis of proteins has been the advent of short columns containing micropellicular stationary phases."^^ These short columns contain nonporous particles of the order of 2 fim that are capable of attaining high flow rates ( ^ 4 mL/min) at elevated temperatures ( ^ 80° C). Such a configuration leads to very rapid and highly • Separations in RP-HPLC can be highly dependent upon the column utilized. Corran"^^ and Johns"^"^ list many commercially available reversed-phase columns suitable for protein analysis.

36

DONALD O. O'KEEFE

efficient protein separations using conventional HPLC equipment. Separations in less than 5 min are not uncommon.'^^''^^ As with any method of protein impurity analysis, RP-HPLC does have its limitations. Certain impurities might not adsorb to the column, v^hile others might not elute from the column under one set of conditions. These impurities will either not be quantitated accurately or they will not be detected, respectively. It is critical to remember that more that one method of analysis must be employed to give assurances about the level of impurities. With RP-HPLC, however, analysis can be carried out using two different solvent systems or different columns to help provide more assurance. ii. Hydrophobic Interaction Chromatography

Columns for hydrophobic interaction chromatography (HIC) are similar to those for RP-HPLC in that both types of stationary phases consist of hydrophobic ligates. A critical difference, however, is the density of these bonded groups. RP-HPLC ligates are generally very densely packed for re versed-phase particles leading to a hydrophobic surface that may be denaturing to a protein. On the other hand, the hydrophobic ligates on HIC columns are much less densely packed with the intention of having them interact with only surface hydrophobic regions on proteins. This, along with the use of more aqueous phases of high ionic strength, makes HIC generally a nondenaturing technique that primarily separates proteins based on their surface hydrophobicity. Because of this property, HIC is widely used for separating product-related impurities of highly pure recombinant proteins such as conformational isomers. HIC is generally carried out near neutral pH with a starting mobile phase high in salt, often (NH4)2804, that promotes protein interaction with the stationary phase. As the salt concentration is lowered, the less hydrophobic proteins are eluted first. The pH of the mobile phase can affect separation and it should be adjusted if results prove dissatisfying at neutral pH. Protocol 4 is a general procedure for performing HIC analysis of proteins. Isopropanol can be included in both mobile phases for the analysis of more hydrophobic proteins. If these conditions are not sufficient, then the analyst can adjust the pH (6.5 to 8.0) or increase the amount of (NH4)2S04 up to 2.0 M. Protocol 4: Hydrophobic Interaction Chromatography

• Column TSKgel Phenyl-5PW(TosoHaas)* 1000 A pore size 75 X 7.5 mm i.d. 10 ^tm particle size • Mobile phases A: 1 M (NH4)2S04 100 m M Na2P04 pH adjusted to 7.0 (±5% isopropanol) • Different column ligates can dramatically affect the separation. A list of several commercially available HIC columns suitable for protein analysis is given in Ref. 44.

37

PROTEIN IMPURITY ANALYSIS

• • • •

B: 100 m M Na2P04 pH adjusted to 7.0 ( ± 5% isopropanol) Detection UV at 220 nm Linear gradient 0 to 100% B over 15 min Flow rate 1.0 mL/min Injection 50 fiL

iii. Size-Exclusion Chromatography

Size-exclusion chromatography (SEC) is the most straightforward of the HPLC techniques presented. Separation is based on the protein's hydrodynamic volume and its interaction with the porous stationary phase. The greater the hydrodynamic volume, the greater the probability that the protein will be excluded from the interior of the particles and the sooner it will elute. Smaller proteins enter the particles more frequently and thus are retained longer. The mobile phases are aqueous buffers containing a sufficient amount of salt to prevent protein retention due to ionic adsorption. The mobile phase utilizes an isocratic gradient. The technique has only moderate resolution and it is mostly used to detect aggregates of the recombinant product, which can be immunogenic. Often two size-exclusion columns are placed in tandem to increase resolution but with a concomitant increase in back pressure. Protein size standards are injected onto the column separately from the protein drug samples for calibration. Protocol 5: Size-Exclusion Chromatograptiy

• Column Bio-Sil SEC 250-5 (Bio-Rad)* o

• • • •

250 A pore size 300 X 7.8 mm i.d. 5 ^tm particle size Mobile phase: 20 m M Na2PO4-150 m M NaCl-pH 7.0 Detection UV at 210 nm Flow rate 1.0 mL/min Injection 50 IJLL

iv. Ion-Exchange Chromatography

Ion-exchange chromatography (lEX-HPLC) is intended to separate proteins based on differences in their total charge. Both anionic and cationic exchange modes are available. This technique can potentially detect and • Additional columns are described by Johns."^"^

38

DONALD O. O'KEEFE

quantitate product-related impurities, such as deamidated proteins, and product-related substances, such as heterogeneously glycosylated proteins. The resolution of this technique is generally only moderate. lEF provides much greater resolution for detecting these variants. lEX-HPLC may prove to be useful in some impurity analyses, but the method must be specifically tailored to a particular application. Numerous variables must be estabhshed for the separation, and therefore lEX is often the most difficult HPLC method to develop."^^ As such, it is difficult to give a brief generic starting protocol. Interested readers are encouraged to look at references at the end of this chapter for more information on this technique."^^'"^^ V. Detection in HPLC

The most common mode of detection in HPLC impurity analysis is UV absorbance monitoring. Generally, UV v^avelengths in the 210-220 nm range are used. At these wavelengths, the major chromophore in proteins is the peptide bond. The absorbance signal for a given protein is proportional to the number of peptide bonds. Therefore, protein impurities can be quantitated with reasonable accuracy using a known protein as a standard. However, slight errors will occur because the side chains of several amino acids (trp, phe, his, tyr, gin, asn) also contribute to absorbance in the low UV range.'*^ Using low UV wavelengths, sensitivities for detecting protein impurities can be as low as 20 ng, depending on the particular detector. Utilizing an upper linear limit of 49 jjig for a recombinant protein in a valid RP-HPLC assay, the detection limit for protein impurities can be as low as 0.04% (400 ppm)."^^ Caution should be taken in selecting the exact wavelength because certain organic modifiers, such as isopropanol, do absorb in the low UV range. An inherent problem in using a single low UV wavelength is its inability to discriminate a protein impurity from a nonprotein impurity. Monitoring for absorbance at 257, 275, or 280 nm can circumvent this lack of specificity. At these wavelengths, the predominant protein chromophores are phenylalanine, tyrosine, and tryptophan, respectively. If possible, two wavelengths are monitored simultaneously to assess a peak's purity and identity. The ratio of the absorbance of a protein at two wavelengths is equivalent to the ratio of the protein's molar absorptivites at those wavelengths. This ratio is independent of protein concentration. Hence, this ratio should be the same throughout a chromatographic peak if that peak represents a pure protein. If an impurity coelutes precisely with the recombinant protein of interest, then this technique will fail if a standard of the pure protein is unavailable. Also, different conformational isomers of the same protein might have different absorbance ratios, so this technique might be limited to RP-HPLC where the protein is mostly denatured. This method of purity analysis can be extended further using photodiode array detection (PDA) where the entire UV spectrum can be monitored simultaneously.^^ Although PDA detection is a very powerful technique for assessing the purity of protein pharmaceuticals, its sensitivity is often lower than that of absorbance at a single UV wavelength. Increased sensitivity of detection in HPLC can often be gained by monitoring the column effluent for intrinsic protein fluorescence by excitation

PROTEIN IMPURITY ANALYSIS

39

at 280 nm and emission at 310-350 nm. This sensitivity can be increased further by excitation at 220 nm. The major fluorophore in proteins is tryptophan with tyrosine contributing a minor signal. Monitoring impurities for fluorescence at these wavelengths almost assuredly identifies them as proteins, however, quantitation of impurities by this method is unlikely. Proteins may not be detected fluorescently because they lack tryptophan residues or those impurities that do contain these residues have them in unknown numbers. Furthermore, the quantum yield of all tryptophan residues will not be identical owing to their diverse molecular microenvironments in their native proteins. Hence, a standard curve from a known unrelated protein is not useful. Another sensitive technique for monitoring HPLC of proteins is to utilize fluorescent derivatization. These methods are often based on the reaction of small fluorescent compounds with specific functional groups in proteins.^^'^^ Most often protein amines and sulfhydryl groups are the targets of these agents.^^ Amine-reacting reagents are more popular since the rarity of proteins lacking amine groups is less than those that lack sulfhydryls. Fluorescamine is a common reagent for this purpose. A stock solution of fluorescamine is dissolved in acetone and added to the protein in an alkaline buffer (pH 9).^"* The reagent labels the a-amino group at the N terminus and the e-amino group on lysine residues. The reaction is completed in less than 1 min and unreacted fluorescamine shows little fluorescence. The labeled products can then be separated by RP-HPLC. Post-column-derivatization procedures are also used.^^'^^ However, amine groups are not exclusive to proteins and the detection of a fluorescent impurity peak does not guarantee the impurity is a protein. Furthermore, quantitation is difficult for the same reasons discussed earlier in the chapter regarding tryptophan fluorescence. Nevertheless, fluorescently labeling proteins in HPLC impurity analysis has found some applications (see the Case Studies at the end of the chapter).^^ vi. Peptide Mapping

The most powerful method available for evaluating primary structural variants of recombinant proteins is peptide mapping. This technique is applicable to the analysis of product-related impurities. Variants that are amenable to this type of analysis include deamidated asparagine or glutamine residues, oxidized methionine residues, N-terminal variants, disulfide isomers, substituted amino acids due to mistranslation or point mutation, and protein variants chemically modified during processing. Although many of these modifications are detectable by other techniques, such as IFF and lEX chromatography, peptide mapping by RP-HPLC is generally used to identify the site of modification. The peptide containing the modification is first identified by a shift in retention time compared to that of a reference peptide map. However, the technique is generally only capable of detecting a modification at the 2 - 5 % level. The methodology of peptide mapping by RP-HPLC is straightforward. The protein is first denatured and reduced with urea and dithiothreitol (DTT), respectively, to unfold the protein. Cysteine residues are then blocked

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DONALD O. O'KEEFE

with a small sulfhydryl-reactive reagent such as iodoacetamide. This minimizes the number of peaks in the chromatogram from peptides containing cysteine residues. Alternatively, DTT can be omitted if disulfide isomers need to be examined. Next, the protein is fragmented completely. Generally, this is carried out using proteolytic enzymes that cleave specific amino acid sequences. Trypsin, which cleaves after arginine and lysine residues, and V8 protease from Staphylococcus aureus, which cleaves after glutamate residues, are most often used. Chemical cleavage methods can also be employed. Cyanogen bromide cleaves proteins after methionine residues,^^ o-iodosobenzoic acid cleaves after tryptophan residues,^^ and hydroxylamine^^ cleaves between asparagine and glycine residues. The exact method of protein fragmentation will depend on the sequence of the recombinant protein under study. The objective is to fragment the protein into peptides no greater than 25 amino acids in length. The fragmented protein is then separated by HPLC. Reversed-phase is the mode most often chosen because of its high resolving capabilities. However, these separations can be difficult because of the enormous number of peaks that must be separated. For example, the peptide map of recombinant tissue plasminogen activator has over 50 chromatographic peaks after trypsin digestion. A multiphasic linear gradient was employed to achieve this separation.^^ Protocol 6 for peptide mapping utilizes trypsin to fragment the protein enzymatically.^^ The trypsin used should be tosylphenylalanylchloromethyl ketone (TPCK) treated to eliminate residual chymotrypsin activity that, if present, would cleave the recombinant protein after phenylalanine, tryptophan, and tyrosine residues. The fragmented protein is then chromatographed via RP-HPLC using a gradient of acetonitrile containing TFA Replacing TFA with 50 m M sodium phosphate as the ion-pairing agent reportedly has given better resolution of peptide peaks.^^ It is likely that the gradient given in the following will have to be modified to achieve optimal separation of a particular peptide mixture. Protocol 6: Tryptic Peptide Mapping Peptide Mapping—Trypsinolysis

• Samples should be prepared in triplicate including a reference standard, if available, and a buffer blank. • Dry separate 200 jx g samples of the protein under vacuum (i.e., using a Savant Speed Vac). Once dried, the samples can be stored at — 20° C. • Resuspend the dried protein in 50 /xL of fresh 8 M urea-80 m M methylamine-0.5 M ammonium bicarbonate. Avoid heating this buffer during preparation. Heat increases the formation of cyanate in the urea that can lead to the carbamylation of the e-amino group of lysine residues. Methylamine helps eliminate cyanate in urea. • Add 5 JJLL of 45 m M DTT and mix thoroughly. Heat the samples at 50°C for 5 min. • After cooling to room temperature, add 5 /xL of 100 mMiodoacetamide to each sample. Incubate for 15 min.

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PROTEIN IMPURITY ANALYSIS

• Bring each sample to 200 ^LL with 0.5 M ammonium bicarbonate and one unit of TPCK-treated trypsin. The TPCK-treated trypsin stock is prepared in 0.5 M ammonium bicarbonate and can be frozen in single use aliquots at -20°C. • Incubate the samples at 37°C for approximately 18 hr. • Stop the proteolysis by adding 60 juL 2% TFA. The samples are now ready for chromatography or they can be stored at — 20°C. Peptide

Mapping—Cliromatograpliy

• Column Vydac 218 TP (Cig Hgate) 300 A pore size 150 X 4.6 mm i.d. 5 iJLtn particle size • Mobile phases A: 0.1% TFA in water B: 0.05% TFA in acetonitrile • Detection UV at 210 nm • Linear gradient 0 to 100% B over 100 min • Flow rate 1.0 mL/min • Injection 80 fjiL

The detection modes for a peptide map are the same as those described earlier for HPLC. Absorbance at low UV wavelengths is most often used. Absorbance at other wavelengths, tryptophan fluorescence, or fluorescence after derivatization might provide additional information on the location of a modification (see Section IV). The use of electrospray mass spectrometry on-line with HPLC is a powerful tool for peptide mapping that can identify the modification without further analysis.^^

C. Capillary Electrophoresis

One of the most powerful separation techniques for purity and impurity analysis for the bioanalyst is capillary electrophoresis (CE). However, it is a relatively new analytical tool and its methodology is evolving at a rapid pace, so there is limited reference to its application to protein impurity analysis in the literature. Nonetheless, this is only a temporary respite. In the future, CE will become a standard and routine analytical technique for the analysis of protein impurities in recombinant pharmaceuticals. Capillary electrophoresis combines the separation principles of gel electrophoresis with the throughput and detection methods of HPLC. It overcomes the disadvantages of slab gel electrophoresis, including slow and labor intensive procedures and the difficulty and inaccuracies of quantitation. CE is

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DONALD O. O'KEEFE

a high-resolution technique. Its resolution surpasses that of slab gel electrophoresis and in many cases that of HPLC. In practical terms it is able to separate closely related protein species present as impurities including those having aspartic acid in place of asparagine (deamidated species). The speed and high resolution of CE is a direct result of the high voltages that are applied. The higher the voltage, the faster the separation, v^hile resolution increases with the square root of the applied voltage. The high heat created by these voltages is effectively dissipated by the use of narrow diameter capillaries of 50-100 /xm. The capillaries are most often made of fused silica surrounded by a polyimide sheath. The internal walls of the capillary will bear a negative charge at pH 3 or greater due to the ionization of the silanol groups. When an electric current is applied to the capillary, cations in the electrolyte move past the immobilized silanol anions towards the cathode buffer and cause a bulk movement of the buffer termed electroendoosmotic flow (EOF). Regardless of charge, all analytes will eventually migrate past the detector due to the EOF. The EOF, in addition to electrophoretic mobility, influences the separation of the analytes, but it can be modified to varying degrees by adjusting the p l i or by using either a coated capillary or buffer additives. The choice of pH in CE separations is critical. It may be the single most important variable in a CE separation. Lowering the pH to 2 or less neutralizes the silanol residues and prevents ionic interactions with proteins. Alternatively, using a pH higher than the isoelectric point (pi) of the most basic protein in the analysis creates repulsion between the negatively charged proteins and the silanol residues. Unfortunately, for analysis of host protein impurities the pi of the most basic protein is unknown so that can limit this approach. Furthermore, a higher pFi leads to higher EOF, and a lower pH might protonate all proteins thus sacrificing differences for their separation. Some widely used buffers and their respective pK^ values include formate (3.75), acetate (4.75), MES (6.15), imidazole (7.00), HEPPSO (8.00), morpholine (8.49), borate (9.24), and CHAPSO (9.60).^^ If pH extremes cannot be utilized, then either a coated capillary or buffer additives can be used. Without either of these, proteins might adsorb to the inner capillary wall. Adsorption will affect the separation and in some circumstances this may be beneficial. Additives that are often added to CE buffer systems include surfactants, zwitterionic salts, ethylene glycol, methylcellulose, organic modifiers, and quaternary amines. Capillary coatings include polyacrylamide, polyethyleneglycol, polyvinylpyrrolidone, and methylcellulose. Mazzeo and KruU^^ discuss capillary coatings and buffer additives further. One significant limitation of CE regarding protein impurity analysis is its inability to be a routinely preparative technique owing to the small injections of sample (-^2-10 nL). Generally, numerous runs must be performed to gather enough samples for subsequent analysis. The use of a fraction collector to automate such a system can be problematic if consecutive runs are not precisely reproducible, which is not an uncommon feature of CE. However, one interesting system was developed by Eriksson et aL^^ The authors designed a moving polyvinylidene difluoride (PVDF) membrane that collected

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43

proteins and peptides as they eluted from the capillary past the detector. The membranes were stained with common protein reagents and they were also identified immunologically with antibodies. This approach can be a significant advance for the separation and identification of minor protein impurities. CE has many separation modes that are beneficial to protein impurity analysis. Within the many thousands of potential protein impurities in a recombinant product there will be several that have only minor physicochemical differences from the drug product. The application of different CE modes can potentially resolve these impurities. CE methods can be divided into four principle modes that are applicable to recombinant protein impurity analysis: capillary zone electrophoresis, capillary isoelectric focusing, capillary gel electrophoresis, and micellar electrokinetic capillary chromatography. Each mode will be discussed briefly. Since the technology is so young and still very exploratory, CE methods are developed empirically for specific separations. It is difficult to provide standard protocols for CE impurity analysis. Instead, protocols that can be used as a starting point for impurity analysis will be provided as well as the citation of examples of impurity analyses from the literature to provide additional sources of protocols for interested readers. i. Capillary Zone Electrophoresis

Capillary zone electrophoresis (CZE), also known as free-solution CE, is the most widely used mode of CE essentially because of its versatility. Protein separation in CZE is based on the differential electrophoretic mobility of the analytes. This mobility is primarily dependent on a protein's size and net charge, the charge-to-mass ratio. Solvent properties that influence the size and charge of a protein include pH, ionic strength, viscosity, and dielectric constant.^^ Manipulation of these properties, most notably pH, dictates the selectivity in CZE. Maximizing the charge difference between two proteins via pH modification optimizes their separation. CZE is an attractive method for the analysis of process-related protein impurities because its mechanism of separation is different from that of RP-HPLC. The former is based on a charge-to-mass ratio, while the latter is based on hydrophobicity. CZE has been used to monitor the impurity profile of recombinant proteins during downstream processing. This includes recombinant hepatitis B surface antigen^^ and recombinant hirudin.^^ However, for impurity analysis, CZE has most often been employed to analyze productrelated impurities, such as deamidated products. Examples include recombinant growth hormone,''^''^^ recombinant insulin^^ recombinant tumor necrosis factor beta,^^ and recombinant interleukin-4.^^ Nielsen et al7^ separated recombinant insulin from its deamidated degradate (desamido-A21) and two incorrectly processed impurities. However, the misprocessed impurity desthreonine-B30 was not resolved by this method but was separated by RP-HPLC. Likewise, these researchers used this same method to separate recombinant growth hormone from its deamidated derivatives but were unsuccessful in resolving a methionine sulfoxide degradate. The latter was also resolved by RP-HPLC. These results underscore the complementary

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DONALD O. O'KEEFE

nature of RP-HPLC and CE, both of which exploit different protein physicochemical properties to affect separation. CZE is a method for the rapid analysis of peptide mixtures and therefore has particular application to peptide mapping and identification of productrelated impurities/"^ Unlike peptide mapping by RP-HPLC, trypsinolysis might not be the method of choice for fragmentation because most of the peptides generated have a net charge of -h 2 at low pH. Alternative methods of fragmentation should be considered or separations utilizing higher pH buffers. Peptide mapping of recombinant proteins by CZE include the following along with their method of fragmentation: growth hormone (chymotrypsin, V8 Protease, trypsin),^^'''^'^^ insulin-like growth factor (trypsin),""^ and interleukin-4 (trypsin).""^ Protocol 7 for CZE was adapted from Gordon et al7^ These authors used this protocol to analyze a variety of proteins including a complex protein mixture. It may provide a good starting point for protein impurity analysis. If insufficient separation is achieved then the analyst is advised to change the pH by using one of the other buffers discussed above. Such an approach showed markedly different separations for a number of recombinant proteins and product-related impurities.^^'""^ Protocol 7: Capillary Zone Electrophoresis

• Running buffer: 50 m M sodium borate, pH 10.0 • Sample preparation: in 20 m M sodium borate, pH 4.0, containing 20% ethylene glycol • Capillary: 37.5 cm X 75/>tm i.d. fused silica; length to detector: 30.5 cm • Voltage: 10 kV • Temperature: 20°C • Injections: variable • Detection: 200 nm • Wash the capillary between runs with 0.1 N NaOH (5 min), then deionized water (3 min), followed by running buffer (5 min) ii. Capillary isoelectric Focusing

Capillary isoelectric focusing (CIEF) separates proteins based on differences in their isoelectric points. CIEF has been used to separate product-related impurities of recombinant proteins, mainly deamidated species, such as those of human growth hormone.''^ There are two basic forms of CIEF that differ based on the method for mobilizing the focused proteins. One method of CIEF is to fill a coated capillary with the sample and the ampholytes and then apply the voltage. Estabhshment of a pH gradient occurs quickly and subsequent focusing of the proteins at their pi values soon follows. The focusing generally takes 15-20 min. Since it is a coated capillary the EOF is negligible and the focused proteins must be mobilized to move past the detector. Mobilization is accomplished by reapplying the voltage after either replacing the acidic anolyte with base or the alkaline catholyte with acid or adding salt to either the anolyte or the catholyte.^^ As with slab gel IFF, the focused proteins can

PROTEIN IMPURITY ANALYSIS

45

precipitate, but incorporating urea, detergents, or ethylene glycol into the running buffer can minimize this problem/^ A second method does not eliminate the EOF but instead uses it to force the focused proteins past the detector.^^ Hence, a postfocusing mobilization step is not required. Meaningful results can be obtained only if protein focusing is faster than the EOF. Protocol 8 uses this one-step method and it is adapted from that of Mazzeo and KruU.^^ Due to run-to-run variability, standards of known pi should be included in the sample. Protocol 8: Capillary Isoelectric Focusing

• Catholyte: 20 m M NaOH • Anolyte: 10 m M phosphoric acid • Sample preparation: wash the capillary with 10 m M phosphoric acid and then fill it with sample (100 /ig/mL) and standards containing 5% ampholytes (Pharmalyte, pH 3-10, Pharmacia), 0.1% methylcellulose, 1% TEMED • Capillary: uncoated fused silica 60 cm X 75 fim i.d., 40 cm from anode to detector • Vohage: 30 kV • Detection: 280 nm (the ampholytes absorb at low UV wavelengths) iii. Capillary Gel Electrophoresis

The separation principle in capillary gel electrophoresis (CGE) is the same as that of slab gel electrophoresis. Most often CGE is used in a denaturing mode with the incorporation of SDS and is referred to as SDS-CGE. As such, separation is based on the protein's molecular mass and, due to the sieving mechanism of the gel, smaller proteins migrate past the detector first. The use of a gel material and SDS decreases the EOF and eliminates protein adsorption to the capillary walls further ensuring that migration is based on molecular mass. This precludes the need for additives and coated capillaries. One of the earliest successes with CGE was that of Tsuji in the analysis of recombinant proteins.^^ In that study, acrylamide gels were polymerized inside and attached to a fused-silica capillary measuring 7 cm X 50 /^m i.d. Utilizing a running buffer of 300 m M Tris, pH 8.8, 0.1% SDS with ethylene glycol and a sample buffer consisting of 15.6 m M Tris, pH 6.8, 0.1% SDS essentially created a discontinuous electrophoresis system. In two recombinant protein preparations, this system detected a dimer in one and a 1500 Da smaller impurity in the other.^^ In recent years, however, gel polymerization within capillaries has been abandoned. Instead linear polymer "gels" have been developed for CGE that eliminate the difficulty and tedium of preparing polymerized gels within capillaries. These gels also provide longer column lifetimes. In this method, a viscous linear polymer solution, or a mixture of polymers, is incorporated into the running buffer. The running and sample buffers of these methods are similar to those of the discontinuous Laemmli SDS-PAGE system. During electrophoresis, protein migration in the capillary is retarded based on the protein's molecular mass. When the technique is used without SDS under

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DONALD O. O'KEEFE

nondenaturing conditions it provides a means of assessing the aggregation state of a recombinant protein. Polymers consisting of dextran, linear polyacrylamide, polyethylene oxide, and polyethylene glycol have been utilized at concentrations of 1-6%. Benedek and Thiede^^ utilized polyethylene oxide (PEO) polymers to analyze recombinant proteins such as erythropoietin, interferon, granulocyte stimulating factor, brain-derived neurotrophic factor, and platelet-derived growth factor. Protocol 9 describes an SDS-CGE method adapter from their work. Protocol 9: SDS ~ Capillary Gel Electrophoresis

• A PEO (M^ = 100,000) stock solution is made in 0.1% ethylene glycol and passed through a 5.0 fim membrane filter • Running buffer: 100 m M Tris-2-(N-cyclohexylamino)ethansulfonic acid (CHES), pH 8.5, 0.1% SDS, 3 % (w/v) PEO • Sample preparation: The samples are placed in a buffer with a final composition of 60 m M Tris-HCl, pH 6,6, 5% (v/v) j8mercaptoethanol, 1% (w/v) SDS, with a trace amount of Orange G as a tracking dye and heated at 95°-100°C for 2 - 5 min. • Capillary: 27 cm X 100 /xm i.d. (20 cm to the detector) fused-silica washed with 1 M NaOH, then deionized water, and then 1 M HCl before conditioning with the running buffer • Voltage: 8.1 kV • Injections: variable • Detection: 214 nm (the detector is placed at the anode end) • Wash the capillary between runs with 1 M HCl, then deionized water, followed by running buffer. Separations in Protocol 9 are comparable to those of SDS-PAGE but each sample run is less than 20 min and more accurate quantitation can be achieved. Limits of protein impurity detection in CGE systems with absorbance detection at a low UV wavelengths are approximately 0 . 1 % (1000 ppm). This is about 10-fold lower than the detection limit of SDS-PAGE. Commercially available linear polymers and CGE kits are available. The chemical composition of some of these polymers is proprietary but others are not. Companies marketing these linear polymers for use in CGE include Bio-Rad,^"^ Perkin Elmer/ABI (linear polyacrylamide),^^ and Beckman Instruments (PEO).^^ The Beckman SDS-Protein Gel Column Kit includes many of the supplies and reagents required for Protocol 9. iv. Micellar Electrokinetic Capillary Chromatography

CE can also separate neutral analytes via micellar electrokinetic capillary chromatography (MEKC). This mode of CE is similar to CZE except a micellar solution, often anionic in nature (e.g., SDS), is incorporated into the running buffer. Neutral analytes partition between the micelles and the running buffer based on their relative hydrophobicity, which greatly influences the separation. Charged analytes also partition according to their relative hydrophobicity but their charge also affects their separation. Charged analytes can potentially interact with the anionic micellar surface to affect

PROTEIN IMPURITY ANALYSIS

47

their separation further. In many ways MECC is analogous to RP-HPLC, and as such it has been mostly used for peptide mapping with rare uses for proteins. For further information and applications, the interested reader is directed to the review by Mazzeo.^^ V. Detection in CE Detection methods in CE are similar to those used in HPLC. Much of the discussion presented early for detection in HPLC can be applied to CE. Low UV absorbance detection is the most popular form of detection in protein CE. Other modes of protein detection in CE include photodiode array detection, mass spectrometry, both intrinsic and derivatized protein fluorescence, and indirect fluorescence. In indirect fluorescence the capillary is filled with a small fluorescent compound having the same charge as the analytes. The analytes are detected as a nonfluorescent band within a fluorescent electrolyte. The sensitivity of this detection is between that of UV absorbance and direct fluorescence .^^ One additional mode of detection in CE showing great promise and increasing usage is laser-induced fluorescence (LIE) detection. This mode almost always involves derivatization of the protein so that it can be excited by the wavelength of the laser. Helium-cadmium lasers emit at 325 and 442 nm while argon-ion lasers emit at 458, 488, and 514 nm. These wavelengths dictate the type of fluorophore used in the derivatization. Fluorophores commonly used to tag protein amines and their excitation maxima include o-phthaldialdehyde (OPA) (340 nm), naphthalenedialdehyde (440 nm), 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde (CBQCA) (456 nm), 4-chloro-7-nitrobenz-2-oxa-l,3-diazole(470 nm), and fluorescein isothiocyanate (488 nm).^^ Detection limits with LIE are reported to be as low as the attomole and zeptomole range .^^'^^ As with all modes of fluorescence detection, LIE only has the potential to be a qualitative technique for the analysis of unknown impurities. The mode of detection selected for a CE method is significant in determining the sensitivity of the method. In CZE using UV absorbance detection at 200 nm, Nielsen and Rickard^^ conservatively estimated the minimum and maximum protein detection levels of their linear range to be 0.1 and 25 ng, respectively. This translates to an impurity sensitivity of 0.4% or 4000 ppm. Coupling CE with electrospray ionization mass spectrometry (MS) can potentially be a very powerful tool for detecting and identifying product-related impurities in recombinant pharmaceuticals. Proteins can be detected in the femtomole range with this mode. Conceivably, the bioanalyst could perform a peptide map with CZE-MS and detect, identify, and sequence aberrant peptides derived from degradates. D. Immunoassays Immunoassays are the most specific and sensitive techniques available for detecting protein impurities. There are two classes of protein impurities that are most often analyzed with these techniques: host-cell proteins and protein additives, both of which are process-related impurities. Although protein additives are known entities and therefore amenable to other quantitative

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analyses such as HPLC, the use of immunoassays is more sensitive. There are two separate formats commonly employed for immunodetecting protein impurities: enzyme-linked immunosorbent assays (ELISA) and Western blot analysis. Both have advantages and disadvantages and both suffer from the potential difficulty of obtaining a suitable antiserum. Both methods v^ill be discussed in turn. With a properly prepared antiserum, immunoassays are capable of detecting protein impurities in the lov^ ppm range. More significantly though, they can do this in the presence of milligram quantities of the recombinant pharmaceutical. For known protein impurities, such as serum proteins, nuclease additives, and chromatography ligands (e.g., monoclonal antibodies or protein A) commercial ELISA or antisera are generally available and these will not be discussed further. For host-cell protein impurities, however, achieving a ppm level of detection requires an appropriate reference standard comprised of potential host protein impurities to use as an immunogen. The reference standard is generally obtained from a mock manufacturing run of the production process. Host cells containing a plasmid identical to the production strain, but lacking the gene for the recombinant product, are grown and processed through the normal purification procedure. Two important assumptions are made regarding this approach. First, the absence of recombinant protein expression does not alter the expression of host proteins. Evidence suggests that there is little difference from the normal process.^^ Second, the absence of the recombinant protein does not alter the behavior of host proteins in the purification steps. Throughout the mock process, fractions are combined, as they would be if the recombinant product was present. At the process step where the product is normally 9 5 - 9 9 % pure, the material in the pooled fractions is collected and this comprises the reference standard.^^ After concentrating this material, either rabbits or goats are immunized. It is best to immunize several animals with the preparation to reduce the effects of immunological diversity among animals of the same species. Immune antiserum is collected, pooled, and an immunoglobulin fraction is prepared by ammonium sulfate precipitation. Passing it through an affinity column made from the reference standard preparation processes this fraction further. The resulting fraction contains antibodies more selective for potential host-cell protein impurities and thus increases the sensitivity of the assay.^^ Anicetti and coworkers^^"^^ and Eaton^^ provide in depth discussions on the preparation of these host-cell protein antibodies. i. ELISA For use in an ELISA, a portion of this antibody preparation is conjugated to a moiety that is capable of eliciting a detectable signal, generally an enzyme, such as alkaline phosphatase. The format of a host-cell protein ELISA is a double-antibody sandwich. The assay begins by coating the bottom of a 96-well microtiter plate with an excess of the purified antibodies to capture the protein impurities. After blocking the remaining binding sites on the plate surfaces, the recombinant product is added. Without the initial antibody layer and subsequent blocking most of the plate's surface would be coated with the recombinant protein and very little binding would be avail-

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49

able for the impurities. A calibration curve is also constructed simultaneously using the impurity reference standard prepared above. After the incubation, the wells are washed and the conjugated antibody is added. The plate is then washed and processed to record the detectable signal. Extensive details on the methodology of host-cell protein ELISA are found in references by Anicetti and coworkers.^^'^^ Host-cell ELISA have been used to detect low ppm protein impurities in a number of recombinant proteins including human growth hormone (18 ppm) [95] and human insulin ( < 4 ppm).^^ Protocol 10 is a host cell ELISA modified from that presented by Anicetti et al.^^ Protocol 10: Host-Cell Protein ELISA

• Add 100 /x,L of 2.5 ^ig/mL of the purified anti-host-protein antibody in 10 m M carbonate buffer, pH 9.6, to the wells of a 96-well microtiter plate and incubate overnight at 4°C. • Wash the wells twice with Tris-buffered saline containing 0.05% Tween 20. • Add 200 fjiL of Tris-buffered saline containing 0.1% gelatin and 0.05% Tween 20 to each well to block the remaining binding sites on the plate surfaces and incubate for 1 hr at 37°C. • Wash the wells twice with Tris-buffered saline containing 0.05% Tween 20. • Add 100 IJLL of the serially diluted samples or calibration standards to individual wells and incubate for 2 hr at room temperature. • Wash the wells twice with Tris-buffered saline containing 0.05% Tween 20. • Add 100 fjiL of the antibody-alkaline phosphatase conjugate (at 0.3 ^tg/mL) and incubate for 1 hr at 37°C. • Wash the wells twice with Tris-buffered saline containing 0.05% Tween 20. • Add 100 /xL of p-nitrophenyl phosphate at 1 mg/mL in 50m M Tris-HCl, pH 9.0, containing 0.5 m M MgCl2. Incubate for 30 min at room temperature. • Add 25 /xL of 3 M NaOH per well to stop color development. • Read the absorbance at 405 nm for each well using a microtiter plate reader. • Determine the concentration of host-cell proteins from the calibration curve. If the samples do not show linear dilution then the capture antibody was not in excess and the assay results are invaUd. ii. Western Blot Analysis

Unlike ELISA, which can detect and quantitate host-cell proteins as a group. Western blots detect single protein impurities. Western blot analysis starts with an SDS-PAGE protocol but does not include a final colorimetric staining step. Instead after electrophoresis the proteins are electrotransferred (blotted) from the gel onto a thin membrane. Membranes made of nitrocellulose or PVDF are most often used. Once the transfer is complete, the membrane is incubated with a nonspecific protein solution to saturate and to

50

DONALD O. O'KEEFE

block the membrane. The blocked membrane is then probed with the specific antiserum (the primary antibody) followed by a secondary antibody that recognizes the primary antibody. The secondary antibody is conjugated to a moiety capable of producing a detectable signal after development. Reports document as little as 30 pg of a protein can be detected by this method.^^ If the total protein analyzed by this method was 10 jitg, then the sensitivity of the assay for a single protein impurity can be as low as 3 ppm depending on the quality of the antiserum employed. Nevertheless, Western blotting is not a quantitative technique. It is a qualitative technique and useful for comparing the protein impurity profile from lot to lot, but in some circumstances it can provide a semiquantitative estimate of a protein impurity if a standard is available (see Section IV).^^ Western blot analysis of host-cell protein impurities should not stand alone. Using recombinant human growth hormone, Gooding and Bristow found £. coli protein impurities with greater sensitivity by Western blotting then with silver-stained polyacrylamide gels but in lesser numbers.^^ For this reason, regulatory agencies insist that immunoassays will not replace silver-stained polyacrylamide gels, but will complement them."* A ppm level of sensitivity is not universal for all host-cell protein impurities in Western blots. It is limited by the shortcomings of the antiserum preparation discussed previously and the Western blot protocol. The efficiency of transfer from the gel to the membrane is variable for different proteins. Electrotransfer from the gel to the membrane is inversely related to the size of the protein, i.e., smaller proteins transfer much more efficiently than larger ones. Including 0 . 1 % SDS in the blotting buffer or partial proteolysis of the protein within the gel facilitates the transfer of larger proteins.^^^ For smaller proteins, the membrane might bind them less effectively and may be permeable to some proteins, causing them to escape detection. Using a membrane with a smaller pore size might ameliorate this, while others have reported overcoming this phenomenon by using crosslinkers to trap the proteins on the membrane.^^^ The electrotransfer described in Protocol 11 is a modification of Burnette's method^^^ as adapter from Towbin et al}^^ Protocol 11: Western Blot Analysis SDS-PACE

• Samples are prepared and electrophoresed as described in Protocol 1 except mini SDS-polyacrylamide gels are used that measure approximately 5.5 cm X 9 cm X 0.75 mm. Suitable gels are supplied by Bio-Rad for use with the Mini Protean II electrophoresis apparatus. • The gels are electrophoresed at 20 mA/gel until the tracking dye reaches the bottom of the gel. Electrotransfer • The electrophoresed gel is washed in transfer buffer (192 m M glycine, 25 m M Tris, 20% methanol, 0.1% SDS) for 10 min to remove protein not within the gel. This lowers the background signal once the membrane is developed.

PROTEIN IMPURITY ANALYSIS

51

• A piece of nitrocellulose membrane (pore size 0.45 fim^ Schleicher and Schuell) is cut to the size of the gel and then equilibrated in transfer buffer. Always wear gloves when handling nitrocellulose to reduce the background on the developed membrane. • The gel is overlaid with the nitrocellulose membrane and any trapped air bubbles are removed. • The gel and the membrane are then sandwiched between sheets of Whatman 3MM paper and the entire assembly is placed in a Genie Electrophoretic Blotter (IDEA Scientific) according to the manufacturer's instructions. • The Genie Electrophoretic Blotter is filled with transfer buffer and connected to the power supply. Transfer is for 1 hr at 24 A with a Schauer battery charger (IDEA Scientific). Other electroblotters can be used but the author has obtained unparalleled success with the Genie. Proteins are completely transferred to membranes in 30-60 min.^^"^ Immunodetection

• All steps are performed at room temperature with gentle agitation. • The nitrocellulose membrane is remove from the Genie Electrophoretic Blotter and incubated for 1 hr in buffer A [10 m M Tris-HCl, pH 8.0, containing 50 m M NaCl, 2 m M EDTA, 1% (w/v) bovine hemoglobin]. • Add the appropriate dilution of the primary antibody to the blocked nitrocellulose membrane. Generally, 1:1000 to 1:5000 dilutions are suitable. Incubate overnight. • Remove the primary antibody and wash the nitrocellulose membrane three times with buffer B [100 m M Tris-HCl, pH 8.0, containing 200 m M NaCl, 1% (v/v) Igepal CA 630 (Sigma Chemical Co.)] for 5 min each time. • After the third wash, place the nitrocellulose membrane in buffer A. • Add the secondary antibody, which recognizes the primary antibody and is coupled to alkaline phosphatase, to a final dilution of 1:3000. Incubate for 2 hr. • Remove the secondary antibody solution and wash the nitrocellulose membrane three times with buffer B for 5 min each time. • Prepare the color development solution by adding the BCIP [25 mg of 5-bromo-4-chloro-3-indolyl phosphate, p-toluidine salt (Sigma Chemical Co.) in 2.5 mL of N,N'-dimethylformamide] and NBT (50 mg of nitro blue tetrazolium in 5 mL of 50% N,N'-dimethylformamide) solutions to 100 mL of 1.0 M Tris-HCl, pH 9.0. • Incubate the nitrocellulose membranes in the color development solution for 10-30 min. • Stop the color development solution by removing the solution and thoroughly rinsing the nitrocellulose membrane in 1 m M EDTA. Certain circumstances might arise where the bioanalyst wants to examine a recombinant protein preparation for low level protein impurities but a suitable antiserum is unavailable and the variability and nonspecificity of silver staining is unacceptable. An additional assay would be a Western blot analysis after derivatization of the protein mixture with Sanger's reagent

52

DONALD O. O'KEEFE

(2,4-dinitro-l-fluorbenzene,DNFB). DNFB reacts with protein amine groups. The subsequent DNP-protein derivatives are detected in a Western blot using anti-DNP antibodies as the primary antibody. How^ever, this procedure suffers from the disadvantage of also detecting the recombinant protein, w^hich is in excess, and producing a strong band at its location. Silver staining polyacrylamide gels has the same drav^back. Nevertheless, the author has detected bacterial protein impurities at levels less than 0.1 ng using this method. Protocol 12 describes this procedure in further detail. Protocol 12: Western Blot Analysis after Derivatization with Sanger's Reagent Derivatization

Prepare the follov^ing solution in an amber-colored tube: 100 fiL protein at 2 mg/mL in phosphate-buffered saline 32 fjiL 0.5 M sodium bicarbonate 160 jLtL DMSO 32 IJLL 0.1% DNFB in DMSO Incubate at room temperature in the dark for 10 min then add 36 ixL 0.1 M lysine SDS-PAGE

Samples are prepared and electrophoresed as described in Protocol 11. Electrotransfer

The proteins separated by SDS-PAGE are transferred to nitrocellulose as described in Protocol 11. Immunodetection

DNP-derivatized proteins are detected on the nitrocellulose membrane as described in Protocol 11 except the primary antibody is a 1:200 dilution of rabbit anti-DNP antibodies (Sigma Chemical Co.). Protocol 12 w^as adapted from Wojtkov^iak et aL w^ho used a similar procedure to react the proteins w^ith DNFB after the electrophoresed proteins w^ere bound to a nitrocellulose membrane.^^^ A disadvantage to derivatization prior to SDS-PAGE is the potential of altering the migration of some proteins or causing aggregation that prohibits protein entry into the gel. This potential problem is avoided w^ith postelectroblotting derivatization using the method of Wojtkov^iak et aL A similar procedure using other reagents is described in detail by Kittler et al}^^ Hi. Immunoligand Assay

In addition to ELISA and Western blots for detecting host-cell protein impurities there is a third immunoassay nov^ available to the bioanalyst for this purpose: the immunoligand assay (ILA). Like the ELISA, it is configured as a double-antibody sandv^ich. The anti-host-cell protein antibodies are separately conjugated to biotin and to fluorescein. A tripartite immune complex is formed betvvreen host-cell protein impurities and these two anti-

PROTEIN IMPURITY ANALYSIS

53

bodies. These immune complexes are then trapped on a biotinylated nitrocellulose membrane by the action of streptavidin. Subsequently, the complex is incubated with an anti-fluorescein-urease conjugate. The urease hydrolyzes urea to ammonia in a volume of 0.5 fiL and the resulting pH change alters the surface potential, which is then measured by a silicon sensor.^^^'^^^ Reportedly, this technology can detect as little as 0.05 ppm of host protein impurities in recombinant biopharmaceuticals.^^'^ This procedure has been used to measure host protein impurities both in recombinant human erythropoietin derived from mammalian cells^^^ and recombinant bovine somatotropin synthesized in £. coli}^^ iv. Detection in Immunoassays

The secondary antibody in immunoassays is conjugated to a moiety capable of eliciting a detectable signal. There are three different types of signals that can be elicited: isotopic, colorimetric, and chemiluminescent. Isotopic signals are derived from labeling the antibody with radioactive iodine. The use of this method is on the decline because of the difficulty working with radioactive hazards and the short shelf life of the reagent due to radioactive decay. Nevertheless, there may be instances where isotopic detection is desirable. The interested reader is directed to references at the end of the chapter describing the preparation of radioactive reagents.^^^"^^^ Chemiluminescence is the production of light from a chemical reaction. The emitted light is detected with either a luminometer or on photographic film. There are several substrates capable of producing these light emissions and luminol (5-amino-2, 3-dihydro-l, 4-phthalazinedione) and adamantyl 1,2-dioxetane aryl phosphate are two of the most popular.^^"^'^^^ The former is used with horseradish peroxidase (HRP) coupled antibodies and the latter is used with alkaline phosphatase (AP)-coupled antibodies. Using an AP-coupled antibody and adamantyl 1, 2-dioxetane aryl phosphate, Bronstein et al}^^ were able to detect as little as 125 pg of protein in a Western blot. Colorimetric detection is the most widely used method of detection in immunoassays. It is based on the action of an antibody-coupled enzyme on substrates to produce a colored product. The two most common enzymes used are HRP and AP. Of these two, HRP has more serious disadvantages. HRP activity is inhibited by azide, certain substrates are suspect carcinogens (e.g., 3,3'-diaminobenzidine), and the results with some substrates on blots can fade with time. HRP activity and sensitivity are also strongly dependent both on the concentration of hydrogen peroxide, which itself is quite unstable, and the pH. AP, on the other hand, is not inhibited by azide but it is inhibited by free phosphate ions. AP-antibody conjugates display excellent stability over time. The color development of the AP substrate p-nitrophenyl phosphate is linear over time. It is stable, nonhazardous, and obtainable in a tablet form to minimize handling. The AP substrate BCIP produces a colored precipitate on blots that does not fade appreciably with time. Many AP-antibody conjugates are commercially available for Western blots. However for the host-cell protein ELISA described in Protocol 10, the analyst has to prepare their own AP conjugate. Two methods are generally employed to create these conjugates. One method utilizes glutaraldehyde and can lead to a

54

DONALD O. O'KEEFE

high molecular weight crosslinked product.^^^ A second method utilizes a heterobifunctional maleimide that produces a better-defined product lacking undesirable crosslinking. A procedure for this method adapted from Ishikawa et al}^^ is described in Protocol 13. Protoco/ / 3 : Conjugation of Alkaline Phosphatase to Antibodies

• Dissolve 8 mg of calf intestinal alkaline phosphatase (CIP) in 1.0 mL of 50 m M Na-borate, pH 7.6, containing 1 m M MgCl2 and 1 m M ZnCl2. • Prepare 80 m M N-succinimidyl 4-(N-maleimidomethyl) cyclohexane1-carboxylate (Pierce Chemical Company) in N,N'-dimethylformamide. • Add 50 IJLL of the maleimide reagent to 1.0 mL of the CIP and

incubate for 1 hr at 30°C with constant moderate agitation. • Separate the derivatized CIP from the reagent by desalting on a Sephadex G-25 column equiUbrated in 100 m M Tris-HCl, pH 7.0, containing 1 m M MgCl2, and 0.1 m M ZnCl2. • Concentrate the maleimide-CIP to 2 mg/mL. • Add 0.5 mL (1 mg) of the maleimide-CIP to 0.5 mL (9.2 mg/mL) of purified antibody Fab' fragments (see reference 117 for the preparation of Fab' fragments) in 50 m M Na-acetate, pH 5.0, and incubate for 20 hr at 4°C. • Stop the reaction by adding 2-mercaptoethylamine to 1 m M and incubating for 20 min at room temperature. • Separate the conjugate from the uncoupled proteins by chromatography on Sephadex G-200 equilibrated in 10 m M Tris-HCl, pH 7.0, 100 m M NaCl, 1 m M MgCl2, 0.1 m M ZnCl2, and 0.02% NaN^, Pool the fractions containing the conjugate and add bovine serum albumin to 1 m g / m L and store at 4°C. • The conjugated CIP-antibody can also be chromatographed on the affinity column of the host-cell protein reference standard to eliminate antibody conjugates that may have lost their binding activity in the coupling procedure.

E. Identification of Host-Cell Protein Impurities

Host-cell protein ELISA have the advantage of quantitating host protein impurities. The disadvantage, hov^ever, is that the quantitation is of a group of impurities. Western blot analysis, on the other hand, provides the analyst w^ith a relative level of an individual impurity compared to other impurities. If the level of one or more host protein impurities appears to be excessive based on the intended use of the drug product then it may be necessary to identify those impurities. This can provide assurance that the impurity is innocuous and it can also define the physicochemical properties of the impurity such that the process can be modified to reduce its presence in future production lots. The identification can also lead to the development of a quantitative assay for monitoring the individual impurity in every lot. For the majority of host-cell protein impurities, the most direct w^ay of identification is through N-terminal sequence analysis. The automated

PROTEIN IMPURITY ANALYSIS

55

gas-liquid protein sequencer can routinely determine the N-terminal sequence of as little as 10 pmole of protein. For a 50 kDa protein this is equivalent to 50 ng, which is 50 ppm for 1 mg of drug product. The isolated impurity is first immobilized on a glass fiber disk followed by repetitive rounds of Edman degradation. The released PTH amino acids are separated by RP-HPLC and identified through the use of an amino acid standard.^^^ Two criteria determine the steps required prior to N-terminal sequence analysis: the level of the impurity and the ability to separate it from the drug product. A high-level impurity that is separable from the drug product by SDS-PAGE can be sequenced directly from a membrane after electroblotting. The procedure is similar to that used for preparing membranes for Western blot analysis but there are critical differences. The electrotransfer buffer is instead 10 m M 3-[cyclohexylamino]-l-propanesulfonic acid (CAPS), pH 11.0, with 10% methanol. The glycine and Tris in the Western transfer buffer would cause high background readings during sequencing. SDS can be added to this buffer as before to facilitate protein transfer. The membrane used is PVDF instead of nitrocellulose because of higher protein binding capacity and superior chemical resistance.^^^ Other types of membranes have also been used; notable is carboxymethylcellulose membranes for more basic proteins.^^^ After SDS-PAGE, the migration of the drug product is determined so that the recombinant protein can be excised from the gel. Since the drug product will be in great excess over any impurity, it is likely that the recombinant protein will overwhelm the membrane after transfer and its sequence will be detected everywhere.^^ The recombinant protein is most easily removed by cutting off the extreme ends of the gel and staining the gel slices with Coomassie Blue to identify its location, and then excising the unstained protein from the gel. Protocol 14: Isolation of Host-Cell Protein Impurities by Electroblotting SDS-PAGE

• Samples are prepared and electrophoresed as described in Protocol 11. Electrotransfer

• The ends of the electrophoresed gel are removed with a scalpel and stained with Coomassie Brilliant Blue R-250 (1 m g / m L in 50% methanol-10% acetic acid). Staining for 10-20 min should be sufficient to see the recombinant protein. If necessary, the gel strips are destained briefly with 50% methanol-10% acetic acid. • The identified region containing the recombinant protein is excised from the gel using a scalpel and discarded. • The electrophoresed gel is then washed in transfer buffer (10 m M CAPS, pH 11.0, 10% methanol) for 10 min to remove protein not within the gel and to wash away Tris and glycine. • A piece of PVDF membrane (ProBlott, Applied Biosystems) is cut to the size of the gel and equilibrated first in methanol and then in transfer buffer for 5-10 min each time. Always wear gloves when handling the PVDF membrane to reduce background sequences.

56

DONALD O. O'KEEFE

• The gel is overlaid with the PVDF membrane and any trapped air bubbles are removed. • The gel and the membrane are then sandwiched between sheets of Whatman 3MM paper and the entire assembly is placed in a Genie Electrophoretic Blotter according to the manufacturer's instructions. • The Genie is filled with transfer buffer and connected to the power supply. Transfer is for 1 hr at 24 A with a Schauer battery charger. Detection and N-terminal Sequencing Preparation

• The PVDF membrane is washed in deionized water and then stained briefly with Coomassie Brilliant Blue R-250 (1 m g / m L in 50% methanol-10% acetic acid) to the point where the protein impurity is visualized but not more than 5-10 min. Alternatively, the membrane is stained with Amido Black or Ponceau S. Destain the membrane with 50% methanol-10% acetic acid as much as possible and then let the membrane air dry. If the impurity is not visualized, then end slices of the PVDF membrane are immunodetected as in Protocol 11 to localize it. • The stained impurity is cut from the membrane on as small a portion as possible. • The membrane strip is cut into small fragments and placed directly into the sequenator for analysis. If no sequence is revealed by the preceding procedure, then it may be because the sequence is blocked at the N terminus or the protein level is too low. Proteins that are blocked at the N terminus can be enzymatically digested on the membrane, the peptides isolated, and an internal sequence can be determined.^^^ If the protein level is too low, then the impurity might not be amenable to the blotting approach for sequence analysis discussed earlier. In these instances, the protein must to be isolated from upstream process intermediates to obtain a fraction enriched in the impurity. This is done by first identifying these fractions by Western blot analysis and then chromatographically purifying the impurity from these fractions. The identification of an £. coli protein impurity in preparations of recombinant acidic fibroblast growth factor was accomplished in this way (see Section IV).^^ Determination of the 10 N-terminal amino acids is generally sufficient to identify the protein. The sequence obtained is compared to published databases using homology search engines.* However, determination of the N-terminal sequence does not ensure that the protein will be identified. With the recent deciphering of the entire genome of £. coli^^^ any protein impurity sequence found in biopharmaceuticals derived from this recombinant bacterium should be identified. The entire protein sequence can then be used to develop peptide antigens for antibodies useful in future assays of the impurity. Unfortunately, protein databases and genome analyses are less complete for other recombinant hosts such as yeast and mammalian cells, so alternative strategies need to be developed. One alternative method is to perform • An Internet link to protein sequence databases and search engines is found at www.sdsc.edu/ResTools/biotools/biotoolsl9.html and a Hnk to Internet resources for sequence analysis is www.sdsc.edu/ResTools/biotools/biotoolsl.html.

57

PROTEIN IMPURITY ANALYSIS

two-dimensional gel electrophoresis. After staining with an appropriate method, the pi and mass coordinates of the impurity are compared to those of two-dimensional gel databases from the host organism.* If the corresponding protein in the database is known, then specific antibodies can be obtained to identify and quantitate that impurity.

III. EXPERIMENTAL SUMMARY Numerous methods exist for determining the protein purity and impurity profile of biopharmaceuticals derived from recombinant DNA. These methods are both qualitative and quantitative in nature and span four different types of analytical technologies. The reasonable detection limits for protein impurities via these technologies are presented in Table 2. No single method or technology is sufficient to give a complete purity assessment or impurity profile for any particular recombinant therapeutic. Instead, several methodologies must be utilized orthogonally, i.e., those that separate and detect protein impurities based on different physicochemical properties. A prudent approach would be to utilize RP-HPLC, CZE, and an immunoassay. This tripartite attack would resolve protein impurities based on hydrophobicity, charge-to-mass ratio, and antibody recognition, respectively. With validated assays in each of these disciplines, it is somewhat unlikely that any detectable impurity would not be resolved from the protein drug product or other impurities. However, impurities below the sensitivity limits will remain undetected. The tripartite strategy proposed is not in complete alignment with that currently recommended by regulatory agencies, many of which still advocate silver staining of polyacrylamide gels as an indispensable and sensitive impurity test. Nevertheless, it is the author's opinion that this will eventually change. As the proposed technologies are developed further, as their usage becomes more widespread, and as their sensitivities become greater and differ * Two-dimensional gel databases are located on the Internet. One for 5. cervisiae is found at www.proteome.com and one for both S. cervisiae and £. coli is found at http://expasy.hcuge.ch/ch2d/.

T A B L E 2 Approximate Protein Impurity Detection Limits for C o m m o n Bioanaiytical Techniques Impurity level Technique SDS-PAGE RP-HPLC CE Immunoassay

%(w/w)

ppm

1 0.04 0.04 < 0.001

10,000 400 400 <10

The levels are based on validated quantitative methods for these techniques.

58

DONALD O. O'KEEFE

less from each other, their utilization in protein impurity analysis will become a mandate.

lY. CASE STUDIES A. Identification of a Host-Cell Protein Impurity in Recombinant Acidic Fibroblast Growth Factor Acidic fibroblast growth factor (aFGF, M^ = 15,997) possesses both mitogenic and chemotactic activity toward endothelial cells. These properties have focused attention on this protein as a potential therapy for wound healing and vascular repair.^^^'^^^ The recombinant form of the protein is produced in E, coli and is indistinguishable in its physicochemical and biochemical properties from aFGF derived from human brain.^^"^"^^^ Early large-scale preparations of recombinant aFGF were evaluated for purity by reversedphase HPLC and SDS-PAGE stained with either Coomassie Blue or silver [98], None of these methods were able to detect protein impurities. However, Western blot analysis of several lots of recombinant aFGF using antiserum raised against £. coli proteins revealed an immunoreactive band of approximately 26,000 Da (Fig. 1). The initial strategy to identify this impurity was to separate the protein by SDS-PAGE, isolate it by blotting onto a PVDF membrane, and subjecting it to N-terminal sequencing.^^^ Unfortunately, this approach was problematic. The great excess of aFGF both prevented complete separation of the proteins during SDS-PAGE and interfered with blotting onto the PVDF membrane. The impurity was instead isolated from side fractions of process

1 2

3

4 ^43.0

<Mlifc'»«<wwfc ii(miii)ni)ip 0mm

^ 2 9 . 0

-18.4 *-14.3 •* 6.2

F I G U R E I Western blot analysis of aFGF preparations probed with anti-£ coll protein antiserum. Each sample contained 10 /ng of aFGF. The lanes represent (I) the reversed-phase product of lot 2, (2) the reversed-phase product of lot 3, (3) the reversed-phase product of lot 4, and (4) lot 6 of the drug substance. The migration of protein markers is shown on the right, with masses expressed in kilodaltons. (Reprinted from O'Keefe et a/.^^ Copyright 1993 with permission from Plenum Publishing.)

PROTEIN IMPURITY ANALYSIS

59

F I G U R E 2 Process side cuts contain the 26 kDa £. coli impurity. Column fractions after preparative HPLC of recombinant aFGF were examined by Western blot analysis using an anti-E. coli protein antiserum (insets). Top: High-performance lEX chromatography. Bottom: High-performance affinity chromatography. The main peak in each chromatogram is aFGF. Chromatographic fractions containing an immunoreactive protein migrating at ~ 2 6 kDa are marked by the hatched areas. Fraction 4 was used to isolate the impurity. The migration of protein markers is shown at the right of each blot, with masses expressed in kilodaltons. (Reprinted from O'Keefe, et o/.'® Copyright 1993 with permission from Plenum Publishing.)

chromatography steps (Figs. 2 and 3) and then subjected to N-terminal sequence analysis. Sequencing tentatively identified the impurity as the S3 ribosomal protein from E, coli and later this was confirmed using monoclonal antibodies to S3 in a Western blot.^^ A semiquantitative Western blot assay was developed with these monoclonal antibodies and the level of the S3

60

DONALD O. O'KEEFE

Time (min) F I G U R E 3 R P - HPLC to isolate the £ coli protein impurity. Column: Poros R/ H (100 X 4.6 mm i.d., Perceptive Biosystems). Mobile phases: A, 0 . 1 % TFA in water; B, 0 . 1 % TFA in 80% acetonitrile. Gradient: 0 - 1 0 sec, 10% B; 10 - 50 sec, 10 - 35% B; 50 sec to 10 min, 35 - 55% B. The flow rate was 4 m L / m i n . Absorbance was monitored at 280 nm with 0.06 AUFS. Inset: Western blot showing the isolated impurity is reactive to anti-£ coli protein antiserum. The migration of protein markers is shown at the right of the blot, with masses expressed in kilodaltons. (Reprinted from O'Keefe et o/.^^ Copyright 1993 with permission from Plenum Publishing.)

impurity in several lots of recombinant aFGF was established at less than 100 ppm(<0.01%).^^ B. Selective Resolution of a Protein Impurity Using RP-HPLC and Fluorescence Derivatization Transforming growth fsiCtor-oi-Pseudomonas aeruginosa exotoxin A 40 (TGFa-PE40, M^ = 44,960) is a recombinant fusion protein synthesized in E. coli}^'^'^^^ The growth factor moiety binds to surface epidermal growth factor (EGF) receptors on cancerous cells and is internalized where it releases the exotoxin domain into the cytosol.^^^ The toxin subunit catalytically inactivates the protein synthesis machinery of the cancer cell and the cell subsequently dies.^^^ It is a recombinant protein intended to be an anticancer therapeutic. During the developing process for TGFa:-PE40 a product-related impurity was copurifying with the drug substance. This impurity lacked the TGFa portion of the fusion protein and only had the PE40 portion. Hence, the impurity was nonreactive in an anti-TGFa Western blot but could be detected in a blot using anti-PE40 serum (Fig. 4). The resolution of SDS-PAGE was insufficient to quantify this impurity.^"" Numerous RP-HPLC methods were also unable to separate these two proteins sufficiently for quantitation.^^

61

PROTEIN IMPURITY ANALYSIS

Anti-PE 1 2

3

4 200 kD 97.4 68 ^ TGFa-PE40 ^ PE40 ^

43 29 18.4 14.3

F I G U R E 4 Western blot analysis of T G F Q ; - P E 4 0 in process samples. Proteins transferred to nitrocellulose paper were probed with either anti-PE or anti-TGFa antisera. The lanes for each blot correspond to: (I) a TGFa-PE40 enriched sample, (2) a PE40 enriched sample, (3) a cell lysate from £ coli that produced TGFa-PE40, and (4) a partially purified TGFa-PE40 sample. Lanes I, 3, and 4 within each blot contained the same amount of TGFa-PE40, while lane 2 contained an equivalent amount of PE40. The gels were overloaded to highlight TGFa-PE40 aggregates and degradates in the samples. The migration of protein markers is indicated for each blot with masses expressed in kilodaltons. (Reprinted from O'Keefe et al}^ Copyright 1992 with permission from Elsevier Science.)

Knowledge of the primary structure of TGFa-PE40 revealed the presence of six cysteine residues in the TGFa moiety and none in the PE40 portion. Derivatization of the cysteine thiols with monobromobimane (mBBr, Molecular Probes) according to Protocol 15 yielded a fluorescent TGFa-PE40 product and a nonfluorescent PE40 impurity (Fig. 5). This enabled the selective discrimination of the two proteins by RP-HPLC with simultaneous fluorescence and UV absorbance detection.^^ Hence, the amount of the impurity was determined by subtracting the amount of T G F Q : - P E 4 0 determined by fluorescence from the total amount of T G F Q : - P E 4 0 plus PE40 determined by UV absorbance.^^ This assay was instrumental in modifying the process for subsequent elimination of the PE40 product-related impurity. Protocol IS: Derivatization of Protein Thiols with mBBr Prepare the following solution and incubate at 90°C for 5-10 min: 10 nmoles of protein thiol 100 fiL 2.0 M Tris-HCl, pH 8.0 100 /xL 10% SDS 100 fiLSOmM EDTA 100 /xL 30 m M DTT (made fresh) Adjust the volume to 1.0 mL with water

62

DONALD O. O'KEEFE

F I G U R E 5 Reversed-phase chromatograms of TGFa-PE40 and PE40-labeled with mBBr. Approximately 7 fig of each protein were injected onto a HY-TACH nonporous C|8 column (30 X 4.6 mm i.d., Glycotech) using a linear gradient of 34 - 64% acetonitrile in 0.1 % TFA over 6 min at 1.0 mL / min. The column was maintained at 80°C. Fluorescence was monitored with a 470 nm filter after excitation at 382 nm. The top chromatogram represents T G F Q ; - P E 4 0 and the bottom chromatogram represents PE40. (Reprinted from O'Keefe et al}^ Copyright 1992 with permission from Elsevier Science.)

Cool to room temperature and add 8 jxL 100 m M mBBr in acetonitrile Incubate for 2 min away from direct light. Terminate the reaction by adding fresh cysteine to 10 m M .

C. Detection of yV-terminal Variants Using Peptide Mapping and Fluorescence Detection The problem of N-terminal variants in recombinant proteins is not uncommon. £. coli synthesizes proteins with a formylated methionine at the N terminus. In vivo, E. coli often removes N-formyl methionine with the action of a deformylase followed by methionine amino peptidase. This removal is not always exact and neighboring amino acids in the peptide chain influence the removal.^^^ This can yield recombinant products lacking a number of encoded amino acids at the N terminus. For smaller proteins, these product-related impurities generally are detected and quantitated by RP-HPLC. However, large proteins differing by only one or two N-terminal amino acids may be difficult to resolve by RP-HPLC. In these instances, peptide mapping by RP-HPLC is a valuable tool.

63

PROTEIN IMPURITY ANALYSIS

UUJ 20'

30'

40'

50' Time

60'

70'

80'

90'

Cmin)

F I G U R E 6 Tryptic peptide mapping of a recombinant protein. A recombinant protein was labeled with mBBr as in Protocol 15, enzymatically cleaved with trypsin as in Protocol 6, and then chromatographed by R P - H P L C on a Vydac 2I8TP column. Mobile phases: A, 0 . 1 % TFA in water; B, 0.1 % TFA in 80% acetonitrile. Gradient: 0 - 5 min, 0% B; 5 - 90 min, 0 - 50% B. The flow rate was 1.0 mL / min. Detection was UV absorbance at 218 nm (top chromatogram) and fluorescence with a 470 nm filter after excitation at 382 nm (bottom chromatogram).

A recombinant protein with an approximate molecular mass of 50 kDa containing N-terminal variants was subjected to tryptic peptide mapping according to Protocol 6. The resulting chromatogram with UV absorbance detection at 218 nm is shown at the top of Fig. 6. Greater than 35 separate peaks are seen in this map. Initially, none of these peaks were characterized and a full-length standard was unavailable for comparison. Therefore, identifying the N-terminal variant peptides in a short time would be a formidable task. However, within this protein there are three tryptic peptides containing cysteine residues, and the N-terminal peptide is one of them. Labeling this protein with mBBr according to Protocol 15 followed by tryptic peptide mapping with fluorescence detection yielded a simpler map (Fig. 6, bottom). However, this method is expected to detect only three fluorescent peptides in this protein, any additional peaks are due to the N-terminal variants. Subsequent process changes reduced the presence of the N-terminal variants and simplified the peptide map with fluorescence detection (Fig. 7, bottom). This enabled the detection of at least four N-terminal variant peptides in the original drug substance (Fig. 7, top) that could be isolated for further study.

64

DONALD O. O'KEEFE

145'

150

155' TimQ

160'

165'

170'

(min)

F I G U R E 7 Detection of N-terminal variants by tryptic peptide mapping with fluorescence detection. The final purified product of two different lots of the same recombinant protein were labeled with mBBr according to Protocol 15 and then subjected to tryptic peptide mapping as described in Protocol 6. The conditions of chromatography were the same as those described in Fig. 6 except the gradient was made shallower by extending it to 175 min. Fluorescence was monitored for both chromatograms, as described in Fig. 6.

ACKNOWLEDGMENTS The author is very indebted to his colleagues Tony Paiva, Tim Smith, and Mark Will, who participated directly, intellectually, and supportively in the development of much of this work.

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PHYSICOCHEMICAL FACTORS IN POLYPEPTIDE AND PROTEIN PURIFICATION AND ANALYSIS BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC TECHNIQUES: CURRENT STATUS AND CHALLENGES FOR THE FUTURE MILTON T. W . HEARN Centre for Bioprocess Technology, Department of Biochemistry and Molecular Biologyy Monash University, Clayton, Victoria, Australia

I. INTRODUCTION II. BASIC CHROMATOGRAPHIC TERMS A N D CONCEPTS III. THE CHEMICAL STRUCTURE OF POLYPEPTIDES A N D PROTEINS IV. PHYSICOCHEMICAL FACTORS THAT UNDERPIN LIGATE INTERACTIONS W I T H POLYPEPTIDES A N D PROTEINS IN HPLC SEPARATION SYSTEMS A. Hydrophobic Interactions B. Hydrogen Bond Interactions C. Electrostatic Interactions D. van der Waals Interactions and Weak Polar Interactions E. Metal Ion Coordination Interactions F. TT ^ 77 Dipole -^ Dipole Interactions and Charge -» Dipole Interactions G. Combined Effects V. STRATEGIC CONSIDERATIONS BEHIND THE HPLC SEPARATIONS VI. SPECIFIC PHYSICOCHEMICAL CONSIDERATIONS O N THE INDIVIDUAL CHROMATOGRAPHIC MODES

Separation Science and Technology, Volume 2 Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

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VII. THE EFFECT OF TEMPERATURE A N D THE THERMODYNAMICS OF POLYPEPTIDE- OR PROTEIN-LIGATE INTERACTIONS VIII. FACTORS THAT CONTROL PERFORMANCE A N D EFFICIENCY IX. SCALING-UP POSSIBILITIES: HEURISTIC APPROACHES AND PRODUCTIVITY CONSIDERATIONS X. EFFECT OF MASS TRANSFER RESISTANCES IN PREPARATIVE HPLC OF POLYPEPTIDES A N D PROTEINS XI. SUMMARY REFERENCES

INTRODUCTION This chapter presents an overview of the physicochemical factors that impact on the analysis and purification of polypeptides and proteins by high-performance liquid chromatography (HPLC) techniques. The current status and some of the future challenges facing this major field of the separation sciences are considered from both didactic and practical perspectives. Deliberately, this chapter does not attempt to develop the full theoretical treatments behind the use of HPLC procedures w^hen operated under linear and nonlinear elution conditions in analytical or preparative overload situations. Nor does this chapter embark on a detailed discussion of the ways computer science, biochemical engineering, and industrial process development interface with HPLC capabilities for the purification of polypeptides and proteins. To examine these issues and their various applications, interested readers are referred to other publications and books.^"^^ Rather, this chapter attempts to provide an overview, particularly for our biological and life sciences based colleagues, of the terms, concepts, principles, practical aspects, and primary references that underpin these recent developments in this field. Where appropriate, key relationships and dependencies that describe the interactive behavior of polypeptides and proteins with chemically immobilized ligands are introduced and discussed. This understanding is central to any subsequent exploration of the alternative avenues now available for further research and development in the field of polypeptide or protein purification and analysis by these high-precision and high-resolution separation methods. During the past 20 years, the so-called high-resolution or high-performance separation techniques have evolved considerably from the methods developed with the less sophisticated antecedents. The development of these powerful techniques, pioneered with much innovation and vigor by a host of talented, but often underrecognized analytical chemists, biochemists, and materials scientists, has set the stage for integration of orthogonal high-resolution separation methods as essential, pivotal components of functional genomes and proteomics in the next century. Just as molecular biology has come to provide the tools to decipher the genetic organization of the cell at a one-dimensional level, advances in high-resolution separation science provide

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the means to turn this opportunity into physical, chemical, and biological realities. Without the modern separation sciences, modern biology, functional genomics, and proteomics would not exist. It is thus appropriate to consider these pioneers of modern high-resolution separation procedures as the unsung champions of the modern biological sciences, the pathfinders who have proved the tools that have enabled the molecular road maps of cellular biology to be at last discriminated in detail. In practically every niche of life science research and development as now practiced, high-resolution separation techniques and, in particular, the hyphenated combinations of high-performance liquid chromatography with other technologies, such as mass spectrometry (MS), capillary electrophoresis (CE), field flow fractionation (FFF), or surface-mediated biosensor interaction (SMID), hold pervasive and often preeminent positions. These techniques can be employed individually, but their full power becomes apparent when they are employed in the preferred tandem mode, as orthogonal separation or analysis procedures par excellence. The power of these techniques to address the molecular diversity of biological phenomena is most impressive, ranging from their ability to enable characterization of mass differences as little as 1 Da in a molecule with a molecular mass of 5 X 10"^ Da such as the singly deamidated or aspartimide-rearranged isoforms of posttranslationally modified proteins^^'^"*; through to the assignment of subtle pK^ differences in compositionally identical but topologically different polypeptide isomers^^; to the resolution of the different tertiary U-shaped and /-shaped conformational structures of long chromosomal DNA fragments^^; to the identification of unique sites of methionine oxidation, lipidation, or other types of posttranslational modification of commercially valuable, therapeutical proteins expressed in mammalian cell culture systems.^^'^^. Irrespective of whether the investigative scientists are employed in the biopharmaceutical or biotechnological industries in quality control laboratories; are engaged in a taxing environmental problem associated with degradation of biowaste or the removal of biocontaminants from water; are carrying out the analysis of metalloproteins in the effluents from the microbial recovery of precious metals from mine tailings; are studying the role of protein allergens in the food industry; are actively pursuing within a molecular biology laboratory the cloning and expression of new genes; are responsible for vaccine development programs for use with animals, humans, or plants; or are developing new classes of chemical or peptidomimetic substances from synthetic combinatorial libraries in drug discovery, the exquisite sensitivity, the speed, and the impressive resolving power of the high-performance separation procedures now available finds repeated application. At the center of this frenetic activity are the HPLC techniques. These techniques have occupied a dominant position for over 2 decades in peptide and protein chemistry, in molecular biology, and in biotechnology. This is not surprising when viewed from the perspective of the ease of their utilization, the robustness and reproducibility of the separation, and their potential for scalability over at least a 1000-fold range or more; all of which have resulted in HPLC-based technologies occupying a significant percentage of

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total share of the instrumentation and biosciences market. In fact, HPLC techniques in their various selectivity modes can rightly be considered the experimental bridges that link, on the one hand, cellular and molecular biology as practiced today v^ithin the emerging fields of biological research that are collectively called structural proteomics and its sibling atomic biology or as more appropriately called zeomics, a term recently introduced by the author to describe the integration of molecular and cellular events in biological systems, thus encompassing and including supramolecular interactions and the information content of a species as revealed or predicted from genomic or proteomic analysis and, on the other hand, industrial process development associated w^ith the recovery and purification technologies that turn these opportunities into realities. Concurrent with the development of nev^ types of sorbents and new^ concepts on how^ to use them and interpret the separation data in terms of molecular properties of the analytes has been the emergence of sophisticated nevv^ instrumental methods. This combination of the conceptual, theoretical framework with the practical has meant that HPLC instrumental procedures and the other high-resolution separation methods now enable the quantitative characterization of changes in composition, topology, and dynamics of biomacromolecules as they interact at liquid-liquid and liquid-solid interfaces. The appropriate integration, optimization, and application of these methods provides the essential experimental strategies for scientists to now evaluate biological phenomena at a level of molecular precision and discrimination not thought attainable even 10 years ago. Central to these research developments is the link offered by high-resolution separation methods such as HPLC and the function of biological molecules at the precise structural level. Elaboration of the biophysical behavior of polypeptides and proteins, for example, in terms of the fundamental structural basis of interaction dependencies, the characterization of the physically relevant isothermal binding properties, and the opportunity to iteratively design and tailor new types of interactive surfaces of greater selectivity and molecular recognition power, have all combined to provide the basis of a new subdiscipline in the high-resolution separation sciences, increasingly known^^'^^ as molecular chromatography and its satellite molecular electrophoresis. Extension of these high-resolution separation capabilities can be anticipated to provide a very significant catalyst for a revolution in our understanding of the physicochemical basis of complex biological relationships. Progressively, the current one-dimensional perspectives of biological phenomena that has been offered by molecular biology will be transcended over the next decade or so by more descriptive and quantitatively more precise interpretations of the cellular function and supramolecular or supermolecular processes by which proteins and other biomacromolecules interact at the three-dimensional atomic level within the ambience of the cell. It can thus be strongly argued that during the next decade high-resolution separation procedures will continue to provide the most potent expression of the role of pervasive instrumental and analytical capabilities in the life sciences. This opportunity, already leading to a bonanza of experimental outcomes and information, will have many practical and pragmatic consequences. For those young scientists who take the plunge enthusiastically, who rise to the

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challenge and commit themselves to the conceptual as well as technical-engineering problems posed by these developments at the interface of high-resolution separation sciences and modern biology, they will have, as prophetically stated^^ over 40 years ago in a related context by Oliver Lowry, a biochemist whose name is synonymous with one of the most widely used "classical" techniques in quantitative life sciences analysis, "all the fun and get paid too!" At one level, the reason for the versatility and importance of HPLC is relatively simple to appreciate. In practical terms, HPLC in its different dominant interactive modes—ion-exchange (HP-IEX), reversedphase (RP-HPLC), hydrophobic interaction (HP-HIC), metal ion affinity (HP-IMAC), hydrophilic interaction (HP-HILIC), biospecific and biomimetic affinity (HP-BAC), normal or polar phase (NP-HPLC), and the various mixed mode combinations that can be generated through the participation of more that one dominant interaction process—represent a cornucopia of analytical and preparative methods that now require relatively little experience on the part of the practitioner to initially achieve a significant result. In this context, our scientific colleagues of the mid-1970s through the early 1990s did their job well, since the experimental methodologies enunciated during this period can in many cases be directly applied by endocrinologists, animal physiologists, or plant biologists to indicate but three groups of current end-users, in their studies on the functional properties of a particular protein, or a gene that encodes it. In conceptual terms, HPLC also provides an often-untapped treasure trove of physicochemical information, since buried within the chromatographic retention data and peakwidth characteristics are Aladdin's caves of quantitative data on the size, shape, and electronic characteristics of these biomacromolecules and how they interact with chemically modified surfaces. This opportunity provides a profitable avenue to approach the interaction thermodynamics; the kinetics of mass transfer in porous and nonporous media, the nature of linear free energy dependencies, and the molecular descriptor relationships that determine retention and separation selectivity; or the origin at a molecular level of the different types of secondary chemical equilibrium that occur during the passage of a biomacromolecules over the interactive surface of a chemically modifies particle use in an HPLC column. Deciphering this information enables important insights to be gained into many quantitative structure-retentionfunction relationships (QSRFs); the nature of protein folding and domain assembly in different types of chemical environments; the processes by which proteins and other biopolymers aggregate and isodesmically self-associate; and the fundamental rules of molecular biorecognition at solid-liquid and hquid-liquid interfaces. The 1980s can be considered the decade when the more generafist and mainly empirical application of HPLC in the life sciences occurred. The 1990s have been a decade of consolidation in the various application fields. The early part of the next century can be expected to provide the quantitative platform whereby HPLC procedures facilitate major advances in our understanding of the relationships between the three-dimensional structure of biosolutes, their interactive behavior, the molecular attributes of the contact regions generated when a biomacromolecule docks with immobilized chemical (or biological) ligands (immobilized ligands, in contradistinction to free

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ligands in solution, are subsequently referred to in this chapter as ligates) on support materials such as chromatographic media, and the rules that govern at the atomic level the maintenance of the biological function of these molecules in these systems. Recognition of the opportunities that HPLC thus offers is what now drives synthetic chemists to prepare new classes of tailored, "molecularly engineered," or combinatorial ligands as part of new sorbent development; the theoreticians who are interested in the computational modeling of the chromatographic and associated interactive processes; the various multidisciplinary groups of investigators who are engaged at the interface between surface chemistry, protein engineering, information science, and bioinformatics; the laser physicists who are interested in new instrumental developments exploiting advances in spectroscopy; the engineers who are creating microfabricated devices and equipment; the biochemists who are utilizing nanochemistry procedures in the development of artificial proteins; or the process engineers who are employing more efficient and productive high-resolution separation procedures for the recovery of a valuable biomolecules at an industrial scale.

II. BASIC CHROMATOGRAPHIC TERMS AND CONCEPTS The 1980s and the early years of the 1990s represented a period of important advances and consolidation in chromatographic theory and its application to biomacromolecular separations by high-resolution procedures. The full impact of these developments on biopolymer HPLC has yet to be realized, although their underlying consequences are providing directions ranging from instrument miniaturization and construction of micromachined devices to the use of integrated modeling procedures for purification processes. Developments associated with nonlinear elution and displacement chromatography are finding a place of special relevance for process HPLC, while combinatorial nanochemistry is offering new opportunities for ligand design and selection. It is not the intention of this chapter to describe in detail all of these various developments in chromatographic theory, but rather to introduce at several places within the chapter examples that have found useful practical expression. Readers who are interested in further exploring these and alternative theoretical treatments, can see several monographs^"^'^'^^"^^'^^"^"^ and other publications^'^'^^'^^'^^""^^ for different perspectives on these important aspects. Thus, in the following section, the basic chromatographic terms are introduced as practical application aides to investigators wishing to understand and improve the separation performance of different HPLC procedures. In a packed chromatographic column, the separation of a mixture of polar, ionizable biomacromolecules, such as peptides or proteins, is a consequence of two events. The first event, which controls the average retention behavior of the biosolutes, is embodied in the concept of mass distribution as the biosolutes migrate along a chromatographic bed (or column) of length L, operated at a mobile phase flow rate F. As the biosolutes move down the column, individual components interact to different extents with the chemical entities within the stationary phase and the mobile phase. When the interac-

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tion of a specific biosolute P^ with the stationary phase is very strong, the equihbrium distribution coefficient Kassoc, i is large, then P^ will be retained to a greater extent than other components that interact less strongly. Solute concentration zones corresponding to each component will therefore migrate through the column with different intrinsic velocities. This differential migration is a function of a variety of related physicochemical properties, including the equiUbrium distribution coefficients established by the biosolutes between the stationary and mobile phases, the effective diffusivities of the biosolutes, and their intrinsic linear flow (or so-called superficial) velocities. The isothermal characteristics, the kinetics of the various mass transfer stages, and the level of heterogeneity of the sorbent will all impact of the mass transport processes, which consequently can assume a differential retention behavior approximated by the assumptions of linear or nonlinear packed-bed chromatography. When fluidized or expanded-bed conditions prevail, an additional parameter arises in so far as the stationary phase particles themselves become distributed in a state of fluidization due to the higher superficial velocities employed. However, the same theoretical considerations are applicable, although the pressure drop AP in these systems will no longer be proportional to l / ( J p ) , where dp is the particle diameter, over the complete range of superficial fluid velocities as occurs in a packed bed, but rather proportional to the terminal fluidization velocity up to a plateau value corresponding to complete fluidization. Finally, selective capture of particular components in a feedstock under the static conditions of batch (or tank) adsorption can similarly be interpreted in terms of similar theoretical approaches, although in this case the longitudinal components of the mass transfer coefficients do not exist. From practical considerations, the modeling of adsorption processes with biomacromolecules usually commences with the batch (tank) systems and then proceeds to the packed-bed and fluidized-bed-expanded-bed systems as a logical interrogative approach. Applications of these approaches are discussed later in the chapter. In the case of gel permeation or size-exclusion HPLC (HP-SEC), selectivity arises from differential migration of the biomolecules as they permeate by diffusion from the bulk mobile phase to within the pore chambers of the stationary phase. Ideally, the stationary phase in HP-SEC has been so prepared that the surface itself has no chemical interaction with the biosolutes, with the extent of retardation simply mediated by the physical nature of the pores, their connectivity, and their tortuosity. In this regard, HP-SEC contrasts with the other modes of HPLC, where the surfaces of the stationary phase have been deliberately modified by chemical procedures by (usually) low molecular weight compounds to enable selective retardation of the biosolutes by adsorptive processes. Ideally, the surface of an interactive HPLC sorbent enables separation to occur by only one retention process, i.e., the stationary phase functions as a monomodal sorbent. In practice with porous materials, this is rarely achieved with the consequence that most adsorption HPLC sorbents exhibit multimodal characteristics. The retention behavior and selectivity of the chromatographic system will thus depend on the nature and magnitude of the complex interplay of intermolecular forces

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MILTON T. W. HEARN

established between the solutes, the stationary phase, and the mobile phase. In addition, retention behavior will be affected by the hydrodynamic and interactive characteristics of the biosolutes themselves, the fluid dynamic properties of the chromatographic system and whether a porous or nonporous sorbent is employed. Concomitant with the equilibrium distribution process that occurs in packed-bed or fluidized-bed-expanded-bed systems, dynamic events are also involved, associated with the diffusional kinetics of how the biosolutes migrate through the chromatographic beds. These processes give rise to the dispersion or broadening of all solute peaks or zones in packed-, fluidized-, or expanded-bed systems. These dispersion processes occur in both static and flowing liquid systems and are mediated, inter alia, by the respective diffusivities of the various biosolutes in the feedstock. The concept of zone dispersion thus incorporates all the kinetic processes associated with the mass transport motions of the biosolutes around the exterior interstitial spaces of the stationary phase, within the pore regions (if they exist), and as part of the adsorption and desorption process. For example, as the biosolute zones move down the column, a number of dispersive effects come into play as a consequence of the nonhomogeneity of the packing of the column bed, from nonlinear flow characteristics, from pore or liquid film resistances to diffusion, and from nonidealities in the kinetics of biosolute distribution between the mobile and the stationary phases. These dispersion effects collectively give rise to zone broadening, which progressively increases throughout the total period of time that the solute spends traversing the chromatographic bed. Compounding these events will, in some cases, be effects due to conformational changes that can occur with biomacromolecules in such environments. As a consequence, the residency time for biosolutes within a chromatographic milieu can impact considerable on the nature of the zonal dispersion, even leading to the emergence of two or more peak zones for otherwise compositionally identical substances. The time taken for a biosolute Pj to completely pass through a chromatographic bed is called the retention time t^^. Traditionally, t^^ is taken as the time for the peak maximum of the concentration zone of the biosolute to move from the inlet end of the column following injection to the exit end of the column and be immediately detected as an eluted zone, assuming that the interconnecting tubing has "zero dead" volume. The ^^ • value of a biosolute is usually compared to a reference void time t^ taken by an inert solute (or solvent) molecule to move through the column bed without any interaction and elute in the void volume of the column. The void volume VQ of the chromatographic bed represents as a first approximation the total volume occupied by the mobile phase and typically corresponds to 0.3-0.6 of the total column volume. In addition, the retention time can be related to the elution volume of the biosolute V^ • through the relationship V.., = ? e , , X f

(1)

where F is the flow rate (in milliliters per minute). Columns of different dimensions or selectivities can thus be compared, while normalization of the retention behavior of any biosolute can be formalized in terms of a dimen-

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

79

sionless capacity factor k'l for a particular sorbent, such that

^; = (^.,,-*o)Ao = (V,,,-Vo)/Vo

(2)

The capacity factor fe^ can also be defined in terms of Nernst Law, i.e., in terms of the ratio njn^^ where n^ is the total number of moles of the biosolute associated with the stationary phase, and n^ is the total number of moles of the biosolute in the mobile phase. Hence, k\ can be expressed as

L ^iim^m where [ X-]^ and [ X j ^ refer to the concentrations (in moles per liter) of the biosolute present in the stationary and mobile phases, while V^ and V^ refer to the volumes of the stationary and mobile phases respectively within the column of length L and internal column diameter d^. The ratio [ X j 5 / [ X J ^ is the equilibrium distribution coefficient i^assoc,/ while the ratio V^/V^ defines the phase ratio O of the chromatographic system. Hence, the capacity factor also takes the form

fe; = * [ X , ] y [ X , ] ^ =
(4)

The selectivity a of all chromatographic modes can be defined as the relative separation achieved between adjacent solute peaks and thus reflects the overall performance in relative selectivity of a chromatographic system. In particular, selectivity a is given by the ratio of capacity factors for adjacent peaks PI and P^^ i.e., a = k\/k)

(5)

Extensive research over the past 2 decades has focused on the development and evaluation of a very large number of different types of stationary phases and elution conditions for the separation of peptides, proteins, and other classes of biomacromolecules in attempts to maximize column selectivities. The essential task in all of these studies has been attainment of an optimal k\ value by primarily selecting conditions which generate the most appropriate K^^^^^j values. Manipulation of the phase ratio O enables additional fine-tuning, e.g., through adjustment of the ligand densities. In this manner, further control over selectivity and throughput can be achieved. III. THE CHEMICAL STRUCTURE OF POLYPEPTIDES AND PROTEINS The chemical organization (i.e., the primary amino acid sequence) and the folded structure (i.e., the secondary, tertiary, and quaternary structure), including the generation of a surface topography as a discrete three-dimensional object, are the essential features of a polypeptide and protein around which a high-performance separation can be designed. Two sets of factors must come into play for these separation skills to be developed. The first set

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MILTON T. W. HEARN

relates to the fundamental structural properties of the amino acid entities themselves, which form the building blocks of all polypeptides and proteins, irrespective of whether they have been modified posttranslationally with carbohydrate, lipid, or other structures. The second set relates to the chemical and physical attributes of the separation system per se, which impact directly on the molecular properties of the polypeptide or protein when it is placed in solution or exposed to the surface of a chromatographic sorbent. Table 1 lists some of the fundamental properties of amino acids in terms of their mass, volume, accessible surface area, partial specific volume, pK^, and relative occurrence in proteins. Table 2 lists a range of chemical and physical factors that are known to influence the chromatographic stability and the resolution of polypeptides and proteins in HPLC systems. Depending on the nature of the amino acid sequence, unique regions within a polypeptide or protein can adopt preferred secondary structures such as a-helical, /3-sheet, or j8-turn motifs. This process sets the stage in solution for different sequence regions within an unfolded polypeptide or protein to progressively nucleate, form secondary structures, and finally to adopt a native three-dimensional structure. Along the way to this energetically favored folded structure, the protein or polypeptides can, in principle, explore in solution a relatively large sample of its conformational space. When carried out as a biophysical study, important information on the changes in Gibbs free energy, enthalpy, or entropy associated with this folding process can be ascertained. Analogous methods can be used to examine the reverse of this process, namely, the unfolding trajectories. A critical factor to constantly recall when undertaking a separation procedure is that the choice of conditions and the experimental manipulations that are attempted will inevitably cause perturbation of the conformational status of these biomolecules. The question is by how much. Polypeptide and protein solubility and conformational stability can be manipulated in a number of ways, including techniques based on salt precipitation, organic solvent precipitation, organic polymer precipitation, isoelectric precipitation or extraction-partitioning in aqueous or nonaqueous twophase or multiphase liquid-liquid systems. All of these options take advantage of changes in the bulk hydration and molecular cavity properties of polypeptides and proteins when they are placed in solution. All polypeptides and proteins are capable of forming finite a n d / o r infinite intermolecular networks of solvated monomeric structures or alternatively self-associated (isodesmic) species or aggregates. Whether the polypeptide or protein exists as a monomer or as an aggregate will depend on its concentration under a particular set of ionic strength or solvent dielectric conditions. The participation of these processes thus clearly involves solution chemical equilibria. Hence, the potential exists to rationally modulate separation selectivity and zone-broadening processes of polypeptides and proteins in high-performance chromatographic systems by taking advantage of the molecular characteristics at the same time as the conformational status is adjusted. To elucidate the molecular forces involved, a composite biophysical experimental strategy is required. The current generation of instrumentation, particularly high field ^H nuclear magnetic resonance (NMR) or solid-state hetereonuclear NMR,

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82

MILTON T. W. HEARN m

T A B L E 2 Chemical and Physical Factors Contributing to Variation in the Resolution and Recovery of Polypeptides, Proteins, and Other Biomacromolecules in H P L C Systems Mobile-phase contributions

Stationary-phase contributions

Organic solvents pH Metal ions Chaotropic reagents Oxidizing or reducing reagents Temperature Buffer composition Ionic strength Loading concentration and volume

Ligand composition Ligand density Surface heterogeneity Surface area Pore diameter Pore diameter distribution Particle size Particle distribution Particle compressibility

mass spectrometry in its different modes—e.g., electrospray ionization mass spectroscopy (ESI-MS) or matrix-assisted laser desorption-ionization timeof-flight mass spectrometry (MALDI-TOFL), polarization fluorescence, diffuse reflectance Fourier transform infrared spectroscopy (DRIFT), laseractivated light scattering (LALLS), and circular dichroism-optical rotary dispersion spectroscopy (CD-ORDS)—provide a basis to explore these aspects at the molecular level. Until recently, the interaction of solvent molecules, ions, and free or immobilized ligands with polypeptides and proteins has, however, been largely explored only by methods that distinguish bulk differences in the induced physical characteristics of these solutes. Precise information on the microenvironment changes that occur in response to variations in the physical state or chemical properties of the solvent or the chromatographic surface have usually not been examined. The research of the next decade with these spectroscopic procedures and more powerful computational modeling methods will certainly provide insight at the submolecular level into the precise mechanisms of interaction and the forces involved in these molecular recognition events as a polypeptide or protein docks with immobilized chemical ligands, thus greatly expanding our knowledge and capabilities for the design improved sorbents and the implementation of new separation principles. For example, with the availability of powerful instrumental techniques such as surface-activated ionization mass spectrometry (SAI-MS) or heteronuclear two- and three-dimensional NMR, coupled with molecular footprinting experimental approaches,"^"^ the ability to precisely define the molecular features of a polypeptide or protein when in contact with a chromatographic surface can now be achieved. Illustrative of this molecular footprinting approach are the recentfindings'*^""^^ on the manner that different cytochrome c's (Fig. 1) and other proteins dock to lipophilic surfaces where the respective binding site for equine cytochrome c on capture by w-butyl ligates has been elucidated. Other examples, based on the use of molecular modeling, including molecular dynamic procedures, have led"^^"^^ to insight into the organization of different classes of ligates at the surface of an HPLC sorbent and the

83

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Free Solution Enzymic Fragments

Nonbound Enzymic Fragments ^

Bound = i > Enzymic K^^ J^ggglS^^^ Fragments F I G U R E I Schematic representation of the /n %Mu molecular footprinting approach used for the characterization of the contact sites or regions of a polypeptide or protein when it docks with a chromatographic surface. In this example, a proteolytic digestion procedure has been employed with a polypeptide or protein in the adsorbed and free states to delineate the sites that are protected from cleavage when the solute is bound from the sites that remain accessible to cleavage. Consequently, an experimental design with a proteolytic enzyme enables all of the enzymatic fragments corresponding to the polypeptide or protein in the free solution state to be obtained, as well as fragments that arise (in independent experiments) from the nonbounds regions of the polypeptide or protein on the surface of the sorbent and the "so-called" bound fragment regions. The binding site characterization of equine cytochrome c on capture by n-butyl ligates provides an illustrative example of this approach, whereby the regions associated with the docking of this protein to lipophilic surfaces has been elucidated. Analogous methods can be employed using other types of ligates - sorbents, by chemical modification procedures, as well as via protein - protein binding protection assays or footprinting approaches with, for example, monoclonal antibodies. See also Refs. 44 and 45,

possible pathways by which polypeptides and proteins can dock or partition with the immobilized ligands. The differential migration of biopolymers in gravitational or thermal fields, which is the basis of all centrifugation, gravitational or thermal field flow fractionation, and thermal denaturation purification methods, largely reflects the physical characteristics of the solute in terms of its hydrodynamic volume, molecular weight, or conformational status, or alternatively its propensity for self-aggregation, precipitation, or association with other biopolymers. Variations in bulk parameters such as the global size, shape, average molecular weight, average Stokes hydrodynamic radius, etc. of biopolymers, reflecting differences in molecular weight and sequence, form the basis of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), ultrafiltration and gel filtration-size-exclusion chromatographic (HP-SEC) methods. Although the various manifestations of these bulk properties of biopolymers in these separation procedures are often taken to imply a fixed physical feature, in fact all biomacromolecules undergo dynamic changes in shape, self-association, and mobility in response to variations in the surrounding liquid environment. In all cases, these environmental changes affect the chemical potential of the biopolymer and involve reversible solution equilibrium processes. When such phenomena are rapid (i.e., with time scales in the range of nanoseconds to milliseconds) they have minimal influence on purification selectivities, other than for inducing the molecular "breathing" events noticed when small ions or allosteric substrates bind-dissociate from biopolymers. However, when these phenomena have long relaxation times (i.e., of the order of seconds) the consequences lead to

84

MILTON T. W. HEARN

major variations in the conformation during the separation. In some cases, these variations may be catastrophic, i.e., resuh in subunit dissociation, denaturation, and very complex adsorption isotherms. Knov^ledge of the characteristics of biosolute-solvent interactions and their optimization are particularly pertinent in the development of general strategies for biopolymer separation, irrespective of v^hether noninteractive or interactive chromatographic media are employed. This know^ledge can be gained from a combination of thermodynamic and kinetic investigations, some facets of v^hich are discussed later in this chapter. What then are the physicochemical factors that underpin the current extraordinary popularity of HPLC for the analysis and purification of biomacromolecules? What aspects set HPLC apart from the earlier, more classical aspects of liquid chromatography in its various modes of separation selectivity? This chapter examines these issues and from an assessment of current status attempts to identify several future challenges. Moreover, in this chapter, the focus is placed on examples of the essential concepts of HPLC techniques that underpin their application in polypeptide and protein purification and analysis. These experimental methods in the broadest context enable many fundamental questions in modern biology to be approached. As quantitative analytical techniques, HPLC methods provide essential data for us to enunciate with molecular descriptors the complex myriad of biological processes that are involved in integrated cellular responses, from the most simple prokaryotic cell system to the most complex of eukaryotic organisms —humans. At the process scale, these same techniques provide in many cases the essential purification procedures to ensure the recovery in high purity of commercially valuable biomacromolecules for use in the food, health, biotechnology, and environmental industries. In the next sections of this chapter, the physicochemical basis for these developments will be examined. Different aspects of the use of this knowledge in the characterization of the molecular basis of ligate interactions with polypeptides and proteins will also be illustrated. lY. PHYSICOCHEMICAL FACTORS THAT UNDERPIN LIGATE INTERACTIONS WITH POLYPEPTIDES AND PROTEINS IN HPLC SEPARATION SYSTEMS The interaction between a solvated biopolymer and a chemically modified stationary phase in a fully or partially aqueous solvent environment can be discussed in terms of weak physical bonds. Interactions that involve covalent bonds are rare in high-performance separation procedures, although examples are known in the literature, including the participation of disulfide exchange and related redox processes that occur^^ with some activated thiopropyl or thiopyridyl ligates with cysteine-rich polypeptides and proteins. The main types of physical interactions that are involved in the establishment of the selective recognition and binding between a polypeptide or protein ligand and the ligate are: (1) hydrophobic interactions, (2) hydrogen bond interactions, (3) electrostatic interactions, (4) Lifshitz-van der Waals and weak polar interactions, (5) metal ion coordination interactions, (6) TT -> TT and TT -^ dipole interactions, and (7) interactions that involve a combination

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

8i

of the phenomena 1 to 6. Ahhough not strictly a bond in the sense of involving an interaction mediated by a polarized electron donor or electron acceptor process, the hydrophobic effect can also be included in this set. A. Hydrophobic Interactions The hydrophobic effect has fascinated experimental scientists since the concept vv^as first discussed^^ in 1954 by Kauzmann, who laid the foundations to our current understanding of a dominant force that contributes to polypeptide or protein folding. Both entropic and enthalpic processes drive the hydrophobic effect. The influence of enthalpic and entropic processes vary considerably w^ith temperature, with the hydrophobic effect changing from an entirely entropic process at temperatures near to 25°C to an entirely enthalpy driven process at ^ 113°C, assuming that the heat capacity (AC° ^) for the different states of the polypeptide or protein remain constant at higher temperatures. The concept that the magnitude of the hydrophobic effect can be equated with the free energy of transfer of a hydrophobic species to water has gained considerable currency over the past 20 years. However, solubility determinations as well as RP-HPLC measurements indicate^^ that the hydrophobic effect is strongest when only the entropic component is involved in the process—observations that are discordant with classical thermodynamic interpretations. Although hydrophobic effects have also been interpreted^"^ in terms of van der Waals interactions between nonpolar groups on a polypeptide or protein, with the solvation of these groups by water leading to a favorable van der Waals effect, the contributions from hydrogen bond formation within water may negate this type of van der Waals interaction. Alternative explanations of the hydrophobic effect have been proposed,^^"^^ based on the flickering cluster model, the random net work model, or the continuum model for liquid water. The hydrophobic effect can then be equated with the change in the order-disorder of the water surrounding a cavity of the dimensions of the polypeptide or protein when it is placed in solution. These models satisfactorily describe the entropy component at low temperatures, based on the formation of clathrate cages of highly structured water surrounding the nonpolar moieties of polypeptides or proteins. As the temperature increases, the hydrophobic effect becomes more enthalpic in character, with disruption of hydrogen bonds, dipole interactions, and van der Waals close contacts, as the highly ordered water structure water collapses, enabling the cavity to expand and the polypeptide or protein to commence to unfold. These models thus indicate that the hydrophobic effect will provide a significant driving force for even polar polypeptides or highly charged proteins to be excluded from aqueous environments and to interact with nonpolar ligates, such as those used with RP-HPLC or HP-HIC sorbents. Solvophobic theory provides a theoretical framework to evaluate hydrophobic effects. To place a polypeptide or protein into a solvent, a cavity of the same molecular dimensions must first be created. The amount of energy or work required to create this cavity is related to the cohesive energy density or the surface tension of the solvent. The fusion of cavities reduces the total surface area in the combined cavity, and thus the free energy of the

86

MILTON T. W. HEARN

system. Consequently, a driving force exists to aggregate or sequester nonpolar moieties away from a highly polar solvent environment. This process leads to energetically favorable interactions of biosolutes with nonpolar ligates in RP-HPLC or HP-HIC. At the macroscopic level, the magnitude of the hydrophobic effect can be assessed from the change in surface tension of the solvent that is required to affect desorption of the biosolute from a nonpolar surface. At the microscopic level, the surface area dependence of the free energy of transfer can be employed to describe the magnitude of the hydrophobic effect(s). The duality of these macroscopic and microscopic phenomena thus provide a rigorous basis to elaborate the hydrophobic processes involved in the molecular interaction of polypeptides and proteins with nonpolar surfaces in terms of both linear free-energy concepts as well as molecular parameters. The magnitude of the hydrophobic effects decays exponentially up to a distance of ^ 1000 A. In both reversed-phase and hydrophobic interaction HPLC, the change in free energy, the equilibrium association constant Kassoc,/ ^^^ ^he retention behavior (expressed as the logarithm of the capacity factor k]) following an increase in concentration [Solvent]^ or volume fraction ^solvent ^^ ^^ organic solvent, or a decreasing concentration of a salt species [Salt]^ in the mobile phase can be empirically evaluated from the following expressions: In k'i = In k]^Q - Si/f,^i^,^, + S'{ilj^^^^^^y - S"{iff^^^^^^y + •••

(6)

In k'i = In fe; 0 + S, ln(l/[Solvent]^) + S^ ln(l/[Solvent]^)^ + S';in(l/[Solvent]^)' + - .

(7)

In k/ = Infe;.0 + H, l n ( l / [ S a l t ] ^ ) + H^ l n ( l / [ S a l t ] ^ ) ' + H ; i n ( l / [ S a l t ] ^ ) ' + ..-

(8)

where k'^ Q is the capacity factor when «Asoivent "^ ^5 In(l/[Solvent]^) ^ 0 or ln(l/[Salt]^) -^ 0 (or when [Solvent] -^ 0 in RP-HPLC and [Salt] ^ 00 in H P - H I C ) ; and the coefficients S, S', S", . . . ; S„ S',, S;', . . . ; and H^, H^, H", . . . are the tangents of the plot of In k'^ versus i//^5oi^gn^.l/ln([Solvent]^), or l/ln([Salt]^), respectively, at defined values of ^solvent? [Solvent]^ or [Salt]^. For these conditions to prevail, and the above empirical relationships to take on a physical meaning, [Solvent]^ will need to approach a value near to unity for RP-HPLC separations. This condition will be achieved for acetonitrile and methanol when ^^acetonitriie ^ ^-^^ and ^methanol ^ 0.03, respectively. Such conditions equate with good experimental practice as far as the selection of the minimum concentration of an organic solvent for a RP-HPLC mobile phase system. Similar considerations also apply to HP-HIC separations, with the similar notional intercept value of the In k'^ versus ln(l/[Salt]^) occurring at [Salt]^ -> 1.0, or in terms of the mole fraction dependency when i/^^^j^ ~ 0.02 for ammonium sulfate or sodium sulfate, respectively. Obviously, when "ideal" first-order dependencies prevail between In k] and iAsoivent. ln(l/[Solvent]^) or ln(l/[Salt]^) in RP-HPLC or HP-HIC separations with polypeptides or proteins, then these empirical dependencies reduce to the famifiar linearized form given by Eqs. (6)-(8).

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

8l

In fe;. = In ^ ; . 0 - S o l v e n t

(9)

In k'i = In k'io + 5 , l n ( l / [ S o l v e n t ] „ )

(10)

lnfe;. = ln^;.o + H , l n ( l / [ S a l t ] ^ )

(11)

Alternatively, changes in the magnitude of In k] with solvent or salt concentration in interactions involving hydrophobic effects can be related^^'^^ to the chemical potential for the process, A/x^, or to the surface tension, y, of the solution (solvent) through the expressions In k', = In O +

A/i«

Z^mol

RT

RT

W

W -w "

W

+^^ "

2

(12)

2

and In k] = In k'o - y^ — , .^..^^ (13) ^ ^ 2.3033RT ^ ^ v^here Z^^^ is the number of the nearest neighboring (solvent) molecules that surround the polypeptide or protein in solution; W^^, W^^, W^^, and W^^ are the reversible v^ork required for partition of the biosolute into a liquid film of the desire composition; R is the gas constant; N is Avogadro's number; V is the partial molar volume; AA^^y^r i^ ^he hydrophobic contact surface area; and K^ is defined as the ratio of the energy require to form a cavity w^ith surface area equal to the surface area of the solute and the energy required to extend the planar surface of the liquid by the same are amount. Equation (12) assumes that the interaction can be described in terms of a partition model, while Eq. (13) accounts for only the solvent cavity formation-deformation in the mobile phase and not in the stationary phase, i.e., for a specific sorbent, the retention is assumed to be independent of the structural organization of the nonpolar ligates on the stationary phase surface. As documented elsewhere,^^'^^ this assumption is sustainable only when the ligate density is > 2.7 /imole/m^ with nonpolar w-alkylsilica sorbents and similar considerations apply with the other types of adsorptive HPLC sorbents of different ligate densities. A very large number ( > 400) of different types of silica-based hydrophobic interaction and reversed phase HPLC sorbents, varying in «-alkyl chain length (e.g., up to C30) and branching, surface density, immobilization chemistries, or production procedure are available commercially as well as from various laboratories interested in their development. Typically, these w-alkyl ligates are introduced using either w-alkyldimethylchlorosilanes or more recently the w-alkyldiisopropylchlorosilanes and other types of shielded chlorosilanes or alternatively with the corresponding «-alkylfc/ssilazines, followed by end-capping the silica support material. Other types of «-alkylphenyl, polyoxyether, and phospholipid ligates can be immobilized onto hydrothermally treated silica in an analogous manner. Moreover, an increasing number of polymeric nonpolar hydrophobic interaction or reversed-phase sorbents are also gaining considerable popularity. Significant differences have been observed when «-alkylsilicas of different chain length or structure are employed for the resolution of polypeptides and proteins, and similar obser-

88

MILTON T. W. HEARN

vations have been found between polymer-based nonpolar sorbents. Some of these effects are clearly mediated by the propensity of the nonpolar ligate surface to induce conformational transitions with polypeptides and proteins, although other effects appear to be associated with the partitional rearrangement of the ligate-biosolute complex once adsorption and conformational reorganization has occurred. However, when well bonded, high-coverage «-alkylsilicas of «-alkyl chain length between C4 and C^g are employed, solvophobic predictions^^'^^'^^ have proved to be remarkably precise with polypeptides that lack secondary structure, i.e., linear relationships have been observed between In k] and y. Illustrative of these dependencies are the plots of In k'l versus ^solvent ^^d In k'^ versus y for several hormonal polypeptides analyzed by RP-HPLC procedures shown in Figures 2 and 3. As discussed elsewhere in this chapter, the variations that arise in these In k] versus ^solvent? ln(l/[Solvent]^), ln(l/[Salt]^), or y dependencies as the temperature T of the system is changed systematically provide fundamental information on the thermodynamic, biophysical, and physicochemical properties of these biosolute at nonpolar interfaces such as those used in RP-HPLC or HP-HIC, and analogous methods are applicable for the other interactive modes of HPLC. B. Hydrogen Bond Interactions In polypeptides and proteins, hydrogen bonds are of polar origin and are usually stronger than other noncovalent bonds. Both backbone amides and

11

1.5 1.0

2 4

4

1,2

:^^ 0.5

^

~^ \ i \

3

L7 j 'f6

lyj

0.0

-0.5

^

^^^^•^

-1.0 L_

0.2

.

1

0.4

.

1

0.6

•

1

0.8

y/-value F I G U R E 2 Plots of Ink,- versus
89

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

1

2.0 -

1.0 -

-0.5 -

7

/ / /

// / li^

v/ ^fy/ // lUm/

\ ^ 0.5 -

^0.0-

?

/ /./ /ft/ i-^

1.5 -

^

1

/ / / ;/ / / ^1/2/

2.5 -

5

III / III 1

•^

/"^

-1.0 -1.5 -2.0 30

40

50

60

70

Surface Tension, y, mNm" F I G U R E 3 Plots of Ink,- versus y for several peptide homologues analyzed by RP-HPLC procedures using methanol as the organic solvent modifier. The experimental conditions were column, piBondapak C|8 (4.0 X 300 mm; dp 10 /im); flow rate 2.0 m L / m i n ; mobile phase, w a t e r - 2 0 m M H3PO4, with the volume fraction ip of methanol adjusted from 0.0 to 0.56. The pH value for the 100% water experiment was 2.2. The key to the peptide structures is: /, pentaphenylalanine (F5); 2, tetraphenylalanine (F4); 3, triphenylalanine (F3); 4, diphenylalanine (F2); 5, phenylalanine (Fl). Also shown are the linear regression lines for each plot, where the corresponding correlation coefficients were obtained: r}^ = 0.982; r^^ = 0.998; r}^ = 0.991; r^^ = 0.988; r^^ = 0.967. Data taken from Ref. 197.

side-chain moieties bearing OH, NH2, CO2H, or SH groups can contribute as hydrogen bond donors or acceptors. Typically, all of these common electronegative species in polypeptides and proteins are capable of hydrogen bonding, either as intramolecular events or with the surrounding solvent, and are of the type N - H • • • O, O - H • • • O, N - H • • • N or O - H • • • N, etc. Although dissociation energies for these bonds are high, they do not appear to play a significant role in the primary stages of the interactions betv^een most biosolutes and adsorptive stationary phases due to the very short distance of o

the order of 1.5-5 A over which they act. The distance dependence of this bond is proportionate to 1/r^, having a weaker and shorter range than charge-dipole interactions. Hydrogen bond effects involving ligand-ligate interactions in aqueous media can be strongly attenuated by hydrogen-bond formation between hydrogen donor and hydrogen receptor moieties and the ambient water molecules. Intramolecular hydrogen bonding effectiveness is greatest among internalized amino acid residues of proteins and polypeptides, and becomes more limited as the accessibility of these residues to the surrounding water environment increases. For this reason, profound differences can be observed with water-organic solvent mixtures when different organic solvents are employed in HPLC separations. Because hydrogen bond effects contribute significantly to the enthalpy of association, AH^^^^^j, when the solute and sorbent interact, measurement of the A H^^soe, i values, either directly from the van't Hoff dependencies as revealed^'^^'^^'"^^'^^'^^ from the

90

MILTON T. W. HEARN

In k'l versus 1/T chromatographic data or from differential scanning microcalorimetric measurements,^^ provides valuable insight into this effect, which contributes about 2.2-6.4 kj/mol per hydrogen bond to the stabilization of polypeptides and proteins. In comparison, the enthalpy for a-helix formation is in the neighborhood of —4.2 kJ/mol per residue, w^hich has largely been attributed^"^ to the enthalpy of the hydrogen bond itself. Hydrogen bonds play an integral role in the maintenance of a-helical and /3-sheet structures of polypeptides and proteins and their folded tertiary structures in solution and at the surface of a sorbent. The N - H group of a polypeptide or protein can form a single hydrogen bond, v^hile the C = 0 group typically (partially) accepts two and sometimes three hydrogen bonds through contributions from the two lone pair electrons of the s/?^ orbitals of the oxygen atom. On average, 68% of the hydrogen bonds in proteins arise from interactions of the amide backbone dipoles, while > 90% of the carbonyl and amino groups are hydrogen bonded.^^ As the hydrogen bond has many features in common with electrostatic or polarized dipole interactions, it follows that the hydrogen bond strengthens at lower temperatures. The recent development^^'^"^ of H ^ H isotope exchange procedures based on electrospray ionization mass spectrometric (ESI-MS) techniques has provided a particularly exciting opportunity to characterize the number and stability of hydrogen bonds in polypeptides or proteins under various solvent conditions or when bound to chemically modified surfaces. Perturbation of hydrogen bond effects can be achieved in a number of ways, ranging from changes in solvent characteristics, temperature or type of support matrix employed in the sorbent preparation. Illustrative of this approach are the data (Fig. 4) for the unfolding at different temperatures of hen egg white lysozyme (HEWL) and horse heart myoglobin (HMYO) under low pH conditions, analogous to those employed in RP-HPLC. Hydrogen bond effects fulfill important roles in the hydrophilic interaction (HP-HILIC) and in polar- or normal-phase (NP-HPLC) modes. In both HPLC modes, the separation selectivity can be significantly modulated through the use of solvents that strongly hydrogen bonding, such as methanol. Thus basic or amphipathic polypeptides with a propensity for self-association can be resolved with good recovery from hydrophilic HP-HILIC sorbents, such as the PolySulfoethyl A sorbent.^^ With silica-based sorbents, the participation of type I and type II silanols can represent a significant source of hydrogen bond contributions, particulatly when a low density of an introduced ligate is present. In the extreme case of a naked silica surface, these effects can lead to irreversible binding of polypeptides or proteins. However, with protected synthetic polypeptides, or amphipathic polypeptides, such NP-HPLC or HP-HILIC procedures can be very useful, particularly when used^^ as "flash chromatographic" methods. With bonded, polar-phase HPLC sorbents, the distribution constant ^abs,i ^^^ be equated with the solubility parameter, 5-, which in turn is a measure of the intermolecular interaction energy per unit volume of the polypeptide or protein in a pure liquid such that [{8^-8j'-i8,-8f] In k', = In $ + V , J ^ „^ RT

-^-^

(14) ^ '

9i

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

14550

^^ B

A

b O 14500

^

__

A^-'-'^A

/^

4 17200 ^^^"^^ --

- 17150 bi) (D 17100 ^

II

14450

53 14400

VH

II

170^0

cd ^

o

CJ ' O 14350

S

17000

I

I 14300

o

16950 1

1

1

1

1

1

i

1

1

1

1

i

1

1

290 300 310 320 330 340 350

290 300 310 320 330 340 350

Temperature (K)

Temperature (K)

F I G U R E 4 Plot of molecular weight versus temperature for the unfolding of hen ^'^ white lysozyme (A) and horse heart myoglobin (B) at different temperatures utilizing the hydrogen exchange H -^ H approach. In this experiment, the constant mass of these proteins at different temperatures under the proton exchange condition is shown as I, while the progressive increase in molecular mass as the protected and less accessible hydrogen bonds are disrupted and exchanged by deuterons are shown as II. The difference between the molecular weight obtained under these conditions of ' H ^ ^ H exchange at any specified temperature equates with the number of exchangeable hydrogen bonds for HEWL or HMYO under the selected pH and solvent conditions. Data taken from Ref. 66.

where 5-, 5^, and 5^ are the solubiHty parameters of the solute ?^ the mobile phase m, and the stationary phase s; and V^ is the molar volume of P^. As i) becomes larger, the polarity of s becomes more similar to that of P^, while when K^^s,/ becomes smaller, the polar characteristics of P^ are more similar to those found for the mobile phase. Alternatively, K^y^^ , can be expressed in terms of the general relationship I n X ads, i

lnV,,, + ^ ( 5 < ' - A , € « )

(15)

where V^ ^ is the volume of the mobile phase m, adsorbed to the sorbent, j8 is the activity coefficient of the sorbent, i^^abs,/ i^ the ratio (mole P-/g sorbent)/(mole P-/mL mobile phase), e^ is the solvent strength parameter of the mobile phase, SQ is the adsorption energy of P^ for a standard sorbent where j8 = 1 from a standard mobile phase where e^ = 0, and A- is the molecular surface area of V-. The variables V^ • and ^ represent characteristic properties of sorbents that predominantly function via hydrogen bond effects. These properties will vary markedly with the amount of adsorbed water or the effective "hydroxylated" or "hydrophilated" content by bonding the hydrophilic ligates to the sorbent. If these hydrophilic ligates have no interaction with the biosolutes per se, but only with the water in the mobile phase, then these chromatographic materials fulfill some of the desirable properties of an ideal HP-SEC sorbent.

92

MILTON T. W. HEARN

In size-exclusion (gel permeation) HPLC separations, the retention can be described in terms of volume units (V^ ^) such that V.,, = Vo + K,,,V,

(16)

where VQ is the void volume and Vp is the total pore volume of the column, and Kp^i is the permeation coefficient of P^, which is assumed in ideal cases to be 0 < Kp^^ < 1. However, when hydrogen bonding effects occur in HP-SEC, then displaced dependencies arise, with K^ • values > 1. Because V^ ^ is related empirically to the logarithm of the molecular weight of a polypeptide or protein [i.e., to In(M^)] over a suitable range of molecular weights, the selectivity in HP-SEC between two proteins or polypeptides P^ and P^ will be controlled by the slope w^ y, expressed by A log MW

Alternative expressions linking V^ ^ to M^ have been developed,"^^ based on the relationship between the radius of gyration R and the molecular mass M^, i.e., Rg cc M", where a = 1 for prolate or rodlike proteins, a = 1/2 for flexible coiled coils, and a = 1/3 for spherical proteins. For a compact globular protein, the hydrodynamic volume, Vf^ can be calculated from

where 17 is the intrinsic viscosity, expressed as the volume of molecules per unit mass; N is Avogadro's number, and v is the Simhas factor. For spherical globular proteins, v = 2.5, while for ellipsoidal proteins v > 2.5. Moreover, according to Tanford,^^ V^ = 1.5444M^, while the Stokes radius of compact globular protein is given by R — 0.81 X My^. Thus, application of the modified Himmel-Squire dependency''^ and related relationships'"^ enables a useful alternative expression to be employed for the characterization of HP-SEC selectivities, namely, -b =

— = ^^^— dlnR dlnM'-''

(19) ^ ^

where Kp^^^ a-

felnM,

(20)

and a/b is related to the pore size distribution of the HP-SEC sorbent, while the slope of the selectivity curve b depends on the pore size distribution. The molecular dimensions of most globular and structural proteins can be readily described in terms of their major elliptical or major-minor prolate axes.'^^''^^ Thus, the major elliptical axis of many globular proteins falls in the range of 50-500 A, while for prolate proteins, e.g., [Q:I]2a!2 collagen, the major and minor axes fall in the ranges 2200-3000 and 120-140 A, respectively. This

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

93

means that most globular proteins with molecular masses up to 7 X 10^ Da can be successfully separated by HP-SEC sorbents with pore sizes greater than 500 A but below '^ 2000 A. Because hydrogen bonding and hydrophobic interaction effects can give rise to anomalous retention behavior for some polypeptides or proteins with HP-SEC sorbents, a common practice is to add a small amount of a suitable organic solvent to the mobile phase in these cases. C. Electrostatic Interactions Coulombic or electrostatic interactions are, like hydrogen bonds, of polar origin. The interactions occur between charged or ionizable groups, which form part of the immobilized ligate, or alternatively are an inherent component of the stationary phase and the complementary charge groups accessible at the surface of the biosolute. Many different types of electrostatic ligates are available^"^ for use as HP-IEX systems, ranging from the familiar (CH3CH2)2N-, (CH3)3N+-, -CH2SO3H, and - C H 2 C O O H moieties to the more exotic malachite green carbinol ligates, such as ((Me)2N-C^H4)2 (NH2-C5H4)COH, which in general are very strong anion exchangers as the corresponding immobilized carbonium species^^ at neutral or slightly acidic pH values. Electrostatic interactions between oppositely charged species are o

effective over distances of the order of 100 A. In the simplest case, the magnitude of the electrostatic force F-^^^ between two point charges Q^ and Q25 separated by a distance r, in a medium of dielectric constant e^ is given by ^iex = — - T

(21)

The amphoteric nature of polypeptides and proteins significantly complicates their treatment as single-charge-state species. The presence of multiple charge states can nevertheless be addressed through the use of average distribution coefficients and weighted mole fractions of individual charge species. Empirically, the dependency of the equiHbrium association constant ^assoc, / o^ ^^^ concentration of the displacing ion can be described in terms of nonmechanistic, stoichiometric models, whereby the mass distribution of the protein, P, of charge state + x is given by

( P ± ^ ) ^ + {x/y)iD^y),

^ ( P ± - ) , + (x/y)iD^y)„

(22)

where D - ^ is the displacing salt counter ion, and the subscripts m and s refer to the mobile and the stationary phases, respectively. The equilibrium association constant i^assoc, i ^^^ ^he ion-exchange process can thus be represented as

94

MILTON T. W. HEARN

where 7.^ represents the ratio ( x / y ) of the effective charge on the polypeptide or protein to that on the displacer counter ion. When near-equihbrium conditions prevail for the chromatographic distribution process, i.e., v^hen T, P, V, O, and the flow^ rate V are constant, and the adsorption process approximates a linear (Langmuirean) isotherm, then this dependency of ^assoc, i ^^d hence the logarithm of the corresponding capacity factor In ¥.\^^on [ D - ^ ] ^ can be v^ritten in terms of a Taylor series, similar to that employed for RP-HPLC or HP-HIC separations, as follow^s:

In^L,,, = a + j8(ln[l/C]) + y ( l n [ l / C ] ) ' + 5 ( l n [ l / C ] ) ' + -

(24)

v^here C is the concentration of the displacing ion, i.e., [ D - ^ ] ^ , and a , j8, y, 6 , . . . , are coefficients dependent on the solubility parameter of the solute, 6-; the zeta potential ^ of the stationary phase: the mobile phase buffer composition, pH, polarizability, and dielectric properties. Over a narrow range of mobile phase compositions, this relationship has often be approximated to a linear dependency, i.e., given in the familiar form lnfe;,,, = lnKai,,, + Z , ( l n [ l / C ] )

(25)

where i^aist,/ i^ ^^e distribution coefficient, a term that includes the Kassoc o the phase ratio O, and the stationary phase electrostatic ligate concentration [ L - ^] in the following manner: jjr _ ^dist,/ -

^assoc,/^L^ -—

J

{'\r\ \^^)

where the constants z^ and z^ adjust for the valency of the solute and the salt species and ZQ is the theoretical maximum number of charges on the protein surface associated with the adsorption process. Thus under linear elution conditions, the slope coefficient Z^ and In K^^^^^ can be determined from the plot of In k[^^i versus l n [ l / c ] . Note, however, that Z^ reflects the apparent number of ionic charges associated with the adsorption of the polypeptide or protein at the Coulombic ligate surface, and is not formally equivalent in mathematical or physicochemical terms to ZQ or Z^. Moreover, curvilinear, rather than linear, plots are more likely to occur for these Infe-^^^ versus l n [ l / C ] dependencies due to the anisotropic nature of the charge distribution on the polypeptides or proteins and the involvement of secondary binding processes, mediated for example by hydrophobic effects. Figure 5 illustrates'^^'^'^ such a case with the plots of In k[^^j versus l n [ l / C ] for the seven proteins using the strong anion exchanger, MonoQ, with the buffer system of 20 m M piperazine, pH 9.6, containing different concentrations of the displacing salt, NaCl. As is apparent for proteins 1 to 5 in Fig. 5, the expected ion exchange adsorption-desorption behavior is clearly evident, while for proteins 6 and 7 ion exclusion effects prevail, consistent with their charge and pi characteristics. Because polypeptides and proteins are amphoteric, Z^ is also expected to show nonlinear dependencies on pH. This behavior has been observed^^'^^

95

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

ln[l/[NaCl]] F I G U R E 5 Plots of Ink,' versus In [I / C ] for seven proteins using the strong anion exchanger, Mono-Q, with the buffer system 20 m M piperazine containing the displacing salt, NaCI. These plots were derived from isocratic measurements using various concentrations of NaCI at pH 9.6 and a flow rate of 1.0 m L / m i n . The protein key is as follows: /, human albumin, pi = 5.85, hA^ — 69,000; 2, hen e^ white ovalbumin, pi 4.70, M,. = 43,500; 3, bovine hemoglobin, pi = 6.80, M,. = 64,500; 4, bovine erythrocyte carbonic anhydrase, pi = 5.89, M,. = 30,000; 5, sperm whale muscle myoglobin, pi = 7.68, M,. = 17,500; 6, hen Q^ white lysozyme, pi = 11.0, M,. = 14,300; 7, horse heart cytochrome c, pi = 9.4, hA^ = 12,400. Data selected from Ref. 76.

for various globular proteins with both strong anion and strong cation HP-IEX sorbents. As a consequence, the minima in the In k[^^ versus pH plots at a defined concentration of displacing salt will not usually occur at the pi value of the polypeptide or protein, but rather at a pH value above or below the pi value. This behavior is a further reflection of the distribution differences of charged groups accessible on the surface of the polypeptide and protein. Thus, the ion-exchange processes of polypeptides and proteins are dependent on the microlocality and extent of ionization of the surface accessible amino acid side chains, or the N- and C-terminal amino and carboxyl groups, respectively, with the retention behavior in the electrostatic modes of HPLC are dependent on the pH of the buffer. This effect can be evaluated for a polypeptide or protein of charge Zi separated under normal HP-IEX conditions, from the dependency

k'

= $

c ([^^' [H:.])1 [[£'..] ^+ ^ a v ii

\

(27)

96

MILTON T. W. HEARN

where [El^^] is the ionic strength of the displacing counterion in the mobile phase, K^^^- is the average distribution coefficient for the various ionized species of the polypeptide or protein, and C is a system constant. Since small changes in pH v^ill result in large changes in k-^^^j, this property can be easily employed in HP-IEX as part of a buffer optimization routine. Unlike other types of interactions, Coulombic forces can be attractive in the case of oppositely charged groups or repulsive in case of identically charged groups at the surface of the interacting molecules. This property can be exploited in charge exclusion effects in some modes of HP-IEX. In various HP-IEX systems, attraction betv^een negatively charged biomacromolecules can also be affected through the use of chelate development with divalent cations like Ca^^, Mg^"^, Cu^"^, Zn^"^, etc., where the interactions can lead to ionic crosslinking processes between the participating biosolutes and the ligate that change the overall selectivity of the system. Thus, the slopes of the In ^'iex,^ versus l n [ l / C ] plots for different salts will not be parallel but be conditional on the polypeptide or protein examined as well as the position in the Hofmeister series where the anion and the cation reside. As noted, the retention of a polypeptide or protein with HP-IEX sorbents primarily arises from electrostatic interactions between the ionized surface of the polypeptide or protein and the charged surface of the HPLC sorbent. Various theoretical models based on empirical relationships or thermodynamic considerations have been used to describe polypeptide and protein retention, and the involvement of the different ions, in HP-IEC under isocratic and gradient elution conditions (cf. Refs.^'^^'^^''*^'"'^"^^). Over a limited range of ionic strength conditions, the following empirical dependencies derived from the stoichiometric retention model can be used to describe the isocratic and gradient elution relationships between the capacity factor In k[^^ I and the corresponding salt concentration [CJ or the median capacity factor In k[^^^, and the median salt concentration [ Q ] of a polypeptide or protein solute, namely, 1 In feU = In i^iex + Z , In - ^

(28)

1 In k,,, = In K,,, + Z , In - ^

(29)

where In K-^^^ is the intercept value of the In k[^^ (or In ~k^^^) versus In ( l / [ CJ) or l n ( l / [ Q ] plots, and is related to the association constant Xassoc ^^^ ^^^ protein-ligand interaction when [CJ -^ 10"^ mol/L (or [ Q ] -> 10"^ mol/L), and Z^ or Z^ represents the slope of the plots derived from the isocratic or gradient data at a defined salt concentration. As expected from molecular surface area arguments, small molecules such as dansyl amino acid derivatives exhibit relatively small Z^ (or Z^) and In Kjgx values, and these values do not change significantly with increasing temperature. Polypeptides and proteins, on the other hand, exhibit much larger Z^ (or Z^) and In K-^^^ values, and these values have a profound temperature sensitivity. Moreover, it is well known that different salts can

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

97

influence the ion-exchange chromatographic behavior of polypeptides or proteins due to the chaotropic or cosmotropic nature of the component cations and anions.^^'^^'^^"^^ The ionic radius and electronegativity of the monovalent and divalent ions can significantly influence the retention behavior of polypeptide or protein w^ith HP-IEX sorbents. Illustrative of these effects are the plots of ^n k[^^ versus [CJ, shown in Fig. 6 for several polypeptides and globular proteins eluted^^ from the cation-exchange HPLC sorbent Lichrospher 1000 SO^ using as the displacing salts CaCl2 and NaCl in the concentrations range 0-1.0 M.

ln[l/ C] F I G U R E 6 Plots of lnk[ versus [I / C ] for a selection of proteins separated on the strong cation exchanger LiChrospher SO3" at 25°C with different concentrations of (a) CaCi2 and (b) NaCI as the displacer salt. Inthese studies, the experinnent data were obtained utilizing linear gradients from 20 to 100 min duration at a flow rate of I m L / min with the buffer system 50 MAI sodium acetate to 50 M M sodium acetate containing I M CaClj or I M NaCI. The protein key is as follows: - • - , angiotensin I; - • - , angiotensin II; - A - , angiotensin III; -w-, arglnlne; - • - , horse heart cytochrome c; - • - , bovine insulin; - ® - , hen egg white lysozyme; - @ - , hen egg white ovalbumin; - A - , bovine pancreatic ribonuclease; - • - , soyabean trypsin inhibitor. Data adapted from Ref. 96

98

MILTON T. W. HEARN

This influence of the valence and activity coefficients of the displacer salt on the retention behavior of polypeptides and proteins can be anticipated from theoretical treatments of the ion-exchange chromatographic separation of proteins. According to the nonmechanistic stoichiometric model of protein retention behavior in HP-IEX^^'^^~^^ the influence of a divalent cation salt such as CaCl2 on the retention behavior of a protein in HP-IEC can be evaluated in terms of the following relationships: As

k'

= K —^

D hi hibiPoCi

(1 - f)

(30)

where K^, A^, and V^ are the equilibrium constant for the interaction of the polypeptide or protein with the ion-exchange sorbent, the accessible surface area of the adsorbent in square meters per gram and the volume of the mobile phase, while z is the number of charge groups on the polypeptide or protein associated with the adsorption and desorption processes, respectively. The term D^- relates to the initial ligand concentration, DQ is the displacing ion concentration in moles per liter and Q is the concentration of counterions associated with the polypeptide or protein, i.e., in the case of cation exchange binding events the concentration of H"^ ions involved with the polypeptide or protein that are substituted by other cations. The relative elutropic strength and activity coefficients of the displacing ions and counterions for the ion-protein interactions and the ion-ligand interactions are represented by the terms a^^ andfc-y,while the fraction of the adsorbent surface covered by the protein following the adsorption interaction is given by the term f. If it is assumed that "near-equilibrium" conditions apply and the amount of the polypeptide or protein loaded onto the HP-IEX sorbent is small, i.e., if only a small percentage of the adsorption capacity ^* is involved in the binding and the polypeptide-ligate or protein-ligate interaction occurs within a linear region of the adsorption isotherm, then the term f ^ 0 and D^^ will remain essentially constant. Under such conditions, the dependence of k[^^ on the concentration of the participating ions can be represented by K. = K^^DI,[a,^b,^D,C.y'

(31)

For a displacing salt with a divalent cation but a monovalent anion, each cation (e.g., Ca^^) will cause the neutralization of two charge group interactions between the polypeptide or protein and the electrostatic ligate, with the concentration of the M^"^ cation DQ (in moles per liter) required to maintain electroneutrality exactly equal to half of the concentration of the accompanying monovalent (e.g., Cl~) counterions, C^ (in moles per liter), associated with the positively charged polypeptide or protein when it is desorbed from the cation exchange HP-IEX sorbent, and hence K.-K,^Di\a,^b,^Dl']~'

(32)

99

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

The logarithmic form of Eq. (32) can be expressed, for the specific illustrative case of CaCl2 as the displacing salt, in the following terms, although analogous expressions can be derived for anion or cation exchange HP-IEX systems with polyvalent ions: In kl^ = In

As

1

+ zln

^,,&,,[Ca2+]

1.5

(33)

or, alternatively. In k[^^ = In K, + zln

«,;^-;

+ 1.52 In

[Ca^-]

(34)

where

K^ =

Ka^DI,

(35)

According to Eqs. (31)-(35), the magnitudes of the slope terms, i.e., the Z^ values, of the isocratically derived In k^^^ versus l n ( l / [ Q ] ) , or the case of gradient derived data In ~k[^^ versus l n ( l / [ Q ] ) , are predicted to be dependent on the valency of the displacing salt. It can also be seen from Eqs. (33)-(35) that the relationship betv^een the slope term Z^ and the effective charge term z can be represented in the form (n X z = Z^) provided the valencies of the displacing ion and counterion are unity, the activity coefficient terms a^^ and bii are independent of the concentrations of the polypeptide or protein, the ligate groups, the displacing ion, the counterion, or the temperature, and the magnitude of the product of a^j and b^^ is close to 1. When the valency n of the displacing ion is > 1, but the counterion remains monovalent, then the relationship depicted by Eqs. (30) and (35) follows the dependency [(w + 1) X z]/n = Z^, again assuming that the value of a^^ Xfc-y-> 1 and the other "ideal" criteria already listed prevail. D. van der Waals Interactions and Weak Polar Interactions van der Waals interactions arise betw^een all atoms that are brought into very close proximity. In these cases, electrodynamic attractions arise betw^een fluctuating dipoles in one atom and other dipoles induced in a neighboring atom. In addition, attractive contributions are generated from permanent dipole-dipole and permanent dipole-induced dipole interactions. Analogous attractive forces are generated betv^een any two macroscopic bodies whose surfaces are separated by very small distances.^^'^^ With weakly polar interactions, segregation of the electronic charges within an aromatic ring gives rise to electron-poor aromatic hydrogens and electron rich 7r-orbitals. This process tends to favor aromatic residues packing edge to face, with a distance dependence of 1/r^. Oxygen and sulphur atoms can also interact with aromatic ring structures via electron-poor hydrogen atoms, with distance dependence oi\/r^ for the uncharged atoms and 1/r^ for the thiolate (S")

I 00

MILTON T. W. HEARN

and carboxylate oxygens. These weakly polar interactions clearly will not represent a significant contribution to a ligand-ligate interaction, or influence the folded status of a polypeptide or protein significantly, although as a secondary contributor, their effects amplified through dipolar interactions with the solvent then become more evident. In the liquid state, the three types of electrodynamic interactions (London, Debye and Keesom dipole interactions) can be treated completely differently from purely macroscopic points of view, in which the interacting bodies are considered as continuous media. The dispersive London forces involve interactions of induced dipoles with a distance dependence of 1/r^. The strength of these forces depends on the polarizabilities of the interacting molecules, with nonpolar atoms such as aliphatic C or H having stronger interactions than polar atoms such as N or O atoms. Moreover, these dispersive forces will favor like groups coming into contact, such as aromatic side chains adopting where possible preferred contacts. These contributions are associated with long-range effects, often called Lifshitz-van der Waals forces or bonds. The energy of Lifshitz-van der Waals interactions decrease monotonically to the distance separating the interacting species (in the configuration of the two parallel slabs). These effects are operative up to 1000 A, in contrast to hydrogen bonds that are effective over only 1.5-5.0 A with an exponential decay of the interaction energy of ^ 1 0 0 A in the case of electrostatic 99

mteractions. E. Metal Ion Coordination Interactions Metal ions can be considered as Lewis acids, with the formation of complexes rationalized in terms of Lewis acid-base interactions. This interaction can be visualized in terms of the ability of a metal atom or ion M to accept a pair of electrons (and thus act as a Lewis acid) from a ligand : X, which is an electron donating base with an accessible lone pair of electrons, i.e., M+ :X^M:X

(36)

The distribution of the electrons as a coordination bond in this Lewis acid-base complex largely dictates the character of the complex, thus the nature of the atoms involved, their ability to form coordination complexes of specific geometry, and their hydration-hydroxylation state in water systems, enable these processes to be categorized^^^ as "hard" or "soft." Typically, a metal ion that acts as a "soft" Lewis acid has outer shell electrons that are easily polarized, low electronegativity characteristics, is easy to oxidize, and contains unshared pairs of electrons in the valence p or d shells. Thus Cu^^ would be considered a "soft" metal ion. In contrast, a metal ion that acts as a "hard" metal ion has a high electronegativity, is difficult to polarize, is hard to oxidize, and contains only high-energy empty orbitals. In this context, Al^"^, Ca^^, and Fe^"^ would all be considered as hard metal ions. The TT-bonding theory of Chatt^^^ adequately rationalizes many of the characteristic features of the coordination interactions of metal ions with polypeptides and proteins. Thus, "soft" metal ions, such as Hg^"^, have a preference for

101

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

"soft" electron donor groups on a polypeptide or protein, such as SH or SMe of Cys or Met residues; borderline metal ions, e.g., Cu^"^, Zn^"^, or Ni^^, have preferences for borderline donor groups associated with Tyr, Trp, or His residues; and "hard" metal ions a preference for "hard" donor groups within Asp, Glu, Lys, and Arg residues, as well as phospho, sulfonato, and sulfate groups. By immobilizing a suitable chelating compound to a support material, immobilized bi- to pentadentate metal ion complexes (IMICs) can be generated as chromatographic sorbents of the type M"'^<=> CheUH20)^(0U~)j, where m i- j with 0 < m < 4 and 0 < / < 2. The equihbrium association constant, ^u""^ ^cheu often represented as the overall metal ion stability constant j8„, associated with the binding of a metal ion M""^ of valence n"^ to an immobilized monovalent chelating compound CheL can be represented as [M"+<=^ CheL] ^M-<.c/..L= [ M - ] [ C A . . L ] "

^^^^

If any coordination sites of the metal ion are not taken up by the electron donor groups of the chelate, they will then available for binding to water, hydroxyl groups of other counter ions, X, or alternatively to electron donor groups (EDGp) accessible at the surface of the polypeptide or protein ?^. Thus, M"+ + (w - a)CheL + ( ^ ) X ^ M " + ^ Che\^_,^X^,^

(38)

and hence the equilibrium association constant i^solvate? ^^^ ^he complex containing hydrate, hydroxide, or counterion species is given by

^solvate

r,.«-nr^i

ri(«-^)r-^i^

[M«+][Cfe^L]^"-^^[X]'

^

^

while the equilibrium association constant Kp, for the complex formed following the coordinative interaction with the polypeptide and protein P, can be represented by M " + « CheL,„_,,X,^, + P,,EDG(,) -- M«+ + /X

(40)

Several scenarios can be considered with immobilized metal affinity chromatography (IMAC) systems in terms of the relative influences of these different equilibria. Thus, if i^soivate ^ ^ P P ^^e polypeptide or protein will not bind to the IMAC sorbent. Similarly, if K^^ > Kj^n+ ^ ^^^^^ the polypeptide or protein will strip the metal ion from the immobilized chelating complex, such as observed, e.g., with native or recombinant fibroblast growth

I 02

MILTON T. W. HEARN

factor j8. Finally, if ^solvate ^ ^w^ <^cheL'> ^he buffer species or added counterion will cause the metal ion to leach from the IMAC sorbent, as commonly observed with many structurally unconstrained bi- and tridentating systems. Metal ion leakage has in fact been one of the major limitations of IMAC procedures with recombinant proteins when structurally unconstrained chelating compounds have been employed to form the metal-ion complexes. This technical facet appears to have now been solved with the development of novel macrocyclic chelating ligand systems with large log j8„ values (see later). The concept of using low molecular weight chelating compounds covalently bound onto chromatographic supports was suggested over 50 years ago by Meinhardt^^^ and subsequently adapted as ligand exchange chromatography to separate metal ions and low molecular weight compounds, predominantly amines, amino acids, mono- and dinucleosides, and nucleotides.^^^'^^"^ The ability of metal ions to selectively bind through coordinative interactions to proteins was turned into practical chromatographic procedure in 1974, when 8-hydroxyquinoline (8-HQ), covalently immobilized to agarose and chelated with Zn^"^ ions, was used to isolate metalloproteins.^^^ Subsequently, Porath and co-workers^^^ recognized the potential of the metal ionbinding properties inherent to immobilized iminodiacetic acid (IDA) and adapted this mode of metal ion interactions with several proteins under the rubic of "immobiHzed metal affinity chromatography (IMAC)." Since then, IDA-based sorbents have been widely employed by many investigators, resulting in this mode of biomimetic chromatography becoming a viable experimental approach for the purification of globular, structural, and membrane proteins at the laboratory scale. In common with many tridentating ligands, significant leakage of border line metal ions occurs under mild elution conditions in HPLC processes with im-M"^-lDA complexes due to their relatively low metal ion stability constants (i.e., 7 < log j8n < 10.5^^^"^^^). As noted earlier, a large number of proteins are, in addition, able to strip metal ions from /m-M"'^-IDA complexes.^^ ^'^^^ To circumvent metal ion leakage, other chelating ligands have been investigated, including ^ns(carboxymethyl)-ethylenediamine (TED),^^^'^^^ a pentadentate ligand that coordinates metal ions via two nitrogen atoms of the secondary amino groups and three oxygen atoms from the three car boxy 1 groups. When Ni^"^ and other M^"^ ions are chelated to the /m-TED ligand, only one vacant coordination site remains available within the coordination sphere of the metal ion for interaction with donor groups on a polypeptide or protein, while three coordination site are available with the corresponding im-Ni^^-IDA complex. The /m-Ni^^-TED complex, therefore, has weaker affinity for proteins, i.e., lower Kp^ value, than the corresponding im-Ni^^-lDA sorbent, although the propensity for metal ion leakage is reduced under comparable elution conditions because of the higher Kj^.^ « chei value. Other chelating ligands have been identified with more optimal association constants for protein binding as well as lower metal ion leakage per se. The combination of hard and soft Lewis acid-base interactions^^^'^^"^ between the acceptor metal ion and donor N - or O-groups of the protein or

103

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

pseudo-cation exchange properties has been of particular interest. Thus, bidentate chelators, e.g., aminohydroxymates (AHM)^^^ or 8-HQs^^^'^^^; tridentate ligands, e.g., O-phospho-serine (OPS), ^^'^ cis-/trans-C2irhoxymethylproline (CMP),^^^ diaminomethyl-pyridine (DAMP)^^^ or dipicolylamine (DPA)^^^; tetradentate ligands, e.g., nitrilotriacetic acid (NTA)^^^'^^^ or carboxy-methylated aspartic acid (CM-ASP)^^^; and pentadentate ligands, e.g., TED or tetraethylene-pentamine (TEPA),^^"^ have all been examined for their potential use in IMAC applications with proteins. Figure 7 illustrates several of the structures of several of these HP-IMAC ligates. Typically, these additional classes of immobilized metal ion-chelating complexes (IMICs) have shown only modest increases in metal ion stability constants (i.e., in the range of 9 < log j8„ < 14), although nevertheless they offer alternative separation options. Recently, a totally different class of IMAC sorbents have been derived from 1,4,7-triazacyclononane (tacn), a macrocyclic chelating ligand, and used in various applications for the purification of human serum proteins and other wild-type proteins.^^^"^^^ Compared to other tri- or tetradentating chelating ligands, these new IMAC systems have much larger stability constants for many borderline metal ions, with values typically higher by at least 4 to 10 orders of magnitude, i.e., in the range of 16 < log j8„ < 30.^^^"^^^ The homologues, fc/s(l,4,7-triazacyclononyl)ethane (dtne) and fc/s(l,4,7-triaza-cyclononyl)-propane (dtnp), involve two tacn macrocyclic rings linked via either an ethyl or a propyl bridge, respectively. Consequently, dtne and dtnp can form two metal ion-binding centers of the type MN3 separated by

OH,

CH^"CO i OH,

(I) CO

CH,-CO

:.^-"^ g ^'--'-

CH

9^2

B t - - -.-.•iOH2

-i^

F I G U R E 7 Representative examples of the structures of different H P - I M A C ligate systems containing a borderline or soft metal metal ion M, such as Ni^"*" or Cu^"*": (I), iminodiacetic acid (IDA); (II), tr/s(carboxymethyl)ethylene-diamine (TED); (III), nitrilotriacetic acid (NTA); and (IV) c/s-trons-carboxymethylproline (CMP).

104

MILTON T. W. HEARN

between 5.6 and 6.8A or alternatively a sandwich-like structure of the type MN6.^^^'^^^ Because of their very high log P„ values, metal ion dissociation does not complicate the solution chemistry of these im-M^^-[tacn]2 species in the presence of proteins. In addition, the spatial orientation of these ligands potentially enables the formation with proteins of either monomeric coordination complexes of the type im-M^^ [ tacn]2-AA [where AA represents a suitable electron donor group of participating amino acid residue(s) of the incoming protein] or alternatively dimeric coordination complexes of the type im-M^^-[tacn]2-[AA]2. Depending on the electronic and steric properties of the functional groups of the participating amino acid residue(s) of the protein and the J-orbital characteristics of the M"^ ion, the protein-ligate interaction with IMAC sorbents derived from dtne and dtnp can involve one or both of the im-M^^-[tacn]2 moieties. Figure 8 illustrates the structures of

[CR,RJ

F I G U R E 8 Representative structures for the im-M"'^-bis-[tacn] ligate in the sandwich MN6 coordination stereochemistry (A) (where M represents a first row or subsequently row transition metal ion) and in the extended MN3 core stereochemistry (D) in the presence of a N - or O - donor group, i.e., an electron-donating nitrogen, oxygen, or sulfur atom within a side chain of a participating amino acid residue of a protein. The relative equilibrium association constant of the protein — im-M"'^-bis-[tacn] complex will depend on the properties of the metal ion, the participating donor solvent, and the size, shape, and the nature of the electron donor group(s) or other surface characteristics of the participating protein. For the Cu^"*" ion, a more extended structure (C) is favored'^'''^° compared to the case of the Ni^"*" ion, where the more compact, sandwich structure (B)I29.I30

105

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

1.5

H 15

in

o^ o

18

300 H • •«—GST-5ATPase-His,

12

1.0

9

o

6 <

0.5

3 O

O PH

0.0

0 10

15

20

Fractions

25

35

F I G U R E 9 Chromatographic separation of the GST-SATPase-His^ from other proteins in the crude Escherichia coli extract using the /m-NI^'^-tocn sorbent. The /m-Ni^"^-tocn Sepharose CL-6B sorbent was equilibrated with 50 m/VI NaH2PO4-300 m M N a C I - 10% glycerol, pH 8.0, at a flow rate of 0.5 m L / min. The crude £. coli extract (2 mL per I mL of sorbent) was loaded onto the packed column. Nonbound or weakly bound proteins were eluted with 10 volumes of the equilibration buffer, while bound proteins were eluted with a linear gradient from 0 to 250 m M imidazole in the equilibration buffer. Following completion of the gradient the column was eluted with 200 m M EDTA in the elution buffer. The GST activity in the recovered fractions was determined by the CDNB assay.^"*^ Other experimental details are provided in Ref. 127.

several of these macrocyclic IMAC systems in the different MN3 and MN6 binding modes, while Figure 9 shows an example of their application in protein fractionation.

F. 7T ^ 77 Dipole -^ Dipole Interactions and Charge -^ Dipole Interactions lonizable groups within a polypeptide or protein can also interact with polarizable groups. Such charge-dipole interactions produce relatively week interactions, with distance dependence of 1/r^. Moreover, positively charged groups within the folded structure of a polypeptide or protein can stabiUze the C-terminal region of a-helical secondary structures and negatively charged groups can stabilize the N-terminal end. Analogous effects can arise through 77 -^ 77 dipole-dipole interactions involving the side chains of the aromatic nonpolar amino acids. In the presence of chemically modified sorbents, equivalent ligand-ligate interactions can occur, particular with ether of phenyl-type sorbents. These effects are of the order of 2.5 kj/mol, and

106

MILTON T. W. HEARN

reinforce other types of interactions rather than act as a dominant contribution to the energetics of the interaction between polypeptides and proteins with these HPLC sorbents. G. Combined Effects As illustrated in Figure 10, during the very early stages of an adsorption process when the distance between the interacting species is relatively large in atomic units, the respectively orientated primary bonds are based on Lifshitz-van der Waals interactions a n d / o r hydrophobic interactions. As the ligand-ligate interaction complex develops, Coulombic effects due to the chemical nature of the interacting ligand or ligate species, or alternatively due to electrostatic characteristics of the support matrix itself, will become more significant. These effects will become particularly evident when the molecular distant between the interacting species are separated by < 100A. Coulombic, hydrophobic, and Lifshitz-van der Waals interactions will thus represent the dominate forces that lead to the develop of the primary interactions between biosolutes and the immobilized groups on the stationary phase surface. Hydrogen bond effects are manifested over relatively short atomic distances. For this reason hydrogen bond effects are often associated with the emergence of secondary bond processes that involve strong matrix-ligand interactions, particularly with the higher energy class I and class II silanols^^^'^^"^ of

o

HydrophobicLifshitz \van der Waals Interactions

hElectrostaticinteractions ^V—-H Hydrogen Bonds

w

w

\&

w

Distance [A] F I G U R E 10 Plot of the energy of interaction versus the distance between the interacting species during the very early stages of an adsorption process. When the distance is relatively large in atomic units, the respectively orientated primary interactions are based on long-range Lifshitz - van der Waals interactions and / or hydrophobic interactions. As the approach distance decreases, the energy term exhibits a greater electrostatic component, with a dependency of \ I r^. On further closure of the approach distance, hydrogen bond effects occur, with a distance dependency of I / r ^ , while on closer association the short-range van der Waals attractions mediated by fluctuating electrical charges will occur, followed by strong repulsion and a larger increase in the energy of interaction at very small approach distances.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

I 07

various silica-based matrices, zirconals^^^ of zirconia-based sorbents, or hydroxyl clusters in some hydrophilic organic polymers. Secondary bond effects can also evolve after a certain time lapse at a closer distance and mainly involve Lifshitz-van der Waals and hydrophobic interactions.^"""^^ Moreover, the surface organization of the ligates and the route employed for their immobilization can affect the selectivity of the interactive HPLC sorbent, as found, for example, v^ith different types of RP-HPLC sorbents.^^^ In some cases, secondary bonds can develop after structural changes of the interacting biosolute generating very strong hydrogen bonds. These effects are particularly noticeable w^hen polypeptides or proteins unfold in hydrogen bonding solvents such as methanol or isopropanoP^^'^^^ v^here large changes in the enthalpy of interaction can become evident. Under these circumstances it can be essential to let as little time as possible elapse betv^een the adsorption and elution step. In so achieving this outcome, the system residency time is kept as short as possible, minimizing the formation of reinforced interactions involving secondary bond formation, which in the case of globular proteins may be associated with denaturation or binding effects so strong that the mass recovery is significantly impaired.

Y. STRATEGIC CONSIDERATIONS BEHIND THE HPLC SEPARATIONS The extraordinary popularity of HPLC for the analysis and purification of polypeptides, proteins, and other biomacromolecules can be attributed to a number of factors: • The experimental ease with which selectivity can be manipulated for a particular sorbent through variations in the composition of the mobile phase. Subtle changes in selectivity and retention can be affected by changes in the concentration of a displacing ion, the water content, the pH, the type of buffer species, or whether specific cosolvent additives have been added to the mobile phase. • The relatively rapid nature of the separation, e.g., routinely can take as little as seconds with analytical systems and between minutes to hours for semipreparative or preparative systems with high-performance, pressurestable, and mechanically stable sorbents with particle diameters in the range of 3-65 fim rather than days with the classical types of soft gels, where the particle sizes are usually greater than 150 /im. • The excellent resolution that can be achieved for closely related as well as structurally disparate polypeptides and proteins under a large variety of HPLC conditions, particular when gradient elution methods are employed. Two exemplars illustrate this point. With suitable optimization, polypeptide diastereoisomers can be easily resolved^^'^^ with RP-HPLC methods, while separation of deamidated or mono-methionine sulfoxide forms and subtle structural variants of recombinant globular proteins can be readily achieved^^'^^'30'i^^-i^^ with RP-HPLC, HP-HIC, HP-IMAC, or HP-IEX techniques.

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MILTON T. W. HEARN

• The excellent reproducibility that can be achieved in repetitive separations carried out over long periods of time, due in part to the stability of the various stationary phases to many aqueous mobile phase conditions. Thus, it is not uncommon v^ith the current generation of pressure-stable HPLC sorbents for little change in the resolution to arise after more than 1000 repetitive analytical separations. • The potential, v^hich is now finding increasing application, for evaluating different physicochemical aspects of solute-eluent or solute-stationary phase interactions and their structural consequences from chromatographic data. In particular, the thermodynamics of the interaction can be easily examined,"^^'"^^'^^'^^ w^hile derivation of linear free-energy "molecular descriptors"and structure-retention correlations enabling the retention behavior to be adequately interpreted^"^^"^"^^ is now^ essentially a task of data acquisition, rather than being limited by the lack of suitable theoretical models for the simulation or analysis of the interactive process. • The generally high recoveries that can be obtained, even at ultramicropreparative levels. Many polypeptides and proteins can be recovered in bioactive form from crude microbial or mammalian cell culture systems or from biological extracts by RP-HPLC procedures, e.g., human transforming grow^th factor-a (TGF-a)^'^^ (see also Table 3 and references cited therein) provided adequate care is given to the residency time issues, recovery, and handling of the fractions, despite the obvious potential for the hydrophobic surface to cause unfolding and denaturation under these lowr pH, organic solvent-v^ater conditions. Similar considerations are also pertinent to the ultramicroisolation-purification of polypeptides or proteins in the buffer and ligate systems employed in the other interactive modes of HPLC. • The high productivity that can be achieved in terms of cost parameters. Various studies have documented that the throughput and productivity of HPLC systems with mechanically stable, high-performance sorbents are intrinsically higher than the conventional soft gel systems. Whether a highperformance sorbent rather than a soft gel material is employed is primarily determined by the cost to market for the product. Thus, in many cases with recombinant proteins, the cost of the sorbent is not the dominant factor. Rather, it is the ability of the manufacturer to produce the product reproducibly, in good purity, and to the stringent standards set by national registration-regulation authorities such as the U.S. Food and Drug Agency, the Australian Therapeutic Goods Committee, etc. • The opportunity provided by all modern high-performance chromatographic procedures to lend themselves to the requirements of either analytical or "scale-up" preparative separations. In many cases, "scale-up" opportunities can be based on suitable algorithmic modeling of the mass transfer processes with experimental data obtained from the corresponding "scaledown" systems.^'^^'^"^^"^^^ Moreover, such "scale-down" procedures have proved useful in assessment of the extent of removal of DNA or viral load from crude recombinant protein preparations.^^^ Also, from economic perspectives such "scale-down"-"scale-up" strategies enable reliable, rapid screening of different sorbents or elution conditions without the heavy financial burden of carrying out the studies initially at the process-scale level.

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j j j ^ H T A B L E 3 Selected Examples of the Use of Laboratory-Scale RP - H P L C Procedures for the Isolation of Biologically Active Polypeptides and Proteins Acidocin-A/bacteriocin from Lactobacillus acidophilus^^^ Alpha-amylase inhibitors from wheat"^^^ Amyloid polypeptide expression in human insulinomas Angiogenin-2, an angiogenic protein from bovine serum and milk"^^^ Atrial natriuretic peptide"^^^ Bitistatin, a disintegrin isolated from the venom of the viper Bitis arietans^^'^ Bone resorptive polypeptides'^^ ^ Bursicon, a cuticle sclerotizing neuropeptide from Tenebrio molitor^^^ Comparison and characterization of the venoms of the Parabuthus transuaalicus, P. granulatus, and P. Villosus scorpion species occuring in southern Africa"^^^ Cytochrome ba(3) subunit polypeptides from Thermus thermophilus hb8^^^ Fibronectin type II domains (Cys^'^-Cys^^ Cys^^-Cys^^^) of the major heparin-binding protein (PDC-109) of bull seminal plasma"^^^ Glucagon-like peptide-1(GLP-1^"^^) amide, a potent insulin releasing hormone"*^^ Glutathione transferase Triticum aestivum GST 1 (Tagst 1) variants^^^ Haemoglobin Hb melusine [aii^gh2)Pro^Ser]47^ Heavy- and light-chain polypeptides of the prophenoloxidase-activating isoenzymes (PPAE-1 and PPAE-2) of the silWorm, Bombyx mori^^^ HIV-1 protease from Escherichia colt inclusion bodies"^^^ Honey bee venom polypeptides'^^ ^ Human fibrinogen chains Human growth hormone antagonist"^^^ Human hemoglobins variants"^^ ^ Kunitz-type trypsin inhibitor from Enterolobium contortisiliquum seeds"^^"^ Low molecular weight glutenin subunits"^^^ Maurotoxin, a four-disulfide-bridges scorpion toxin from the venom of the Tunisian chactoid scorpion Scorpio maurus"^^^ Neuroparsin A, a polytropic neurohormone of the locust, Locusta migratoria"^^^ Peptide YY^-^^ and peptide YY3-^^ from human blood'^^^ Pituitary adenylate cyclase-activating polypeptide (PaCap)"^^^ Polypeptides fractionated from crude plant biomass"^^^ Prolamin from Kodo millet ( Paspalum scrobiculatumY^ ^ Prolamin, is the major storage protein from selected varieties of foxtail millet (Setaria italica (I) beauvY^^ Proteinase inhibitor from the hemolymph of a solitary ascidian, Halocynthia roretzi'^^^

Rat pancreatic polypeptide hormones"^^^ Recombinant human factor VIII "^^^ Recombinant human factor proinsulin"^^"^ Relaxin-like molecule from the male atlantic stingray, Dasyatis sabina^^^ Retinal pigment epitheUal protein"^^^ Scrapie prion proteins (PrPSc)'^^'' Sea anemone toxin"^^^ Subunits of giant extracellular hemoglobins from earthworms Pontodrilus matsushimensis and Pheretima communissima^^^ Thymopoietin II (BTP-II)^^<^ Type I fish antifreeze protein^^^ Venom protease with )8-fibrinogenase activity from the Taiwan habu (Trimeresurus mucrosquamatusy^'^ White muscle 65 kDa protein from warm temperature-acclimated goldfish Carassius auratusY^^ Xenopus laevis oocyte immunoactive insulin^o^ j8-D-Galactoside-bindinglectin from cuttlefish, Todarodes pacificus^^^ a- and /^-Polypeptides from the peripheral light-harvesting pigment-protein complex II of Rhodobacter sulfidophilus^^^ Brain-derived neurotropic factor ^^"^ Mabinlin Il-related polypeptides^^^ Gliostatin and platelet endothelial cell growth factors ^^^ Transforming growth factor-a^^^ Murine epidermal growth factor mutants^^^ Heparin-binding growth factors from procine, bovine brain and pituitary extracts^^"^"^^"^ Epidermal growth factor receptor^^^ Insulin receptor ^ ^ ^' ^ ^ ^ Bovine, procine, and sheep inhibin from follicular fluid extracts^^^'^^^ Hemopoietic growth factors^^^ Human platelet-derived growth factor ^^^'^-^^ Interleukin-2^^^ Colony stimulating factor-1^^^ POMC-peptides derived from rat pituitaryn extracts ^^ Neurophysins from rat pituitaries^^^ Cholecystokinin-related polypeptides^^ ^ FSH-releasing protein from bovine follicular fluid^29,535' /3-Transforming growth factor^^ ^' ^^"^ Nerve growth factor^^^ Interleukin-S^^"^ Gonadotropin-releasing hormone ^ ^ ^ a-Transforming growth factor ^^^ Uterine milk protein from ovine allantoic fluid^^^ Recombinant activin )3^ j8^ from CHO cell supernatents^^^ CRISP-related proteins from Pichia pastoris extracts^^^

MILTON T. W. HEARN

At the micropreparative (i.e., 0.1-10 /x,g level) through to the semipreparative scale (i.e., 1-250 mg), it is now feasible, by combining the capabilities of several HPLC separation techniques, to achieve purification factors between 100,000- and 500,000-fold for bioactive substances. Implementation of these capabilities is obviously essential if the biomacromolecule of interest is present in only trace amounts in biological fluids. Numerous examples can be cited where such levels of separation performance have been achieved. Table 3 provides a representative sampling of examples of some of the small laboratory-scale isolations of important new biologically active polypeptides and proteins where the very high resolving power of RP-HPLC has been exploited to obtain purification levels in this range. What is not evident from Table 3 is the exquisite resolving power of these separation procedures at these micro- or ultramicropreparative levels. The question can also be asked what lessons have emerged from these research investigations which are relevant to the large scale or process scale purification task? Although extremely high selectivities (i.e., large a-factors) and efficient bandwidths can be achieved by these methods, the possibility arises, as already noted, for the biological activity of the biosolutes to be lost. The conventional approach to overcome this problem with analytical HPLC or HP-CEC^^^ procedures as well as in the ultramicro- and small-scale HPLC purification of polypeptides and proteins and other biomacromolecules has largely been predicated on the use of short separation times, columns of narrow internal diameter, and optimized elution conditions, thus reducing the "residence" time of the biomolecule on the surface of the chromatographic sorbent. Increasingly, chromatographic scientists have also attempted to design and develop laboratory systems that exhibit improved recovery for particular classes of biomacromolecules by using, for example, shielded phases^^^'^^^"^^^ or ligate-engineered stationary phases,^^^'^^^'^^^"^^^, exploiting combinatorial phage display or synthetic library approaches to achieve enhanced resolution. Obviously, with preparative or process approaches, it is essential that the issue of bioactivity recovery be specifically addressed as part of the selection of any process strategy. By proper attention to the physicochemical consequences of the dynamic behavior of biopolymers in bulk solution and at liquid-solid interfaces, these requirements can be systematically addressed. Problem solving associated with loss of bioactivity or mass recovery in process HPLC is still largely based on empirical, or in a limited number of cases (e.g., large-scale purification of human albumin^^^"^^^, on more specific experience of the behavior of a particular biomacromolecule in a chemically defined solution or within the macro- or microenvironment of the stationary phase surface. Since preservation of the bioactivity at high mass recovery levels represents the major goal in process purification, any limitations in instrumental design, system engineering, or process development that result in impaired resolution, or the ability of contaminants to adulterate the final product and thus to reduced the overall purification factors, will confuse the quality analysis data. Although these issues are relatively straightforward to identify, they can be difficult to remedy. Central to being able to overcome these difficulties is a proper understanding of the purpose for which the

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(bio)product is being separated. Different criteria will apply if the purified products are intended to be used as industrial biocatalysts compared to a product that will be used in a human biopharmaceutical formulation. This definition of the purpose of the separation task thus defines the level of sophistication that will be required for the chromatographic procedures to achieve their stated objective in terms of the minimum level of resolving power, the plant cost, and the level of expertise that will be compatible with the task. Analytical chromatographic options, based on linear and nonlinear elution optimization approaches, have a number of features in common with the preparative methods of biopolymer purification. In particular, both analytical and preparative HPLC methods involve an interplay of secondary equilibrium and within the time scale of the separation nonequilibrium processes. The consequences of this plural behavior are that retention and band-broadening phenomena rarely (if ever) exhibit ideal linear elution behavior over a wide range of experimental conditions. First-order dependencies, as predicted from chromatographic theory based on near-equilibrium assumptions with low molecular weight compounds, are observed only within a relatively narrow range of conditions for polypeptides and proteins. Identification of the secondary parameters that contribute to the nonideality thus represents an important set of objectives for optimizing both analytical and process-scale HPLC applications. In process applications, a further complication can arise, associated with the high concentrations or loading volumes of the feed used. These overload effects in process HPLC impact on product throughput in a manner not experienced in most analytical HPLC procedures. By operating in the overload mode, rather than within a linear region of an adsorption isotherm, whatever its shape may be, then complex peak shapes, concentration- a n d / o r volume-dependent shifts in the retention and effects on bioactivity and mass recovery can accrue. Preservation of biological function and achievement of maximal product bioactivity and mass yield with minimal degradation are the dominant requirements and endpoints in all HPLC tasks, but particularly in preparative process separations. Attainment of this "bioactivity" property as part of a preparative HPLC task introduces another dimension to the well-known separation optimization triangle (speed, capacity, and resolution) not usually experienced with the process chromatography of low molecular weight organic molecules. The use of biotechnological procedures for protein production and the application of more precise deterministic models that fuse the chromatographic behavior of biopolymers with their biophysical-structural behavior is thus even more pressing than ever before. Various approaches to such deterministic models have been established around modern chromatographic theory and experimental practice, which differentiates the thermodynamics and kinetics of the separation process. Rational improvements in separation performance can thus arise from analysis of the consequences of changing the physical (e.g., particle size, flow rate, column configuration, pore size, bed-packing quality) or chemical characteristics (e.g., sorbent type, eluent composition) of a particular chromatographic system.

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MILTON T. W. HEARN

Traditionally, the improvement in separation performance with proteins has arisen from data accumulated on the one hand from nonchromatographic measurements such as spectroscopic studies on the structure-function, the stability of the biosolute in different solvent environments, and from conformational investigations. Batch or bath adsorption procedures have often been employed to evaluate changes in the biological or immunological activities of a protein in response to changes in the separation variables. Experimental methods that enable the nature of the contact sites to be elucidated, the kinetics of polypeptide or protein adsorption-desorption behavior measured or the kinetics associated v^ith conformational, ion-binding, or solvent hydration phenomena in the bulk mobile phase or at the liquidsolid interface determined, have special importance in these evaluations.^'^'^"^'^^'^^'"^^""^^'^^^"^^^ Such approaches are based essentially on the concepts of chemical equilibrium with the major challenge being the proper understanding of the physicochemical factors that control the interactive behavior and the stability of the tertiary structure(s) of biopolymer(s). Experimental control over resolution will be achieved if the biosolute(s) manifest only a limited set of preferred interactions, conformations, and orientations in the stationary phase-mobile phase distribution process during the separation. When these criteria are achieved, high mass and bioactivity recovery can often be realized following desorption. In this regard, attention to the extent of the molecular heterogeneity of the ligates in the distribution process,^^^"^^^ the impact of system residency and dwell effects, and the opportunity to minimize large changes in the entropy of the interaction associated with the binding of the biosolute(s) to the stationary-phase surfaces or as they permeate through the pores of the sorbent are all important parameters, which impact on and find expression in preparative HPLC separation procedures. It is also evident from recent trends in the scientific literature that empirical models describing the retention and mass transfer ^^^"^^^ of polypeptides and proteins with microparticulate HPLC sorbents require modification to address more precisely the propensity of biomacromolecules to undergo slow conformational equiUbrium^'^^'^^''"^^^ in solution or at Uquid-solid interfaces. The combination of these theoretical investigations with experimental studies on biopolymer folding increasingly forms the basis for the development of new types of tailored HPLC sorbents,^^'^^'^^^'^^^'!^^-^^^'^^^-^^^ some of which have already found their way into the biotechnology industry. Modern chromatographic methods draw on the further utilities provided by new detection methods, e.g., on-line photodiode array detection, optical rotary dispersion-circular dichroism (ORD-CD), laser-activated light scattering, derivative spectroscopy, and increasingly mass spectrometry, either in the electrospray ionization (ESI-MS) mode or in the matrix-assisted laser desorption time-of-flight (MALDI-TOFL) mode to assess^^'^'^ the purity and conformational integrity of biomacromolecules. The application of these powerful spectroscopic procedures in an integrated, orthogonal fashion will have profound ramifications in the future on how biochemists and process engineers deal with the vagaries of biorecovery and select alternative HPLC strategies to achieve protein purification. A further consequence of recent

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developments is the rate at which quantitative data can be acquired w^ith HPLC systems. Illustrative of these advances is the manner that HP-IEX optimization studies can now be carried out at the laboratory-scale level to investigate the influence on protein recovery and resolution of three different pH conditions in the presence of two different salts with 10 different gradient options. Typically, as little as 48 hr of experimental time may be required with modern HP-IEX sorbent materials and suitable computer-assisted optimization equipment, e.g., BioCad- or BioSys-based instrumentation. To perform the corresponding studies with soft gel ion exchangers could require in excess of 800 hr of experimental time. In common with the classical modes of liquid chromatography, HPLC procedures can be classified according to the selectivity of the sorbent. In size-exclusion chromatography (HP-SEC), the biosolute, in principle, does not interact with the sorbent but permeates through the pores of the material. Retention is thus controlled solely according to the hydrodynamic and fluidic properties of the system. In all the other modes of HPLC separation, an adsorption isotherm dictates the nature of the mass transfer of the biosolute in the presence of the sorbent. This adsorption involves a combination of both specific interactions, such as protein-ligand binding, or nonspecific interactions mediated by other classes of binding sites, often associated with the chemical nature of the support material. In an ideal system, a single class of interactions will prevail, i.e., only protein-ligand binding occurs. Even in these ideal cases, binding site heterogeneity must be anticipated^^^"^^^ due to the asymmetric distribution of the immobilized ligands, or the proclivity of the biosolute to self-associate or bind to other components or contaminants in the feedstock.^'^'^^^'^^^'^^"^ The dominant migratory processes involved in the separation of biopolymers with many types of HPLC sorbents are controlled in fluidic terms by diffusion-convection mass transfer events, although with the increasing use of macroporous sorbents, perfusive events can also contribute, particularly at high linear flow velocities. All of these processes are generally associated with high mass conservation. However, when biopolymer folding and unfolding become significant events, or alternatively inappropriate control over secondary equilibrium processes has arisen, the chromatographic system effectively behaves as an adsorption-desorption chemical reactor in which mass and activity conservation of a particular species cannot be assumed. From a microscopic point of view, such secondary processes lead to adsorption isotherms that are more complex than predicted by, e.g., the more simpfistic Langmuirean descriptions of adsorption, and require much more sophisticated numerical analytical methods to evaluate the relevant mass balance equations and accommodate the influence of nonlinear isothermal behavior, particularly with multicomponent samples. For these reasons a number of other isothermal representations have been favored, including the Temkin and the Jovanovic-Freundlich isothermal models. High-resolution chromatographic methods per se are only part of the arsenal available for the analysis and purification of a particular biomacromolecule. Table 4 summarizes some of the comparative features that HPLC offers compared to these other separation alternatives in terms of process

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i ^ H T A B L E 4 Comparative Features between the Separation Procedures Used in the Preparative H P L C Purification of Polypeptides and Proteins with Alternative Approaches and Typical Purification Factor Ranges and T i m e Required per Unit Operation

Parameter

Process

Temperature Solubility

Heat denaturation Salt precipitation Solvent precipitation Isoelectric precipitation Polymer precipitation Aqueous partitioning Two-phase systems Size-exclusion chromatography-gel filtration Centrifugation Ultrafiltration Gel electrophoresis Free electrophoresis Ion-exchange chromatography Chromatofocusing Hydrophobic interaction chromatography Reversed-phase chromatography Hydrophilic interaction chromatography Biospecific affinity chromatography Biomimetic affinity chromatography Immunosorption (e.g., monoclonal antibodies) Lectin affinity chromatography

Size and shape

Net charge Isoelectric point Hydrophobicity Hydrophilicity Function Antigenicity Carbohydrate content Thiol content Exposed histidine Exposed metal ion Group specific

Covalent chromatography Metal chelate affinity chromatography Chelate affinity chromatography Hydroxyapatite chromatography Dipolar chromatogaphy Dye affinity chromatography Charge transfer chromatography

Typical purification factor ranges (x-fold)

Typical Time per run (h)

2-20

1-40

2-20

10-100

2-20

40-80

2-20 2-5 2-10 2-5 2-40 2-10 2-30 2-200 2-20 50-1000 20-400 20-100

10-60 4-20 100-h 100-h 2-50 10-20 2-10 2-10 2-10 2-10 2-10 2-20

2-10

2-10

2-10 2-10 2-10 2-10 2-40 2-40 2-40

2-10 2-10 2-10 2-20 2-10 2-10 2-10

Based on fractionation data for hen egg white proteins including hen egg white lysozyme, ovalbumin, and avidin.

time and relative purification factors. What thus sets HPLC apart in its various analytical and preparative modes, e.g., ultramicropreparative through process scale applications, is the speed of the separation that can be achieved w^ithout necessarily sacrificing the magnitude of the purification factors and resolution. It is, however, an ideal scenario, rarely attainable in practice, for the full knowledge on the mechanistic processes underlying the selectivity and the kinetics of biosolute adsorption, desorption, and mass transport under the separation conditions to be available. To achieve this stage of understanding in the high-resolution chromatographic analysis and purification of a target protein, a considerable amount of research effort is required.

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Nevertheless, with the development of new HPLC separation media, more advanced instrumentation, and faster computation procedures, fundamental questions central to the physicochemical nature of the separation selectivity and kinetics of different classes of biopolymers can, however, now be addressed. With adsorptive chromatographic systems, in particular, the molecular dynamics, associated with multisite interactions between the biopolymers and the stationary phase surface take on a very significant dimension and control not only the retention and zone-broadening behavior but also the mass and bioactivity recovery. For several practical reasons, mainly associated with cost or difficulties with column regenerability, highresolution chromatographic procedures are usually not brought into play with sub-25 fim microparticulate adsorption media of narrow particle diameter distributions and narrow pore size distributions in a specific purification methods until clarification of the feed stock is complete and partial fractionation has been carried out. The considerable activity underway at both academic and industrial centers, exploring different options to improve stationary phase characteristics, based on ceramic^^^'^^^'^^^"^^^ or polymeric sorbents^"^'^^^'^^^"^^^ with the purpose to enhance selectivity and improving kinetics with macro- or gigaporous, mesoparticulate (dp ^ 5 - 3 0 ^j^y88,i90,i9i^ or monolithic sorbents^^^"^^^ will undoubtedly lead to greater opportunities for the microscale, semiscale, as well as process-scale isolation of polypeptides and proteins by HPLC procedures in the near future. Interactive sorbents have the potential to probe the topography of biopolymers. This attribute is particularly useful in assessing the molecular nature of surface accessible regions or binding sites unique to the protein or other biomacromolecules of interest. The development of purification stratagems based on a rational combination of HP-IEX, HP-BAC or HP-BMC, and HP-HIC currently represents the core concept behind the evolution of a specific high-resolution chromatographic separations for the purification of globular proteins. Because of their "simpler" structures, polypeptides are much more amenable to process purification by tandem RP-HPLC approaches utilising different ion-pairing strategies.^^^'^^"^ Figure 11 illustrates, as a schemata, the more common modes of proteinligate interaction used in these adsorption modes of HPLC. As is apparent from the preceding considerations, separation selectivities can also be generated through combinations of these different interaction processes. Thus, exploitation of the physicochemical interplay between hydrophobic and Coulombic interactive phenomena forms the basis of many of the so-called "mixed-mode" HPLC procedures. Under appropriately chosen eluent conditions, proteins can be efficiently separated on sorbents containing immobilized electrostatic ligands with hydrophobic selectivity,^^^'^^^ i.e., in order of increasing hydrophobicity under conditions of decreasing displacing salt concentration from a high ionic strength condition typically of /i, - 3-5 M down to a low ionic strength condition such as /x ~ 0.2 M or lower. Similarly, under appropriately chosen solvent conditions hydrophobic supports such as w-alkylsilicas can be induced to exhibit polar phase selectivity with polypeptides and proteins eluting in order of increasing polarity. Thus, hydrophobic polypeptides and proteins can be separated on RP-HPLC

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MILTON T. W. HEARN

(1)

L^m^

(2)

(3)

WQ^^

(4)

F I G U R E I I Schematic illustration of several common modes of protein - ligate interaction used in these adsorption modes of HPLC, namely the electrostatic interaction (I), immobilized metal ion affinity interaction (2), hydrophobic interaction and reversed phase (3), and b/s-macrocyclic metal ion affinity interaction v/ith the psuedo-cation exchange features typical of the MN6 system.

sorbents with polar-phase selectivity^^'^^'^^'^^^'^^^'^^^"^^^ with retrogradients derived from eluents of high organic solvent content down to lower organic solvent content, i.e., from volume fractions oi \p ^ 0.9 to t/f - 0.5. Adaptations of this concept have lead to the generation of the mixed-mode hydrophilic HP-HILIC sorbents,^^'^^"^ where again good selectivity features can be generated through the use of the polar interaction capability of mixed-mode interactions. Finally, the sequential use of these different physicochemical principles can be employed to guide the order and selection of different HPLC techniques. Thus, for simultaneous separation^^^'^^^ of the glycoprotein hormones human follitropin (hFSH), human lutropin (hLH), and human thyrotropin (hTSH) from pituitary preparations, the sequential use of size-exclusion chromatography, preparative isoelectric focusing, anion-exchange HPLC, and HP-IMAC was very successful in the purification of individual isoform species. Three observations relevant to the general applicability to the protein fractionation area arise from this study. First, by employing ion exchange HPLC after preparative isoelectric focusing, it was feasible to resolve components with the same pi value but different surface charge anisotropic features. Second, by employing a multidimensional buffer selection approach with the same HPIEX sorbent via eluent composition optimization, considerable improvement in recovery were achieved. Third, by employing HP-IMAC after the ion-exchange HPLC step, the different surface accessibilities of histidine residues in isoforms of the same pi and similar surface charge anisotropy but overall different glycosylation state or extent of internal cleavage could be probed and the specific isoform resolved. Finally, the use of RP-HPLC enabled the individual subunits of the individual isoforms to be separated.

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thus enabling more detailed molecular characterization. The enhancement in resolution thus achieved permitted many glycosylated isoforms of these protein hormones to be characterized functionally for the first time in terms of their specific activities, receptor binding behavior, immunological properties, and subunit structures. The composite interplay betv^een size exclusion phenomena, hydrophobic effects, hydrogen bonding processes, van der Waals contributions, and Coulombic interactions is thus a feature of all current high-performance chromatographic materials used for the analysis and purification of biopolymers such as peptides and proteins. The retention and kinetic behavior of biosolutes in such interactive systems can be formalized (see Section VI) in terms of the summated contributions from these fundamental physicochemical processes. Depending on the magnitude of these interrelationships, and the magnitude of their relative contribution to the to the overall retention and kinetic processes, the separation process can be classified as a size-exclusion, hydrophobic interaction, Coulombic, polar-phase, etc. HPLC method. The parameters that make the greatest overall contribution(s) to the chromatographic behavior of biopolymers in a particular sorbent system will thus depend not only on the permeability, ligand composition, and ligand density of the stationary phase, but also on the mobile phase characteristics in terms of the water content, the pH, the ionic strength, the presence-absence of an organic solvent, the buffer composition, and whether additives such as ion-pairing reagents, dissociating reagents, or surfactants and detergents have been added to the eluent.

Yl. SPECIFIC PHYSICOCHEMICAL CONSIDERATIONS ON THE INDIVIDUAL CHROMATOGRAPHIC MODES The selection and chemical modification of the current generation of chemically and physically robust stationary phases with narrower particle and pore size distributions has been based on the developmental effort that has occurred over the past 20 years. Initially chemically modified, deformable polymeric gels were used, such as the crosslinked agaroses, dextrans, or acrylate-based copolymers, but more recently various classes of highly refined type I and type II silicas and other ceramic materials, or new classes of controlled porosity polymeric organic materials have found increasing application. Criteria now recognized as essential for the selection of chromatographic support materials are fisted in Table 5. Many of these criteria apply equally to sorbent materials intended for analytical separations as they do to largescale preparative separations. Clearly, in the latter case the issues of column productivity, in terms of kilograms of product resolved at a defined purity level per unit time per unit cost for the operation of the overall purification system, is of fundamental importance. The potential of the purification approach to satisfy good manufacturing practice (GMP) scale-up procedures, and thus meet governmental regulatory agency guidelines, is also of major relevance in the selection of a particular sorbent for use in an industrial

I8 B B

MILTON T. W. HEARN T A B L E 5 Criteria N o w Recognized as Essential for the Selection of Chromatographic Sorbents for the Purification of Polypeptides and Proteins Chemical and physical stability of the sorbent under various flow rate and mobile phase regimes Mechanical strength and resistance to deformation, particularly when operated at higher temperatures, but also at relatively high superficial velocities Particle uniformity in terms of the particle size distribution as well as the notional pore size distribution Reproducibility between different batches of the sorbent High capacity in terms of dynamic loading in preference to static loading of the desired component(s) in the feedstock High resolution and selectivity under linear and partial overload chromatographic conditions High mass and biological recoveries of the target product(s) High product throughout in terms of productivity per cycle Hydrophilicity, wettability, and a general chemical inertness to relatively forceful eluent conditions Sterilizability and regenerability, thus ensuring that the separations can be duplicated in terms of loadability and resolution Lack or minimal occurrence of leachables Characteristics that enable GMP scalability to be achieved on the basis of the scaledown-scale-up considerations Cost of sorbent per cycle, amortized as a percentage of the investment cost for the production process Opportunity for the knowledge gained with the particular sorbent to be readily adapted and/or transferred to other recovery problems and tasks Potential for the sorbent to be used under batch (tank) and packed-bed scenarios interchangeably Adequacy of masterfile documentation for the sorbent when the purified product is to be used in the food industry or as a human or animal therapeutic

application for the purification of a peptide or protein. Although sorbents used in production process can be expensive, i.e., an average cost for a process immunoaffinity sorbent used at the process-scale level for the recovery of a recombinant protein could easily exceed lOOK U.S. dollars per column bed, this cost is amortized over at least 500 runs and thus hardly represents an impediment to its use. The biotechnology service industry has long appreciated this reality, and has been able in many cases to commercially position themselves accordingly by introducing nev^ classes of sorbent materials based on biomimetic affinity, hydrophobic interaction, and ion-exchange chromatographic selectivities that are essential cost-independent as far as the overall production cost, and certainly an insignificant percentage of the total cost to market of the product. The misconception that modern chromatographic sorbents are expensive and therefore not applicable in many processes simply does not stand up to careful productivity analysis. The cost of labor, plant investment, and quality control of the product represent much larger percentages of the manufacturing cost, while the age-old rubric that a manufacturer v^ho cannot pay the bills for water and power does not have a manufacturing process applies equally well here. The production of highpurity, pyrogen-free water and the service costs for the power to run the

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

I I 9

refrigeration plant, the positive pressure-ducted air-conditioning, the lyophilizing equipment, or the myriad of other equipment and service items that determine a viable production process frequently represent the real costs behind the use of HPLC procedures in the recovery of protein products in biotechnological processes. Not unexpectedly, the so-called "noninteractive" HPLC modes of separation (e.g., the size-exclusion, gel permeation, and hydrodynamic chromatographic modes) do not exhibit the same level of resolution or capacity as chromatographic techniques based on polypeptide- or protein-ligate interactions. Consequently, the most successful separation techniques are those capable of achieving a selective interaction by probing the topography of the desired biopolymer, and the nonuniformity of distribution of charge, hydrophobic, metal ion-binding groups, or other chemical functionality. For example, the charge distribution and anisotropy of the charge potential over different regions of the surface of the biopolymer is exploited in ion-exchange chromatography separations. A number of other separation techniques are also capable of resolving biopolymers on the basis of differences in net charge or alternatively the anisotropy of the charge distribution. These alternative procedures, mainly analytical or micropreparative, include capillary zone electrophoresis, isotachophoresis, micellar or affinity electrophoresis (HP-CZE, HP-ITP, HP-MECK, or HPACE), and capillary electrochromatography (HP-CEC). Under conditions in which a transient or static pH condition can be generated, such that the pH at a point within the separation system corresponds to the pi of the protein of interest, further extensions of the net charge separation approach are found in analytical and preparative chromatofocusing and isoelectrofocusing. However, ion-exchange HPLC methods have assumed a dominant position for the purification of many enzymes, and other proteins in their bioactive state. From a process perspective, ion-exchange HPLC methods, as a consequence, have occupied for the past decade a central position in the repertoire of separation methods for the purification of different classes of polypeptides and proteins. Hydrophobic interaction chromatography and its counterpart, reversedphase chromatography, in comparison exploit the accessibility and surface distribution of lipophilic or nonpolar residues of a biomolecule. The term "hydrophobic interaction chromatography" is frequently associated with separations that are affected by a decreasing salt concentration, while the term "reversed-phase chromatography" has become identified with separations involving an increasing concentration of organic solvent in the eluent. The physicochemical basis for these hydrophobic interaction and reversedphase separation methods is, however, common, and largely results from an incremental changes in the microscopic surface tension associated with the biosolute-solvent-stationary phase interaction. Further examples of separation techniques that exploit the asymmetric distribution of amino acid residues at the surface of folded proteins include metal ion affinity chromatography (HP-IMAC), ligand exchange chromatography (HP-LEC), immunoaffinity chromatography (HP-IAC), hydrophilic chromatography (HP-HILIC), and the various modes of biospecific (HP-BAC), and biomimetic (HP-BMC) chromatography. For example, the

I 20

MILTON T. W. HEARN

binding of coordinated metal ions to exposed histidines (or other specific amino acid residues), either within the sequence of the protein or as part of a fusion peptide tag introduced by genetic engineering methods, forms the basis of IMAC procedures. Similar regioselective discrimination can also be achieved with group-specific affinity methods such as triazine-dye affinity, hydroxyapatite interaction, or borate affinity HPLC as well as other forms of ligand interactions which use generic biological ligands, i.e., lectin-oligosaccharide systems, biotin-avidin systems, and protein A-IgG systems. The final group of separation procedures, and the ones that potentially give the highest selectivity, represents methods that exploit functional properties of a biopolymer such as a specific ligand-binding site, an antigenic determinant, or a structural element such as a lipid-binding amphipathic (nonpolar) domain in lipoproteins or subunit contact region of a multimeric protein complex. With appropriate immobilization chemistries and choice of the ligand, biospecific affinity chromatography and immunoaffinity chromatography both have the potential to generate separation peak capacities more than two orders of magnitude greater than observed with adsorption methods based on simple chemical ligands such as those typically employed for ion-exchange or reversed-phase chromatography. Examples of the separation parameters that can be used in protein purification with the ranges of purification factors that can be expected as a typical single-stage procedure are summarized in Table 4. For most purification tasks, it is now routine to utilize combinations of all of the separation parameters listed in Table 4 with different objectives in mind during the different stages of the isolation and analysis procedures. The sequential application of two or more different high-resolution chromatographic separation modes in a particular study is known as multidimensional HPLC. The example described earlier for the resolution of the pituitary glycoprotein hormones is one such case. Manifestation of more than one mode of separation process or selectivity with a particular class of sorbent is known as multimodal HPLC. There is no doubt that greatly expanded databases on the behavior of proteins and other biomacromolecules in various physical or chemical environments are urgently required if the predictive abilities of both multidimensional and multimodal HPLC systems are to be fully integrated into high-resolution separation strategies. Related considerations apply also to the assembly and interpretation of the mass transport and associated mechanistic-physical parameters that are associated with the fluidic and surface interactive behaviour of biopolymers in batch (tank), packed-bed or fluidized-expanded-bed systems. The progressive assembly and collation of such databases, as a prelude to the establishment of so-called "expert systems" are currently underway in this and other academic laboratories. These results are already finding usage in first-generation "knowledge-based expert" systems,^^^'^^^ neural network systems,^^^'^^^ and other computerassisted^^'^^'^^'^^^"^^^ separation strategies, thus enabling various algorithmic methods to be applied as part of the development of a suitable strategy for the HPLC separation of biomacromolecules. These intuitive knowledgebased procedures are, however, still in their infancy, but will undoubtedly grow in importance and sophistication over the next several years. With

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

I2 I

sufficient attention to the assembly and organizational arrangement of reliable databases on biopolymer behavior under different solution and sorbent regimes, these computer-assisted predictive tools w^ill find significant application during the next tv^o decades. From practical considerations, probably the most important decision an investigator can make v^ith regard to the selection and optimization of a particular chromatographic system is the choice of the k'l range over which the separation is to be achieved. One of the initial actions should be an examination of different HPLC conditions that provide the smallest value of k'l without compromising selectivity. A range of 0 < k'^ < 20 has been preferred by many investigators for high-performance chromatographic separations with peptides and proteins, since these conditions avoid excessively long retention times and the concomitantly greater band-broadening of the peaks. Even when gradient elution conditions are employed the corresponding range of 0 < ^^ < 20 (where ~kj is the median capacity factor of the polypeptide or protein the gradient HPLC system) should be the target. As the a value approaches unity, selectivity declines. Thus, the choice of a values for a defined range of k'l values represents an equally important task in the selection of a particular set of HPLC conditions. Since the equilibrium distribution coefficient i^assoc,/ i^ related to the overall energy change in Gibbs free energy AG^^gg^c,/ ^^^ ^he separation process carried out at constant pressure P and constant molar volume V of the solvent, then the capacity factor K-, also takes on the well-known fundamental thermodynamic dependency through the relationships: AGa°ssoc.,= - ^ T ^ l n X a s s o c , i

(42)

AG'' lnfe;. = l n < D - - - ^

(43)

where JR is the gas constant, and the T is the absolute temperature (in degrees Kelvin). The capacity factor k'^, thus enables a particular HPLC process operated at a constant flow rate and temperature to be defined both in empirical as well as thermodynamic terms. It is, however, important to recall that under conditions of changing T, P, or V, the more general form of Eq. (43) should be employed, i.e.,

In k'l = Incl)

AGl,, I RT ^ ^ + In 1^77 RT PV

(44) ^ ^

Differences in the molecular characteristics and interactive behavior of biosolutes can thus be revealed from these HPLC separations, through quantitative evaluation of the thermodynamic and extrathermodynamic differences manifested in the interaction of the biosolutes with the sorbent, under a defined set of mobile phase conditions at specified temperature and pressure. Exploitation of these principles forms the basis for the evaluation of the retention coefficients of polypeptides^'^^'^^'^"^^"^"^^'^^"^ (through the dependency of linear free energy relationships given by group retention indices such

I 22

MILTON T. W. HEARN

as T = In a on the distribution coefficients P^, fragmental constants f^, or other physicochemical parameters). Similar considerations apply to the characterization of binding sites"^^""^^'^^'^^^ of polypeptides and proteins in contact with sorbent surfaces, and the determination of folding or unfolding pathways associated with biopolymer conformational behavior in the presence of HPLC sorbents.^^'^^'^2'1^^'1^^-1^^'2^0'^^1 In fact, evaluation of the ^^assoc,i dependencies under different conditions, particularly under different temperature regimes, permits the composite effects of all of the individual unitary free-energy changes associated with electrostatic, hydrophobic, sterimolar, solvational, hydrogen-bonding, and van der Walls interactions involving self-association-aggregation to be examined. These various contributions to the overall free-energy change for the ligand-ligate interaction, given by AG^s^o^ •, in all modes of HPLC of polypeptides and proteins can thus be expressed in terms of the relevant solvophobic considerations^^'^^'"^^'"^^'^^'^^'^^^'^^^ such that AGl,,,,. = AGii,,. + A G ^ , , . + AG,V,. + AG^,, + A G ^ , , , + AG,«,a,, (45) where AG,^^,,, A G ^ , , . ( = AG,^,,,,-), A G , V . , AG^^,,, A G ^ , , , , and AG,«,a,. are the free-energy differences associated with electrostatic effects (subscript es, /), hydrophobic processes including the formation of a solvent cavity of dimensions of the biosolute (subscript hydr, / or cav, /), van der Waals interactions (subscript vdw, /'), solvational effects (subscript sol, /), self-association or heterogeneous association of the biosolute in the presence and absence of solvent (subscript aggr, /), and the reduction in free energy due to nonideal effects (subscript red, /), respectively. The fundamental relationship between ^G^^^^^j and In fe^ thus provides the basis for understanding the composite interplay between size exclusion phenomena, solvophobic, Coulombic, and the other interaction processes that are evident as dominant features of all current HPLC stationary phases. Depending on the magnitudes of these individual Gibbs free-energy capacity factor dependencies, the retention behavior of any biosolute with an interactive ligand system can be formalized in terms of the incremental contributions to the overall retention process and thus to the isocratic retention factor ^^ through the relationship given as Eqs. (43)-(45). Under conditions of electrostatic interaction, the magnitude of the AG^^^ , term varies with the salt concentration m, and hence, the dependence of the protein activity coefficient in the mobile phase can be expressed through the extended Debye-Boltzmann equation as B

m^-^

-•• "^ ^ m p , / ^

where JUL^ is the dipole moment of the polypeptide or protein, and the coefficients A^^ and B^^ (where the subscript mp refers to the mobile-phase term) are both proportional to the net charge on the protein. The coefficients

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

I 23

Cjj^p and Djjjp are specific constants related to the dimensions of the macromolecule, while the coefficient A^^ is inversely dependent on protein size. A similar relationship can be derived for the stationary phase component of the change in the Gibbs free energy due to electrostatic effects (AGg^^ ^p ^), although the precise relationship betw^een salt concentration in the mobile phase and activity coefficient of the protein vv^hen bound to the sorbent may take a more complex form. The electrostatic free-energy change associated v^ith the chromatographic retention process then can be expressed as AG°,, = A G « , , p , , - A G ° , , p , ,

(47)

v^here the subscript sp refers to the stationary phase. Under ideal conditions, i.e., when only electrostatic interactions prevail in the ligand-ligate interaction, Eqs. (46) and (47), in association with Eqs. (23)-(35), represent a theoretical basis to interpret the interaction of polypeptides and proteins in the ion-exchange chromatographic mode, in effect treating the electrostatic interactions as a series of independent point-charge molecular recognitions. If multiple electrostatic interactions take place at the stationary surface, or alternatively when the displacing salt species carries a charge > 1, then an expanded Debye-Huckel equation must be employed to enable an appropriate interpretation. In these more complex electrostatic interaction cases, mobile-phase conditions must also be chosen with some care to suppress other nonideal behavior, such as charge shielding or salt induced chelation effects. According to the Debye-Boltzmann equation, the form of the free-energy change due to electrostatic effects can be represented by with the magnitude of the force decaying according to a 1/r^ dependency (Eq. 21), such that the relationship between the electrostatic free energy AG^^^ , the charge on the protein q, and the electrical potential if/ is given by AGl,=

f%dq

(48)

The potential at the surface of the protein where the charge is located is given by

*=i^][i DrJI

"

(49)

I + Ka

If it is assumed that the final effective charge at the macroion surface of a polypeptide or protein can be represented by Z^, where Z is the magnitude of the charge, q is the sign of the charge, r is the protein radius [calculated""^ from the relationship r = (0.81 M/^^), then

-^..=mihT^]=

KY

IDr

I + Kr

(50)

124

MILTON T. W. HEARN

Hence, the average distance a between the polypeptide or protein macroion and the charged Ugates at the hquid-soUd sorbent interface is given by 2^2 Z^q

Z^q^

(51)

-lDr^Gl

The quantity K ' , also known as the Debye length, has the dimensions of distance and is an approximate measure of the thickness of the ionic atmosphere over which the electrostatic field of the ion extends with an appreciable strength. The K term can be calculated from the following relationship lOOONe^ e.DkT

Lc^Z,

(52)

where N is Avogadro's number, e is the protonic charge, Q is the sah concentration, EQ is the permitivity in vacuo, k is the Bohzmann constant, and T is the absolute temperature (in degrees Kelvin). As the value of the Zterm can be achieved from the plot of the logarithm of the capacity factor In ^^, versus 1 / Q , then the value of a can be derived directly from Eqs. (50)-(52). Shown in Fig. 12, are the plots of the approach distance a (in angstoms) versus k'^ for the protein human serum albumin measured under isocratic conditions at a constant flow rate of 1.0 mL/min and at four different pH values.

k' F I G U R E 12 Plots of the approach distance a (in angstroms) versus k,' for the protein human serum albumin (HSA) (pi = 5.85, M^ = 69,000) measured with the strong anion exchange sorbent, Mono-Q, under conditions of varying NaCI concentration to achieve smaller kj values at a flow rate of I m L / m i n and at a temperature of 298 K with (I) 20 m M piperazine buffer, pH 9.6; (2) 20 m M triethanolamine, pH 7.5; (3) bistris buffer, pH 6.5; and (4), 20 mAi piperazine buffer, pH 5.5, as the eluents. Data selected from Ref. 542.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

I 25

In the case of the hydrophobic component of the interaction, the energy of cavity formation in the mobile phase is related to the surface tension y and surface area of the molecule A^^^^i •, according to AG°.,„p,, = [NA„„,,, +

4.8NV3(K;

- 1)VV3]^

(53)

where K/ is a constant that corrects for the curvature of the cavity, and N is Avogadro's number. The surface tension of aqueous salt solutions is a function of the molal salt concentration m and can be given by y = 7^ + crm

(54)

where y^ is the surface tension of pure water and a is molal surface tension increment, a constant characteristic of each salt. Table 6 provides details of the molal surface tension increments and other properties of salt systems commonly used in HP-HIC of polypeptides and proteins. Assuming that the magnitude of the salt concentration has no effect on Acf, A^^^^i • or V, the free-energy change associated with cavity formation in the mobile phase (AG^^a^ n,p ^) can be evaluated from

+ [NA„,„,, + 4 . 8 N V 3 ( K / - 1)VV3]^^

(55)

which can be expressed in a simplified form as AG,^av,mp,. = ^ ^ c o n t a c t ^ ^ + COUStaUt

T A B L E 6 Characteristic Molal Surface Tension Increments of Different Salts Used in H P - H I C

Salt Calcium chloride Magnesium chloride Potassium citrate Sodium sulfate Potassium sulfate Ammonium sulfate Sodium dihydrogen phosphate Potassium tartrate Sodium chloride Potassium perchlorate Ammonium chloride Sodium bromide Sodium nitrate Sodium perchlorate Potassium thiocyanate

Molal surface tension Increment a X lO^dyne • g • c m " ' • mole~ 3.66 3.16 3.12 2.73 2.58 2.16 2.02 1.96 1.64 1.40 1.39 1.32 1.06 0.55 0.45

(56)

I 26

MILTON T. W. HEARN

where AA^^ntact i^ ^^^ difference in surface area the protein exposed to mobile phase in the bound and unbound states, i.e., equivalent to the molecular contact area on binding. In a system of constant P and V, the net free-energy change due to van der Waals interactions (AG^^j^ ^) is assumed to be unaffected by the addition of exogenous salts or solvents and therefore, is expected to follow^ the relationship

Combining the equations for the different changes in free energy associated v^ith the changes in the salt or solvent concentration leads to the foUow^ing expression

+ AA,o,^iO-m -\- vm -{- a

(58)

where k'Q is the retention factor at zero salt or solvent concentration and H is a constant. In the case of electrostatic separations, at sufficiently high ionic strength the -B^^-m^'^H -\- C^^jfn^'^) term approaches a constant value and then the logarithmic retention factor becomes linear with regard to the salt molality, i.e., ln(feyfe'o)=A,,,m33,,

(59)

where A is a parameter that measures the retentive strength of the salt and is similar to the salting-out constant. When electrostatic-ionic interactions can be excluded in the retention process with polypeptides or proteins, the free-energy changes associated with the cavity formation and the Lifshitz-van der Waals interaction are the basis of hydrophobic interaction (HP-HIC) and reversed phase HPLC separations. These differences in free energy between the stationary and mobile phase components of the interaction of a polypeptides or proteins with an immobilized ligand associated with cavity formation AG^!^^^ j^p,/? electrostatic charge AGg^s ^, and van der Waals interactions AG^^^ • correspond to solute specific parameters and are thus related to the slope of the plots of the total free-energy change AG^^s^^^ ^ for a particular biopolymer versus the reciprocal of the logarithm of the concentration of the organic solvent modifier in the case of RP-HPLC separations, or versus the reciprocal of the logarithm of the concentration of the displacing ion in the case of hydrophobic interaction (HP-HIC) and Coulombic (HP-IEX) separations (see Eqs. 6-11). Which of these terms make the greatest overall contribution to retention of the biopolymers depends not only on the permeability, ligand composition, or density of the sorbent, but also on the mobile phase characteristics in terms of water content, pH, ionic strength, buffer composition, and whether additives such as an organic solvent, ion-pairing reagents, dissociating reagents, or surfactants-detergents are present in the eluent.^^'^

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

127

The manner in which the relative retention of several proteins can change as a function of the concentration of the displacing reagent in the HP-HIC mode is illustrated in Fig. 13. When analyzed in terms of the preceding solvophobic framework, increased retention of different polypeptides or proteins in HP-HIC is associated with an increase in salt molality in the mobile phase or change of one salt to another with a greater molal surface tension increment. As evident from the selection of such salt systems and their respective molal surface tension increments detailed in Table 6, a significant range of elutropicities can be generated with these slats selected on the basis of their position in the Hofmeister series of chaotropic and cosmotopic ions. Implicit to these solvophobic considerations are the assumptions that the surface area A^, the cavity curvature K% the net charge coefficients Ajjjp and B^^, and the topographic coefficients D^^ and C^^ are not time-dependent and show monotonous change, with changing eluent pH, salt or solvent concentration, or eluent dielectric properties. However, transitions in conformation or the formation of ion pair species with polypeptides or proteins that occur as the mobile-phase composition is manipulated clearly are manifestations of time domain dependencies. The consequence of this behavior are condition and time dependent changes in A^, the cavity curvature K% the net charge coefficients A^^ and B^^ and the topographic coefficients D^^ and C^^ are which are translated experimentally into

Ammonium Sulphate (M) F I G U R E 13 Plot of the logarithm of the retention volume (In V^) versus the concentration of the displacing salt, ammonium sulphate, in the H P - H I C mode with the proteins I, insulin B-chain; 2, bovine trypsin inhibitor; 3, bovine trypsinogen; 4, insulin A-chain; 5, ribonuclease; 6, sperm whale myoglobin; 7, horse heart cytochrome c. Data from Ref 42.

128

MILTON T. W. HEARN

discontinuities or changes in slope of the plots of In ^^ versus l n ( l / [ d i s placer]) or changes in the band-broadening relationships again as a function of In k'j a f {ln(l/[displacer])}. As noted, the stationary phase itself can additionally affect the magnitude of these thermodynamic and retention terms in a major way (i.e., through the Nernst equation k'- = Q / C ^ ) . Besides exerting a direct influence due to the chemical nature of the support material per se, the chemistry of the ligate surface and the density of introduced functional groups affect retention. In view of this dual dependencies, there are thus many ways to modulate retention behavior of a given mixture of biosolutes if one of the phase conditions is found unsuited to preservation of biorecovery or recovery. Such modulation is often achieved through an analysis of the incremental retention terms that contribute to the overall distribution process, linking AG^SSQ^ ^ to In k'^. A common relationship^'^^^'^^^ by which the relationship between In k'^ and AG^sso^, ^ can be evaluated in terms of all of the incremental retention contributions, thus all of the incremental free-energy contributions, to the overall capacity factor can be expressed as In ^;. = In [ XseciKec^i

+ A:hydr,.^hydr,/ + X.s,iKs,i

+ •*• ]

(60)

or alternatively by In k', = In [ A - s e c ^ ' s e c + A ' h y d r . / ^ O , , ^ " ' ' + ;^es, , ^ . , , ^ " ° ^ ' " ^' + -

]

(61)

where A'sec,/^'sec,/ corresponds to the size exclusion component, A'hydr,/^Mr,/ corresponds to the solvophobic component, A'es,/^'es,/ corresponds to the Coulombic component, etc. of the retention; while the coefficients Afsec,/. A'hydr,/. Xes,i'" ^nd the parametersfe;,,^^,fe'^ydr,/.Ks,n • • •. correspond to the molar fraction and the implicit capacity factors for the respective size exclusion, the solvophobic, the electrostatic, etc. component of the processes. The ^0 i^ ^'z /5 • • • 5 terms correspond to the to the (hypothetical) capacity factors of the biosolute P, in neat water, e.g., at zero organic solvent content in an RP-HPLC system or zero ionic strength in HP-IEX. correspond to biosolSimilarly, the coefficients S, Z, H, M, P, D,,.., ute-specific parameters associated with the solvophobic, electrostatic, hydrophobic interaction, metal ion coordination, polar-hydrophilic interaction, solvational, etc. processes that can, in principle, occur as part of the ligand-ligate interaction. These coefficients are conventionally derived from the slope of the plots of the logarithmic capacity factor. In k'- for a particular polypeptide or protein versus the reciprocal logarithmic concentration of organic solvent modifier in the case of RP-HPLC separations, or In ^^, versus the reciprocal logarithmic concentration of displacing ion in the case of HP-HIC or Coulombic HP-IEX separations, and analogous plots can be generated for the remaining modes of interactive HPLC. Depending on the magnitude of the S, Z, H, M, P, D,.,,, parameters and the corresponding kQ-,k'^i,,.., terms, a variety of retention versus mobile phase eluotropic strength scenarios can be calculated for any biomacromolecule in any HPLC separation mode.

129

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Figure 14 represents examples of four limiting cases of such retention dependencies (cf. Eqs. 6-11). Case a represents a typical scenario for the behavior of polypeptides and globular proteins in the presence of reversed-phase and hydrophobic interaction HPLC sorbents or with most polymerand silica-based anion and cation HPLC sorbents.i'"'i^'25.53,58,62,68,i46,i54,i97,200,20i,2i8-223 A steep dependence of In k] on the change in the volume fraction of the displacing species ^, leading to a minimal value at a specific value of ^, followed by an increase in In k\ as ^ is changed further. With RP-HPLC and HP-IEX methods the volume (or mole) fraction of the displacing species increased from a low value to a higher value. In HP-HIC the reverse occurs with the value of the volume (or mole) fraction of the displacing species decreased to achieve elution. Case b is typified by shallow In k\ versus ^ [or l n ( l / [ C J ) ] dependencies with small values of In ^^ when ^ [or I n ( l / [ C J ) ] ^ 0 . Case b thus represents a

10.0

1 1

i

: ;

1 1'/

*K

<

1

1

1 1 1 1

1 1 1

1 1 1 1 1 1

5.0

V (b)

\

' i' • ; \ ; ^

%i

1 1 1 1 1 1 1

!

0.0

c

= 0-^1.0

F I G U R E 14 Schematic illustrating four limiting cases of the retention dependencies manifested between In k\ (proportional to the change in free energy AG*ssoc) ^ " ^ ^^^ rc\o\Q fraction of the displacing species ^,=o-> i.o» o^®"" ^^^ rc\o\Q fraction range of 0 to 1.0 (cf. Eqs. 6 - I I ) . As the contact area and the retention factor k\ of the polypeptide or protein increase, the slope of the plot of In k\ versus ^/=o-> i.o increases, resulting in a narrowing of the elution window over which the biosolute can be desorbed from the HPLC sorbent. Cases (a) and (b) are typically observed in RP-HPLC, H P - H I C , H P - I E X , and the other interactive modes of HPLC with small peptides and globular proteins, while cases (c) and (d) are more representative of the behavior evident with more hydrophobic polypeptides, membrane proteins or other nonglobular proteins.

I 30

MILTON T. W. HEARN

commonly observed situation with small peptides separated by RP-HPLC or polar polypeptides and some small proteins in HP-IEX, where relatively small free-energy changes arise over the range of elution strength employed/^'^^'14^'^^4-^^^ Shallow dependencies in terms of the In fe^ versus ^ (or l o g l / [ C J ) dependencies can also be observed under some conditions with more retentive biosolutes, but in this case much larger values of In k'^ are the norm. Such a situation is illustrated by case c, which is representative of the behavior of middle molecular weight proteins and very hydrophobic polypeptides under some RP-HPLC conditions; in HP-IMAC with histidine-rich polypeptides or proteins^^^'^^^; in substrate-analogue affinity displacement HP-IEX of proteins^^^"^^^ where the displacing ionic species or substrate-analogue is typically of low molecular weight. Some examples of polypeptide or protein displacement RP-HPLC or HP-HIC^^^'^^^ also fall within the boundary of case c. Because the ^Q, /? ^'z P • • • ? terms are large with case c biosolutes, small changes in a secondary mobile phase component or condition, e.g., pH or salt type, can lead to significant secondary retention effects. When such behavior is evident, the limiting chromatographic conditions are frequently chosen from practical considerations so that the minimum of the plot of In k] versus ^ (or l n [ l / C j ) corresponds to k'^ values equal to unity. Typically, this criterion is easier to achieve in HP-IEX than RP-HPLC or HP-HIC separations, although sufficient empirical information is now at hand to enable sensible choices to be made for these latter HPLC modes. At this stage of development, insufficient data have been accumulated to enable similar a priori estimations of suitable ranges of ^ to achieve a particular In k] value in the other modes of HPLC, such as HP-IMAC, HP-BMC, etc. In situations associated with the purification of large globular multisubunit proteins or hydrophobic membrane proteins, retention behavior typified by cases a-c with smaller globular proteins and polypeptides in the R P - , IEX-, or BAC-HPLC modes are rarely seen. With the more hydrophobic protein classes retention dependencies approaching case d are often observed, resulting in a narrow desorption window, with evidence of significant secondary high-affinity sorption effects and associated poor mass balance and recovery of bioactivity. Illustrative examples of these different types of retention dependencies are shown in Figs. 2 and 15 as plots of the experimentally determined In fe^ (proportional to AGfsso^ ^) versus the volume fraction of organic solvent if/ for the several hormonal polypeptides and lower molecular weight amino acid derivatives in the presence of an «-octylsilica sorbent, which clearly demonstrate this behavior. From the perspective of a generalized analytical or preparative purification strategy, it is obviously desirable to select chromatographic conditions in which the In k'^ at ^ (or ln(l/[C-])) retention dependencies approximate case a or case b, rather than cases c and d. For the latter two cases the i^assoc, i ^^ the biosolute for the stationary phase is obviously too high, the desorption window suitable for elution is too narrow, and the mass (or bioactivity) recovery potentially is at risk. However, with crude feed stocks implementation of the case a or d scenario should not necessarily be excluded out of hand from a selectivity point of view. For example, situations have been

131

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

0.5 0.0 -0.5

-

-1.0

p-Endorphin 0.26

0.28

0.30

+

0.26

Glucagon 0.28

+

0.30

+

0.32

Volume Fraction, y/ F I G U R E 15 Illustrative examples of the retention dependencies for the polypeptide hormones, j8-endorphin and glucagon, as plots of the experimentally determined In k\ (proportional to AG^jj^^ ,) versus the volume fraction of organic solvent i// measured with a n-butylsilica reversed-phase sorbent at temperatures from 5° to 85°C under isocratic elution conditions at a flow rate of I mL/mIn, encompassing the range of acetonitrile concentrations from 0.25 < \p < 0.30. Data from Ref. 237.

identified where the potential offered by the case a and d scenarios can be exploited, such as the removal of undesirable contaminants during the purification of therapeutic proteins by taking advantage of the so-called "negative-adsorption", "roll-over" adsorption and "feedstock" displacement strategies.^^^'^^"^'^^"^"^^^ For example, in this laboratory, useful adaptations of these approaches have been developed for the removal of trace components of Hageman factor and associated plasminogen activator-prekallikreinrelated proteins from therapeutic grade human immunoglobulins based on a tandem dye-affinity and anion-exchange HPLC method, and similar procedures based on these concepts have been employed^^^ in the fractionation of human a^-plasminogen inhibitor. Gradient or step elution procedures represent the two most commonly adopted methods for the elution of biopolymers from adsorptive HPLC sorbents. These choices are often dictated because of the pronounced dependencies of retention and zone broadening phenomena on the chromatographic conditions. In gradient elution procedures, advantage can be taken of the severity of the In ^^ at ^ (or I n ( l / [ C J ) ) retention dependencies. However, in common with elution carried out under isocratic conditions, these procedures do not necessarily address the important requirements mandated by the conformational dynamics or the desorption kinetics of the biosolute. Significant progress has been made over the past 15 y^^^^i,6,17,32,168,191,208,210,237,238 j ^ ^^^ application and interpretation of the gradient elution data for the simulation of the retention behaviour of polypeptides and proteins in R P - and lEX-HPLC systems. Furthermore, it is often feasible in circumstances of so-called "regular" elution behavior, e.g., with polypeptides that satisfy the case b scenario above, or small globular proteins (case a) to apply experimental data derived^^^'^^^'-^^^'^^^ from analytical, small-scale experiments as normalized integrals of the elution volume.

132

MILTON T. W. HEARN

with the column performance characteristic optimized using computer-assisted techniques, algorithmically based on the use of BioCAD, ProSys, instrumentation, etc., to scale-up the chromatographic bed configuration and to make informed choices of the physical characteristics of the separation media, to achieve appropriate process-scale purification levels. For packed-bed systems, the extent of chromatographic zone broadening of a biosolute can be discussed in terms of the column efficiency, v^hich usually is expressed as the number of theoretical plates N^ or the height equivalent of a theoretical plate H^ • (where H^ ^ value = L/N- and L is the column length). The N- value is thus dependent on a variety of solute and chromatographic parameters including the diffusivities D^f ^, I^sp,/5 ^int,/? D^ ^, and D^ • of the solute within the stagnant film layer, the pore environment, the surface interactive regions of the stationary phase, or the bulk mobile phase, respectively; the column length L; particle diameter dp\ and linear flow velocity u- (equivalent to L/t^J)^ respectively. The theoretical plate number N-, of a solute P^ in a packed bed column can thus be defined in terms of the retention time t^ ^; the peak variance cr/^ of the eluted zone in time units; the peak width at baseline response ^^ ^; the capacity factor k\\ and the column dead time ^Q? according to

N.

= 16

16

(1 + k\)

(62)

When the eluted peak zones assume a Gaussian distribution, the peak width at baseline for a particular biosolute ^^ ^ approximately corresponds to 4cr- (4 X standard deviation), although in reality only ~ 95% of the true peak area of a Gaussian peak will be integrated using this approximation. A major task of current practice with HPLC techniques is to select experimental conditions that maximize the N^ value or minimize the H^ ^ value. To achieve this outcome, it is essential that control is maintained over the different mass transport processes that characterize the zone broadening of a particular biosolute in a packed bed of defined column configuration, flow rate, temperature, sorbent type, or eluent composition. The interplay of these different mass transport processes, which include (i) eddy diffusion, (ii) mobile phase mass transfer, (iii) longitudinal molecular diffusion, (iv) stagnant mobile phase mass transfer, and (v) stationary phase mass transfer (see Fig. 16) have posed some of the more significant challenges to the separation scientists over the past two decades. Nevertheless, many of these challenges have been addressed, initially with the development of well-packed columns containing particles of more uniform size and pore diameter distributions, and subsequently with the development of improved procedures for the chemical modification of the surfaces of the particles. However, with biomacromolecules, some but not all of these band-broadening processes can be controlled from a practical standpoint by the quality of the column bed-packing procedures or the use of small particles of narrow particle size and pore size distributions. Compared to low molecular weight organic analytes, most biopolymers have relatively small effective molecular

133

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Inject Band J^ %%%% Width

@0 ©®

Brownian Motion

• %••• •

Final Band Width

^^ ^^ ^^

Mobile Phase Mass Transfer

i^^^fp Diffusion

t^.

Stagnant Mobile Phase Mass Transfer Stationary Phase Mass Transfer

F I G U R E 16 Schematic representation of the origins of zone-broadening behavior and mass transfer effects of a polypeptide or protein due to Brownian motion, eddy diffusion, mobile phase mass transfer, stagnant fluid mass transfer, and stationary-phase interaction transfer as the polypeptide or protein migrated through a column packed with porous particles of an interactive HPLC sorbent.

diffusivities in HPLC systems. As a consequence, the major problems experienced in the separation of biomacromolecules in the adsorption modes of HPLC as low efficiencies, i.e., small N- or large H^ ^ values, can invariably be traced to inadequate control over three mass transfer aspects, namely, the stagnant mobile phase mass transfer, the film diffusion component, and the stationary phase mass transfer kinetics. Since these kinetics are associated with the diffusive, convective, and perfusive components of the mass transport events, improvements in the physical characteristics of the sorbent and better control over the extent of heterogeneity of the ligates on the surface of the sorbent are essential requirements. In the extreme case of sorbents with very large pores (i.e., with 2000 A < p^ < 5000 A) convective flow conditions can in principle be employed with very high linear flow velocities without sacrificing column capacity and biosolute retention. The development of sorbents with interconnecting macro- or even gigaporous structures^"*^"^"*^ (i.e., with p^ up to 10,000 A) as part of the physical architecture of the sorbent has provided one possible solution to these limitations. Such large-pore materials loosely fall into the category of perfusive sorbents. Similarly, the surface modified so-called "tentacular" sorbents have provided^^'^^^'^^"^'^"^^ an another avenue to address the issue of slow adsorption kinetics or reduced capacity due to restricted diffusion in pores of inappropriately small size. Comparison of peak efficiencies between columns of identical bed dimensions but packed with sorbent particles of different physical or chemical

I 34

MILTON T. W. HEARN

characteristics, or alternatively comparison of columns of different dimensions packed with the same sorbent under different procedures, can be achieved by redefining the height equivalent H^ • in terms of the reduced plate height h^j , while the linear flow velocity u-(= L/IQ) can also be expressed in terms of a reduced velocity such that f^e,^-H,^i/dp

and

v.^u-dp/D^

(63)

These contributions from the various mass transport effects have been formalized in terms of the dependency of h^^^ on u^ for a particular biosolute Pj through the well-known van Deemter-Knox relationships, which take the form h^^. = Avy^ + B/Vi + Cv,

(64)

where the A term expresses the eddy diffusion and mobile phase mass transfer effects and is a measure of the packing quality of the chromatographic bed, the B term encompasses the longitudinal molecular diffusion effects, while the C term incorporates mass transfer resistances within the microenvironment of the stationary phase. Thus, if the diffusional processes prevail, the plots oi h ^ • versus v- will reach a minimum value at a unique v^ value. With well-packed columns, operating at optimal flow rates under carefully selected elution conditions, h^^ values approaching two to five times the particle diameter dp can be achieved. Similarly, at high values of the reduced velocity i;^, the diffusional model predicts that h^ • will become linearly dependent on v^ with slope proportional to C, because under these conditions the Av]^^ and B/v^ terms both are small in comparison to Cv^. The major challenge today for very high efficiency separations of peptides, proteins, and other biomacromolecules with available HPLC packing technology thus remains proper control of the C term effects. The challenge here is to decrease the impact of the C term on Z?^ -, either through the use of advanced nonporous sorbents or by using sorbents that exhibit very shallow h^ • versus v^ dependencies, such as the advanced perfusive or high-flux sorbents.^^^'^46-250 In this regard, sorbents capable of exhibiting little change in h over a wide range of v^ values would in principle enable very high superficial velocities to be employed within a practical separation process. The availability of various nonporous silica-based sorbents in the particle size range of 0.7 to 2.5 jxm. has enabled such separations to be achieved routinely at the analytical scale, while macroporous HP-IEX and RP-HPLC sorbents, i.e., HyperD or Poros RPIO, fulfill similar roles at more preparative scales. As expected, nonporous sorbents of small particle diameters exhibit very high efficiencies and very short analysis times but have unfavorable column backpressure characteristics. Porous materials of similar particle diameters, or alternatively the monolithic rod materials,^^^"^^^ are also now becoming available for researchers to investigate, and similar efficiency criteria have been established with these newer classes of interactive HPLC sorbents. At the process level, the demand for very high efficiency is not as compelling. Selectivity, rather than the number of theoretical plates is often perceived as a

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

135

more important issue here, although clearly particles of better size uniformity or narrower pore size distributions will result in packed columns that are usually more robust, with higher resolution factors and greater productivities. The zone broadening of a biosolutes on elution from a chromatographic system and visualized with an appropriate detector arises from two contributions, the intracolumn contribution that has been discussed and an extracolumn contribution that is due to the characteristics of the instrumentation, tubing, and detector cell. For this reason, in a practical context, the theoretical minimum h^^ value anticipated solely on the basis of the chromatographic bed and flow characteristics can rarely be achieve. Rather, system effects due to the interconnecting tubing, the consistency of the flow rate of the buffer delivery pumps, the characteristics of the detector cell and related instrumental design features contribute to the band-broadening according to the relationship: ^total ^

^column + ^system

V^^^)

where o-^oiumn ^^^ ^system ^^^ ^^^ peak variances arising from zone broadening induced by column effects and by the extracolumn system effects, respectively. With careful attention, the extracolumn system effects can be kept small, i.e., by the appropriate design of the flow through detector cell, with tubing suitable for the chosen separation task, optimal type of injector, and the maintenance of the system at constant temperature, free of flow irregularities or pressure changes, etc. When these precautions are achieved, the impact of o-^ystem o^ ^he overall h^j value can be minimized. Such considerations are particularly important when high-sensitivity analytical microbore HPLC systems are employed, where it is essential that o-^ystcm ^^ ^column? otherwise the opportunity to achieve high-speed, sensitive microbore HPLC analyses will become a futile exercise. Even with preparative HPLC systems, attention to extracolumn system effects is required, since peak overlap may be the consequence due to poor control over fluid mixing in the effluent stream or during elution development. Yll. THE EFFECT OF TEMPERATURE AND THE THERMODYNAMICS OF POLYPEPTIDE- OR PROTEIN-LIGATE INTERACTIONS For the HPLC separation of low molecular weight organic compounds and various biomacromolecules, the "near-equihbrium" criterion has generally been assumed for the binding and desorption behaviour. Changes in thermodynamic parameters due to polypeptide- or protein-ligate interaction can thus be depicted in terms of the Gibbs-Helmholtz relationship, namely, A G ° _ , , = AHl„,,, - TAS«_,,

(66)

Under such binding conditions, the properties of the polypeptide or protein, the surrounding bulk and structured solvent, and the interactive ligate surface have also been usually assumed to be invariant with regard to

I 36

MILTON T. W. HEARN

the temperature T. Hence, the contributions from the corresponding changes in enthalpy AH^^^^^ ^ or entropy AS^^^^^^ to the overall Gibbs free-energy change AG^gg^^, • associated with the solute-sorbent interaction, as well as the phase ratio of the system ^ will also be independent of temperature. When such conditions prevail, then the dependency oiln k'j on 1 / T takes the form of the well-known linear van't Hoff plot. Although this linear van't Hoff plot behavior has been observed experimentally with some small peptides,^'^^'^^'^^^'^^^"^^^ in an increasing number of investigations with polypeptides and proteins studied under such conditions significant divergences from this ideal behavior have been observed.^'^^'^^'^^'^^'^^^'^^^'^^^"^^^ As noted earlier, the interaction of a polypeptide P^ with a chemically defined ligate(s) on a chromatographic sorbent can be described in terms of the logarithm of the capacity factor In fe^, which can be related from fundamental thermodynamic considerations to the temperature T through the expression: In k', = - A H , ° _ , , / R T + A S « _ , , / R + In <&

(67)

where R is the gas constant, AHj^^^^ • and AS^ggo^ ^ are the changes in enthalpy and entropy associated with the interaction, and In $ is the logarithm of the phase ratio of the system, i.e., the logarithm of the ratio of the stationary phase volume V^ to the mobile phase volume V^ within a column for a defined chromatographic sorbent and mobile phase composition. For a polypeptide- or protein-ligate interaction where the phase ratio and the properties of the biosolute and the sorbent surface are invariant with temperature, then the values of AH^^^^^^ and AS^gs^^, ^ associated with the interaction of Pj with the ligates can be derived in the traditional manner by linear regression analysis of the In k] versus 1 / T van't Hoff plots from the slope and intercept values, respectively. In the case of a polypeptide or protein interaction with nonpolar ligates, e.g., in RP-HPLC or HP-HIC, based on the use of w-alkylsilicas or nonpolar polymers, the extent of fit of the experimental data to the linearized form of the van't Hoff dependency can be employed to gain insight into whether linear (Langmuirean) adsorption-desorption conditions prevail or whether additional factors are involved in the polypeptide (or protein) interaction with the ligates, e.g., whether stabilization-destabilization of the secondary or tertiary structure of P^ occurs under the perturbing conditions of the interaction. Comparable measurements can also be achieved with the other interactive modes of HPLC. Detailed analysis of the characteristics of the In k] versus 1 / T dependency as a function of T thus has the potential to provide insight into the mechanism of the interaction of PI with the ligate surface of the sorbent and whether participation of secondary equilibrium processes occur that are dependent on temperature. In the case of RP-HPLC and HP-HIC of polypeptides and proteins, three situations can be contemplated^^'^^'"^^^'^^^ for the interaction of a polypeptide or protein with an hydrocarbonaceous ligate, whereby the change in heat capacity AC^ , of the system (i) is zero and remains invariant with regard to temperature (the isothermic scenario), (ii) is not zero and is linearly

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

137

dependent on temperature (the homothermic scenario), or (iii) is not zero and shows a nonhnear dependency on temperature (the heterothermic scenario). In the bulk state, the heat capacity C^ • of a substance is usually defined as the quantity of heat necessary to raise the temperature of a unit mass of the substance by 1 K. Related scenarios can be derived for the HP-IEX, HP-IMAC, HP-BAC, or HP-BMC modes with polypeptides and proteins, leading to the application of analogous expressions for all ligate-ligand system, including the interaction of a polypeptide with immobilized «-alkyl chains (the example described in the following), provided the characteristics of state of a unit cell of the solvated polypeptide-ligate complex can be defined. Under these conditions, when a unit mass of the ligand-ligate-solvent system initially in a state defined by P, V, and T undergoes a small change defined by SP, S V, and ST, a change in a particular parameter or quantity U associated with the ligate-ligand interaction can be represented by the equations of state as a function of P, V and T such that dU AU =

dU AP +

dP

dU AV + -—AT dV dT

(68) ^ ^

where P is the pressure (in pascals), V is the molar volume, and T is the absolute temperature in degrees Kelvin. Accordingly, the change in the internal energy A £ of the system can be described in terms of the change in entropy AS^S^Q^, ^, by the perfect differential dE = TdS - PdV

(69)

where ^£\

ldE\

ldT\

ldF\

Similarly, the change in enthalpy of association A H^^ssoc, / ^^ ^^e system can be defined in terms of the internal energy £ such that

H = E + PV

(71)

dH = TdS + VdP

(72)

ml - \

(73)

where

In the case of RP-HPLC or HP-HIC, the change in heat capacity AC^ , for a polypeptide- or protein-nonpolar ligate system as a function of T can

I 38

MILTON T. W. HEARN

be expressed according to the Kirchhoff relationships^^^'^^^ as ACl,

= TSSl,/dT

(74)

^Sl^., = f—^dT

(75)

AC,«,. = ^ H l , , , / ^ T

(76)

^H^lo.^,==f^Cl,dT ^Cl^dT

= dE + PdV-

(77) ii-dii,

(78)

where o>A £ represents the incremental difference in the internal energy of the system. According to Eq. (78), AC^ ^ is also related to the change in the standard enthalpy and the standard pressure-volume product (o>PV) terms, while the term /x • takes into account contributions from processes not defined by the criteria of the Stefan-Boltzmann-Maxwell law. If the interaction of ?• with the chromatographic ligates satisfies these criteria, i.e., no effects other than standard changes in P, V, and T are involved, then Eq. (78) becomes A q . ^ T = dE + PdV

(79)

and hence ^C'dT=dHl,,^^

- d{?V)

+ FdY

(80)

This relation enables the change in heat capacity AC^ • for the polypeptide or protein interaction with the ligates to be evaluated from the change in enthalpy AH^^^^c n assuming that the change in pressure for the system is known, i.e., ^cl,^^=^Hl,,^,-v^F

(81)

Evaluations of the interaction of P, with the hydrocarbonaceous ligates in terms of Eqs. (74)-(81), however, do not represent complete descriptions of the binding process, because the condition(s) under which the thermal energy of the system is increased has not been specified. If the heat capacity of the system is increased under experimental conditions where the pressure P does not change significantly, then Eq. (80) can be simplified and the specific heat capacity can be redefined as follows: C;,,
(82)

C°,, = (
(83)

In RP-HPLC, as well as the other HPLC modes, when the temperature of the system is changed, the viscosity 7] of the mobile phase will also change. This variation in viscosity will result in change in pressure (AP) according to

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

139

the relationship UovL ^P = T1T

(84)

where UQ is the Hnear flow velocity of the eluent, L is the length of the column, €p is the specific permeability of the sorbent, and dp is the average particle diameter. Over the T range usually employed in most laboratories, i.e., 5°-65°C, the AP variations for well-packed columns will typically not exceed ^ 15% of the original pressure drop, i.e., from '^ 240 bar at 10°C to 210 bar at 50°C for the water-based eluents for an analytical RP-HPLC column, with modern pressure-damped instrumentation and standard HPLC columns of dimensions 150 X 4.6 mm i.d. containing spherical «-alkylsilica sorbents of average particle diameter of 6 /xm. Although constant pressure conditions can be achieved by adjusting the linear flow velocity of the eluent by a corresponding percentage, in practice this approach experimentally requires special flow constriction or stream-splitting equipment, which is usually unavailable to most investigators. A further factor must also be considered when the linear flow velocity is adjusted to compensate for pressure changes. Because of the dependency of the column void time fg on WQ, i.e., tQ = (L X F)/UQ, where L is the column length and F is the flow rate, changes in UQ will, most importantly, affect directly the k] value of a polypeptide or protein P- eluted under isocratic or gradient conditions. Isocratic conditions at constant flow rate are thus the preferred experimental option^^'^^ for the measurement of thermodynamic parameters associated with the interaction of polypeptides or protein with the nonpolar ligates in RP-HPLC systems, or the corresponding ligates in the other HPLC modes. Even when extremely high pressure changes exceeding 2 kbar are generated from an initial value of « 4 kbar with specialized systems, e.g., microbore columns with inner diameter of 30 ^im packed with 1.5 fim nonporous particles,^^^ variations in the k] values of moderately retained solutes are still relatively small (i.e., Ak'^ < 2)}^^ Such large pressure variations are outside the operational range employed in most laboratories. Moreover, at pressures of in the range of ~ 220-250 bar, the partial molar volumes V of many polypeptides and proteins are expected to vary by less than 10% under these conditions. Based on these considerations, the effect of the product terms, namely, VdF and FdY (e.g., Eqs. 79-83) on the thermodynamic parameters ^^a^ssoc,/5 ^'^rssoc,/? o^ ^^p,i ^^'^ ^^ Small or tend to zero in most analytical HPLC systems. Application of Eqs. (74)-(83) thus provides a useful and appropriate approximation of the ligate interaction with polypeptides and proteins in interactive HPLC, enabling evaluation of the relationship between k\'^ T, and the thermodynamic parameters AH^^^^^,^ •, AS^sso^,/? ^^^ ^ ^ P , / ^^^ a defined polypeptide or protein, flow rate, and mobile phase composition. As discussed earher, the equilibrium association constant K^^^^^i, for the interaction of P^ with a ligate is a function of temperature, with the dependency taking the form K.ssoo,i = e-^''--'^''''

(85)

I 40

MILTON T. W. HEARN

where the value of the Gibbs free-energy change for the interaction ^G^^^^^^is a function of temperature only. By substituting and rearranging the relevant terms of Eq. (85), the foUow^ing thermodynamic dependencies can be derived: ^ In K3,3„,,, = - 1 / R X [(T X ^ G , V , ) / T 2 ]

(86)

d In K , _ , , . / ^ T = A H l „ , , , / R T ^

(87)

The equilibrium association constant K^^^^^^^ for the binding of the polypeptide or protein to the ligate is related to the capacity factor k] through the relationship (cf. Eq. 4): lnX,33„,,, = lnfe;. + l n $

(88)

and hence d In k]/dT=

AH'

,/RT'

(89)

In situations v^here AHj^g^^ • and AS^ss^^, ^ are both invariant with temperature, i.e., under the conditions of the isothermic binding scenario, where over a range of temperatures linear van't Hoff plots are observed, the following familiar expression is obtained: lnfe;.= - A H l < „ , , / R T + J

(90)

with the term / incorporating contributions from both the entropic and the phase ratio terms in the form I = \S^^^^^j/R + In $ . If the phase ratio is assumed (or found) to be independent of temperature, then the plots of In k'l versus 1 / T will generate a straight-line characteristic of linear van't Hoff plots. Thermodynamic data for a large number of low molecular weight solutes have been measured by these chromatographic procedures, interpreted in terms of this linear van't Hoff relationship (Eq. 67) and the integrity of the derived data firmly established^^^"^^^ from independent measurements, such as partition coefficient with octanol-water systems or aqueous solubility parameter determinations. For the assessment of the extent of change of the phase ratio $ of a HPLC column system with temperature or another experimental condition, several different experimental approaches can be employed. Classical volumetric or gravimetric methods have proved to be unsuitable for the measurement of the values of the stationary phase volume V^ or mobile phase volume V^, and thus the phase ratio ( $ = V,/V^). The tracer pulse method^^^'^^^ with isotopically labeled solutes as probes represents a convenient experimental procedure to determine V^ and VQ, where VQ is the thermodynamic dead volume of the column packed with a defined chromatographic sorbent. The value of V^ can be the calculated in the usual manner from the expression V^ = VQ — V^. In addition, the true value of V^ can be independently measured using an analyte that is not adsorbed to the sorbent and resides exclusively in the mobile phase. As a further independent measure, the extent of change of O with T can be assessed with weakly interacting neutral or

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

I4I

nonpolar solutes that show hnear dependencies of In k'^ versus 1 / T over a large T range at a defined mobile phase composition.^^"^'^^^ Experimental data derived from several studies have demonstrated that ^ changes at a specified solvent composition are usually small, i.e., A = ± 10% over the T range of 60 K for the w-alkylsilica reversed-phase systems, suggesting that the contribution from the In $ term to the k'j changes will be small and essentially constant. On the other hand, if the chromatographic conditions are selected such that the corresponding K^^^^^^ • value remains constant for a specified polypeptide or protein P-, to achieve a significant change in the value of k\^ such as the 20-fold or more variations observed with many polypeptides over a 60 K range in temperatures in RP-HPLC systems, then according to Eq. (88), a change in $ of 20-fold or more would also have to occur. For such 4> changes to arise, a simple expediency can be invoked, whereby the surface area-to-volume ratio of the stationary phase support material is changed by a corresponding amount. This effect can only be achieved^^^ by utilizing nonporous spherical particles with a surface coating of the same polarity, ligate composition, and the same nominal mean dp but with surface areas in the range of 0.02-1 m^/g instead of the commonly used, highly porous particles with surface areas of 5-350 m^/g or larger. When AH^^go^ • and AS^s^^^ ^ are dependent on temperature, plots of In k\ versus 1 / T do not follow linear dependencies. According to Kirchhoff's law, when temperature-dependent heat capacity conditions prevail, i.e., when ACp ^ ^ 0, as observed for example with the heterothermic binding scenarios,^^'^^'^^'^^'^^^"^^^ respectively, then the dependency of In ^^ on T can be approximated by a polynomial expression as represented by In k\ = a + b(l/T) + c(l/Tf

+ d(l/Tf

+ . . . +ln $

(91)

and thus d In k]/dT = [^ In k\/d{l/T)]

X[^(1/T)/^T]

= - ( 1 / T ) ^ X [b - h 2 c ( l / T ) -h 3d(l/Tf

(92) + •••] (93)

In these homothermic and the heterothermic scenarios, the change in enthalpy can be thus represented by AHl„,,,= -RX lb + 2c(l/T) + 3d(l/Ty

+ ...]

(94)

while the change in heat capacity can be represented as AC;,,. = ^ A H l , , , / ^ T = m / ( T ) ^ + n/(Tf

(95) + •••

(96)

where m = 1 Re, n ^ 6 Rd, etc. When nonlinear plots of In k'^ versus 1 / T are observed, under some circumstances AH^^goc,^ "^ ^ and In k'^ may thus reach a maximal value at a temperature corresponding to T^^ since the slope of the plots of In k'^ versus

I 42

MILTON T. W. HEARN

1 / T at any temperature for a polypeptide or protein interacting with a HPLC iigate will proportional to the AH^^^g^^ • value at that temperature?^^ In these circumstances, when In k] reaches a maximal value as T is varied and ACp • is constant for the process, the dependency of the AH^^^^^- and AS^gso^, • contributions on temperature can be expressed in terms of AC^ , as A H l , , , , = AH*3,„,,, + AC»,,(T - T*)

(97)

AS«soc,i = AS*,„,,, + A C ; , , l n ( T / T ; )

(98)

and

where the symbols T^ and T^ refer to the isoenthalpic and isoentropic temperatures at which the plots of AH^^s^^ ^ versus T or iiS^^^^^^^ versus T, respectively, for the polypeptides (or proteins) intercept and attain a common value of AH^*3o^ • or AS*^^^^ ^ respectively, under the different experimental conditions. When such ligand-ligate processes prevail, then the dependency of In k\ on T can also be approximated by the logarithmic expression^'^^:

ln^;.= - ^ ( ^ - l n ^ - l ] + l n c I >

(99)

From Eqn. (91)-(99), values of AH,^^^^,,, AS,^,,^,„ or AC^^^ corresponding to homothermic or heterothermic interaction processes for any polypeptide or protein can thus be evaluated from the nonlinear van't Hoff plots using nonlinear least-squares regression and associated curve-fitting procedures. The corresponding values of AG^^SSQ^ ^, the S term. In ^Q, AA^^^^^^^^ ^assoc r? ^^^' ^^^ ^^ similarly derived from the same data set using the appropriate Eqs. (4), (9), (13), (35), etc. Illustrative of this approach are the plots of In k'l versus the volume fraction i/f of the organic solvent modifier for two polypeptides [namely, the all L-a-polypeptide, 1, NH2-DDALYDDKNWDRAPQRCYYQ-COOH and its (all D-a)-retro-inverso isomer, 2, NH2-QYYCRQPARDWNKDDYLADD-COOH] determined byRP-HPLC methods over the organic solvent volume fraction range of ijj = 0,17 to if/ = 0.21 with water-acetonitrile mixtures and from if/ = 0.35 to i/^= 0.40 with water-methanol mixtures, respectively, and from temperatures of 278 to 338 K in 5 K increments shown in Figs. 17a-17d, respectively. As evident from these data, the change in In k'^ values was > 2.5 over this temperature range for most of the mobile phase conditions. For these polypeptides, the trend expected for the hydrophobic effect dominating the interaction mechanism was evident, with In k'- decreasing as the i[/ value was increased. The corresponding three-dimensional grid plots for the S values (derived according to Eq. 9) of these two polypeptides as the temperature and i// values were systematically varied are shown in Figs. 18a-18d. In each case, the S values for 1 and 2 were derived by regression analysis methods from the gradient of the experimental plots of In k] versus if/ at the specified if/ and T values with the regression coefficients > 0.9985. The S value of a polypeptide

143

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

3.0 2.0 1.0.

3.0 2.0 :i^ 1.0

-So.0 -1.0 -2.0

^

^

-1.0^ -2.0 -3.0 3.0 2.0 1.0. 0.0^ -1.0'"* -2.0 -3.0

3.0 2.0 i^ 1.0 ^ 0.0 -1.0 -2.0 0.17 0.18 0.19 0.20 0.21 0.35 0.36 0.37 0.38 0.39 0.40

I/A-Value F I G U R E 17 Representative plots of In k[ versus the volume fraction if/ of organic solvent for two polypeptides (namely the all L-a-polypeptide, I, N H 2 - D D A L Y D D K N W D R A P Q R C Y Y Q - C O O H (A) and its all D-a-retro-inverso isomer, 2, N H 2 - Q Y Y C R Q P A R D W N K D D Y L A D D - C O O H (B) determined by R P - HPLC methods over the organic solvent volume fraction range oi ifj = 0.\7 to i// = 0.21 with water-acetonitrile mixtures and from if/ = 0.35 to if/= 0.40 with water - methanol mixtures (C) and (D), respectively, and from temperatures of 278 to 338 K in 5 K increments. Data adapted from Ref. 62.

or protein in the presence of a reversed phase sorbent can be related^'^^'^^'^^'^"^^'^^"^ to extrathermodynamic parameters, such as the accessible molecular surface area ^A^^^ through the expression S = a'AA^^^ + b'K' + c'

(100)

where A A^^j is the hydrophobic contact area (proportional to the accessible molecular surface area, A Aj^^i), /c^ is a solvophobic cavity factor related to the ratio in energy required for the formation of a cavity w^ith surface area equal to t^A^^^ and the energy required to extend the planar surface of the liquid by the same area; and a\ b', and c' are constants related to the molecular properties of the polypeptide. Several general features relevant to the interaction of polypeptides and proteins v^ith HPLC ligates are evident from Figs. 17 and 18. First, the overall similarity of the In k] versus i/^ data sets for 1 and 2 w^ith acetonitrile or alternatively with methanol (cf. Fig. 17a with Fig. 17b; or Fig. 17c with Fig. 17d) and their derived three-dimensional plots (cf. Fig. 18a with Fig. 18b; or Fig. 18c with Fig. 18d) of S versus T versus i/^ confirms that these two 20 mer polypeptides exhibit similar interactive behavior, and thus they can assume similar conformational properties, when in the same ligate environment. However, note from the comparison of the acetonitrile versus the methanol solvent systems shown in Figs. 17a-17d, and particularly from comparison of the data shown in Figs. 18a-18d, that depending on the

144

MILTON T. W. HEARN

•P-va/ue S versus T versus cp for peptide / with ACN

S versus T versus cp for peptide 1 with MeOH

f'^alue S versus T versus cp for peptide 2 with ACN

S versus T versus 9 for peptide 2 with MeOH

F I G U R E 18 Three-dimensional grid plots for the S values (derived according to Eq. 9) of the polypeptide isomers, the all L-a-polypeptide. I, N H 2 - D D A L Y D D K N W D R A P Q R C Y Y Q - C O O H (A) and (C), and its all D-a-retro-inverso isomer, 2, N H j - Q Y Y C R Q P A R D W N K D D Y L A D D - C O O H (B) and (D) as the temperature and ip values were systematically varied using acetonitrile or methanol as the organic solvent modifier. Data from Ref. 62.

choice of solvent composition significant changes in the shapes of these plots arise at a specific temperature for these polypeptides. As evident from the data shown in Figs. 17a, 17b, 18a, and 18b, with the water-acetonitrile mixtures (as well as with the water-methanol mixtures shown in Figs. 17c, 17d, 18c, and 18d) small but nevertheless significant differences are also evident between the interactive behavior of 1 and 2 under some conditions of temperature and solvent mole fraction. Analogous differences have been observed^^^ in the local conformations of specific amino acid residues in the NOESY two-dimensional ^H NMR spectra of the polypeptides 1 and 2 acquired under different solvent conditions at 500 and 600 MHz in 40% deutero-trifluoroethanol (J3-TFE)/H20 at pH 2.3, or in 300 m M sodium dodecylsulphate in 100% D2O or 90% H 2 O / 1 0 % D2O at pH 2.6, although overall the conformations of both polypeptides were very similar. The predominant conformation observed for 1 and 2 under these solution conditions incorporates a central a-helical region which extends over the same series of amino acid residues in each polypeptide, i.e., Y^DDKNWDRA^^ in polypeptide 1 and A^RDWNKDDY^^ in polypeptide 2. Application of such thermodynamic approaches in combination with spectroscopic measurements provided in this case a definitive demonstration that the conformations of

145

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

retroinversopolypeptides^''^'^'^'^ and their all L-a-amino acid isomers can assume similar topology in the presence of aquo-organic solvent mixtures and lipophilic sohd-liquid interfaces. Shown in Figs. 19a-19d are the corresponding plots of In k'^ versus 1 / T for the L-a- and retro-all D-a-polypeptides, 1 and 2, determined at different il/ values with water-acetonitrile and water-methanol mixtures containing 0.09% TFA. The trends in the In k] versus 1 / T data, involving nonlinear (parabolic) dependencies of the van't Hoff plots, have been observed in investigations^^'^^^ the interactive behavior of the hormonal polypeptides j8-endorphin, glucagon, and bovine insulin at different temperatures with immobilized fz-butyl and w-octadecyl groups. The characteristic shapes for the plots of In k] versus 1 / T for polypeptides and proteins can thus be interpreted in terms of the extended mean value theory that forms the basis of Kirchhoff's law. As a consequence, the values of the coefficients a,b,c,.,. of the polynomial dependency of Infe• versus 1 / T can be readily derived by regression analysis utilising, for example, the Hephaestus software^^^ based on Eqs. (91)-(100). Concomitantly, it is also possible to calculate the corresponding AH^^^^^^j, ^^Lod^ ^^^ ^^p,i values from these nonlinear plots of In k] versus 1 / T for different solvent compositions. In the case of some polypeptides and proteins in RP-HPLC and HP-HIC systems, the In k'^ values increase with decreasing T, but do not reach maximum values over the examined range of T values. Thus, with larger polypeptides and globular 4.0 3.0

3.0

2.0

2.0

1.0^

-^ 1.0

0.0

" ^ 0.0 -1.0

-2.0 -3.0

-2.0 _J

3.0

c

-1.0'"^

•

I

I

I

I

\

L_

\j/=o.n

3.0 2.0 1.0 0.0 1.0

2.0 ; ^ 1.0

r S 0.0 -1.0

-2.0

-2.0

-3.0

3.0 3.1 3.2 3.3 3.4 3.5 3.6

3.0 3.1 3.2 3.3 3.4 3.5 3.6

1/T X 1000 [K"^] F I G U R E 19 Plots of Ink,' versus 1 / T for the L-a-polypeptide, I, N H j - DDALYD D K N W D R A P Q R C Y Y Q - C O O H (a) and (c), and Its all D-a-retro-inverso Isomer, 2, N H j Q Y Y C R Q P A R D W N K D D Y L A D D - C O O H (b) and (d) determined at different ip values with w a t e r acetonitrile and water-methanol mixtures containing 0.09% TFA in the presence of an n-octylsilica sorbent. Data from Ref. 62.

146

MILTON T. W. HEARN

proteins, such as insulin, cytochrome c variants or the transcription factors fos and ]un (Fig. 20) more complex RP-HPLC dependencies have been observed^^'"^^'^^'^^^'^^^"^^^ with corresponding plots of In k!^ versus 1/T, as v^ell as the derived plots of AHj^^^^^ • versus T and AS^^g^^^ ^ versus T, reaching maximum values at different \\f (or in a more generalised case for all HPLC modes, ^ ) values of the mobile phase modifier-displacer. According to Eqs. (91)-(100), in cases of polypeptide or protein interactions w^ith ligates when AC^ • values are not constant, and when AC^ • # 0, this behavior is consistent with the participation of heterothermic processes. Figure 21 (for an acetonitrile-water system) and Fig. 22 (for a methanol-water system) illustrate this behavior, again using polypeptides 1 and 2 as the exemplars. In this case, essentially linear dependencies were observed between AH^^^g^^ •, AS^^ss^^ •, or AC^ ^ and T for the interaction of these 20 mer polypeptides with sorbent. It is interesting to note that although the AH^^53o^ • and AS^^^SQ^ • values were negative, the slopes of these AH^^ss^^ • versus T and AS^ssoc,/ versus T plots were dependent on the volume fraction \\f of the solvent composition, resulting in these plots intersecting at convergent values near to T = 320-330 K. Similarly, the slopes of the AC^ • versus T plots for both the acetonitrile and the methanol systems were also essentially linear. Acetonitrile is known to be a poor hydrogen bond acceptor as a solvent resulting in the generation of solvent clusters with water-acetonitrile mixtures, while in contrast, methanol is a good hydrogen bond acceptor.^^^'^^^'^'^^'^^^ These solvent differences give rise to significant changes in the hydrogen bonding characteristics and solvation state of polypeptides (or proteins) and their ligates, as demonstrated in the preceding case. As also evident from the data shown in Figs. 21 and 22, these differences in solvation properties were reflected as different slopes in the

3.0 3.1 3.2 3.3 3.4 3.5 3.6

3.0 3.1 3.2 3.3 3.4 3.5 3.6

i/r[K"Viooo

i/r[K"^]xiooo

F I G U R E 2 0 Plots of logk,' versus Ml for the transcription factors ^o% and \ux\ measured at different \\f values with water-acetonitrile mixtures containing 0.09% TFA in the presence of an n-octylsilica sorbent. Data from Ref. 257.

147

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Peptide 1

Peptide 2

-70

^-. '^

-70 -80 -90 -100

-80 -90

-100

o

-110

S

-120 -IJO

O

o

§ ^

-140 -150 -160

I

•

I

•

'

•

I

•

I

'

I

•

I

I

•

I

•

I

'

I

•

I

•

I

•

I

•

I

•

I"

-20

,^ O

e J^ 1—5

O

^'

i4 "o

S

•—s

' ^ o

S

*—i

i

<

-20

-25

-25 S^

-30

-30

o

S ^ ""—^ ^ ^' >—s

--?S

-3S

-40

-40

-45

-45

-50

-50

200 JT^

-110 -120 -130 -140 -150 -160

'^

200

100

100

0

0

-100

-100

-700

-700

-300

-300

-400

-400

-500

-500 -600

-600

,^^ ^ "o

a >—»

280 290 300 310 320 330 340 280 290 300 310 320 330 340

Temperature, T^K)

Temperature, T^K)

and AC° versus T for the all L-a-polypeptide, I, F I G U R E 21 Plots of ^H^,, "''assoc N H , - DDALYDDKNWDRAPQRCYYQ- C O O H , and its all D-a-retro-inversoisomer, 2, N H , Q Y Y C R Q P A R D W N K D D Y L A D D - C O O H , determined In the presence of an n-octylsilica sorbent with acetonltrile water mixtures at different if/ values. According to Eqs. (91)-(100), in cases of polypeptide or protein interactions with llgates when AC° , values are not constant, and when AC° , ¥= 0, this behavior is consistent with the participation of heterothermic processes. Data taken from Ref. 62.

plots of AH^^soc,/? ^'^rssoc,/? ^^ ^^p,i versus T. Because of the molecular organisation of acetonitrile, the properties of the derived binary water-acetonitrile mixtures will, moreover, be less constraining at higher T values than for the hydrogen-bonding methanol-based mixtures. This behavior v^ill be reflected in terms of both the magnitudes of the AH^^^^^^-^ ^^assoc,i'> ^^ ^^p,i values per se, as well as the magnitude of the changes in ^^a^soc,/5 ^'^rssoc,/? o^* ^^p,i ^^ ^^^ temperature T is systematically varied. The consequences of the type of thermodynamic behavior manifested by the polypeptides 1 and 2, and other polypeptides and proteins in their interactions with the different classes of ligates found in HPLC sorbents are

148

MILTON T. W. HEARN

Peptide 1

Peptide 2 150

-190

160 170 180 190 200 210

-200 -210

S

-220 -230 -240

Co

220 230 240 750

-250 -260

s

2

J

"4

-55

45

-60

50

-65

55

S

60

3

-70

-65 -75

-70

-80

-75 I

•

I

I

I

I

I

I

I

400 300

}4 'o S

S 1—5

260

-270

o

>d

200 100 0 -100 -200 -300 '^i

I

I

I

I

I

I

400 300 200 100 0 -100 -200 -300 -400 -500 -600

o

S U ^

280 290 300 310 320 330 340 280 290 300 310 320 330 340

Temperature [^K]

Temperature [""K]

F I G U R E 2 2 Plots of AH^ssoa^ AS°s5o^ ,, and ACp , versus T for the all L-a-polypeptlde, I, N H j - D D A L Y D D K N W D R A P Q R C Y Y Q - C O O H , and its all D-a-retro-inverso isomer, 2, N H j Q Y Y C R Q P A R D W N K D D Y L A D D - C O O H , determined in the presence of an n-octylsilica sorbent with methanol - water mixtures at different ij/ values. According to Eqs. ( 9 ! ) - ( 1 0 0 ) , in cases of polypeptide or protein interactions with ligates when AC° ; values are not constant, and when ACp j ¥= 0, this behavior is consistent with the participation of heterothermic processes. Data taken from Ref. 62.

clearly fundamental, since similar manifestations underpin the so-called enthalpy-entropy compensation effect observed for protein folding in bulk solution/^'^^^'^""^"^^^ Enthalpy-entropy compensation represents a further type of extrathermodynamic relationship whereby a linear dependence of ^^Loc,i o^ ^'^assoc,/ is predicted following a change in an experimental variable. When molecular associations between a polypeptide or protein and a ligate systems involve enthalpy-entropy compensation, large enthalpy changes will be associated with stronger binding and a more restricted conformation for the polypeptide in the bound state, i.e., a higher order will be induced with a concomitantly greater changes in entropy. Shown in Figs. 23a-23d are the plots of /iH^^^^^i versus AS^^^^^^ , for polypeptides 1 and 2

149

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

AH^,,^^. (kJmor .) -50 -45 -40 -35 -30 -2

^

10 -75

-70

-65

-60

-55

-90 -100

1-110 w -120 -240

1-130 ^""-140 -150

Peptide 1

Peptide 1

-260

-70 j ^

-80

i^

-90

160

^

-180

B -110

-200

^-120 1-130

^ -50 -45 -40 -35 -30 -25 -20

-75 -70 -65 -60 -55 -50 -45

AH ^ (kJmol"') assoc ^

'

F I G U R E 23 Plots of AH^^SQ^ , versus AS^^^^^ , for polypeptides I and 2, the all L-a-polypeptide, 1, N H j - D D A L Y D D K N W D R A P Q R C Y Y Q - C G O H (A) and (C), and its ail D-a-retro-inverso isomer, 2, N H 2 - Q Y Y C R Q P A R D W N K D D Y L A D D - C O O H (B) and (D), determined for the different solvent systems ranging from i// = 0.17 to i/^ = 0.21 with the water-acetonitrile mixtures and from \\f — 0.35 to «// = 0.40 with the water - methanol mixtures respectively. Data taken from Ref. 62.

for the different solvent systems ranging from (p = 0.17 to (p = 0.21 with the water-acetonitrile mixtures and from ^ = 0.35 to 9 = 0.40 with the water-methanol mixtures, respectively. Such linear relationships revealed from these AHj^g^^ ^ versus AS^^g^^ ^ plots confirm that the thermodynamic behavior satisfies the criteria of entropy-enthalpy compensation. Moreover, the slopes of these plots represent the compensation temperatures T^omp? ^ characteristic parameter of the system. When the T^omp values for eluents of different solvent compositions are very similar, the interactions of the polypeptide or protein with the ligates at different T values can thus be considered to be isoequiUbrium processes driven, in the case of RP-HPLC and HP-HIC separations, by the hydrophobic effect. In HP-IEX, the conformational status of polypeptides and proteins will also be substantially influenced by temperature over the usual operational range of ^ 70 from 278 K. The progress of temperature-induced perturbations of the polypeptide or protein secondary and tertiary structure during the ion-exchange chromatographic separation can thus be monitored from the variations in the magnitude of the Z^ (or Z^) and In K-^^ values under different temperature conditions (see Section IV. C). The effect of T on the chemical equilibrium of various ions and counterions in ion-exchange processes has been the subject of extensive investigation for more than 40 years.^^"^'^^^ Similarly, temperature effects can be used to achieve the resolution of low molecular weight polar compounds with ion-exchange sorbents. Thus, L-proline, L-valine, L-methionine and a mixture of D- and L-leucine can be resolved with two synthetic cation exchangers^^^ at temperatures of 60°

I 50

MILTON T. W. HEARN

and 80°C, while these same amino acids were not resolved at room temperature. Elevated column temperatures have long been widely used to achieve improved resolution and higher sensitivity detection as part of amino acid analysis with microparticulate HP-IEX sorbents.^^^''^^^ In contrast to the preceding representative examples of studies with low molecular weight substances and ions, the role of temperature on the HP-IEX behavior of higher molecular weight biosolutes, and in particular polypeptides and proteins, has not been as extensively investigated. In part, this more limited literature on HP-IEX of proteins at different temperatures can be attributed to the fact that the equilibrium processes for the ion-exchange separation of polypeptides and proteins are more complex than the separation of small molecules, due to the participation of multiple ionization processes that these higher molecular weight amphoteric substances can undergo. In addition, the general concern held by many protein chemists and biologists that elevated temperatures will automatically lead to polypeptide and protein denaturation and precipitation has also been a significant factor. The lower viscosity of the mobile phase and the enhancement of the desorption kinetics at higher temperatures, however, offer the opportunity for improved peak shape and better resolution of closely related polypeptides and proteins components, particularly in the analytical mode.^^^ The use of elevated temperatures for the enhancement of resolution of polypeptides and proteins in HP-IEX has, however, also led to conflicting observations in terms of retention behavior. Thus, the resolution of ovalbumin isoforms from albumin with the strong anion exchange Synchropak SAX sorbent increased^^^ when the temperature was raised from 4° to 25°C. In contrast, a decrease in resolution between /3-lactoglobin and carbonic anhydrase was observed^^^ with an increase in temperature with the Partisil-10 SAX adsorbent. Similarly dichotomous results have been observed with various other polypeptides and proteins.^'^^'^^^'^^^ Therefore, the effect of temperature on the ion-exchange chromatographic resolution of polypeptides and proteins cannot yet be readily anticipated. However, the available evidence from other areas of chromatographic optimization suggests that variation in column temperature represents a useful approach to adjust the chromatographic resolution of polypeptides and proteins and other high molecular weight biomacromolecules. In addition, variations in the column temperature and the chromatographic residence time provides a straightforward avenue to investigate the role of the hierarchal structure of polypeptides and proteins in HP-IEX, particularly since the changes in the secondary and tertiary structures of polypeptides and proteins, which can be induced within the temperature range of 5°-85°C, occur over a temperature increment whereby chromatographic separations can also be readily performed. As a consequence, investigation of the ion-exchange chromatographic behavior of polypeptides and proteins over a wide range of temperatures provides important information on both the role of conformation in HPIEX systems and also allow resolution optimisation per se. Shown in Figs. 24a and 24b are the plots^^ for the Z^ and In K^^^ values versus T for several globular proteins and other lower molecular weight polypeptides interacting with a strong cation exchange HPLC sorbent.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

151

10 20 30 40 50 60 70

Temperature (""C) F I G U R E 2 4 Plots of the (a) Z^ and (b) In K^^^ values versus 7 for several globular proteins and other lower molecular weight polypeptides interacting with a strong cation exchange HPLC sorbent, LiChrospher 1000 S03~, over the temperature range from 278 to 348 K using NaCI as the displacing salt. The polypeptide and protein code is - T - , arginine; - 0 - , angiotensin I; - V - , angiotensin II; - • - , angiotensin III; - D - , cytochrome c; - A - , hen egg white ovalbumin; • , insulin; - A - , lysozyme; - O - , ribonuclease; - • - , soyabean trypsin inhibitor. Data from Ref. 96.

Lichrospher 1000 SO^, demonstrating the types of induced changes in retention which arise from such temperature effects. With the larger polypeptides and proteins, there is a larger number of positively charged residues which are surface accessible and which can interact with the electrostatic ligates, leading to larger values of the Z^ and In K-^^^ parameters. The maximum values of the Z^ and In K^^^ parameters are limited by the accessible area of contact, the distribution of the positive charges within this binding region(s) presented by the surface of the polypeptide or protem 50,77,80,163 and the affinity of the interaction. As the conformation and shape of the polypeptide or protein change with increasing T, the trend lines for the Z^ and In K^^^ parameters will not necessarily parallel each other. Rather these trend lines reflect the progressive molecular transitions whereby amino acid side chain clusters on the surface are destabilized and internalized amino acid residues become exposed as the temperature is elevated. This rearrangement of the surface structure of polypeptides or proteins at the liquid-solid interface of the electrostatic sorbent may induce or reinforce hydrophobic interactions. The addition of an organic solvent to the mobile phase can be used to verify if any additional hydrophobic interactions are involved under these conditions.

I 52

MILTON T. W. HEARN

As apparent from the preceding data as well as other experimental findings, there is no simple additivity relationship between the Z^ value and the pi value of a polypeptide or protein. Similarly, there is no simple dependency existed between the Z^ values for a polypeptide or protein and the corresponding molecular weight. Collectively, these and other results, e.g., the interaction of rat cytochrome b^ with the Mono-Q anion exchanger ^^^'^^^ or bovine serum albumin with Sepaharose Fast Flow Q,^^^ lead to the conclusions that retention models, such as the net charge hypothesis,^^^'^^^ based solely on considerations of the pi value, the net charge, the average surface area and molecular volume, or the molecular weight of polypeptides and globular proteins are poor predictors of the retention behavior of these biosolutes in HP-IEX. The surface of a polypeptide or protein molecule is characterized by asymmetric distribution of charged amino acid residues, and this property results in regions of varying electrostatic potential. As the temperature increases and the conformation of the polypeptide or protein is disrupted, the electrostatic contact regions will become more extended, which in turn will influence the magnitude of the Z^ and In K-^^^ value. Overall, these results reinforce the concept that the contact area established between a polypeptide or protein and the ion-exchange ligates is not directly related to the overall charge of the polypeptide or protein nor to its molecular size per se, but rather to the charge anisotropy and its relationship to the distribution of hydrophobic regions present on the polypeptide or protein surface. The plurality of this interplay of electrostatic and hydrophobic interactions in polypeptide or protein chromatography with ion-exchange sorbents thus forms the basis of various fundamental investigations derived from extensions of the Manning counterion condensation theory^^'^^^'^^^ and thermodynamic considerations.^^'^4'^^'^i^'^^^'^^^'^^^ The preceding approach, in principle, also enables the effect of the valency and the activity coefficients of different displacer salts to be considered in the evaluation of the Z^ or the corresponding z values for any HP-IEX system. For example, if the retention results for a series of polypeptides a n d / o r proteins chromatographed with the same HP-IEX sorbent, column, flow rate, elution conditions, buffer composition, pH, and at the same temperature, but with NaCl or CaCl2 as the displacing salt are compared, then the relative differences between these two salt systems can be evaluated in terms of a free energy selectivity parameter r* such that ^* = In a = In (fe;,, Naci/^'iex,caci,) T* = [AG^caci,) - AG(Vci)l/2.3RT = ^^Gl,.,,^,/23RT

(101) (102)

where AG^Q^Q^ ^ and AG^^aci) ^^^ ^he Gibbs free energy for the polypeptideor protein-ligate interaction in the presence of CaCl2 and NaCl, respectively, AAG(^j^^ jgx) is the difference in Gibbs free energy for the ion-exchange interaction at the same standard state for these two displacing salt systems, R is the gas constant, and T (K in degrees Kelvin) is the temperature. By utilizing Eqs. (25)-(29), (101), and (102), the following dependency for the salt selectivity effect can be obtained for the NaCl (represented below

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

153

as system 1) and the CaCl2 (represented below as system 2) elution conditions (and similar expressions would apply for other monovalent or multivalent salt elution conditions), assuming that the HP-IEX phase ratio remains constant and the same number of charge groups are accessible on the surface of the polypeptide or protein are involved in the interaction with the Coulombic ligates, namely,

(103)

If the same HP-IEX sorbent and column are employed, then the initial ligate concentration D^, will be identical for both sets of displacing salt experimental conditions, i.e., NaCl and the CaCl2 in this illustrative example, and Eq. (103) can be simplified to

= ln

(104)

As noted, from Eqs. (101) and (102), the r* values for a series of polypeptides or proteins, when investigated under identical chromatographic conditions, represent the incremental differences in Gibbs free energies manifested by these two salt systems. Hence, from Eqs. (101)-(104), as the temperature is varied over the same range, linear dependencies of the r* values for a series of different polypeptides or proteins on the reciprocal of T, or the ratio of the Z^ ^ and Z^ 2 values for a particular polypeptide or protein will only be observed under special conditions, i.e., provided the individual a^^ and bjj terms are independent of the temperature and the characteristics (solvated ionic radius, charge density, etc.) of the ions of the displacing salt do not induce conformational changes. On the other hand, if the Uii or 6-y terms associated with the polypeptide- or protein-ligate interactions are dependent either on the ionic characteristics or the temperature relationships of the displacing salt, or are affected by changes in the conformational or aggregation state of the polypeptides or proteins as the temperature is varied, then both salt- and temperature-specific variations in the Z^ i / Z ^ 2 i*atio can be predicted to occur for a series of polypeptides or proteins. In this context the Manning ion condensation theory ^^^'^^^ takes on an important significance. Thus, limited evidence for positive correlation between the retention behavior and the z values of various polypeptides and proteins have been found when di- and trivalent ions have been employed as ionic components of the displacing salt.^^'^^'^"^'^^^"^^^ However, in these cases it is frequently assumed that the mechanism of interaction between the polypeptide or protein solute and the charged ligates were not influenced by the nature of the displacer salt. When such criteria apply, then for different proteins the ratios of the slopes of the^ In fe-g^ versus l n ( l / [ Q ] ) , or the gradient derived In ^J^^ versus I n ( l / [ C J ) , plots determined at the same temperature with a monovalent salt versus a divalent salt is predicted to be

154

MILTON T. W. HEARN

4 / 3 . When secondary chemical equilibrium prevail, such as "salt in" or "salt out" effects involving changes in the self-association or aggregation state of the polypeptide or protein, or alternatively w^hen additional hydrophobic interaction effects are manifested, or finally if conformational transitions are induced due to the choice of a v^ater structuring or w^ater-destabilizing salt species from the Hofmeister series,^^"^^ then divergences from this "ideal" behavior v^ill be evident. Illustrative of this behavior are the data shown in Figs. 25a and 25b and Table 7 for the Z^ and In K-^^^ values for several polypeptides and proteins eluted from same "tentacle-type" LiChrospher 1000 SO^ HP-IEX cation exchange sorbent using NaCl and CaCl2 as the displacing salt at different temperature conditions. These comparative results show^ that the Z^ values of each biosolute was greater when NaCl was used as the displacer salt than for CaCl2. Also, some of the proteins were not eluted at high T values when NaCl was used as the displacer salt, but were eluted when CaCl2 was employed. For example, with the NaCl system, soybean trypsin inhibitor (STI) was not eluted at 65° and 75°C, while cytochrome c, insulin, and ovalbumin were not eluted at 75°C. However, with CaCl2 as the displacing salt, these latter proteins were eluted at both 65° and 75°C. Furthermore, variations in the Z^ values and In K-^^^ values of this selected group of

4.0 3.0

N^

2.0 1.0 H

1

\

\

1

h

20

30

40

50

60

70

20 H

^ 15

^

10

Temperature ("^C) F I G U R E 2 5 Plots of the Z^ and In Kj^^ values versus T for several globular proteins and other lower molecular weight polypeptides interacting with a strong cation exchange HPLC sorbent, LIchrospher 1000 S03~, over the temperature range from 278 to 348 K using CaClj as the displacing salt. The polypeptide and protein code is - 0 - , arginine; - • - , angiotensin I; - V - , angiotensin II; - T - , angiotensin III; - D - , cytochrome c; - A - , hen egg white ovalbumin; - • - , insulin; - A - , lysozyme; - O - , ribonuclease; - • - , soyabean trypsin inhibitor. Data from Ref. 96.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

155

I H l T A B L E 7 Representative Z^ and In Kj^^ Values for Several Polypeptides and Proteins Analyzed with the Strong Anion Exchange H P - 1 E X Sorbent, Mono-Q, Fractogel-TMAE, and LiChrospher 1000 T M A E MonoQ

Fractogel-TMAE

Protein

^c

In Kjex

^c

i" '^iex

Hen egg white ovalbumin Bovine insulin Bovine erythrocyte carbonic anhydrase Porcine recombinant grow^th hormone Sperm w^hale muscle myoglobun Bovine pancreatic ribonuclease Hen egg white lysozyme

9.5 3.9 5.6 3.9 4.8 0.8 3.3

48.9 20.9 28.4 20.2 24.2 3.5 10.2

7.2 2.8 2.4 4.0 1.9 1.6

37.7 14.8 7.0 20.6 8.5 7.1

—

—

LIChrosph er-TMAE In Kje^ 1.6 1.6

— 0.7

7.2 (y.S

— 2.8

—

—

0.4 1.9

1.7 10.1

polypeptides and proteins as a function of temperature were much smaller with the CaCl2 than with the NaCl elution system. The nature of the anion-cation as well as the ionic strength of the displacing salt thus influences the retention behavior and hence the magnitude of the Z^ values of polypeptides and proteins in HP-IEX. The preceding results, for example, show that at most values of T, the Z ^ ^ / Z ^ 2 ^^atios for this NaCl and CaCl2 exemplar are significantly different from 1.33 for all the biosolutes, irrespective of their molecular size, while the and In Xjg^ i / l n i*C-gx 2 ratio was not unity. In addition, the magnitude of the Z^ i / Z ^ 2 ^^^io varied differently as T was increased (with no linear inverse dependency of r* on T). These results, typical of the interaction of many polypeptides and proteins with different HP-IEX sorbents, strongly indicate that the assumptions implicit to the stoichiometric binding model, requiring that the value of z or Z^ are independent of a^ and h^ (and that the product ^i] ^ ^ij ~^ 1) over the range of displacing salt concentrations and temperatures employed, are not physically or thermodynamically consistent. In this context, other investigations^"^'^^ on the thermodynamic basis of the adsorption of polypeptides and proteins to HP-IEX sorbents and related the Hill plot investigations^^^ have indicated that the interaction of large, charged biomacromolecules with various HP-IEX sorbents essentially follows rectilinear isothermal behavior involving a multilayer dissolution mechanism,^^ which represents an extension of the Manning ion condensation theory.^^^'^^^ The thermodynamically favorable interaction^"*'^^^ of a polypeptide or protein with the highly charged ligates leads to partial neutralization of the regional electrostatic charges on the polymer surface, and results in the formation of more closely packed protein-protein or protein-ligate complexes at the ligate-solvent interface. This feature generates with some types of HP-IEX systems, e.g., HyperD sorbents,^"*^'^^^ higher fluxes and more rapid mass transport phenomena. In essence, the interaction of two different types of "flexible" polyelectrolyte structures with complementary types of charge characteristics, results in a reduction in the effective charge density per unit area of the sorbent in the microenvironment of the interaction, reinforced by free-energy changes associated with a decrease of solvation and

I 56

MILTON T. W. HEARN

possibly an entropically driven motion that transiently leads to the entrapment of the polypeptide or proteins. These observations on the effect of temperature on the retention behavior of polypeptides and proteins in the RP-HPLC and HP-IEX modes have parallels in the other interactive HPLC modes, e.g., HP-HIC. In addition, the derivation of these data has important implications for the interpretation of the molecular processes associated with surface-induced conformational changes of polypeptides and proteins in the presence of the perturbing environment of different types of ligates and mobile phase compositions. The application of these approach thus provides both a theoretical as v^ell as an experimental framew^ork to rigorously explore the retention behavior of polypeptides and proteins in different ligate systems in terms of the thermodynamic and extrathermodynamic characteristics associated v^ith the interaction, including the influence of their molecular properties and global conformational effects. As such, temperature studies carried out under nearequihbrium conditions provide half the essentially information on the characteristics of the interaction. The other half of this physicochemical equation, v^hich directly addresses the molecular pathways of the interaction, comes from an examination of the kinetics of the interaction, gained from evaluating the binding processes within the framework of time. Yin. FACTORS THAT CONTROL PERFORMANCE AND EFFICIENCY As is evident from the preceding discussion, the retention behavior of a polypeptide or protein ?• expressed in terms of the capacity factor k] is governed by thermodynamic considerations. Peak dispersion, on the other hand, arises from time-dependent kinetic phenomena, which are most conveniently expressed in terms of the reduced plate height h^^. When no secondary effects, i.e., when no temperature effects, conformational changes, slow chemical equilibrium, pH effects, etc. occur as part of the chromatographic distribution process, then the resolution R^ ^ that can be achieved between adjacent components separated under these equilibrium or nearequilibrium conditions can be expressed as

-''(T^)

""''

or R.„-i(Nr(„-l)(^)

(106)

Equations (105) and (106) provide an important linkage between the three essential parameters that dictate the overall quality of the chromatographic resolution, namely, the relative retention, expressed in terms of the capacity factorfe^the relative selectivity a, and the extent of peak dispersion N- or h^ •, Higher system performances and thus larger values of R^ ^ per unit time

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

I 57

arise when larger peak efficiencies are achieved in analytical separations. When such criteria are met, this translates in the analytical mode to lower cost per analysis. At a process level, performance in HPLC separations can also be evaluated in terms of the throughput per system cost, i.e., the cost of isolating a kilogram of product at a predefined purity level. To these ends, and as is evident from Eqs. (105) and (106), resolution in a HPLC separation can be improved through increases in the N- (and thus decreases in the h^i) values, by adjusting the k] values or by increasing the a values. A large variety of experimental procedures enable the fe^ and a values of an HPLC separation to be readily manipulated, i.e., through the selection of different types of stationary phases through to a systematic screening of mobile phase conditions and the influence of temperature changes. As noted, such adjustments can frequently be achieved using computer-assisted optimization^'^'^'^^'^^'^^'^^^'^^"^ or expert system-knowledge-based method development^^^~^^^ such as the recently established^^^ Q-system. Resolution is thus very responsive to variations in the chemical characteristics of the stationary and mobile phases which affect fe'^ and a. To affect comparable changes in resolution through variations in N^ (or h^^) are in contrast more demanding and often (technically) impractical to address. Thus, if the k'^ and the a values of a pair of polypeptides in a HPLC separation are held constant, to achieve a three-fold increase in resolution (a typical objective during optimization), a ninefold increase in the number of theoretical plates must be achieved. Such increases in N^ require the use of sorbents of smaller particles with better mass transfer kinetics; eluents of lower viscosity by operating at higher temperatures or containing solvents with smaller TJ values; and more highly optimized packing materials and fluidic characteristics. High-resolution separations, i.e., separations where R^ > 1.5 for all components, require the application of carefully thought through strategies, some of which can be based on the use of optimization algorithms such as the DryLab, Pek-n-ese, Q-PeakPlus^ Hephaestus^ etc. software. Since resolution and product purity are seriously compromised through inadequate attention to the extent of peak broadening of adjacent zones in analytical as well as preparative separations, it makes good sense to initiate the optimization of the relevant chromatographic parameters as early as possible whenever this can be achieved. Three strategies have been favored to enhance resolution, e.g., either (i) by varying k'^ over an operational range so that the biomacromolecules elute as discrete zones between -^ 1 < ^^ < 20; (ii) by increasing a, usually through adjustment of the mobile phase or stationary phase chemical composition; or (iii) by increasing N- and thus decreasing h^-. For a particular analytical separation, each biosolute will have an optimal k] value for maximum resolution with a designated column, flow rate, and mobile phase composition. Similar criteria apply in preparative (overload) chromatography with multicomponent mixtures, where resolution is similarly enhanced following optimization of chromatographic selectivity and zone bandwidth. The conventional approach to process purification with low molecular weight solutes has frequently been based on linear scale-up of the performance of an analytical column system. In these cases, high-resolution separations can be achieved often without the burden of conformational or

I 58

MILTON T. W. HEARN

Other secondary equilibrium effects, particularly when high-speed separations (tg of the order of seconds) are carried out. Optimization of process scale HPLC separations of low molecular weight compounds, i.e., with M^ < 500 can be considered a relatively mature area of the separation sciences. With polypeptides, proteins, and other biomacromolecules, similar endeavors are still somewhat in their infancy. However, important progress has been made in the application of gradient elution methods that enable gradient retention data obtained for a particular polypeptide mixture to be applied to the selection of isocratic elution conditions with progressive increases in loading scale and vice versa.^^'"^^'^"^^'^^^"^^^ Furthermore, it is feasible in circumstances of regular retention behavior and high mass recovery to apply data derived from small-scale or analytical systems with, for example, polypeptides as normalized integrals of the elution volume, column performance, etc., to affect scale-up of a chromatographic bed configuration or the choice of the preferred physical characteristics of the sorbent 1^8,149,167,173,307-309 Such approaches underpin the current popularity of RP-HPLC procedures for the purification of synthetic or recombinant polypeptides at the production scale, or analogous approaches employed in the HP-IEX of commercially valuable proteins. However, in some cases when linear scale-up methods are applied to higher molecular weight polypeptides or proteins, their biological activity may be lost due to unfavorable column residency effects and sorbent surface area dependencies. It is thus mandatory that the design and selection of preparative separation system specifically address the issues of recovery of bioactivity. Often some key parameters can be easily controlled, i.e., by operating the preparative separation at lower temperatures such a 4°C, or by minimizing column residency times. A conventional approach applied with for the purification of low molecular weight solutes has been based on the scale-up of analytical column systems that allow very high resolution through optimization of chromatographic selectivity and zone bandwidth. A weakness of this approach with polypeptides and proteins is that sorbents of inappropriate particle size or surface characteristics may initially be employed to acquire the analytical data. Inherent to all purification stratagems with polypeptides or proteins is thus the question of to what end use the purified product will be required. If the task involves purification solely for the purpose of subsequent primary structure determination (i.e., essentially an analytical task carried out at a semipreparative scale to generate ' ^ 1 - 1 0 nmol of the desired polypeptide or protein), then the requirements of adequate control over bioactivity are not necessarily relevant. Obviously, in the case of a new or partially characterized polypeptide or protein, recovery of the component of interest with high mass and bioactivity balance is essential. In preparative approaches where subsequent biological uses are contemplated, it is similarly mandatory that the design of the separation system specifically takes into account these recovery issues. With proper attention to the physicochemical consequences of the dynamic behavior of the target polypeptides and proteins in the bulk solution and at solid-liquid interfaces, this criterion of high recovery of bioactivity can often be satisfied without sacrificing the obvious demands of selectivity.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

I 69

When conformational requirements impinge on a purification strategy, availability of other analytical data gained, for example, from nonchromatographic measurements, such as evaluation of the spectroscopic properties or assessment of the biological-immunological activity of the polypeptide or protein in response to changes in separation variables, fulfill a very useful role. Table 2 lists a variety of factors that influence the mass and bioactivity recovery, and thus the overall resolution, of proteins and other biomacromolecules in HPLC systems. The influence of the binding site heterogeneity of the polypeptide or protein vv^ith the chromatographic sorbent on the overall distribution process; the participation of enthalpic and entropic effects associated v^ith the thermodynamics of the polypeptide or protein interaction w^ith the surfaces of the sorbent or permeation through the internal pores; or the kinetic impact of system residency effects, all represent important parameters which must be controlled in preparative HPLC separations. If these parameters are adequately controlled as part of the optimization process, then quantitative structure-retention relationships can be elucidated and insight into the overall mechanism of the retention process developed. To be implemented, mechanistic approaches based on stochastic models require the acquisition of extensive databases, howrever, before adequate response function. Simplex algorithm or factor design procedures can be carried out. The ultimate success of a HPLC strategy at the analytical-scale as w^ell as processscale applications hinges very much on the ability of the investigator to intuitively integrate these response functions into a robust analytical form of a chromatographic optimization scheme w^ith data obtained on the structure-function and structure-retention behavior of polypeptides and proteins in different solution and interfacial environments. The acquisition of such data and its enunciation within the algorithmic framework of current concepts about a N-dimensional separation space^^ pose an interesting set of challengers for the next generation of scientists, but these challenges will be solved, and the resultant new separation strategies will be greatly benefit from these refinements. Fortunately, the effects of most mobile-phase characteristics such as the nature and concentration of organic solvent or ionic additives; the temperature, the pH, or the bioactivity; and the relative retentiveness of a particular polypeptide or protein can be ascertained very readily from very small-scale batch "test tube" pilot experiments. Similarly, the influence of some sorbent variables, such as the effect of ligand composition, particle sizes, or pore diameter distribution can be ascertained from small-scale batch experiments. However, it is clear that the isothermal binding behavior of many polypeptides or proteins in static batch systems can vary significantly from what is observed in dynamic systems as usually practiced in a packed or expanded bed in column chromatographic systems. This behavior is not only related to issues of different accessibility of the polypeptides or proteins to the stationary phase surface area and hence different loading capacities, but also involves the complex relationships between diffusion kinetics and adsorption kinetics in the overall mass transport phenomenon. Thus, the more subtle effects associated with the influence of feedstock loading concentration on the

I 60

MILTON T. W. HEARN

separation performance with polypeptides or proteins, the competitive binding influences of other protein components at the solid-liquid interface or the influence of ligand density, surface heterogeneity, or effective surface area of the sorbent are much harder to assess from batch determinations. From the view point of the assessment, the quality of an HPLC separation in response to changes in different system variables, such as the stationary phase particle diameter, the column configuration, the flow rate, or mobile phase composition, or alternatively, changes in a solute variable such as the molecular size, net charge, charge anisotropy, or hydrophobic cluster distribution of a protein, can be based on evaluation of the system peak capacity (PC) in the analytical modes of HPLC separations and the system productivity (Pgff) parameters in terms of bioactive mass recovered throughput per unit time at a specified purity level and operational cost structure. The system peak capacity PC depends on the relative selectivity and the bandwidth, and can be defined as PC = (^,„ - ?o)/4cr,„

(107)

where t^ ^ is the retention time for the biosolute, tQ is the column dead time, and 4or^^ is the standard deviation of the peak zone (in time units) for a chromatographic system with an average resolution of R^ = 1. Optimization of peak capacity must of necessity take into account knowledge of kinetic behavior associated with mass transport as well as conformational and other secondary chemical equilibrium processes mediated by the stationary-phase surface or alternatively by components in the mobile phase. Participation of conformational effects due to unfolding or refolding pathways of polypeptides and proteins represents a unique set of challenges, which from both theoretical and experimental aspects are not experienced with low molecular weight, conformationally more constrained compounds. Analysis or scaled-up purification of simple organic substances by HPLC procedures is thus much more straightforward. Although empirical interpretations are still applied at present, for reasons of practical expediency, to account for the conformational effects of polypeptides and proteins in preparative high-resolution HPLC or electrophoretic systems, the trend is already evident for more systematic approaches to be based on the analysis of the retention and kinetic behavior. These more advanced approaches utilize computer-aided analysis of the retention and kinetic data in terms of different mechanistic interaction models that account for the behavior of polypeptides and proteins in the bulk mobile phase or at the solvated liquid-solid interface of different HPLC sorbents. Preliminary work has been described^'^'^^'^^'i^^'^^^'^^^'^^^'^i^'^^^'^i^ on classification of retention and kinetic data in terms of different mechanistic pathways for a number of polypeptides, enzymes or globular proteins interacting in the RP-HPLC, HP-HIC, and HP-IEX modes. The ability of modern HPLC techniques to yield quantitative data on rate constants for polypeptide or protein folding and unfolding transitions as well as to detect conformational intermediates with relaxation half-times of similar (or larger) magnitude to the mass transport time (i.e., r^^^f - T'masstransfer? where Vasstransfer > 10 sec) also has important ramifications in the selection

161

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

of conditions in a purification procedure. For example, if a polypeptide or protein were to undergo a two-stage interconversion in the mobile phase and at the surface of the sorbent then a retention cycle corresponding to a distribution process involving the two chromatographic phases and the protein in its native form, P^ and two unfolded states P^ ^ and Pjj ^ will occur via the corresponding transition states Pu,w ^^d P^^ and can be represented as shown in Fig. 26. Two relevant recent examples where such behavior related to the conformational interconversion of proteins has been documented^^^ are the cases of hen egg white lysozyme (HEWL) and sperm whale myoglobin (SWMYO) in RP-FiPLC systems where intermediates similar to the molten globule states^^^"^^^ of these proteins have been observed. Different retention mechanistic pathways can thus be envisaged, based on composition and complexity of such distribution cycles. Table 8 lists some of these conformational interconversion possibilities. The retention mechanism of a particular polypeptide or protein undergoing conformational changes will thus be conditional on the respective rate constants for the various bulk distribution processes involving the bulk mobile phase and the stationary phase as well as the interconversion pathways. Thus, if the fraction of time the protein P^ spends in one form is represented by t, then the probability that this fraction is in the range t + 8t is given by P^^^^, where / = 1, 2, 3 , . . . , representing each of the intermediate forms on the unfolding trajectory. The simplest case of such conformational unfolding is represented by the six-component cycle with the rate determining step for the pathway determined by with the conformational unfolding P^,^ ~^ ^u,m- ^^ ^^^^ C2LSQ^ the interconversion cycle is involves only the conformational species.

Mobile Phase UM

••^^ip-^^f.' ;

•-'.-'Msfrq

Stationary Phase F I G U R E 2 6 Schematic representation of the conformational interconversion of a polypeptide or protein, Pf^,^ in free solution and in two unfolded states, Pyj^ and P^ 5, which occur in the presence of a liquid-solid interface via the corresponding transition states PJ y^ and P j j . Also shown are the corresponding rate constants k,^ for these interconversions.

I 62 [ l ^ l

MILTON T. W. HEARN T A B L E 8 Classification of S o m e of t h e Different Interconversion Pathway Possibilities T h a t Can Influence t h e Retention Behavior of Polypeptides o r Proteins in A d s o r p t i o n H P L C JJni-uni pathway: Typified by simple distribution process and kinetics, narrow bandwidth with high mass and biological recovery; may be characterized by very rapid interconversion kinetics as the solute transverses the chromatographic bed as a single, time-averaged structure. Isouni-uni pathway: Typified by solvent-induced solute unfolding-refolding phenomena in the mobile phase with all conformational species binding to the sorbent with the same distribution coefficient; elution is characterized as a single zone with high mass recovery but time-dependent loss of biological activity in the mobile phase. Uni-isouni pathway: Typified by ligand-induced solute unfolding-folding phenomena at the stationary phase surface; single elution zone observed but time-dependent loss of mass and bioactivity possible when the rate constants for interconversion, ^23 ^ ^32Isouni-isouni pathway: Typified by solvent- and sorbent-induced solute unfolding-refolding phenomena in both phases; may be characterized by impaired mass recovery of two species; i.e., the native and the nonnative states, with time-dependent loss of bioactivity w h e n ^14, ^23 ^^ ^415 ^32-

Isobi-uni pathway: Typified by solvent-induced, time-dependent biphasic unfoldingrefolding of solute in the mobile phase but stabilization of the tertiary structure by the ligate surface if k^2 ^ ^14? characterized by high mass recovery, elution of a single zone with apparent half-life for loss of bioactivity larger than in the mobile phase alone. Uni-isobi pathway: Typified by ligand-induced, time-dependent biphasic unfoldingrefolding of the solute at the stationary phase surface with further destabilization of structure; characterized by time-dependent loss of mass and activity. Isobi-uni pathway: Typified by mobile-phase-induced and ligate-induced biphasic unfolding-refolding of the solute; characterized by the time-dependent loss of mass and bioactivity with the emergence of a second, often later eluting inactive zone of inactive solute if ki2, ^43 ^^ ^21? ^34-

This classification is based on the concept of the interconversion of different states of a polypeptide or protein, each able to independently bind to the sorbent or undergo conformational changes in the bulk mobile phase or at the surface of the sorbent, i.e., an P„ > P* > P, interconversion. More complex branched interconversions can be similarly described using the notation (/so^Obr etc. The classification of the pathways when the solute in its various conformational forms, binds to the same class of ligand can be noted as an "ordered" pathway. When the binding of the solute to a heterogeneous stationary phase surface occurs, then the notation of a "random" pathway is used. In this manner, an N-dimensional retention network can be identified for different conformational-secondary equilibrium processes that proteins and other biopolymers undergo at adsorptive interfaces. See also Figs. 26-29.

PN,m, PN,S^ Pv,m^ ^u,.' Pu,m, ^^d Pg „ whcre Pj^,„, Pjj,^ and P^ ,„ are the native, unfolded, and partially unfolded species in the mobile phase, and PN,S^ PU,S^ ^^'^ Pu,s ^""^ '^he native, unfolded, and partially unfolded species in the stationary phase, then the probability that P starts as P^ ^ can be given hY"°''''

Pl{t) dt

r.r^il - t)

e x p [ - r i ( l -t)

- r^t\l[Ar^r^t{\

-t)\''t (108)

163

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

where r, = (0^12^23 + ^14^21)** A l l

(109)

ri = {^k,^k,,

(110)

+ k,,k,,)t*/k,,

?* = overall separation time exp2[rir2?(l A7r'/'[r,r,t{l

t)Y^^

- t)]

3/4

(111)

(112)

When the concentration profiles of F^^^^ and Fu.^ centered around ^ = 0 and ^ = 1 follow a Gaussian distributions, and if a single diffusion coefficients can be employed to describe the molecular dispersion of each conformational species in the chromatographic process, both P(f=o) ^^^ P(j^i) can be treated as discrete functions and the probability distributions of the interconverting species, separated by retention time differences of A^i^^ and Afj^ 2? ^^^ be readily computed. The calculated concentration profile for HEWL undergoing such a dynamic interconversion during the RP-HPLC interaction is compared in Fig. 17 to the experimental findings. Equivalent expressions can be derived for multistep conformational perturbations with non-Gaussian concentration profiles associated with several intermediate states or alternatively with heterogeneous interactions at the sorbent surface. When such relatively slow secondary conformational interconversions (i.e., rapid partial loss of the tertiary structure but slow unfolding of the secondary structure), the "near-equifibrium" assumptions do not apply. As this scenario is typified by the generation of molten globule states of a protein, the preceding stochastic probability approach may not be able to accurately accommodate all of these transitions, simply because incomplete data on the respective diffusion coefficients, relative rates of interconversion, or lack of information on the contribution of secondary sorbent effects often exist. In this situation, the individual simultaneous partial differential equations for each of the processes associated with the overall mass conservation must be derived, solved, and used to obtain expressions for the first and second moments of the elution profile and the concomitant plate height arising from the slow kinetics of these types of secondary conformational equilibrium. When the process F^ -^ Fjj —> P^ in the mobile phase or stationary phase can be represented by first-order or pseudo-first-order interconversion kinetics and as a reversible binding event, the resolution of the interconverting species can be evaluated^^^ by treating the column as a "chemical reactor" with properties specified by the corresponding Damkohler number D^ and the corresponding interconversion rate constants derived. Thus,

^^=

4D.(., + .,)

("^)

164

MILTON T. W. HEARN

0.150

0.125

g 0.100

o CO

< >

0.075

0.050

0.025

48 50 52 54 56 Relative Peak Retention Times (mins) F I G U R E 2 7 Typical elutlon profiles for HEWL chromatographed on a Bakerbond Cig reversed-phase sorbent, nominally of 5 fjom particle size and 30 nm average pore diameter, packed into a column of dimensions of 250 X 4.6 mm i.d., using a linear gradient of 0.1 % trifluoroacetic acid in water (buffer A) and 0.09% TFA in 65% aqueous acetonitrile (buffer B) with a gradient time of 90 min and column temperatures within the range 15° - 55°C, at 75° and at 85°C, respectively. For clarity, the elution profiles for the 75° and 85°C experiments have been offset by ~ 2 min with regard to the 5° - 65°C experiments. The dashed arrows refer t o the progressive conversion of peak I t o peak 2, and peak 2 to peak 3. The concentration profile for a two-state interconversion of HEWL, calculated from Eqs. (108)-(144) according to the stochastic model for dynamic interconversion during the interaction of a globular protein with a reversed-phase sorbent, is shown in the insert panel.

and Da =

M*^12^23 + ^43^34)(fel4 + ^4l)

(114)

^14^21^0

where is the phase ratio iV^/V^); fe,^ are the respective rate constants for adsorption, desorption, unfolding, or refolding; a-, is the peak width of component i; L is the column length; and MQ is the linear velocity. The plate height increment due to slow kinetics associated with such an interconversion H.„ can be determined from

H.

lA "D7

,-D„

1 +

- 1 D.

(115)

where

[L(fe;,,-fe:,,)VKj A =

(116)

165

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

and k'l^^ and fe^ 2 ^^^ ^^^ respective capacity factors for the two interconverting species. The Damkohler number D^ represents the ratio of the time taken by the protein to passage along the column in the mobile phase to the overall relaxation time for all conformation interconversions in the chromatographic system. Figure 28 illustrates three different examples of the influence of the Damkohler number on the chromatographic profile. When the D^ ratio is small, and in the limit approaches zero, the chromatogram for a four-component cycle will reflect the average macroscopic behavior of a fully unfolded form or alternatively two peaks separated by a time interval ^;^(fe 12/^21 ~ fe 43 7^34). Conversely, when D^ is very large, kinetic effects associated with conformational interconversion essentially vanish. However, at intermediate reaction rates where 1 < D^ < 10, complex elution profiles will be obtained, which are dependent on the intrinsic column efficiency, the equilibrium distribution and mole fractions of the species P^y, P^ and Pjj; the rate of interconversion of Pv PJJ, and the type of chromatographic condi^u tions used. The influence of the Damkohler number on the bandwidth dependence is demonstrated in Figure 29. Relatively slow kinetics of interconversion, which are characterized by small Damkohler numbers, cause the overall bandwidth to go through a maximum at intermediate retention times, with significantly higher H^^ obtained at relatively small k] values. The following example illustrates how the impact of these kinetic processes can escalate rapidly in multistep purification procedures. Let us assume that the purification of a particular protein to a level of purity of 99.95% required a 10-step procedure. If the average yield of bioactivity-mass per step in this 10-step method was 60% (a relatively favorable situation), predominately due to conformation changes leading to "denatured-unfolded" product then the overall yield of the desired native protein would be only 0.6%

O

u o

<

11

—'—'—'—'—•—//—

Da' «10

,

D,« 0.001

-—y/—'

A

Time F I G U R E 2 8 Three illustrative examples of the influence of the Damkohler number on the chromatographic profile of a polypeptide or protein undergoing conformational interconversion (or another type of secondary equilibrium process). When the D^ ratio is small, i.e., D^ = 0.001 and in the limit approaches zero, the chromatogram for a four-component cycle will reflect the average macroscopic behavior of a fully unfolded form usually a peak of longer retention time, or alternatively as two peaks separated by a time interval t^ikij/kji — ^43/^34), When D^ is very large, i.e., Dg = 1000, the conformational interconversion effects essentially vanish due t o the very rapid interconversions and a single peak of shorter retention time will be observed. Over the range of intermediate interconversion - reaction rates where I < D^ < 10, complex elution profiles will be obtained. These latter profiles will be dependent on the equilibrium distribution and mole fractions of the interconverting species P^, Py and Py; the rate of interconversion of P^ -^ Py ~^ ^u^ ' " ^^® mobile and stationary phases, the intrinsic column efficiency and the nature of the chromatographic conditions used.

166

MILTON T. W. HEARN

D=\,K=10^M

F I G U R E 2 9 The influence of the DamkohJer number on the bandwidth dependence of a polypeptide or protein undergoing conformational changes involving a two-state interconversion, / to j , in a chromatographic system as the relative capacity factors separating the peak maximum for species / and species j is increased. When D^ is large or very small, i.e., D^ < 0.00! or D^ < 0.001 > 1000, the change in peakwidth AHjg ,^ exhibits a relative insensitivity on A k ' , and this behavior will be not significantly influenced by the magnitude of the association constant K^. However, in the intermediate range of I < D^ < 10, larger variations in AH^g ,j occur, reflecting the slow interconversion kinetics. As a consequence, relatively slow kinetics of interconversion, which are characterized by Damkohler numbers in this range and reflect the presence of relatively stable conformational intermediates, cause the overall bandwidth(s) to go through a maximum value at an intermediate D_ value.

at the completion of the process, i.e., 99.4% of the product has been lost (Fig. 30). Should the average yield per step drop to 30% due to partial denaturation, or associated loss of resolution, then the overall recovery would reach a disastrous value. In this latter scenario, 1024 times as much raw material would need to be processed simply to yield the same mass of purified native protein. Neither scenario can be considered efficient, and certainly would create considerable disposal problems, cost overruns, and quality control limitations. From a management point of view, disastrous effect of low repetitive yields due to conformationally induced losses can result in unacceptable purification productivities and cost of goods that make the product of marginal value, i.e., turn a high-value product into manufacturing nightmare. Similar recovery considerations can be applied if the loss of the native protein was due to enzymatic degradation, self-association, aggregation, or precipitation. As noted, and as detailed in Table 2, a large variety of stationary-phase and mobile-phase factors influence the selectivity, recovery, and stability of proteins and other biomacromolecules in the adsorptive modes of HPLC. Batch adsorption pilot experiments provide an expedient approach to ascertain the effect of many parameters, such as the pH, nature, and concentration of organic solvent or ionic additives in the mobile phase, the temperature- or the static-binding capacity with a defined sorbent. Similarly, the influence of

167

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

100

-

• •

«L

80

2^

60

>^

40

95% 90% 80% 60% 40%

A V

•

• 1—1

20 0

^ 1

1

1

I

1

# 1

A L

T

1

8

W 1

9 1

10

1

Step Number F I G U R E 3 0 Plot of the product yield versus the number of steps in a multistep purification procedure. These plots correspond to the situations that apply when the repetitive yield is 95, 90, 80, 60 and 40%, respectively.

many stationary phase variables, including the effects of the chemical composition of the ligand and the support material, the ligand density, the surface heterogeneity of the sorbent, or the surface area and pore diameter distribution of the sorbent can also be ascertained from small-scale batch experiments. More subtle chromatographic effects due to the influence of the sample loading concentration (as a volume or concentration overload) on the efficiency of recovery of the targeted protein, or the influence of other protein components at the solid-liquid interface on the selectivity, particularly w^hen displacement effects are operating, are much harder at this stage to quantitatively anticipate. For these reasons, an iterative approach, by which the static and then dynamic binding capacities of the targeted protein are measured, represents a logical path to foUow^. Various investigations over the past decade have shown that the static binding behavior of many polypeptides or proteins as determined in batch systems can vary significantly from that observed in dynamic systems as employed in packed bed or fluidized column chromatography. This behavior is related not only to issues of accessibility of different polypeptide or protein to the stationary phase surface area,^^^'^^^'^^^"^^^'^^^ but also to whether more complex isotherm relationships are involved with multicomponent systems where nonlinear ef£^^^31,8,9,18,148-151,169,172,320-324 influence the diffusiou and adsorption kinetics of the overall mass transport phenomenon. All of these effects impact on the loading capacity of a particular HPLC sorbent, which can thus exhibit subtly different selectivity-capacity dependencies with different classes of polypeptides and proteins. Such behavior has been documented^'^^'^^^'^^^'^^^"^^^ for enzymes and other proteins in a variety of studies. For example, when conformational reordering of a protein structure occurs in both the mobile phase and the stationary phase, this will

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MILTON T. W. HEARN

lead to multizoning of the component into active a n d / o r inactive zones, v^ith the relative mole fraction abundance of each species time-, concentration-, and temperature-dependent. If the protein contains a cofactor or prosthetic group that is easily displaced under the chromatographic conditions, then the apo-iorm of the protein will result. Such behavior has been observed with various multimeric enzymes and heme-containing proteins such as myoglobins. Figure 31 illustrates a possible unfolding a concomitant loss of the prosthetic group under the influence of inappropriate binding conditions. Other phenomena besides conformational processes can also lead to multizoning effects with polypeptides and proteins when they interact with adsorptive HPLC sorbents. The so-called split peak effect is probably the easiest of these phenomena to be identified and steps taken to remedy. The split peak effect is very often seen in HP-BAC, RP-HPLC, and HP-HIC and to a lesser extent in the HP-IEX of proteins.^^^"^^^ This effect is manifested by the presence of a weakly retained (or occasionally as a nonretained peak) and a more strongly retained peak with the bound-to-free ratio between the weakly retained to strongly retained species dependent on the diffusion and adsorption kinetics. An extreme case of the split peak effect involves the weakly interacting component elution in or near to the column breakthrough volume. In this case, the amount of protein in the breakthrough zone is influenced by the nominal pore diameter and ligand density of the sorbent, the flow rate, and the injection volume. This effect can be circumvented by

(a) >

(b)

F I G U R E 3 I Schematic illustration demonstrating the binding of sperm whale muscle myoglobin (SWMYO) t o an n-ali
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

I 69

the choice of more appropriate loading volumes and concentrations, selection of a sorbent with better surface area and ligand accessibility characteristics for the particular polypeptide or protein, and by lower flow rate. A further type of multizoning phenomenon arises when nonlinear isothermal behavior occurs with polypeptides or proteins, due to matrix heterogeneity and nonuniformity of the ligate distribution over the sorbent surface. This effect can be problematic during the first few cycles of use of a freshly packed column and is most noticeable between "virgin" and "conditioned" columns operated at preparative scales of loading. At the micropreparative level this type of multizoning effect can lead to catastrophic results associated with nonreproducible recoveries. Other forms of multizoning phenomena are associated with equilibrium due to slow aggregation and isodesmic self-assembly between the monomeric and oligomeric forms of the protein, often mediated by conformational effects. Such effects have been noted^^^ in the HP-IMAC and HP-IEX of proteins. All of these multizoning effects are known^''^'^'^^'^'^^'^^^ to significantly affect resolution and recovery. Remedies for each can be achieved by applying the physicochemical principles discussed in the earlier sections of this chapter. Multiple-stage HPLC steps are now the norm in polypeptide and protein purification strategies. As a consequence, the stage at which a particular chromatographic selectivity is introduced requires careful planning. Systematic optimization methods developed for low molecular weight solutes based on adaptations of the selectivity triangle concepts^'^'^^ can be adapted as a basis for a multistep HPLC approach with single-column or multicolumn systems with polypeptides and proteins. Such strategies have formed the basis of several innovative computer-assisted procedures for the selection of elution conditions or generic type of sorbent. Importantly, these approaches offer considerable potential for the use of knowledge-based methods for optimization of resolution of very complex mixtures of proteins and other biopolymers using the same stationary-phase operating under different elution conditions. Resolution contour plots for each of the peak zones can be derived from the experimental data obtained with different binary-ternary mobile phase combinations under either isocratic or gradient elution conditions. By integrating this information with data on spectroscopic contour profiles, derived, for example, from on-line manipulation of spectroscopic data accumulated with multichannel or photodiode array spectrometers such as second derivative or CD spectra^^'^^^'^^^"^^^ from it is feasible to rapidly explore a variety of separation variables with single-column or multicolumn systems. Importantly, these approaches offer considerable potential for the optimization of resolution of very complex mixtures of proteins using the same sorbents operating under different elution conditions. The so-called Q-system of intelligent self-learning computer-assisted optimization^^ ^ is an excellent example of what can be achieved in this field. Such methods have been widely used for a number of years as multidimensional techniques in the HPLC separation of polypeptides and proteins as well as many lower molecular weight compounds within the pharmaceutical industry. Integral to these HPLC approaches has been the application of

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MILTON T. W. HEARN

computer-optimized routines for the selection of mobile phases of different composition, notably containing different types of ionic species selected from the Hofmeister series, ion-pairing systems, or organic solvents.^^ 16,30,196,200-202,217,334-337 ^j^^ji^^ procedures are equally pertinent to HP-IEX and HP-HIC separations using displacing ions of different solvated radii and molar surface tension increment or electronegativi^i^g 6,38,42,50,76,77,79-87,96,215,338-340 j^^^^g^^j ^^ ^^is approach has been the application of mobile phases of different composition, notably the use of different ion-pairing systems in the RP-HPLC of polypeptides. Similarly, by employing an HP-IEX sorbent prior to a HP-HIC step, protein components with the same pi value can be resolved by taking advantage of the Donnan effect on ionization equilibrium v^ithin the microenvironment of the stationary phase and the ability of the Coulombic ligates to act as molecular probes for the asymmetric distribution of charge on the protein surface. The potential for resolution optimization exploiting ions of different electronegativity and solvation state selected from the Hofmeister series has, furthermore, also been routinely utilized in HP-HIC by taking advantage of the "salting-in" and "salting-out" phenomena of different anions and cations. By utilizing mobile phases of different ion compositions, pH value, or organic solvent content in the different HPLC modes, multidimensional separation strategies can be automated and efficiently carried out with tandem columns packed with the same or different sorbents. One avenue that offers considerable versatility in the analytical and particularly preparative application modes with tandem columns is, as described earlier, the so-called "positive-negative" adsorption mode. In this approach, the chemical characteristics of the immobilized ligand are selected to ensure that the components of interest are either adsorbed very strongly, i.e., large Kassoc,/ values are achieved, or alternatively not absorbed and elute in the breakthrough with a series of by sequential or tandem columns. Such approaches are particularly suited to HP-IMAC, HP-BMC, and some forms of HP-BAC approaches. Tandem batch methods when used at the initial stages of purification scheme may also improve overall recoveries because of the early removal of contaminants and due to their ability to produce fractions at a higher concentration level. Typical of this approach has been the integration in this laboratory of large-scale biomimetic affinity and ion-exchange high-performance chromatographic methods as positive-negative capture modes into automated protocols has led to the development of general stratagems for the purification of other therapeutically important human plasma proteins. With such methods, the effective peak capacities may be relatively modest, e.g., PC values of between only 2 and 10 per stage achieved. However, the overall productivity of such systems, their potential for further scale-up and their capability to meet GMP requirements make these approaches attractive for exploration in an industrial setting. Moreover, intercalation of batch methods with gradient elution techniques, where peak capacities in excess of 200 can be routinely realized, has many desirable features for polypeptide or protein fractionation. Because greatly decreased masses of the relevant protein fractions are loaded onto chromatographic beds following batch fractionation, the prior application of batch fractionation methods enables substantially higher peak capaci-

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

I7 I

ties to be achieved at subsequent chromatographic stages. Such batch methods can be readily incorporated into a purification strategy using both chemically modified silica-based sorbents and the organic polymer-based HPLC sorbents. When used at an early stage of a purification scheme, rapid, tandem batch or column methods frequently improve overall recoveries. Whether a particular sorbent functions as a single selectivity mode or as a mixed selectivity mode is clearly not an essential requirement for its successful application in high-resolution purification of a particular polypeptide or protein. High-performance biospecific affinity chromatography (HP-BAC) and its immunological counterpart, immunoaffinity chromatography (HP-IAC), are logical extensions of mixed mode interactions. In both HP-BAC and HP-IAC the composite interplay of Coulombic, hydrogenbonding and hydrophobic forces determine the magnitude and nature of the association and dissociation phenomena. An important area of application that arises from this knowledge on mixed mode (multimodal) interactions is, of course, the design of chemical ligands that mimic HP-BAC and HP-IAC systems. Biomolecular modeling, molecular mechanics, and molecular dynamics methods coupled to computer graphic procedures can be expected to play an increasing role in sorbent design during the next decade, thus expanding the range of selectivities available. Already, such computer modeling methods"^^'^^'^^^"^^^'^^^"^^^'^^^ have been employed to replicate the composite interplay of Coulombic, hydrogen-bonding, and hydrophobic forces that determine the magnitude and nature of the association and dissociation phenomena with the design of several novel ligate systems, e.g., ligates specific for glycoproteins.^"^^'^"^^ The major challenge in the design of these ligands is to obtain sufficient information to enable a detailed understanding of the factors controlling the selectivity during the chromatographic distribution process as a generic event, rather than simply treating them as a parochial event limited to a single biomacromolecule. The application of combinatorial synthetic and screening approaches can fulfill many important niches in the identification of these new types of ligates. As currently manufactured, all micro- and mesoparticulate chromatographic media, irrespective of the nature of the ligand or the chemical functionality of the matrix, exhibit separation features characterized by composite multimodal phenomena. Exploitation of the secondary retention capabilities of a sorbent should not thus be discounted out of hand since it may potentially provide the solution to a difficult set of separation tasks. For example, mixed beds containing hydrophobic interaction and ion-exchange chromatographic media can be successfully used^"^^ for the purification of a variety of proteins under conditions where the relative selectivity was significantly different to that observed with a single type of sorbent. In the case of HP-BMC systems, such as the biomimetic triazine dye affinity,^^^'^^^'^"*^ the combinatorial peptide-based ligates,^^""' ^^^ or the macrocyclic metal ion complexes,^^^"^^'^ the participation of multiple retention mechanisms is again evident, with the features of both cation exchange and hydrophobic interaction effects particularly noticeable under neutral or weakly acidic pH conditions. Consequently, these ligate systems offer a route to explore the

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MILTON T. W. HEARN

physicochemical boundaries of interaction with polypeptides or proteins as "combinatorial" kits, cartridges, or as when immobilized onto biosensor chips, as described recently.^"^"^"^^^ Extensions of these mixed-mode approaches underlie recent developments of the multimodal salt-promoted adsorption media.^"^^"^^^ In these cases, salt-mediated changes in the retention behavior of polypeptides and proteins are largely entropically driven. Changes in the associated water structure or bound ions provide a mechanism to either stabilize or destabilize the three-dimensional structures of the polypeptide of protein. Although much useful information exists^^'^^"^^'^"^"*'^^^'^^^'^^^ on the effects in bulk solution of different salt species on polypeptide of protein conformation as already noted, systematic adaptation of these studies to adsorption chromatography with micro- and mesoparticulate high-performance chromatographic media requires substantial development. For example, the empirical Setchenow equation can be used to quantitatively described the "salting-in" or "salting-out" effects of chaotropic and cosmotropic salts on polypeptides or proteins such that In (solubility) = C^ — S^ p X (salt concentration)

(117)

where S^ p is the so-called Setchenow constant that is a characteristic of the particular salt and protein examined, while C^ is a system constant. The form of the empirical Setchenow equation is thus remarkably similar to the empirical relationships used to describe the retention behavior of polypeptides or proteins in the reversed-phase, hydrophobic interaction, and ion-exchange selectivity modes, namely. In k]j = In k^^ — X^y, where X represents to slope of the plot of In k^^ versus ^y for the selectivity mode / (cf. Eqs. 6-11 etc.). Rigorous thermodynamic demonstration of direct physical relationships between precipitation and interaction parameters Sg p and ^y have yet to be firmly made, nor has a common mechanism been defined. Because of their impact on preparative HPLC separation of polypeptides or proteins, such studies will certainly be fruitful avenues of further research over the next decade. IX. SCALING-UP POSSIBILITIES: HEURISTIC APPROACHES AND PRODUCTIVITY CONSIDERATIONS From pragmatic considerations, the most important step in the establishment of viable analytical separation as well as process-scale purification of polypeptides and proteins by HPLC techniques is the selection of the appropriate sorbent. The sorbent choice represents a separation variable for which the investigator can, or may be able to, introduce some rational selection criteria. Knowledge gained about the molecular features of the target polypeptide or protein come into consideration, i.e., what is the hydrodynamic radius of the polypeptide or protein; what are the surface hydrophobic or charge features as deduced from the composition, amino acid sequence, or other structural data; does it contain glycosylation or other posttranslational

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

1/3

modifications; is it monomeric or multimeric; what are its solubility features in water-organic solvent mixtures etc.? The result of an HPLC separation can never be better than the selectivity that the sorbent and mobile phase allows. On the other hand, the mass transport characteristics of the HPLC system established between the biosolute, the eluent, and the bed material are features which relate more to the quality of the packed, fluidized- or expanded-bed or batch (tank) system and the nature of the fluid flow that can be generated. Obviously, chemically and physically robust sorbents of the correct particle size and porosity with ligate characteristics appropriate for the intended application must be used. The challenge, then, that has to be faced in "scaling-up" of a particular HPLC separation with polypeptides and proteins from the analytical to the semipreparative (laboratory-scale) and finally to the process (industrial) level is how to maintain resolution without sacrificing efficiencies (cost, purity, or peak) as the sample size is increased. Concomitantly concentration or volume overload effects will participate. Again, knowledge of the physicochemical features of the affinant-affinate interaction from thermodynamic and well as kinetic perspectives becomes important. Traditionally, the choice of appropriate sorbent materials and the optimization of the basic operating parameters (eluent composition, flow rate, temperature, etc.) have been made in laboratory trials at the analytical level prior to scale-up. Key issues at the core of the design of a HPLC separation process and subsequent introduction of adequate levels of engineering control have been, and remain, system productivity and maintenance of bioactivity. These issues must be concomitantly addressed if implementation of scale-up procedures is to be effective. To overcome the problems inherent to the design, layout, and construction of optimal separation processes within a production framework, the availability of rapid methods to screen alternative purification pathways are mandatory. These knowledge-based methods now often assisted through the use of computational modeling and simulation inevitably draw on a series of rules —called heuristics. The evolution of heuristic rules rely on scientific intuition, experience, and common sense supplemented by integrative cycles of yes-no decision trees as the process development moves, for example, from the small-scale batch (tank) investigations through to the design and implementation of a large-scale expanded-bed application. Such methods are now being widely applied in industry. An example of such an approach is illustrated in Figure 32. In this case, which is based on investigations carried out in this laboratory for the fractionation of human therapeutic proteins by specific affinity chromatographic systems, heuristic rules and computer-assisted sorbent-eluent optimization methods were used to facilitate the development of a viable process alternative. In the design of particular separation logic trees, heuristic methods drawing on information related to the physicochemical properties of polypeptides and proteins provide very useful guidelines to facilitate the decision of a preferred option over others for exploration as a subsequent scale-up step in a recovery process. Heuristic methods can be subdivided according to whether the rules are based on the two major constraints of the system. The first of these constraints relates to the separation features of the system per se. Thus,

174

MILTON T. W. HEARN

Batch Experiment

q„&K, D&K,

Performance F I G U R E 3 2 Decision tree based on heuristic principles for the utilization of binding and isothermal data for polypeptides or proteins with interactive HPLC sorbents derived from batch equilibrium measurements and column (packed or fluidized - packed bed) investigations. Experimental correlations can be employed initially for 0^,0^ and K^, while the first-pass q^ and K^ values can be iteratively employed in conjunction with a best guesstimate value of the k, in the refinement of the isothermal binding and kinetic mass transfer models. Analogous considerations can be employed to assist in the evaluation of the individual kinetic parameters, leading progressively to a new model, and full implementation within the packed column or expanded - fluidized-bed scenarios.

the physicochemical properties of the components in the feedstock, the nature of the sorbent used, the temperature of the system, or the selectivity and resolution that can be reasonably obtained for the system in repetitive or cycling applications will all impact. The second constraint relates to the nature of the time-to-process considerations. Hence, effects arising from the mass or volume of feedstock to be processed; the product abundance relative to the contaminants in the feedstock; and the volume characteristics of the system can all be expected to affect the productivity and purity levels. Collectively, these two constraints will set the parameter boundaries for the cost of manufacture as well as the overall efficiency, in terms of economic considerations per cycle, as well as the longer term logistical need capital investment for the construction of new plant, of the purification process. The advantage of employing alternative heuristic approaches in the development of alternative process design trees is self-evident. These intuitive and interrogative procedures enable different unit operations of the process to be contrasted or simulated, areas of potential design conflict to be identified and reconciled, deficiencies in knowledge or implementation to be highlighted, and the order of individual unit operations that will ultimately form the basis of the final process pathway to be examined as part of the investigations. These heuristic approaches for the purification of polypeptides, proteins, and other biomacromolecules at the process-scale level have their origins in industrial fermentation and recovery of low molecular weight commodity products, such as citric acid, antibiotics, and other bulk chemicals. Arising out of these developments, the following rules can be proposed for the

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

I 75

fractionation of different classes of biopolymers by HPLC procedures: • Choose a process that has the minimum number of steps, consistent with the expected outcome of purity and productivity. • Use a high-resolution step as soon as possible. This rule is in accord with the preceding "use of the minimum number of separation steps" rule, which anticipates that product yields decline exponentially. • Choose those process steps that exploit the largest differences in the physicochemical properties of the products and impurities in the most economical manner. • Employ sequential separation processes based on different physical, chemical, or biochemical properties that are synergistic, and thus orthogonal, rather than repetitive, i.e., use a metal ion-affinity HPLC procedure prior to a size-exclusion step, a hydrophobic interaction HPLC step after an ion-exchange HPLC procedure, a biomimetic HPLC step before a biospecific affinity HPLC step, etc. This rule is in accord with the "orthogonality" rule, which anticipates that the most efficient separation procedures are ones that take advantage of the anisotropy of molecular physicochemical properties of the target protein or polypeptide rather than the commonality of the molecular features. • Separate out the most abundant impurities first using the positivenegative strategy. This rule anticipates that a major, if not the major, contaminant in a feedstock placed into purification stream may in fact be water. The ability to reduce the operational volume quickly has major ramifications on the product recovery, plant investment costs, and process efficiencies. • Perform the most expensive step last. This rule anticipates "Murphy's law" in so far as process mistakes can and do arise at the production level, and very expensive steps most often require elaborate process monitoring equipment, substantial experience of the supervisory staff, and usually a limited opportunity to be applied to a different recovery problem. Such heuristic approaches thus enable theory to be reconciled with practice. Initially, qualitative information, and subsequently more quantitative data, can be obtained from the application of these heuristic approaches and used directly or indirectly to establish more specific rules that more adequately anticipate the design requirements and limitations of a specific large-scale purification process. Choice of the appropriate sorbent (Table 9) and optimization of basic operating conditions should be made in laboratory trials prior to scale-up. It is very important that favorable mass transport conditions between the eluent and the sorbent must be maintained at the larger process scale. Optimum resolution is expected, according to theory, when a sorbent of the smallest possible average particle size is used. In large-scale HPLC, choice of particle size is, however, more often governed by the practical constrains of cost efficiencies and the necessity to use high and constant flow rates. Especially in large-scale fractionation of polypeptides and proteins when soft or deformable sorbents are used at high superficial

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MILTON T. W. HEARN

T A B L E 9 Comparison of Some of the Different Properties between Soft Gels and Silica-Based Sorbents Used in the Purification of Polypeptides and Proteins Property

Soft gels

Silica-based matrices

Buffer compatibility Mechanical rigidity Limiting pressure drop Separation speed Deformation properties Particle size distribution Pore size distribution Thermal stability Chemical stability pH limitation Specific surface area Resolution Capacity Mass recovery Biological recovery Regeneration in base Cost ($ per g)

Medium-high Low^ 2-10 bar Lov^-high Poor-good Narrov^-medium Narrow-medium Lov^ Good-high Good-high 10-100 m V g Lov^-high Low-high Good-excellent Low-moderate Good Low-high ($10-$200)

Low-medium High 250 bar Low-high Good-excellent Narrow Narrow High Low-good Low-good 20-400 m V g Moderate-high Low-high Low-moderate Low-moderate Poor Moderate-high ($25-$400)

velocities, these conditions will lead to particle deformation even at relatively low hydrodynamic pressures. How can these conflicts be reconciled? For mechanically rigid and physically-chemically robust particles, the relationship between column performance, in terms of the separation impedance or column resistance factor (CRF), such that pressure drop and the flow rate is linear over a wide range of flow rates and inversely proportional to square of the particle size, and can be approximated by the relationships CRF =

t.

AP

N'vil + k]) LTJUQ

where

A? =

(118) (84)

where AP is the pressure drop over the bed, L is the bed height, 17 is the eluent viscosity, UQ is the linear velocity of the eluent, e^ is the specific permeability of the column (typically in the range of 1 X 10 ~^ to 1.3 X 10"^), and dp is the average particle diameter. A more precise dependency linking the porosity and permeability of a HPLC sorbent is given by the Hagen-Poiseuille equation^^^ for a given column and particle size involved evaluation of the column resistance factor a I/EQ - 180(1 - eY/e^ (where e is the void fraction). For columns well packed with spherical silica-based sorbenrs the column resistance factor is typically 500-700.^^"^ The column resistance factor also includes the aspect factor K^^^^^, which for spherical silica-based sorbents is close to 5. For nonrigid or deformable particles, hke the soft polysaccharide gels and some organic polymers of low level of crosslinkage. With "soft" gels, the observed pressure drop relationship fol-

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

I 77

lows a logarithmic rather than a linear dependency and is a function of the bed height and bed diameter as well as the water regain of the sorbent. The following empirical equation has been found^^^ to adequately fit the experimental data: AP = y l n ( K o A )

(119)

where L is again the bed height; ^ is a function of the bed height, bed diameter, aspect factors, and sorbent solvent regain; and KQ and K^ are system dependencies incorporating the influences of different linear flow rates UQ and w^, respectively. Whether a silica-based sorbent, a polysaccharide-based sorbent, or a sorbent, derived from copolymerization of organic monomers is employed in a scaled-up process will be determined by the product characteristics, its end-use, and the performance and productivity criteria set by the designers of the process, e.g., whether the constraint is the need to meet a particular purity level, time per cycle, the cost per cycle, mass throughput, or pressure drop etc. Similarly, when using any porous or nonporous sorbent, the more homogeneous the particles are in terms of their physical and chemical features, the more likely will be the opportunity to simulate their performance in packed columns as weU as fluidized-expanded beds. From practical considerations, there may, however, be limited choices available in many recovery processes due economic reasons and the selection of the sorbent particles will not meet the preceding stringent criteria. Reproducibility of the performance of different batches of the same sorbent in terms of particle and pore size distribution, and chemical selectivity thus become imperative. The best grade possible should be used, within the pressure limits set by chromatography system, irrespective of whether porous or nonporous sorbents are used. This choice will result in some compromises, since the limits set by the allowable pressure drop of the system must not be exceeded. Employing linear flow velocities that are above the maximum operational values for sorbent deformation may result in catastrophic collapse of the bed. Heuristic guidelines again aid in the selection of the particle size of the sorbent. For example, in all HPLC systems, the force that is exerted on the bottom part of the bed is the sum of the weight of the packing material, and the drag force acting on it, minus the friction force component due to the column wall. For this reason, sorbents of the suitable density can be successfully used in fluidized-expanded-bed systems at high superficial velocities, where otherwise they would be unusable in packed beds. The fact that the pressure drop across the bed reaches a asymptotic value with fluidized-expanded-bed systems within the plug flow range of superficial velocities, moreover provides a significant opportunity for high throughput separations and selective product capture. The tendency of nonrigid gel materials to undergo deformation increases with increasing bed height of the column as a consequence of the higher pressure drop (cf. Eqs. 118 and 119). Consequently, "pancake" or "egg" columns of large width-to-depth aspect ratios have been favored in numerous applications with these conventional soft gel sorbents. Sudden increases in hydrostatic pressure with soft gel sorbents packed into these

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MILTON T. W. HEARN

column configurations will nevertheless lead to bed compression and a significant drop in flow rate. Subsequent operational cycles of the column will as a consequence show different performance characteristics, resolution, and capacities. Such effects are even apparent even with very short columns and become particularly significant with noncrosslinked wide pore organic or polysaccharide polymer-based gels. With pressure stable silica-, zirconia- or other metal oxide-ceramic-based sorbents, the selection of column configuration, although still very important, does not assume the same level of burden as found with soft gels when operating at high superficial velocities and high levels of performance and productivity. Other considerations, e.g., cost, pH stability, biocompatibility, regenerability, and sterilizability come under scrutiny. In these cases with pressure stable silica-, zirconia- or other metal oxide-ceramic-based sorbents, the choice of column configuration can still be made on the basis of a set of heuristic rules, where the design priority is dictated by whether the process task hinges on the size of the feed stock volume to be processed, the complexity of contaminants, the need for aseptic operational conditions, the ease of the separation, or the cost of goods.

X. EFFECT OF MASS TRANSFER RESISTANCES IN PREPARATIVE HPLC OF POLYPEPTIDES AND PROTEINS All mass transfer effects have their origins in the physicochemical properties of the target polypeptide or protein, the ligates, the chromatographic support material, and the composition of the feed and the mobile phase. Superimposed on these properties are the mass transfer effects that are influenced as well by the molecular heterogeneity of the affinant and affinate, the design features of the equipment, the flow rate, and the temperature characteristics of the separation system. Thus, the large-scale purification of commercially important polypeptides and proteins to the degree of purity necessary for them to be used as pharmaceutical products requires the availability of procedures, protocols, and research-based information that enable accurate prediction of the mass transport phenomena and bioactivity behavior profiles of these biosolutes under various operational conditions.^^^"^^^ Not only must the engineering scale-up and integration of each purification stage be successfully achieved, but also specialized and dedicated equipment must be purchased, installed, and vaHdated. These tasks collectively lead to the inevitable outcome of a complex manufacturing plant almost exclusively dedicated to one particular product if GMP guidelines are to be fully adhered to during the production. The primary and secondary recovery stages and the various purification steps that are involved with the production of polypeptide- or protein-based biopharmaceuticals from fermentation broth, biological fluids, or chemical reaction vessels frequently require the use of creative combinations of different HPLC separation techniques operated under nonlinear adsorption conditions approaching column overload criteria. In some cases, the concentration of the desired product may be very low; in other cases, very closed related contaminants may be present in the feedstock; while sometimes the stability characteristics or conformational properties of the target polypeptide or protein may be very sensitive to the operational condi-

179

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

tions, presence of degradative enzymes, etc. Moreover, simple imitation of a production process developed for a low molecular w^eight compound will not necessarily lead to a fully optimized preparative procedure even when the surface of the porous sorbent is totally accessible to a specified polypeptide or protein.^'^^'^^^ The involvement of more complex mass transfer resistances associated with individual HPLC step as part of the process scale purification of middle molecular weight polypeptides and proteins, currently can result in very costly scale-up procedures. The performance of the purification process under variable conditions (flow rate, particle and pore size, etc.) is limited by the rates of mass transfer, adsorption, and desorption and by axial dispersion. Plots of the effluent concentration as a function of time or throughput volume contain much of the information needed to evaluate column performance. The shape of such breakthrough curves, however, is a complex blend of equihbrium and nonequilibrium processes (see Figure 33). The position of the breakthrough curve depends on the capacity ^* of the sorbent in the packed or expanded bed with respect to the concentration of the feedstock components. The shape of the actual breakthrough curve is due to the existence of flow nonidealities, axial and longitudinal mixing effects, and finite mass transfer and sorption rates. These nonequilibrium effects depend on the operating conditions and system configuration. Scale-up and optimization of the large scale HPLC purification of polypeptides and proteins thus requires an understanding of these nonequilibrium interactions as well as of the nature of the equilibrium process that is established between the sorbent and the different solutes in the feedstock. The adsorption and elution operations that the biosolute(s) undergo as they proceed along the column bed can thus be considered as a series of diffusion, convection, and reaction steps. In the adsorption process, the solute(s) in the feed must diffuse through a liquid film surrounding

JM • • • Vl^-t-- • • f

1.0-

^

Unused Sorbent^ Capacity < ^

C3 O

1 O

\

1/

Porous

0.5-

a o O Wasted Feed

/

W O.OH 1 ^...§.-:^r^ ^—0_ ^—•

1

Non-Porous

i» 1

1

Normalised Time - t F I G U R E 3 3 Typical breakthrough curves as plots of the normalized effluent concentration (C/CQ) versus normalized time, t for nonporous and porous HPLC sorbents with polypeptide or proteins. As evident from this figure, when the adsorption step is terminated at an effluent concentration of C^-, a small amount of feed has been wasted in both cases. This does not represent a significant loss for either sorbents, although clearly wastage is less with nonporous materials. However, in terms of underutilized column capacity, a more significant difference arises, with the porous HPLC sorbent used significantly below its potential.

I 80

MILTON T. W. HEARN

the sorbent particles and then diffuse (or perfuse) through the pores in the particles before interacting with the immobilized ligands. Each of these mass transfer or reaction steps can be explicitly included in various types of predictive models (see subsequent section on predictive modes), which incorporate the physicochemical features of the affinant(s) and affinate(s). However, the resulting derivations of these predictive models are often cumbersome and require detailed information on mass transfer coefficients and rate constants, while time-demanding numerical methods must be applied to obtain physically and mathematically consistent solutions.^'^^'^^^'^^^"^^^ One of the consequences of the recent advances in protein biotechnology, genetic engineering, and cellular expression systems is that the yield and availability of commercially valuable polypeptides and proteins from biological extracts has been considerably enhanced. A fundamental issue still remains in product manufacture, however, since high recovery of the specific polypeptide or protein in bioactive form must be achieved with a selected sorbent without sacrificing speed, productivity, and process economics. Typically, three or four stages of "downstream processing" are required to preconcentrate and fractionate the crude feedstock into a suitable fraction containing the majority of the desired product. Sequential exploitation of the differential solubility, state of aggregation, or partitioning behavior of the various components in the feedstock or the simultaneous use of a combination of all three of these physicochemical properties are employed as the basis of these prefractionation and concentration methods. In achieving this outcome, the protein chemist, the separation sciences speciaUst, or the process engineer is simply exploiting the different molecular attributes and physicochemical properties of the target polypeptide(s) or protein(s).^^^'^^'^'^^^ In the manufacturing process, a stage is, however, reached irrespective of the success of these initial preconcentration procedures, when the desired wild-type or recombinantly derived polypeptide or protein must to be further purified from a still complex multicomponent mixture by specific adsorption HPLC procedures.To ensure optimal adsorptive capture of the desired polypeptide or protein, or alternatively to maximize the capture of the contaminants and to enable the polypeptide or protein product to emerge in the breakthrough peak using an approach based on the "positive-negative" capture concepts, it is necessary to identify what effects the contaminants may have on the adsorption process. Unwanted contaminants may include inter alia other polypeptides or proteins of very similar composition or three-dimensional structure, such as partially deamidated, phosphorylated, or glycosylated isoforms, or simply be the huge volume of water-buffer that formed part of the initial stages of the recovery process. The way that these contaminants are removed, and their impact on the adsorption behavior of the desired product with the chosen HPLC sorbent, will determine the economic productivity of the procedure. As noted in Section VIII, preparative ion-exchange and hydrophobic interaction chromatographic procedures have come to occupy, from practical considerations, central positions in the fractionation of protein mixtures, typically enabling purification factors in the range of 10- to 100-fold.^'^^"^'^^^

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

I8 I

In the case of polypeptides, preparative RP-HPLC methods tend to have a dominant position with purification factors as high as 200-fold achieved in some cases. For multicomponent mixtures, the maximum effective dynamic adsorption capacity (DAC) and the rate of dynamic adsorption (DAR)^' ^^^ of a specific polypeptide or protein for a specified HPLC sorbent v^ill depend inter alia on the molecular size, composition, surface characteristics, and concentration of the target polypeptide or protein, the total bioload in the feedstock, the viscosity and composition of the feedstock, the composition and pH of the loading-v^ash-elution buffers and the operating temperature. Because HPLC sorbents of generic selectivity, such as HP-IEX, bind to polypeptides or proteins and other components encompassing a wide range of molecular features via less discriminatory primary and secondary interaction processes than those that occur with, e.g., HP-BAC sorbents, a larger range of contaminant substances will be coadsorbed according to their affinities and relative abundances. In this context, the physical properties of the HPLC sorbent, including the pore volume and pore size, have important practical sequelae. Because of their structural and conformational complexity, polypeptides, proteins, and their feedstock contaminants thus represent an especially challenging case for the development of reliable adsorption models. Iterative simulation approaches, involving the application of several different isothermal representations^'^^^"^^^ enable an efficient strategy to be developed in terms of computational time and cost. Utilizing these iterative strategies, more reliable values of the relevant adsorption parameters, such as ^3'*, K j , or the mass transfer coefficients (the latter often lumped into an apparent axial dispersion coefficient), can be derived, enabling the model simulations to more closely approximate the physical reality of the actual adsorption process. The simplest theoretical model that has been employed to describe the adsorption phenomenon of a single polypeptide or protein with an HPLC sorbent is based on the Langmuir equation. Application of this model to experimental data requires that the following assumptions prevail: (i) the binding is reversible; (ii) all binding sites are energetically equivalent and independent; (iii) the protein molecules are adsorbed onto a fixed number of well-localized sites in a single equivalent orientation; (iv) no lateral or isodesmic interactions occurs between the adsorbed molecules, i.e., further adsorption ceases once monolayer coverage has been achieved and independent binding of the proteins has occurred. With multicomponent mixtures, both competitive and noncompetitive binding interactions can occur. As a consequence, modifications to treatments based on the Langmuir model for a single component interaction are required. A variety of other models have been proposed to accommodate the heterogeneity of the binding sites present either at the surface of the adsorbent or at the surface of the adsorbed protein for a single-component system. These additional models include the noncompetitive and competitive bi-Langmuir isotherm,^^'^'^''^ now widely used for the prediction of the elution bands of organic compounds in preparative zonal or displacement chromatography;^^^'^^^ the Freundlich isotherm or

182

MILTON T. W. HEARN

the composite Langmuir-Freundlich isotherm;^^^'^^^ the Jovanovic isotherm;^ ^^'^^"^'^ ^^ the Redhch-Peterson isotherm;^^^ the Temkin isotherm;^^^'^^^ the Fowler-Guggenheim isotherm;^^^ the S-shaped isotherm;^^^ or combinations of these expressions as more complex competitive or noncompetitive representations of binding of multicomponent mixtures to adsorptive HPLC sorbents. The general forms of the more commonly employed isothermal expressions are given in Table 10. Noncompetitive or competitive Langmuirean models have been frequently used as the starting point for such representations of the adsorption behavior for mixtures of polypeptides and proteins w^ith HPLC sor bents. The noncompetitive Langmuir model describes an extreme case of the adsorption phenomena w^ith a multicomponent polypeptide or proteins mixture. This model assumes that there is no competition betvs^een the target polypeptide(s) or protein(s) and other components in the mixture for the available adsorption sites. As a result, the adsorption behavior of each component will be the same as if the other components were absent. The general form of the noncompetitive Langmuir isotherm describing this extreme case of adsorption for a binary mixture can be given in Table 10. In the context of the different HPLC adsorption modes with feedstocks containing mixtures of polypeptides and proteins, this extreme case of noncompetitive adsorption is unlikely to occur. The semicompetitive approach with multicomponent mixtures also assumes that the adsorption phenomena occurs with either no or only partial competition.^^^ In this case, however, it is assumed that the sites are not equally available to all species, rather than being exclusively associated with one or other species. The isothermal expression of this model is given in Table 10. This semicompetitive model can be anticipated to be applicable for mixtures containing components of significantly different molecular sizes and chemical properties. Such circumstances may, for example, arise when lower molecular weight peptide fragments (derived from, e.g., proteolysis of the parent or contaminant proteins) preferentially bind to ligates accessible on inner porous surfaces which are not accessible to the intact, larger protein species.^'^^^ An alternative approach which can be used to describe the adsorption phenomena of a multicomponent system is to assume that there is total competition between target polypeptides, proteins, and other components in the mixture for adsorption to all the accessible sites of the ligate. In this case, the Langmuir approach can be expanded to encompass binary (or more generalized examples) of adsorption iso^[^gj.j^58,15,148-151,167-175,227-236,365,369,372,373,379,382-386

f^^

^^^

^^

^^^^.^

polypeptides or proteins from the mixture. Although this generalized form of Langmuirean adsorption has found application in the purification of a ^-antitrypsin or y-interferon from multicomponent feedstock mixtures with substantial differences in molecular mass, charge, or hydrophobicity,^^"^'^^^ nevertheless this treatment assumes that the adsorbent has the same saturation capacity for all adsorbates present in the system. With most crude feedstocks from biological extracts, this assumption will most likely not be physically or chemically realistic. Polypeptides, proteins, and other components present in the feedstock mixture will have a range of different molecular sizes and

183

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

m T A B L E 10 General Form of the Commonly Employed Isothermal Expressions Used for the Analysis of the Binding Behavior of Polypeptides and Proteins with H P L C Sorbents Freundhch isotherm

Langmuir isotherm X C*

r = K,

(?* =K^

+ C*

Langmuir-Freundlich isotherm

X(C*)"

Temkin isotherm ^* = ^ 7 ^ X l n [ l

+KjC*]

K3+(C*)" Javanovic isotherm

Fowler-Guggenheim isotherm

r = (l„ - ?*)C* X

txp(liq*/q„)/Kj

Redlich-Petersen isotherm

^

q*=q„x[\-exp{-0/C*)] S-shaped isotherm IT =

K, + (c*)"

and for multisite interaction ^m X C *

?2 =

<JT Noncompetitive Langmuir isotherm ^r = 1 + /7C1*

and

?«,2 + K,,2 X C|^+''^^

*;^,2i X C | " + ^„,2i X Cf"

Semicompetitive Langmuir isotherm

a,Ct ^2

=

1 + ^C?

^r

1 + ^iQ* + ^ 2 ^ 1

(1 - ^ i ^ 2 / ^ 1 ^ 2 ) ^2CJ + 1 1 + ^iCi* ^2^1

^2* =

1 + ^iCi* + /?2CJ

where

where 6 i = X , , i , ^ 2 = i^.,2

H = ^ 1 X (3'^, 1,^2 = ^2 X ^ ^ , 2

Competitive Langmuir isotherm ^1 =

1 + h^CX + ^2CJ

and

q*

a,a 1 + h^CX + ^2CJ

where ^^ = iC^ 1, ^2 = ^d,2» ^i = b^ X q^^i, ^2 = ^2 X ^^,2» ^^^^ the various terms as defined earher refer to adsorbates 1 and 2, respectively. The generalized expression of this model for the adsorption of the various components within a multicomponent mixture takes the form

Legend to symbols C* = concentration of the protein in the bulk solution = concentration of the protein species / binding to the adsorption site b on the sorbent q* = amount of protein bound at equilibrium to the adsorbent q^ = maximum capacity of the absorbent for the protein qj = differential increase in the limiting capacity q^ for protein adsorption with increasing binding affinity Kj = maximum binding affinity constant K^ = dissociation constant X j = apparent dissociation constant iC^ = an empirical constant « = an empirical constant j8 = the interaction energy between two adsorbed molecules 6 = reciprocal of the dissociation constant K^ Subscripts 1, 11, 12, and 22 correspond to different binding sites for the interaction of different molecules

184

MILTON T. W. HEARN

characteristics,^^ ^ and hence these different substances will occupy different areas on the adsorbent surface. Moreover, during the adsorption process, as discussed earlier, polypeptides and proteins may undergo significant changes in their conformation. These conformational changes will affect the binding capacities of the sorbent for the target protein and the other components present in the mixture. From a strictly physical perspective, the generalized competitive Langmuir model is better suited to describing the adsorption of enantiomeric mixtures^^^ rather than the adsorption of polypeptides or proteins with widely different molecular and compositional properties. Nevertheless, application of competitive Langmuir treatments of the binding data for multicomponent mixtures of polypeptides and proteins has remained one of the most commonly employed approaches'^'^'^^^'^""^'^^^ for the description of the adsorption behavior, although other isothermal models, e.g., Freundlich-Jovanovic or Temkin isotherm, have gained considerable recent attention. The origins of these different theoretical models, which can be applied to describe the adsorption behavior of a component or multiple components in a mixture with a HPLC sorbent have been de^^ilgji5o,2io,2ii,227-236,308,356-384,390,39i j ^ ^^e Separation scieuce and chemical engineering literature. The combined influences of the different contaminants in a complex mixture of polypeptides and proteins will lead to reduced dynamic adsorption capacities^ and reduced dynamic adsorption rates^ for the polypeptide or protein of interest. In some cases, enhancement of the DAC but with a reduced DAR have also been observed, usually as a consequence of the so-called "protein-protein" multilayer phenomenon.^^^ By monitoring the manner that the equilibrium isotherms or breakthrough curves change for single-component and multicomponent mixtures of polypeptides and proteins when the loading volume, concentration, temperature, or other experimental parameter is changed provides very valuable insight into the nature of the adsorption processes and the extent of competition for the ligate binding sites. Illustrative of these effects are the results shown in Figures 34 and 35 for the binding of human serum albumin (HSA) to Cibacron Blue F3GAFractosil 1000 and also with a^-plasminogen inhibitor (a^-PI) to DEAE Spherodex, a silica-based HP-IEX sorbent.^^^ As is evident from these results, the equilibrium binding and elution behavior of feedstock mixtures containing HSA and a^-PI exhibit features characteristic of competitive interactions, with the ability of a^-PI to act as a protein "displacer" in the development of the HSA zones due to its higher affinity. Consistent with this conclusion, the apparent dissociation constants (K^) extracted from the plot of C* versus C * / ^ * were equal to 1.7 X 10"^ M for a^-PI and 7.6 X 10"^ M for HSA, respectively This displacement behavior is not without precedent^^^'^^^'^^^ and indicates that contaminant polypeptides and/or protein(s) can bind competitively in the presence of the target polypeptide or protein to the same functional groups immobilized onto the HPLC sorbent, thus reducing the number of binding sites. The mass balance for a single solute over a section of a column containing an adsorptive sorbent of monodisperse porous particles, assuming that a

185

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

(a)

0.8 , o 0.6 U

A

0.4

0.2 I3

= 0.64 cm/mirj = 1.28 cm/min = 2.55 cm/min = ^.82cm/min|

6

12

9

Amount applied to the column ( mg / g adsorbent)

1.0 0.8

B

T~, /

(b) / ,

0.6 0.4

/ / /

0.2

= 0.10mg/ml = 0.25 mg/ml = 0.50 mg/ml

0.0 10

5

15

20

25

Amount applied to the column ( mg / g adsorbent)

1.0 0.8

r"?*^'

(c)

/ /

0.6

.Li£ 5

= 4°C = 15°C = 25^C = 37°C

10

15

20

25

Amount applied to the column ( mg / g adsorbent) F I G U R E 3 4 Effect of the flow rate, feed concentration, and temperature on the experimental breakthrough curve for the adsorption of human serum albumin to Cibracron Blue F3G-A immobilized onto Fractosil 1000 against the amount of protein applied to the column.

discrete isothermal profile is generated in the plug flow, can be described in terms of the continuity equation such that for a well-packed bed, dc

dc dq + e—+ ( ! - € ) — = dt dt

E

1^

(120)

where c^z^t) is the solute concentration, with a volumetric flow rate P through a bed of length L, cross-sectional area A and void fraction e, and q is the sorbate concentration in the particle, which includes the solute in the pores. The particles are assumed to be spherical with radius R, porosity /?^, and density p^. The concentration of the solute adsorbed to the particle is ^^(r, z, i) and the solute concentration in the pores is c^Cr, ^, i). The different terms in this equation account for convective transport of the solute, accumulation in the interstitial spaces, solute uptake by the particles, and axial dispersion.

186

MILTON T. W. HEARN

^

150 125 W) 100

' /"^T-^^*^^^'^ -/ /

S 75 ^

50^

• o

25

orI

0.0

•

,

•

.

•

0.2

0.6 K

= HSA

,

0.8

1.0

C(mg.mr') F I G U R E 35 Equilibrium binding isotherms for the batch adsorption of human serum albumin and a I-proteinase inhibitor (a|-PI) onto DEAE-Spherodex in 25 m M sodium acetate buffer, pH 5.2. The experimental data are plotted as milligrams of protein adsorbed per gram of DEAE-Spherodex (q*) against milligram per milliliter protein in solution (C*). The theoretical curves generated for a Langmiurean interaction of HSA and a,-PI are also shown. Data adapted from Ref. 436.

The rate of change in the average concentration of a polypeptide or protein inside a particle is equal to the flux of the solute entering the pores. Thus, (1

ds

3(1 - e)

dt

R

dc:

(121) '=R

where D^ ^ is the effective particle diffusion coefficient based over the entire particle volume and the quantity 3(1 — e)/R is the surface area per unit bed volume of the spherical particles. For a given particle, the diffusion of the solute into the pores and adsorption at the pore surface can be described as D

d^c^ ' dr'

+

Ir dC: dc, T - - / 3 ^ r r - Pt<^rdt dt dr

(122)

The solute concentration in the pores c^ and in the bulk liquid surrounding the particles c^, respectively, are coupled by the rate of mass transfer through the fluid film. Thus, the film transfer rate can be evaluated from

or

(123) r=R

At this stage of developments, most fixed-bed adsorption models assume that film mass transfer resistance is small compared with the other transport resistances in the system and that equilibrium is reached instantaneously between the solute in the pore liquid and at the surface of the sorbent. Even if it assumed that a homogeneous adsorptive HPLC sorbent is used, it can be readily shown, however, that both film and pore diffusion mass transfer resistances cannot be ignored^^^'^^"^ and that the dynamic behavior of the adsorption stage is greatly dependent on the rate of the polypeptide- or protein-ligate interaction.^'^^^'^^^ Breakthrough of solute(s) may thus occur

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

I 87

at a much shorter time in a system where the polypeptide-or protein-hgate interaction rate is finite compared to systems where local equilibrium exists between the adsorbate and the adsorbate-ligate complex. Such early breakthrough results in a very low utilization of the accessible ligates. With nonporous sorbents much more favorable mass transport and adsorption-desorption kinetic behavior occur. Breakthrough curves obtained from surface modified nonporous silica, for example, are much steeper compared to porous silicas using the same ligand functionality and column configurations (see Figure 33). The use of nonporous HPLC sorbents with favorable mass transport behavior avoids underutilization of the ligates and enables improved mass transport and in principle high mass and biological recoveries compared to equivalent porous affinity sorbent. The downside of nonporous sorbents is their low surface areas per unit volume, which translates to small binding capacities. Over the past decade a variety of theoretical approaches have been developed to account for the binding properties and isothermal behavior of polypeptides and proteins during some of the individual stages of a HPLC process (e.g., the individual adsorption, washing, elution, and regeneration steps). In terms of addressing the optimization and scaling-up of the total HPLC process with polypeptides and proteins, no one generally accepted simulation method has yet emerged. The physical validation of the different mathematical models used to describe these interactive HPLC processes with polypeptides and proteins has thus been mainly limited to analysis of the origins of the breakthrough curves and predominantly related to the adsorption step. Recently, however, some effort has been devoted to the application of more comprehensive mathematical models of actual complete processes using HPLC sorbents that take into account the various physicochemical parameters and molecular properties of the polypeptides and proteins under examination. In these more comprehensive models, the influence of the adsorption kinetics, conformational interconversions, and the effect of axial dispersion on the extent of product loss have been examined. In addition, the issue of column capacity and productivity for columns loaded to an effluent concentration of 5% of the influent value, followed by washing^'^^^ can be included. Similarly, the effect of flow rate and the terminating effluent concentration on the production rate, the yield and the column capacity utilization for the adsorption of proteins with different HPLC systems have been examined.i^^'i^^'i^^'22^'^^^'^1^-^^^'^^^-^^^ Over the past decade, the literature on the theory of packed-bed adsorption and its application, in particular, to preparative affinity-and ion-exchange HPLC separations of polypeptides and proteins has undergone significant evolution. Earlier treatments have generally been based on the substantial body of knowledge derived from chemical engineering analysis of (simple) chromatographic systems, primarily involving low molecular weight solutes. As apparent from earlier sections of this chapter, the adsorption and desorption behavior of polypeptides and proteins with HPLC sorbents can be markedly different to low molecular weight organic compounds. Much more complex equilibrium and kinetic processes

I 88

MILTON T. W. HEARN

arise with polypeptides, proteins, and other high molecular weight biopolymers. This behavior leads to greater difficulties in obtaining analytical solutions to the rigorous mathematical models describing the overall mass transport phenomena. Substantial simplifications and assumptions are often necessary to obtain useful solutions. For example, it is commonly found with low molecular weight compounds that linear adsorption isotherm approximations may be used. In common with other complex compound mixtures, RP-HPLC, HP-HIC, HP-IEX, HP-IMAC, HP-BMC, or HP-BAC separations of polypeptides and proteins often display characteristics of non-Langmuirean adsorption isotherms. Moreover, the very limited availability of suitable experimental data for polypeptides and proteins encompassing complete large-scale chromatographic processes for model verification has typically constrained developments. This limitation is not totally unexpected due to the commercial sensitivity that this type of information holds for manufacturers of high value-added products, such as protein biopharmaceuticals, in the market place. Overall, the theoretical models for the separation of polypeptides and proteins in the interactive HPLC modes can be classified into two groups. In the first group of models (group 7), a single mechanism is assumed as the rate-limiting step for all conditions under study, involving pore diffusion,^^^ pore perfusion,^^^'^"^^"^"*^ or surface interaction mechanisms.^^^ Such assumptions greatly simplify the analysis, and enable analytical solutions of the concentration profile-time relationships to be obtained. For preliminary evaluation of column performance, group I models have proved simple to use, but fail to address the impact of important parameters such as particle size, specific mass transfer coefficients, or surface interaction rate constants, which may assume considerable importance if flow rates or loading conditions are changed. In the group II models, on the other hand, the physical basis of the mass transfer phenomena as the polypeptide or protein mixture passes over the sorbent particles in a stirred tank, a packed bed or an expanded bed is examined by more comprehensive theoretical analysis These group II models are much more rigorous than the group I models in a physical sense, with all of the potential rate-limiting steps in the adsorption processes considered, including the external liquid film mass transfer, internal pore diffusion, and surface interaction.^'i^'^^'^^^'^^^'^^^'^^^'^^^-^^i The resuhing complexity of these group II models mean that analytical solutions are not possible, and resort to various numerical methods of analysis is required to solve the relevant mass transport equations. Currently, such numerical iterations involve excessive computation time and specialized software packages, many of which are routinely not available or readily employed in a "user-friendly" manner by process operators. However, by examining the adsorption behavior of polypeptides and proteins with comparable porous and nonporous particles in finite baths, packed columns and expanded or fluidized beds, an iterative simulation approach based on the heuristic principles described earlier and along the lines of the flow diagram shown in Fig. 32 can be developed, leading ultimately to the implementation of useful scale-up criteria. Along the way, computer simulations, generated from the analysis of the concentration-time

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

I 89

profiles, derived from the information gained from the batch, packed-bed and expanded- or fluidized-bed adsorption and washing stage experiments with different sorbents and mixtures of proteins, can be used to form part of the selection procedure. In this manner, the appropriate choice of the fluid velocity, sample loading volume and concentration, the time to terminate a particular stage, the column length and diameter, the temperature, and other relevant conditions can be made. Due to the complexity of the mass transport with polypeptides and proteins with most HPLC sorbents when used in large-scale operations, it is usually not possible to identify a single ideal optimal operating condition from a limited set of analytical or small-scale experiments. For this reason, computer-assisted optimization^^^"^^^ using, for example, instrumental systems such as the BioCAD, ProSys, etc. becomes important for buffer and sorbent selection and a valuable time-saver. It is important with such systems to identify an optimum region or regions of the derived parameter set, which are compatible with the predetermined production criteria. In this context, the application of heuristic guidelines and simple yet rigorous mathematical models are essential, with analytical or semianalytical solutions preferred over numerical methods. In addition, it is important that the selected model(s) take into account the physical and chemical realities of the system, including all of the known physicochemical effects that may influence the major rate-limiting factors, as well as accommodating the influence of different types of adsorption isotherms. Not unexpectedly, the mass transfer coefficients will largely be affected by changes in the physicochemical properties of the polypeptide- or protein-sorbent system. These properties include the particle size, pore size, and pore fraction of the sorbent; the particle rigidity; the size, shape, and molecular complexity of the polypeptide or protein; the bed porosity; the fluid flow rate; and the fluid viscosity. Fluid viscosity, in turn, is a function of the temperature and the fluid composition. Factors influencing the surface interaction behavior of the polypeptide- or protein-sorbent system have their basis in the chemical nature of the polypeptide or protein, sorbent, and mobile phase composition. Finally, the ligand density and distribution, the distribution of charged, hydrogen bonding or hydrophobic sites on the surface of the polypeptide or protein, its conformation and the nature of its interaction with components in the solvent-feedstock, will all contribute to the surface interaction behavior. The influences of these chemical equilibrium effects on the chromatographic behavior, to a large extent, are still less well understood as the effects due to the physical properties of the system. Although the use of some parameters (such as mass transfer coefficients) may be less rigorous due to the simplifications required for their (fast) calculation, they nevertheless provide a starting point for the more detailed (and usually more time-consuming) studies with the group II models. The various chromatographic processes employed in a packed-bed or an expanded-fluidized-bed configuration for polypeptide and protein separations involve four stages (adsorption, washing, elution, and regeneration) as a sequence of events. Typically, large quantities of fiquid must be processed, with the feed concentration of the target polypeptide and protein possibly

I 90

MILTON T. W. HEARN

low. In these circumstances, the time taken to make full use of the adsorptive capacity of the chromatographic bed may become very long, and the flow rates constrained by the pressure drop. Secondary equilibrium events, e.g., denaturation, or chemical-enzymatic degradation may start to represent a significant source of loss of product. In such cases, carrying out the adsorption stage in a stirred tank (i.e., as a finite bath) can reduce the operating time. Moreover, with fermentation broths or biological extracts containing cell debris and other solid contaminants, stirred tank or fluidized-expandedbed systems are preferable for the initial adsorption stages to avoid clogging of the packed bed. During the initial stages of evaluating new protein adsorption processes at the laboratory scale, finite bath systems are often a simple, more efficient, and cheaper tools to use. A useful literature relating to polypeptide and protein adsorption kinetics and equilibrium behavior in finite bath systems for both affinity and ion-exchange HPLC sorbents is now availablei^^'^^^'i^^-i^^'^^^'^^^'^^^-^^^'^^^-^^^ and various mathematical models have been developed, incorporating data on the adsorption behavior of proteins in a finite ba^h.8>i60,i67-i69,i7i-i74,400,403-405,406 Qne such modd, the so-calkd combined-batch adsorption model (BAM^^jj^^,), initially developed for nonporous particles, takes into account the dynamic adsorption behavior of polypeptides and proteins in a finite bath. Due to the absence of pore diffusion, analytical solutions for nonporous HPLC sorbents can be readily developed using this model and its two simplified cases, and the effects of both surface interaction and film mass transfer can be independently addressed. Based on this knowledge, extension of the BAM^^^mb approach to porous sorbents in bath systems, and subsequently to packed-, expanded-, and fluidized-bed systems, can then be achieved. In a finite bath, at time zero a volume of fluid containing the polypeptides or proteins (adsorbates) of interest is brought into contact with a quantity of a nonporous or porous HPLC sorbent. Interaction is then allowed to occur for a period of time t as equilibrium is approached. The basic assumptions for the rate-limiting steps involved in the simplest case of the BAM^^^jj^^ and related models for the adsorption process in a finite bath are: (a) the transport of adsorbate from the bulk fluid to the surface of the particle can be described by a film resistance mechanism; and (b) the interactions between the adsorbate and the adsorption sites at the particle surface can be described by an appropriate adsorption isotherm, i.e., a competitive Langmuir-type model for example. One of the advantages of the BAM^^j^b model approach is that extended versions of this model can be derived to accommodate the case of complex multicomponent mixtures with nonlinear adsorption behavior where Langmuirean isothermal dependencies may not apply (see Table 10 for other types of isothermal representations and the definition of the terms and abbreviations). Implicit to the preceding treatment, the finite bath is assumed to be well mixed, and therefore the concentration of the polypeptide(s) or protein(s) in the liquid phase is uniform throughout the finite bath. With these assumptions in mind, the mass balance for the polypeptide(s) or protein(s) in the liquid phase and the rate equations for mass transfer and surface interaction can be established. Thus, the overall mass balance for the

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

I9 I

adsorption of a protein to nonporous particles in a finite bath can be expressed as eC + (1 - e)q = eC^

(124)

where C is the protein concentration in the bulk of the liquid phase, q is the polypeptide or protein concentration on the solid phase, and e is the volume fraction of liquid phase in the finite bath. The variable Cj is the equivalent concentration w^hen the total amount of the polypeptide or protein in the system is assumed to be only in the liquid phase, and can be calculated from Cj=C, + R,qo

(125)

where CQ is the initial protein concentration in the liquid phase, ^Q i^ the initial protein concentration in the solid phase, and Ru(= (1 — e ) / e ) is the volume ratio of the sofid phase to the Hquid phase. For adsorption with fresh or regenerated adsorbent particles, qo = 0 and Cj = CQ. Similarly, at the washing and elution stages, CQ = 0 and Cj = R^qQ. The differential form of Eq. (125) then can be expressed as dC

dq

In many cases with the polypeptides and proteins the mass transport from the bulk fluid to the surface of the nonporous particle can be described by a film resistance mechanism, such that dq — = aKfiC-C*)

(127)

where a(= 3/RQ) is the interfacial area per unit volume of the adsorbent particles, RQ is the radius of the particle assuming the average shape can be represented as a sphere, K^ is the hquid film mass transfer coefficient, and C* is the intermediate concentration of the protein in the liquid phase at the surface of the particles. The interaction between the protein and the adsorption sites at the particle surface can then be described in terms of a secondorder reversible process, such that ^=k,[iq„-q)C*-K,q]

(128)

where k^ is the forward interaction rate constant, q^ is the maximum adsorption capacity of the sorbent, and K^ is the adsorption equilibrium constant. If, at equiUbrium, in the simplest case Eq. (128) takes the form of the Langmuir isotherm equation, and the values of both the film mass transfer and surface interaction rates are finite, then two simplified cases can be considered. In the first case, the surface interaction rate constant i^j -^ oo, and therefore the external mass transfer becomes the rate-controlling step of the batch adsorption mechanism. The occurrence of this type of interaction implies that equilibrium exists between the adsorbate and the adsorbate-ligate

I 92

MILTON T. W. HEARN

complex at each point on the particle surface. In the second case, Kyr ^ oo and the surface interaction (second-order kinetics) is then the rate-controlling step. In the cases where the value of the liquid mass transfer coefficient K^r is finite, a bisectional method can be adopted to calculate the profiles. Analytical solutions and simulations based on the BAM^^j^i, model can then be used directly to calculate the time-concentration profiles. Since pore diffusion processes with porous sorbents can be described in terms of a linear driving force approximation,^^"^'"^^^ extension of such BAM^Qjj^t, models to cover the case of polypeptide or protein adsorption to porous particles in a finite bath can be achieved. With this approximation, the pore fluid can be treated as a mass transfer medium rather than as a separate phase, thus enabling the pore volume to be combined with the bulk fluid volume in the overall mass balance. In an actual HPLC separation process, the pores of the sorbent particles are normally filled with buffer liquid before the adsorption process commences. As the volume of pore fluid is normally very small in comparison with the volume of bulk fluid in a bath system, it may be neglected or lumped with the bulk fluid term. Hence, two simplifying options can be considered for the evaluation of the differential form of the mass balance equations, which become the same as in the case with the nonporous particles, but now with different phase volume ratios. Again, the dominant rate limiting steps in this adsorption process are the mass transfers and the surface interaction with both processes taken into account with polypeptides and proteins.^^^ Moreover, with porous particles, the internal surface area is normally much greater than the external surface area. The effect of the external surface area may either be neglected (in the case where the external surface area is less than, e.g., 1.0% of the total surface area) or lumped with the internal surface area in the simulation-analysis procedures. Hence, with porous particles, surface interaction will predominantly occur when the polypeptide and protein adsorbates reach the internal surface of the particles, thus enabling the mass balance, rate-limiting steps, and the mass transfer coefficients to be quantitatively and independently described. If it is assumed that the pores of the porous HPLC particles are initially filled with buffer liquid before the adsorption process starts, then the overall mass balance for a polypeptide or protein in a finite bath is given by eC + i l - 6 ) e , C , + (1 - €)(1 - e^)q = [e + {1 - e)e^]C^

(129)

where Cp is the protein concentration in the pore fluid, e^ is the particle void fraction, and q is mass of polypeptide or protein bound per unit volume of the sorbent. In the case of the adsorption of the polypeptide(s) or protein(s) to fresh or regenerated adsorbent particles, ^Q "^ 0, C^ = 0, and Cj can be written as

CT-

eCo ,,

(130)

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

I93

During the washing stage, CQ = 0 and C^ becomes r

(l-^)epC,o

""

+

( l - 6 ) ( l - 6 , ) ^ o

e+(l-e)e,

^'^'^

In circumstances where the pore volume can be neglected, i.e., when e^ ^ 0 such as for pellicular HPLC sorbents, or shielded ligate sorbents, Eq. (131) becomes the same as for the nonporous particles (Eq. 128). The differential form of this equation then becomes the same as Eq. (126), with the same volume ratio, R^ = (1 - e)/£. On the other hand, when the pore fluid is significant, but can be lumped with the bulk fluid, then Cp = C and Eq. (131) becomes

[6 + (1 - 6)6,]C + (1 - e){l - ep)q = [e + (1 - 6 ) € , ] Q (132) The differential form of Eq. (132) is dC da ^ +R „ ^ =0

(133)

which is the same as Eq. (129), except the volume ratio of the sohd phase to the hquid phase, R^, becomes

R. =

1 - [e+(l- e ) e J \ . / ^ e+{l-e)

(134)

P

Because the internal surface areas of porous HPLC particles are normally much greater than their external surface areas, the mass transfer rate of the polypeptide(s) or protein(s) adsorbates from the bulk fluid to the internal particle surface can be expressed as Nflux = Kf(C- C*) = Kp(C* - Cj) = K , ( C - Q )

(135)

where N^^^ is the mass flux of the polypeptide(s) or protein(s) adsorbate into the particle, K^ is the liquid film mass transfer coefficient, Kp is the apparent pore fluid mass transfer coefficient, and K^ is the overall effective liquid phase mass transfer coefficient. The term C* is the intermediate concentration of the polypeptide(s) or protein(s) in the liquid phase at the external surface of the particles, and Q is the intermediate concentration of the protein in the liquid phase at the internal surface of the particles. When the volume of the liquid film is negligible, and there is no accumulation of the polypeptide(s) or protein(s) in the pore fluid, the rate of change in the concentration of the polypeptide(s) or protein(s) in the solid phase then must equal the rate of mass transfer, i.e., -^

= aKf{C-C*)=aKp{C*-C,)

(136)

194

MILTON T. W. HEARN

where the term a = O / R Q ) is the external surface area per unit volume of the adsorbent particles, and RQ is the radius of the particle. The following form of the rate of change of polypeptide or protein concentration with these porous HPLC particles can thus be written as dq — =aKXC-C,)

(137)

and 1 ^e

1 ^f

1 ^p

Hence, the overall resistance to the mass transfer predicted by this variant of the BAM^omb rnodel is the sum of the resistance in the hquid film and the resistance in the pore fluid. The value of the Uquid film mass transfer coefficient K^ can be calculated from literature correlations,"^^^'"^^^'"^^^ while the method for the estimation of the apparent pore liquid mass transfer coefficient Kp can be derived from Eq. (163). The surface interaction between the protein and the immobilized ligand at the internal particle surface can be treated in the same way as for nonporous particles, i.e., ^=k^[iq^-q)C,-K,q]

(139)

where k^ is the forward interaction rate constant, q^ is the maximum adsorption capacity of the immobilized ligand, and K^ is the adsorption equilibrium constant. Based on a similar approach, the adsorption behavior of polypeptides or proteins can be modeled with porous sorbents, with the transport of adsorbate from the bulk fluid to the internal surface of the particle occurring in two stages. In the first stage, the polypeptides or proteins diffuse through a thin liquid film to reach the external surface of the particle where the entrances to the pores are located. In the second stage, the polypeptides or proteins diffuse through the pore fluid, which is stagnant, to reach the internal surfaces of the particle where surface interaction between the polypeptides or proteins adsorbates and the ligate predominantly occurs. As both the liquid film on the external surface of the particle and the pore fluid are the media for mass transfer, both processes can be described by a linear driving force approximation. Therefore, the mass transfer rates of the different polypeptides or proteins adsorbates from the bulk fluid to the internal particle surface can be expressed in terms of the relevant physicochemical parameters within the physical constraints of the interaction. Assuming that the volume of the liquid film is negligible, and that there is no accumulation of the polypeptides or proteins in the pore fluid, the rate of change in the concentration of each polypeptide or protein adsorbate in the solid phase then equals to the rate of its mass transfer. According to Eq. (138), the overall resistance to the mass transfer will be the sum of the resistance in the liquid film and the resistance in the pore fluid. In an

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

I 95

analogous manner, the interaction between the proteins and the immobiUzed Hgands at the internal particle surface can be described by the second-order reversible equation. This outcome is similar to that found for nonporous particles, except that the adsorbate concentration(s) in the liquid phase at external surface of the particles C* is replaced by Q , the concentration of the polypeptide(s) or protein(s) in the liquid phase at internal surface of the particles. The accurate prediction of the elution profiles for polypeptide(s) or protein(s) from columns packed with nonporous and porous adsorbents represents a much more significant challenge. In the case of the use of nonporous sorbents in packed beds, an analytical solution can again be obtained. For porous adsorbents in packed beds, a sectional adsorption model (SAM), based on the (hypothetical) ability to treat the bed as a series of tanks-in-series has found wide application. Optimum performance characteristics can be determined, particularly with regard to the effect of particle size, loading, and flow rates, with nonporous particles in packed columns, by initially assuming that (a) the effect of axial diffusion is negligible, and the fluid velocity is uniform over the cross section of the column; (b) transport of the polypeptide or protein adsorbate(s) from the bulk fluid to the surface of the particle can be described by a film resistance mechanism; and (c) the interaction between the polypeptide or protein adsorbate(s) and the ligates at the particle surface can be described by an appropriate multicomponent adsorption isotherm that reflects the more complex (and more physically realistic cases involving multicomponent systems). Characterization of the physical form of this multicomponent adsorption isotherm, i.e., whether it is a FreundlichJovanovic, competitive Temkin isotherm, or the like, can usually be achieved using the appropriate nonlinear least-squares procedures, based on the analytical methods developed more than 50 years ago by Thomas,"^^^ and subsequently modified by Hiester and Vermeulen"^^^ and Sherwood et al,"^^^ When packed beds or fluidized-expanded beds of defined bed expansion are involved, further consideration must come into play. Thus, by assuming that axial diffusion can be neglected, the continuity equation linking concentration, axial distance and time for nonporous particle HPLC sorbents takes the modified form of Eq. (120), namely^^^'^^^ Uf) dC

—

e dx

dC

+

dt

1 — e dq

+

e

^ =0

dt

(140) ^

^

where UQ is the superficial liquid velocity, e is the interstitial void fraction of the packed bed, x is the axial distance, t is time, C is the protein concentration in the bulk of the liquid phase, and q is the protein concentration adsorbed to the nonporous solid phase. The rate of mass transfer in the liquid film at the particle surface can then be described by

j-^=aKf{C-C*)

(141)

I 96

MILTON T. W. HEARN

where a{= 3 / R Q ) is the external surface area per unit volume of the particle. The term RQ is the radius of the particle, X^ is the Hquid film mass transfer coefficient, and C* is the intermediate concentration of the protein in the liquid phase at the surface of the particles. The surface interaction rate is described by the second-order reversible equation ^-^=kA{qn,-^)C*-K,q\ ot

(142)

v^here k^ is the forward interaction rate constant, q^ is the maximum adsorption capacity of the sorbent particles, and K^ is the adsorption equihbrium constant. Eliminating C* from Eqs. (142) and (143) yields

dt

aKf+k,{q^-q)

Interpretation of the adsorption behavior of polypeptides and protein with nonporous HPLC sorbents can thus be based on Eqs. (140)-(143) in which the film mass transfer and surface interaction rates are both considered finite. Simplified cases can be derived from these two relationships for fixed-bed performance^^^''*^^''*^^ where the equiUbrium relationship can be expressed by the Langmuir isotherm. Under these isothermal conditions, attainable adsorption capacity of the adsorbent ^*, which is the amount of the protein retained by the adsorbent when the column reaches saturation, can be expressed by ^* = T-%^^rn

(144)

where CQ is the protein concentration in the inlet solution. By introducing the dimensionless terms X = C / C Q and Y = q/q*, the position and shape of the breakthrough curve can be expressed in the following form"^^^'"*^^'"*^^: C — = Co J{r-z,

/(r*^,0 — — 0 + [1 - Jiz, r * 0 ] e x p [ ( r * - l ) ( f - z)]

ri45) ^ ^

where K.

(146)

K, + C,

and the dimensionless distance parameter z and the dimensionless time parameter T can be given by z =

^'^

T=KJT-—1

K,

(147)

(148)

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

I 97

The continuity Eqs. (120) and (140) then can be simphfied to dX

dY

with the boundary conditions, assuming that the bed is initially free of adsorbate, given be X = 1

at ^ = 0 for all T

Y= 0

at T = 0 for a l U

The average dimensionless sohd phase concentration Y* can be given"^^^ the numerical value of 0.5, and if the effect of the film mass transfer is negligible, i.e., if Kf -^ oo, then adsorption with the nonporous HPLC sorbent in a well-packed bed is controlled by second-order kinetics.^^^'^^^ When the external film resistance iC^ controls the adsorption, equilibrium is assumed to exist between the polypeptide or protein and the polypeptide- or protein-ligate complex at each point on the particle surface. In the sectional model, the column is hypothetical divided into a series of sections with each section treated as a well-mixed tank, analogous to the discrete cell or discrete stage model.^^^''^^'^''^^^ In each section the fluid containing the polypeptide or protein (adsorbate) of interest is brought into contact with the adsorbent and interaction is allowed to occur for a period of time Af. At the end of each time increment, the contents of the liquid phase in each section is transferred to the next section. As each section is assumed to be weU mixed, the concentration of the adsorbate in the liquid phase will be uniform throughout the section. The sectional model can thus be used for both porous and nonporous particles with the fluid flow then conceptually treated as a noncontinuous parameter. In each section of the column, the fluid containing the polypeptide or protein adsorbate is brought into contact with the adsorbent and interaction is allowed to occur for a period of time A?. At the end of each time increment, the content of the hquid (mobile) phase in each section is transferred to the next section. The time increment A? is calculated as A^ = L/iu^m)^ where L is the column length, UQ the superficial velocity (linear flow rate) of the fluid, and m is the number of the sections. For adsorption of the polypeptide or protein with fresh or regenerated adsorbent particles, the initial polypeptide or protein concentration in the solid (stationary) phase is zero for all the sections at the beginning of the process, and the column can be assumed to be saturated with buffer solution. During the adsorption stage, the initial polypeptide or protein concentration in the liquid phase is equal to Q , the inlet concentration. For washing and elution stages, CQ = 0. In the case of a single component isotherm, the assumptions are: (a) the transport of polypeptide or protein adsorbate from the bulk fluid to the surface of the particle can be described by a film resistance mechanism and (b) the interaction occurs between the polypeptide or protein adsorbate and independent binding sites at the particle surface. As each section of the column bed is assumed to be well mixed, the polypeptide or protein concentration in the liquid phase is assumed to be uniform

I 98

MILTON T. W. HEARN

throughout that section. This treatment permits other types of isothermal behavior, e.g., FreundUch-Jovanovic, competitive Temkin isotherm, muhicomponent Freundhch-Langmuirean isotherms to also be accommodated."^^ ^ The overall mass balance for the adsorption of polypeptides and proteins with nonporous particles in a section / is given by €C, + ( l - e ) ^ , = e Q , ,

(150)

where C^ is the protein concentration in the bulk of the liquid phase, q^ is the protein concentration on the solid phase, and e is the volume fraction of liquid phase in the column, which is assumed a constant throughout the column. The variable C j • can be calculated from CT,i = Co,, + RWo,i

(151)

where CQ^ ^ is the protein concentration in the liquid phase at the beginning of the time increment, q^^, is the protein concentration in the solid phase at the beginning of the time increment, and 1^^; is the volume ratio of the solid phase to the liquid phase, i.e., the phase ratio 4>, 1 -e Rv = - 7 -

(152)

The differential form of Eq. (153) then can be expressed as

da

da:

• ^ ^R „ ^ = 0

(153)

The transport of the protein from the bulk fluid to the surface of the particle can be described by a film resistance mechanism according to the relationship ^

= ^K^(C,-C*)

(154)

where a(= 3/RQ) is the interfacial area per unit volume of the adsorbent particles, RQ is the radius of the particle, K^ is the liquid film mass transfer coefficient, and Cf is the intermediate concentration of the protein in the liquid phase at the surface of the particles. Similarly, the surface interaction rate is described by the second-order reversible relationship: ^

= m^n.-c}dCt-K,q,]

(155)

where k^ is the forward interaction rate constant, q^ is the maximum adsorption capacity of the adsorbent particles, and K^ is the adsorption equihbrium constant. At equifibrium, Eq. (155) becomes the Langmuir isotherm equation when first-order kinetic prevail. In an analogous manner to the behavior of porous HPLC sorbents in a batch stirred bath, a linear driving force approximation can be used with the

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

I 99

sectional model for porous particle in a well-packed column. In addition, the pore fluid can be assumed to be stagnant, therefore only the bulk fluid will be transferred to the next section at the end of each time increment. The overall mass balance in section / is thus given by e Q + (1 - 6)6pC,,, + (1 - 6)(1 - €p)^, = [e + (1 - 6 ) e , ] Q , ,

(156)

where C^ , is the protein concentration in the pore fluid, and e^ is the particle void fraction. Other symbols are the same as defined for Eq. (150). The unit of q^ is taken as the mass of the polypeptide or protein bound per unit volume of solid. Arising from the linear driving force approximation, the pore fluid has been treated as a mass transfer medium rather than a separate phase, thus enabling it to be combined with the bulk fluid in the overall mass balance. Therefore, the pore fluid was lumped with the bulk fluid, i.e., Cpi = Q . Equation (157) then becomes [€ + (1 - €)€p]C, + (1 - €)(1 - e^)q, = [e + (1 - €)e^]Cj^,

(157)

In this case with porous HPLC sorbents, the value of Cj ^ can be calculated by Eq. (157), while the volume ratio of the sohd phase to the liquid phase R^^ becomes 1 - [ 6 + (1 - 6 ) 6 , 1

and the value of Q ^ can be calculated from -

g

(1

-€)ep

^''^ " e + ( 1 - 6 ) 6 , ^ 0 ' ' - ^ "^ € + ( l - € ) € , ^ ' ' ' '

^^^^^

Thus, at the end of each time increment, the pore fluid has in effect remained in the section, and only the bulk fluid in section / — 1 has been transferred to section /. With porous HPLC sorbents, the effective overall liquid phase mass transfer coefficient K^ replaces K^r, and Cf is now defined as the intermediate concentration of the protein in the liquid phase at the internal surface of the particles. The concentrations of the protein in liquid phase for each section can be then calculated with Eq. (159), while the boundary condition of the liquid phase concentration in the last section C„ is the outlet concentration. The concentration-time plot, i.e., the breakthrough curve, can then constructed and compared to the experimental findings. In common with adsorption to nonporous sorbents in packed beds, two simplified cases can also be considered with the sectional model approach with porous HPLC sorbents. When the surface interaction rate constant k^ ^ ^^ the mass transfer through the stagnant fluid will be the rate-determining step. In the case where the mass transfer rate is very high (abbreviated as the kinetic-controlling case, SAMj^j^^), then the surface interaction is considered as the rate-controlling step.

200

MILTON T. W. HEARN

For the case with porous particles, the pore fluid can be treated as a mass transfer medium rather than a separate phase thus enabling it to be combined with the bulk fluid in the overall mass balance. Under plug flow transfer conditions, at the end of each time increment, the pore fluid was assumed to remain stagnant, and only the bulk fluid was transferred to the next section. Based on these assumptions and initial conditions, the concentrations of the polypeptide or protein adsorbate in both liquid and solid phase can be calculated. The hquid phase concentration in the last section C„, is the outlet concentration. The concentration-time plot, i.e., the breakthrough curve, can then be constructed. Utilizing this approach, the axial concentration profiles can also be produced for any particular time since the concentrations in each section for each complete time cycle are also derived. For interpretation and simulation of the adsorption behavior of polypeptides and proteins with HPLC sorbents by the sectional model, one of the key variables is the number of the sections n. Typically, if n > 16, the effect of "numerical dispersion" will be negligible and the breakthrough curves produced by the sectional model and the analytical solution of a packed bed containing nonporous HPLC sorbents become synonymous. The advantage of the sectional model is thus its computational versatility. To predict the behavior of a given system, certain specific information must be obtained experimentally and, in particular, data on the adsorption characteristics of the target polypeptides(s) and protein(s) in the presence of different sorbent particles. As noted earlier, the Langmuir isotherm is still the most commonly used description of the interaction of polypeptides or proteins in RP-HPLC, HP-HIC, HP-IEX, HP-IMAC, HP-BAC, and HP-BMC processes, although other isothermal representations accommodate the adsorption phenomena in a more physically consistent manner.^^^' ^^^'3^9,398,417,418 j j ^ ^ Langmuir relationship for the equilibrium concentration of the polypeptide or protein n the solid phase ^* and the equilibrium concentration in the liquid phase C* can be expressed in the following double reciprocal plot form 1 q*

^Ei± + 1
q„

(160)

where q^ is the maximum adsorption capacity, and K^ is the equilibrium constant. Alternative representations of the Langmuirean equation are the semireciprocal plot (Eq. 161) and the Scatchard plot (Eq. 162) C* C* K. — = — + — q
(161)

11 = 1^ + ^

(162)

C*

^

K,

K,

^

From Eqs. (160)-(162), the characteristic parameters of maximum capacity q^ and equiHbrium constant K^ can be determined^''^'^^^'"^^^''^^^ by a least-squares fit of the experimental adsorption data generated using batch methods from the plot oi l/q* against I / C Q . Due to the nature of the data

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

20 I

acquisition, the double reciprocal plot favors the experimental data obtained with polypeptides and protein at low concentration, while the semireciprocal plot gives more weight to the data obtained at higher adsorbate concentrations. Nonlinearity in the Scatchard plot is an excellent indicator of the extent of deviation from Langmuirean behavior. Similarly, the adsorption isotherm can be generated using packed-column or fluidized-expanded-bed methods^^^ where in this case, the variable q* in Eqs. (160)-(162) becomes the adsorption capacity of the sorbent, which is the amount of the adsorbate retained by the sorbent when the bed reaches saturation. In this case, the equilibrium concentration in the liquid phase, C* in Eqs. (1) to (4) can then be replaced by CQ which is the protein adsorbate concentration in the inlet solution. The values of ^* at given CQ can then be evaluated by numerical integration of the area behind the breakthrough curve.^^^"^^"^ However, it must be recognized in the absence of detailed knowledge on the nature of the actual isotherm that prevails for the particular polypeptide-or protein-HPLC sorbent system, that the values of these parameters may not be physically precise over the whole range of concentrations encountered in a real biochromatographic system. To implement these simulation approaches, the value of the liquid film mass transfer coefficient K^ is required, which for nonporous and porous HPLC particles, can be calculated from literature correlations derived for bath^^^'"^^^'"^^^ or column models."^^^'"^^^ For the case with porous particles, the apparent pore fiquid mass transfer coefficient Kp can be expressed as an effective pore diffusivity over an average effective diffusion path length, such that

where D^ is the effective pore diffusivity, and A* is an area factor. The apparent pore liquid mass transfer coefficient Kp is based on the external surface area a, while D^ is based on the total area perpendicular to the direction of diffusion a*, which is less than the total internal surface area per unit volume of the particle but larger than ^ (A* is the ratio of a""/a). The term cri^Q is the average effective diffusion path length that is expressed as a linear function of the particle radius RQ- The effective diffusivity D^, according to the random pore model,"^^^ can be estimated from ^e = ^U^l

(164)

where D^ is the free molecular diffusivity, calculated from a literature correlation."^^ ^ With chromatographic systems involving polypeptides and protein purification, the physical properties of the system and operating conditions are often predetermined. For polypeptides and proteins of known molecular weight, the liquid-phase mass transfer coefficients can be evaluated as discussed earlier. However, the forward surface interaction rate constant {k^ must be determined from experimental data via a parameter-fitting program

202

MILTON T. W. HEARN

written for this purpose. Once numerical values for these parameters for a specific system having been derived, the opportunity exists to predict the adsorption behavior for any polypeptide or protein w^ith a HPLC sorbent over a range of operating conditions. Illustrative of this approach is the comparisons betv^een the simulated concentration-time profiles'*^ ^ with the experimental adsorption behavior of human serum albumin (HSA) to the weak anion-exchanger DEAE Sepharose Fast Flow adsorbent shown in Fig. 36 for two initial protein concentration values. As is evident from these plots, the agreement between the predicted and experimental determined data for CQ = 0.65 mg/mL data was excellent, but for the CQ = 0.93 m g / m L data, a slower adsorption rate at the early stages of the process was predicted. As the isotherm parameters and rate-limiting parameters in these systems are largely determined by the particular polypeptide- or protein-sorbent combination and their environment (such as temperature, pH, and ionic strength), the parameters that can be varied during an experimental study are the operating parameters. The initial concentration CQ is one such parameter, the solid to liquid volume ratio R^^ value, formerly equivalent to the phase ratio is another. Achievements of high production rates and a high yields are important considerations for any commercial process. The production rate can be defined by dividing the total amount of protein adsorbed when a predetermined value of yield has been reached, by the amount of sorbent in the system, and by the total processing time. In turn, the total processing time can be defined as the sum of adsorption time required to achieve a certain yield, and any preparation time associated with the adsorption stage. The processing time, typically with polypeptides and proteins, tends to increase almost linearly with a decrease in the R^ value on a log-log scale with many types of HPLC sorbents. For each yield requirement, a particular value of R^ must thus be found, offering the maximum production rate.

1.0

a

C^ = 0.93 mg/ml C^ = 0.65 mg/ml

^0.8 C 0.6

.2 I 0.4 a

I* '^B

it

d 0.2 O U 0.0

«

2

4

•

6

" •

-•-..^

10

Time (min) F I G U R E 3 6 Experimental data and theoretically generated concentration curves for the adsorption of HAS to a weak anion exchange sorbent DEAE-Sepharose FF at two different protein concentrations. As is evident from the data shown in this figure, the BAM^-o^i, model used in this case predicted a slower adsorption rate during the earlier stages of the adsorption process when higher protein concentrations were employed.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

203

As discussed elsewhere in this chapter, compUcated mass transfer and surface interaction processes, e.g., aggregation, muhilayer formation, conformational changes, and surface reorientation at high protein concentrations in the microenvironment of the ligates of the sorbent, may lead divergences between the predicted and experimental breakthrough curves. For a large number, but not all, of polypeptide- or protein-ligate systems, the adsorption is a reversible process. During the washing stage, these equilibrating polypeptides or proteins that have already been adsorbed onto the solid phase will reenter the liquid phase and may be carried out of the column. In these circumstances, at the end of the washing stage the amount of the polypeptide or protein retained in the column will not be the same as at the end of the adsorption stage,"*^^ with the binding efficiency ( = yield) dropping significantly below 100%. The effects of a washing step on the position and shape of a breakthrough curve and the amount of protein retained in the column are shown in Fig. 37 using hen egg white lysozyme and Cibacron Blue F3GA immobilized Fractosil 1000 system as the exemplar. In Fig. 37a the time-concentration

§

1 8

8 §

6

•(3

0.0

0.2 0.4 0.6 0.8 Dimensionless Column Length

F I G U R E 3 7 The upper panel (a) shows the time concentration curves for the loading of a H P L C - B M C column (Cibacron Blue F3GA Fractosil 1000 with HEWL) to an effluent concentrations of 2, 20, and 99% of the influent concentration, respectively, followed by washing, (b) The lower panel shows the concentration profiles in the solid phase corresponding to the numbered times the upper for this protein-sorbent combination. Data adapted from Ref. 8.

204

MILTON T. W. HEARN

curves are shown for loading the column to effluent concentrations of 2, 10, and 99% of the influent concentration, respectively, follow^ed by w^ashing. The numbers on the 10% curve correspond to four stages of the operation as follows: the column at the end of loading (curve 1), and the column washed with 1-column volume (curve 2), 10 column volumes (curve 3), and 30 column volumes (curve 4) of buffer. The dimensionless concentration profiles in the solid phase for the 10% curve at these stages of the process are shown in Fig. 37b. Although the protein solution was no longer applied to the column, as evident from Fig. 37 the effluent concentration continued to rise even after the column was washed with 10 column volumes of buffer solution (curve 3). As observed with this HEWL-Cibacron Blue F3GA immobilized Fractosil 1000 system, and numerous other cases of polypeptide or protein interaction with HPLC sorbents, the maximum production rate tends to increase with the increase of the terminating effluent concentration. At fluid velocities lower than the optimum velocities, the effect of the terminating effluent concentration, however, becomes less important. The use of a flow rate at the maximum capacity of the pump (or to the pressure limit of the system as is sometimes practiced) will usually lead to an impaired production rate with HP-BAC, HP-BMC, and HP-HIC sorbents.^^^'^^^'^^^ This conclusion has been also supported by other experimental data on large-scale chromatographic purification of proteins with HP-IEX sorbents.^^^''^^^''^^^''^^'^ Often with large-scale HPLC processes, such as the purification of human serum albumin, thrombin, or a^-proteinase inhibitor from crude feedstocks, the choice of operating conditions for the washing stage such as the amount of washing solution used and the washing flow rate, are constrained by the nature of the contaminants (e.g., in terms of their molecular weights and concentrations) and the purity requirement of the target protein. For example, in the purification of human thrombin present in the Cohn II -h III cryosupernatent fraction of blood plasma using immobilized heparin silicabased affinity sorbents with columns of larger dimensions (e.g., 10-30 cm), enhanced purification factors and higher productivities could be achieved through careful attention to the impact of the forward interaction rate constant k^ the desorption rate constant ^ _ i , the maximum adsorption capacity of the sorbent q^^ the sensitivity of the equiUbrium constant Kj to temperature effects, the initial concentration CQ, the solid to liquid volume ratio R^, and the choice of superficial velocity on the overall recovery. One way that these operation regions can be identified to achieve optimization of the performance and productivity is through the use of contour plots derived directly from the experimental data for a particular polypeptide- or protein-ligate system as illustrated in Fig. 38. In this case, the optimization was based on a primary requirement of 94% yield. If a 60% column capacity utilization was the preferred option, the separation would have to be carried out in the region enclosed by the 94% yield curve and to the left of the 60% capacity curve. It can be seen that the highest production rate in the region occurs at the intersection of these two curves. Similar operational boundaries can be determined for the other cases, such as 70 or 50% as the preferred column capacities, and the values of fluid velocity and

205

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

12 10

u

J /

\ 1

A1 180/ 1 / 1 / 1 (/ \l/ //

\Proauf:tion

Rat^

~v{

i/ \ / / ' \/0.13 . VcoihmnRapacity (9i)^.^^^ ^

J

h

200

T7

400

/>-^ 600

1/0.12

800

1000

Linear Flow Rate (cm / h) F I G U R E 3 8 Operational regions at 94% yield for four different utilizations of the column capacity (50, 60, 70, and 80%) levels for the loading of a H P L C - B M C column (Cibacron Blue F3GA Fractosil 1000 with HEWL). Data from Mao, Q. M. and Hearn, M. T. W., 1996. Biotechnoi Bioeng., 52, 202. Reprinted by permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.

effluent concentration at these points then calculated (by, for example, the BEDSTP program^). Based on these preceding considerations, the influence of the operating range and the constraints imposed by the main operating parameters can then be identified. Typically, in these systems, the highest yield will be restricted with packed columns to the low flow rates and relatively low loading region. This interdependence between flow rate and the column loading is illustrated in Fig. 39, where the maximum production rate at these yields is indicated by open circles. For each yield curve, there is only one set of operating conditions that will provide a maximal production rate. If the yield was allowed to drop from 100 to 99%, this 1% reduction in yield could result in a 6 0 - 7 0 %

0

500

1000

1500

2000

2500

Linear Flow Rate (cm/h) F I G U R E 3 9 Operating regions based on the product yield and processing rate for a human serum albumin (HAS) - DEAE Trisacryl M ion-exchange chromatographic system. The open circles are the maximum production rates (0.87 and 1.46 m g / m L • min) for the 100 and 99% product yield curves derived from the tanks-in-series sectional model approach. Data from Mao, Q. M. and Hearn, M. T. W., 1996. Biotechnoi. Bioeng., 52, 202. Reprinted by permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.

206

MILTON T. W. HEARN

increase of the processing rate as found for the purification of HEWL. Depending on the actual yield requirement of the process, the expected production rates and the operating range of the flow rate and column loading can thus be easily estimated from these plots. Since the choice of optimum fluid velocity is subject to the practical pressure drop limitation,"*^^ the relationships between packed-bed pressure drop, superficial fluid velocity, and particle size can be calculated based on the Blake-Kozeny equation,"^^^ assuming rigid particles. If the predicted optimum fluid velocity exceeds the limit imposed by the system pressure drop, then a shorter column with a larger diameter must be used. These conclusions appear to be generally applicable for most preparative HP-BAC, HP-BMC, and HP-HIC sorbents now available for the separations of proteins at the process level. Several practical criteria thus need to be taken into account when choosing a suitable column packing material for a specific chromatographic process. Since the aim is to achieve a cost-effective preparative fractionation of a complex biological mixture with maintenance of the biological activity and high mass recovery, it is prudent to devote at the initial (smaller scale) end of the process development considerable effort to the evaluation and selection of the most appropriate sorbent from other alternatives. The rapid screening of sorbents can now be easily carried out in a semiautomated or totally automated fashion with robotic systems coupled to computer-assisted HPLC equipment systems. These approaches to process design enable conditions to be contemporaneously established that enable high resolution without sacrificing separation times. Experience has shown^^'^^^'^^^ that sorbents with the highest ligand densities do not necessarily provide the highest throughput, even when other features, such as chemical or pressure stability, compatibility with industrial sterilisation methods, etc., are satisfied. This conclusion applies equally well to tailor-made sorbents based on various agarose, dextrans, polyacrylamide, or trishydroxymethylpolyacryl-amide soft gel sorbents, or based on the more rigid chemically modified silicas and zirconias as well as the newer classes of crosslinked polymer based supports, such as the macroporous, polystyrene-divinyl benzene sorbents. The highest (possible) ligate densities are not synonymous with sorbents that achieve the maximal throughput and productivity. For example, various studies^^^'"*^^'"*^^ have shown that there is little to be gained by attempting to immobilize saturation levels of antibodies onto porous supports to generate very high density immunoaffinity sorbents, and similar observations have been made^'^^^'^^^'^^^'^^^'"^^^'"^^^ with reversed-phase, biomimetic affinity, and biospecific affinity sorbents specifically developed with polypeptide and enzyme purification in mind. Again, criteria developed from the heuristic guidelines can be profitably used to aid the design selection. Table 9 has Usted some of the common characteristics of the soft gels and silica-based rigid sorbents that are used as preparative packing materials in polypeptide or protein purification. New sorbents suitable for large-scale purification of polypeptides and proteins are continuously being developed with enlarged average pore diameters from 300 to 6000 A and particle sizes between 10 and 150 fim to freely accommodate biomacromolecules with molecular weights of 200 kDa and more. For a specific preparative HPLC

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

207

system to be successfully applied with these new classes of sorbents (as is also the case with the more traditional types of HPLC sorbents), it is essential that the relationship between mass loadability and productivity, i.e., the product throughput in terms of grams (or kilograms) per cycle per currency unit per unit time, is determined. Early identification of unacceptable system effects associated with time-dependent denaturation or degradation events with the target polypeptide or protein or the participation of secondary chemical equilibrium processes due to isodesmic self-association or heteroprotein aggregation will also have major impacts on the quality and outcome of the purification approach. Although proteolytic degradation can to some extent be minimized by the use of generic inhibitors of serine, thiol, and some metalloproteases, the inhibition of much more specific degradative enzymes may not be addressed by such procedures. With many feedstocks obtained from fermentation, cell culture, or tissue extraction, such specific enzymes are often present in trace amounts and packaged as copurifying cell debris, often adsorptively bound to residual lipid membrane micelles or to the protein of interest. Careful inspection of the analytical data generated during the initial stages of the purification stratagem, e.g., objective assessment of the SDS-PAGE, analytical HPLC, HP-CEC, or HP-CZE data, can, however, give early warning of about such occurrences and suggest likely remedies. The use, for example, of the multiple "tea bag" affinity chromatographic approach^^'^^^'"^^^"^^^ or the modified 96-well microtiter plate adsorption reactor for the identification of cell proteases, antitoxins, nucleic acid fragments, or other types of contaminants can be particularly effective at the initial stages of procedures involving scaling up regimes. Once again, the evolution and usage of such "microscreening" methods have their origins directly in the application of the heuristic rule 4. As evident from the preceding discussion, equilibrium adsorption parameters describing the batch (or bath) behavior of polypeptides and proteins with HPLC sorbents can be used to predict the corresponding behavior with packed-bed column systems.i^^'i^^'^^i-^^^'i^^'^^^-^^^'^^^-^^i'^^^'^o^ Data extracted from laboratory- and pilot-scale column procedures based on frontal analysis methods with mechanically rigid sorbents have been found in some circumstances to yield similar equilibrium parameters to those obtained from batch techniques.^'^^^'^''^'^^^'^^^ In other cases, particularly with sorbents derived from mechanical soft, organic polymeric materials, the binding data derived from frontal breakthrough procedures have not always correlated well with those obtained from batch experiments.^^^'^^^ One reason that can be put forward to account for this divergence involves the pressure-related compression of soft gels, causing a reduction in pore volume of the particle and thus the capacity of the sorbent.^^^'^^^ One way to avoid pressure-induced deformation of the particles, as well as to bypass the requirement for reduced flow rates with packed columns with viscous fluids, is to carry out batch adsorption procedures in stirred tanks. From an economical perspective, the isolation of the target polypeptide or protein from a crude feedstock using batch systems in stirred tanks might not be the ideal solution if production quantities of the product to be isolated from derived from large volumes of a feedstock of complex composition.

208

MILTON T. W. HEARN

An alternative solution involves the use of fluidized or expanded-bed columns.^'^^^'"^^"^""^^^ In the case of fluidized-bed systems operated under laminar flov^ conditions, reproducible bed expansion characteristics can be achieved v^ith particle Reynolds numbers in the range of 0.05 < R^ < 1 w^ithout significant loss of the HPLC sorbent from the column. The advantage of fluidized- or expanded-bed systems over the bath adsorption lies in the capability of this approach to process large amount of the feedstock in a short period of time, thus in principle increasing the performance of the system in terms of overall productivity. In addition, removal of cell debris by centrifugation steps may not necessarily be required w^hen fluidized- or expanded-bed systems are employed as found in our investigations^^'^''^^'^''^^^ on the recovery of grow^th factors from very viscous mammalian amniotic fluid using expanded beds of the HP-BAC sorbent, heparin immobilized onto porous silica of ^ 65 jitm in particle diameter. Similar observations have been made for the recovery of proteins from bacterial or yeast extracts by other investigators.'^^^"'*'*^ Moreover, v^hen such fluidized- or expanded-bed systems are properly designed, the adsorption process can also be made continuous.'^'^^''^'^^ For example, in whey protein isolation,'^'*"^ recycling systems v^ere employed once the sorbent has been saturated v^ith protein, w^ith both the protein-laden sorbent and the interparticulate fluid removed from the column by changing the mobile phase to the loading buffer alone, increasing the flow^ rate and introducing fresh adsorbent into the column through a tangential opening located at the bottom of the column above the distributor frit. Alternatively, w^ith high-density particles, e.g., surface modified zirconia,^^'^^^'^^^ the bed can be allov^ed to rapidly settle, the nonbound proteins contained within the intra- and interparticulate fluid of the column flushed away using a wash step, and the desired component(s) eluted in the usual fashion of conventional packed column techniques. According to the tanks-in-series model of m stages, the concentration profile of the feed stock containing polypeptides and proteins continuously introduced from time t = 0 onward and leaving a column at time t is given by C 1 1 -Jill = 1 - e - ^ n i + me] -h —mO^ + ••• + —m^^^-i> Co,. 2! (m-1)!

(165)

where 6 equals t/'t; ~t is mean average residence time for all components within the feedstock fluid to traverse the column, i.e., t = L/u^; and CQ / is the initial concentration and C^ ^ is the instantatneous concentration if the polypeptide or protein species /. When m is large, i.e., m > 20-50 and the fluid flow within the column is close to plug or laminar flow, the axial dispersion coefficient ^ , for rigid sorbents of 40-65 /xm diameter typically falls within the range of 0.01-0.02 cm^ min^ Under such conditions, plots of the bed voidage versus the logarithm of the superficial velocity UQ for different sorbents will be linear. Scaling can then be achieved from knowledge of the effects of the ratio of the particle diameter to column diameter on the dispersion coefficient. In an alternative approach, based on the axial

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

209

dispersion model, the concentration versus time profile is given by Q,;

1

Co,;

2

1 -erf

1 2

I UQL X 1 / - ^ X V ^

1- e V^

(166)

The dimensionless term (^/UQ L , where ^ is the axial dispersion coefficient, UQ is the superficial fluid velocity, and L is the expanded-bed height) is the column-vessel dispersion number, F^, and is the inverse of the Peclet number of the system. Two limiting cases can be identified from the axial dispersion model. First, when ^/UQL -^ 0, no axial dispersion occurs, while when ^/UQ L ^ 00 an infinite diffusivity is obtained and a stirred tank performance is achieved. The dimensionless term F^, can thus be utilized as an important indicator of the flow characteristics within a fluidized-bed system."^"*^ To optimize these fluidized or expanded-bed systems, it is highly advantageous if the performance of several different sorbents of similar selectivities are examined in the presence of different concentrations of the crude feedstock using initially continuously stirred bath systems, and the corresponding dissociation constants (K^s) and the maximum binding capacities ((^'max^) oi these sorbents determined for the target polypeptide of protein under these conditions. Moreover, the rheological properties and mixing characteristics of these sorbents should be assessed in the fluidized- or expanded-bed columns in terms of the fluid flow distribution and the extent of dispersion at different superficial velocities in columns of different dimensions. Compared to packed-bed systems, where polypeptide- or protein-laden feedstocks can be loaded into the column in either an upward or a downward flow direction; with fluidized- and expanded-bed systems the feed is usually introduced in a continuously manner in an upward direction causing expansion of the bed materials inside the column. Bed expansion will thus depend on the physical properties of the sorbent, the distributor design, and the superficial velocity Under low superficial velocity conditions, the feedstock zones will move up fluidized bed in a plug flow manner, with the fluid flow profile initially ensuring that only the HPLC sorbent particles at the bottom of the bed are exposed to the polypeptides or proteins in the feedstock. When the values of UQ are high, on the other hand, less laminar flow conditions will occur in the fluidized bed with the particles within the column effectively exposed to polypeptides or proteins very shortly after the feedstock has been introduced through the distributor at the bottom of the column. Under conditions of extremely high superficial velocities, turbulent flow conditions will occur and the sorbent particles inside the column will not have sufficient time to interact with the biosolute molecules present in the bulk fluid, resulting in the product(s) being swept out of the column with a breakthrough curve being very rapidly generated. Since the vessel dispersion number F^, represents the overall extent of dispersion and incorporates dispersive and convective transport effects as the value of UQ becomes smaller, the convective contribution will become reduced and the dispersive contributions will become more important* As a consequence, depending on the magnitude of the UQ value of

2 I0

MILTON T. W. HEARN

the feed stock fluid, axial dispersion effects can make important and even dominant contributions to the shape of the breakthrough curve with both fluidized- and packed-bed systems. Such axial Hquid dispersion effects have been observed, for example, w^hen HSA in an intermediate ionic strength elution buffer (such as 0.15 M sodium acetate, pH 4.5) was loaded at different superficial velocities in fluidized-bed columns containing HP-IEX sorbents.^^^'^2^ With fluidized or expanded beds of HPLC sorbents, as the superficial velocity increases and the flow becomes less laminar, the Hquid axial dispersion coefficient can also increase. When this occurs, the unused column capacity will become larger. For this reason, a choice must be made as to maximum value of WQ as well as the maximum binding capacity q^^^ for a particular bed configuration. Typically this limit is often set at the nominal saturation level of the bed at '^ 10%. Under such conditions the breakthrough capacity Q^ can be determined, with the reduction in the binding capacity of the sorbent for the polypeptide or protein at higher UQ values compensated by a reduction in the loss of bed efficiency. Use of such conditions can lead^'^'^^^''^^'^''^^^''^'^^ with various types of ion-exchange and biomimetic affinity HPLC sorbents to overall enhancement of the productivity when multiple runs are employed with complex feedstocks. With several of the current generation of silica-based preparative HPLC sorbents, column configuration, superficial velocity, and bed expansion conditions can be readily selected such that the value of T^ is close to the threshold Reynolds value of the system, so that the polypeptide or protein interaction with the appropriate ligate occurs essentially under laminar flow conditions. Thus, in a recent study on the adsorption behavior of HEWL or HSA with the immobilized Cibacron Blue F3G-A-Fractosil 1000 sorbent, a F^ value of 0.033 (corresponding to column Peclet numbers of 30) was used, which approximated the threshold Reynolds value of 0.02 for this type of sorbent. A number of parameters affect the position and shape of the breakthrough profile with fluidized- or expanded-bed systems with a designated HPLC sorbent, including the superficial velocity, bed configuration, feedstock concentrations, temperature, and buffer composition. Figure 40a shows the effect of changes in the superficial velocity as plots of C / C Q versus processing time t for the adsorption of HEWL onto a triazine dye biomimetic affinity adsorbent, immobilized Cibacron Blue F3G-A-Fractosil 1000. As expected, at low superficial velocities, e.g., at a UQ value of 0.64 cm/min, the breakthrough curves were significantly shifted to the right in the temporal context (i.e., to larger t values). As the superficial velocity was increased from 0.64 to 3.82 c m / m i n \ the position and shape of the breakthrough curves progressively converged. When these data for the binding of HEWL to Cibacron Blue F3G-A immobilized onto Fractosil 1000 are replotted to show the effect of superficial velocity as plots of C / C Q versus the amount of protein (in milligrams per gram of sorbent) applied to the bed, it is evident (Fig. 40b), that the relative position of the capacity of the sorbent as measured as a q^Q value of these plots was unaffected by the change in the superficial velocity. However, as the UQ value was increased from 0.64 to 3.82 c m / m i n \ the shape of the breakthrough curves as expected from Eqs.

21

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

•

= 0.64 cm/min .28 cm/min

= 2.55 cm/min = 3.82 cm/min

J 20

30

40

50

Time (min) L

1.0

B

0.8

*

j •

"= 0.64 cm/min == 1.28 cm/min

^ •

= 2.55 cm/min = 3.82 cm/min

[(b)

0.6

nA

0.4 0.2 0.0

1

0

4

8

12

16

20

Amount applied (mg/g sorbent) F I G U R E 4 0 Effect of changes in the feed superficial velocity on the experimental breakthrough curve for the adsorption of HEWL to Cibacron Blue F3G-A immobilized onto Fractosil 1000 as a function of time and the amount of protein added to the column. Data adapted from Ref. 436.

(165) and (166) became shallower, indicating that the column efficiency of the fluidized bed was reduced at the higher UQ values. Inlet protein concentration also affects the shape of the C/C^ versus t profile in fluidized, expanded, and packed beds of HPLC sorbents. At lower protein concentrations, greater processing times will be required for the effluent concentration to reach a value equal to the inlet concentration. These differences in the shapes of the breakthrough curves at different protein concentration presumably reflect the dependency of protein diffusivity on the concentration of the polypeptides and proteins in the feed stock over the range of flow rates studied.^'^^^ Presentation of the binding data as plots of C/CQ versus ^, a dimensionless time parameter, or as plots of C / C Q versus Q^, at different protein concentrations enables the binding features of the different polypeptides and proteins to be identified. For example, data reported"^"*^ for the binding behavior of HEWL or HSA in crude feedstocks to different HP-IEX sorbents confirmed that the slopes of the plots of C/C^ versus ~t and C/C^ versus Q^ because both proteins became shallower as the protein concentrations were increased. Moreover, the extent of shallowness was more pronounced in the case of HSA, indicating slower adsorption kinetics either due to steric hindrance and restricted transport within the pores of the HP-IEX sorbents at higher protein concentrations or to increased lateral interaction between protein molecules. Both HEWL and HSA are known to undergo isodesmic interactions at higher concentrations in free solution and at chromatographic surfaces.^^^'"*"^^'^"*^ The formation of

212

MILTON T. W. HEARN

polypeptide or protein dimers and oligomers, leading to significant geometrical constraints as the affinate monomers adsorb to porous HPLC sorbents, could represent one of the contributing effects leading to decreases in the capture efficiencies. Temperature can also have a profound effect on the shape of the breakthrough curve with fluidized- and expanded-bed systems. Figures 41a and 41b are show^n tv^o representative examples of the effect of temperature on the plots of C / C Q versus the processing time t for the adsorption of HEWL and HSA v^ith the fluidized beds containing Cibacron Blue F3G-A immobilized to Fractosil 1000. Typically, for the interaction of globular proteins v^ith these types of biomimetic HP-BMC sorbents, a shift to larger t values has been observed when the temperature was increased from 4° to 37°C, indicating that the binding capacity of the biomimetic ligate for each protein becomes at higher temperature. Such behavior would be consistent with the interaction having a dominant hydrophobic component over this temperature range. However, the slopes of the breakthrough profiles for proteins, e.g., HEWL or HSA, are often steeper at lower temperatures. One advantage of increasing the temperature with fluidized- or expanded-bed systems is thus to reduce the bed voidage. The higher viscosity of the feedstock Hquid in a fluidized or expanded-bed at lower temperatures will be reflected as a greater bed voidage if the same flow rate was applied. The changes in the steepness of the breakthrough curves for globular proteins such as HEWL or HSA at the different temperatures can be considered to have their origin in the combined effects of temperature-dependent variations

• • ^ V 5

10

15

T = 277K T = 288K T = 298K T = 310K

_i_

_L_

20

25

30

Time (min) F I G U R E 41 Effect of temperature on the experimental breakthrough profile obtained for the adsorption of (a) HEWL and (b) HSA to Cibacron Blue F3G-A immobilized onto Fractosil 1000 as a function of time. Data from Finette, G. M. S., Mao, Q. M., and Hearn, M. T. W., 1998, biotechnol Bioeng., 58, 35. Reprinted by permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

2 I 3

in the protein conformation, the change in viscosity of the surrounding liquid, as well as the pK^ values and ionization states of the proteins and the ligate species. In particularly, the reduced viscosity at higher temperatures v^ill affect the diffusivity of the protein in solution and improve the transport of the protein molecules within the stagnant fluid boundary layer and within the pores of the adsorbent. In the absence of conformational changes or self-self aggregation events, an increased efficiency in the sorption process for globular proteins can be anticipated, reflected by steeper breakthrough curves and an increased ratio of breakthrough capacity to static capacity as the temperature is increased. This behavior has been observed"^^^'"^^^ with bovine serum albumin (BSA) with HP-IEX sorbents in fluidized beds. In contrast, other proteins have been found to exhibit more complex behavior. For example, in the case of the adsorption of HSA to biomimetic sorbents, a more significant broadening of the breakthrough curve has been observed at higher temperatures, and this may be due to either a change in the conformation of the protein or the occurrence of protein self-association. Both phenomena have been documented"^"^^ for HSA in preparative HP-IEX. The participation of these secondary equilibrium processes at elevated temperatures lead to molecular species with increased hydrodynamic radii (which results in increased steric restrictions within porous sorbents) and the exposure of additional hydrophobic amino acid residues (which reinforces the potential for protein-protein aggregation a n d / o r hydrophobic interactions to occur during the adsorption process). In this context, differences in adsorption and self-aggregation behavior of different albumins,"^"^^ such as HSA, BSA, and horse serum albumin in the presence of different types of HPLC sorbents can be attributed to the subtle differences in the surface accessibility and distribution of nonpolar amino acid residues of these molecules and analogous behaviors can be anticipated for other polypeptides and proteins. Provided that the polypeptide or protein can gain full access to the ligates within the pores of a HPLC sorbent, and that the binding equilibrium is relatively rapid, then similar values of the binding capacity measured under static and dynamic conditions can be achieved. A convenient way to determine whether this equivalence exists between the batch-derived and columnderived Kj and q^^^ values is to measure the DAC defined in terms of the Qg value or the amount of protein adsorbed per unit mass of adsorbent when the effluent concentration reaches 10% of the influent concentration at different superficial velocities with beds of different dimensions.^'^^^ A typical finding is that the percentage of the column capacity utilized will decrease as the superficial velocity is increased. Although the vessel or column dispersion number F^, will usually be larger for globular proteins when higher superficial velocity conditions are used,"*^^""*^^ other factors can also contribute to the observed dispersion effects evident from these breakthrough curves of these proteins in the fluidized-bed columns. These factors are related to the rate at which the polypeptide or protein binds to the HPLC sorbent (i.e., the forward rate constant for the interaction k^), the rate at which the polypeptide or protein diffuses inside the porous network of the sorbent (i.e., the effective diffusivity D^) or a combination of both these parameters.

214

MILTON T. W. HEARN

As note earlier, the DAR associated with the adsorption of a polypeptide or protein to a HPLC sorbent can be defined as the amount of polypeptide or protein adsorbed per unit mass of sorbent per unit processing time. Figure 42 shows the DAR changes for HEWL and HSA with Cibacron Blue F3G-A immobilized onto Fractosil 1000 at different UQ values. Adsorption was terminated at 10% of the influent concentration in this example, with the DAR values increasing as the superficial velocity increased. This trend is generally followed with polypeptides and small globular proteins. However, with higher molecular weight proteins more complex behavior becomes evident. If HEWL was replaced by HSA, the DAR values for HSA with the immobilized Cibacron Blue F3G-A sorbent showed only modest increases up to relatively low superficial velocity values, i.e., up to a UQ value of 2.54 cm/min, but subsequently decreased. This type of behavior, associated in part with restricted pore exclusion effects appears to be a general phenomenon, i.e., similar observations related to the variation of DAR as a function of flow rate have been made for the adsorption of trypsin onto Sepharose-4B soyabean trypsin inhibitor."^^^ Although the DAC for the adsorption of a polypeptide or protein to a HPLC ligate(s) is a function of the inlet protein concentration, this general finding does not imply that higher polypeptide or protein concentrations per se will automatically alter the shape of the breakthrough curve. It is well known that the breakthrough curve profile can be represented in terms of the dissociation constant when C/CQ is plotted against the volume of protein feedstock applied to the column. Only when the magnitude of the inlet concentration is smaller or in the same range as the magnitude of the dissociation constant, will a change in the position and shape of the break-

Superficial velocity (cm.min" ) F I G U R E 4 2 Influence of superficial velocity on the DAC and the DAR obtained for the adsorption of (a) HEWL and (b) HSA to Cibacron Blue F3G-A immobilized onto Fractosil 1000. Data from Finette, G. M. S., Mao, Q. M., and Hearn, M. T. W., 1998, Biotechnoi Bioeng., 58, 35. Reprinted by permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

2 I 5

through curve be observed.^^^ Contrary to the influence on the DAC, the percentage of bed capacity utihzation is often unaffected by a change in the concentration when polypeptides or low molecular weight proteins are involved. When the process purification of high molecular weight proteins is undertaken, however, an increase in the percentage of bed capacity utilization is frequently observed if the concentration or relative abundance of the protein of interest is increased. This finding suggests that slow transport within the porous adsorbent is limiting sorption efficiency with the high molecular weight proteins. Consistent with this conclusion, the breakthrough curves show significantly more tailing in the late phase of the sorption, whereas the initial region of the sorption, where dispersive effects would be expected to be more dominant, are not as affected. Such behavior is typical of transport-limited adsorption with HPLC sorbent. For recombinant proteins, this behavior thus creates the additional imperative for achieving as high an expression rate as feasible to ensure that the relative concentration of the protein of interest is maximized. The outcome will then be an increase in the amount of protein processed per unit time as the protein concentration in the feedstock increases. Depending on the physicochemical properties of the targeted polypeptide or protein, an increase in the DAC at 10% saturation can be achieved at higher temperatures. This behavior at higher temperatures may be due to either a change in the molecular conformation of the polypeptide or protein, the occurrence of polypeptide or protein aggregates, or a combination of both. As discussed in earlier sections of this chapter, at higher temperatures, polypeptides and proteins are known to unfold and expose additional buried hydrophobic amino acid residues to the surrounding solvent or to the HPLC ligates. As a result, the hydrophobic surface area of the polypeptide or protein molecule is increased. A difference in temperature dependence between different polypeptides and proteins is, however, expected due to their molecular characteristics. These differences in temperature dependencies also suggest that different adsorption mechanism may occur either due to the interplay of different conformational species for each polypeptide or protein at different temperatures or alternatively due to the ability of the polypeptide or protein to bind to several different classes of ligates a n d / o r matrix sites accessible on the HPLC sorbents. The higher increase in the DAR values can be mainly accounted for in terms of faster mass transfer kinetics. An increase in temperature reduces the viscosity, thus enabling greater mixing between the polypeptide or protein molecules, solvent, and adsorbent. Moreover, the effective diffusivities of the respective solutes will increase with an increase in temperature. Because of the influence of greater axial dispersion and mixing of the polypeptide or protein zones in fluidized or expanded-bed systems, vis-a-vis packed bed systems, the position of the breakthrough curves for fluidized or expanded beds will typically be more delayed (and broader) than the breakthrough profiles for packed columns containing the same sorbent and under these same eluent, temperature, etc. conditions. Figures 43a and 43b illustrate these characteristic differences, comparing the position and shape of the plots of C/CQ versus the processing time t as breakthrough curves for both the

216

MILTON T. W. HEARN

1.0

• O

• O

= Fluidised Column = Packed Column

= Fluidised Column = Packed Column

0.8 o 0.6

O

0.4 0.2 0.0

0—Q--<>--P-—Q-

10

20

30

40

0

Time (min)

2

4

6

10

12

^ CHo-00-0 0-0-0-Q

1.0

r

/•

0.8

f'

o 0.6

.

I /

/

^

8

Amount applied (mg/g)

0.4

k

0.2 /jrt

0.0 j) . ^ r ^ y • t

Fluidised Column = Packed Column

O • • _i

4.

-^•^rlW

I

i_,_L__i

L

12

15

18

Time (min)

J L

Column, • = Fluidised Flui( O = Packed Column

12

16

20

Amount applied (mg/g)

F I G U R E 4 3 Comparison of the shape and position of the breakthrough curves in terms of time or the amount of protein applied and generated for the corresponding packed and fluidized columns for the adsorption of HEWL (a) and (b), respectively, and HSA (c) and (d), respectively, to Clbacron Blue F3G-A immobilized onto Fractosii 1000. The superficial velocities applied were 0.64 cm / min in (a) and (b) and 3.84 cm / min in (c) and (d), respectively.

packed and the fluidized beds, with HEWL and the Cibacron Blue F3G-A immobilized Fractosii 1000 sorbent. At low superficial velocities (e.g., u^ = 0.64 cm/min), the time taken for the first breakthrough point to be detected with the fluidized-bed system was slightly longer than that of the corresponding packed column. This difference in the relative position of the breakthrough curve can be related to the greater distance the polypeptides or proteins must travel before emerging at the bed outlet. In many cases, however, for both packed and fluidized or expanded beds similar DAR values can be achieved following optimization of the superficial velocity. However, the conclusion can be drawn from a number of studies that higher capture efficiencies occur with packed-bed systems compared to the fluidized or expanded beds at higher u^ values. Changes in column efficiency are reflected in the shape of the breakthrough curves, which themselves are a function of the liquid axial dispersion. Since greater axial dispersion will always occur in the fluidized or expanded beds vis-a-vis packed beds at any superficial velocity of the feed; as a consequence of this behavior, bed efficiency should not be the only parameter employed to assess performance of a particular

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

2 I 7

fluidized or expanded bed in the large-scale purification of a specific polypeptide or protein. Rather, the bed performance should be judged on how fast a product can be processed at a defined level of purity and economic efficiency, i.e., in terms of productivity. With HPLC sorbents in fluidized or expanded bed, smaller DAC and Q^ values w^ill thus be typically obtained for polypeptides or proteins at higher superficial velocities than with packed beds. Significant temperature effects can be achieved with both packed and expanded beds in terms of the position and shape of the breakthrough curves for the polypeptides or proteins with different types of HPLC sorbents. Moreover, at low flow rates, both column efficiency and column productivity have been found to be greater in the packed column. However, an important compromise can be reached with fluidized and expanded beds for the optimal DAC and the DAR values, resulting in a maximum in the bed utilization efficiency at higher flow rates. With fluidized- or expanded-bed systems, lower column efficiency can be compensated by a greater column productivity at higher flow rate. On a preparative scale, column productivity^^^''^^^"'^^^''^^'^"'^^^ in fluidized- or expanded-bed systems can be greater at higher feed superficial velocities, despite showing a lower column efficiency. Thus, when crude feedstock mixtures are involved, the ability of packed columns to achieve the same level of productivity as fluidized or expanded beds often becomes compromized at higher flow rates by the fluid viscosity as well as the presence of cell debris, protein aggregates, or microparticulate matter, all of which will result in elevated back pressure effects. Although the viscosity can be modulated through changes in temperature at which an adsorption is carried out, the use of high temperatures is often dictated by the relative resistance of the target and contaminant polypeptides and proteins to thermal denaturation, and as a consequence the use of stirred-tank or fluidized- or expanded-bed systems represent attractive alternatives, at least in the initial stages of the purification of a polypeptide or protein at the process scale when the heuristic rules described earfier are applied. Undoubtedly over the next decade, the extension of heuristic approaches into knowledge-based algorithmic procedures will provide an avenue from which computer intuitive and computer interrogative methods for the modeling, simulation, and de novo prediction of chromatographic processes including scale-up requirements will evolve. Already some of the essential stages of these developments exist in the form of the so-called expert computer-driven systems^^^"^^^'"^^^""^^^ capable of controlling on-line process streams. The powerful Q-system developed^^^ at Pfizer is an excellent example of these developments. To be effective, such expert systems must be capable of exploiting advances in chemometrics, multicomponent error function analysis, principal component analysis, as well as the more mundane aspects of system monitoring and control-loop feedback. The availability of greatly expanded databases on biopolymer adsorption behavior will facilitate these developments even further. Collation and classification of the existing knowledge base, initially by empirical approaches and subsequently by knowledgebased computational mining in a manner analogous to that now used in bioinformatics, will in its own right become a formidable undertaking.

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MILTON T. W. HEARN

worthy of an international cooperative effort. Although such an undertaking may not engender a similar presence in the popular press as, for example, the human genome or proteomic projects, the practical consequences of these research initiatives w^ould be of enormous benefit to industry and academe alike, linking together essential threads of research and development in the life sciences. The ability of scientists to develop and adapt HPLC procedures over the next decade into these knowledge-based systems will certainly drive the fields of protein engineering, structural genomics and computational biology, and bioinformatics in a manner analogous to how HPLC has been an engine that powered molecular biology and protein chemistry over the past two decades.

XI. SUMMARY The success of a particular analytical or preparative HPLC strategy with polypeptides or proteins is predicated by the ease of resolving to a predefined level the desired component from other substances, many of which may exhibit similar separation selectivities but are usually present at different abundance levels. For high-resolution purification procedures to be carried out efficiently, it is self-evident that rapid, multistage, high-recovery methods must be utilized. To minimize losses and improve productivity, on-line, real-time evaluation of each of the recovery stages is an essential objective. Furthermore, overall optimization and automation of the individual unit operations must be achieved. Similar criteria but with different endpoints apply in high-resolution analytical application. Just as major advances and greatly expanded utilization can be anticipated during the next 2 decades for the preparative purification of mixtures of polypeptides and proteins by HPLC procedures, there will also be a comparable, or even larger, enhancement of understanding per se of the molecular basis of the interaction of these biomacromolecules with various classes of ligates. The fundamental nexus that links the thermodynamics of interaction with extrathermodynamic physicochemical properties of all polypeptides and proteins in interactive HPLC systems will thus take on a greater significance, providing an avenue to more precisely interpret the molecular binding, the docking events, and the atomic forces involved. This nexus for polypeptides and proteins in all of the HPLC modes starts, of course, with the well-known relationship AGl,,=

-RT

In k' + RT In ^

(167)

In terms of the impact and directions that these forms of "molecular HPLC" will take, let us consider the RP-HPLC separation of a specific polypeptide as an exemplar. If, for example, the free energy of association ^^Issoo where $ in this case refers to the RP mode of interaction, was

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

2 I 9

differentiated with respect to the mole fraction of the organic solvent modifier il/ we have

and

^i/f

#

^i/r

^

^

In circumstances where linear dependencies between In k' and r/^ prevail, di\nk')j -S

(170)

and hence

^ ^ 1 ^(AH,LJ, _ 1 ^(A5LOC)T RT

dijj

R

dip

^

^

Equations. (167)-(171) thus predict that a relationship exists for the S value of a polypeptide or protein on interaction with a nonpolar ligate in the presence of an organic solvent-water mixture, through the thermodynamic virial coefficients d(AH^^^^^)j/dil/ and d(^.Sl^^^^)j/dil/ to the free-energy change AG^^^^^, If the extrathermodynamic relationship between the S value and the hydrophobic contact area AA^^^j as well as the cavity factor K^ is recalled (Eq. 100), then clearly this linkage also extends to a number of other molecular-physicochemical properties. Studies have already documented^^'^^'^^'^^^'^^^ that underlying molecular-structural basis of these thermodynamic and extrathermodynamic linkages can be revealed from the experimental HPLC data and related to the physicochemical properties of these biomolecules. Thus, the virial coefficients d{AH^^^^^)j/dip and d(ASf^^Q^)j/dil/ can be employed to derive^^ the corresponding S values for polypeptides and proteins and can provide a basis to interpret nonlinear dependencies of the S value on T, such as found for bombesin, j8-endorphin, and glucagon, with acetonitrile-water mixtures as a function of T at different ip values with «-alkylsilica sorbents. Clearly, in cases of nonlinear dependencies of S on T, where the hydrophobic contact area A A^^^ as well as the cavity factor K^ change with T, and where homo- or heterothermic interactions (Section VII) are manifested due to the participation of conformational processes from, e.g., a-helical states at low T to more unfolded states at higher T, or alternatively due to other secondary equilibrium effects, such as preferential solvational changes, the more usual curvilinear form of these dependencies prevail such that

—

— = -S + 2 S > - 3 S ' > •

(172)

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MILTON T. W. HEARN

Nevertheless the general conclusions discussed here, as well as the overall experimental design for their validation, still follov^ the same unifying trends. For example, linear extrathermodynamic expressions can be proposed between the free energy change of a polypeptide or protein molecule involved in such hydrophobic interactions and particular molecular property parameters ^y. This relationship takes the form of A G * _ = t AG* = Za^aj ^/ + ^hcj

(173)

where a^Qt is the respective free-energy change per unit value of a molecular property ^y, and b^Qt is the free-energy change when ^y = 0. In this context, both a^Qt and bj^Qt can be considered as group molecular parameters. Each group molecular paremeter set forms a series of constants for a particular class of molecular property. Thus, the nonpolar surface area of a polypeptide or protein that is exposed to the solvent can be discussed in terms of ^y. Similarly, other molecular properties such as the free energy of amino acid side chain partitioning for a group of polypeptide or protein analogues between an octanol-water interface, the electrostatic charge distribution parameters, the dipole-polarizability vectors, group fragmental constants f, solvochromic parameters associated with hydrogen bonding effects, molecular volume or partial specific volume terms, etc. can also be described in terms of these linear free-energy relationships on molecular structure. In this regard, the change in preferential solvation that is associated with the binding of polypeptides and proteins in RP-HPLC, has its equivalent in HP-HIC, namely. ^(Ini^') d{lnm^')

= 3

= H

(174)

where H is the first-order slope of the plot of In fe' versus In m^,^ m^ is the molal concentration of water, ^ 3 is the molal concentration of the salt, AL'^ is the number of moles of water, and AL'3 is the number of moles of salt from condition A to condition B. Moreover, the change in surface tension that is achieved in an HP-HIC separation of a polypeptide and protein can be related to the surface tension of pure water y^ = 72 dynes/cm, the molal surface tension increment of the salt cr, and the molal concentration of the salt ^ 3 , by the relationship y=7o + ^^3

(175)

From strictly formalistic considerations, much larger surface tension changes are achieved in RP-HPLC than in HP-HIC separations of polypeptides and proteins, and these differences underpin the relative facility of these techniques to follow the same types of molecular binding events (and their perturbations) to the same level of physicochemical discrimination. Obviously, analogous approaches linking the interaction thermodynamics and the extrathermodynamic physicochemical properties or enthalpy-entropy compensation phenomena can be developed for the other major HPLC modes.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

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i.e., HP-IEX, HP-IMAC, HP-HILIC, etc. These unifying developments will enable greatly enhanced molecular descriptions of the interaction phenemena associated with these modes of high-resolution separation to emerge over the next few years. For these steps to be properly integrated, detailed assessment of the retention and kinetic data using computer-aided methods for factor analysis are (ideally) required at each stage. This knowledge provides the essential insight into the atomic and molecular basis of the separation selectivity and biorecovery contours of the preparative a n d / o r analytical procedures. The development and application of new generations of on-line detectors capable of monitoring these structure-function-retention characteristics of biopolymers represent a pressing challenge for spectroscopists and chromatographic scientists alike. Instrumentation is available already for on-line laser activated light scattering (LALLS), optical rotary dispersion (ORD), enhanced laser fluorescence (ELF), and laser Raman spectroscopy (LARS) for use in the analytical modes of HPLC of biopolymers. The development of on-line electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOFL-MS), and surface-enhanced laser desorption ionization mass spectrometry (SELDI-MS) over the past decade has provided very powerful and essentially universally applicable procedures for the characterization of the molecular features of biomacromolecules and their interactions with chemically defined surfaces. The next decade will certainly witness further significant developments in this area. Finally, if the progress of the past decade is any prognostic indicator, further significant development in sorbent design and their use in high-resolution HPLC analysis and preparative isolation of bioproducts will be a hallmark of the next decade. These developments will be driven in part by the public and government perception of the requirements of product purity and quality, by underlying commercial considerations of greater efficiency and reproducibility in process purification, and also by the lay curiosity and scientific inquisitiveness of researchers themselves. To a significant extent, the "winners" in modem biotechnology have largely been associated with the health industry-biomedical research sectors of this biotechnological revolution. Manufacturers and service providers of equipment and chemical suppUes used by these researchers and their colleagues in the aligned industries have also profited from these breakthroughs. Currently, subtle shifts in this pattern are occurring, thus ensuring further novel HPLC products and procedures will be around to tantalize and assist the researcher, the engineer, or the business executive in the biotechnological, pharmaceutical, food, chemical, and environmental industries well into the 21st century. In this chapter, the physicochemical basis of polypeptide and protein retention in adsorption HPLC has been examined. It is evident from this treatment that significant developments are underway with regard to both the theory and practice of high-resolution chromatographic methods, particularly at the preparative level with new classes of adsorptive materials. The recognition that most purification strategies must be based on multidimensional multistage procedures presents numerous challenges for the protein chemist.

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the chromatographic scientist, and the biochemical engineer. The urgency for these developments has been stressed by the potential of modern biotechnology to produce large quantities of new polypeptides and proteins that must be obtained in highly purified form. The capabilities which these advances present will prove catalytic in the materials sciences as new solid phases are examined with particular properties for large-scale utilization. Similarly, new initiatives in protein engineering will lead to greater utilization of the fusion handle approach for selective isolation and recovery of recombinant proteins. Realization of these capabilities will provide innovative opportunities that will revolutionize the role of bioprocess development over the next decades. Certainly, the ability to design and interpret the interactive behavior of polypeptides and proteins in terms of a full understanding of their physicochemical properties will have a profound effect on how high-resolution separation science, and HPLC in particular, is practiced in the next millennium. The challenge before us is whether these goals are reached in a decade or less.

ACKNOWLEDGMENTS The support received by the author from the AustraUan Research Council, the National Health and Medical Research Council of Australia, the Alexander von Humboldt Foundation, and the Japan Society for the Promotion of Science is gratefully acknowledged.

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512. Bertolini, J., and Hearn, M. T. W. (1987). Mol. Cell. Endocrinol. 21, 187. 513. Ensch, F., Baird, A., Ling, N., Meno, N., and Gospodarowicz, D. (1985). Proc. Natl. Acad. Set. U.S.A. 82, 6057. 514. Shing, Y., Feldman, J., Sullivan, R., Butterfield, C , Murray, J., and Klagsbrun, M. (1984). Science 223, 1296. 515. Downward, J., Yarden, Y., Mayes, E., Scare, J. G., Totty, N., Stockwell, P., Ullrich, A., Schessinger, J., and Waterfield, M. D. (1984). Nature (London) 307, 521. 516. Pilch, P. F., and Czech, M. P. (1980). / . Biol Chem. 255, 1722. 517. Ullrich, A., Bell, J. R., Chen, E. Y., Gerra, R., PetruzzeUi, L. M., Dull, R. J., Gray, A., Coussenc, L., Liao, Y. C , Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfeld, C , Rosen, O. M., and Ramachandran, J. (1985). Nature (London) 313, 756. 518. Forage, R. G., Ring, J. M., Brown, R. W., Mclnerney, B. V., Bobon, G. S., Gregson, R. P., Robertson, D. M., Morgan, F. J., Wettenhall, R. E. H., Findlay, J. K., Burger, H. C , Hearn, M. T. W., and de Kretser, D. M. (1986). Proc. Natl. Acad. Sci. U.S.A. 83, 3091. 519. Robertson, D. M., Foulds, M. L., Leversha, L., Morgan, F. J., Hearn, M. T. W., Burger, H. G., Wettenhall, R. E. H., and de Kretser, D. M. (1985). Biochem. Biophys. Res. Commun. 126, 220. 520. Leversha, L. J., Robertson, D. M., de Vos, F. L., Morgan, F. J., Hearn, M. T. W., Wettenhall, R. E. H., Findlay, J. K., Burger, H. G., and de Kretser, D. M. (1987). / . Endocrinol. 13, 1. 521. Whetton, A. D., and Dexter, T. M. (1986). Trends Biochem. Sci. 11, 207. 522. Waterfield, M. D., Scrace, G. T., Whittle, N., Stroobant, P., Johnsson, A., Wasteson, A., Westermark, B., Heldin, C. H., Huang, J. S., and Deuel, T. F. (1983). Nature (London) 304, 35. 523. Chesterman, C , Walker, T., Grego, B., Chamberlain, K., Hearn, M. T. W., and Morgan, F. J. (1983). Biochem. Biophys. Res. Commun. 116, 809. 524. Robb, R. J., Kutny, R. M., and Chowdry, V. (1983). Proc. Natl. Acad. Sci. U.S.A. 80, 5990. 525. Nicola, N. A., Metcalf, D., Matsumoto, M., and Johnston, G. R. (1983). / . Biol. Chem. 258, 9017. 526. Bennett, H. P. J. (1986). / . Chromatogr. 359, 383. 527. Kanmera, T., and Chaiken, I. M. (1985). / . Biol. Chem. 260, 8474. 528. Reeve, J. R., Eysselein, V., Welsh, J. H., Ben-Avram, C. M., and Shively, J. E. (1986). / . Biol. Chem. 261, 16392. 529. Robertson, D. M., and Hearn, M. T. W. (1987). Mol. Cell. Endocrinol. 44, 271. 530. Vale, W., Rivier, J., Vaughan, J., McClintock, R., Corrigan, A., Woo, W., Kart, D., and Spiess, J. (1986). Nature (London) 321, 776. 531. Roberts, A. B., Anzana, M. A., Meyers, C. A., Wideman, J., Blacher, R., Pan, V. C , Stein, S., Lehrman, S. R., Smith, L. C , Lamb, L. C , and Sporn, M. (1983). Biochemistry 22, 5692. 532. Frolik, C. A., Dart, L. L., Meyers, C. A., Smith, D. M., and Spore, M. B. (1983). Proc. Natl. Acad. Sci. U.S.A. 80, 3676. 533. James, R., and Bradshaw, R. A. (1984). Annu. Rev. Biochem. 53, 259. 534. Clark-Lewis, L, and Schrader, J. W. (1981). / . Immunol. 127, 1941. 535. Seeburg, P. H., and Adelman, J. P. (1984). Nature (London) 311, 666. 536. Derynck, R. (1986). / . Cell. Biochem. 12, 293. 537. McFarlane, J. R., Foulds, L. M., O'Connor, A. E., Phillips, D. J., Jenkin, G., Hearn, M. T. W., and de Kretser, D. M. (1999). Endocrinology (Baltimore) (in press). 538. Li, S., and Hearn, M. T. W. (1999). Submitted for publication. 539. O'Bryan, M., de Kretser, D. M., and Hearn, M. T. W. (1999). In preparation. 540. Hearn, M. T. W. (2000). Anal. Biochem. (in press). 541. Hearn, M. T. W., and Grego, B. (1983). / . Chromatogr. 255, 125. 542. Hearn, M. T. W., Hodder, A. N., and Aguilar, M. I. (1988). / . Chromatogr. 443, 97. 543. Mannervik, B., and Danielson, U. H. (1988). CRC Crit. Rev. Biochem. 23, 283.

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CAPILLARY ELECTROPHORESIS OF COMPOUNDS OF BIOLOGICAL INTEREST S. AHUJA Ahuja Consulting, Calabash, North Carolina 28467

I. INTRODUCTION II. CAPILLARY ZONE ELECTROPHORESIS A. Operation B. Comparing CE with High-Performance Liquid Ch ronnatography III. MIGRATION BEHAVIOR OF PEPTIDES A N D PROTEINS A. Relationship of Protein Mobility to Protein Mass and Valence B. Buffer Selection C. Buffer Additives D. Adsorption of Proteins IV. MODIFICATIONS OF FUSED SILICA CAPILLARIES A. Surface Modification of Capillaries V. EFFECT OF TEMPERATURE O N SEPARATIONS VI. STRATEGY FOR PROTEIN SEPARATIONS VII. CAPILLARY GEL ELECTROPHORESIS VIII. MICELLAR ELECTROKINETIC CHROMATOGRAPHY IX. CAPILLARY ELECTROCHROMATOGRAPHY X. APPLICATIONS A. Serum Proteins B. Glycoproteins C. Lipoproteins D. Hemoglobins E. Recombinant Proteins F. Milk Proteins G. Protein Folding H. Genomics REFERENCES

Capillary electrophoresis has been found to be quite useful for resolving a very large number of compounds of biological interest, including peptides and proteins. The primary advantage of capillary electrophoresis is that it can Separation Science and Technology, Volume 2 Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

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offer rapid, high resolution of water-soluble components present in small volumes. Various approaches to peptide and protein separations with capillary electrophoresis (CE) are discussed in this chapter. Also discussed are various considerations in protein and peptide separations, strategies for protein separations, and a variety of applications. INTRODUCTION Electrophoresis is defined as the transport of electrically charged particles in a direct-current electric field.^ The particles may be simple ions, complex macromolecules such as proteins or colloids, or particulate matter such as living cells (erythrocytes or bacteria). Electrophoretic separation is based on the differential migration rate in the bulk of the liquid phase rather than the reactions occurring at the electrodes. Resolution can be significantly improved when an element of discontinuity is introduced into the liquid phase, such as a pH gradient, or with the sieving effect of high-density gels. Membrane barriers can also be introduced into the path of migrating particles. In this chapter, various approaches to peptide and protein separations with CE are discussed. Also discussed are various considerations in protein and peptide separations, strategies for protein separations, and a variety of applications. Interested readers should also read reference 1 for a basic introduction to this technique and books ' for more in-depth coverage. Peptides containing 10 or fewer amino acids are called small peptides, or oligopeptides. Peptides with 50 to 70 amino acids with a molecular mass of 5000 to 7000 Da lie on the borderline between polypeptides and proteins. The analyses of peptides constitute an important part of protein characterization schemes. Capillary electrophoresis has been found to be quite useful for resolving a very large number of compounds, including peptides and proteins. The primary advantage of capillary electrophoresis is that it can offer rapid, high-resolution of water-soluble components present in small volumes. The separations are based, in general, on the principles of the electrically driven flow of ions in solution. Selectivity is accomplished by alteration of electrolyte properties, such as pH, ionic strength, and electrolyte composition, or by the incorporation of electrolyte additives. Some typical additives include organic solvents, surfactants, and complexing agents. The separation of small peptides is fairly well understood; however, it appears that no single approach can be applied to the separation of large peptides or proteins. To a great extent, this is because of the wide diversity and complexity associated with these molecules. As a result, different techniques must be employed for different protein separation problems. Various modes of electrophoresis, along with the separation mechanism, are shown in Table 1. At times, capillary electrophoresis is called high-performance capillary electrophoresis to reflect the high number of theoretical plates offered by this technique; as a result, superior resolution is possible. The original contributor(s) to high-performance capillary electrophoresis and the basis of separation in various modes are shown in Table 2^~^

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TABLE I

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Modes of Electrophoresis

Mode

Separation mechanism

Free solution SDS-PAGE Isoelectric focusing MECC and solution phase

Based Based Based Based

on on on on

solute solute solute solute

size and charge at a given pH size isoelectric point partitioning between micelle "phase"

Here free solution indicates capillary electrophoresis carried out in a solution without any additives; SDS-PAGE is sodium dodecyl sulfate-polyacrylamide gel electrophoresis; MECC is micellar electrokinetic capillary chromatography, at times, also called MEKC (micellar electrokinetic chromatography).

Note that high-performance CE blossomed in the 1980s, and it is still a relatively new technique compared with most other separation techniques. A number of interesting techniques have evolved in this field; they can be relatively easily performed with commercially available equipment: • • • • •

Capillary zone electrophoresis (CZE, or more simply CE) Capillary gel electrophoresis (CGE) Micellar electrokinetic capillary chromatography (MECC) Capillary electrochromatography (CEC) Isoelectric focusing (lEF)

The first four techniques are discussed in this chapter; isoelectric focusing is covered in Chapter 5. It is important to note that a major advantage of these techniques is that it is possible to analyze small samples.^^ Of course, this proves to be a limitation if a larger sample size is desirable, as for example, in the case of preparative separations. II. CAPILLARY ZONE ELECTROPHORESIS Capillary electrophoresis can be classified into two groups on the basis of whether it is carried out on a free solution or on support media. When a support medium is used, the technique is called capillary zone electrophoresis. Capillary electrophoresis that is commonly carried out today fits into this

TABLE 2

Contributors to High-Performance Capillary Electrophoresis

Name

Basis of separation

Reference

Free-zone CE MECC

Electrophoretic mobility Partitioning into detergent micelles, charge Isoelectric point Enantiomeric structure Size, charge Size

Jorgensen and Lukacs"^ Terabe et al.^

Isoelectric focusing Chiral separation Gel electrophoresis: polyacrylamide SDS-PAGE

Hjerten and Zhu.^ Gassman et al7 Cohen et al} Cohen and Karger^

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category and at one time was called capillary zone electrophoresis. It is simply called capillary electrophoresis today, so these terms are used interchangeably in this chapter.

A. Operation The mode of operation of CE can be described in a simplified form as follows: A block diagram of a CE instrument is shown in Fig. 1. A relatively short capillary attached to a reservoir is subjected to high voltages—around 30 kV. The capillary tube has a diameter of 75 ixm or less, enabling an easy dissipation of generated heat through the wall of the tube. The sample can be drawn inside the capillary tube by a short exposure to high voltage. The zone breadth is proportional to the appHed voltage. In the continual development of CE since the 1980s, the critical issues affecting quantitative precision are better understood and system design has been changed to address these issues. Instruments today range from very simple designs to models that include capillary and sample-tray temperature control, high-sensitivity flow cells, fraction collectors, constant current operation, voltage-ramping capabilities, and operations under constant pressure. Various sample preconcentration techniques have been utilized. A new approach^ ^ to sample preconcentration before separation entails membrane preconcentration with CE (mPC-CE). Several detectors are now available that provide a choice of conductivity, fluorescence, laser-induced fluorescence, photodiode array, and mass spectrometry. A number of separations of peptides and proteins have been reported with mPC-CE-MS and mPC-CE-MS-MS, and some are listed next.

CE Capillary

riirvvvv;! V

Computer

FIGURE I

Block diagram of a CE instrument.

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Peptides • • • •

Peptide standards Trypsin digest of a-casein Cell media MHC class I compounds

Proteins • • • • • • •

Protein standards Aqueous humor Bence-Jones urine proteins Blood dialysis proteins Cerebrospinal fluid proteins Proteins in tears Renal dialysate proteins

Additionally, specialized systems have been introduced to target the growing fields of combinatorial chemistry and genetic sequencing.

B. Comparing CE with High-Performance Liquid Chromatography Strictly speaking, free-solution capillary electrophoresis is not a chromatographic technique because two phases are not involved in the separation process. Recall that the two phases in chromatography are designated as the stationary phase and the mobile phase, based on their roles in the separation process. Technically, there is no stationary phase in capillary electrophoresis unless the capillary walls are assigned that role. Some chromatographers promote this concept, but it is not entirely correct. Capillary zone electrophoresis, on the other hand, does have some similarities to chromatography especially when some of the similar additives are used for improving separations, e.g., cyclodextrins, crown ethers, or other surfactants. As a result, most chromatographers are comfortable in using CE because of its similarities to chromatography in that some of the manipulations used to optimize chromatographic separations are also suitable for CE. And symposia on CE are often included in the major chromatographic meetings. Capillary zone electrophoresis is also complementary to high-performance liquid chromatography (HPLC), as is shown by the application of these methods in purity assessment of peptides (see Fig. 2)}^^ In Fig. 2a, we see that a 7.5 kDa peptide purified by reversed-phase HPLC shows several peaks by CE, indicating that at this stage in the purification process, the sample is not pure. Further fractionation by cationexchange HPLC yielded a much purer sample, as shown in Fig. 2c. However, this sample shows a minor component just before the main component; the same component is seen after the main component by cation-exchange HPLC. This is explained by the fact that positively charged analyte appears first in the electropherogram, whereas, the same peak would elute last from a

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.020-

A

1 w .010 -

1

S .005 -

JIIJIL

0 -

20 Time (min)

15

20

25

Time (min)

20 Time (min)

F I G U R E 2 Purification of a 7.5 kD natural product peptide"*: (a) CE of peptide purified by reversed-phase HPLC, (b) preparative HPIEC of tiie peptide purified by reversed-phase HPLC, and (c) CE of the peptide purified by HPIEC.

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cation-exchange column. A relatively new technique, capillary electrochromatography, further bridges the gap effectively between HPLC and CE (see Section IX). Biomolecules such as proteins, nucleic acids, and polysaccharides are often present in small quantities, and sample sizes are often limited—requiring highly selective and sensitive techniques. Since samples of biological origin are often complex, two or more different yet complementary techniques are often used to perform qualitative or quantitative analysis. The use of complementary techniques provides greater confidence in the analytical results. HPLC and CE, which represent chromatography and electrophoresis, fulfill this requirement. For example, in reversed-phase HPLC (see Chapter 3), the species are separated on the basis of hydrophobicity; in CE, chargeto-mass ratio plays a key role. The difference in separation mechanism is helpful in characterization or elucidation of the structure of complex molecules of biological origin. Furthermore, these techniques provide high resolution with a short analysis time, as well as fully automated microprocessor-controlled quantitative results.

III. MIGRATION BEHAVIOR OF PEPTIDES AND PROTEINS The peptides, which are composed of only a few amino acids, exhibit predictable behavior in CZE in that their mobility (electrophoretic migration) can be predicted on the basis of their size (mass) and their charge characteristics. The charge of such peptides can be predicted from the pK^ values of individual amino acids contained in them. For larger peptides and proteins, the calculation of charge based on ionization constant is not trivial and can not be easily calculated based on the pK^s of the free amino aids. Besides the mass-to-charge ratio, the other factors that affect mobility are hydrophobicity, primary sequence, conformational difference, and the chirality of amino acids. This point has been demonstrated by an investigation on several nonapeptides of identical composition but with different primary sequences: • • • • • •

NH2-ALDYALAHR-COOH NH2-ALDYARLAH-COOH NH2-ALDYHALAR-COOH NH2-HALDYARLA-COOH NH2-ALDYHARLA-COOH NH2-DHAYLAAR-COOH

The results showed that these peptides could be separated from each other at different pH values. It is obvious that these peptides exhibit different charge and a small change in the pH of the buffer, particularly near the pK^s of the amino terminal or car boxy terminal or side groups. Since all of the investigated peptides contained the same amino acids, the amino acid sequence can influence the pK^s of the ionizable side groups and thus influence the migration behavior of the peptides.

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A. Relationship of Protein Mobility to Protein Mass and Valence In 1966, Offord^^ proposed the following equation relating the mobility (m) of peptides to their valence ( Z ) and molecular mass ( M ) : m = kZM-^/^ where fe is a constant. The plot of mobility versus ZM~'^^^ yields a straight line. Since mobility is defined as velocity per unit field strength, this equation can be written in the form of relative migration time (versus a known reference peptide): t/to = KoZ-^M^/^ where t/tQ is the relative migration time, and KQ is a constant pertaining to the reference peptide. A plot of relative migration time versus M'^^^/Z also gives a straight line. The relative migration time of proteins has been found to relate linearly to the isoelectric point (pi) of the given proteins. This conclusion has been drawn from studies of a large number of collagen proteins that vary considerably in molecular weight. The relationship has been studied in untreated fused silica capillaries (see Section IV for details relating to various treatments of these capillaries) within the pH range of 6.9 to 10.5. A number of investigators have studied the mobility of proteins in CE and applied their studies to microheterogeneity analysis. Mobility has been found to be a continuous function of M~^^^ to M~^/^ depending on the magnitude of M and the ionic strength of the buffer (see Section III.B). Research on the modeling of mobility of peptides and proteins is ongoing, and new modes of separations are continuously evolving out of this research. The analysis of small or large peptides constitutes an important part of protein characterization schemes. As mentioned before, peptides containing 10 or fewer amino acids are called small peptides and peptides having 50 to 70 amino acids with a molecular mass of 5000 to 7000 Da lie on the borderline between polypeptides and proteins. For example, in peptide mapping applications proteins are cleaved with enzymes or other suitable chemicals into smaller units and are subsequently analyzed by a variety of methods including CE. The biopharmaceutical industry's interest also stems from the need for synthetic peptides in process development of new therapeutic agents. Peptide mapping is also important in the area of quality control, where sequencing is not performed routinely. B. Buffer Selection The choice of a buffer is very important in CE. Some of the characteristics of a useful buffer are as follows: • Good buffer capacity

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• Good UV transparency • Low conductivity To optimize a particular separation, the type of buffer, its ionic strength, and its pH can be varied. For the majority of peptide appHcations with untreated fused siHca capillaries, the use of high pH generally produces fast separations because the electroosmotic flow (EOF) is high. At low pH values, the peptides migrate primarily on the basis of their charge characteristics. The buffers based on acetate, borate, citrate, and phosphate, or combinations of these are frequently used. However, conductivity associated with these buffers is frequently high, necessitating smaller capillaries or lower field strengths. Such limitations can be minimized by employing efficient cooling techniques, or low conductivities can be achieved by utilizing zwitterionic buffers.

C. Buffer Additives

In addition to buffer selection and effective pH control, a number of buffer additives can be used to optimize a separation (for more detailed discussion, see Section IV.A). i. Surfactants

Both ionic and nonionic surfactants have been used. a. Ionic Surfactants

Addition of micelle-forming agents to the buffers can help improve selectivity. The agents can be either cationic (e.g., CTAB) or anionic (e.g., SDS). As mentioned before, this technique is called MECC. It is similar to reversed-phase HPLC in that an analyte partitions between a mobile phase, i.e., the background electrolyte, and a pseudo-stationary phase, i.e., the micellar phase like SDS, which migrates against the EOF. In MECC, the detergent is added to the buffer above the micellar concentration, e.g., 50-100 m M of SDS can be used. b. Nonionic Surfactants

When peptides with subtle differences in hydrophobicity are being separated, nonionic surfactants may provide a better balance between the electrostatic and hydrophobic forces influencing the separation. Polyoxyethylene-10 (20 m M ) has been found useful for separating two analogues of growth hormone-releasing peptide (composed of six amino acids) with the same mass-to-charge ratio. ii. Ion-Pairing Reagents

Short-chain ion-pairing reagents, e.g., hexane sulfonic acid, have been used in HPLC for protein and peptide separations. This reagent can also be used in CE for hydrophobic peptides that are difficult to separate.

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The use of these additives was first explored in HPLC and later extended to CE. Separation of peptides can be improved by the addition of cyclodextrins. Addition of cyclodextrin can improve sensitivity of detection by fluorescence of derivatized amino acids and peptides. The use of these additives can also help resolution. iv. Organic Solvents

Small amounts of organic solvents (e.g., up to 2% THF or 20% methanol or acetonitrile has been used) are added to improve solubility of analytes in buffer. The addition of organic solvents to the buffer solution causes a decrease in the EOF due to a decreased zeta potential, resulting in a lower current and less joule heat generation. This can lead to better resolution. V. Divalent Amines

Large peptides or proteins can be irreversibly adsorbed on the capillary wall. Addition of small amounts (0-5 m M ) of cationic divalent amines such as 1,4-diaminobutane, 1,5-diaminopentane, or morpholine can help minimize this problem. Larger amounts are necessary for separation of proteins. vi. Complexing Agents

Metal ions can enhance the resolution of nucleic acids and amino acids in MECC. Zinc perchlorate (0-30 m M ) has been found useful as a complexing reagent for histidine-containing peptides.

D. Adsorption of Proteins

A variety of surfaces such as metals, plastics, and glass can retain proteins during a separation process. In CE, a problem is manifested in the adsorption of proteins by fused silica capillaries (see Section IV). This problem is attributed to the adsorption of positively charged sites of proteins on negatively charged sites (silanol groups) on the capillary wall—a process that leads to band broadening and a much lower number of theoretical plates than would be expected on the basis of theory. The band width of an analyte zone as migration progresses through the CE capillary is affected by the specific rate constants of adsorption and desorption, k^ and k^. Protein adsorption may also occur through hydrophobic forces if the stability of the protein in solution is affected by changes in its environment (e.g., by changes in pH or temperature). They are held together in solution by hydrogen bonding and hydrophobic forces. The hydrophobic areas are exposed when the protein unfolds, which can result in aggregation or hydrophobic adsorption. Urea or guanidine hydrochloride can be used for protein denaturation. The disulfide bonds that hold the polypeptide chain together can be broken with j8-mercaptoethanol. Various investigators have utilized high pH to overcome the electrostatic adsorption of proteins on the capillary wall. For example, at pH 10, the pi of most proteins is less than the pH of the buffer; as a result, both the protein and the capillary

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wall are negatively charged, and thus the adsorption process is minimized. This approach is useful in some cases; however, the use of high pH is frequently undesirable for a number of reasons.

lY. MODIFICATIONS OF FUSED SILICA CAPILLARIES Appropriate modification of fused silica capillaries can lead to a lot of very successful separations. The subject of modification of fused silica capillaries has been given excellent coverage by Regnier and Lin.^^ The readers are encouraged to read this reference. Discussed here is a short summary. The internal surface of fused silica capillaries is rich in silanol groups that ionize over a broad pH range from 3.5 to 9. At high pH values, these weakly acidic groups may be present in concentrations ranging up to 8 / i M / m ^ . With increasing pH, the zeta potential of capillary walls and the concomitant EOF grows in direct proportion to the ionization of surface silanols. This suggests that the voltage-driven transport in fused silica capillaries may occur in the following three ways: • Electrophoresis of analytes directly or indirectly (by association with an ionic species) • EOF • A combination of these two The surface charge makes additional contributions to the separation of proteins. For example, some proteins are adsorbed at silanol-rich surfaces, denatured, and then rapidly released into solution. The resulting change in conformation generally impacts electrophoretic mobility, i.e., the denatured protein elutes differently from the native protein. When there is partial denaturation, multiple conformers of the same protein may be observed. Another problem is that silanol groups can make fused silica capillaries behave like a cation exchange column. Severe band spreading and diminished recovery occur when a protein with an isoelectric point higher than the buffer pH interacts with the capillary wall. Still another problem is that adsorbed proteins change zeta potential at the capillary wall, having an impact on both the EOF and column efficiency. This affects elution time and thus compromises reproducibility. It has been shown that an axially heterogeneous distribution of zeta potential can diminish the separation efficiency of a column by triggering nonuniform flow along the length of capillary. This axially heterogeneous distribution of zeta potential results when the inlet of a capillary is fouled with the sample protein. A. Surface Modification of Capillaries The techniques that have been employed successfully are as follows: • Dynamic modification (using extreme values of pH or competing ions) • Static modification (e.g., adsorbed oligomers, polymer coatings, or covalent derivatization)

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It is important to assure that protein recovery is optimal after these procedures because a symmetrical peak does not necessarily provide this assurance. The use of plate count (efficiency) alone suffers from the fact that factors other than silanols affect efficiency. i. Dynamic Modification

Some information on various buffer additives v^as given in Section III.C. A more detailed discussion of dynamic modification of fused silica capillaries is provided here. This entails transitory modification of the capillary v^all by utilizing certain buffer additives. Some examples are • • • • • • • • •

High salt or high ionic strength buffers Zwitterions Divalent cationic amines Nonionic surfactants Charge reversal reagents Zeta potential suppressors Organic solvents Denaturants Surfactants

A simple method of modifying the surface of capillaries utilizes mobile phase (buffer) additives. The objective is to negate the predominantly electrostatic interactions of protein w^ith the w^alls of capillary. This can be accomplished in three v^ays: • Modification of the charge of the capillary w^all • Modification of charge on the protein • Combination of these two effects a. Effect of pH

When the operating pH of fused capillaries is reduced to less than 3.0, silanol ionization drops to almost zero and the EOF is drastically reduced. Both peptides and proteins can be separated under these conditions. Note hov^ever, that they are partially denatured at extremes of pH range. This may cause dissociation of multimeric proteins into subunits, or multiple conformers may be formed. Belov^ pH 3 or above pH 10, only basic or acidic amino acids are ionized, respectively. The reduction in charge diversity of proteins observed at or near physiological pH v^ill diminish selectivity of electrophoretic separations. Polypeptides undergo ion pairing w^ith the acid used to control the pH under acidic conditions. The relative hydrophilicity or hydrophobicity of this acid will have a strong influence on selectivity. b. Competing Ions

The negative charge of silanols can be minimized by utilizing competing ions, such as salts, amines, or zwitterions. The tailing peaks frequently observed with fused silica capillaries can be sharpened by increasing mobile phase salt concentration to 100 m M or more. However, the limitation of this approach is that joule heating increases in direct proportion to the ionic

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strength. Dynamic modification with amines provides an effective means of minimizing silanol effects. Some of the amines that have been used are^^ Hydroxylamine Ethylamine Triethylamine Triethanolamine Morphohne Guanosamine Galactosamine Chitosan Ethylendiamine Putrescine Cadaverine Hexamethonium bromide or chloride Decamethonium bromide Agmatine Spermidine Spermine The amines appear to function by ion pairing with anionic silanol groups at the silica surface. Ion pair formation increases resolution by decreasing the EOF and competing with proteins for ionic groups at the surface. In general, the decrease in the EOF is inversely proportional to the concentration of amine additive. Since diamines and poly amines bind with higher affinity than monoamines, they are more effective in reducing the EOF. It has also been shown that the EOF is reduced in direct proportion to C H 2 - N H ratio of the amine additive, total number of CH2(CH3) groups, and the molecular weight of the amine. c. Surfactants

An attempt has been made to solve silanol problems by titrating the surface charge with counterions. Another strategy is to covalently sequester many of the surface silanols with octadecyl or dimethylsilane and then dynamically coat the hydrophobic surface of the column with nonionic surfactants during use. This coating acts in such a manner that the charge from residual silanols projects through hydrophilic coating and is used to drive the EOF at a velocity substantially less than that with the uncoated capillaries. The EOF is relatively constant across the pH range of 4 to 10. Better than 95% recovery of a broad spectrum of proteins has been achieved in the CZE mode from Brij35-coated capillaries. With pluronic surfactants, good separation efficiency has been achieved by using dimethysilane-derivatized capillaries. Surfactants have also been used with uncoated capillaries. It has been observed that electrophoretic mobility increases with anionic detergents such as sodium deoxycholate (SDC) or sodium dodecyl sulfate (SDS) and decreases in the presence of nonionic surfactants such as Triton X-lOO.The surfactant is presumably adsorbed onto the silica surface and increases the surface charge.

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The electrophoretic mobility of apolipoprotein is very different in SDS and SDC because it binds four times as much with SDS than SDC.^"^ d. Neutral Polymers

Polymers such as poly(vinyl alcohol), dextrans, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, and poly(ethylene oxide) can be used as dynamic or permanent coatings. The latter mode is generally more effective. ii. Static Modification Approach

This entails permanent modification of the capillary vvrall by suitable coatings. Some examples are given in Table 3.^^"^^^ iii. Adsorbed Coatings

These coatings are essentially permanently adsorbed. The permanence is achieved either by the binding affinity of the coating agent or by cross-linking adjacent adsorbed species into a continuous, permanent film. a. Neutral Polymers

A number of neutral polymers have been used. Some of these are discussed in the follov^ing. Poly(vinyl alcohol). Deposition is achieved by flushing the capillary w^ith a 10% aqueous solution of 50 kDa PVA (99% or more hydrolyzed), pushing the coating solution out of the column with a stream of nitrogen, and then heating the capillary at 140°C under a stream of nitrogen. This produces capillaries v^ith an efficiency of greater than a million plates, essentially no EOF, stability up to pH 10, and the ability to separate basic or acidic proteins (see Fig. 3^^). A 50 m M sodium phosphate buffer at pH 3.0 is used, and CE is carried out at 30 kV at 20°C. Cellulose Acetates. Fused silica and polypropylene capillaries have been coated v^ith 1% (w/v) solution of CD A in acetone, pushing the coating solution out v^ith a stream of helium, and drying for 30 min with helium. The coating thickness depends on the viscosity of the coating solution and the speed with which the solution is expelled from the capillary. Excellent separations of basic proteins, with efficiencies up to 1 million plates/m, have

TABLE 3 Capillary Coatings for Separations of Proteins Coating

Researcher

Reference

Methylcellulose Polyacrylamide Polyacrylamide Polyethylene glycol Polyether a-Lactalbumin Ion exchangers

Hjerten Hjerten Novotny Poppe El Rassi Swerdberg Engelhardt

15 15 16 17 18 19 20

251

CAPILLARY ELECTROPHORESIS

N = 1,851,000 m1,239,000

.1.204,000 '861,000 1,245.000

"I

Ulu_JUU 1

r

10

T

r— mln

F I G U R E 3 Efficiency of PVA-coated capillary. (Reprinted with permission from Gilges et o/.^' Copyright 1994 American Chemical Society.)

been achieved at pH 4.0-4.3. The coating is not suitable for use above pH 7.5. Polyiethylene oxide). This ether-rich polymer (PEO) apparently forms hydrogen bonds with silanol groups, with concomitant adsorption of a PEO layer at silica surfaces. In the case of CZE, the fused silica capillaries are pretreated with 0.1 M NaOH and 0.1 M SDS at the beginning of each day. The typical coating protocol is to flush the capillary with 1.0 M HCl, followed by a solution of 0.2% PEO, then washing with an electrophoretic buffer. The coating process has to be repeated before each run. The EOF is reduced by 60-70%, and the columns thus treated work well for basic proteins. b. Polyamines

Polyethyleneimines (PEI) with high molecular weights can be coated on silica by flushing the capillary with an aqueous solution of polymer. The coated capillaries can be used in the pH range of 3 to 11. With these capillaries, the EOF is toward the anode. This means that proteins will elute in the order of increasing pi. The EOF is not constant; it is relatively large at pH 3 - 5 and approaches zero at pH 10. When the PEI layer is quaternized during cross-linking, the EOF is almost constant for pH range of 3 to 11. c. Polymers An excellent solution to the surface deactivation problem of silica is provided by polymers because they can form a continuous surface coating that is sufficiently thick to prevent proteins from reaching the surface. Cellulose. Hydrolytically stable cellulose-derived coatings for CZE have been prepared in a number of ways. Methyl cellulose or hydroxypropyl

252

S. AHUJA

cellulose is generally derivatized with a vinyl, allyl, or methyacyl group and then grafted to a fused silica capillary through an organosilane coupling agent carrying a vinyl or methacryl group. Columns of dextran have been prepared with a more elaborate procedure.^^ It is claimed that the EOF has been eliminated and that columns are very stable under very harsh conditions, e.g., 0.05 M NaOH with 5% SDS or 1 M HCl. Cellulose-coated capillaries have been prepared by coupling 2-hydroxyethyl methacrylate (HEMA)-derivatized hydroxypropyl cellulose (HPC) to columns derivatized with 7-oct-l-enyltrimethoxysilane.The coating was stable for several weeks in the pH range from 2 to 10 and diminished the EOF, and it has been found useful for separation of peptides, glycoproteins, and derivatized oligosaccharides. Acrylates. Acrylamines are widely used to create polymeric coatings on fused silica capillaries in a two- to three-step process. Linear polyacrylamide is then grafted to the appropriately prepared surface. The problem of acrylamide stability can be solved by using a monomer that is inherently more hydrolytically stable. Epoxy Coatings. Epoxy polymers are easily attached to fused silica capillaries through gamma-glycidyltrimethoxypropylsilane and then reacted with diglycidyl ethylene glycol under either anionic or cationic catalysis to produce a polymeric coating. This coating eliminates protein adsorption and reduces the EOF up to 80% and enables separation of proteins in a pH range from 2 to 10. The major application of coatings are in CZE and isoelectric focusing (see Chapter 5). All coatings discussed above have been used in CZE, with only a few exceptions. These coatings help solve the protein adsorption problem, with the EOF approaching zero. A large database is available to further assist the readers with their coating problems.^^

Y. EFFECT OF TEMPERATURE ON SEPARATIONS The separation of proteins is also influenced by temperature. The broad peaks observed at higher than ambient temperatures may be due to the fact that folded and partially unfolded species are encountered under the electrophoretic conditions that have been used for a given separation.

Yl. STRATEGY FOR PROTEIN SEPARATIONS A number of principles governing the separation of proteins that can help optimize the strategy for protein separations^ are given below. The intrinsic pK^s of the proteins depend on the local environment. They are further influenced by ionic strength, dielectric constant, and temperature. Mobility estimates should account for the effective mass-to-charge ratio and molecular shape contributions. The denaturation processes produce sets of

CAPILLARY ELECTROPHORESIS

253

confomers and different aggregation states. The protein concentration can adversely affect peak shape and the estimation of mobiUty. The charge of proteins is equal to the sum of the estimated charge and the charge of the bound ions for a given pH, temperature, and ionic strength. Adsorption occurs by both electrostatic and hydrophobic forces. Proteins tend to fold in a simple first-order process. Relatively small changes in charge, pH, ionic strength, temperature, and denaturant can initiate protein unfolding. Resolution is enhanced by titration across a common pK^ region or a region where either charge or side-chain modification has occurred that will emphasize charge difference. The resolution is increased when the EOF and mobility are matched. Protein mobility changes in a sigmoidal fashion as a function of pH. Successful CZE of proteins often depends on appropriate sample handling that stabilizes native structures. The following information can be very useful in this regard: • Membrane proteins require surfactants to stay in solution. • Sulfhydryl reagents, which are often added to prevent oxidation, should be replenished. • For a given protein mixture with diverse pis, electrophoresis across an isoelectric boundary can result in aggregation and precipitation. • The assessment of solubility, stability, isoelectric point, and matrix requirement is important to assure successful resolutions.

VII. CAPILURY GEL ELECTROPHORESIS The separation of sodium dodecyl sulfate-protein complexes according to molecular weight by slab-gel electrophoresis has played an important role in biochemistry. The gel structure creates a molecular sieving effect, allowing separations on the basis of the size of molecules. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is a popular gel electrophoretic technique, especially in a two-dimensional (2-D) mode, since it can offer resolution of a large number of proteins. SDS binding to polypeptide chains leads to similar charge densities and constant mass-to-charge ratio of different proteins. As a result, the separation takes place mainly on the basis of the size of the proteins. SDS-PAGE is frequently used to analyze the purity of proteins and peptides and to determine the apparent molecular mass by comparison with the calibration standards. CGE offers an alternative that provides fast separations, quantitative analysis, and automated operation. High-level sensitivity detection (500 n g / m L protein) is possible with this technique.^^ Compounds such as nucleic acids and SDS-protein complexes that possess the same mass-to-charge ratios cannot be resolved by CZE. To separate these species, electrophoresis must be performed in a medium in which the rate of migration depends on molecular size. CGE is useful for these separations. A material such as

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acrylamide is polymerized in the presence of catalyst and cross-linked inside the capillary. The porosity of the gel can be controlled by varying the concentration of monomer and crosslinker. Polyacrylamide gel-filled capillaries provide very high resolution of nucleic acids, and they are used for separation of oligonucleotides, PCR products, and DNA sequencing. Polyacrylamide gel-filled capillaries are not useful for protein separations, since the strong UV absorbance of the gel restricts their use to longer w^avelengths where proteins have poor sensitivity. Cross-linked polyacrylamide gel utilized in SDS-PAGE interferes in CE with the UV absorbance detection of proteins at 214 nm. Widhalm et alP used linear polyacrylamide as a sieving medium for proteins. This low-viscosity medium is preferable in that capillary gels are easily prepared. They are convenient to use since they can be readily rinsed out of capillaries. Linear polymers such as celluloses and polyethylene glycols have also been used for separations requiring sieving in CE, e.g., DNA fragments. A discontinuous buffer system can be applied to increase the resolution and loadability of protein solutions. The application of larger volumes of the sample solution helps improve detectability of the system. The samples can be introduced by means of pressure injection, and after running the experiment, the gel matrix can be replaced by using pressure rinsing. Proteins of fairly high molecular weights can be separated by this approach. Dynamic sieving capillary electrophoresis (DSCE) employs a polymer solution as the sieving agent. It is introduced into the capillary under pressure and replenished after each analysis. The UV transparency of polymer solutions enables determination of native proteins. DSCE can be used for molecular weight determination of SDS-protein complexes because they migrate proportional to their molecular size. Polymer systems have been developed that provide good resolution of proteins in the 14-200 kDA range. There are several advantages of utilizing CGE over traditional PAGE for quality control and stability studies of recombinant monoclonal antibodies (Table 4)}"^ Another important point to note is that CE is virtually automated, whereas PAGE is labor-intensive.

TABLE 4 Advantages and Disadvantages of CE Parameter

CE

PAGE

System preparation Sample preparation and loading Maximum number of samples Separation Detection

30 min 60 min 36/tray 30 min/sample Direct (UV)

Data analysis Waste generation Analysis throughput

Simultaneous Minimal (milliliters) 108 samples/3 days

30 min 30 min 13/gel 105 min/gel Indirect (staining); long lag time 1-2 hr Substantial (liters) 13 samples/3 days

Data from Bowen and Schnerman.^"^

255

CAPILLARY ELECTROPHORESIS

VIU. MICELLAR ELECTROKINETIC CHROMATOGRAPHY In MEKC the separation medium is a buffer containing surfactant at a concentration higher than critical micelle concentration (CMC). Surfactant micelles constitute a pseudo-phase into which analyte molecules are partitioned by hydrophobic interactions with the lipophilic micelle interior. The EOF is used as a pump to transport micelles and the bulk electrolyte to the detection point. SDS is commonly used as a surfactant. Since SDS micelles are anionic, they migrate electrophoretically in a direction opposite that of the EOF, thus extending the separation range. MEKC exhibits higher efficiency than RPLC because the flat flow profile of the EOF eliminates band broadening caused by laminar flow in HPLC and high mass transfer kinetics due to the rapid exchange of surfactant between the monomer and micelle phase. Denaturation of proteins under reducing conditions with SDS causes them to assume a globular shape and have a constant mass-to-charge ratio. The only remaining difference then is their size, which relates to molecular weight (My). Linear plots of electrophoretic mobility versus log M^ are obtained across one log of M^ (Fig. 4).^^

IX. CAPILLARY ELECTROCHROMATOGRAPHY Earlier investigations by Mould and Synge in the 1950s and by Pretorius and his associates in the 1970s showed that EOF would act as a pump for chromatography. Jorgensen and Lukacs adapted this concept to capillaries

Molecular mass calibration curv«

LJL tfj, [min] FIGURE 4 CGE of a test mixture of proteins on a eCAP SDS 14-200 gel. Key: I, a-lactalbumin; 2, carbonic anhydrase; 3, ovalbumin; 4, BSA; 5, phosphorylase b; 6, /3-galactosidase; 7, myosin. (Reprinted from Guttman et al}^ Copyriglit 1994, with permission from Elsevier Science.)

256

S. AHUJA

(see Table 2) and called the technique capillary electrochromatography, or CEC. This hybrid technique of capillary electrochromatography couples the separation power of reversed-phase HPLC and the high efficiency of capillary electrophoresis. In CEC, separation of an uncharged molecule is achieved on the basis of differential partitioning into the stationary phase. The mobile phase is pumped electrically; as a result, the analytes are carried through the column by electroosmotic flow. Basically, CEC differs from CE in that the capillary is packed with stationary-phase particles and requires a suitable frit to contain the packing material. Because there are no pressure limitations in CEC, the stationary-phase particles can be reduced theoretically to a submicrometer level. This should minimize two sources of band broadening in chromatography, namely, eddy diffusion and resistance to mass transfer. As mentioned earlier, CEC couples the separating power of HPLC and the high efficiency of CE. The packed capillary can be considered the heart of the CEC system because it acts as a pump and provides chromatographic selectivity. The design of the stationary phase; related parameters such as mobile phase and pH; and instrumental parameters such as pressurization, injection modes, temperature, and voltage polarity play an important role in this technique. A CE instrument that is capable of operating at high pressures such as 12 bar at each end of the capillary can be used. A potential difference applied across both ends of the capillary generates an EOF that drives the mobile phase through the column. The observed EOF in packed CEC is generated from both the silica-based stationary phase and the capillary, and as a result of dissociation of silanol groups. The capillaries are packed with 1-3 fim stationary phase particles. Both reversed-phase and ion-exchange types of stationary phases can be used. Selectivity can be enhanced by changing the pH of the mobile phase and varying the type of organic solvent in the mobile phase. Detection is possible with UV, fluorescence, or mass spectrometry. Solvent gradient CEC can also be performed. A potentially useful block diagram is shown in Fig. 5. X. APPLICATIONS A large variety of proteins and other materials of interest are discussed in this section. A. Serum Proteins The classical five-component serum protein fractions designated as albumin a^, 0^2, j8, and y can be easily resolved by CZE.^^ Although many of these separations have been achieved in borate buffer, the resolution can be increased to 10 protein zones by switching to an operational electrolyte containing 0.1 M methylglucamine and 0.1 M 6-aminocaproic acid or 0.1 M methylglucamine and 0.1 M y-aminobutyric acid. The analysis of cerebrospinal fluid (CSF) has been used as an aid in the diagnosis of central nervous system disorders, even though the composition

257

CAPILLARY ELECTROPHORESIS

Computer

High-voltage power supply 1

High-voltage power supply 2

'TP

Caplltarv 1

I

CapiKary 2

M. Mobti&'phase 1

Separation column

Lans

Moblie*-plias0 2

-Microseope objactrve

4--CI]

.-^"'

Rlters

H I

Lens Photomultiplier

Q

O

Slit

Data acquisition

Las^r

FIGURE 5

A block diagram of solvent gradient CEC.

of CSF is related to serum. The adsorption of proteins in uncoated capillaries has been controlled by the use of Beckman electrophoresis buffer and a commercial column with hydrophilic coating, along with incorporation of methylcellulose into the operating buffer.^'^ With a borate buffer at pH 10 in the latter case, substantial EOF has been noted toward the cathode. The elution order of proteins and peptides as a result is cations, neutral ions, and finally anions. A surprisingly large number of CSF-specific acidic species have been noted relative to serum. As many as 25 peaks have been observed in a total run time of approximately 30 min. The resolution of clinical grade human serum albumin (HSA) increases as the operating pH of the column approaches the pi of HSA.^^ Eight peaks have been resolved with a neutral coated capillary. The mass spectral analysis of these fractions indicated that major component is HSA with a free Thiokol at cystein 34, a variant having cystein 34 blocked cysteine, amino-terminally

258

S. AHUJA

degraded HSA, and probably a combination of these variations. The glycosolated forms of HSA were not resolved from HSA. B. Glycoproteins Glycoproteins are being developed as potential pharmaceuticals because of their activity in biological systems. These proteins require special consideration because their function and specificity may be dependent on carbohydrate moieties. Differences in a protein's glycosylation can affect its biological activity; therefore, heterogeneity generated in its production needs to be evaluated and controlled. The analysis of microheterogeneity in glycoproteins is a challenging problem because a single polypeptide can exist in several to a hundred different glycoforms, which may vary in the position of glycosylation along the peptide chain, the size of oligosaccharide attached to the protein, the sequence of specific oligosaccharide, the degree of branching, and end-group substitution. Glycoforms vary sufficiently in structure and charge and are easily separated by CZE. Glycosylation variants of RNases A and B, horseradish peroxidase, and ovalbumin have been separated in cellulosecoated capillaries at an acidic pH, with a small amount of 1-propanol added to the buffer.^^ As mentioned earlier, the mobile phase additives can improve the separations of proteins. In the case of glycoforms, this occurs by pairing with monosaccharide residues in the protein. The di- and polyamine mobile phase additives, such as decamethonium bromide and spermine, susbstantially enhance the separation of ovalbumin variants in the presence of 100 m M borate buffer.^^ Removal of N-acetylneuraminic acid from the oligosaccharide portion of human recombinant factor Vila (rFVIIa) reduced the number of peaks, thus confirming the importance of ion-pair formation with the amine additives. The determination of the position and the number of glycosylation sites is an important part of characterizing a glycoprotein. In the case of O-linked oligosaccharides of bovine submaxillary mucin, j8elimination occurs at O-linked threonine residues in alkaline sulfite with the formation of cysteic and a-amino and /3-sulfonylbutyric acid residues in the polypeptide.^^ After hydrolysis and derivatization with naphthalene-2,3dicarboxyaldehyde, the stoichiometry of sulfonated derivative was determined by CZE. The selective cleavage in the glycoprotein with glycosidic enzymes, accompanied by electrophoretic analysis of products, provides an even more effective way to study structural and immunogenic properties of glycoproteins.^^ The structure of glycoproteins is generally too complex for direct resolution and analysis of glycoforms. To deal with this problem, at times oligosaccharides are cleaved from the protein and analyzed individually. For example, N-glycans, which are liberated from distinct glycoproteins by either polypeptide N-glycosidase (PNGase) F treatment or hydrozinolysis and isolated by anion exchange chromatography, have been analyzed by CZE with 80 m M ammonium sulfate and 20 m M phosphate buffer (pH 7.0) with 2 m M 1,5-diaminopentane additive.^^ N-linked oligosaccharide mixtures from re-

CAPILLARY ELECTROPHORESIS

259

combinant human urinary erythropoietin, a ^-acid glycoprotein, and bovine fetuin have been resolved into 15-20 components and compared against an N-glycan-mapping database. C. Lipoproteins These proteins are of great interest because of their relationship to coronary heart disease (CHD). Lipoproteins, a group of macromicellar complexes of lipids and proteins, are closely associated with the risk of developing CHD. Structurally, lipoprotein particles contain a nonpolar lipid core of triglycerides and cholesterol esters and a polar surface that is comprised of apolipoproteins and unesterified cholesterol and phospholipids. There are three principle classes of lipoproteins: • High-density lipoproteins (HDL) • Low-density lipoproteins (LDL) • Very low density lipoproteins (VLDL) The preliminary separation of lipoproteins is achieved with ultracentrifugation. LDL and HDL are then resolved by CZE in < 2 0 min in uncoated capillaries with 50 m M borate buffer (pH 10) containing 3.5 m M SDS and 20% acetonitrile.^"* Both lipoprotein a [Lp(a)] and a reduction product Lp(a~) and apolipoprotein a [apo(a)] are separated. HDL is generally determined indirectly via apolipoprotein A-I (apoA-I), which is the major protein constituent of HDL. It has been observed that Biorad LLV buffer is suitable to determine apoA-I in serum directly. The method offers significant advantages over immunoassays or SDS-PAGE in terms of its time, efficiency, and sensitivity. D. Hemoglobins CZE can provide a fast high-resolution method. The major hemoglobin (Hb) species of interest in sickle cell anemia are Hb A^ (pi = 7.10), Hb A 2 (pi = 7.40), Hb F (pi = 7.15), and Hb S (pi = 7.25). CZE separations have been obtained in uncoated capillaries between pH 8 and 9 by using 1.0 M Tris, 50 m M vernol, or 20 m M borate buffer with detection at 415 nm.^^ The best separations have been obtained at pH 8.5 in borate buffer. Hb E, Hb C, and Hb G have been distinguished by their altered mobility in CZE under denaturing conditions using linear polyacrylamide-coated capillaries.^^'^"^ Hemoglobins are denatured and separated in 10 m M phosphate buffer (pH 2.5) using 7 M urea and 0.1% Triton X-100 E. Recombinant Proteins The proteins produced by recombinant DNA technology are frequently accompanied by structural variants resulting from expression errors, in vivo modifications, posttranslational errors, improper folding, aggregation, and chemical modifications that occur during purification. The presence of vari-

260

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ant forms of proteins in a human therapeutic product is undesirable because they may diminish biological activity or be immunogenic. CZE has been utilized to determine the absence of hGh variants in various preparations. Isoforms resulting from primary structure cleavage, monodeamidation at two different positions, amino terminal succinylation, and a HIS to GLN replacement have been found at position 18 in hGH produced by Escherichia coli?^'^^ The separations have been achieved in uncoated capillaries with 100 m M phosphate buffer (pH 6.0). Recombinant human interleukin-4 (rhIL-4) is a monomeric protein with a molecular weight of 15,400 Da and pi of 9.2, with three intrachain disulfide bonds. It is a cytokine that has been investigated for cancer therapy. CZE of rhIL-4 mixtures prepared by in vitro degradation has been performed in uncoated capillaries with 50 m M 1,3-diaminopropane and phosphate buffers with pH ranging from 4.5 to 8.0."^^ The resolution of degradation products by CZE appeared to be superior to HPLC. Human antithrombin III (haT III) is a single chain of glycoprotein that inhibits serine proteins of blood-clotting cascade. After removing immunoglobulins from the cultured supernatant by protein G chromatography, haT III has been analyzed by CZE in an uncoated capillary at pH 2.0 with 50 m M phosphate buffer and 0.1% hydroxypropyl methylcellulose."^^ CZE is useful for monitoring both the biosynthesis and purification of haTIII. Hirudin is a potent thrombin inhibitor that is used as an anticoagulant. During production of the recombinant desulfatohirudin variant (r-Hr) for anticoagulation therapy in humans by recombinant technology, seven variants were observed as either by-products or degraded forms of r-Hr. All the major variants have been separated in an uncoated capillary with 60 m M acetate buffer (pH 4.4) containing 0.3% PEG 20,000 and 0.1 m M Zn^+.^^

F. Milk Proteins Bovine milk contains 3-3.5% proteins. Caseins comprise 80% of these proteins; the serum proteins make up the rest. The casein fraction can be divided into a^-, a2~5 i^"? ^^^ K-casein components. The major serum proteins are j8-lactoglobulin (A, B, and C) and a-lactoglobulin. Uncoated capillaries can be used with either 10 m M phosphate (pH 7.4) or 150 m M borate (pH 8.5) with 0.05% Tween 20. It is possible to partially resolve whey into five major components: a-lactoglobulin, j8-lactoglobuhn A, j8-lactoglobulin B, bovine serum albumin (BSA), and immunoglobin (IgG)."^^ Although j8-lactoglobuhn variants and BSA are only partially resolved in this case, it is possible to separate all of the variants of j8-lactoglobulin (A, B, and C) from other whey proteins in the absence of BSA by changing the separation conditions slightly. This is accomplished in an uncoated capillary with 50 m M 2-morpholinoethane sulfonic acid buffer at pH 8.0 with 0.1% Tween 20.^"^ Note that j8-lactoglobulin A and B vary by an Asp to Gly substitution at the 64 position and Val to Ala substitution at the 118 position. j8-Lactoglobulin C is similar to the B variant with an additional Gin

CAPILLARY ELECTROPHORESIS

261

to His substitution at position 59. Other examples of food proteins resolved by CZE include fish proteins, cereal proteins, and soy proteins. G. Protein Folding In vitro folding of a polypeptide generally occurs immediately after it leaves the ribosome during biosynthesis or is excreted from the cell. One of the reasons for the great interest in protein folding and unfolding stems from the fact that polypeptides are frequently stored as inactive structural conformers in genetically engineered host cells. They must be refolded in vitro to reach their native 3-D structure and become biologically active. The 3-D structure may be altered during purification, formulation, or storage. The main problem associated with studying protein folding using separation systems is that such folding can occur in a time frame shorter than the analysis time. CZE is attractive for studying folding because it is much faster than the commonly used methods. For example, it has been shown in trypsinogen that fully reduced protein at neutral pH will eventually re-form the native 3-D structure with the accompanying disulfide bonds. CZE has been found to differentiate native trypsinogen from both the reduced and partially folded intermediates."*^ H. Genomics A human genome consists of 3 billion base pairs of nucleic acids. The magnitude of this problem is immense. At present, a good molecular laboratory can sequence about 3 million base pairs per year. This means it could take 1000 years to sequence the genome once. There is a need for a set of technologies that can function in concert to achieve the final goal of highspeed, high-throughput DNA sequencing. Several research groups have shown that CGE can be a useful tool for DNA sequencing."*^ The medium used (cross-linked polyacrylamide), buffer composition, separation mechanism, sequencing chemistry, and tagging chemistry for CGE are all derived from proven slab gel electrophoresis (SGE) schemes. A 25-fold increase in sequencing rate per capillary has already been demonstrated. The use of parallel sequencing runs in a set of 24 capillaries has been demonstrated successfully. To provide sensitive laser-excited fluorometric detection, a confocal illumination geometry has been coupled to a single laser beam to a single photomultiplier tube. However, there are subtle features inherent to the confocal excitation scheme that may limit its use for a very large number (hundreds) of capillaries. Of the various mutiplexed CE schemes that have been proposed, the machined channel format seems to be most interesting presently. Novel sieving media and acceleration of electrophoretic runs coupled with suitable data processing are likely to provide excellent approaches to DNA sequencing by multiplexed CE. The technology can be readily scaled to 1024 capillaries, which is a convenient number for available array detectors. Such a system can be optimized to accelerate the sequencing process enormously.

262

S. AHUJA

REFERENCES 1. Schwartz, H. E. Palmeiri, R. H. Nolan, J. A., and Brown, R. (1992). "Separation of Proteins and Peptides by Capillary Electrophoresis." Beckman Instruments, Fullerton, CA. 2. Khaledi, M. G. (1998). "High Performance Capillary Electrophoresis.'* Wiley, New York. 3. Wehr, T. and Rodriguez-Diaz, R. (1999). "Capillary Electrophoresis of Proteins." Marcel Dekker, New York. 4. Jorgensen and Lukacs (1981). Anal. Chem. 53, 1298. 5. Terabe et al. (1984). Anal. Chem. 56, 113. 6. Hjerten and Zhu (1985). / . Chromatogr. 346, 265. 7. Gassman et al. (1985). Science 242, 813. 8. Cohen et al. (1988). Natl. Acad. Set. 8, 9660. 9. Cohen and Karger (1987). / . Chromatogr. 397, 409. 10. Ahuja, S. (in press). "Chiral Separations by Chromatography." Oxford University Press, New York. 11. Yang, Q., Tomlinson, A., and Naylor, S. (1999). Anal. Chem. 183A. lla.Guarrino and Philips (1991). Am. Lab., p. 68. 12. Offord, R. E. (1966). Nature, 111, 591. 13. Regnier, F. E., and Lin, Shen (1998). "High Performance Capillary Electrophoresis" (M. G. Khaledi, ed.), p. 683. Wiley, New York. 14. Tadey, T., and Purdy, W. C. (1993). / . Chromatogr. 652, 131. 15. Hjerten (1985). / . Chromatogr. 347, 191. 16. Novotny (1990). Anal. Chem. 62, 2478. 17. Poppe (1988). / . Chromatogr. 471, 429. 18. El Rassi (1991). / . Chromatogr. 559, 367. 19. Swerdberg (1991). / . High Res. Chromatogr. 14, 65. 20. Engelhardt (1991). / . Microcol. Sep. 3, 491. 21. Gilges, M., Kleemis, M. H., and Schomburg, G. (1994). Anal. Chem. 66, 2038. 22. Dittman, M., and Rozing, G. (1999). LCGC, p. 132. 23. Widhalm, A., Schwer, C , Blass, D., and Kendler, E. (1991). / . Chromatogr. 549, 446. 24. Bowen, S., and Schnerman, M. (1998). Biopharm, November, p. 42. 25. Guttman, A., Shieh, P., Lindahl, J., and Cooke, N. (1994). / . Chromatogr. A 676, 111. 16. Dolnik, V. (1995). / . Chromatogr. 709, 99. 27. Cowdry, G., Firth, M., and Firth, G. (1995). Electrophoresis 16, 1922. 28. Denton K. A., and Harris, R. G. (1995). / . Chromatogr. 705, 335. 29. Huang, M., Plocek, J., and Novotny, M. (1995). Electrophoresis 16, 396. 30. Klausen, N. K., and Kornfeh, T. (1995). / . Chromatogr. 718, 195. 31. Weber, P. L., Bramich, C. J., and Lunte, S. M. (1994). / . Chromatogr. 680, 225. 32. Schmerr, M. M., and Goodwin, K. P. (1993). / . Chromatogr. 652, 199. 33. Hermentin, P., Doenges, R., Witzel, R., Hokke, C. H., Viegenthart, J. F. G., Kamerling, J. P., Conradt, H. S., Nimitz, M., and Brazel, D. (1994). Anal. Biochem. Ill, 29. 34. Hu, A. Z., Cruzado, I. D., Hill, J. W., McNeal, C. J., and Macfarlane, R. D. (1995). / . Chromatogr. 717, 33. 35. Sahin, A., Laleli, Y. R., and Ortancil, R. (1995). / . Chromatogr. 709, 121. 36. Zhu, M., Wehr, T., Levi, V., Rodriguez, R., Shiffer, K., and Cao, Z. A. (1993). / . Chromatogr. 652, 119. 37. Casagnola, M., Messana, L, Cassiano, L., Rabino, R., Rosetti, D. V., and Giardina, B. (1995). Electrophoresis 16, 1492. 38. Frenz, J., Wu, S. L., and Hancock, W. (1989). / . Chromatogr. 480, 379. 39. Dupin, P., Galinou, F., and Bayol, A. (1995). / . Chromatogr. 707, 396. 40. Bullock, J. (1993). / . Chromatogr. 633, 235. 41. Rief, O. W., and Freitag, R. (1994). / . Chromatogr. 680, 383. 42. Dette, C , and Watzig, H. (1995). / . Chromatogr. 700, 89. 43. Kinghorn, N. M., Norris, C. S., Paterson, G. R., and Otter, D. E. (1995). / . Chromatogr. 700, 111. 44. Paterson, G. R., Hill, J. P., and Otter, D. E. (1995). / . Chromatogr. 700, 105. 45. Strege, M. A., and Lagu, A. L. (1993). / . Chromatogr. 652, 179. 46. Yeung E. S., and Li, Q. (1998). "High Performance Capillary Electrophoresis" (M. G. Khaledi, ed.), p. 767. Wiley, New York.

ISOELECTRIC FOCUSING DAVID E. GARFIN Bio-Rad Laboratories, Life Science Group, Hercules, California 94547

I. INTRODUCTION II. THE PRINCIPLES OF ISOELECTRIC FOCUSING A. The Mechanism of Isoelectric Focusing B. Practical Considerations C. Establishing pH Gradients III. ANALYTICAL ISOELECTRIC FOCUSING A. Carrier Ampholyte lEF in Gel Slabs B. lEF with Immobilized pH Gradients C. Electrofocusing D. Determining pH Gradients and pis E. Detection of Protein Bands F. Preservation of Gels IV. TWO-DIMENSIONAL GEL ELECTROPHORESIS V. PREPARATIVE ISOELECTRIC FOCUSING A. Removal of Ampholytes from Proteins VI. CAPILLARY ISOELECTRIC FOCUSING VII. SUMMARY VIII. APPENDIX A. Band Shape in lEF B. Factors Affecting Resolution REFERENCES

I. INTRODUCTION Isoelectric focusing (lEF) is unique among separation methods. As its name implies, lEF makes use of the electrical charge properties of molecules to focus them in defined zones in the separation medium. It is the focusing mechanism that distinguishes lEF from other separation processes. In all other methods of separation, diffusion and interactions with the medium act to disperse the bands of separated materials. In contrast, the basic mechanism of isoelectric focusing imposes forces on molecules that directly counteract the dispersive effects of diffusion. During separation, molecules in the sample accumulate in specific and predictable locations in the medium regardless of their initial distribution. The focusing mechanism distinguishes lEF from other forms of electrophoresis as well. With all other forms of electrophoresis an applied electrical field moves molecules through the separation medium at Separation Science and Technology, Volume 2 Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

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fixed rates. The applied field in lEF establishes and maintains a steady-state distribution of sample molecules. This distribution collapses once the field is discontinued. Isoelectric focusing is applicable only to the fractionation of amphoteric species, such as proteins and peptides, that can act as both acids and bases.^'^ Nonamphoteric species, nucleic acids in particular, cannot be resolved by lEF. Both analytical and preparative modes of lEF have been developed that are of value in studies of proteins. The basis for the electrofocusing mechanism lies in the properties of the charge-bearing constituents of proteins. The information that lEF provides is important not only in itself but also because it complements information on other physical parameters such as molecular weight. In comparison to some other separation methods, lEF is easy to understand and relatively easy to use. Most methods are straightforward and the results are readily interpretable.

II. THE PRINCIPLES OF ISOELECTRIC FOCUSING Proteins, as amphoteric molecules, can possess positive, negative, or zero net charge, depending on the pH of their local environments. At low pH, proteins carry net positive charge and at high pH they are negatively charged. The overall charge of a particular protein is determined by the ionizable acidic and basic side chains of its constituent amino acids, various posttranslational modifications, and prosthetic groups. Carboxylic acid groups (-COOH) in proteins are uncharged in acidic solutions and dissociate to the anionic form (-COO~) at pH values above about pH 3. Amines (-NH2) and other basic functions of proteins, such as guanidines, are uncharged at alkaline pH, but are cationic below about pH 10 (e.g., - N H 3 ) . The overall compositions and structures of proteins and the properties of the separation media affect the pH at which individual ionizable side chains actually dissociate. As a result, each individual ionizable group in a protein has a nearly unique dissociation point. The key to understanding lEF is the recognition that the net charges carried by proteins are pH dependent. The net charge on a protein is the algebraic sum of all its positive and negative charges. There is a specific pH for every protein at which the net charge it carries is zero. This isoelectric pH value, termed the isoelectric pointy or pi, is a characteristic physicochemical property of every protein. The definition of pi for molecules as complex as proteins is more or less an operational one and is taken to be that pH at which a protein has zero electrophoretic mobility in an isoelectric focusing run. Nevertheless, it has been shown that the pis of some acidic proteins (up to about pH 7) can be calculated from their amino acid compositions.^"^ If the number of acidic groups in a protein exceeds the number of basic groups, the pi of that protein will be at a low pH value and the protein is classified as being acidic. On the other hand, if basic groups outnumber acidic groups, the pi will be high, with the protein classified as basic. Proteins show considerable variation in isoelectric points, but pi values usually fall in the range of pH 3 to pH 12 with a great many having pis between pH 4 and

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pH 7 / " ^ Some peptides are too small to have a balance of positive and negative charge groups. These peptides do not focus into sharp bands. Isoelectric focusing v^as developed to separate proteins on the basis of differences in their pi values. The mechanism of lEF can be treated mathematically and the classical analysis is presented in the Appendix. On the other hand, the mechanism of lEF is relatively easy to conceptualize and lends itself to a simple description. The historical, theoretical, and practical aspects of lEF are well documented. Many experimental details can be found in several monographs and review^ articles.^'^'^"^^ Refs. 1 and 2 are particularly recommended. They provide valuable insights from one of the pioneers in the field. A. The Mechanism of Isoelectric Focusing

Proteins are positively charged in solutions at pH values below their pi and negatively charged above their isoelectric points. In electrophoresis, the net charge on a protein determines its direction of migration (electrophoretic mobility). When a protein is subjected to an electric field, it will move toward the electrode with the opposite charge. At pH values below the pi of a particular protein the electric field will cause it to migrate toward the cathode, or negative electrode. Conversely, at pH values above its pi a protein will be driven toward the positively charged anode. A protein at its pi does not respond to the electric field because it is uncharged (Fig. 1). Ignoring for now how a pH gradient is established, consider a protein under the dual influences of an electric field and a changing pH environment where the pH increases monotonically from the anode to the cathode (Fig. 2a). During its movement through the pH gradient, the protein will either pick up or lose protons and its net charge will change accordingly (Fig. 2b). The direction of migration is always in the direction of decreasing charge and mobility. Eventually, the protein will arrive at the position in the gradient where the pH equals its pi. There, being uncharged, it will stop migrating in

Cathode

net charge F I G U R E I The basic elements of isoelectric focusing. A protein is depicted in a pH gradient in an electric field. The pH gradient increases from acidic (pH 3) at the anode to basic (pH 10) at the cathode. The hypothetical protein in the drawing carries net charges of + 2 , 0, or —2 at the three positions shown. The electric field drives the protein toward the cathode when it is positively charged and toward the anode when it is negatively charged, as shown by the arrows. A t the isoelectric point (pi), the net charge on the protein is zero and it does not respond to the field. The protein loses protons and becomes progressively less positively charged as it moves toward the cathode. Conversely, the protein gains protons and becomes less negatively charged as it moves toward the anode. A t the pH where the number of positive charges on the protein equals the number of negative charges, the uncharged protein ceases to move in the field.

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a

o

a3 "

O

o

O

a (D

o

O C

o

o

x= 0 Distance, x, in Focusing Chamber F I G U R E 2 The mechanism of isoelectric focusing. (A) In a typical isoelectric focusing run, an electric field £ generates a pH gradient by one of the electrolytic processes described In the text. The pH gradient Increases from an acidic pH at the anode to a basic pH at the cathode. The pH denoted as pi represents the isoelectric pH of the protein of interest. (B) A t pH values below its pi, the protein of interest is positively charged and experiences a force driving it toward the cathode (F+ ). Above Its pi, the protein is negatively charged and it is forced toward the anode (F — ). The protein is uncharged at its pi and experiences no electrical force. (C) The protein of interest eventually accumulates at its pi. The resultant Gaussian mass distribution centered at x = 0 has a maximum concentration CQ and a standard deviation (T. See the Appendix.

response to the field. If a protein at its pi should happen to diffuse to a region of lower pH, it will become protonated and be forced toward the cathode by the electric field. If it diffuses into a pH higher than its pi, the protein will become negatively charged and will be driven toward the anode (Fig. 2b). Protein molecules accumulate, or focus, into sharp bands at their characteristic pi values. As shown in the Appendix, the concentration profiles of proteins in focused bands are represented by Gaussian distributions (Fig. 2c). B. Practical Considerations IFF is a high-resolution technique generally carried out under nondenaturing conditions, in which proteins maintain most of their physical and chemical

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characteristics. Resolution of proteins differing in pi by as little as 0.02 pH units, or less, is common^ and separation of proteins with pis as little as 0.001 pH unit apart has been achieved.^ Because of this high resolution, protein samples that appear to be homogeneous by other methods can often be separated into several components. Such heterogeneity may be indicative of differences in primary structure, posttranslational modifications, or denaturation. Maintenance of both the pH gradient and the separation of focused bands depend on the presence of a sustained electric field. Once the applied voltage is turned off in an lEF cell, the pH gradient quickly dissipates and the protein bands begin to spread. In addition, convective and gravitational disturbances can cause focused bands to disperse. Some means of band stabilization must be incorporated into the system to verify and utilize the focusing pattern. At one time, all lEF was carried out in vertical columns using density gradients of sucrose or glycerol to stabilize the pH gradient against convection during the run and to support separated zones until they could be collected.^'^ However, these density gradient columns are cumbersome and difficult to operate. Focused zones in vertical columns are inherently unstable (because they are denser than the surrounding medium) and not adequately maintained by the density gradients. In addition, the resolution obtained by focusing is often lost during recovery of the focused materials by draining the columns from their bottoms. As a consequence, other stabilizing media have, for the most part, replaced density gradients. Most analytical lEF is currently carried out in continuous polyacrylamide ggjgi,2,9-i7 (^pjg 3) Polyacrylamide provides a virtually uncharged support matrix for IFF. Gels are formed with large pores, which enable the relatively unimpeded motion of proteins. The most common configuration for an IFF gel is the slab format. This configuration provides good cooling efficiency, makes sample application relatively easy, and allows for the comparison of multiple samples. Agarose gels are sometimes used for IFF of very large protein molecules, but residual charges on commercial agarose cause unwanted instabilities in focusing. These bound residual charges attract small mobile ions in the sample solution that carry the opposite charge. Under the influence of an applied electrical field, the mobile ions move from one bound charge to another, carrying with them their waters of hydration. The resultant mass transportation of water through the medium, called "electroosmosis," disrupts the focusing pattern. In practice, electroosmosis becomes a factor in IFF only as a consequence of an improper choice of materials or during very long IFF runs. Preparative electrofocusing is now most commonly done in free solution in specially designed chambers made up of interconnected cells that hold the focused, separated protein zones until they are coUected.^^"^^ The separation axes of the most popular preparative cells are horizontal to minimize the effects of gravity on focused bands. Very often, electrofocused proteins show patterns of multiple bands in places where only a single band is expected. This phenomenon is called "microheterogeneity." Farly discussions of microheterogeneity attempted to explain it in terms of denaturation or of variable interactions between carrier

i

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DAVID E. GARFIN

1

2

3

4

5

6

7

8

9

10

F I G U R E 3 Isoelectric focusing in a polyacrylamide gel. In this demonstration gel, three different types of protein samples were separated. The pH gradient extends from pH 3 at the bottom of the figure to pH 10 at the top. Lanes I and 10, contain marker proteins with known pis of 5.1, 6.0, 6.5, 6.8, 7.0, 7.1, 7.5, 7.8, 8.0, 8.2, and 9.6 (bottom to top; major bands only). Lanes 2 - 5 contain increasing amounts of a preparation of snake venom and lanes 6 - 9 contain increasing amounts of a preparation of the enzyme horseradish peroxidase. This particular gel was run at relatively low resolution in an "inverted" cell"^^ with the (5%T, 3.3%C) polyacrylamide gel suspended between two carbon rod electrodes (see the text). Focusing was carried out in a stepped fashion starting at 100 V for 15 min, then increasing the voltage to 200 V for 15 min, and completing the run at 450 V for I hr (525 volt-hours). Following focusing, protein bands were made visible by staining them with the dye Coomassie Brilliant Blue R-250.

ampholytes and proteins.^^ However, the accumulated evidence suggests that it is a result of compositional differences betv^een protein isomers (or denaturation). It is now believed that microheterogeneity is a consequence of mutations and variable posttranslational modifications to the polypeptide chains of some proteins.^^

C. Establishing pH Gradients lEF became widely useful only when simple methods for establishing and maintaining pH gradients were developed. There are three distinct ways in which pH gradients can be set up. 1. Mixtures of synthetic molecules called carrier ampholytes are used to estabhsh pH gradients under the influence of applied voltages.^ This is the most common and perhaps simplest means for gradient formation. It is the method of choice for most lEF runs.

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269

2. Synthetic buffer compounds containing reactive double bonds (acrylamido groups) are covalently incorporated into the lattices of poly aery 1amide gels that form the separation media. The pH gradients are fixed in place in the separation media (polyacrylamide gels) with this technique.^ These immobilized pH gradients (IPCs) are more cumbersome and expensive to cast than carrier ampholyte gels. However, the commercial availability of consistent IPGs has made them the method of choice for the lEF first dimension of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). 3. Pairs of specially selected buffers will form very narrow pH gradients in some lEF devices and are useful in preparative lEF.^"^'^^ All three methods for generating pH gradients depend on electrolytic, or protolytic, mechanisms to form pH gradients. The pH gradients obtained with carrier ampholytes and the buffer pairs are linear, but IPGs can be constructed that are nonlinear.^ i. Electrode Reactions

When an aqueous solution of a weak acid, HA, in a suitable container is connected to the terminals of a direct current power source (through inert platinum electrodes), electrolysis takes place. At the cathode, where reduction or the gain of electrons occurs, water dissociates and the anion A~ is Hberated (ignoring the hydronium ion H30"^): 2e-+ 2H2O ^ 2 0 H - + H2

(a)

HA + O H - - ^ A - + H 2 O

(b)

These are the predominant reactions when the environment at the cathode is kept alkaline by the addition of base as is the case in lEF. At the anode, where oxidation or the loss of electrons occurs, the electrolysis of water leads to protonation of the anion 2H2O ^ 4 H + + O2 + 4e~ H++A--^HA

(c) (d)

Anolyte solutions in lEF are made acidic to ensure these reactions. These reactions apply as well to the electrolytes at the electrodes, the ampholytes or buffers in the medium, and the proteins to be separated. Protein molecules can be thought of as being deprotonated toward the cathode (basic end of the separation medium) and protonated toward the (acidic) anode. ii. Carrier Ampholytes

The first practical lEF experiments were carried out with the use of synthetic molecules, called carrier ampholytes, to generate the pH gradients.^'^^ Carrier ampholytes are amphoteric electrolytes that carry both current and buffering capacity. Much of the early theoretical activity in electrofocusing dealt with the properties required of carrier ampholytes and is more or less irrelevant to a current discussion.^'^'^ Different varieties of

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carrier ampholytes fulfilling the fundamental criteria are commercially available from several sources. To function in lEF, carrier ampholytes must first of all be amphoteric and capable of being focused by an electric field into a well-defined, monotonic pH gradient. They should also provide an even conductivity throughout the gradient to maintain the flow of current while proteins are undergoing focusing. In addition, carrier ampholytes must have high buffering capacity at their individual pis to limit local variations in pH in any part of the gradient. Moreover, carrier ampholytes must be soluble at their pis, interact weakly, or not at all, with proteins, and be small enough to be easily separated from proteins by ordinary means such as dialysis or gel filtration.^'^ The exact compositions of carrier ampholytes are proprietary to the different manufacturers. In general, they are mixtures of oligomers (about 300-1000 Da in size) containing multiple aliphatic amino and carboxylate groups, although some types contain sulfonic and phosphoric acid residues (Fig. 4). Depending on the source, carrier ampholytes will be mixtures of hundreds (maybe thousands) of molecules of the types shown in Figs. 4a, 4b, or 4c, with some products containing more complex types of molecules.^''^^ They are formed in the aggregate by reactions of the Michael type, then fractionated (also by proprietary methods) and blended to give mixtures that will generate gradients in the desired pH ranges. Protein pi values can be estimated in gradients formed with wide-range carrier ampholytes covering up to 7 pH units (e.g., pH 3 to 10) and more closely estabUshed with narrow pH ranges, down to 2 pH units. Commonly available ampholyte mixtures are designed to generate pH gradients in the following ranges: pH 3 - 5 , pH 4 - 6 , pH 5 - 7 , pH 5 - 8 , pH 6 - 8 , pH 7-9, and pH 3-10. From an experimental perspective, it is important to note that with commercial ampholytes pH 3 and pH 10 are nominal designations. In practice, these ampholytes reach about pH 3.5 and pH 9.5, respectively. The products from the different manufacturers are not necessarily interchangeable, but they may be blended if experimentation shows that to be desirable. Gradient formation in a mixture of carrier ampholytes can be thought of as taking place in a stepwise fashion^ (Fig. 5). Initially, the solution is at some intermediate pH value depending on the ampholytes involved (Fig. 5a). Once a voltage is applied to the ampholyte mixture, the electrode reactions begin and at the same time ampholytes start to move toward their attracting electrodes. The region in the vicinity of the anode becomes acidified while close to the cathode the solution becomes basic (Fig. 5b). The most acidic ampholytes (lowest pi) migrate to positions close to the anode, while the most basic ones (highest pis) move to the cathode. There, being buffers, the two sets of ampholytes estabUsh small plateaus of pH at their pis (Fig. 5c). This process continues, from the ends toward the center, until the many hundreds of ampholyte molecules focus to produce a nearly linear pH gradient from low pH at the anode to high pH at the cathode (Fig. 5d). Although they move more slowly than the carrier ampholytes, proteins, too, begin to migrate when voltage is applied. Proteins with pis at the extremes of the pH gradient begin to form distinct bands first before those with pis in the middle of the gradient.^'^^'^^ The separation of proteins

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A

... CH2

N

CH2

CH2 - — CH2

NH

CH2 .

CH

I

CH

+NH

/ \

CH2 CH2

COO-

CH2

CHo

coo

B ... CH2

N

CH2

CH2

CH2

CH2.

CH2

I

I

COO-

coo-

CH2

... CHo

N

N

CH2

CHo

CHo

NH

CH,

CH2

CH2

NH

CH2

CH2

CH2

SO3-

F I G U R E 4 Compositions of three types of carrier ampholytes. Short segments of some representative carrier ampholytes are shown. The different types of molecules arise from different manufacturing processes. Most carrier ampholytes are mixtures of oligomers (about 300 - 1000 Da in size) containing multiple aliphatic amino and carboxylate groups (A and B), although some types contain sulfonic and phosphoric acid residues (C). See Ref. I.

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DAVID E. GARFIN

pH

I A

pH

O

o

pH

pH

W

A

F I G U R E 5 Creation of a pH gradient with carrier ampholytes. (A) An aqueous solution of carrier ampholytes has a uniform pH that is an "average" determined by the particular species of ampholyte molecules in the mixture. (B) When an electric field is applied to the separation chamber, hydrogen ions are produced at the anode and hydroxyl ions are produced at the cathode. The region in the vicinity of the anode becomes acidified while the cathodic end of the separation chamber becomes basic. (C) The carrier ampholytes begin to move in response to the field. The directions of motion are determined by the pis of the individual species of ampholytes. The most acidic ampholytes (lowest pi) migrate to positions close to the anode and the most basic ampholytes (highest pi) move to the cathode. Since the ampholytes are good buffers and they stop migrating at their pis, plateaus of pH develop at the ends of the separation chamber. (D) The great many ampholyte species in the chamber form a multitude of pH plateaus at their respective pis (curved line) to eventually form a nearly linear pH gradient that increases in pH from the anode to the cathode (straight line).

Starting at the two ends of the focusing column becomes readily apparent in certain situations (such as in the Rotofor apparatus described later) when mixtures of colored proteins are fractionated. a. Gradient Instability

For most practical purposes, pH gradients generated with carrier ampholytes are stable once the steady state has been reached. During extended focusing runs (longer than about 3 hr under standard analytical conditions),

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ISOELECTRIC FOCUSING

however, gradients are found to slowly deteriorate.^'^'^^~^'^'^'' This decay is characterized by a drift of the gradients toward the cathode and is accompanied by acidification at the anode, flattening of the gradient in the neutral pH region, and a loss of alkaline bands. The mechanism of instability, which has been called "cathodic drift," is not completely understood. Cathodic drift is probably caused by a combination of factors including CO2 absorption into the hquid in the gel,^'^'^^ nonzero, pl-dependent electrophoretic fluxes,^'^^ and electroosmosis.^^ The practical consequence of cathodic drift is that excessively long carrier ampholyte lEF runs should be avoided. Hi. Immobilized pH Gradients Gradients formed with carrier ampholytes are subject to cathodic drift (see earlier). They are also subject to regions of relatively low conductivity and low buffering capacity. In addition, because of the complexity of the manufacturing process, carrier ampholyte preparations show some batch-tobatch variability. None of these inconsistencies poses a major problem in most applications of lEF. However, when very precise pi positioning and reproducible patterns are required, as is the case with some 2-D PAGE experiments, any imprecision in focusing patterns can negatively impact a study. The chemicals and methods for casting IPGs were developed to provide a means for obtaining predictable and reproducible lEF j-uns.^'^'^^'^^'^^'^^"^^ This kind of precision is important for 2-D PAGE where literally thousands of proteins can be separated and compared first by pi then by molecular weight.^^ High resolution and consistent comparisons require that all of the proteins in different samples focus completely at their true pis and moreover that the positions of proteins in the lEF gels be identical from gel to gel. This requires focusing times long enough for all proteins to reach their steady state positions. It appears to be the case that this can only be accomplished by immobilization of the pH gradient, which is done by grafting it directly into a polyacrylamide matrix. The sample load capacity of IPGs is much greater than that of carrier ampoholyte systems because the proteins cannot displace the background buffering compounds.^ This property is important in studies of low-abundance proteins. IPGs also appear to provide the best method available for separating basic proteins (with pis up to pH ll).^""'^^ A set of nonamphoteric buffers that are derivatives of acrylamide were synthesized specifically for casting IPG gels.^'^^ They are blended with the monomer mixtures and copolymerized into polyacrylamide gel matrices. In properly oriented electric fields, IPG gels develop pH gradients that increase in pH value from the anode to the cathode. Acrylamido buffers for IPGs have the general structure CH2 = C H - C O - N H - R , where R is a compound containing either a carboxyl (-COOH) or a tertiary amino group [e.g., -N(CH3)2]. For attaining very high and low pHs, R contains either a quaternary ammonium [-N"^(C2H5)3] or sulfonate group (-SO^), respectively (see Table 1). Acrylamido buffers are synthesized by

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DAVID E GARFIN

j j j j ^ l TABLE I

Acrylamido Buffers'*

Compouna

Mr

PK^

2-Acrylamido-2-methylpropane sulfonic acid N-acryloyl glycine 4-Acrylamido butyric acid 2-Morpholino ethylacrylamide 3-Morpholino propylacrylamide N, N-dimethyl aminoethyl acrylamide N, N-dimethyl aminopropyl acrylamide N, N-diethyl aminopropyl acrylamide QAE-acrylamide

207 129 157 184 198 142 156 184 198

1 3.6 4.6 6.2 7.0 8.5 9.3 10.3 >12

See Refs. 2 and 33. Commericially available. pK values are nominal. The effect of temperature on pK is ignored. See Ref. 2.

acylation of amino buffers with acryloyl chloride then purified by crystaUization or distillation.^ Acrylamido buffers have been extensively studied. Seven of the possible compounds are commonly used for casting IPGs. The pK values of the seven key acrylamido buffers are at pH 3.6, 4.6, 6.2, 7.0, 8.5, 9.3, and 10.3 (Table 1). Theoretical and experimental considerations led to formulations and protocols for casting immobilized pH gradients spanning ranges of from 1 pH unit to 7 pH units.^'^'^^'^^'^^ The two most commonly used gradients cover the pH ranges of 4 - 7 and 3-10. Very high resolution separations are possible v^ith 1 pH unit gradients [see Eq. (11) in the Appendix]. In the casting of IPGs, some of the acrylamido buffers serve as buffering compounds and others function as titrants. Monomers are mixed in the proper proportions as determined by published recipes in standard tv^o-chamber gradient formers. Polymerization mixtures consist mostly of acrylamide and bisacrylamide that constitute the gel matrices. Initiator and accelerator compounds in the mixtures bring about gelation. The mixing chambers, but not the reservoir chambers, of the gradient formers usually also contain glycerol or some other dense substance. The purpose of the glycerol is to form density gradients in the gel mixtures that stabilize the chemical gradients during gelation. The most efficient polymerization is achieved when the two monomer solutions are adjusted to about pH 7 and the final stages of polymerization are done at an elevated temperature.^'^'^^'^^'^^'^^ iv. pH Gradients Formed by Buffer Pairs

The third method of pH control in lEF is with use of pairs of specially selected buffers. Rather than using multiple buffers (a multitude in the case of carrier ampholytes) to generate pH gradients for lEF, this method creates very shallow gradients by using common buffers in a novel way. A set of 13 buffer pairs has been selected that can generate stable pH gradients covering

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the range of pH 3 to pH 10 in increments of less than 1 pH unit.^"*'^^ The components of the system were chosen from among amino acids, biological buffers, weak acids, and weak bases. These compounds are of known chemical composition, are nontoxic, and do not form complexes with proteins. They are usable in preparative lEF devices of the Rotofor type (see later)^^ and address the need for preparative methods that do not employ poorly defined carrier ampholytes. Each member of a buffer pair complements the other. One component is more acidic than the other and the two compounds titrate each other. When the two buffers in one of the pairs are mixed together in water, the solution assumes a particular operating pH. When voltage is applied to the focusing chamber, the buffers act to maintain this operating pH. The electrode reactions create acidic and alkaline regions near the anode and cathode, respectively. As a consequence of the polarization at the electrodes, a very shallow pH gradient, centered on the operating pH, is established in the focusing chamber. To hold the material in the separation chamber at the operating pH in the presence of the applied electrical field, the buffers must not migrate out of the chamber as a result of the field. The buffers must, thus, have low electrophoretic mobilities. This requirement is met by the choice of suitable compounds that remain largely undissociated. Appropriate buffer pairs are available under the trade name of RotoLytes (Table 2). The net mobility of a weak electrolyte, such as one of the components of a buffer pair, is the product of the absolute mobility of the ionized form and the degree of ionization. (The net mobility of a weak electrolyte is the average mobility of the ionized form: /JL^^^ = /JLX, where x is the fraction of molecules ionized at a particular pH, and jn is their mobility. Each molecule can be thought of as being ionized x% oi the time and uncharged the rest of the time.) The two buffers in a RotoLyte pair are only partially ionized at the operating pH. In a binary mixture, the ionization of either of the components is only at the 1 to 10% level. So, under working conditions the net mobilities of the buffers are less than 10% of their absolute mobilities and the molecules migrate only slowly in the electrical field. Moreover, there is a large surplus of undissociated molecules to replace ions that migrate out of the chamber. At ionization levels of less than 1%, or so, the buffering capacity and conductivity are too low to be useful. lEF buffer pairs are intended for use as a final purification step when it is desirable to obtain protein preparations that are demonstrably free of carrier ampholytes, such as for use as pharmaceuticals or in sensitive bioassays. With the buffer pairs, it is not necessary to carry out lengthy dialysis or other procedures for ampholyte removal. The correct choice of buffer pair is that combination for which the pi of the protein of interest falls in the middle of the pH gradient (which, of course, means that the pi must be determined beforehand). The desired proportion of the buffers (Table 2), at 100 m M final total concentration, is mixed with the sample solution prior to being placed the separation chamber and the lEF run is conducted according to the instructions of the manufacturer of the chamber.

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TABLE 2

p H Gradients Obtainable with p H Gradient-Forming Buffer Pairs°

Buffer pair components

Overall pH range

(A) Acidic component (B) Basic component (A) Propionic acid (B) DL-serine (A) MES (B) Gly-Gly (A) MOPSO (B) j8-Alanine (A) MOPS (B) y-Amino-w-butyricacid (A) TAPS (B) e-Amino-«-caproicacid (A) HEPPS (B) Creatinine (A) CAPSO (B) e-Amino-w-caproicacid (A) AMPSO (B) j8-Picoline (A) AMPSO (B) Bis-Tris (A) Hydroxyproline (B) Bis-Tris (A) e-Amino-«-caproicacid (B) Bis-Tris (A) e-Amino-«-caproicacid (B) Triethanolamine (A) e-Amino-w-caproicacid (B) Tris Base

Approximate pH gradients 80%A:20%B

50%A:50%B

20%A:80%B

2.9-4.1

2.9-3.7

3.2-3.9

3.4-4.1

3.9-5.6

3.9-4.7

4.5-5.0

4.8-5.6

4.5-6.1

4.5-5.7

5.2-5.7

5.4-6.1

4.9-6.2

4.9-5.6

5.4-6.0

5.8-6.2

5.5-6.8

5.5-6.3

5.7-6.4

6.2-6.8

6.0-7.2

6.0-6.5

6.4-7.0

6.6-7.2

6.4-7.5

6.4-7.1

6.7-7.2

7.2-7.5

6.9-8.2

6.9-7.6

7.4-7.9

7.8-8.2

7.2-8.6

7.2-8.1

7.8-8.3

8.2-8.6

7.8-8.9

7.8-8.4

8.3-8.7

8.5-8.9

8.5-9.5

8.5-9.0

8.8-9.3

9.0-9.5

9.2-10.4

9.2-9.8

9.6-10.1

9.8-10.4

9.5-11.0

9.5-10.1

9.6-10.6

9.9-11.0

Commerically available as RotoLytes. In each buffer pair, component A is the more acidic member of the pair and component B is the more basic of the two constituents of the pair. When the acidic (A) and basic (B) components of a particular buffer pair are mixed in the proportions shown (80%A:20%B; 50%A:50%B; or 20%A:80%B) and used for pH control in the Rotofor cell, the tabulated approximate pH gradients will be generated. Other ratios of the A and B components can also be used. See Refs. 24 and 25.

III. ANALYTICAL ISOELECTRIC FOCUSING A. Carrier Ampholyte lEF in Gel Slabs i. Gels Of the various support matrices that have been used for lEF, only polyacrylamide and agarose gels have come into widespread use."*^ The structural features of these two types of gels are very similar. a. Polyacrylamide

Gels

Polyacrylamide gels are formed by the copolymerization of acrylamide (CH2 = C H - C O - N H 2 ) and a cross-linking comonomer, N, N'-methylene-

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bisacrylamide (bisacrylamide: CH2 = C H - C O - N H - C H 2 - N H - C O CH = CH2). Bisacrylamide is a bifunctional reagent that covalently links adjacent chains of poly aery lamide to form the gel structure. The gel-forming reaction is a vinyl addition polymerization initiated by a free-radical-generating system. For lEF gels containing carrier ampholytes, free radicals are formed by use of a combination of three compounds, riboflavin, ammonium persulfate, and tetramethylethylenediamine (TEMED). Riboflavin is a photoinitiator. Light generates the riboflavin free radical, v^hereas persulfate chemically decomposes to the sulfate radical. It is these free radicals that initiate polymerization. TEMED, as the free base, is an accelerator for persulfate and riboflavin decomposition. The combination of the tw^o initiators, riboflavin and persulfate, results in more complete polymerization in gels containing low-pH ampholytes than does chemical polymerization alone. The efficiency of the riboflavin-TEMED pair as a catalyst of polymerization falls rapidly above about pH 6, v^hile the efficiency of persulfate-TEMED is at its best above pH 6."^^ It is common to use all three compounds together as a routine practice to avoid different setups for different ampholyte sets. Suitable initiator concentrations are 0.015% ammonium persulfate and 5 jjig/mL riboflavin with 0.03% TEMED. Polymerization is continued for 1 hr under direct lighting from a nearby fluorescent lamp. Riboflavin is not needed in IPG gels because in the preferred protocols the monomer solutions are adjusted to about pH 7 before polymerization. During polymerization, acrylamide and bisacrylamide monomers couple together across their double bonds. Linear chains of poly acrylamide form v^ith bisacrylamide molecules cross-linking adjacent chains. The pendant carboxamide groups ( - C O - N H 2 ) from the acrylamide monomer are subject to hydrolysis to carboxyls (-COO~) as gels age."^^ Only freshly made gels should be used in most lEF w^ork. At the microscopic level, gels take on the appearance of three-dimensional networks of solid, polymeric material separated by open, liquid-filled spaces, called pores. Gels with high concentrations of polyacrylamide have smaller-sized pores than low-concentration gels. During electrophoresis, proteins move through the pores. At total monomer (acrylamide plus bisacrylamide) concentrations above about 7 gm/100 mL (denoted as 7%T), gel pores are small enough to impede the motion of most proteins. These gels act like three-dimensional sieves and should be avoided in lEF. The gel matrix for lEF must be both nonsieving and mechanically stable. A suitable gel composition for electrofocusing is 5% (w/v) total monomer (acrylamide -h bisacrylamide) with the cross-linker, bisacrylamide, accounting for 3.3% ( w / w ) of total monomer. By convention, this gel composition is denoted by the pair of figures (5%T, 3.3%C). The rate of polymerization is dependent on the net concentration of monomers and free radicals, the temperature, and the purity of the reagents. All of these items must be controlled for reproducible gels. Because the reaction requires free radicals, any contaminants that can act as free radical traps will inhibit polymerization. Since oxygen is the most abundant radical trap, proper degassing to remove dissolved oxygen from acrylamide solutions

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is essential for absolute reproducibility. Nevertheless, completely acceptable gels can be obtained without degassing. b. Agarose Gels

Agarose is a natural polysaccharide obtained by treatment of certain types of agar."^^ Agarose gels consist of long-chain, complex-sugar molecules cross-linked by hydrogen bonds. These gels are formed w^hen molten mixtures of agarose and carrier ampholytes are poured onto glass plates, or other supports, to sohdify. The procedure is similar to those used in preparing agarose gels for immunoassays or for the electrophoresis of DNA. The overall structures of agarose gels are similar to those of polyacrylamide gels, but their pores are appreciably larger. Large proteins ( > 200 kDa) that do not migrate very far in polyacrylamide gels v^ill move through agarose gels. Suppliers provide several different types of agarose, qualified for particular electrophoretic purposes. Agarose varieties differ in their physical and chemical properties, such as gelling temperature, gel strength, porosity, and electroosmosis. To minimize electroosmotic solvent flow^s, only agarose prepared specifically for lEF (designated "zero — m / ' ) should be used and the viscosity of the medium should be increased by incorporating glycerol into the gels. Since the gels are held together by hydrogen bonds, chaotropic denaturants, such as urea, that are commonly used to increase the solubility of proteins, can impair the quality of agarose gels. Consult Refs. 1, 9, 14, and 17 for procedures for carrying out agarose gel lEF. ii. General Considerations for Carrier Ampholyte lEF

The proper choice of ampholyte range is very important to the success of an lEF fractionation. Ideally, the pH range covered by the focused carrier ampholytes should be centered on the pis of the proteins of interest. This insures that the proteins of interest focus in the linear part of the gradient v^ith many extraneous proteins excluded from the separation zone. Carrier ampholyte concentrations of about 2% (w^/v) are best. Concentrations of ampholytes belov^ 1% (w/v) often result in unstable pH gradients. At concentrations above 3 % (w/v) ampholytes are difficult to remove from gels and can interfere with protein staining. lEF w^ith carrier ampholytes is a high-resolution technique that can resolve proteins differing in pi by less than 0.05 pH unit. The technique is rapid and nondenaturing. Typical analytical runs take 90 min. Enzymes, antibodies, and antigens usually retain their biological activities during lEF. However, lEF does require relatively careful sample preparation. For best results, low ionic strength sample buffers are necessary and nonionic detergents or urea are often included in IFF runs to minimize protein aggregation. Some proteins, especially membrane proteins, require detergent solubilization during isolation. Ionic detergents, such as sodium dodecyl sulfate (SDS), are not compatible with lEF, but nonionic detergents, such as octyl-glucoside, and zwitterionic detergents, such as CHAPS and CHAPSO, can be used."^"^ Triton X-100 and NP40 may be less satisfactory due to the slight charge content of some commercial preparations. Ampholytes sometimes form com-

ISOELECTRIC FOCUSING

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plexes with proteins that may necessitate steps for their removal. In addition, under some conditions proteins may precipitate at their pis. Narrow pH ranges and high applied voltages give highest resolution in lEF (small A pi in Eq. 11 in the Appendix). In addition to the effect on resolution, high electric fields also result in shortened run times. However, high voltages in electrophoresis are accompanied by large amounts of generated heat (joule heating). Thus, there are limitations on the magnitudes of the electric fields that can be applied. It is common to increase applied voltages in a stepwise manner from low initial values to high final voltages. This electrophoretically clears the system of contaminant current-carrying ions and enables focusing to progress before the high focusing field is applied and results in less heating than in runs at constant high voltage. Because of their higher surface-to-volume ratio, thin gels are better able to dissipate heat than thick ones and are therefore capable of higher resolution. Electrical fields used in carrier ampholyte lEF are generally of the order of 100 V/cm. iii. Experimental Methods

Detailed procedures for carrying out isoelectric focusing in polyacrylamide and agarose gels can be found in Refs. 1 and 9-17. Equipment and reagents for lEF are available from many suppliers. For best results, follow the manufacturer's instructions and recommendations, especially when working with high-voltage equipment. It is acceptable and advisable to prepare reagents for lEF as concentrated stock solutions. All water used for lEF should be deionized or distilled. As an analytical tool, lEF is carried out in large-pore polyacrylamide (5%T, 3.3%C) or agarose gels (1%) which serve mainly as anticonvective matrices.^^ The recommended acrylamide monomer concentrate is (30%T, 3.3%C). It is composed of 30% total monomer (w/v; acrylamide plus bisacrylamide; 30%T), of which 3.3% (1/30) of the total monomer weight is the cross-linker bisacrylamide (3.3%C). Acrylamide solutions of this type are available in already-made form from several suppliers, as are solutions of (40%T, 3.3%C). Also available are commercial blends of acrylamide and bisacrylamide at a 29:1 ratio (3.3%C). Alternatively, mix 29 g acrylamide with 1 g bisacrylamide. Dissolve 30 g of an acrylamide-bisacrylamide blend in 72.5 mL of water. The final volume will be 100 mL (specific gravity 1.025). Filter the solution through a 0.45 /xm filter and store it protected from light at 4°C for up to 1 month. Caution: Acrylamide monomer is considered to be a neurotoxin. Work with it in a fume hood. Avoid breathing acrylamide dust, do not pipette acrylamide solutions by mouth, and wear gloves when handling acrylamide powder or solutions containing it. For disposal of unused acrylamide, add bisacrylamide (if none is present), induce polymerization, and discard the solidified gel (which is considered safe). The most common configuration for analytical lEF is the horizontal polyacrylamide slab gel. Gels are cast on glass plates or specially treated plastic sheets with one exposed face. They are placed on cooling platforms and run with the exposed gel face upward. Electrolyte strips, saturated with 1 N phosphoric acid at the anode and 1 N sodium hydroxide at the cathode, are placed directly on the exposed surface of the lEF gel. Contact between the

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DAVID E. GARFIN

electrical power supply and the electrolyte strips is maintained by electrodes of platinum wire. In another possible configuration, the gel and its backing plate are inverted and suspended between two carbon rod electrodes without the use of electrolyte strips."^^ Ultrathin gels ( < 0 . 4 mm) enable the highest field strengths and, therefore, the highest resolution of the analytical methods. Electrofocusing can also be done in tubes and is often used as the first dimension of two-dimensional gel electrophoresis."^^ ""^^ Precast lEF gels in the popular "mini" formats (6 cm long by 8 cm wide and 1 mm thick) are available for carrying out electrofocusing in vertical configurations. These gels have the advantages that the electrophoresis equipment for running them is available in most laboratories and they can hold relatively large sample volumes. Because vertical electrophoresis cells cannot tolerate very high voltages, this orientation is not capable of the ultrahigh resolution of horizontal cells (cf., Eq. 11 in the Appendix). To protect the materials of the electrophoresis cells (mainly the gaskets) from caustic electrolytes, alternative catholyte and anolyte solutions are substituted in vertical lEF runs. As catholyte, 20 m M arginine, 20 m M lysine is recommended in vertical slab systems (0.34 g arginine free base and 0.36 g lysine free base in 100 mL of water). The recommended anolyte is 70 m M H3PO4, but it can be substituted with 20 m M aspartic acid, 20 m M glutamic acid (0.26 g aspartic acid and 0.29 g glutamic acid in 100 mL of water). a. Casting Gels

For best results when casting gels for use in a particular lEF cell, follow the manufacturer's instructions. The use of gel support film for polyacrylamide is highly recommended, especially with thin gels, which cannot be easily handled unless supported. Polyacrylamide binds covalently to these sheets of treated polyester, simplifying gel handling in all steps, from running gels through drying and storing them. Although polyacrylamide gels adhere to well-cleaned glass plates and remain bound through the lEF runs, gels will often come off of the backing plates during the staining or destaining steps. Thin lEF gels are very difficult to manipulate once they become detached from their backings. Basic ampholytes (pH > 8) may interfere with adhesion of gels to support films. Increasing the ammonium persulfate (APS) concentration in the final gel to 0.7 m g / m L should alleviate this problem. Prolonged soaking in the acidic staining and destaining solutions can also affect adhesion of polyacrylamide gels to the support films. Do not soak the gels any longer than necessary in the staining and destaining solutions. Carrier ampholytes are used as supplied with no pretreatment. They are usually supphed as aqueous solutions containing 40% or 20% (w/v) of the compounds. The pH range used depends on the protein(s) of interest. Other chemicals required are APS, N^N^N', N'-tetramethylethylenediamine (TEMED), riboflavin 5'-phosphate (FMN; the phosphate is much more soluble than riboflavin itself), and glycerol. Recommended stock solutions are 25% (w/v) glycerol (25 g of glycerol and water to 100 mL total volume), 10% (w/v) APS (100 mg APS in 1 mL of water, freshly made before gel casting), and 0.1% (w/v) FMN (50 mg FMN in 50 mL water). TEMED is used neat.

ISOELECTRIC FOCUSING

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The final concentrations in the monomer solution delivered to the cassette are (5%T, 3.3%CX 5% glycerol, 2% ampholytes, 0.015% APS, 0.03% TEMED, 5 fig/mL FMN. Position a fluorescent lamp about 3-4 cm from the gel cassette and illuminate the gel solution for about an hour. Gels may be used immediately or covered in plastic wrap and stored at 4°C for several days. Best results are sometimes obtained by letting a gel "cure" overnight at 4°C before use. b. Sample Preparation

Many protein samples require the use of detergents for their solubilization. For lEF work, the zwitterionic detergents CHAPS and CHAPSO, or the nonionic detergent octyl-glucoside, at concentrations of 1-2% in the gel are recommended. Even in the presence of detergents, some samples may have stringent salt requirements and may aggregate in salt-free environments. Urea is a common solubilizing agent, especially for those proteins that precipitate at their isoelectric points; 3 M urea is often satisfactory for maintaining protein solubility, but concentrations up to 8 M urea have been employed. To avoid carbamylation of proteins by cyanate breakdown products of urea, only fresh solutions of urea should be used. Salt should be present in a sample only if it is an absolute requirement. Carrier ampholytes contribute to the ionic strength of the solution and can help to counteract a lack of salts in a sample. Small samples (1 to 10 ^LL) in typical biochemical buffers are usually tolerated, but better results can be obtained with solutions in deionized water, 2% ampholytes, or 1% glycine. Suitable samples can be prepared by dialysis or gel filtration. Good visualization of focused protein bands generally requires a minimum of about 50 ng of each protein with dye staining or about 1 ng of protein per band with silver staining. c. Sample Application

One of the simplest methods for applying samples to horizontal gels is to place filter paper strips impregnated with sample directly on the gel surface. Up to 20 fiL of sample solution can be conveniently applied after absorption into 1 cm squares of filter paper. A convenient size for applicator papers is 0.2 X 1 cm, holding 5 fjuL of sample solution. Alternatively, 1-2 fiL samples can be placed directly on the surface of the gel. Some lEF cells have movable cups that can be placed on the gels to aid sample application. There are no fixed rules regarding the positioning of the sample on the gel. In general, samples should not be applied to areas where they are expected to focus. To protect the proteins from exposure to extreme pH, the samples should not be applied closer than 1 cm from either electrode. Preforming the pH gradient before sample application will also limit the exposure of proteins to pH extremes. A good strategy when focusing a protein for the first time is to apply samples to three different areas of the gel, one near each electrode and one near the middle of the gel. This strategy is also valuable in estimating the approach to steady state focusing. When the patterns obtained on applying the sample at opposite ends of the gel become identical, it can be assumed

282

DAVID E. GARFIN

that the steady state has been reached. Steady-state conditions should be duphcated when determining the pi values of particular proteins. Note that samples apphed at opposite ends of lEF gels v^ill not alv^ays focus into identical patterns. The reason for this in not entirely clear, but may simply be because some proteins require very long times to reach steady state focusing.^^ Vertical slab and tube gels have fixed orientations, with samples usually loaded at the cathode ends (the top) of the gels. Samples are mixed with glycerol to 5 or 10% (w/v, final concentration) and deposited in the wells of the gels. Here, too, about 50 ng of each protein is required with dye staining and about 1 ng with silver staining for good visualization of individual bands. B. lEF with Immobilized pH Gradients The casting of IPG gels requires gradient-forming equipment and is more complicated than the use of carrier ampholytes. It is not covered here. For detailed procedures consult Refs. 2, 9, 12, 17, 32, 33, and 50. The simplest approach is to purchase ready-made IPGs. They are available in several sizes and pH ranges. IPGs are supplied as dehydrated gels on polyester support sheets. They are rehydrated before use and run in horizontal formats. All common solubilizing agents can be used with IPGs. Samples can be applied directly to the surfaces of rehydrated gels, either with pipettes or with sample-loading cups. A very effective methods for loading IPG strips, especially for 2-D PAGE, is to rehydrate them with the sample itself.^^'^^ C. Electrofocusing The actual running conditions will vary with the apparatus, the gel thickness, the sample solution, and the ampholytes. Gels should always be run at the highest voltage compatible with the heat-dissipation capabilities of the electrophoresis cell. Check the manufacturer's recommendations for proper power settings. At the start of a run, when voltage is first applied, the current will be at its highest value, because the carrier ampholytes have not yet focused and the ions in the sample solution have not yet migrated to the electrode ends of the gel. As the run progresses, the conductivity of the gel will drop and the current will fall. For most carrier ampholyte IFF, it is recommended that the run be started in the constant power mode set at the heat-dissipation limit of the cell. The power supply should be maintained in this mode until the current drops to its lowest value and the highest voltage is reached. Thereafter, the run should be continued at the high-voltage setting. The constant power mode is not recommended when the run consists of several individual lEF-gel strips, as is often the case for 2-D PAGE. Failure of even one strip will subject the others to adverse electrical conditions. Fields as high as 300 V / c m interelectrode distance have been used with thin gels (0.2 mm) and the final fields reached with IPGs for 2-D PAGE often exceed 500 V/cm. An advantage of the constant power mode is that the voltage applied to the IFF cell automatically increases as the current drops. However, constant

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applied power can lead to excessive heating. An alternative approach is to gradually increase the voltage applied to the cell. Many power supplies incorporate programmable voltage control as standard features. Exact running voltages depend on the sizes of the lEF gels and the recommendations of the manufacturers of the cells and the gels should be followed. A short, 15-30 min run at a low voltage, such as 150 V, is usually sufficient to drive salts in the samples to the ends of the gels and into the electrode wicks. An intermediate voltage of about 1000 V is then maintained for an additional 15-30 min as focusing proceeds. Once focusing is near completion and the number of current carriers is at a minimum, as evidenced by a steady current, the voltage is increased to about 3000 V. With IPGs and their stable pH gradients, voltages are often increased further to 5000 V or more. Vertical minigels are run for 1 hr at 100 V, 1 hr at 250 V, and 30 min at 500 V. I. Volt-Hours It is customary to characterize the extent of focusing in lEF gels with the time integral of the appfied voltage, expressed as "volt-hours." The volt-hour (V-hr) designation is meant as a standard for reproducing focusing conditions. Although this designation has no real physical meaning, it is a valuable "bookkeeping" tool with which to establish conditions of electrophoresis for achieving reproducibility between runs. The conditions for attaining steady state focusing, once determined, are reproducible. However, many factors, especially temperature, affect the absolute reproducibility of focusing. Thus, although the volt-hour quantity is a convenient indicator of the extent of focusing, it is not a definitive measure of the lEF process. Runs for short times at high voltage result in better separations than low voltage runs for long times even though the volt-hours of the two runs are identical.^"^ D. Determining pH Gradients and pis When focusing is completed, pH gradients can be determined in various ways. The most straightforward method is to base pH profiles on the positions of focused marker proteins. Any one of many commercially available protein standards can be used for this purpose. lEF protein standards are combinations of proteins with well-characterized pi values blended to give uniform staining. The blends often contain naturally colored proteins, which enable focusing runs to be continually monitored. They often also are made up of proteins that achieve steady state focusing in relatively short times. Marker proteins usually reach the steady state in about 2500 V-hr. Gels are calibrated with one or two lanes of lEF protein standards. Unknown isoelectric points can be estimated by interpolation between the pis of flanking marker proteins. Some gel analysis software contains programs for analyzing lEF gels. The pH gradients in carrier ampholyte gels can be directly determined with surface electrodes or by elution of ampholytes (before staining). In the latter method, gels are sliced into small pieces with a blade. Then, each piece is individually soaked in a minimum volume of 10 m M KCl for 1-2 hr and the pH is measured.

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DAVID E. GARFIN

Regardless of the method used in determining the gradient, what is actually measured is the pH of the focused carrier ampholytes, not the proteins themselves. Ideally, pH measurements should be made at the same temperature as the lEF run. Nevertheless, temperature and solvent effects and interference from absorption of atmospheric CO2 are usually neglected in most pH determinations unless accurate pi measurements are required. Discussions of the effects of these factors in Refs. 1, 9, 10, 12, and 29 should be consulted. It is acceptable to assume that the pH gradient in an IPG gel extends linearly betw^een the pH extremes and to infer pis from this. In reality, other factors of the run affect protein focusing. High-accuracy pi determinations require complicated calibration of the IPG gels for the particular electrophoretic conditions of the run."^

E. Detection of Protein Bands Protein staining is the most general method of detecting proteins in gels. Discussions of other detection methods and means for quantitating protein bands in gels can be found in Refs. 1, 2, and 9-17. Carry out staining and destaining steps at room temperature w^ith gentle agitation (e.g., on an orbital shaker platform) in any convenient container, such as a glass casserole or photography tray. Carefully peel off sample paper strips and electrode v^icks (if possible) before beginning the procedure. The most common stain makes use of the w^ool dyes Coomassie Brilliant Blue R-250 (at 0.04%) and Crocein Scarlet (0.05%) in 27% ethanol, 10% acetic acid. This staining solution is available commercially. Crocein Scarlet rapidly binds and fixes proteins in the gel. The ethanol and acetic acid in the solvent also have a fixative effect. Gels are soaked in staining solution for at least 1 hr. Destaining is done w^ith several changes of a large excess of 40% ethanol, 10% acetic acid until a clear background is obtained. Proteins appear as thin blue or maroon bands in a slightly tinted gel (Fig. 3). The relative thickness of a band is an indication of the comparative amount of protein in the sample (Eq. 8 in the Appendix). lEF gels can also be silver stained for increased detection sensitivity using any one of several commercially available kits. The manufacturer's recommendations should be followed w^hen silver staining gels that are cast on gel support films, otherwise the silver might turn the support into a mirror. Several types of imaging systems and associated software are commercially available for analyzing stained gels. These instruments greatly simplify data acquisition and analysis and the archiving of gel patterns.^^ F. Preservation of Gels lEF patterns can be preserved by photographing them or by capturing an electronic image of them. Gel images on support film can also be preserved by simply allowing them to dry overnight in air in a dust-free location or more rapidly with a heat gun at a low setting. To dry unsupported gels, such

ISOELECTRIC FOCUSING

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as those from glass plates, first soak them in 7% acetic acid, 5% glycerol for 1 hr, then smooth them on water-wetted cellophane or filter paper and dry them in a gel dryer.

lY. TWO-DIMENSIONAL GEL ELECTROPHORESIS Combining two different techniques to produce a two-dimensional separation of the components in a sample can increase resolution. The best approach is to use two different physical principles for the two different separations. This has been done most successfully, as already alluded to, by combining lEF with SDS-polyacrylamide gel electrophoresis in 2-D PAGE. The two separation principles are orthogonal and complementary. Proteins are first separated on the basis of their pis by lEF in thin tube gels or gel strips, then they are further fractionated according to molecular weight in SDS-PAGE gels. Following the lEF run, the first-dimension gels are laid across the tops of polyacrylamide slab gels and the second-dimension SDS-PAGE gels are run at right angles to the IFF direction. Very high resolution methods have been developed, enabling thousands of polypeptides to be resolved in a single slab gel (Fig. 6). Proteins appear as distinct spots in the final gel. The positions of spots give information about both the pis and the molecular weights of the protein in the sample and the sizes of spots indicate the relative amount of each protein. The technique works best with soluble proteins, such as those from serum or cytoplasm, but methods are being developed for 2-D PAGE analyses of less soluble proteins.^"^'^^ Because 2-D PAGE is relatively labor intensive for an electrophoresis method, it requires comparatively high skill levels for best results. The IFF dimension of 2-D PAGE is run either in glass capillary tubes with inner diameters of from 1.5 to 2.5 mm^^ or in IPG strips with gels 0.5 mm thick and 3 mm wide.^^'^^ The IFF gels contain urea and various detergents to maintain the solubility of the proteins in the sample. Tube gels are run in tanks and IPG strips are run in horizontal apparatus. After the run, capillary gels are extruded from their glass tubes into an equilibration buffer to prepare the proteins for the second-dimension electrophoresis while IPG strips are placed directly into the buffer. The equilibrated IFF gels are affixed to the tops of the SDS-PAGE slabs with agarose and the second-dimension gels are run under standard conditions. As done in capillary tubes with carrier ampholytes, 2-D PAGE is inherently variable. As a consequence, there is a major shift in technique to the use of IPGs. Future advances in the methodology of 2-D PAGE are likely to be in the areas of sample preparation^"^'^^ and means for simpfifying the mating of the two different gels.^^ This chapter cannot do justice to the important technique of 2-D PAGE. Consult Refs. 32, 36, 46-50, 59, and 60 and the articles cited therein for details of 2-D PAGE.

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pH4

pH7

100

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i i p i i illlrii • i iiii Hi iilli iri

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l!^fc 11111:1

18% T

F I G U R E 6 Two-dimensional gel electrophoresis pattern of the proteins from ^c\\^r\dn\Q coli. A whole-cell lysate of £ coli was prepared by a method similar to that presented in Ref. 54. Proteins from a sample of £. coli were extracted into 5 M urea, 2 M thiourea, 2% CHAPS (detergent), 2% SB 3 - 1 0 (detergent), 2 m M tributylphosphine (reducing agent), 40 mM Tris base, and 0.2% carrier ampholytes (pH 3 - 10). An aliquot of the protein mixture, containing I mg of total protein, was used to rehydrate an 18 cm long IPG strip, pH 4 - 7 . The proteins were separated by two-dimensional gel electrophoresis as described in Refs. 54 and 55. The first dimension of separation was by isoelectric focusing in the IPG strip. After the completion of eiectrofocusing, the IPG strip was transferred to a denaturing polyacrylamide gel (18 cm wide X 20 cm long) for an orthogonal, second-dimension separation on the basis of molecular weight (MW). The second-dimension gel contained a gradient of increasing polyacrylamide concentration from 8%T to I8%T (2.7%C) from top to bottom. This type of gel separates proteins on the basis of molecular weight from approximately 10 to approximately 100 kDa. The proteins in the gel were stained with colloidal Coomassie Brilliant Blue G-250 by a procedure designed for the second-dimension gel and not discussed in the text. The positions of the indices for pH, MW, and gel concentration are only approximations. This gel image was provided by the courtesy of Dr. Ben Herbert, of Proteome Sciences Limited, Sydney, Australia, and used with his permission.

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Y. PREPARATIVE ISOELECTRIC FOCUSING While the objective of analytical lEF is the separation and identification of proteins in complex mixtures, the purpose of preparative lEF is the purification of one protein, or a small number of proteins—the protein(s) of interest. Laboratory-scale preparative electrofocusing^^ is accomplished in devices such as the Rotofor cell (Fig. 7)}^'^^ Other types of preparative lEF devices that operate on somew^hat more complicated principles than the Rotofor have been described and revievvred elsewhere.^^' ^^'^^ With many of the devices, preparative fractionations are possible on the scale of from hundreds of milligrams to grams of protein and v^ith recoveries of greater than 90%. Purifications of 10- to 100-fold place IFF procedures intermediate betw^een ion-exchange and ligand-binding chromatographies as preparative methods. IFF is well suited for use at any stage of a preparative scheme, and is particularly effective in the early stages of purification. In many cases, simple sequential fractionation and refractionation on the same device provide the desired purity. It is not necessary to attain steady-state focusing in preparative IFF, since adequate separations may be achieved before then. The operation of the Rotofor cell is conceptually very simple. In fact. Fig. 1 w^as originally draw^n not so much as a description of IFF in general but as depicting IFF in the Rotofor. The Rotofor cell achieves IFF in free solution. Stabilization of focused protein zones in the Rotofor cell is achieved by rotating the separation chamber about its (horizontal) axis to inhibit gravitationally induced convection. The separation chamber is divided into compartments by means of screens of woven polyester. The screens offer resistance to fluid convection, but do not hinder the flow of current or the transport of proteins. Proteins, which are initially dispersed uniformly throughout the chamber, migrate to the one or more compartments that are at pH values nearest to their isoelectric points. The combined effect of compartmentalization and rotation is superior to either method alone in maintaining the stabihty of focused zones.^^ The segmentation of the column also facilitates fraction collection. Two focusing chambers are available, capable of holding up to 18 or 55 mL of sample, respectively. Fach chamber is divided into 20 compartments by cores made up of 19 disks of polyester screen (6 /xm pores). Ceramic cooling fingers run through the centers of the focusing chambers to dissipate the heat generated during focusing runs. Two electrode assemblies hold the anolyte (0.1 M H3PO4) and catholyte (0.1 M NaOH) solutions. Appropriate ion-exchange membranes and gaskets isolate the electrolytes from the sample in the focusing chambers while allowing electrical contact with the sample solutions. Vent caps provide pressure relief from the gases that build up in the electrode chambers by electrolysis during the run. Rotation inhibits convection, maintains even cooling and efficient electrical contact, and prevents the screens from becoming clogged by precipitated protein. Runs are done at 4°C at constant power (12 W) for 4 hr or less. Samples are collected by aspiration through tubing lines connecting the 20 individual compartments with corresponding test tubes in a specially de-

=

FIGURE 7 Preparative isoelectric focusing in cell. A mixture of naturally colored proteins consisting of phycocyanin (pl 4.6), myoglobin (pl 7.0). hemoglobin C (pl 7.5), and cytochrome c (pl 9.6) was combined in water containing I% carrier ampholytes, pH 3 - 10. The mixture was loaded directly into a Rotofor cell and focused for I hr at 12 W constant power, as described in the text. Bands of focused proteins can be seen in the segmented focusing chamber (the large dark region in the middle of the chamber contains the myoglobin and hemoglobin of the sample; phycocyanin is at the left of the chamber and cytochrome is at the right). The anode (pH 3) is at the left and the cathode (pH 10) is at the right (cf. Fig. I).

ISOELECTRIC FOCUSING

289

signed vacuum chamber. The individual test tube fractions are easily sampled for assay or measured for pH with standard electrodes. Samples for the Rotofor need not be completely desalted before fractionation. Ions in the sample solution are electrophoresed into the two end compartments in the early stages of the run. Two percent (w/v) carrier ampholyte in the initial sample solution supplies enough ampholyte for refractionation of pooled material. After the tubes containing the protein of interest have been identified, they can be pooled for a second run. The pooled material need only be diluted enough (usually with water) to fill the separation chamber. The ampholytes in the pooled fractions cover pH ranges centered on the pis of the proteins of interest and are generally less than 1 pH unit. Thousand-fold purification has been achieved on refractionation. When RotoLyte buffer pairs are used to establish the pH, the operation of the Rotofor cell is the same as with carrier ampholytes. The choice of the proper buffer pair requires that the pi of the protein of interest be known. The sample is diluted with water to half the chamber volume and then mixed with an equal volume of the appropriate blend of buffers (Table 2). Refractionation is usually not required with buffer pairs. A single run often gives the protein of interest in the desired degree of purity. A representative lEF purification with the Rotofor that demonstrates the effectiveness of refractionation is shown in Fig. 8. The starting material for this fractionation was 150 mg of crude snake venom containing 2% (w/v) pH 3-10 carrier ampholytes. Aliquots of each of the 20 fractions from the run were analyzed in polyacrylamide lEF gels in pH 3-10 gradients. The focusing patterns of the proteins in the odd-numbered fractions are shown in Fig. 8a. The protein of interest (outlined with an oval) was found in fractions 10, 11, and 12. These three fractions were pooled, diluted with water to the volume of the separation chamber and refractionated. No additional ampholytes were added to the pooled material. IFF analysis of the refractionated material (Fig. 8b) revealed that fraction 13 contained the protein of interest in nearly pure form. The ideal sample run on the Rotofor cell would contain only the protein mixture, water, and ampholytes or buffers. However, pi precipitation may require that 3 M urea be included for solubility. When higher urea concentrations are needed, the Rotofor cell is run at 12°C. Detergents ( 1 - 2 % w / v ) may also be added to samples. Zwitterionic detergents, such as CHAPS, CHAPSO, and nonionic octyl-glucoside are satisfactory. The Rotofor has been incorporated into two-dimensional purification schemes based on the principles of 2-D PAGE. Fractions collected from the Rotofor were further purified by 2-D PAGE^^ or by preparative polyacrylamide gel electrophoresis.^^"^^ Highly purified proteins were obtained by this scheme, even low-abundance proteins, to allow for multiple analyses and for use as antigens. A. Removal of Ampholytes from Proteins There are a number of ways to separate ampholytes from proteins. Electroelution; ammonium sulfate precipitation; and gel filtration, ion exchange, and

290

DAVID E. GARFIN

& ^% ^^

fc^^

ff^Su^^^^

- ^

9 11 13 15 Fraction Number

17

19 crude

^

1

3

5

7

9

crude 11

13

15

17

19

Fraction Number

B F I G U R E 8 Preparative purification of a protein in the Rotofor cell. (A) Initial fractionation of crude snake venom; 150 mg of total protein was fractionated using the 55 mL focusing chamber. Focusing was carried out for 4 hr at 12 W constant power using 2% carrier ampholytes, pH range 3 - 1 0 . Fractions I - 20 were analyzed by electrofocusing in a polyacrylamide gel through a gradient from pH 3 (bottom) to pH 10 (top). The odd fractions are shown in an image of the silver stained analytical gel. The last lane of this gel contained the crude snake venom starting material. The protein of interest (denoted by the oval) was found in fractions 10, I I , and 12. These fractions were pooled for refractionation. (B) Fractions 10, I I , and 12 from the initial separation were pooled and diluted with water to fill the 18 mL separation chamber. No additional ampholytes were added. Focusing was for 4 hr at 4 W constant power. The 20 fractions were analyzed by polyacrylamide gel electrofocusing as in (A). The odd fractions are shown in the silver stained gel image. The apparent difference in the position of the protein of interest in (A) and (B) is a consequence of the way the two images were cropped. A nearly pure sample of the protein of interest (pi 6.5; oval) was obtained in fraction 13.

hydroxylapatite chromatographies have all been used. Dialysis is a simple and effective method for removing ampholytes from solutions of proteins. Pooled fractions are first adjusted to 1 M NaCl to disrupt v^eak electrostatic complexes betv^een ampholytes and proteins then dialyzed against appropriate buffers. Extensive dialysis is required for thorough removal of am-

ISOELECTRIC FOCUSING

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pholytes. There is no good way to demonstrate complete absence of ampholytes in a protein solution, but for many applications they need not be entirely removed. For those applications where the complete absence of ampholytes is a requirement, the use of RotoLytes is recommended.

VI. CAPILLARY ISOELECTRIC FOCUSING Capillary electrophoresis is an instrumental technique for automating electrophoretic analyses.^^ It is carried out in fused silica capillaries with internal diameters of 25-100 fim. The capillaries are coated with an external layer of polyimide for mechanical strength. The small internal diameter and high surface-to-volume ratio allow for efficient heat dissipation from the capillaries, enabling separations to be carried out at very high field strengths (up to 1000 V/cm). Samples are introduced at the inlet ends of capillaries by electrophoretic injection or hydraulic injection. Electrophoretic injection is accomplished simply by dipping the capillary inlet into the sample solution and applying high voltage for a short time (typically several seconds). In electrophoretic injection, only ionic species are loaded into the capillary and analytes are injected in proportion to their electrophoretic mobilities. Hydraulic injection is accomplished by dipping the capillary inlet into the sample solution and briefly applying pressure at the inlet or vacuum at the capillary outlet for a short time. Samples loaded by hydraulic injection contain analytes in the same concentrations as the sample solutions. Injection volumes are in the nanoliter range, which usually requires relatively high concentration samples to ensure enough protein for good detection. Detection of separated components is done directly in the capillaries. A section of the polyimide coating is removed at one end of the capillary and this "window" is positioned in the light path of an optical detector. Bands of separated compounds are recorded as they migrate past the detector window. Capillary isoelectric focusing is analogous to conventional IEF.^^~^^ Carrier ampholytes are used to estabfish stable pH gradients within the capillaries and proteins form focused zones at their isoelectric points. The capillary approach differs from conventional lEF in that focused zones must be transported past the monitoring point to detect the separated proteins. This has been achieved by hydraulic mobilization of zones using gravity, pressure, or vacuum to move the entire plug of liquid in the capillary past the detector. Electroosmotic flow has also been used as a pump for mobilization of the focused band pattern. An alternative method, termed chemical or electrophoretic mobilization, induces a pH shift in the gradient by changing the composition of the catholyte and (or) anolyte, which causes proteins to migrate past the detection point in sequence. Capillary lEF has been used successfully for the characterization of related proteins with subtle differences in structure—e.g., hemoglobin genetic variants, immunoglobulins, and glycoforms of recombinant biopharmaceutical proteins.

292

DAVID E. GARFIN

VII. SUMMARY Isoelectric focusing is a mature separation technique that has a place in any laboratory doing work with proteins. The analysis of a protein is not complete without a determination of its isoelectric point and all protein databases have at least estimates of the pis of the represented proteins. Proteins thought to be pure by other methods are often found to be mixtures of several proteins when analyzed by lEF. Isomeric forms of the same protein that are revealed by lEF are valuable indicators of mutations or differences in posttranslational modifications. lEF plays a crucial role in 2-D PAGE and preparative lEF allows for high-purity fractionations of unparalleled resolution. A simple keyword search of literature databases shows that about 500 journal articles are written per year referring to IFF. This amply attests to the value of lEF as a tool for protein analysis and purification. Yin. APPENDIX A. Band Shape in lEF i. Electrophoretic Mobility A molecule with charge q in an electric field E experiences a force F=qE

(1)

where ^ is a signed ( + or — ) scalar quantity and £ is a vector whose sense is that of the direction of motion of a positively charged particle. Frictional, viscous drag opposes the electrical force with a magnitude and direction directly proportional to the velocity v of the molecule. The magnitude of viscous drag is fv, in which f, the friction coefficient, reflects the size and shape of the molecule and the viscosity of the medium. Under the two opposing forces, molecules rapidly reach steady-state, terminal velocities with the two forces equal

fnet =

qE-fV=0

or qE = fv

(2)

The electrophoretic mobility of the molecule ^t is defined as the steady-state velocity per unit field, or V

a

" = 1 =7

<''

In Eq. (3), the vector quantities have been replaced by their scalar magnitudes because electrophoresis is a one-dimensional process with the direction of motion x, defined by the direction of the field. Mobility )Lt is a characteristic property describing the response of a molecule to electric fields. The units of mobility are cm^/V • sec, since v is expressed in centimeters per second and

ISOELECTRIC FOCUSING

293

£ in volts per centimeter. The magnitude of £ is simply the applied voltage divided by the shortest-line distance between the electrodes in the electrophoresis chamber. In lEF, mobilities are largely determined by the net charges on proteins, w^hich in turn are determined by the pH of the immediate environment. Since the pH varies (usually linearly) in lEF, /x is a function of position in the separation medium (Fig. 2a). ii. Transport Equations The continuity equation for a single species of molecule undergoing an electrophoretic process is as follows^'^^""^^i dc dt

= -

d 1 \cvdX \

dc D — dx

(4)

This equation is a statement of the conservation of mass. It states that the rate of change of the concentration c: of a molecular species {dc/dt) in a volume element between x and x + dx is equal to the net flow gradient (or flux) of molecules into and out of the volume element. The direction of migration x is in the direction of the electrical field, which in IFF is also the direction of the pH gradient. The origin of the coordinate system for this discussion is at the center of the focused band (the pi). The quantity within parentheses on the right side of Eq. (4), often denoted by the letter / , is the net flow of molecules (flux) through the volume element. The first term, cv^ shows the flow of material into the volume element and the second term, Ddc/dx^ represents the diffusional migration out of the volume element (this is Fick's first law of diffusion); D is the diffusion coefficient for the protein under investigation and is dependent on the shape of the molecule and the properties of the separation medium. IFF is a steady-state mechanism in which the net flow of molecules ceases on attainment of focusing. This means that the time derivative in Fq. (4) is zero when focusing is achieved: dc/dt = 0. Thus, dc cv = D—

(5)

dx From Fq. (3), v = jnE, so that rearranging Fq. (5) gives dc -—dx = — (6) D c ^ ^ At focusing, it is reasonable to assume that mobility fi is a linear function of x,^'^ fi = -px (7) LLE

The negative sign in Fq. (7) comes about because the charges on proteins in IFF are inversely proportional to the pH gradient (Figs. 2a and 2b). Inserting

294

DAVID E. GARFIN

Eq. (7) into Eq. (6) and solving the resultant differential equation gives

As is the case with all good separation methods, lEF lends itself to an analysis v^hereby a Gaussian distribution (Fig. 2c) can describe band shape. Here, concentration c is expressed in moles per cubic centimeter, and CQ is the concentration of the molecule in question at the center of the focused band. There is a similar equation for the distribution of each and every component species in the sample and the overall behavior of the separation system is described by the sum of these equations. This classical analysis provides a description of the principal features of isoelectric focusing that is adequate for most purposes. How^ever, since it is a steady-state approach this type of analysis does not provide details of the dynamics of focusing. Certain assumptions are made for the analytical solution of the differential equations describing electrofocusing (Eqs. 4 and 6). In the classical analysis, the pH gradient is assumed to form rapidly enough that the proteins migrate in a more or less preexisting gradient, the electrophoretic mobilities of proteins are assumed to vary linearly vv^ith position, and the effect of the proteins on the pH gradient is ignored. With computer analyses, these assumptions are not necessary and the dynamics of focusing are more thoroughly described.^'^^'^^''^^'^'^"^^ Computer models incorporate the dissociation rates of the components into the transport equations, generalize the discussion to multicomponent systems, and are not restricted to the steady state. The models successfully predict concentration, pH, and conductivity profiles as functions of time. Computer models describe transient states in the formation of the steady state distributions of ampholytes and proteins. In addition, sources of asymmetry in concentration distributions as wtW as mechanisms of instability in distribution profiles can be studied relatively easily v^ith computer simulations of lEF. B. Factors Affecting Resolution The standard deviation of the Gaussian distribution of Eq. (8) is

If tv^o similar molecules v^ith similar distributions are to be resolved, their bands must be separated enough to be clearly distinguished one from the other. For IFF, the separation allov^ing tw^o bands to be clearly distinguished. Ax, is usually taken as three standard deviations, or 3cr, although the numerical factor (3 here) is not important for the follow^ing discussion. In terms of pH units, w^hich, after all, is the relevant separation parameter in IFF, JpH Apl = ApH = ——^x ax

JpH = 3——oax

(10)

ISOELECTRIC FOCUSING

295

The pH interval between the pis (band centers) of two resolved protein bands is given by this expression (ApH = A pi). Recalling from Eq. (7) that the term p in Eq. (9) is djji

dfi

JpH

dx

JpH

dx

Eq. (10) becomes

,

D{dpH/dx)

^^' = '^1 Ei-d^/dpH)

^''^

As an expression for the difference in pi of two resolved bands, Eq. (11) shows a very important fundamental property of lEF. The interrelationship between the variables of the run that affect resolution, shown in Eq. (11), is almost intuitive. Any change that decreases Apl improves resolution. Two of the variables are under experimental control. Shallow pH gradients (low values of dpH/dx) and high applied fields (large E) give high resolution (low Apl) in lEF. Thus, utilizing a narrow range of pH or increasing the appHed voltage will improve resolution. In addition, good resolution is favored by proteins which have high rates of change of mobility with pH (d/ji/dpH) and low diffusion coefficients D near their pis. These latter two factors are properties of proteins that cannot be changed, but most proteins satisfy these latter two criteria. It is important to note that carrying out lEF runs in gels rather than in free solution can effectively minimize D.

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DAVID E. GARFIN 10. Lias, T. (1989). Isoelectric focusing in gels. In "Protein Purification: Principles, High Resolution Methods, and Applications" (J.-C. Janson and L. Ryden, eds.), pp. 376-403. VCH Press, Weinheim. 11. Garfin, D. E. (1990). Isoelectric focusing. In "Methods in Enzymology" (M. P. Deutscher, ed.). Vol. 182, pp. 459-477. Academic Press, San Diego, CA. 12. Righetti, P. G., Gianazza, E., Gelfi, C., and Chiari, M. (1990). Isoelectric focusing. In "Gel Electrophoresis of Proteins: A Practical Approach" (B. D. Hames and D. D. Rickwood, Eds.), 2nd ed., pp. 149-216. IRL Press, Oxford. 13. Dunn, M. J. (1993). "Gel Electrophoresis: Proteins." BIOS Scientific Publishers, Oxford. 14. Allen, R. C., and Budowle, B. (1994). "Gel Electrophoresis of Proteins and Nucleic Acids: Selected Techniques." de Gruyter, Berlin. 15. Bollag, D. M., Rozycki, M. D., and Edelstein, S. J. (1996). "Protein Methods." Wiley-Liss, New York. 16. Hawcroft, D. M. (1997). "Electrophoresis: The Basics." IRL Press, Oxford. 17. Westermeier, R. (1997). "Electrophoresis in Practice: A Guide to Methods and Applications of DNA and Protein Separations," 2nd ed. VCH Press, Weinheim. 18. Bier, M. (1986). Rotating apparatus for isoelectric focusing. U.S. Pat. 4,588,492. 19. Egen, N. B., Thormann, W., Twitty, G. E., and Bier, M. (1984). A new preparative isoelectric focusing apparatus. In "Electrophoresis ' 8 3 " (H. Hirai, ed.), pp. 547-549. de Gruyter, Berlin. 20. Bier, M. (1998). Recycling isoelectric focusing and isotachophoresis. Electrophoresis 19, 1057-1063. 21. Righetti, P. G., Bossi, A., Wenisch, E., and Orsini, G. (1997). Protein purification in multicompartment electrolyzers with isoelectric membranes. / . Chromatogr. 699, 105-115. 22. Cann, J. R. (1979). Multibanded isoelectric focusing patterns produced by macromolecular interactions. In "Methods in Enzymology" (C. H. W. Hirs and S. N. Timasheff, eds.). Vol. 61, pp. 142-147. Academic Press, New York. 23. Gooley, A. A., and Packer, N. H. (1997). The importance of protein co- and post-translational modifications in proteome projects. In "Proteome Research: New Frontiers in Functional Genomics" (M. R. Wilkins, K. L. Williams, R. D. Appel, and D. F. Hochstrasser, eds.), pp. 6 5 - 9 1 . Springer, Berlin. 24. Bier, M., Ostrem, J., and Marquez, R. B. (1993). A new buffering system and its use in electrophoresis and isoelectric focusing. Electrophoresis 14, 1011-1018. 25. Bier, M., and Marquez, R. B. (1994). High-resolution large-scale isoelectric focusing of proteins in a chemically defined buffer system. Tech. Protein Chem. 5, 249-258. 26. Just, W. M. (1983). Synthesis of carrier ampholytes for isoelectric focusing. In "Methods in Enzymology" (C. H. W. Hirs and S. N. Timasheff, eds.), Vol. 91, pp. 281-298. Academic Press, New York. 27. Thormann, W., Mosher, R. A., and Bier, M. (1986). Experimental and theoretical dynamics of isoelectric focusing: Elucidation of a general separation mechanism. / . Chromatogr. 351, 17-29. 28. Thormann, W., and Mosher, R. A. (1988). Theory of electrophoretic transport and separations; the study of electrophoretic boundaries and fundamental separation principles by computer simulation. Adv. Electrophor. 2, 45-108. 29. Delincee, H., and Radola, B.J. (1978). Determination of isoelectric points in thin-layer isoelectric focusing: The importance of attaining the steady state and the role of CO2 interference. Anal. Biochem. 90, 609-623. 30. Mosher, R. A., Thormann, W., and Bier, M. (1986). An explanation for the plateau phenomenon in isoelectric focusing. / . Chromatogr. 351, 31-38. 31. Rilbe, H. (1977). Stable pH gradients—a key problem in isoelectric focusing. In "Electrofocusing and Isotachophoresis" (B. J. Radola and D. Graesslin, eds.), pp. 35-50. de Gruyter, Berlin. 32. Gorg, A., Fawcett, J. S., and Chrambach, A. (1988). The current state of electrofocusing in immobilized pH gradients. Adv. Electrophor. 2, 1-43. 33. Righetti, P.G., Gelfi, C., and Chiari, M. (1996). Isoelectric focusing in immobilized pH gradients. In "Methods in Enzymology" (B. L. Karger and W. S. Hancock, eds.). Vol. 270, pp. 235-255. Academic Press, San Diego, CA.

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DAVID E. GARFIN 55. Herbert, B. R., Molloy, M. P., Gooley, A. A., Walsh, B. J., Bryson, W. G., and Williams, K. L. (1998). Improved protein solubility in two-dimensional electrophoresis using tributyl phosphine as reducing agent. Electrophoresis 19, 845-851. 56. Harrington, M. G., Gudeman, D., Zewart, T., Yun, M., and Hood, L. (1991). Analytical and micropreparative two-dimensional electrophoresis of proteins. Methods: Companion to Methods Enzymol. 3, 98-108. 57. Gorg, A., Postel, W., and Giinther, S. (1988). The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 9, 531-546. 5S. Bjellqvist, B., PasquaU, C., Ravier, F., Sanchez, J.-C., and Hochstrasser, D. (1993). A nonlinear wide-range immobilized pH gradient for two-dimensional electrophoresis and its definition in a relevant pH scale. Electrophoresis 14, 1357-1365. 59. Dunn, M. J. (1987). Two-dimensional polyacrylamide gel electrophoresis. Adv. Electrophor. 1, 1-109. 60. Link, A. J., ed. (1999). "2-D Proteome Analysis Protocols." Humana Press, Totowa, NJ. 61. Egen, N. B., Twitty, G. E., Thormann, W., and Bier, M. (1987). Fluid stabilization during isoelectric focusing in cylindrical and annular columns. Sep. Sci. Technol. 22, 1383-1403. 62. Hochstrasser, A.-C., James, R. W., Pometta, D., and Hochstrasser, D. (1991). Preparative isoelectrofocusing and high resolution 2-dimensional gel electrophoresis for concentration and purification of proteins. Appl. Theor. Electrophor. 1, 333-337. 63. Sanchez, J.-C., Paquet, N., Hughes, G., and Hochstrasser, D. (1995). "Preparative 2-D Purifies Proteins for Sequencing or Antibody Production," Bull. No. 1744. Bio-Rad Laboratories, Hercules, CA. 64. Masuoka, J., Glee, P.M., and Hazen, K.C. (1998). Preparative isoelectric focusing and preparative electrophoresis of hydrohobic Candida albicans cell wall proteins with in-line transfer to polyvinylidene difluoride membranes for sequencing. Electrophoresis 19, 675-678. 65. Davidsson, P., Westman, A., Puchades, M., Nilsson, C.L., and Blennow, K. (1999). Characterization of proteins from human cerebrospinal fluid by a combination of preparative two-dimensional liquid-phase electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal. Chem. 71, 642-647. 66. Wehr, T., Rodriguez-Diaz, R., and Zhu, M. (1998). "Capillary Electrophoresis of Proteins." Dekker, New York. 67. Wehr, R., Zhu, M., and Rodriguez-Diaz, R. (1996). Capillary isoelectric focusing. In "Methods in Enzymology" (B. L. Karger and W. S. Hancock, eds.). Vol. 270, pp. 358-374. Academic Press, San Diego, CA. 68. Rodriguez-Diaz, R., Wehr, T., Zhu, M., and Levi, V. (1997). Capillary isoelectric focusing. In "Handbook of Capillary Electrophoresis" (J. P. Landers, ed.), 2nd ed., pp. 101-138. CRC Press, Boca Raton, FL. 69. Rodriguez-Diaz, R., Wehr, T., and Zhu, M. (1997). Capillary isoelectric focusing. Electrophoresis 18, 2134-2144. 70. Tanford, C. (1961). "Physical Chemistry of Macromolecules." Wiley, New York. 71. Shimao, K. (1987). Mathematical simulation of steady state isoelectric focusing of proteins using carrier ampholytes. Electrophoresis 8, 14-19. 72. Weiss, G. H., Sokoloff, H. Zakharov, S. F., and Chrambach, A. (1996). Interpretation of electrophoretic band shapes by a partition chromatography model. Electrophoresis 17, 1325-1332. 73. Svensson, H. (1961). Isoelectric fractionation analyses and characterization of ampholytes in neutral pH gradients. Acta Chem. Scand. 15, 325-341. 74. Bier, M., Palusinski, O. A., Mosher, R. A., and Saville, D. A. (1983). Electrophoresis: Mathematical modeling and computer simulation. Science 219, 1281-1287. 75. Mosher, R. A., Bier, M., and Righetti, P.G. (1986). Computer simulation of immobilized pH gradients at acidic and alkaline extremes: A quest for extended pH intervals. Electrophoresis 7, 59-66. 76. Stoyanov, A. V., and Righetti, P. G. (1998). Steady-state concentration distribution of ampholytes in isoelectric focusing in a linear immobilized pH gradient. Electrophoresis 19, 1596-1600.

MASS SPECTROMETRY OF BIOMOLECULES DAN GIBSON Mass Spectrometry Resource, Boston University School of Medicine, Boston, Massachusetts 02118 and Department of Pharmaceutical Chemistry, School of Pharmacy, Hebrew University of Jerusalem, Jerusalem, Israel

CATHERINE E. COSTELLO Mass Spectrometry Resource, Boston University School of Medicine, Massachusetts 02118

Boston,

I. INTRODUCTION A. The Coming of Age of Mass Spectrometry in Biological Research B. Mass Spectrometry — What It Is and How It Works II. EXAMPLES OF APPLICATIONS OF MASS SPECTROMETRY T O BIOLOGICAL RESEARCH A. Protein Analysis B. Protein Folding C. Noncovalent Interactions III. CONCLUSIONS REFERENCES

I. INTRODUCTION A. The Coming of Age of Mass Spectrometry in Biological Research The advances in technology which occurred in the last decade transformed mass spectrometry from an analytical tool for the study of small and relatively stable molecules to an ubiquitous and indispensable technique for studying biomolecules. The new "soft" ionization methods, electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), coupled with advances in instrumentation, laser and computer technologies, and data processing algorithms, enable routine detection and structural analysis of biomolecules.^ In addition to molecular mass determination of biomolecules, it is now possible to sequence peptides, proteins, oligonucleotides, and oligosaccharides; probe protein folding; and study inter- and intramolecular noncovalent interactions.^"^ The new commercial mass specSeparation Science and Technology, Volume 2 Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved,

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trometers can have one or more of the following characteristics: a large accessible mass range ( > 300,000 Da), high sensitivity (the sample can be in the low^ femtomole range), high accuracy ( < 0.01% error in determination of the molecular weight), and mass resolution that can exceed 1:100,000 (an ion with a mass of 100,000 can be distinguished from an ion with a mass of 100,001). There is no one mass spectrometer that possesses all these traits and different instruments are used for different purposes. In contrast with some of the older instruments, which required an experienced mass spectrometrist to operate them, the new generation instruments are user-friendly and can be successfully operated by researchers in the scientific and medical communities. Unlike other spectroscopic techniques, mass spectrometry (MS) does not require the analytes to possess any special physical properties such as fixed charge, electric or magnetic moments, radioactivity, etc. This, in conjunction with the properties already mentioned and with the short measurement times, places it in the unique position of being able to answer a broad range of questions in a multitude of biological and medical research areas. In addition to its traditional role as an analytical tool used to solve a specific research problem, MS has become an enabling technique in the emerging field of proteomics. Mass spectrometry plays a central role in the attempts to isolate and characterize the 100,000 human proteins. It is increasingly used by biotech companies in conjunction with two-dimensional poly aery lamide gel electrophores (2D-PAGE).^ The goal of this review is to familiarize investigators in biological and medical research with mass spectrometry and its potential applications in these fields, in the hope that this will encourage and enable them to utilize MS in their research. We have tried to provide a clear basic description of the modern mass spectrometers and of the most relevant types of experiments that they can perform. We will discuss the advantages and drawbacks of the different methods in the context of biological research and provide examples of the ability of mass spectrometry to solve problems in biological research. Since this review is geared for a general audience, we have limited the discussion to methodologies that are accessible to the nonspeciafists and that can be carried out on commercially available spectrometers without special modifications. Most of these techniques are already in widespread use; to indicate an important future direction that is just beginning to reach applications laboratories, a discussion of Fourier transform ion cyclotron resonance (FT-ICR) MS is also included. The specific examples used reflect some of the interests of the authors and are not intended to cover the entire field.

B. Mass Spectrometry—What It Is and How It Works A mass spectrometer provides the molecular mass of a given compound or of a mixture of compounds. Since all mass spectrometers carry out the mass analysis on gas-phase ions, the analytes, whether solid or liquid, neutral or charged, must be converted to gas-phase ions before they can be mass analyzed. Thus, all mass spectrometers are comprised of several key compo-

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nents: Ion source—the compartment where the (usually uncharged) analyte molecules are transformed into gaseous ions (positive or negative) which can be manipulated by ion optics (electric and magnetic fields) and then mass analyzed Mass analyzer—a unit whose role is to differentiate between the various gaseous ions on the basis of their mass-to-charge ratio (m/z) Detector-the instrument that registers the abundance of the ions which reach it and converts the ion current, directly or indirectly, to an output signal Data station-a unit for instrument control and storage and processing of the experimental data There are several types of ionization sources [MALDI, ESI, FAB (fast atom bombardment), PD (Cf-252 plasma desorption), EI (electron ionization), CI (chemical ionization) etc.], different types of mass analyzers [combinations of magnetic and electric sectors, quadrupolar filters (Q) and ion traps (IT), time-of-flight (TOP) and FT-ICR] and different detectors, each with its own advantages and drawbacks. We describe herein only the systems that presently have widespread use for the study of biomolecules: ESI coupled to a quadrupole (or triple quadrupole, QqQ) mass analyzer or an ion trap, the MALDI source with the linear or reflectron TOP analyzer, and the PT-ICR system which can be equipped with both ESI and MALDI sources. Earlier ionization methods (such as EI) suffer from two major drawbacks: They cannot generate intact gas-phase ions from high molecular weight compounds; and the gas-phase ions of the lower molecular weight compounds contain a large amount of internal energy that causes extensive fragmentation of the molecular ion. Thus, in many cases, the molecular ion cannot be observed in EI mass spectra. The development of PAB in the early 1980s addressed these problems, but the major breakthrough occurred about 6 years later when two new ionization methods were introduced. These were ESI and MALDI.^'^ Both these methods can generate gas-phase ions from high molecular weight compounds and both are "soft" ionization processes, in that the gas-phase ions have low internal energy and undergo only minimal fragmentation, enabling facile observation of molecular ions. i. ESr-MS In this ionization method, the gas-phase ions are generated in a continuous fashion directly from dilute solutions of the analyte, at atmospheric pressure. A solution of the analyte containing some additives (electrolytes, acid, or electron traps) flows at a slow rate (up to several microliters per minute) through a very fine metal capillary which is held at a high (positive or negative) voltage ( ^ 4 kV). Prom the tip of the capillary emerges a fine mist of charged droplets, which contain molecules of the analyte, solvent molecules, as well as the electrolyte ions. The solvent molecules are stripped from the droplets by a stream of heated gas and, as a result, the size of the droplets continues to shrink, bringing the ions closer together. When the repulsive Coulombic forces that the ions exert on each other exceed the

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surface tension of the droplet, the droplet explodes, releasing charged analyte molecules into the gas phase. Often, when the molecule is large ( > 1000 u), this procedure yields molecular ions with multiple charges [e.g., (M + nHY^], which result from protonation of n basic sites on the analyte. These charged molecules contain very little excess internal energy and undergo little (if any) fragmentation. The charged molecules are directed toward a small orifice, and enter the evacuated mass analyzer. The fact that the ionization occurs at atmospheric pressure in a continuous fashion, coupled with the high sensitivity of the technique, enables interfacing the mass spectrometer to column-based purification systems such as high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE).^^'^^ Often the ESI source is used in conjunction with a quadrupole mass analyzer. A quadrupole mass analyzer acts as a mass filter that, at a given setting, transmits only one specific m/z ratio. Scanning the quadrupole results in the sequential transmission of ions with increasing m/z ratios, generating the typical mass spectrum (signal intensity as a function of the m/z ratio). Figure l a presents a schematic view of the ESI mass spectrometer. Several properties of the quadrupole make it suitable for interfacing with an ESI source: (a) the quadrupole tolerates the relatively high pressure (10"^ torr) which results from the atmospheric pressure ionization, (b) it has good resolution (unit resolution of m/z 2000 or more), and (c) it is relatively inexpensive. The limited mass range (m/z up to 4000) of the quadrupole is compensated for by the multiple charging of the molecular ion in the ESI source, which reduces the mass-to-charge ratio (m/z) of the biomolecules and brings it into the range of the quadrupole analyzer (i.e., a protein with a mass of 15,000 Da with a +15 charge will appear at m/z 1001).

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scannmg F I G U R E I (A) An ESI source attached to a single quadrupole analyzer. The instrument can be interfaced to an HPLC, CE or a syringe pump. The ionization is done at atmospheric pressure and the mass analysis in vacuum. This configuration is used for molecular weight determination. (B) An ESI source attached to a triple quadrupole analyzer. In a typical MS - MS experiment the precursor ion (Mp) is selected by the first analyzer ( Q l ) , reacts with an inert gas in Ql to yield the product ions ( M I , M2, M3), and the product ions are analyzed by scanning Q3. This yields the product (fragment) ion spectrum.

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ESI often results in multiple charges accumulating on the intact molecule.^ A protein that has 15 accessible basic residues can be protonated at any of its basic sites and at the N-terminal amino group. Since we are looking at a large ensemble of molecules, some will be singly protonated, others will be doubly protonated, etc., leading to the observation of a distribution of charge states. A singly protonated protein will have an m/z ratio of (M + D / l , while a doubly charged protein will have an m/z of (M + 2 ) / 2 , etc. Each charge state will appear at a different m/z ratio and the ESI mass spectrum of a pure protein thus exhibits many peaks. Yet, the pure protein has only one molecular mass. The molecular mass of the protein can be calculated from the values observed for each of its several charge states and, from these, an accurate value for the molecular mass can be obtained. Figure 2a depicts the mass spectrum of denatured (unfolded) horse muscle apomyoglobin showing 12 charge states ranging from 13 + to 24 + with the most abundant being 20 + . A more "straightforward " spectrum can be obtained by using the maximum entropy algorithm for calculating the mass of the protein (Fig. 2b). The ESI source is usually attached to a single or triple quadrupole analyzer or to a triple quad. The single quadrupole system is used mainly for molecular mass determination, while the triple quadrupole is used for both molecular mass determination and for tandem mass spectrometry (MS-MS). The principle of tandem mass spectrometry is simple: The first quadrupole is used as a mass filter, which transmits only ions with a preselected m/z ratio (precursor ion). The second "quadrupole" is not a mass analyzer, but is a reaction chamber containing an inert gas such as argon. The precursor ions, selected by the first quadrupole, enter the reaction chamber and collide with molecules of the inert gas. These low-energy collisions induce fragmentation of the precursor ions giving rise to a spectrum of lower mass product ions. This process is known as CID (collision-induced dissociation). The distribution of the product ions is obtained by scanning the third quadrupole. In a sense, the entire region up to and including the second quadrupole serves as an ion source for the product ions, while the third quadrupole is the mass analyzer. Figure l b describes how the triple quad (with an ESI source) is used for tandem mass spectrometry (MS-MS, using CID). This is a very powerful technique since it provides direct information on fragmentation pathways and it is now used routinely for structural analysis.^^'^^ If only a single quadrupole mass analyzer is available, product ions can still be obtained by manipulation of the ion source voltages to achieve decomposition of the molecular ions. The disadvantage of this approach is the sacrifice of precursor ion selection. ii. M A L D I - T O F MS Desorption and ionization of the analyte molecules are achieved through the mediation of the matrix molecules. The "matrix" usually is an organic acid with a chromophore that can absorb radiation at the wavelength of a specific laser (usually in the UV or IR range). ^"^ While the MALDI process is not completely understood, it is clear that the matrix is crucial for both the desorption and ionization processes. The analyte is mixed in solution with a

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large excess (10"^) of the matrix and the mixture is cocrystaUized by air evaporation. The sample is then irradiated with a pulsed laser and the energy absorbed by the matrix breaks down the matrix-analyte lattice, causing the desorption of both matrix and analyte molecules into the gas phase. Ionization of the analyte may occur either in the condensed phase or in the gas phase but, in either event, it is believed to involve proton transfer between the matrix molecules and the analyte molecules. In contrast with ESI, the most abundant ion formed by MALDI, for analytes below about 10 kDa, is the singly charged species, and, even for analytes over 100 kDa, low charge states predominate. Collisions with matrix molecules cause some fragmentation of the molecular ions; the decompositions may occur promptly (in the ion source) and, as a metastable process, in the mass analyzer. The MALDI source is usually coupled to a TOP analyzer, which is composed of a flight tube with a detector attached at the opposite end. The ions that are generated in the ionization chamber are accelerated into the analyzer by an electric potential, and all the ions (except for those with multiple charges) acquire the same kinetic energy (E = 1/2 mv^). Since they have the same kinetic energy, the velocity of the ions is inversely proportional to the square root of their mass, and so, the lighter ion reaches the detector before the heavier ion does. Recording the signal intensity registered by the detector as a function of time (in hundreds of microseconds) gives rise to the mass spectrum.^^ There are several reasons why MALDI is coupled to a TOP analyzer: 1. The TOP works in a pulsed fashion. This is perfectly suited to the pulsed MALDI source. 2. TOP analyzers have no theoretical upper limit on the m/z ratio which they can analyze. This is important because MALDI produces ions with high m/z values which cannot be measured by other types of mass analyzers. 3. TOP analyzers are inexpensive. One of the biggest disadvantages of MALDI-TOP MS is its low resolution. Por small proteins, the resolution of a linear TOP MS is around 1:500 (i.e., an ion with m/z of 10,000 can be distinguished from an equally abundant ion with a m/z of 10,020). The low resolution is due partly to the fact the ions produced by MALDI do not have uniform kinetic energy, but a distribution of kinetic energies. Thus, ions with the same m/z ratio arrive at the detector at different times giving rise to broad peaks and hence reducing the observed resolution. There are two ways in which the resolution can be increased. a. Ref/ectron - TOF hAS

One approach is to change the geometry of the analyzer to achieve focusing, at the detector, of ions with the same mass but with different energies. The reflectron MALDI-TOP spectrometer is schematically depicted in Pig. 3. This is what the reflectron-TOP- MS does. While the linear TOP MS uses the detector at the end of the straight flight tube (detector 1 in Pig. 3), in the reflectron-TOP mode, an energy-focusing reflector deflects the ions.

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Pulsed laser Detector 1 low resolution spectrum

reflector high resolution spectrum Detector 2 F I G U R E 3 A MALDI source attached to a TOP analyzer. The sample - matrix mixture is placed on the probe tip and irradiated with a pulsed laser. The ions are accelerated into the flight tube. In the linear mode, the reflector is turned off and the detection is done at detector I, yielding the low-resolution mass spectrum. If a potential is applied to the reflector, then ions with the same mass but slightly different energies [high (H), medium (M), and low (L)] are focused by the reflector and arrive simultaneously at detector 2, giving a higher resolution spectrum.

by nearly 180°, to the second detector (detector 2 in Fig. 3). The reflector ensures that all ions with the same mass, regardless of their initial kinetic energy, arrive at detector 2 at the same time. This causes a narrowing of the peaks and increased resolution. b. Delayed Extraction

Another approach is to reduce the initial kinetic energy distribution of the ions before they leave the ion source region. This is done by using delayed extraction (DE), which amounts to inserting a brief delay pause (in the nanosecond range) between the production of the ions in the source and the time the acceleration potential is turned on. During the delay, the faster ions drift closer to the accelerating plate than the slower ions, and experience a slightly weaker accelerating potential than the slower ions resulting in velocity-based focusing at the detector. DE, in conjunction with the linear TOE MS, increases the resolution to 1:10,000 (i.e., an ion with m/z of 10,000 can be distinguished from an ion with an m/z of 10,001).^^ Figure 4 attempts to demonstrate the meaning of mass spectral resolution in the context of the different experimental techniques. MALDI-TOF MS does not have the type of double analyzer geometry that, in an ESI-triple quadrupole system, confers capability for MS-MS. However, product ions can be related to their metastable precursor (parent) ions by using a technique called postsource decay (PSD), which is now done performed routinely with reflectron TOF MS instruments. Metastable ions are those ions that fragment in the flight tube. Since the ions have acquired their velocity before the fragmentation, both the precursor ions and the fragments have the same velocity and will arrive at first detector 1 at the same time. However, due to the mass difference between them (the fragments have lower masses than their precursors), they have different kinetic energies. The reflectron focuses the ions on second detector 2 according to their energies. The PSD spectrum results from a series of experiments in which the

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101

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F I G U R E 4 A theoretical calculation of the appearance of the molecular peak of a peptide as it would be observed at different resolutions which correspond to the typical performance of a linear TOF without DE (250), a quadrupolar analyzer (2500), and an FT-ICR ( > 250,000). (Reproduced with permission from Siuzdak.' Copyright 1993 National Academy of Sciences, U.S.A.)

reflectron potential is decreased stepwise, yielding spectra optimized in stages for transmission of the lower energy ions that were formed from a given precursor ion. At the end of data acquisition, a complete PSD spectrum is assembled by combining segments of the spectra recorded at the various steps, retaining from each only the optimized portion. A more detailed description of PSD is beyond the scope of this review, but the technique was recently summarized.^^ iii. Comparison between ESI and MALDI MALDI differs from ESI in two major aspects: (a) the MALDI ionization process is not continuous, but is a pulsed event, and (b) while the analytes for ESI are dissolved in the appropriate solvent and introduced into the ionization source, the MALDI analytes are dissolved and mixed with a solution of the appropriate matrix and then are cocrystallized with the matrix. Sensitivity for both techniques enables analyses at the low femtomole range (and even below), although most analyses are conducted at the low picomole range because of the greater convenience of sample handling at this level. Mass measurement accuracy, as indicated elsewhere in this discussion, depends on the choice of analyzer. These techniques are complementary and neither has exhibited dominance in the analysis of biological molecules. The main disadvantages of ESI are: (a) the difficulty in handling mixtures of compounds in the case of direct infusion-coelution and (b) the low tolerance of for electrolytes. Thus, the samples must be purified (by on-line chromatography where possible) and desalted before they can be analyzed. The main advantages of ESI are: (a) the mass measurement accuracy, (b) the ability to interface directly with HPLC and CE, and (c) the soft ionization, which allows for observation of protein folding and noncovalent interactions. The major disadvantages of MALDI are: (a) the strong dependence of the quality of data obtained on the sample preparation, (b) the low resolution of linear TOF instruments (without DE), and (c) the inability to interface the MALDI ion source directly to HPLC or CE. The advantages of MALDI are:

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•

•

TABLE I

ESI MS

MALDI MS

Comparison between ESI-QqQ MS and MALDI-TOF MS Advantages

Disadvantages

Mass range— < 150 kDa

Not suitable for analysis of complex mixtures Needs low salt concentration (< mM)

Resolution—2000 Sensitivity—femtomole Accuracy—0.01% Compatible with HPLC-CE Good for study on noncovalent interactions Mass range— > 300 kDa Sensitivity—femtomole Good for analyzing mixtures High tolerance for salts

Resolution— < 500 (without DE) Accuracy—0.1-0.01% Not compatible with HPLC-CE

(a) the ability to detect high m/z ratios, (b) the tolerance for relatively high salt concentration, and (c) the ability to analyze mixtures of compounds. The advantages and drawbacks of each of the techniques are summarized in Table 1. iv. Ion Trap - MS

A quadrupolar ion trap is an analyzer system that is capable of multiple stages of consecutive mass spectrometry experiments (MS") with a single analyzer system. The basic principle behind the ion trap mass spectrometer is that all the ions that are generated by an external ionization source (ESI or MALDI), are captured in the ion trap, ejected from it one by one according to their m/z values, and then detected. The ion trap is filled with He gas which serves to reduce the kinetic energy of the ions entering from the ionization source (cooling), and also serves (under defined conditions) to cause the ions to decompose due to collision-induced dissociation, which generates the MS-MS spectra. The detailed mechanism of how ion traps work is well beyond the scope of this review. Jonscher and Yates^^ recently reviewed the theoretical and practical aspects of ion trap mass spectrometry. While the quadrupole ion trap is not a new invention (it was first introduced in the 1950s), until recently, it was not suitable for biological, biochemical, or biomedical applications. Recent technological developments (in addition to the emergence of ESI and MALDI), especially in the software control of the instruments, have made the new generation of ion trap mass spectrometers an extremely useful tool for the study of biomolecules. The modern systems can perform up to 12 stages of tandem mass spectrometry, have a molecular weight range from 650 to 70,000 Da, and can have fairly high resolution and mass accuracy of 0.3 Da at m/z 1000.^^"^^ These qualities, coupled with their great sensitivity, compact size, and low cost, will make ion trap mass spectrometers major players in future biomedical and biological research.

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V. F T - I C R MS-High-Resolution Mass Spectrometry Two of the most important parameters in mass spectrometry are resolution and accuracy. The mass analyzers that are used in most ESI and MALDI mass spectrometers (quadrupole and TOP, respectively) compromise to a certain extent, these two parameters (see Table 1). The FT-ICR analyzer has ultrahigh resolution (> 100,000), which results in increased accuracy (in the low parts per million range) in the analysis of biological molecules, high sensitivity, and the capability for multistage MS" with precise control of the ion populations at each stage. Detailed explanation of FT-ICR MS is beyond the scope of this review but suffice it to mention here that FT-ICR can be coupled to both ESI and MALDI sources thus offering two distinct advantages: (a) ultrahigh resolution and (b) multiple tandem spectrometry MS" (w < 5). The use of FTMS is increasing rapidly with more than 225 installations worldwide by mid-1997, and this technique promises to play an important role in future biological research.^^

II. EXAMPLES OF APPLICATIONS OF MASS SPECTROMETRY TO BIOLOGICAL RESEARCH A. Protein Analysis The goal of protein analysis is to obtain sufficient information to be able to assign the identity of the protein (if it has previously been reported) or to determine its primary sequence, if it is unknown. The initial stage of the analysis is composed of two sequential tasks: (a) determining the accurate molecular mass of the protein and (b) obtaining its primary structure (amino acid sequence). The molecular mass of a protein can be estimated from gel electrophoresis and its amino acid sequence can be obtained by Edman degradation analysis. The problem is that molecular mass determination by gel electrophoresis is not very accurate, and, while Edman degradation works well with natural amino acids, it does not work well with modified amino acids and cannot be used for proteins having a blocked N terminus. Another problem with Edman degradation is its slow rate—about half an hour per amino acid. In the era of proteomic research, which is predicated on highthroughput protein analysis, it is simply too slow.^ Mass spectrometry is more than 100 times more accurate than gel electrophoresis in molecular mass determination, and obtaining the data for sequence analysis requires only a fraction of the time needed for Edman degradation. Moreover, mass spectrometry is well suited for analysis of posttranslational modifications, which cannot be determined by Edman degradation. Posttranslational modification of proteins plays an important role in many physiological processes; identifying and localizing these modifications is an essential step in the quest to understand protein function. Posttranslational modifications are not limited to few and select proteins, but occur in

3 I0

DAN GIBSON AND CATHERINE E. COSTELLO

the vast majority of eukaryotic proteins. There are over 200 reported modifications of amino acids that occur in vivoP Since posttranslational modifications can interfere w^ith both chemical and enzymatic protein analysis, and since amino acid modification is almost always accompanied by a mass change (increase or decrease), mass spectrometry is the method of choice for studying posttranslational modifications.^"^ The first task is to obtain an accurate molecular mass for the protein. This is straightforward, and can be done by either MALDI or ESI-MS, though for purified intact proteins, ESI-MS provides more accurate results. The second task is to obtain the amino acid sequence of the protein. Neither the amino acid composition nor the amino acid sequence can be inferred from the molecular mass measurement of the protein. There are several reasons for this: (a) the simple mass spectrum of a protein does not provide sequence information; (b) in large proteins, there are several combinations of amino acids that can give rise to the same molecular mass, especially noting that there are amino acids that have the same mass or differ from each other by only 1 u and; (c) co- and posttranslational modifications make data interpretation impossible. To obtain the desired information, the protein is subjected to protease digestion (usually by trypsin) and a mixture of tryptic peptides is obtained. A mass spectral (usually MALDI) analysis of the mixture of the tryptic peptides maps the molecular mass of each peptide ("peptide map"). This information (peptide mapping) can now be used to search existing protein databases and to try and match the experimental peptide map with computer generated tryptic profiles of known proteins.^^'^^ If the protein is not heavily modified, the database search will yield matches for some of the fragments while others may show deviations. These deviations must then be analyzed to see if they originate from posttranslational modifications, mutations or perhaps from sequencing errors. If it turns out that there are no matches, the protein has to be sequenced completely. This is done by chromatographic separation of the tryptic peptides, followed by a tandem mass spectral analysis of each of the peptides (CID or PSD). Collision-induced dissociation results in predictable fragmentation of the peptide backbone, usually at the amide linkage between the carbonyl and the amine.^^ For singly charged precursors, this dissociation generates a positive ion and a neutral fragment; for multiply charged precursors, each product may retain some of the charge. If the charges reside on the amino side, the ions are called b ions and if the charges are stay on the carboxy sideend they are called y ions. A subscript indicates the position of each with respect to the relevant terminus. The mass difference between two adjacent y (or b) peaks, corresponds to the residue mass of the intervening amino acid. On this basis, the amino acid sequence of each peptide is assigned. This is illustrated in Fig. 5 for a tetrapeptide with amino acids AA1-AA4. Modifications to amino acids will clearly show up as mass shifts in this analysis and thus they can be easily localized and identified. This is probably the best method for localization and identification of posttranslational modifications. After the all peptides have been sequenced, they must be ordered to yield the complete primary structure. This process is not always easy and often

31

MASS SPECTROMETRY OF BIOMOLECULES

H2N

CH

C-|-HN

-OH

CH

B y3

y2

yi

bl

b2 AA2

AAl

AA2

AA3

precursor

b3 AA3

AA4 m/z

F I G U R E 5 (A) Scheme of the fragmentation expected for a tetrapeptide which has been subjected to CID. The b ions have the charge located on the amino side of the bond rupture, and the y ions retain the charge on the carboxy side. Theoretically we expect to observe three b ions and three y ions. (B) Sketch of the expected MS - MS spectrum, showing the two series of b and y ions and the way by which the amino acids can be sequenced.

requires repeating the sequencing procedure using different site-specific enzymes to generate additional sets of sequence ladders which overlap the original ladders. The procedure is illustrated in Fig. 6. An unknown protein is digested by enzyme A to yield six fragments (I-VI), and each of the fragments is sequenced by tandem mass spectrometry. There is not yet enough information to enable us to put the fragments together in the correct order. If we repeat the procedure with enzyme B, it is clear that by knowing the sequence of fragment 1, we can deduce that fragment II follows fragment I, and from the sequence of fragment II, we know that fragment 2 follows fragment 1 and fragment 3 follows 2, etc. In this manner, the entire primary structure of the protein can be obtained. In principle, this is a straightforward procedure for sequencing proteins. Like any other methodology, this procedure has its pitfalls and care should be exercised, especially in the analysis of novel proteins and peptides for which other information (e.g., cDNA data) is not available. The application of these methodologies to the characterization of a previously unknown mutant of a clinically relevant protein is described below as an example of the application of this approach to protein sequencing.^^ Familial transthyretin amyloidosis (ATTR) is a hereditary degenerative disease which is closely associated with single amino acid substitutions in the plasma protein transthyretin (TTR), a 127 amino acid protein (M^ 13,761 Da) that is tetrameric in its native state.^^ The clinical manifestations of ATTR are related to specific mutations of TTR (e.g., Val30 -^ Met and Thr60 -^ Ala). The definitive diagnosis is often estabHshed on the basis of the

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DAN GIBSON AND CATHERINE E. COSTELLO

unknown intact protein

I

II

III

IV

V

VI

^AAA/V^AAAAAJ^A^AAAAAAAAAAAAAAAAAAAAAAAAAAAA/VV^VV^AJ^/VVVVVAAAAAAA/^^

digestion by enzyme A

1

2

3

4

5

\A'VVVV\AAAAAAAAAAAAAAAAAAAAM/VVAA/V\AAA'VWVVVV\AAA/VV\A/^^

digestion by enzyme B F I G U R E 6 Sketch showing the need for two enzymatic digestions, which contain different cleavage sites to enable the construction of the primary sequence from the sequenced peptide maps.

detection of TTR variants using isoelectric focusing (lEF) a n d / o r molecular biology,^^ but these procedures are most effective for the detection of known variants, whereas MS offers the possibility for direct determination of both known and novel mutations. For one patient, a new mutation was suspected but the regular screening methods (lEF and restriction enzyme analysis of most of exons 2, 3, and 4) failed to identify the mutation. ESI and MALDI MS were utilized to characterize the novel variant. The strategy used by the authors for the analysis of the variant protein consisted of three steps: 1. Detection of variants through ESI MS mass measurement of the intact protein obtained by immunoprecipitation from the patient's serum 2. Location of the variant peptides through "peptide mapping" of the tryptic digest of the protein using LC-ESI MS or MALDI 3. Use of MS-MS techniques to obtain sequence information from the variant peptide Determination of the molecular weight difference between the wild- type and the variant TTR obtained by ESI MS limited postulated amino acid substitutions to a relatively small number. Analysis of the tryptic digest of the protein by LC-ESI MS was used to locate more precisely the site of the mutation. Comparison of the tryptic map obtained for the variant with that of a "normal" sample facilitated detection of the modified tryptic peptide. MS-MS was carried out on the variant tryptic peptide contained in the appropriate chromatographic fractions collected during the LC-ESI MS experiment. The ESI MS analysis of the fraction obtained from the patient's serum showed two distinct peaks corresponding to the wild-type ( M^ 13,839.0) and

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MASS SPECTROMETRY OF BIOMOLECULES

I

13750

14000

F I G U R E 7 Deconvoluted ESI mass spectrum of transthyretin obtained by immunoprecipitation from the serum of a patient found to have a novel form of ATTR: A, wild-type TTR (mass 13,759.1); B, variant TTR (mass 13,786.5); C, sulfated TTR (mass 13,839.0); and D, sulfated variant TTR (mass 13,864.9).

variant (M, 13,864.9) sulfated TTR (Fig. 7). The + 2 6 ( ± 1 ) Da mass shift is most likely caused by a single base mutation. Inspection of the UV chromatogram from the LC-ESI MS analysis of the tryptic digest of the protein revealed a peak not present in the digestion of wild-type TTR, at retention time 25.95 min. The [M + H]"^ of this variant peptide {m/z 1392.6) was consistent with a 27 Da increment to the "normal" tryptic peptide T4, GSPAINVAVHVFR34. The location of the mutation was confirmed by MALDI MS analysis of the chromatographic fraction collected between retention times 25.0-27.5 min. In the MALDI mass spectrum, peaks at m/z 1365.2 (wild-type T4) and m/z 1392.1 (variant T4) are both present (Fig. 8). This observation confirmed that the mass difference between the variant and the wild type peptides is indeed +27 Da. The only possible amino acid substitution that would give rise to a +27 Da shift in that peptide is Ser23 -^ Asn. A more definite assignment of the mutation was obtained by measuring the MALDI PSD spectrum of the HPLC fraction containing both the wild-type and the variant T4. A mass window {m/z 1350-1410) covering both molecular masses was selected for the PSD experiment. The position of the modification was immediately apparent because the resulting PSD spectrum exhibited limited fragmentation in the form of a h^_^ and h^_^ -\-T7 ion series originating from the wild-type and variant peptides, respectively. The significance of the h^_^ -\-T7 ion series is that the amino acid substitution resulting in the +27 Da shift must occur before or on the third amino acid in the peptide. This incomplete fragmentation information did not provide ultimate confirmation of the nature of the mutation but supported the proposed substitution of Ser23 -^ Asn. The MS results were confirmed by DNA sequencing of the primer of the PCR product of the second exon. The

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DAN GIBSON AND CATHERINE E. COSTELLO

Tj (Normal) m/z 1365.2 '10

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F I G U R E 8 MALDI TOF mass spectrum of the HPLC fraction containing the T4 tryptic peptides of the wild-type and mutant TTR.

fact that Ser23 -^ Asn substitution requires only a one base change (AGT -> AAT) increases the Ukelihood of this mutation. This example not only demonstrates how mass spectrometry is a powerful tool in the analysis of proteins, but it also sets a good example of how the different mass spectrometric techniques should be combined to give the best answers possible. The molecular mass was determined by the more accurate ESI-MS analysis, and the initial tryptic digest was studied by exploiting the ability of ESI-MS to be directly interfaced with an HPLC. Then MALDI-TOF MS was used efficiently to obtain the MS-MS (PSD) data.

B. Protein Folding The three-dimensional structures of proteins and enzymes are the key to understanding many of the most fundamental biological processes. Thus, the mission of structural biology is to obtain high-resolution structural parameters for biological molecules. Due to technological advances, the study of protein structures by X-ray crystallography and nuclear magnetic resonance (NMR) has increased tremendously in the past 15 years. The tertiary structure of a protein is determined by its primary structure (the sequence of the amino acids). The linear sequence of amino acids folds into its specific, biologically functional conformation in a process (which is not thoroughly understood) called protein folding. Protein folding has been studied extensively for many years by circular dichroism, NMR, fluorescence, viscometry and theoretical calculations.^^ The advantages of ESI-MS for studying conformational changes in proteins were recognized by Katta and Chait in 1991?^ ESI-MS is used not only to distinguish between native (folded) proteins and denatured (unfolded) proteins but also to follow the dynamics of the protein (un)folding process. In one case, an interesting conformational phenomenon that went unnoticed

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both in X-ray crystallography and NMR studies was detected by mass spectrometry.^^ There are several good reviews on the role of mass spectrometry in the study of protein folding.^'^^ In the following examples, we will focus on three mass spectrometric approaches to the study of protein folding: • Monitoring conformational changes by charge state distribution (CSD) • Study of conformational changes by hydrogen-deuterium (H-D) exchange • Monitoring protein folding dynamics by time-resolved ESI-MS i. Monitoring Conformational Changes by Charge State Distribution

Multiple charging of proteins in ESI-MS is the result of the protonation of the accessible basic sites. Thus, a protein with n accessible basic sites can accumulate as many as n positive charges [corresponding to a molecular mass of (M -h nHY ]. In the highly folded state, many basic sites are not exposed on the surface and only low charge states are observed. As the number of accessible basic sites increases (due to conformational changes), the protein acquires a higher positive charge. This means that the charge states have higher values and that the charge state distribution becomes broader. The connection is now clear. When a protein is tightly folded, some of its basic residues (e.g., Arg, Lys, and especially His residues) are buried in the hydrophobic interior and are not available for protonation. As the protein denatures (as a result of a change in temperature, pH, or addition of denaturing agent or organic solvents), more of its basic sites can be protonated. Thus, there are three ways to distinguish between tightly folded and denatured proteins: • Denatured proteins will exhibit a higher molecular mass {M =^ M '\nH). • Denatured proteins will exhibit higher charge states. • Denatured proteins will have a broader charge state distribution. All these are easily detected by ESI-MS on small quantities of proteins (<100pmol). Protein systems whose folding has been studied by ESI-MS include lysozyme, ubiquitin, myoglobin, and cytochrome c (see Ref. 6, and references therein). The early work done by Katta and Chait on ubiquitin is one of the first examples for the use of both CSD and H - D exchange to monitor conformational changes in proteins.^^ We will use this example in both this section and the next section. Bovine ubiquitin is a protein that has 76 residues (of which 13 are basic residues) and has a molecular mass of 8565 Da. In its native conformation, it is tightly folded. It can be denatured by addition of 50% methyl alcohol at a low pH (2.5). In the original experiment, the native protein was dissolved in CD3C02D:D20 (1:99) and its spectrum was measured after 20 min. The spectrum of the denatured protein was obtained by dissolving it in a denaturing solution of CD3C02D:D20:CD30D (1:99:100). Figure 9 shows the results of the ESI-MS measurements of the folded (Fig. 9a) and the denatured (Fig. 9b)

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D A N GIBSON A N D CATHERINE E. COSTELLO

8+

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F I G U R E 9 (A) ESI-MS of native bovine ubiquitin obtained after 20 min in a nondenaturing solution. (B) ESI-MS of native bovine ubiquitin obtained after 23 min in a denaturing solution. (Reproduced with permission from Katta and Chait.^' Copyright John Wiley & Sons Limited.)

conformations. In the folded conformation, the most prevalent charge state is 8 + and the charge state distribution is narrow, ranging from 6 + to 8 + . In the denatured protein, the charge state distribution becomes much broader and ranges from 6 + to 13 + and the most abundant charge state is 10 + . The data shown indicate that in the folded conformation, there are four to five basic residues that are not accessible to protonation, while in the denatured ubiquitin, all 13 basic residues can be protonated. These results are in agreement with both solution CD studies and with the solid-state X-ray crystallographic analysis.^"^'^^ This example demonstrates that ESI-MS is a straightforward and efficient way to examine protein folding. ii. Study of Conformational Changes by H - D Exchange

Hydrogen-deuterium exchange experiments have been used extensively to study both structural parameters and dynamic and mechanistic aspects. They are utilized in conjunction with both NMR and IR studies. Mass spectrometry, with its high sensitivity and resolution, is ideally suited for isotopic exchange studies. Just as the accessibility to the basic residues of the protein is the key for CSD analysis, the accessibility to exchangeable protons (both on the side

MASS SPECTROMETRY OF BIOMOLECULES

3 I 7

chains and on the backbone) is the key to analysis by H - D exchange studies. H - D exchange rates for peptides vary as a function of temperature and pH, but even the more slowly exchanging backbone amide protons are fully exchanged in a matter of minutes. The situation in native (folded) proteins is more complex. Some protons are highly labile and exchange rapidly, w^hile others are buried in hydrophobic regions or are involved in hydrogen bonding or in formation of salt bridges and are not easily accessible. Thus, in a folded protein, different hydrogen atoms have different exchange rates, which can vary by many orders of magnitude, and in case of tightly folded proteins, complete (100%) exchange may be a very slow process.^^ As the protein unfolds (denatures), it behaves more like a peptide and the proton exchange becomes more rapid. Increasing H - D exchange results in higher molecular masses and broader charge distributions. Ubiquitin has a total of 144 exchangeable hydrogen atoms (72 on the backbone and 72 on the side chains) and a molecular mass of 8565. The spectrum obtained after 20 min in deuterated solvents appears in Fig. 9a. The molecular mass (obtained from the 7 + and 8 + charge states) is 8654, which is higher by 89 Da than the original mass. This indicates that 89 protons (62%) exchanged rapidly with deuterium. After 90 min, 105 protons exchanged (73%) and after a week 130 protons (90%). Under denaturing conditions (Fig. 9b), 131 protons (91%) exchanged after 23 min and 96% in an hour. iii. Monitoring Protein Folding Dynamics by Time-Resolved ESI MS

To follow the dynamics of protein folding, the conformational changes of the protein must be measured as a function of time. Moreover, it would be advantageous to be able, simultaneously and independently, to detect the different conformers rather than to obtain a measured value that reflects the average state of all the conformers in solution. ESI-MS is suitable for this kind of a study. Konermann et al?^ studied the folding kinetics of cytochrome c by time-resolved ESI-MS. Cytochrome c has 5 pH-dependent conformational states. The reversible transition between the largely unfolded conformation (II) and the folded one (III) has a pK^ of 2.5. The authors used a continuous flow apparatus in which the protein (at pH 2.4) was mixed with a tetramethyl ammonium chloride solution. The mixing resulted in an abrupt jump in the pH from 2.5 to 3.0, providing the initial time point for the protein folding process. By controlling the flow rate and by changing the distance between the mixing point and the electrospray source, they were able to change the time that elapsed from the initiation of the folding until the beginning of beginning of detection. They were able to obtain a time resolution of 0.1 sec. The ESI mass spectra of the unfolded state (pH 2.5) and of the folded protein (pH 3.0) are shown in Fig. 10. The 8 + and 9 + charge states are characteristic of the folded protein, while 15 + to 1 7 + states are due to the unfolded structure. The intensities of each of the charge states were plotted as a function of time (see Fig. 11), providing the kinetic data. Analysis of the data yielded two exponential lifetimes, which are in good agreement with those measured by other methods.

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DAN GIBSON AND CATHERINE E. COSTELLO 1

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F I G U R E 10 (A) ESI - MS of native bovine cytochrome c at pH 2.4. (B) ESI - MS of cytochrome c at pH 3.0 (Reproduced with permission from Konermann et al}^ Copyright 1997 American Chemical Society.)

While ESI-MS is an extremely useful technique for studying protein folding, it cannot provide information on the role of specific residues (or protons) in the protein folding process. The information that can be obtained by mass spectrometry is limited to those conformational changes that produce changes in the CSD of the protein. C. Noncovalent Interactions Noncovalent interactions, whether specific or nonspecific, intermolecular or intramolecular, play a major role in the most fundamental biological processes. These interactions determine macromolecular structure and are responsible for the formation of DNA duplexes, triplexes, and quadruplexes, for protein subunit aggregation and for protein folding. In addition, noncovalent interactions also play an important role in molecular recognition events such as enzymatic catalysis (enzyme-substrate and enzyme-inhibitor interactions); host-guest complexes; drug-receptor, protein-ligand interactions; protein-DNA binding; and immune system response.

319

MASS SPECTROMETRY OF BIOMOLECULES

10^

;10° tn

c

m c CO

:^ CO

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15 1000 FIGURE II A plot of the mass spectral intensity of the 13+ and 16+ charge states of cytochrome c (reflecting the denatured protein) as a function of time (B) A plot of the mass spectral intensity of the 8 + and 9 + charge states of cytochrome c (reflecting the folded protein) as a function of time. (Reproduced with permission from Konermann et al?^ Copyright 1997 American Chemical Society.)

Noncovalent interactions are weak inter- or intramolecular interactions that result from a combination of electrostatic interactions (ionic), hydrogen bonding, hydrophobic interactions (stacking or intercalation), and van der Waals interactions (dipole-dipole or induced dipole-induced dipole). Complexes formed by these types of interactions are usually fragile. This property is often essential to their biological function, which depends on the equilibria between the associated and free forms of these molecules. A variety of experimental methods both in solution and in the solid phase have been employed to study noncovalent complexes. The most detailed structural information is obtained from NMR spectroscopy and from X-ray crystallography. The major drawbacks of these two techniques are: (a) both require relatively large amounts of samples that are not always available, (b) NMR is limited to the study of macromolecules with molecular weights that

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DAN GIBSON AND CATHERINE E. COSTELLO

are less than 40 kDa, (c) the high concentrations required by NMR (low millimolar) may induce aggregation which does not occur in the biological system, and (d) X-ray crystallography is limited to systems that yield large enough single crystals. Other techniques that are commonly used are spectroscopic (circular dichroism, fluorimetry and light scattering), ultracentrifugation, calorimetry (differential scanning calorimetry and titration calorimetry), surface plasmon resonance, and electrophoresis-based assays. While mass spectrometry cannot provide the detailed structural information that is obtained by NMR and X-ray crystallography, it can, in principle, provide valuable information on the formation and stoichiometry of noncovalent complexes. There are several key questions that need to be addressed before we can decide whether the advantages of mass spectrometry (sensitivity, speed, and specificity) can be successfully applied to the study of noncovalent interactions. These questions are • Can the fragile noncovalent complexes survive the ionization process.^ • Do the structures of the noncovalent complexes in the gas phase truly reflect the structures in solution? • Do the affinity constants in the gas phase correlate with those obtained in solution? There are no unanimous clear-cut answers. In an excellent recent review of ESI-MS of noncovalent complexes, Loo^ states that, "There are three camps of opinion: believers, non-believers and undecided," based on their personal experience. There are reports in the literature that demonstrate a good correlation between the solution and gas phase properties, while others report on discrepancies between the results obtained in solution and by mass spectrometry. We provide next two examples that demonstrate the ability of ESI-MS to tackle problems involving noncovalent interactions. While mass spectrometry can provide valuable information on noncovalent complexes, we would like to stress that this technique is by no means routine or straightforward and a great deal of caution must be exercised in the design of the experiments and in selection of the appropriate control experiments. There are over 100 publications dealing with this subject but no generalization can be drawn regarding the best approaches or experimental conditions that should be employed, as these vary from case to case. Following are two examples of the use of mass spectrometry in the analysis of noncovalent complexes. i. The Binding of Biotin to the Avidin T e t r a m e r

Avidin is a glycoprotein that is isolated from egg white. In its active form, it exists as a tetramer which is composed of four identical subunits that are held together by noncovalent interactions. This tetramer, which has a molecular weight of 64 kDa, binds four biotin ligands (each with a molecular weight of 244 Da). Four biotin molecules bind the tetrameric avidin at sites that are partially created by the quaternary structure of avidin. The avidin-biotin complex is one of the strongest known non-covalent com-

MASS SPECTROMETRY OF BIOMOLECULES

321

plexes, with a dissociation constant (K^) of 10~^^M. Due to this strong biotin-avidin binding, this "couple" is extensively used in immunochemistry, affinity chemistry and biochemistry. This system is an obvious choice for study of noncovalent interactions. We chose to use this system as an example because it demonstrates the ability of ESI-MS to observe a complex with eight components, which has both protein-protein interactions (the tetrameric avidin aggregate) as well as protein-ligand interactions (avidin-biotin complex). Smith et al, studied the binding of biotin and some biotin derivatives to avidin and strepavidin.^^ In the initial experiment, they measured the ESI mass spectrum of avidin (Fig. 12A) observing four charge states (15 + to 18 + ) corresponding to a molecular mass of 63,915 Da which correlates well with the expected mass of 63,870 Da. The spectrum obtained for a mixture of avidin-biotin (lower plot) shows that peaks are shifted to higher m/z values that correspond to a mass increase of 973 u. This indicated that four biotin molecules are indeed bound to avidin. The mass increase is accompanied by a reduction of one unit in the charge states, which now range from 1 4 + to 17 + . This study demonstrated that the ESI process transfers the solution complex to the gas phase. A second study by this group demonstrated that the specificity of the binding, which is observed in solution, is carried over to the mass spectral analysis (Fig. 12B).^^ The active form of strepavidin was reacted with both biotin ( K j - 10"^^ M ) and with iminobiotin (K^ - 10"^ M). The data indicated that four molecules of biotin bind to the strepavidin after short incubation times and at low strepavidin-biotin ratios, while under the same conditions, no strepavidin-iminobiotin complexes were observed. This result provided evidence that, for this system, the solution properties (relative binding constants) of the stepavidin-biotin interactions were carried over to the gas phase. ii. Protein - D N A Complexes

Due to the strong avidin-biotin interaction, the previous example represents the most favorable case for studying noncovalent interactions. To be able to utilize ESI-MS for more common biological systems, the usefulness of the technique must be demonstrated in cases where the non-covalent bonding is weaker. We selected as examples for presentation here studies relating to protein-DNA interactions because these interactions are extremely important in cellular processes and are widely studied. Until recently, there were no reports of mass spectrometric studies of such complexes. This is mainly due to the difficulty in maintaining the integrity of such elaborate complexes on transferring them from aqueous solution to the gas phase. In 1996, Smith and coworkers published two papers on mass spectrometric studies of protein-DNA complexes."^^'"^^ The first mass spectral analysis of a protein-DNA complex studied the oligonucleotide-binding stoichiometry of the gene V protein. This system was extensively studied in solution, and it is known that, under physiological conditions, the gene V protein forms a dimer which is known to bind single-stranded DNA with high affinity

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7500

3500

4500

5500

7500

F I G U R E 12 (A) ESI mass spectra of avidin (top) and of avidin - biotin mixture (bottom). Both the shift of the peaks to higher miz values and the decrease in the charge state values for the avidin - biotin mixture, indicate complex formation. (Reproduced by permission of Elsevier Science from Schvy^artz et al?^ Copyright 1994 by American Society for Mass Spectrometry.) (B) ESI mass spectra of streptavidin - iminobiotin (top) and of strepavidin - biotin (bottom). The spectra demonstrate the selectivity in binding toward the biotin. (Reproduced with permission from Schwartz et o/.^' Copyright John Wiley & Sons Limited.)

(iiCj= 10"^^ M ) and cooperativity, showing a distinct preference (two orders of magnitude) for poly(dT) over poly(dA). Gene V protein was believed to bind ssDNA at a stoichiometric ratio 3-5 ohgonucleotides per protein monomer, although it was not known whether this reflects a mixture or a single complex with that ratio. Mixtures of the protein with different oligonucleotides, at different protein-oligonucleotide ratios, were analyzed by negative-ion ESI-MS. The mass spectral analysis, the results of which

323

MASS SPECTROMETRY OF BIOMOLECULES

Streptavidin-Iminobiotin

4500

5500

6500

7500

Streptavidin-Biotin

il^ll•di<4^^LJ^iA 500

1500 FIGURE

12

2500

3500 ^ ^

4500

I

5500

rtA

*lf

6500

7500

{Continued)

appear in Fig. 1 3 , yielded the following information:

• A definite preference for the binding of ssDNA over dsDNA • A significant preference for binding poly(dT) over poly(dA) • An oligonucleotide with more than 15 bases forms a 4:1 complex with the protein while a 13 base oligonucleotide forms a 2:1 complex with the gene V protein An ESI mass spectrum of a mixture of 1:1 d(pT)^3:d(pA)i4 with the gene V protein (Fig. 13a) shows the existence of a double-stranded dimer and of a complex between the protein and a single d(pT)i3 oligonucleotide. Complexes of the protein with the dsDNA, or with d(pA)i4 do not appear in the spectrum. This clearly points to the preference for binding ssDNA over dsDNA and poly(dT) over poly(dA). When the mixture of d(pT)i3: d(pT)i5:

324

DAN GIBSON AND CATHERINE E. COSTELLO

B (4:1)TI5"

glOO S

80

1

60

3

40

^ (4:1)T15^^

I 20 IMM

li»ip>iPfi|l»HHl|i|i»ii|iii if iOP>

1000

2000

3000

4000

(1:1 )w^-

8(1:1 )w'

m/z

Dm^-

(1:1)m^-

(1:1)m«-

°m^' Jm 2600

2800

3000

t

D„8-

VX,...AT 1000

1500

2000

2500

3000

3500

m/z F I G U R E 13 (A) ESI mass spectrum of a 1:1 mixture of d(pT)|3:d(pA)|4 with the gene V protein showing peaks for the dsDNA around m/z 1400 and 1700 and for the 2:1 d(pT)|3:protein complex at m/z 2900 and 3300. (Reproduced with permission from Cheng et o/.'*° Copyright 1996 National Academy of Sciences, U.SA) (B) ESI mass spectrum of the mixture of d(pT)|3: d(pT)|5: d(pT)|8 with the protein showing the 2:1 complex of d(pT)|3:protein and the 4:1 complexes of d(pT)|5 and d(pT)|8 with the protein (Reproduced with permission from Cheng et o/. Copyright 1996 National Academy of Sciences, U.SA) (C) ESI mass spectrum of the protein with a 20:1 excess of the mutant relative to the wild-type. The inset shows that only the I: I complex with the wild-type is detected. (Reproduced with permission from Cheng et o/.'*' Copyright Academic Press Inc.)

MASS SPECTROMETRY OF BIOMOLECULES

325

d(pT)i8 with the protein was studied, the two longer ohgonucleotides formed a 4:1 complex with the protein while the shorter one formed a 2:1 complex (Fig. 13 b). The ESI-MS analysis is in very good agreement with the solution Studies, and these results taken in conjunction with the control experiments, strongly suggest that there is an intact transfer of the solution complexes into the gas phase. Therefore, the data of the ESI-MS analysis does not reflect properties which are unique to the gas phase and which may be different than those studied in solution. The second publication describes the sequence-specific complexes of dsDNA with a DNA-binding domain of a transcription factor (PU.l). Briefly, the experiment consisted of measuring the binding of a 17-base-pair doublestranded oligonucleotide containing the consensus sequence (wild-type) and of a 19-base-pair double-stranded oligonucleotide without the consensus sequence (mutant) to the protein. The results clearly demonstrate that the wild-type oligonucleotide formed a 1:1 complex with the protein while the mutant did not. Moreover, even with a 20:1 excess of the mutant relative to the wild-type, no protein-mutant complexes were detected while 1:1 protein-wild-type complexes were observed (Fig. 13c). This data is in agreement with a gel shift assay, which was run as a control experiment. This demonstrates the potential of using ESI-MS as a probe to screen for specificity in the solution binding of proteins to DNA. III. CONCLUSIONS Our main goal in this review was to acquaint the general audience with the basics of modern mass spectrometry, its capabilities, and its potential application in biological and biomedical research. We tried to accompfish this by focusing on the principles rather than the details, and by acquainting the readers with the basic terminology used in this field, in the hope to encourage researchers to follow the progress in this field, and to harness the fast-increasing potential of mass spectrometry to their own work. The examples that we chose were intended to demonstrate how the various techniques (MALDI, ESI, MS-MS, etc.) can and should be appHed to biological and biomedical research. While the examples we used were all from protein research, the mass spectrometric techniques are being successfully applied to many other fields, and their usefulness is general. Mass spectrometry continues to evolve, and new and more powerful spectrometers are making their way to academic institutes and to industry. This will undoubtedly result in new opportunities for mass spectrometry in biological research. REFERENCES 1. Siuzdak, G. (1994). The emergence of mass spectrometry in biochemical research. Proc. Natl Acad. Set. U.S.A. 9 1 , 11290-11297. 2. Yates, J. R. (1998). Mass spectrometry and the age of the proteome. / . Mass Spectrom. 33, 1-19.

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3. Nordhoff, E., Kirpekar, F., and Roepstroff, P. (1996). Mass spectrometry of nucleic acids. Mass Spectrom. Rev. 15, 67-138. 4. Rudd, P. M., Guile, G. R., Kuster, B., Harvey, D. J., Opdenakker, G., and Dwek, R. A. (1997). Oligosaccharide sequencing technology. Nature (London) 388, 205-207. 5. Loo, J. A. (1997). Studying noncovalent protein complexes by electrospray ionization mass spectrometry. Mass Spectrom. Rev. 16, 1-23. 6. Winston, R. L., and Fitzgerald, M. C. (1997). Mass spectrometry as a readout of protein structure and function. Mass Spectrom. Rev. 16, 165-179. 7. Ogorzalek Loo, R. R., Mitchell, C., Stevenson, T. L, Martin, S. A., Hines, W. M., Juhasz, P., Patterson, D. H., Peltier, J. M., Loo, J. A., and Andrews, P. C. (1997). Sensitivity and mass accuracy for proteins analyzed directly from polyacrylamide gels: Implications for proteome mappimg. Electrophoresis 18, 382-390. 8. Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F., and Whitehouse, C. M. (1989). Electrospray ionization for the mass spectrometry of large biomolecules. Science 246, 64-71. 9. Karas, M., and Hillenkamp, F. (1988). Laser desorption ionization of proteins with molecular masses exceeding 10000 daltons. Anal. Chem. 60, 2299-2301. 10. Figeys, D., Durcet, A., and Aebersold, R. (1997). Identification of proteins by capillary electrophoresis tandem mass spectrometry. Evaluation of on line solid-phase extraction device. / . Chromatogr. A 763, 295-306. 11. Figeys, D., and Aebersold, R. (1998). High sensitivity analysis of proteins and peptides by capillary electrophoresis tandem mass spectrometry—recent developments in technology and applications. Electrophoresis 19, 885-892 12. Eng, J., McCormack, A. L., and Yates, J. R. (1994). An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database / . Am. Soc. Mass Spectrom. 5, 976-989. 13. Papayannopoulos, I. A. (1995). The interpretation of collision-induced dissociation tandem mass spectra of peptides. Mass Spectrom. Rev. 14, 4 9 - 7 3 . 14. Hop, C. E. C. A., and Bakhtiar, R. (1997). An introduction to electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry—essential tools in a modern biotechnology laboratory. Biospectroscopy 3, 259-280. 15. Weickhardt, C., Moritz, F., and Grotemeyer, J. (1996). Time of flight mass spectrometry— state of the art in chemical analysis and molecular science. Mass Spectrom. Rev. 15, 139-162. 16. Bahr, U., Stahl-Zeng, J., Gleitsmann, E., and Karas, M. (1997). Delayed extraction time of flight MALDI mass spectrometry of proteins above 25,000 Da. / . Mass Spectrom. 32, 1111-1116. 17. Kaufmann, R., Kirsch, D., and Spengler, B. (1994). Sequencing of peptides in a time-of flight mass spectrometer: Evaluation of post source decay following matrix-assisted laser desorption ionization (MALDI). Int. } . Mass. Spectrom. Ion Processes 131, 355-385. 18. Jonscher, K. R., and Yates, J. R. (1997). The quadrupole ion trap mass spectrometer—a small solution to a big challenge. Anal. Biochem. 244, 1-15. 19. Kaiser, R. E., Cooks, R. G., Stafford, G. C., Syka, J. E. P., and Hemberger, P. H. (1991). Operation of a quadrupole ion trap mass-spectrometer to achieve high mass charge ratios. Int. J. Mass Spectrom. Ion Processes 106, 79-115. 20. Louris, J. N., Broadbelt-Lustig, J. S., Cooks, R. G., Glish, G. L., Van Berkel, G. J., and McLuckey, S. A. (1990). Ion isolation and sequential stages of mass-spectrometry in a quadrupole ion trap. Int. J. Mass Spectrom. Ion Processes 96, 117-137. 21. Cooks, R. G., Hoke, S. H., Morand, K. L., and Lammert, S. A. (1990). Mass spectrometers —instrumentation. Int. J. Mass Spectrom. Ion Processes 1 1 8 / 1 1 9 , 1-36. 22. Dienes, T., Pastor, S. J., Church, S., Scott, J. R., Yao, J., Cui, S. L., and Wilkins, C. L. (1996). Fourier Transform Mass spectrometry—advancing years (1992-mid 1996). Mass Spectrom. Rev. 15, 163-211. 23. Krishna, R., and Wold, F. (1997). Identification of common post-translational modifications. In "Protein Structure: A Practical Approach" (T. Creighton, ed.), 2nd ed., pp. 91-116. Oxford University Press: New York.

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24. Kuster, B., and Mann, M. (1998). Identifying proteins and post-translational modifications by mass spectrometry. Curr. Opin. Struct. Biol. 8, 393-400. 25. Feistner, G. J., Faull, K. P., Barofsky, D. P., and Roepstorff, P. (1995). Mass spectrometric peptide and protein charting. / . Mass Spectrom. 30, 519-530. 26. Yates, J. R. (1998). Data base searching using mass spectrometric data. Electrophoresis 19, 893-900. 27. Theberge, R., Skinner, M., Connors, L., Skare, J., and Costello, C. E. (1999). Characterization of transthyretin mutants from serum using immunoprecipitation, HPLC/Electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry, Anal. Chem. 71, 452-459. 28. Benson, M. D. (1995). Amyloidosis. In "The Molecular Basis of Inherited Disease" (Scriver, C. R., Beaudet, A. K., Sly, W. S. and Vallo, D., eds.), 7th ed.. Vol. 3, pp. 4159-4191. McGraw-Hill, New York. 29. Connors, L. H., Ericsson, T., Skare, J., Jones, L. A., Lewis, W. D., and Skinner, M. (1998). A simple screening test for variant transthyretins associated with Familial transthyretin amyloidosis using isoelectric focusing. Biochim. Biophys. Acta 1407, 185-192. 30. Ghelis, C , and Yon, J. (1982). "Protein Folding." Academic Press, New York. 31. Katta, V., and Chait, B.T. (1991). Conformational changes in proteins probed by hydrogenexchange electrospray-ionization mass spectrometry. Rapid Commun. Mass Spectrom. 5, 214-217. 32. Anderegg, R. J., and Wagner, D. S. (1995). Mass spectrometric characterization of proteinligand interaction. / . Am. Chem. Soc. 117, 1374-1377. 33. Miranker, A., Robinson, C. V., Radford, S. E., and Dobson, C. M. (1996). Investigation of protein folding by mass spectrometry. FASEB J. 10, 9 3 - 1 0 1 . 34. Gary, P. D., King, D. S., Crane-Robinson, C , Bradbury, E. M., Rabbani, A., Goodwin, G. H., and Johns, E. W. (1980). Structural studies on two high-mobility-group proteins from calf thymus, HMG-14 and HMG-20 (ubiquitin), and their interaction with DNA. Eur. } . Biochem. 112, 577-580. 35. Vijay-Kumar, S., Bugg, C. E., and Cook, W. J. (1987). Structure of ubiquitin refined at 1.8 A resolution. / . Mol Biol. 194, 531-544. 36. Roder, H. (1989). Structural characterization of protein folding intermediates by proton magnetic resonance and hydrogen exchange. In "Methods in Enzymology" (N. J. Oppenheimer and T. L. James, eds.). Vol. 176, pp. 446-473. Academic Press, Orlando, PL. 37. Konermann, L., Collings, B. A., and Douglas, D. J. (1997). Cytochrome c folding kinetics studied by time resolved electrospray ionization mass spectrometry. Biochemistry 36, 5S54-5559. 38. Schwartz, B. L., Light-Wahl, K. J., and Smith, R. D. (1994). Observation of noncovalent complexes in the Avidin tetramer by electrospray ionization mass spectrometry. / . Am. Soc. Mass Spectrom. 5, 201-204. 39. Schwartz, B. L., Gale, D. C , Smith, R. D., Chilkoti, A., and Stayton, P. S. (1995). Investigation of non-covalent ligand binding to the intact strepavidin tetramer by electrospray ionization mass spectrometry. / . Mass Spectrom. 30, 1095-1102. 40. Cheng, X., Harms, A. C , Goudreau, P. N., Terwilliger, T. C , and Smith, R. D. (1996). Direct measurement of oligonucleotide binding stoichiometry of gene V protein by mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 93, 7022-7027. 41. Cheng, X., Morin, P. E., Harms, A. C , Bruce, J. E., Ben David, Y., and Smith, R. D. (1996). Mass spectrometric characterization of sequence specific complexes of DNA and transcription factor PU.l DNA binding domain. Anal. Biochem. 239, 35-40.

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7 LIQUID-LIQUID

PARTITIONING METHODS FOR BIOSEPARATIONS

TINGYUE GU Department of Chemical Engineering, Ohio University, Athens, Ohio 45701

I. INTRODUCTION II. SOLVENT EXTRACTION FOR BIOSEPARATIONS A. Solvent Selection B. Effect of pH C. Effect of Ion Pairs D. Solvent Extraction of Amino Acids E. Solvent Extraction of Alcohols from Fermentation Broths F. Solvent Extraction of Aliphatic Carboxylic Acids G. Solvent Extraction of Antibiotics H. Liquid - Liquid Extraction with Reversed Micelles I. Electrically Enhanced Solvent Extraction J. Solvent Extraction Equipment and Operational Considerations III. AQUEOUS TWO-PHASE PARTITIONING FOR BIOSEPARATIONS A. Effect of Polymer Molecular Weight and Concentration B. Effect of Temperature C. Effect of Salt D. Affinity Partitioning E. Large-Scale Aqueous Two-Phase Partitioning of Biomolecules F. Equipment and Operational Considerations IV. SUMMARY REFERENCES

I. INTRODUCTION Various unit operations are used in the downstream processing of biomolecules. These recovery and purification methods include cell disruption, centrifugation, micro- and ultrafiltration, precipitation, liquid-liquid partitioning, and various forms of liquid chromatography. Among them, liquid-liquid partitioning methods are w^ell established, often inexpensive, and suitable for steady-state large-scale operations. There are tw^o main categories in liquid-liquid partitioning. One is the conventional solvent Separation Science and Technology,

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Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

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TINGYUE GU

extraction which is used for the separations of many metabohtes from fermentation such as alcohols, carboxylic acids, amino acids, and antibiotics.^'^ The other is the aqueous two-phase partitioning using water soluble polymers such as polyethylene glycol (PEG) and dextran, and salts such as potassium phosphate. The latter method is very attractive for the separation of biomolecules such as proteins and peptides including many enzymes^"^ that may be denatured by solvents. As the scale of bioseparation processes goes up, liquid-liquid partitioning becomes more and more competitive because it is easy to scale up and it permits continuous steady-state operation.^ The cost for liquid-liquid partitioning is much lower than other more sophisticated bioseparations method, such as Uquid chromatography.

II. SOLVENT EXTRACTION FOR BIOSEPARATIONS The International Union of Pure and Applied Chemistry (lUPAC) recommends the use of liquid-liquid distribution over the traditional term solvent extraction.^^ However, solvent extraction is still used prevalently in the literature. Solvent extraction utilizes the partition of a solute between two practically immiscible liquid phases: one solvent phase and the other aqueous phase.^^ A separatory funnel can be used in a lab to carry out solvent extraction. Of course, a simple test tube can also be used in conjunction with a glass pipette. The organic phase (solvent phase) is usually the top phase and the aqueous phase bottom phase. However, some organic solvents are heavier than water (for example, methylene chloride's specific gravity is 1.33 at 20°C) and in such cases the organic phase becomes the bottom phase. Thousands of papers and dozens of books and book chapters have been published on solvent extraction. Most of them deal with extraction compounds that are not biologically derived. The chemistry of solvent extraction is extensively investigated in a book edited by Sekine and Hasegawa^^ entitled "Solvent Extraction Chemistry: Fundamentals and Application." Many operational aspects of extraction are investigated in a book edited by Rydberg, Musikas, and Choppin^^ entitled "Principles and Practices of Solvents Extraction." Various extraction equipment and their operations are discussed in a book edited by Godfrey and Slater ^"^ entitled "Liquid-Liquid Extraction Equipment." For practitioners, the "Handbook of Solvent Extraction" by Baird et al}^ should be very helpful. Schiigerl^ wrote a book entitled "Solvent Extraction in Biotechnology: Recovery of Primary and Secondary Metabolites." This is a book exclusively dealing with solvent extraction for bioseparations. There are some book chapters dealing with solvent extraction for the separation of biomolecules. Thornton's book of "Science and Practice of Liquid-Liquid Extraction" contains a chapter dealing with solvent extraction of pharmaceuticals (such as antibiotics) and a chapter on extraction of food products.^ Aires-Barros and Cabral^^ contributed a chapter on hquid-Uquid extraction to a book edited by Kennedy and Cabral^"" entitled "Recovery Processes for Biological Materials." Weatherley^^ discussed the operational consideration of extraction of fermentation broth in a chapter of the book entitled "Downstream Processing of Natural Products"

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edited by Verrall.^^ Wheelwright^^ summarized solvent extraction briefly in his downstream protein purification book. Some theoretical and practical aspects of solvent extraction were discussed by Scopes^^ in his book on protein purification. Belter et alP' discussed the basics of solvent extraction in an easy to read chapter in their bioseparations textbook. They studied the following factors in solvent extraction. A. Solvent Selection The partition coefficient K is defined as follows: (1) in which x and y are the solute concentrations in the lighter phase (usually the organic phase) and the solute concentration in the heavier phase (usually the water phase), respectively. Table 1 shows the partition coefficient of some biomolecules.^^ We can also express K in terms of chemical potentials in standard reference states for the lighter phase [ /t°( L)] and heavier phase [M°(H)]:

K = x / y = exp{[ ^J.\H)

TABLE I

Partition Coefficients of Some Bionnolecules in Solvent Extraction

Category

Solute

Solvent

Amino acids

Glycine Alanine Lysine Glutamic acid a-aminobutyic acid a-aminocaproic acid Celesticetin Cycloheximide Erythromycin

«-Butanol «-Butanol «-Butanol «-Butanol «-Butanol «-Butanol «-Butanol Methylene chloride Amyl acetate

Lincomycin Gramicidin Novobiocin

«-Butanol Benzene Chloroform-methanol Butyl acetate

Penicillin F

Amyl acetate

Penicillin K

Amyl acetate

Glucose isomerase

PEG 1550-potassium phosphate PEG 1550-potassium phosphate PEG-crude dextran

Antibiotics

Proteins

(2)

- ^«(L)]/(RT)}

Fumarase Catalase

K 0.01 0.02 0.2 0.07 0.02 0.3 110 23 120 0.04 0.17 0.6 17 100 0.01 32 0.06 12 0.1 3

Comments 25°C

At pH 4.2

At pH At pH At pH At pH At pH At pH

7.0 10.5 4.0 6.0 4.0 6.0

0.2 3

Source: Reprinted with permission from Belter et alP" Copyright 1988 John Wiley & Sons, Inc.

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In Eq. (2), R is the universal gas constant and T is the absolute temperature in degrees Kelvin. The concept of solubility parameter can be used to predict the partition coefficient qualitatively in the selection of solvents.^^ We can write K as InK =

= RTVj^

(3) ^ ^

in V with different subscripts indicates partial molar volumes of lighter phase (subscript L), heavier phase (subscript H) and solute A (subscript A); S indicates the solubility parameter. Table 2 shows the solubility parameter values for some solvents.^^ Obviously, some solvents are better than others for the extraction of a particular compound. A solvent system should provide a suitable partition coefficient, preferably much greater than unity or much less than unity, depending on whether you want the solute to migrate to the solvent phase or to stay in the aqueous phase. The following factors must be considered during the selection of a suitable solvent system^^'^"^: 1. There must be no irreversible reaction with solutes. 2. The solvent phase after extraction must allow ready recovery of the solutes from it. 3. The interfacial tension should be appropriate for solvent-water contact. High values for interfacial tension result in an increased energy requirement to maintain sufficient contact time, while low interfacial tension result in the formation of stable emulsions that make separation considerably more difficult.

T A B L E 2 Solubility Parameters for Some Solvents

Solvent Amyl acetate Benzene Butanol Butyl acetate Carbon disulfide Carbon tetrachloride Chloroform Cyclohexane Hexanol Acetone Pentane Perfluorohexane Toluene Water

Solubility parameter (cal^^cm-^'^) 8.0 9.2 13.6 8.5 10.0 8.6 9.2 8.2 10.7 7.5 7.1 5.9 8.9 9.4

Source: Reprinted with permission from Belter et al?^ Copyright 1988 John Wiley & Sons, Inc.

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333

4. The density difference between the solvent and water should be sufficiently large. 5. The solvent should be immiscible with water or has a very limited solubility. 6. The solvent should be readily available at a reasonable cost. Above all, the solvent system must provide a selectivity good enough for practical applications. Additional factors such as volatility and toxicity also require attention. The "Handbook of Organic Solvent Properties" by SmalIwood^^ contains information on many solvents. In the extraction of fragile biomolecules such as peptides and proteins, one must know whether the solvent system will denature the peptide or the protein. As a rule of thumb, the larger a peptide or a protein, the more complex its stearic structure and the higher possibility for denaturation. As a result, larger peptides and proteins cannot usually be separated using solvent extraction. The more biocompatible aqueous two-phase partitioning method is used instead of solvent extraction. B. Effect of pH pH is an important parameter in extraction that is often adjusted to achieve desirable results.^^'^^ The partition coefficient of many solutes in solvent-water systems can be altered by changing pH. If the solute is a weak acid, the following relationship^^ can be used to calculate partition coefficient K at a given pH value of the aqueous phase, logio(X,/K-l)=pK,-pH

(4)

where Kj is the intrinsic partition coefficient (not dependent on pH) defined as the ratio of the acid concentration in the solvent phase to that in the water phase. K. = [ R C O O H ] y [ R C O O H ] ^

(5)

The pK^ values for some biomolecules are fisted in Table 3}^ They are also available from biochemistry reference books. Similarly, for weak bases, logio(KyK-l)=pH-pKb

(6)

Based on Eq. (4), the selectivity between solutes A and B can be obtained via the following relationship k^^:

P=

K(A)/K(B)

= [K,(A)/K,iB)]-{l+K,(B)/[Hn}/{i+K,(A)/[Hn}

(7)

The dramatic effect of pH on K for the extraction of some antibiotics can be seen in Table 1.

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TINGYUE GU

TABLE 3

T h e pK^ Values of Some Biomolecules

Simple acids and bases Acetic acid Propionic acid H3PO4 H2PO4HPO|-

NHJ CH3NHJ Amino acids Glycine Leucine Glutamine Aspartic acid Glutamic acid Histidine Cysteine Tyrosine Lysine Arginine Peptides Gly-Gly Gly-Gly-Gly Ala Ala-Ala-Ala-Ala Gly-Asp Ala-Ala-Lys-Ala Antibiotics Celesticetins Cephalosporin C Lincomycin (free base) Monensin (carboxyl) Novobiocin (enol, phenolic) Penicillamin (carboxyl) Rifamycin B Spectinomycin

pKa 4.76 4.87 2.14 7.20 12.4 9.25 10.6 pKi(COOH) pK2(aNHp pK, ( (R Group) 2.34 9.6 2.36 9.6 2.17 9.13 9.82 2.09 3.86 2.19 9.67 4.25 1.82 9.17 6.0 1.71 10.78 8.33 2.20 9.11 10.07 2.18 8.95 10.53 9.04 12.48 2.17 pKi(COOH) pK2(aNHj) PK;5 (R Group) 3.06 8.13 3.26 7.91 2.34 9.69 7.94 3.42 8.60 4.45 2.81 10.58 3.58 8.01 pKa values 7.7, 9.8 3.9, 5.3, 10.5 7.6 6.65 (in 66% dimethylformamide) 4.3, 9.1 1.8 2 , 1 , ,6.7 6.95S 8.70

Source: Reprinted with permission from Belter et al.^^ Copyright 1988 John Wiley & Sons, Inc.

C. Effect of Ion Pairs If the solute to be separated is ionic, counterions from organic soluble salts (greasy salts) may be used to increase the solubility of the solute in the organic phase greatly. For example, sodium acetate can increase the solubility of tetrabutylammonium cation in chloroform by 10-fold. Some common counterions for ion-pair extractions are acetate, butyrate, tetrabutylammonium, perfluorooctanoate, dodecanoate, linoleate, cholate, and tetraphenylboride.^^ SchiigerP systematically studied various applications of solvent extraction in his book on solvent extraction of biological metabolites. He discussed the solvent extraction of alcohols, carboxylic acids, amino acids, and antibiotics in fermentation broths. Operational strategy and equipment w^ere also investigated. There is a growing interest in v^hole broth ex-

LIQUID-LIQUID PARTITIONING

335

traction because of its economical and environmental benefits according to Weatherley.^^ D. Solvent Extraction of Amino Acids Amino acids are zwitterions with amino and carboxyl groups. They act as cations at low pH values, as anions at high pH values and are dipolar at intermediate pH values. Their solubility in nonpolar solvents are very low thus extraction with nonpolar solvents is not feasible. Partition coefficients of amino acids in solvent-water systems are usually very small. An organic carrier, for example trioctylmethylammonium chloride (TOMAC), can be used to enhance extraction."^^ Amino acids are usually extracted with phosphorus-bonded, oxygen-bearing solvents or with large quaternary aliphatic amines. Not much was done for extraction at low pH values due to excessive coextraction of other inorganic cations. TOMAC is used in xylene to extract amino acids from an aqueous phase. Table 4 lists the isoelectric points and extraction equilibrium constants of several amino acids with TOMAC at high pH values.^ The separation of amino acids from each other is not an easy task according to SchiigerP who reviewed the following cases. Isoleucine and leucine can be separated from lysine that has a positive charge at pH 6 with TOMAC and 6% decanol in methylcyclohexane at pH 10 to 11. Isoleucine and leucine are transferred to the organic phase because they have nonpolar side chains. After reextraction with HCl or NaCl solution, isoleucine and leucine are separated by extraction using cyclohexane with the addition of an anion exchanger (such as tetra-docylammoniumbromide). Isoleucine will stay in the aqueous phase while leucine migrates to the organic phase. An alternate method is to use 6% decanol in xylene at pH 12 to separated

T A B L E 4 Extraction Equilibrium Constants K and Isoelectric Points of Some A m i n o Acids Amino acid

Pl

Glycine Alanine Valine Leucine Isoleucine Methonine Phenylalanine Tryptophan Tyrosine Histidine Arginine Serine Threonine

6.0 6.0 6.0 6.0 6.0 5.7 5.5 5.9 5.7 7.6 10.8 5.7 6.2

K 0.036 0.038 0.089 0.29 0.24 0.21 0.97 8.89 0.40 0.083 0.062 0.049 0.017

Source: Reprinted with permission from Schiigerl.^ Copyright 1994 Springer Verlag.

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isoleucine and leucine. Tryptophan and tyrosine, two aromatic amino acids, can be extracted into the organic phase from the aqueous phase with 15 m M TOMAC in xylene at pH 10 to 11. They can then reextracted with 1.5 M NaCl solution. Tyrosine migrates to the aqueous phase while tryptophan stays in the organic phase because tryptophan's side chain is nonpolar unlike tyrosine's polar side chain. To separate arginine or lysine from aspartic acid, 0.3 M TOMAC in xylene at pH 12 is used. Aspartic acid migrates to the solvent phase while arginine and lysine stay in the aqueous phase. The extractions of some other amino acids are covered by several European patents.^ E. Solvent Extraction of Alcohols from Fermentation Broths Ethanol and butanol are the most important alcohols produced by fermentation. They are also produced from petroleum. Ethanol and butanol from fermentation are considered renewable resources since they can be produced with renewable agriculture feed stocks. Because of product inhibition in yeast fermentation, ethanol concentration is limited to 5 to 10% (v/v) in the broth.^ According to Essien and Pyle,^^ solvent extraction of ethanol is about 60% higher in capital-related costs than distillation. This more than offsets any advantage in energy and utility even without considering the cost of the solvent. Distillation is the preferred method. Emphasis has been on in situ extractive fermentation to reduce product inhibition.^^'^^ It is difficult to find a suitable solvent with a high partition coefficient, that does not form stable emulsion to hamper extraction, and that does not have harmful effects on cells.^ The following solvent systems have been reported in the literature: dodecanol, higher iso-alcohols, higher ^-alcohols, tributylphosphate, dibutylphthalate, dodecane, fluorocarbons, and higher iso-acids.^ Aqueous two-phase systems can also be used for alcohol recoveries.^^ F. Solvent Extraction of Aliphatic Carboxylic Acids Kertes and King^^ reviewed the extraction chemistry of carboxylic acids from fermentation broths. The following methods were reviewed by Kertes and King: • Extraction with carbon-bonded oxygen-bearing extractants • Extraction with phosphorous-bonded oxygen-bearing extractants • Extraction by proton transfer or by ion-pair formation using high molecular weight aliphatic amines Table 5 shows the partition coefficient of some common aliphatic carboxylic acids produced from fermentation.^^ Aromatic carboxylic acids are not produced from fermentation because they are rarely formed by microorganisms. One noteworthy exception is the production of salicylic acid formed by Pseudomonas aeruginosa from naphthalene through biotransformation.^ The extraction of salicylic acid with and without amine in xylene was reviewed by Schiigerl^ in detail including equipment and operational parameters.

337

LIQUID-LIQUID PARTITIONING

TABLE 5

Partition Coefficients of Sonne Carboxylic Acids at 2S°C

Acid and solvent Propanoic acid «-Hexane Cyclohexane Benzene Toluene Xylene Carbon tetrachloride Chloroform Nitrobenzene Diethyl ether Diisopropyl ether Methylisobutyl ketone Cyclohexanone «-Butanol «-Pentanol Lactic acid Diethyl ether Diisopropyl ether Methylisobutyl ketone «-Butanol Isobutanol «-Pentanol «-Hexanol w-Octanol Pyruvic acid Diethyl ether Succinic acid Diethyl ether Methylisobutyl ketone «-Butanol Isobutanol «-Pentanol «-Octanol

Partition coefficient 0.005 0.006 0.043 0.034 0.030 0.015 0.11 0.16 1.75 0.80 2.15 3.30 3.20 2.95 0.10 0.04 0.14 0.73 0.66 0.40 0.37 0.32 0.16

Partition coefficient

Acid and solvent Fumaric acid Diethyl ether Methylisobutyl «-Butanol Isobutanol Maleic acid Diethyl ether Methylisobutyl Isobutanol Malic acid Diethyl ether Methylisobutyl Isobutanol Itaconic acid Diethyl ether Methylisobutyl Isobutanol Tartaric acid Diethyl ether Methylisobutyl «-Butanol Citric acid Diethyl ether Methylisobutyl w-Butanol Isobutanol

ketone

1.50 1.40 3.30 4.60

ketone

0.15 0.21 0.92

ketone

0.02 0.04 0.36

ketone

0.35 0.55 1.80

ketone

0.003 0.02 0.16

ketone

0.009 0.09 0.29 0.30

0.15 0.19 1.20 0.96 0.66 0.26

Source: Reprinted with permission from Kertes and King.^"^ Copyright 1986 John Wiley & Sons, Inc.

G. Solvent Extraction of Antibiotics Antibiotics are microbial compounds that inhibit and even destroy other organisms.^ Antibiotics are usually secreted by microbial cells. After clarification using centrifugation or microfiltration, solvent extraction can be used to extract antibiotics from the clarified broth. The extract is further purified using reextraction, precipitation, and ion-exchange chromatography, or crystaUization.^ If the antibiotic is a weak acid with a low pK^ value, the pH used in the extraction should be lowered to below the pK^ value to obtain the antibiotic in its free acid form. One the other hand, if the antibiotic is a weak base with a high pK^ value, the pH should be increased to above the pK^ value to obtain a free base after extraction. If the antibiotic is highly soluble in water, the broth must be saturated with salt to enhance extraction.^ Table

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TABLE 6

Solvent Extraction of Sonne Antibiotics

Product

Medium

Solvent

PH

Actinomycin Adrianimycin Bacitracin Chloramphenicol Clavulanic acid Cyloheximid Erythromycin Fusidic acid Griseofulvin Macrolides Nisin

Cake Cake Broth Broth Broth Broth Broth Broth Cake Broth Broth Mash Broth Broth Cake Broth Broth Broth

1 MeOH + 2 methylene chloride Acetone «-Butanol Ethylacetate «-Butanol Methylene chloride Amylacetate Methylisobutylketone Butylacetate, methylene chloride Methylisobutylketone, ethylacetate CHCI3 + sec-Oct.alc. CHCI3 1-Butanol Butylacetate, amylacetate Butylacetate 1-Butanol Butylacetate, amylacetate Methylisobutylketone

2.5 Acidic 7.0 N-alkaline 2.0 3.5-5.5 Alkaline 6.8 Neutral AlkaUne 4.5 2.0

Oxytetracycline Penicillin G Salinomycin Tetracycline Tylosin Virginiamycin

2.0 9.0

Acidic

Source: Reprinted with permission from Schiigerl.^ Copyright 1994 Springer Verlag.

6 is a list of antibiotics extracted with different solvent systems.^ Table 1 also contains some antibiotics purified using solvent extraction.^^ Solvent extraction of penicillin from fermentation broths has been well documented in the literature. Penicillin G and penicillin V can be efficiently extracted with amyl acetate or butyl acetate at pH 2.5-3.0 and at 0° to 3°C.^^ Schiigerl^ systematically reviewed solvent extraction of different forms of penicillin from fermentation broths. Figure 1 shows an integrated process for the extraction of penicillin G from clarified broth of Fenicillium chrysogenum fermentation.^ Penicillin G is converted to 6-amino penicillanic acid and phenylacetic acid at pH 8 in a 10 L Kiihni extractor by penicillin G-amidase immobilized in an emulsion Hquid membrane. The 6-amino penicillanic acid is subsequently converted to ampicillin at pH 6 and the enzyme is recycled. A book edited by Vandamme^"^ entitled "Biotechnology of Industrial Antibiotics" studied the production of many antibiotics. The recovery and purification of the majority of them involved a step using solvent extraction. Verrall^^ also reviewed solvent extraction of some antibiotics. The antibiotic clavulanic acid is a fused bicyclic j8-lactam which is produced naturally by Streptomyces calvuligerus. Clarified broth was first extracted with nbutanoliHCl ( 3 / 4 vol.) at pH 2 and at 5°C. It was reextracted back to aqueous phase with 1/20 vol. of water at pH 7 and 20% (w/v) aqueous N a O H . Ion-exchange chromatography was then used for further purification.^^ The antibiotic tetracycline is a polyketide produced by Streptomyces aureofaciens. Tetracycline was extracted from an acid or alkaline medium by 1-butanol. There are also other methods such as extraction with a methylalkyl ketone.^'^ Nowadays tetracycline is purified by precipitation

339

LIQUID-LIQUID PARTITIONING MICRO-/ULTRAFILTRAT10N CELL-FREE BROTH ELECTROCOALESCENCE UNIT

PH ADJUSTMENT ]PRECURSOR

EMULSION PREPARATION

EN2YME PHASE

MEMBRANE PHASE

F I G U R E I An integrated process for the recovery of penicillin G. (Reprinted with permission from Schijgerl.' Copyright 1994 Springer Verlag.)

replacing the old extraction method.^ The antibiotic erythromycin is a macrolide secreted by Saccharopolyspora erythraea?^ Figure 2 depicts an industrial process for the recovery of erythromycin from fermentation.^^ It involves three extraction steps. Gramicidin D is a linear-chain peptide antibiotic produced from Bacillus brevis?^ Filtered broth is adjusted to pH 4.5-4.8 to precipitate a mixture of gramicidins. The dried solid mass is extracted with alcohol. Saline was added to precipitate the gramicidins again. Further extraction with an acetone-ether mixture produces a neutral fraction containing the linear gramicidins. A system with benzenexhloroform: methanohwater = 15:15:23:7 (v/v) can separate gramicidin D from gramicidins A, B, and C in a countercurrent extraction.^^ The antibiotic cycloheximide is an effective protein synthesis inhibitor used as an agricultural fungicide. It was discovered in a streptomycin-yielding culture of Streptomyces griseus."^^ Several purification schemes involving solvent extraction were reviewed by Jost et al."^^ Ethyl acetate, cyclohexane, amyl acetate, and methylene chloride were used as solvents in those schemes. Fusidic acid is a steroid antibiotic particularly useful in treating staphylococcal infections. It is produced by fermentation of the fungus Fusidium coccineum^^ It belongs to a group of tetracyclic triterpenoic acids. Von Daehne et al^^ described an industrial recovery process for fusidic acid. It involved sequential extractions using methyUsobutylketone (MIBK), benzene, and acetone. Antimycin A is an antifungal and piscicidal antibiotic produced intracellularly by Streptomyces kitasawaensis and S. griseus?^ Its recovery involved extractions with CH3CI and CH2Cl2.'^^ Chloramphenicol is an antibacterial agent produced by Streptomyces venezuelae. It is a rather simple aromatic compound. It is easily extracted from a clarified broth by a solvent such as

340

TINGYUE GU

Broth 40 m^ (preheated/cooled to SO'C in Fcrmenter) Concentration 5000-6000 (im\ — K4Fe(CN)620 kg Benkol 40 kg Ammonium Chloride

To precipitate proteins

— 1 0 % NaOH 1501 pH7.0 — Dicalite Filter Aid 800 kg Agitate (Volume 41 m^)

I Drum Filter (Dicalite precoat 400 kg)-wash

^ First filtrate 37.4 m'

Wet mycelium cake 4000 kg I

Water 13 m^

Total filtrate 60 m^ - E . D . T . A . 1501 (30 kg)

Slurry

t

Cake 4000 kg

Precipitate protein

Drum filter • -Wash 9 m' (precoat 400 kg)

.

Ammonium

^

Sulphate 500 kg

h-10% NaOH 8001 Second filtrate 22.4 m^Filter press Distill spent filtrate ^^_ "60m^ ^

Primary extraction

pH9.5

- Fresh butyl acetate 15 m

Rich butyl acetate 15 m^ ^10%CH3COOH3501 pH5.0 _ Distill lean 3 -4 "Butyl aceetate 15 m"

[ Secondary extraction]

Water 5 m^

1

Rich buffer (-10% NaOH 501 pH9.5 Lean buffer 5 m < -

Tertiary extraction

• Fresh butyl acetate 2.5 m^

Rich butyl acetate e- 20% NaSCN 220 1 E- 10% CH3COOH pH7.0 Agitate Mother liquor ^^_ Wet bu. ac. 2.5 m^ ^

Wet EMT 250 kg - • Vacuum dry @50'*C 176 kg

F I G U R E 2 Recovery of erythromycin from fermentation. (Reprinted from VerralP^ by permission of Oxford University Press.)

ethyl acetate at a slightly alkaline pH."^^ The antibiotic virginiamycin is produced by Streptomyces virginiae. It consists of two synergistic components, factors M and S which are two cyclic lactone peptolides. Biot"^"* reported that virginiamycin was produced from fermentation broth using a three-stage extraction process with MIBK. Addition of hexane to the MIBK

LIQUID-LIQUID PARTITIONING

341

precipitated virginiamycin which was further separated by centrifugation under a positive CO2 pressure to prevent fire. Biot"^"^ presented a flowchart of the process. H. Liquid-Liquid Extraction with Reversed Micelles Organic solvents have the tendency to denature larger peptides and proteins irreversibly and render them biologically inactive. To overcome this drawback of solvent extraction, liquid-liquid extraction with reversed micelles is used. Reversed micelles are formed when surfactant molecules in a nonpolar solvent aggregate in such way that the polar head groups of the surfactant molecules turn inward to form a polar inner core."^^ Water and host molecules such as proteins can be solubilized inside the micellar cores. Biomolecules in an aqueous phase such as a fermentation broth are first transferred to an organic micellar phase and then reextracted back into a new aqueous phase."^^ Figure 3 illustrates the transfer of a protein from a fermentation broth to an organic micellar phase."^^ Figure 4 is a typical flowchart of liquid-liquid extraction with reversed micelles."^^ Experimental results suggest that hydrophillic proteins tend to be solubilized within the water core of the reversed micelles, while lipophilic biomolecules can either stay in the interface or even partially exposed to the organic phase."^^ Because of the protection offered by the reversed micelles, proteins were shown to maintain their functional properties.'^'' The retention of bioactivity depends strongly on the solvent system and it is usually not 100%.^^ The most commonly used anionic surfactant in reversed micelles for the extraction of proteins is AOT [sodium bis(2-ethylhexyl)sulphosuccinate]

F I G U R E 3 Protein transfer between an aqueous phase and an organic micellar phase. (Reprinted with permission from Rahaman et 0/.'*^ Copyright 1988 American Chemical Society and American Institute of Chemical Engineers.)

342

TINGYUE GU reversed mi cellar phase

mixer 1

settler 1

mixer 2

settler 2

FORWARD

BACK

EXTRACTION

EXTRACTION

F I G U R E 4 Combined forward and back extraction involving reversed micelles. (From M. Dekker, K. Van't Riet, S. R. Weijers, J. W . Baltussen, C. Laane, and B. H. Bijisterboch, Enzyme recovery by liquid-liquid extraction using reversed micelles. Chem. Eng. J. 33, B27-33, 1986, with permission from Elsevier Science.)

which aggregates spontaneously in hydrocarbon solvents such as iso-octane, forming tiny water pools with radii more than 170 A."^^'"^^ TOMAC is a commonly used cationic surfactant. When a cationic surfactant is used, a cosurfactant usually an aliphatic alcohol such as octanol is needed to stabilize the reversed micelles by partitioning between the micelles and the continuous phase."*^ The driving forces in the transfer of proteins into reversed micelles are electrostatic interactions because proteins are dipolar molecules. The phase transfer depends on the isoelectric point, size and shape, hydrophobicity, and charge distribution on the protein surface. It also depends on the pH, ionic strength, type of electrolyte, and surfactant concentration of the two-phase system."^^ Figures 5 and 6 show that pH and ionic strength have a dramatic effect on the transfer of cytochrome c, lysozyme, and ribonuclease-a to the reversed micelles."^^ The advantage of reversed micelles in solvent extraction is the selective solubilization of proteins inside reversed micelles which provides a logical aqueous environment protecting the bioactivities of the proteins. The disadvantage is mainly the often necessary need to reextract the proteins into a aqueous phase. In some cases, the yield of reextraction is very low and the transfer of proteins into the organic phase seems to be irreversible. Hu and Gulari^^ studied the reverse micelle system of sodium di-2-ethylhexyl phosphate (NaDEHP, an anionic surfactant) for the extracton of two aminoglycoside antibiotics, namely neomycin and gentamycin. The antibiotic molecules are first transferred from the aqueous phase to the polar core of reverse micelles. During back extraction to the aqueous phase, the antibiotics in the micelle phase are released back to an aqueous phase by breaking up the reverse micelles using divalent cation solutions, such as Ca^"^ solutions. Cabral and Aires-Barros"^^ showed a double extraction process (Fig. 4) for the continuous extraction of a-amylase using TOMAC in iso-octane. Goklen and Hatton"^^ described an experimental procedure to separate a ternary mixture of ribonuclease-a, cytochrome c, and lysozyme with 50 m M AOT in iso-oc-

343

LIQUID-LIQUID PARTITIONING

F I G U R E 5 Effect of pH on protein solubilization in AOT-iso-octane system with 0.1 M KCI: ( O ) cytochrome c, ( D ) lysozyme, ( A ) ribonuclease-a (from Goklen and Hatton"*' by courtesy of Marcel Dekker, Inc.).

tane. In the solubilization step at pH 9 with 0.1 M KCI, cytochrome c, and lysozyme were transferred to the organic phase while ribonuclease-a remained in the aqueous phase. Cytochrome c was then back extracted to a 0.5 M KCI aqueous phase. Lysozyme was subsequently transferred to a 2.0 M KCI solution at pH 11.5. Recovery of intracellular enzymes directly from bacterial cells using reversed micelles was investigated by Giovenco et al,^^

0.2

0.4 0.6 KC I C o n c e n t r o t i o n , M

0.8

1.0

F I G U R E 6 Effect of ionic strength on protein solubilization in AOT-iso-octane system with no pH control: ( O ) cytochrome c, ( D ) lysozyme, ( A ) ribonuclease-a (from Goklen and Hatton'*' by courtesy of Marcel Dekker, Inc.).

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When cells are disrupted by surfactants, intracellular enzymes are transferred to the reversed micelles directly. This may provide a more efficient alternative to the conventional multistage purification method."^^ I. Electrically Enhanced Solvent Extraction Electrical fields have been applied to solvent extraction. The follov^ing mass transfer enhancements v^ere mentioned by Weatherley et aL^^: • Small oscillating droplets are generated v^ith a high interfacial area. • Dispersion phase transfer is greatly improved due to sustained droplet oscillation. • The continuous phase film mass transfer rate can be increased by electrostatic acceleration of charged droplets of the dispersed phase in the continuous phase. Improved mass transfer leads to smaller extraction equipment v^ith shorter residence times. Weatherley et aL^'^ studied ethanol extraction v^ith decanol under a range of electrically enhanced experimental conditions. J. Solvent Extraction Equipment and Operational Considerations Figure 7 shoves four common single-stage devices used in solvent extraction.^^ The most common laboratory device for extraction is the separatory funnel. Craig extraction, also knov^n as fractional extractions w^ith a stationary phase, can be used to extract a solute out of an aqueous phase (or an organic phase) repeatedly with a solvent (or water) for a very a high recovery yield.^^ Figure 8 is an illustration of the extraction scheme.^^ The net effect of Craig extraction resembles that of elution chromatography. Figure 9 shows Craig extraction tubes. These tubes are tipped between two positions: at the left to mix the two phases till an equilibrium is reached and at the right to empty

Liquid-Liquid Dispersion Separatory Funnel

/T

Vinyl Acetate-Acetic Acid Solution • Water

Ip V)o

Mixer Vinyl Acetate Water

Settler

t:

Vinyl Acetate Product Water Product

Light Phase Dispersion

^1 ^

Light Phase vL

44-

Dispersion Band

y

Heavy Phase

F I G U R E 7 Separatory funnel and mixer-settlers for extraction. (Reprinted with permission from Belter et al}'^ Copyright 1988 John Wiley & Sons, Inc.)

345

LIQUID-LIQUID PARTITIONING

All tubes except the zeroth contain only heavy solvent.

The light in the zeroth tube is moved to the first, and replaced with fresh light. The light phases are again moved, and new light added.

Zero Transfer

^

1/2

ti ti

One Transfer

Two Transfers

L2I/8

H 1/4

1/8

1/8

1/4

1/8

After each step, equilibrium is allowed to occur.

3/16

1/16

3/16

1/16

The result is a solute peak moving through the tubes.

3/16 3/16

1/32 1/8

1/32

F I G U R E 8 Craig extraction scheme. (Reprinted with permission from Belter et al}^ l988John Wiley & Sons, Inc.)

Copyright

the two phases.^^ Various laboratory and industrial devices and equipment for liquid-liquid extracted were discussed in a book entitled "Liquid-Liquid Extraction Equipment" edited by Godfrey and Slater.^"^ They include plate and packed columns, mixers, gravity settlers, centrifugal extractors, and so on. Figure 10 shows four types of reciprocating plate column.^^ The openings on each plate are designed for countercurrent flow. Figure 11 is the schematic

F I G U R E 9 Craig extraction tubes. (Reprinted with permission from Belter et al}^ l988John Wiley & Sons, Inc.)

Copyright

346

TINGYUE GU

(a)

(b)

KRPC

PRPC

(d)

( c ) PRPC (counterphase)

MVDC

F I G U R E 10 Some types of reciprocating plate columns. (From Baird et al.^^ Reproduced by permission of John Wiley & Sons Limited.)

of a Westfalia countercurrent centrifugal extraction decanter.^^ Centrifugal force is used for mixing and separation of the lighter and heavier phases. Various operational topics including mass transfer and extraction equipment in solvent extraction of fermentation broth were discussed by Weatherley.^^ In his mass transfer textbook, Treybal^"^ devoted one chapter to various industrial extraction equipment and calculations. King's^^ textbook on separation processes is also a good source for solvent extraction calculations. Figure 12 shows a multistage extraction process with simple individual mixers and settlers in series.^"^ Three extraction towers are illustrated in Fig. 13.^^ The left one is a packed bed extractor. The middle one is Mixco Lightnin CMC contactor with flat-blade turbine impellers to disperse and mix

Clarifying zone

Separating disc

Countercurrent extraction

Clarifying zone

Scroll Aqueous feed extract phase _ • Enriched extract phase Centripetal pump

Raffinate phase F I G U R E I I Westfalia countercurrent extraction decanter. (Baker-Perkins technical literature. Reprinted from VerralP^ by permission of Oxford University Press.)

LIQUID-LIQUID

347

PARTITIONING

Finol extroct

Stoge 3

Stage 2

Stoqe 1

M:\

M:\

<^ Mixer

1

Mixer

H / Settler j

Settler

^

t

Feed

FIGURE

12

Three-stage

A

Mixer Settler p T '^i^

^

Ju_ countercurrent

mixer-settler

^Finol ^roffin Solvent

extraction

cascade.

Reprinted

from

Treybal^"* w i t h p e r m i s s i o n o f T h e M c G r a w - H i l l C o m p a n i e s .

the two phases and horizontal compartmenting plates to reduce axial mixing. The right one is a rotating-disk contactor. Figure 14 is a sieve-tray extraction tower arranged for light liquid dispersed.^"^ These extractors are multistage extractors. Multistage extraction is required for extraction systems with partition coefficients close to unity or when the partition coefficients are close for the product and impurities. The calculations of multistage extraction are well documented in chemical engineering textbooks such as McCabe et al.'s^^ unit operations textbook and Wankat's ^ staged separations textbook, apart from Treybal's and Kings textbooks on mass transfer and separations.^"^'^^ Belter et al}^ presented calculation methods for extractions of biological compounds such as antibiotics in their textbook on bioseparations. Mass transfer calculations and operational parameters for various extractors were investigated in a book on extraction equipment edited by Godfrey and Slater.^"* The selection, design, pilot testing, and scale-up of

Light ^ Liquid Outlet

J|| X.

I Light Liquid Outlet

-oeja. JH Heavy. Liquid inlet

—^

5"

Light Liquid Outlet

• Stator Ring - Rotor Disk

Light • Liquid Inlet Heavy Liquid Out

Light Liquid Inlet FIGURE

13

Differential

McGraw-Hill Companies.

extractors.

^-JL^

Heavy Liquid Outlet Reprinted

from

Treybal^"^

with

permission

Heavy Liquid Outlet

of

The

348

TINGYUE GU Light liquid out

Principol interfoce

- Heavy liquid - Coolesced dispersed liquid

- Perforated plate

- Downspout

:T
J C ^ ^ Light "U

liquid

Heavy liquid out P-

F I G U R E 14 Sieve-tray extraction tower arranged for light liquid dispersed. Reprinted from Treybal^"* with permission of The McGraw-Hill Companies.

various extraction equipment were investigated by Pratt and Stevens/^ They classified extractors into about 20 types. A comprehensive extractor selection chart was presented.

III. AQUEOUS TWO-PHASE PARTITIONING FOR BIOSEPARATIONS According to Albertsson^ aqueous two-phase partitioning systems were first reported by Beijerinck in 1896 who described that gelatin, agar, and water mixture within a certain concentration range separated into two aqueous phases, the top phase being gelatin rich, and the bottom phase, agar rich. As a matter of fact, for polymer mixtures miscibility of different aqueous phases is an exception rather than the rule.^ Dobry and Boyer-Kawenoki^^ systematically tested 35 pairs of polymers soluble in solvents, only 4 gave homogeneous solutions. The remaining pairs all exhibited phase separations.^ Similar results were obtained for water soluble polymers.^^

LIQUID-LIQUID PARTITIONING

349

A popular aqueous two-phase system is the PEG-dextran-water system. For example,^ a 5 w t % dextran 500 and 3.5% PEG 6000 water solution partitions into two aqueous phases at 20°C. The top phase contains 4.9% PEG, 1.8% dextran, and 93.3% H2O, and the bottom phase 2.6% PEG, 7.3% dextran, and 90.1% H2O. Solutes can have different solubilities in the two phases, thus providing a basis for separation. The high water content in aqueous two-phase systems, typically greater than 70% (w/w), provides a biocompatibility with biomolecules not attained in solvent extraction.^^ Aqueous two-phase systems have been used to separate biomolecules and bioparticles such as proteins including enzymes, peptides, nucleic acids, and viral and cell particles.^'"^ Several thousand research papers have been published on this subject. A comprehensive 49-page bibliography on aqueous two-phase systems was provided by Sutherland and Fisher^^ covering 1956 to 1985. There are also several monographs dealing theories and applications of aqueous two-phase systems. The most prominent monograph, now in its third edition is the one by Albertsson^ entitled "Partition of Cell Particles and Macromolecules." It is a comprehensive and most cited work on aqueous two-phase systems. Albertsson^ presented a systematic mechanistic study of aqueous two-phase partitioning. He also attached more than 50 phase diagrams of PEG-dextran-water, methylcellulose-dextran-water, and polyvinylalcohol-dextran-water systems for practical applications. More recently, Zaslavsky^ authored a monograph entitled "Aqueous Two-Phase Partitioning-Physical Chemistry and Bioanalytical Applications." It contains more than 160 phase diagrams of PEG-dextran-water, PEG-polyvinylmethylether-water, PEG-salt-water, Polyvinylpyrrolidone-dextran-water, polyvinylalcohol-dextran-water, and FicoU-dextran-water systems. Both monographs have sections dealing with separations of biomolecules. Aqueous two-phase systems are routinely used for enzyme purification. Walter and Johansson^ edited a comprehensive book on the subject entitled "Aqueous Two-Phase Systems" as Vol. 228 of the series Methods in Enzymology published by Academic Press. Another very useful book is the one edited by Walter, Brooks, and Fisher^ entitled "Partitioning in Aqueous Two-Phase Systems: Theory, Methods, Uses, and Applications to Biotechnology" which deals with separations of a variety of biomolecules and bioparticles. The most popular aqueous two-phase systems in use today are the PEG-dextran-water system and the PEG-potassium phosphate-water system."^ Both PEG and dextran are fully water soluble, yet the two polymers are incompatible and separate into two aqueous phases in certain concentration ranges. Table 7 is a list of various aqueous two-phase partitioning systems compiled recently by Zaslavsky.^ Figure 15 is a phase diagram of PEG 3400-dextran 500-Water'^ in which the average molecular weight of PEG is 3400 and that of dextran 500 is 500,000. At point A, the system is a homogeneous liquid. Point P in the phase diagram is the critical point at which the compositions of the two liquid phases are identical. Above the phase envelop, the system splits into two separate phases. The PEG-rich phase is the top phase and the dextran-rich phase is the bottom phase. The four straight lines above the phase envelop

350

TINGYUE GU

TABLE 7 Aqueous Two-Phase Systems A. Nonionic polymer-nonionic polymer-w^ater Polypropylene glycol Methoxypolyethylene glycol Polyethylene glycol Polyvinyl alcohol Hydroxypropyldextran Dextran Polyethylene glycol Polyvinyl alcohol Polyvinylpyrrolidone Dextran Arabinogalactan Hydroxypropyl starch Ficoll Polyvinyl alcohol Methylcellulose Hydroxypropyldextran Dextran Polyvinylpyrrolidone Methylcellulose Maltodextrin Dextran Methylcellulose Hydroxypropyldextran Dextran Ethylhydroxyethylcellulose Dextran Hydroxypropyldextran Dextran Ficoll Dextran B. Polymer-low molecular weight component-water Polypropylene glycol Potassium phosphate Glycerol Methoxypolyethylene glycol Potassium phosphate Polyethylene glycol Inorganic salts, e.g., K^, Na"*", Li^, N H 4 , etc. and P04^-,S0|-,etc. Glucose, maltose, cellobiose, iso-maltose, maltotriose, iso-maltotriose, j8-cyclodextrin Polyvinylpyrrolidone Butylcellosolve Potassium phosphate Butylcellosolve Polyvinyl alcohol Butylcellosolve Dextran Propyl alcohol, isopropyl alcohol Sodium chloride (0°C) Na dextran sulfate C. Polyelectrolyte-nonionic polymer-water Na dextran sulfate Polypropylene glycol Methoxypolyethylene glycol NaCl Polyethylene glycol NaCl Polyvinyl alcohol NaCl Polyvinylpyrrolidone NaCl Methylcellulose NaCl Ethylhydroxyethylcellulose NaCl Hydroxypropyldextran NaCl Dextran NaCl Polypropylene glycol NaCl DEAE dextran HCl Polyethylene glycol Li2S04 Polyvinyl alcohol Methylcellulose (Continues)

351

LIQUID-LIQUID PARTITIONING T A B L E 7 (Continued) C. Polyelectrolyte-nonionic polymer-water Casein Dextran Pectin Ficoll Amilopectin Na carboxymethyldextran Methoxypolyethylene glycol NaCl Polyethylene glycol NaCl Polyvinyl alcohol NaCl Polyvinylpyrrolidone NaCl Methylcellulose NaCl Ethylhydroxyethylcellulose NaCl Hydroxypropyldextran NaCl Polypropylene glycol NaCl Na carboxymethylcellulose Methoxypolyethylene glycol NaCl Polyethylene glycol NaCl Polyvinyl alcohol NaCl Polyvinylpyrrolidone NaCl Methylcellulose NaCl Ethylhydroxyethylcellulose NaCl Hydroxypropyldextran NaCl D. Poly electrolyte-poly electrolyte-water Na carboxymethyldextran Na dextran sulfate DEAE dextran HCl NaCl Na carboxymethylcellulose Na carboxymethyldextran Na carboxymethylcellulose Casein Sodium alginate, 0.1 M NaOH Na carboxymethylcellulose, 0.1 M NaOH Ovalbumin (pH 6.6) Soybean globulins Ovalbumin thermotropic aggregates Casein Source: Reprinted from Zaslavsky^ by courtesy of Marcel Dekker, Inc.

"o o 2^ o c 0)

o CL 10

15

20

25

Dextran %(w/w) FIGURE 15 PEG 3400-dextran 500-water phase diagram at 4°C. (Reprinted with permission from Diamond and Hsu.'* Copyright 1992 Springer Verlag.)

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are tie lines just like those in phase diagrams for solvent extraction. If the solution is mixed with compositions at point B, two phases will result at equilibrium. The top phase has PEG and dextran concentrations at point C and the bottom phase at point D. Like in other phase diagrams, the lever rule apphes. The ratio of the weight of the top phase (point C) to that of the bottom phase (point D) is equal to the ratio of the distance between B and D to that between C and E. The distances can be measured either using the points' X axis readings or y axis readings. A complete experimental procedure on how to obtain the binodial curve, the tie lines and the critical point was described by Albertsson.^ The procedure is based on observing turbidity change, while adding a polymer solution to another polymer solution dropwise. The partition coefficient K for a solute in aqueous two-phase partitioning is defined as

K = eye,

(8)

in which C^ and C^ are concentrations of the solute in the top and bottom phases, respectively.^ The partition coefficients for proteins generally fall within the range of 0.1 to 10. For large molecules (such as high molecular weight DNA and RNA) and particles (such as cells and viral particles), partition coefficients > 100 to < 0.01 are observed.^'^ Small ions tend to partition equally between the two phases. According to Albertsson,^ the mechanism for aqueous liquid-liquid partitioning is complicated and largely unknown. Hydrogen, ionic, hydrophobic, and other weak forces may be involved. The following equation was proposed by Albertsson^ to describe the various influences on partition coefficient K: In X = In X« + In X,i + In K^fob + In K^osep + In Ks.e + In i^conf (9) in which subscripts el, hfob, biosp, size, and conf denote the electrochemical, hydrophobic, biospecific, size, and conformational contributions to the partition coefficient and K^ lumps all other factors. The Bronsted^^ equation may be used to describe the partition coefficient K qualitatively [5]: lnK = \M/(kT)

(10)

in which M is the molecular weight of the solute, k is the Boltzmann constant, T is the absolute temperature, and A is a parameter characteristic of the aqueous two-phase system and its interaction with the solute. Among the many factors that affect the partitioning of biomolecules and bioparticles, there are the polymers and their molecular weights and salts used in the system, concentrations of the polymers and salts, ionic strength, pH, and temperature."^'^'"^^ A. Effect of Polymer Molecular Weight and Concentration Albertsson"^^ found that the molecular weight of the polymers strongly affects the partitioning of a protein in PEG-dextran-water systems. If it is desirable

LIQUID-LIQUID PARTITIONING

^ H

353

T A B L E 8 Effect of Molecular W e i g h t of Dextran on Partition Coefficient of Proteins with Different Molecular Weights^ Dextran Protein Cytochrome Ovalbumin Bovine serum albumin Lactic dehydrogenase Catalase Phycoerythrin P-galactosidase Phosphofructokinase Ribulose diphosphate carboxylase

Molecular weight 12,384 45,000 69,000 140,000 250,000 290,000 540,000 800,000 800,000

Dex40 0.18 0.58 0.18 0.06 0.11 1.9 0.24 <0.01 0.05

Dex70

Dex 220

Dex 500

Dex 2000

0.14 0.69 0.23 0.05 0.23 2.9 0.38 0.01 0.06

0.15 0.74 0.31 0.09 0.40

0.17 0.78 0.34 0.16 0.79 12 1.59 0.02 0.28

0.21 0.86 0.41 0.10 1.15 42 1.61 0.03 0.50

— 1.38 0.01 0.15

Source: Reprinted with permission from Albertsson.^ Copyright 1986 John Wiley & Sons, Inc. In phase systems with 6% ( w / w ) PEG 6000 and 8% dextran having different molecular weights; 10 mM sodium phosphate at pH 6.8.

to have a higher partition coefficient, lowering the average PEG molecular w^eight may help/ The effect of polymer molecular weight depends on molecular weight of the solute as demonstrated by Tables 8 and 9.^ Higher molecular weights and higher concentrations for the polymers usually bring higher viscosities to the liquid solutions. To provide a discrimination basis, the polymer concentrations in the two phases should differ sufficiently. As expected, the larger the difference between the polymer concentrations of PEG in the two phases, the better the partition coefficient (i.e., deviating farther away from unity) as demonstrated in Fig. 16.^ Several factors influence the difference including starting polymer concentrations, temperature, etc. In PEG-dextran-water systems, increased PEG concentration will result

T A B L E 9 Effect of PEG Molecular W e i g h t on the Partition Coefficient of Proteins with Different Molecular Weights^

Protein

Molecular weight

12,384 Cytochrome c Ovalbumin 45,000 BSA 69,000 Lactic dehydrogenase 140,000 Catalase 250,000

Dex 500 (9%), PEG 4000 (7.1%) 0.17 1.25 0.52 0.13 0.82

Dex 5000, PEG 600

Dex 500, PEG 20,000

Dex 500, PEG 40,000

0.17 0.85 0.34 0.08 0.38

0.13 0.50 0.14 0.05 0.16

0.12 0.50 0.11 0.03 0.10

Source: Reprinted with permission from Albertsson.^ Copyright 1986 John Wiley & Sons, Inc. Phase systems contain 8% ( w / w ) dextran and 6% PEG unless marked otherwise; phosphate, 10 mM sodium phosphate at pH 6.8.

354

TINGYUE GU

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APEG. %wt. F I G U R E 16 Effect of PEG concentration difference in the two phases on partition coefficient K in PEG 6000-dextran 7 0 - w a t e r system containing 0.15 mol/l
in compositions of phases deviate from the critical point where the two phases have identical concentrations.^ Albertsson"^^ found the following fair relationship for proteins: -logioi^ = ^M^/^

(11)

in which a is a constant depending on the concentration of polymers and M is the molecular weight of the protein. In PEG-dextran-water and PEGmethylcellulose-water systems, most proteins prefer the lower (dextran-rich or methylcellulose-rich) phase.^' ^"^ B. Effect of Temperature Temperature affects the shape of phase diagrams. When temperature is decreased, phase separation occurs at lower polymer concentrations for PEG-dextran-water systems, meaning less PEG and dextran are needed to achieve phase separation. The opposite is true for PEG-salt-water systems.^^ Temperature also changes the partitioning of biomolecules. Table 10 shows the partition coefficients of lysozyme and catalase and their ratios in different PEG-dextran-water systems at different temperatures.^ It seems that a lower temperature tends to provide a better separation of the two proteins. Kula^ also pointed out that K values are usually higher at a lower temperature. This trend is consistent with the aforementioned Bronsted equation. Using a lower temperature will cause the viscosity to be higher. The use of hy-

3i>i>

LIQUID-LIQUID PARTITIONING

T A B L E 10 Partition Coefficient of Lysozyme and Catalase in Four Aqueous Two-Phase Systems

TCC)

•^lysozyme

'^catalase

Separation factor

System composition

4.0 25.0 40.0

0.54 0.44 0.85

0.046 0.063 0.174

11.7 7.0 4.9

12.2% ( w / w ) Dex-10 8.4% PEG-4000

4.0 25.0 40.0 4.0 25.0 40.0

0.35 0.31 0.53 0.65 0.47 0.98 0.58 0.36 0.72

25 19.4 26.5 3.0 2.2 2.3 11.2 8.6 10.6

10.0% Dex-10 5.6% PEG-20,000

4.0 25.0 40.0

0.014 0.016 0.020 0.22 0.21 0.43 0.052 0.042 0.068

11.3-11.5% Dex-500 7.9% PEG-4000 10.3% Dex-500 7.65% PEG-20,000

Source: Reprinted from Zaslavsky^ by courtesy of Marcel Dekker, Inc.

drophilic polymers enhances enzyme stability so that room temperature can be used with minimal bioactivity losses. This means chilling aqueous twophase systems is usually not required/ unless very fragile proteins are involved. Grimonprez and Johansson^^ achieved enhanced bioactivity for some enzymes, especially phosphofructokinase, from baker's yeast partitioned at subzero temperature with the addition of ethylene glycol in PEG-dextran-water systems.

C. Effect of Salt Salts at moderate concentrations have only marginal effects on the phase diagram of nonionic polymer-polymer-water systems. However, systems containing polyelectrolytes, such as DEAE-dextran-water systems, are strongly affected. Usually a much lower polymer concentration is required for phase separation when the salt concentration increases.^^ Salt can be used rather effectively to change the partition coefficient of biomolecules. At low salt concentrations (0.1 to 0.2 M), the effects of salt type and concentration can be dramatic for proteins at pH far away from their isoelectric points."^ As a rule of thumb, the decrease of partition coefficient for negatively charged proteins in PEG-dextran-water systems is sulfate > floride > acetate > chloride > bromide > iodide and lithium > ammonium > sodium > potassium. Positively charged proteins follow the opposite trend."^'^"^ Albertsson^ reported that increasing NaCl concentration in the range of 0 to 5 M greatly increased the partition coefficient of several proteins (phycocyanin, phycoerythrin, gamma globulin, ceruloplasmin, and serum albumin) in a phase system containing 4.4% PEG 8000 and 7% dextran at pH 6.8. Salt has little effect on proteins close to their isoelectric point.

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T A B L E 11 Affinity Partitioning of Biomolecules in Aqueous Two-Phase Systenns Biomolecule

Affinity ligand

Trypsin Serum albumin /3-Lactoglobulin S-23 myeloma protein Histones 3-Oxosteroid isomerase Formaldehyde dehydrogenase Formate dehydrogenase Colipase Myosin Phosphofructokinase Interferon Pyruvate kinase Glutamate dehydrogenase Glycerol kinase Flexokinase Lactate dehydrogenase Malate dehydrogenase Transaminase a-Fetoprotein Pre-albumin Glucose-6-phosphate dehydrogenase

Diamino-a, w-diphenyl carbamyl-trypsin inhibitor Fatty acid Fatty acid Dinitrophenyl Fatty acid Estradiol NADH NADH/procion red Lecithin Fatty acid Triazine dye Phosphate Triazine dye Triazine dye Triazine dye Triazine dye Triazine dye Triazine dye Triazine dye Triazine dye Remazol yellow Triazine dye-triazine dye and charged groups (DEAF, sulfate) Triazine dye

Glyceraldehyde phosphate dehydrogenase 3-Phosphoglycerate kinase Alcohol dehydrogenase Nitrate reductase Acid proteases Thaumatin IgG Human hemoglobin Vancomycin Cytochromes Myoglobins Hemoglobins Q;2-Macroglobulin Tissue plasminogen activator Superoxide dismutase Monoclonal antibodies Membranes from calf brain synaptosomes Albumins Thylakoid membranes

Triazine dye-ATP Triazine dye Triazine dye Pepstatin Glutathione Protein A CudDlDA D-ala-ala-ala CudDlDA CudDlDA CudDlDA Metal-IDA Metal-IDA Metal-IDA Metal-IDA Procion yellow^ HE-3 Alcohols Alcohols

Source: Reprinted with permission from Diamond and Hsu."^ Copyright 1992 Springer Verlag.

LIQUID-LIQUID PARTITIONING

357

D. Affinity Partitioning Addition of affinity ligands to an aqueous two-phase partitioning system can greatly enhance the partitioning of biomolecules. The biospecific binding of the biomolecule with the Hgand preferentially move the biomolecule to the desired phase. To do so, it is believed that the ligand must be covalently coupled to the target phase polymer.^' ^ Affinity ligands can, in some cases, even reverse the partitioning behavior of certain proteins.^ Compared to affinity chromatography, affinity binding does not require an expensive stationary phase and there are no problems such as loss of ligands on the stationary phase, nonspecific binding which are commonly seen in affinity chromatography media. Stability of biomolecules is usually increased in an aqueous two-phase system. The drawbacks of adding ligands include the added cost of ligands and coupling of the ligands to the polymers. The two commonly used types of ligand are fatty acids and triazine.^ Metallated iminodiacetic acid (IDA) derivatives of PEG including Cu(II)IDAPEG were also used for binding with proteins rich in surface histidines. Table 11 is a fist of biomolecules purified using affinity ligands in aqueous two-phase systems."^ The partitioning of penicillin acylase from Escherichia coli^ human hemoglobin, myoglobin, cytochrome c, monoclonal antibodies, horseradish peroxidase, porcine lactate dehydrogenase isoenzyme 5, phosphofructokinase from rate erythrocyte haemolysate, isoenzymes of lactate dehydrogenase from rabbit tissues, human alkaline phosphatase isoenzymes, and glucose-6-phosphate dehydrogenase from yeast extract were reviewed by Zaslavsky.^ Various polymer-ligands used in aqueous two-phase affinity partitioning were reviewed by Harris and Yalpani.^"" Kopperschlager^^ reviewed the pH, temperature, and competition effects in affinity partitioning for the separation of various enzymes using dye ligands. E. Large-Scale Aqueous Two-Phase Partitioning of Biomolecules Aqueous two-phase partitioning has been used widely in bioseparations especially separations of proteins. It is an attractive addition or alternative to other bioseparation methods."^'^^ Table 12 is a list of biomolecules purified at large-scale using aqueous two-phase systems."^ PEG-dextran-water and PEG-salt-water are most commonly used in large-scale applications because they possess a general applicability, relatively suitable viscosity, and density difference and they are nontoxic and biodegradable and are certified in the pharmacopoeias of most countries."^ The cost of purified dextran is very high (in the lower hundreds of dollars per kilogram). Crude dextran or hydrolyzed crude dextran, and more recently, hydroxypropyl starch have been used to cut cost."^^ Crude dextran causes a rather high viscosity in water. Hydrolyzed dextran reduces the viscosity.^ F. Equipment and Operational Considerations The operational modes for aqueous two-phase partitioning are similar to those used in solvent extraction. Single-stage partitioning, repeated batch

358

TINGYUE GU m H

T A B L E 12 A List of Biomolecules Purified at Large-Scale Using Aqueous Two-Phase Systems Acyl aryl amidase Alcohol Dehydrogenase a-Amylase Aspartase Aspartate /3-decarboxylase Chlorophyll a/b-protein (LHPC) Chromatophores Formaldehyde dehydrogenase Formate dehydrogenase Furnarase j8-Galactosidase Glucose dehydrogenase Glucose isomerase Glucose-6-phosphate dehydrogenase a-Glucosidase Hexakinase D-2-hydroxyisocarproate dehydrogenase L-2-hydroxyisocarproate dehydrogenase Interferon Isoleucyl-tRNA synthetase Isopropanol dehydrogenase D-Lactate dehydrogenase Leucine dehydrogenase NADkinase Pencillin acylase Phosphofructokinase Phospholipase Phosphorylase PuUulanase Staphylococcal protein A-)3-galactosidase hybrid Whey proteins Source; Reprinted with permission from Diamond and Hsu."^ Copyright 1992 Springer Verlag.

extraction, continuous countercurrent extraction using extractors-settlers in series and liquid-liquid column extraction can be used.^ The same equipment used in solvent extraction may be used for aqueous two-phase partitioning. The use of large extraction equipment for PEG-dextran-water systems is limited due to low feed rates owing to the longer time required for partitioning. Phase separation is easier in PEG-salt-water systems because relatively large bubbles are generated during mixing. They disappear faster than tiny bubbles. The relatively large density difference between the two aqueous phases and lower viscosity of the salt phase are also favorable factors."*^ Bamberger et al7^ discussed details of laboratory preparation of phase systems and measurement of their physiochemical properties. The time needed for phase separation depends on polymer type and concentration, the volume ratio, the rate of coalescence of droplets, and the presence of particles. Typical times required for PEG-dextran-water systems are 5 to 30 min. Low-speed centrifugation cuts the time down to 1 min.^^ In

359

LIQUID-LIQUID PARTITIONING

acceleration field

F I G U R E 17 Section of a centrifugal countercurrent distribution apparatus with several test tubes shown: a, the lower phase; b, the upper phase; c, the outer ring with cavities for the lower phase; d, the inner ring with cavities for the upper phase; e, the lid ring; and f, an O-ring for sealing. (Reprinted with permission from Albertsson.^ Copyright 1986 John Wiley & Sons, Inc.)

many cases, a single-stage partitioning does not provide an adequate separation. As in solvent extraction, multistage aqueous two-phase partitioning is used. Figure 17 is an anatomy of a centrifugal countercurrent distribution apparatus designed for such an application.^ The separation results resemble elution chromatography. Figure 18 shows the partitioning results from the apparatus for the partitioning of enolase from baker's yeast.^ Enolase is known to have three isoenzymes. Two peaks are shown in the figure. There are several ways in which polymers can be removed after aqueous two-phase partitions. If the desired protein is partitioned to the salt phase in a PEG-salt-water system, the salt can be removed easily using dialysis with an

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r e s o l u t i o n o f C C D [ c o u n t e r c u r r e n t d i s t r i b u t i o n ] curves. Acta Chem. Scand. B 2 8 , 5 6 - 6 0 , 1974.)

360

TINGYUE GU Stripping

Loading Top PEG-rich phase

Disrupted cells PEG4phosphate

Ultrafiltration

Products

>

(Cell debris, contaminants) phosphate-rich phase F I G U R E 19 Illustration of a two-stage aqueous two-phase partitioning process with integrated phase recycling. (From Huddleston and Lyddiatt.^' Copyright John Wiley & Sons Limited. Reproduced with permission.)

ultrafiltration membrane. If the protein ends up in the PEG phase, ultrafiltration can be used to remove PEG, which typically has a relatively small molecular v^eight of a few thousands. Another approach is to add a salt to partition the protein again into a new salt phase.^'"^^ Figure 19 illustrates a process with integrated phase recycling.^^ The majority of the target protein (denoted by stars in the figure) in a disrupted broth is first partitioned to the top PEG-rich phase. This phase is then contacted with a fresh bottom phase first in the second extractor in the stripping extractor. Most of the protein ends up in the bottom phase, which then undergoes an ultrafiltration step to recover the protein. Precipitation of the protein using the salting out method or solvent is also an option. Size exclusion chromatography should be an effective method to separation PEG from the protein. However, it is more expensive and time-consuming. Ion exchange for the PEG protein solution suffers from a high-pressure drop due to the high viscosity of the solution. lY. SUMMARY Liquid-liquid partitioning methods including solvent extraction and aqueous two-phase partitioning are very useful tools for bioseparations. This chapter provided the methodology with various examples and principles as guidelines for the liquid-liquid partitioning in a practical application. Although the engineering aspects of equipment design and operation for liquid-liquid partitioning in industrial applications are firmly established owing to the long history of solvent extraction, the science of predicting partition coefficient accurately in solvent extraction and aqueous hquid-hquid partitioning is not fully developed. Trial and error is still a needed approach in many applications to locate an optimal partitioning system and its operating conditions. With rapid advances in biotechnology and a growing emphasis on products

LIQUID-LIQUID PARTITIONING

361

from renewable resources, the need for efficient downstream processing is increasing. Liquid-liquid partitioning methods will become more and more popular because of their low cost, versatility, and facility of large-scale continuous operations. They can be used in research laboratories or in industry as part of an integrated downstream process for the recovery and purification of biomolecules. Whole broth extraction can even be carried out to cut down the number of stages in downstream processing for a higher overall process yield.

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46. Rahaman, R. S., Chee, J., Cabral, J., and Hatton, T. A. (1988). Recovery of an extracellular alkaline protease from whole fermentation broth using reversed micelles. Biotechnol. Prog. 4, 218-224. 47. Pires, M. J., Aires-Barros, M. R., and Cabral, J. M. S. (1996). Liquid-liquid extraction of proteins w^ith reversed micelles. Biotechnol. Prog. 12, 2 9 0 - 3 0 1 . 48. Albertsson, P.-A. (1958). Partition of proteins in liquid polymer-polymer two-phase systems. Nature (London) 182, 709-711. 49. Goklen, K. E., and Hatton, T. A. (1987). Liquid-liquid extraction of low molecular-weight proteins by selective solubilization in reversed micelles. Sep. Set. Technol. 22, 831-841. 50. Hu, Z., and Gulari, E. (1996). Extraction of aminoglycoside antibiotics with reversed micelles. / . Chem. Technol. Biotechnol. 65, 4 5 - 4 8 . 51. Giovenco, S., Laane, C., and Hilhorst R. (1987). Recovery of intracellular enzymes from bacterial cells using reverse micelles. Proc. Eur. Congr. Biotechnol. 4th, Amsterdam, Vol. 2, p. 503. 52. Weatherley, L. R., Campbell, L, Slaughter, J. C , and Sutherland, K. M. (1987). Electrically enhanced solvent extraction of biochemicals. In "Separations for Biotechnology" (M. S. Verrrall and M. J. Hudson, eds.), pp. 32-332. Ellis Horwood, Chichester. 53. Baird, M. H. I., Rao, N. V. R., Prochazka, J., and Sovova, H. (1994). Reciprocating-plate columns. In "Liquid-Liquid Extraction Equipment" (J. C. Godfrey and M. J. Slater, eds.), pp. 307-362. Wiley, New York. 54. Treybal, R. E. (1980). "Mass-Transfer Operations." McGraw-Hill, New York. 55. King, C. J. (1980). "Separation Processes," 2nd ed. McGraw-Hill, New York. 56. McCabe, W. L., Smith, J. C , and Harriott, P. (1993). "Unit Operations of Chemical Engineering," 5th ed. McGraw-Hill, New York. 57. Wankat, P. C. (1988). "EquiHbrium Stages Separations." Elsevier, New York. 58. Pratt, H. R. C , and Stevens, G. W. (1992). Selection, design, pilot-testing, and scale-up of extraction equipment. In "Science and Practice of Liquid-Liquid Extraction" (J. D. Thornton, ed.), Vol. 1, pp. 492-589. Oxford University Press, New York. 59. Dobry, A., and Boyer-Kawenoki, F. (1947). Phase separation in polymer solutions. / . Polym. Sci. 2, 90-100. 60. Dobry, A. (1948). Sur I'incompatibilite des macromolecules en solution aqueuse. Bull. Soc. Chim. Belg. 57, 280-285. 61. Huddleston, J. G., and Lyddiatt, A. (1996). Two-phase aqueous systems for the recovery of intracellular microbial products. In "Downstream Processing of Natural Products" (M. S. Verrall, ed.), pp. 53-69. Wiley, New York. 62. Sutherland, L A., and Fisher, D. (1985). Partitioning: A comprehensive bibliography. In "Partitioning in Aqueous Two-Phase Systems: Theory, Methods, Uses, and Applications to Biotechnology" (H. Walter, D. E. Brooks, and D. Fisher, eds.), pp. 627-676. Academic Press, Orlando, FL. 63. Bronsted, J. N. (1931). Molecular magnitude and phase distribution. Z. Phys. Chem., Abt. A (Bodenstein Festband), pp. 257-266. 64. Johansson, G. (1985). Partitioning of proteins. In "Partitioning in Aqueous Two-Phase Systems: Theory, Methods, Uses, and Applications to Biotechnology" (H. Walter, D. E. Brooks, and D. Fisher, eds.), pp. 161-226. Academic Press, New York. 65. Albertsson, P.-A., and Tjerneld, F. (1994). Phase diagram. In "Methods in Enzymology" (H. Walter and G. Johansson, eds.). Vol. 228, pp. 3 - 1 3 . Academic Press, San Diego, CA. 66. Grimonprez, B., and Johansson, G. (1995). Liquid-liquid partitioning of some enzymes, especially phosphofructokinase, from Saccharomyces cerevisiae at sub-zero temperature. / . Chromatogr. 680, 5 5 - 6 3 . 67. Harris, J. M., and Yalpani, M. (1985). Polymer-ligands used in affinity partitioning and their synthesis. In "Partitioning in Aqueous Two-Phase Systems: Theory, Methods, Uses, and Applications to Biotechnology" (H. Walter, D. E. Brooks, and D. Fisher, eds.), pp. 589-626. Academic Press, New York. 68. Kopperschlager, G. (1994). Affinity extraction with dye ligands. In "Methods in Enzymology" (H. Walter and G. Johansson, eds.). Vol. 228, pp. 313. Academic Press, San Diego, CA.

364

TINGYUE GU 69. Hustedt, H., Kroner, K. H., and Kula, M.-R. (1985). Applications of phase partitioning in biotechnology. In "Partitioning in Aqueous Two-Phase Systems. Theory, Methods, Uses, and AppHcations to Biotechnology" (H. Walter, D. E. Brooks, and D. Fisher, eds.), pp. 529-587. Academic Press, New York. 70. Bamberger, S., Brooks, D. E., Sharp, K. A., van Alstine, J. M., and Webber, T. J. (1985). Preparation of phase systems and measurement of their physiochemical properties. In "Partitioning in Aqueous Two-Phase Systems: Theory, Methods, Uses, and Applications to Biotechnology" (H. Walter, D. E. Brooks, and D. Fisher, eds.), pp. 589-626. Academic Press, New York.

SEPARATION OF NUCLEIC ACIDS AND PROTEINS ROHIT HARVE Wyeth Ayerst Research, Marietta, Pennsylvania 17547

RAKESH BAJPAi Department of Chemical Engineering, Department of Biological and Agricultural Engineering, University of Missouri-Columbia, Columbia, Missouri 65211

I. II. III. IV. V.

INTRODUCTION PRECIPITATION OF NUCLEIC ACIDS NUCLEASE TREATMENT AQUEOUS TWO-PHASE EXTRACTION A CASE STUDY A. Precipitation by Streptomycin Sulfate, Protamine Sulfate, and Manganous Sulfate from Cell Homogenate B. Removal of Nucleic Acids from Cell Homogenate Using Aqueous Two-Phase Extraction VI. CONCLUSIONS REFERENCES

INTRODUCTION

A large number of biological molecules are extracted from naturally occurring plant and animal resources. Advances in biotechnology over the past several decades enable production of many desired compounds under controlled conditions using engineered and unengineered microorganisms and cells (animal, plant, and insect). Even v^hole plants and animals are being engineered for specialized properties. Recovery of the desired products from these sources involves sequence of operations vv^hose final aim is to obtain the desired product at a prespecified level of purity. Another goal is to achieve high yield (recovery) of the product, thus improving the utilization of resources and reducing production cost(s). The various steps in recovery of biological products from their natural environment have been divided into four categories.^ These are separation of solubles from insolubles, isolation, purification, and polishing. Separation of solubles from insolubles is a common chemical engineering operation and Separation Science and Technology, Volume 2 Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

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ROHIT HARVE AND RAKESH BAJPAI

extensive discussions of this operation exist in literature.^ Polishing is a product- and application-specific step. Another step, grinding a n d / o r disruption to release the product(s) from organs-cells-organelles, is also commonly encountered depending on the source of the desired compounds. Harve^ and Sadana"* have reviewed this operation. Sadana"^ has also reviev^ed the complications that occur during the isolation and purification steps. The selection and design^ of the sequence of isolation and purification operations is done either heuristically^'^ or by using an expert system.^'^ Eventually, economics controls the final purification sequence.^^ Isolation, steps that involve getting a small volume of concentrate from a large volume of fluids,^ is conducted first. Isolation operations often employ relatively low resolution methods, such as precipitation, crude adsorption, etc., characterized by low cost per volume processed. Isolation is followed by purification, which involves methods with successively higher resolution, such as ion-exchange, affinity, and gel chromatography. In either case, an understanding of the physical-chemical nature of the constituents and their interactions is necessary for informed selection and design of the individual operations.^^ Recent advances in such understanding has blurred the boundaries of isolation and purification operations'^ by increasing specificity of separation in complex matrices. Differences between characteristics of the desired product and the impurities can be used for suggesting successive steps in the isolation and purification scheme.^ A classification of the different separation methods by the underlying physical-chemical basis is presented in Table 1. A number of books and review articles have described the different techniques listed in Table 1. Many recent publications have furthered the state of the art concerning these technologies.

^ ^ H T A B L E I Classification of Separation Processes on the Basis of Physical - Chemical Characteristics Property Broad category

Density

Binding

Ionic charge

Centrifugationsedimentation

Isopycnic bonding

Membrane chromatography

Electrodialysis

Electrokinetics

Dead-end and crossflow filtration; reverse osmosis Electrophoresis

Chromatography

Gel permeation

Affinity chromatography

Precipitation Extraction

Membrane-based separations

Solubility

Size Decantation; settling; gradient centrifugation

Salting out Liquid-liquid extraction; aqueous biphasic extraction

Isoelectric focusing Ion exchange ion exclusion

367

SEPARATION OF NUCLEIC ACIDS AND PROTEINS

This chapter will focus on a unique problem encountered during recovery of intracellulary produced proteins. Disruption of cells produces a mixture of nucleic acids and proteins in the solution from which the desired proteins must be fractionated. Typical separation schemes involve first the removal of nucleic acids from solution by precipitation. The desired protein is then isolated and purified from the mixture of remaining nucleic acids and proteins. A scheme for recovery of intracellular bacterial enzyme tartrate dehydrogenase from cell paste is shown in Fig. 1. Material balance at the different stages of the scheme in two different experiments showed that 53-60% loss in enzyme activity took place during precipitation of nucleic acids by protamine sulfate and during ammonium sulfate fractionation of proteins (Table 2). Reduction in volume, removal of major nonprotein

Cdl Paste

NudeicAdd Removal

Cell Disruption

^

Protease Inhibitors

L^

Buffer

EDTA

Heat Treatment & Ammonium Sulfete precq>itation

<

NaCl Gradient Ultrafiltration

^

• L

1 ^ r ^r

y

Centrifii

F P L C I O N E X

c H A N G E

^ ^

1

1r Dialysis

T FINAL PRODUCT FIGURE (TDH).

I

Flow diagram for recovery of intracellular bacterial enzyme tartrate dehydrogenase

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ROHIT HARVE AND RAKESH BAJPAI

^ ^ H T A B L E 2 Material Balance at Different Stages of Recovery of Tartrate Dehydrogenase from Cell Paste

Sample (after)

Volume (mL)

Runl Cell disruption 60 Protamine sulfate 75 Ammonium sulfate (40%) 64 12 Ammonium sulfate (70%) Dialysis 12 Anion exchange 120 Ultrafiltration 7 Gel filtration 54 Ultrafiltration (final sample) 0.9 Run 2 Cell disruption 80 Protamine sulfate 93 Ammonium sulfate (40%) 85 Ammonium sulfate (70%) 18 Dialysis 18 Anion exchange 105 Ultrafiltration 8 27 Gel filtration Ultrafiltration (final sample) 1.3

TDH(U/mL) 0.67 0.42 0.14 0.38 1.27

+ 0.13 + 0.38 ± 0.01 + 0.008 + 0.20 NA 2.16 ± 0.08 0.20 + 0.005 7.62 + 0.55 0.89 0.53 0.28 0.63 1.88 0.303 3.49 0.85 15.3

± ± + ± + + ± ± +

0.034 0.022 0.025 0.019 0.07 0.16 0.08 0.063 0.25

Protein (mg/mL)

Specific activity (U/mg protein)

22.98 19.85 11.78 53.57 28.79 NA 4.92 0.44 16.15 35.13 31.18 18.40 77.7 72.36 0.58 5.97 0.82 10.30

Yield

(%)

Fold purification

0.029 0.021 0.013 0.007 0.044 NA 0.438 0.458 0.472

100 78 24 11 38 NA 38 29 17

1.00 0.72 0.43 0.24 1.51 NA 15.0 15.7 16.2

0.025 0.017 0.015 0.008 0.003 0.52 0.59 1.041 1.481

100 70 33 16 47 45 39 32 28

1.00 0.67 0.59 0.32 1.02 20.5 23.1 41.1 58.3

impurities, and little increase in fold purification of the desired enzyme characterize these steps. Accordingly, these steps represent the classical isolation steps.^ Separation of proteins has been reviewed in the following. Recent literature dealing with this subject is a rarity, although techniques for purification of proteins continue to be a subject of continued interest.^^"^^ Results of selected methods in the context of recovery of TDH from cell paste are also presented.

II. PRECIPITATION OF NUCLEIC ACIDS In systems containing proteins (as desired biomolecules) and nucleic acids (contaminant), the goal is to remove the contaminant while maintaining the biological activity of the protein. The nucleic acids can be precipitated by exposure to nucleases,^^ shear,^^'^^ low ionic strength,^^ and high pH.^"^ However, these techniques (with the exception of nucleases) affect proteins as well and are not very selective. Since nucleic acids possess negatively charged phosphate residues, precipitating agents with positively charged groups have potential for selective removal of nucleic acids from solution. This has been the principle behind search for several precipitating agents for nucleic acids. Jones^^ described the isolation of bacterial nucleic acids from Mycobacterium tuberculosis, My, phlei, and Sarcina lutea by precipitation with the

SEPARATION OF NUCLEIC ACIDS AND PROTEINS

369

cationic detergent cetyltrimethyl ammonium bromide (cetavlon). Guerritore and Ballelli^^ showed that the precipitation of RNA from aqueous solution was a function of ratio of nucleic acid-cetavlon, pH, and salt concentration. Sodium chloride, sodium sulfate, and sodium citrate all interfered with precipitation at concentrations above 0.2 M, while glycine, glucose, and urea had no effect. Atkinson and Jack^^ effectively precipitated nucleic acids from cell extracts using polyethyleneimine (a long chain cationic polymer with a molecular weight of '-'24,000). Nucleic acids and cell debris can be removed simultaneously by adding polyethyleneimine to a cell extract following cell disruption. By adding 0.294% polyethyleneimine, 89% RNA, and 96% DNA was precipitated at 4°C from a synthetic DNA-RNA mixture in 0.2 M NaCl at a pH of 7.0. Bingham et al?^ did selective purification of restriction endonuclease EcoRI using polyethyleneimine. Depolymerization of nucleic acids during preparation of cell extracts adversely affects their precipitation by polyethyleneimine.^^ Melling and Atkinson^^ also reported that only 85% of nucleic acids were removed when extracts were produced under conditions of high shear (these conditions result in depolymerization of nucleic acids) compared to 95% removal when extracts were prepared by nonshear methods. Other precipitants for nucleic acids are streptomycin sulfate,^^ protamine sulfate, and manganese chloride.^^ Presence of salts interferes with precipitation of nucleic acids.^^'^^ As a result, Oxenburgh and Snoswell^^ found it necessary to dialyze ammonium sulfate extracts before adding streptomycin sulfate to precipitate nucleic acids. In presence of proteins, these too precipitate when the precipitant is added. The extent of protein precipitation is dependent on the pH of the solution and on the ratio of precipitant to protein. Oxenburgh and SnoswelP^ reported only 24% loss of protein from an extract of Lactobacillus plantarum containing 10 m g / m L protein, pH 7.0, conductivity 0.38 mS, and a streptomycin sulfate-protein ratio of 1.0. Use of streptomycin sulfate compared favorably with other precipitants and was more reproducible. Protamine sulfate and manganese chloride often proved difficult to reproduce.^"* Higgins et al?^ used manganous sulfate for precipitation of nucleic acids in continuous production of j8-galactosidase from Escherichia coli. The precipitation was fast (most of the nucleic acid was precipitated in 10 min of contact time). At 0.05 M manganous sulfate, only a small amount of nucleic acid precipitated. Precipitation was improved at 0.1 and 0.5 M concentrations of the precipitant, but as much as 70% enzyme activity was also lost under these conditions. With an extract of £. co//, the effectiveness in precipitating nucleic acids decreased in the order polylysine > polyethyleneimine > cetavlon > streptomycin sulfate > protamine sulfate > MnCl2 > spermine. With MnCl2, precipitation was relatively inefficient and as much as 75% nucleic acids remained in solution. Extracts of Micrococcus lysodiekticus treated with protamine sulfate showed a loss of catalase activity and ineffective removal of RNA. In contract, treatment of a similar extract with polyethyleneimine resulted in 90% precipitation of nucleic acids and 70% recovery of catalase

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ROHIT HARVE AND RAKESH BAJPAI

and oxaloacetate carboxylase. However, some enzymes (such as dihydrofolate reductase from L. casei) form complex with polyethyleneimine. Excessive enzyme loss is to be expected when the proteins show a high affinity with nucleic acids (triosephosphate isomerase from Bacillus stearothermophilus^^). A major drawback in use of polyethyleneimine is its suspected carcinogenic nature. Streptomycin sulfate is also toxic^^ and its long-term use may result in development of resistant bacterial strains in the environment. III. NUCLEASE TREATMENT Bacterial nucleic acids were hydrolyzed using bovine pancreatic ribonuclease and deoxyribonuclease.^^ Deoxyribonuclease treatment of disrupted bacterial suspensions has also been reported.^^ Melling and Atkinson^^ investigated nuclease treatment as a method for the removal of nucleic acids from bacterial suspensions. Two strains of E. coli were used and for both the strains the nuclease treatment was effective in depolymerizing nucleic acids and, hence, in recovery of supernatant after centrifugation to remove cell debris from disrupted cells. The nucleotide content in the supernatant was found to be 15-20% of total proteins and nucleic acids. The nucleotide content in the supernatant was reduced to a very low level by ammonium sulfate precipitation followed by dialysis of the redissolved precipitate. As stated earlier, direct precipitation of nucleotides resulted in significant residual nucleotides in the proteins. For effective removal of nucleic acids, both ribonuclease and deoxyribonuclease need to be used.^^ The cost of nucleases is a major hindrance in use of this method in removal of nucleic acids from mixtures during protein purification. An organism's own nucleases may provide an often-unrecognized nuclease treatment. lY. AQUEOUS TWO-PHASE EXTRACTION An aqueous two-phase (ATP) system is formed when a water-soluble polymer becomes incompatible with other polymer or salts present in the solution.^^ Mixing polyethylene glycol with dextran or with salts such as ammonium sulfate or potassium phosphate in appropriate concentrations can create these systems. Cells, organelles, and macromolecules partition differentially between the two phases thus formed."^^""^^ Partitioning of macromolecules between the two phases depends on the concentration and molecular weights of the polymer(s), the molecular weight and nature of the partitioning macromolecule, temperature, pH, ionic strength, and the presence of polyvalent salts in the mixture."^^ Carlson"^"^ has reviewed factors influencing the use of ATP extraction for purification of proteins. ATP extraction has been used for the separation and large-scale purification of enzymes."^^'"*^ Proteins and nucleic acids partition between the two phases differently due to differences in their surface charges and hydrophobic-hydrophilic domains. Proteins generally prefer the aqueous phase rich in

SEPARATION OF NUCLEIC ACIDS AND PROTEINS

371

polyethylene glycol (PEG), where as the nucleic acids prefer the dextran-rich phase."^^ The operating conditions in terms of pH and salt concentration may be manipulated to fractionate the proteins as well."^^'"^^ By coupling a ligand to PEG, partitioning of a specific protein can be enhanced/^ An advantage of the aqueous two-phase method is that the polymers stabilize the tertiary structure and biological activities of the biomolecules being separated.^^ In spite of this advantage, this method has not become very popular because of the high cost of phase-forming polymers. Use of crude dextran and other less expensive polymer systems has also been explored with success.^^'^^ Another major problem with aqueous two-phase extraction is lack of general methods for selection of phase forming polymers and prediction of phase equilibrium. Noteworthy efforts in this direction were made by Baughman and Liu,^^ Cohen and Gainer,^"^ Haynes et al,^^^ and King et al.^^ In ATP extraction, the interfacial tension between the two phases is generally low (-^10"^ N / m ) . As a result, mixing the phases is generally not a problem; power input levels achieved in passage through static mixers suffice."^^ However, low-density differences and high viscosity of the polymer solutions result in long separation times under gravity.^'''^^ Several different type of extracting devices have been explored: gravity separations using stacked-disk liquid-liquid separators,^^ continuous countercurrent ATP extraction in a packed column,^^'^^ Graesser Raining Bucket Contactor,^^ sieve plate extractor,^^ and spray extraction column.^"^'^^ Lee et aL^^ investigated recovery and purification of intracellular enzyme, aspertase, from E. coli using multiple aqueous two-phase extractions in a system of polyethylene glycol-phosphate. During three successive extractions, the cell debris, nucleic acids, and most contaminating proteins were removed and the enzyme was recovered in the phosphate-rich phase. This stream was passed through an affinity chromatography column containing ligand L-aspartate, resulting in 72% recovery of enzyme, 32-fold increase in purity, and high specific enzyme activity. Similar high yields were also reported by Guan et al.^^ for recovery of penicillin, by Guan et al.^^ for recovery of recombinant a-interferon, and by Sebastiao et al.^^ for recombinant Fusarium solani pisi cutinase. In all of these cases, the product could be extracted directly from crude paste and sometimes even without cell disruption, with a high yield and good-to-excellent purification factor. Coimbra et al,^'^ reported that ATP extraction was effective in separation of isomers from crude. Even though a number of studies refer to protein purification from crude extracts by ATP extraction, specific studies dealing with separation of proteins and nucleic acids are few. Johansson^^ conducted such studies and reported that virtually nucleic acid free proteins can be obtained using ATP extractions. On the other hand, Cascone et al7^ reported problems in separating their target protein in presence of cell homogenate and suggested that system parameters such as pH, salt concentration, polymer types, and concentrations, need to be modified to achieve adequate separation using ATP extraction. Interactions between proteins and nucleic acids when present together in cell homogenate were suspected to be the causes of the problems faced by Cascone et al7^ Bajpai et al."^^ reported that presence of nucleic

372

ROHIT HARVE AND RAKESH BAJPAI

acids does indeed reduce the partitioning of proteins in the PEG phase (Table 3). Presence of affinity Hgand, Cibacron blue FGF, helped, but could not effectively counter the effect of yeast RNA on partition coefficients of the proteins.

V. A CASE STUDY As shown in Table 2, the net loss of enzymatic activity in the purification scheme (Fig. 1) ranged from 72 to 84%. Of these, 5 2 - 6 5 % of the enzymatic activity v^as lost in the initial stages in which the enzyme was separated from nucleic acids and other undesirable enzymes. As a result, several precipitation methods and aqueous two-phase extraction were evaluated for removal of nucleic acids from cell homogenate of tartrate dehydrogenase (TDH) producing strain Pseudomonas putida ATCC 17642. Harvey^ has presented preparation of cell homogenate and experimental methods. The precipitation agents chosen were streptomycin sulfate, protamine sulfate, and manganous sulfate. The ATP system selected was PEG 8000-dextran 70; its composition was the same as specified in Table 3. The details of this case study were published by Harve and Bajpai.^"^ A. Precipitation by Streptomycin Sulfate, Protamine Sulfate, and Manganous Sulfate from Cell Homogenate The base amounts of the precipitating agents were obtained from published literature. Linn and Lehman^^ have suggested that 0.48 g of streptomycin sulfate should be added per gram of RNA in the crude homogenate. Based on the RNA content of cells (7% of cell dry weight^^), a value of 4.09 mMole of streptomycin sulfate per 1 liter of cell homogenate was calculated.^ Tipton and Piesach^"^ have recommended using 5 mg protamine sulfate per gram of wet paste used for homogenization. Higgins et al?^ recommended using ^HH

TABLE 3

Effect of Nucleic Acids on Partition Coefficients

Protein

Single-component system

In the presence of yeast RNA

In the presence of affinity ligand

Trypsin Chymotrypsin Bovine albumin Yeast RNA

3.21 2.74 2.46 0.46

0.89 + 0.14 0.56 ± 0.13 0.93 + 0.12

3.45 2.98 3.89 0.35

+ + + +

0.35 0.26 0.24 0.04

+ + + +

0.35 0.27 0.33 0.03

In the presence of affinity ligand and yeast RNA 1.31 + 0.15 0.74 ± 0.15 1.52 + 0.12

These measurements were conducted in ATP system consisting of 5.1% ( w / w ) PEG 8000 and 10% ( w / w ) dextran 70 in phosphate buffer at 4°C. Under these conditions, the volume ratio of PEG (light) to dextran (heavy) phases vv^as 3:7. The composition (w^/v^) of PEG phase was 9.25% PEG and 0.52% dextran. The composition of the dextran phase w^as 0.54% PEG and 21.1% dextran. The phase composition w^as unaffected by NaCl concentration in the range of 0-5 M. The partition coefficient is defined as ratio of concentration in PEG phase to that in dextran phase. The triazine dye, Cibacron blue FGF, v^as ligated to PEG for affinity partitioning.

373

SEPARATION OF NUCLEIC ACIDS AND PROTEINS

MnS04, H2O at a concentration of 100 mmol/L homogenate. In the experiments, the actual amounts were varied around these values. Streptomycin sulfate and manganous sulfate were added to cell homogenate as a powder; protamine sulfate was added as aqueous solution. After addition of the requisite amount of a precipitant to cell homogenate, pH of solution was adjusted to 7.0-7.1, and the solution was stirred for 10 min. The precipitate was removed by centrifugation in a cold room and the supernate was analyzed for TDH activity and for the relative contents of proteins and nucleic acids. The results are presented in Figs. 2 - 4 . In all the cases, increasing the concentration of precipitant in solution increased the precipitation of nucleic acids. Similarly, the precipitation of TDH also increased with increasing precipitant concentration. As a means of resolving this monotonous trend in precipitation, ratio of percentage of loss of TDH from solution to percentage of nucleic acid precipitation was calculated for each experiment. These ratios are also plotted in each figure. Based on the least value of this ratio, optimal concentrations of precipitants were established at 4 mmol streptomycin sulfate per liter of homogenate, 9 mg protamine sulfate per gram of wet paste, and 100 mmol manganous sulfate per liter homogenate. Deviations on both sides of these concentrations extract a higher penalty in terms of enzyme loss for unit increase in nucleic acid removed by precipitation. Of all the precipitants, streptomycin sulfate is the most effective precipitant since the minimum ratio with this salt is 0.41 compared to values around 1.0 for protamine and manganous sulfates. In

120

% TDH in supernate

100 80 60 40 20

% Nucleic acid precipitated

0 1

2

3

4

5

6

Streptomycin sulfate, mmol/L homogenate

1 0.8 0.6

&

0.4 0.2 +

B

0 0

1

2

3

4

5

6

7

Streptomycin sulfate, mmol/L homogenate F I G U R E 2 Precipitation of nucleic acids witli streptomycin sulfate: (A) effect on nucleic acid precipitation and residual T D H activity in the supernate and (B) effect on the ratio of T D H lost t o nuclei acid precipitated.

374

ROHIT HARVE AND RAKESH BAJPAI

% TDH in supernate

100

200

300

400

500

600

Manganous sulfate, mmol/L homogenate

2.5 2 1.5 (0

1 0.5 0 0

100

200

300

B

+

H-

400

500

600

Manganous sulfate, mmol/L homogenate F I G U R E 3 Precipitation of nucleic acids with manganous sulfate: (A) effect on nucleic acid precipitation and residual T D H activity in the supernate and (B) effect on ratio of T D H lost to nucleic acid precipitated.

5

10

15

Protamine sulfate, mg/g wet paste

3 2.5 2

S.

1.5 1 0.5 0

H-

5

H-

10

15

B 20

Protamine Sulfate, mg/g wet paste F I G U R E 4 Precipitation of nucleic acids with protamine sulfate: (A) effect on nucleic acid precipitation and residual T D H activity in the supernate and (B) effect on ratio of T D H lost to nucleic acid precipitated.

SEPARATION OF NUCLEIC ACIDS AND PROTEINS

375

any case, only 63% nucleic acids are precipitated from solution and as much as 26% enzyme is lost under the best conditions (4 mmol streptomycin sulfate per liter of cell homogenate). Manganous sulfate did not even achieve this level of removal of nucleic acids from solution within the concentration range investigated. B. Removal of Nucleic Acids from Cell Homogenate Using Aqueous Two-Phase Extraction Harvey^ investigated the effect of yeast RNA on partitioning of proteins in an ATP system of PEG 8000-dextran 70 and concluded that both pH and concentration of NaCl strongly influence the partitioning and selectivity of solutes in the two phases. The effects of pH and salt concentration on partition coefficients of total proteins, TDH, and nucleic acids in crude cell homogenate of Pseudomonas putida ATCC 17642, are presented in Table 4. As the solution pH v^as lov^ered from 7.2 to 6.5, partitioning of all the components, total protein, TDH, and nucleic acids improved. Further lowering of pH from 6.5 to 5.5 resulted in improvements in partitioning of total proteins and of nucleic acids, but none in the enzyme TDH. On the other hand, increasing NaCl concentration in the system from 0 to 5 M favors the partitioning of TDH and nucleic acids, without changing the partition coefficient of total proteins. This essentially suggests improved protein fractionation. Under these conditions, the selectivity (ratio of partition coefficient of TDH to that of nucleic acids) was 32, suggesting a high extent of removal of nucleic acids from the crude homogenate. These conditions correspond to ^ 80% recovery of TDH in the PEG phase and '^ 89% nucleic acids in the dextran phase. It compares very favorably with the optimal precipitation of nucleic acids with streptomycin sulfate (74% protein recovery and removal of 63% nucleic acids). The PEG phase containing the tartrate dehydrogenase was then backextracted with phosphate buffer (pH 7.2) and the results of the extractionbackextraction are presented in Table 5. For comparison, a control in the form of protamine sulfate precipitation was also conducted and is also reported in Table 5. The optimal conditions for protamine sulfate precipitation (Fig. 4) were selected for the control. On the whole, a single-stage T A B L E 4 Partition Coefficients of Total Proteins, T D H , and Nucleic Acids from Crude Cell Homogenate in the T w o Phases of A T P System Partition coefficients

pH 7.2 6.S

s.s

6.5

NaCi concentration (M)

Proteins

0 0 0 5

5.28 9.30 11.03 9.51

+ + + ±

TDH 0.15 0.07 0.56 0.81

2.03 4.26 4.61 9.20

+ ± + +

0.38 0.21 0.03 0.04

Nucleic acids

Percentage TDH recovered in PEG phase

0.91 0.72 0.49 0.29

47 65 66 80

+ + + +

0.03 0.04 0.08 0.02

376

ROHIT HARVE AND RAKESH BAJPAI ^ • 1

T A B L E 5 A T P E x t r a c t i o n of T D H f r o m Cell H o m o g e n a t e ( p H 6.5, 5 M NaCI) and B a c k e x t r a c t l o n of t h e PEG Phase w i t h Phosphate Buffer ( p H 7.2); C o n t r o l : P r o t a m i n e Sulfate P r e c i p i t a t i o n " Total activity added to the ATP system Activity in PEG phase Activity in dextran phase Activity in buffer backextracted from PEG phase Percentage yield in the backextracted buffer Total activity used for protamine sulfate control Activity in supernate after precipitation Percentage yield for precipitation control

32 units 27 units 5 units 25 units 77% 32 units 16 units 50%

Loading of protamine sulfate 9 mg per gram of vv^et paste: w^eight of wet paste, 48 g; dry weight, 10.7 g.

ATP-extraction followed by backextraction showed a considerable improvement over purification with protamine sulfate precipitation. A multistage ATP extraction followed by multistage backextraction should further improve the enzyme recovery. This would be in keeping with the observations of Lee et aL^^ and of Sebastiao et al,^^

Yl. CONCLUSIONS Aqueous two-phase extraction has been shown to be an efficient method for separation of nucleic acids from proteins. By proper selection of an ATP system and optimization of pH and salt concentration, it was possible to achieve a high degree of purification using a single-stage extraction-backextraction for an intracellular enzyme. The enzyme yield and purification achieved by ATP process was significantly higher than from a comparative precipitation process.

REFERENCES 1. Cussler, E. L. (1989). Ber. Bunsenges. Phys, Chem. 93(9), 944-948. 2. Fair, J. R. (1989). Chem. Eng. Prog. 85(12) 38-44. 3. Harve, R. H. (1994). MS Thesis, submitted to the Faculty of the Graduate School, University of Missouri—Columbia, Columbia. 4. Sadana, A. (1998). "Bioseparation of Proteins—Unfolding/Folding and Validations," Chapter 2. Academic Press, San Diego, CA. 5. Singh, P. C , and Singh, R. K. (1996). Trends Food Set. Technol. 7(2), 4 9 - 5 8 . 6. Nadgir, V. M., and Liu, Y. A. (1983). AIChE J. 29(6), 926-934. 7. Wheelwright, S. M. (1989). / . Biotechnol. 11(2-3), 89-102. 8. Asenjo, J. A., Parrado, J., and Andrews, B. A. (1991). Ann. N.Y. Acad. Set. 646, 334-356. 9. Prokopakis, G. J., and Asenjo, J. A. (1990). In "Separation Processes in Biotechnology," (J. A. Asenjo, ed.), pp. 571-601. Dekker, New York. 10. Bonnerjea, J., Oh, S., Hoare, M., and Dunhill, P. (1986). Bio/Technology 4(11), 954, 956, 958.

SEPARATION OF NUCLEIC ACIDS AND PROTEINS

11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.

41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54.

377

Belfort, G. (1989). Ber. Bunsenges. Phys. Chem. 93(9), 939-944. Ramirez-Vick, J. E., and Garcia, A. A. (1996-1997). Sep, Purif. Methods 25(2), 85-129. Fang, B., and Jiang, T. (1998). Chin. } . Chem. Eng. 6(1) 7 3 - 7 8 . Harve, R. and Bajpai, R. K. (1998). Appl. Biochem. Biotechnol. 70-72, 677-6S6. Hayes, D. G., and Marchio, C. (1998). Biotechnol. Bioeng. 59(5) 557-566. Agarwal, R. and Gupta, M. N. (1996). Protein Express. Purif. 7(3), 294-298. Sinha, R., Singh, S. P., Ahmed, S., and Garg, S. K. (1996). Bioresour. Technol. 55(2), 163-166. Powers, D. D., Carbonell, R. G., and Kilpatrick, P. K. (1994). Biotechnol. Bioeng. 44(4), 509-522. Conroy, M. J., and Lovrien, R. E. (1992). / . Cryst. Growth 122(1-4), 213-222. Gierer, A. (1957). Nature (London) 179, 1297-1299. Davidson, P. F. (1959). Proc. Natl. Acad. Set. U.S.A. 45, 1560-1568. Trim, A. R. (1959). Biochem. J. 73, 298-304. Marmur, J., Roland, R., and Schildkraut, C. L. (1963). Prog. Nucleic Acids Res. 1, 231-300. Ralph, R. K., and Bergquist, P. L. (1967). Methods Virol. 11, 465-538. Jones, A. S. (1953). Biochim. Biophys. Acta 10, 607. Guerritore, D., and Ballelli, L. (1969). Nature (London) 194, 1638. Atkinson, A., and Jack, G. (1973). Biochim. Biophys. Acta 308, 4 1 - 5 2 . Bingham, A. H. A., Sharman, A. F., and Atkinson, A. (1977). FEBS Lett. 2, 250-256. Melhng, J., and Atkinson, A. (1972). / . Appl. Chem. Biotechnol. 11, 739-744. Oxenburgh, M. S., and Snoswell, A. N. (1965). Nature (London) 203, 1416-1417. Heppel, L. A. (1955). In "Methods in Enzymology" (S. P. Colowick and N. O. Kaplan, eds.). Vol. 1, pp. 137-138. Academic Press, New York. Moskowitz, M. (1963). Nature (London) 200, 335-337. Dinovick, R., Bayon, A. P., Canales, P., and Pansy, J. (1948). / . Bacteriol. 56, 125. Snoswell, A. M. (1963). Biochim. Biophys. Acta 77, 7-19. Higgins, J. J., Lewis, D. J., Daly, D. H., Mosqueira, F. G., Dunhill, P., and Lilly, M. D. (1978). Biotechnol. Bioeng. 20(2), 159-182. Atkinson, A., Phillips, B. W., Callow, D. S., Stones, W. R., and Bradford, P. A. (1972). Biochem. } . 127, 63-64. Davidson, J. N. (1965). "The Biochemistry of Nucleic Acids," 5th ed. Methuen, London. Burgess, R. R. (1969). / . Biol. Chem. 244, 6160-6167. Mattiasson, B. (1983). Trends Biotechnol. 1, 16-20. Albertsson, P.-A. (1985). In "Partitioning in Aqueous Two-Phase Systems: Theory, Methods and Uses, and Applications in Biotechnology," (H. Walter, D. E. Brooks, and D. Fisher, eds.), pp. 1-10. Academic Press, New York. Todd, P., Hawker, D. T. L., Davis, R. H., and Owens, R. B. (1993). Fluid Phase Equilib. 82(1), 225-235. Xiu, Z., Jiang, W., and Su, Z. (1993). Huagong Xuebao (Chin. Ed.) 44(6), 757-760. Dissing, U., and Mattiasson, B. (1994). Bioseparation. 4(6), 335-342. Carlson, A. (1988). Sep. Sci. Technol. 23(8-9), 785-817. Hustedt, H., Kroner, K. H., and Kula, M. R. (1984). Proc. Eur. Congr. Biotechnol, 3rd Vol. 1, pp. 597-605. Takahashi, T., and Adachi, Y. (1982). / . Biochem. (Tokyo). 9 1 , 1719-1724. Weuster-Botz, D., Paschold, H., Striegel, B., Gieren, H., Kula, M.-R., and Wandrey, C. (1994). Chem. Eng. Technol. 17(2), 131-137. Bajpai, R. K., Harve, R., and Tipton, P. (1995). Appl. Biochem. Biotechnol. 54, 193-205. Johansson, G. and Joelsson, J. (1989). In "Separations Using Aqueous Two-Phase Systems" (D. Fisher and L A. Sutherland, eds.), p. 33. Plenum, New York. Kopperschlager, G., and Johansson, G. (1982). Anal. Biochem. 124, 117-124. Szlag, D. C , and Guiliano, K. A. (1988). Biotechnol. Tech. 2(4), 277-282. Venancio, A., Teixeira, J. A., and Mota, M. (1993). Biotechnol. Prog. 9(6), 635-639. Baughman, R. D. and Liu, Y. A. (1994). Ind. Eng. Res. 33(11), 2668-2687. Cohen, L. M., and Gainer, J. L. (1994). Sep. Sci. Technol. 29(5), 1925-1942.

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55. Haynes, C. A., Blanch, H. W., and Prausnitz, J. M. (1989). Fluid Phase Equilib. 53(2), 463-474. 56. King, R. S., Blanch, H. W., and Prausnitz, J. M. (1988). AIChEJ. 34(10), 1585-1594. 57. Flygare, S. Wilkstrom, P., Johansson, G., and Larsson, P.-O. (1990). Enzyme Microb. Technol. 12(2), 95-103. 58. Raghava, R. K. S. M. S., Stewart, R., and Todd, P. (1991). Sep. Sci. Technol. 26(2), 257-267. 59. Kroner, K. H., and Hustedt, H. (1986). ISEC '86 Int. Solvent Extr. Conf. Prepr. Vol. 3, pp. 703-711. 60. Yan, G., Zhu, Z., Mei, L., and Guan, Y. (1996). / . Chem. Eng. Ind. (China) 47(4), 495-499. 61. Patil, T. A., Jafarabad, K. R., Sawant, S. B., and Joshi, J. B. (1991). Can. J. Chem. Eng. 69(2), 54S-556. 61. Coimbra, J. D. R., Thommes, J., Meirelles, A. J., and Kula, M.-R. (1995). Bioseparation 5(5), 259-268. 63. Bhawsar, P. C. M., Pandit, A. B., Sawant, S. B., and Joshi, J. B. (1994). Chem. Eng. J. 55(1-2), B1-B17. 64. Rostami, J. K., Sawant, S. B., Joshi, J. B., and Sikdar, S. K. (1992). Chem. Eng. Sci. 47(1), 57-68. 65. Pawar, P. A., Jafarabad, K. S., Sawant, S. B., and Joshi, J. B. (1993). Chem. Eng. Commun. Ill, 151-169. 66. Lee, C.-K., Wang, N.-H., and Ju, Y.-H. (1995). Sep. Sci. Technol. 30(4), 509-519. 67. Guan, Y., Zhu, Z., and Mei, L. (1996). Sep. Sci. Technol. 31(18), 2589-2597. 68. Guan, Y., Lilley, T. H., Treffry, E., Zhou, C.-L., and Wilkinson, P. B. (1996). Enzyme Microb. Technol. 19(6), 446-455. 69. Sebastiao, M. J., Cabral, J. M. S., and Aires-Barros, M. R. (1996). Enzyme Microb. Technol. 18(4), 151-160. 70. Johansson, G. (1974). Acta Chem. Scand. Ser. B 28(8), 873-882. 71. Cascone, O., Andrews, B. A., and Asenjo, J. A. (1991). Enzyme Microb. Technol. 13, 629-635. 72. Linn, A., and Lehman, J. K. (1964). / . Biol. Chem. 240, 3. 73. Wang, D. I. C., Cooney, C. L., Demain, A. L., Dunhill, P., Humphrey, A. E., and Lilly, M. D. (1979). "Fermentation and Enzyme Technology." Wiley, New York. 74. Tipton, P. A., and Piesach, J. (1990). Biochemistry 29, 1749-1756.

BIOSEPARATIONS BY DISPLACEMENT CHROMATOGRAPHY A B H I N A V A.

SHUKLA

ICOS Corporation, Bothell, Washington 98021 S T E V E N M.

CRAMER

Department of Chemical Engineering, Rensselaer Polytechnic Institute, New York 12180

Troy,

I. INTRODUCTION II. PURIFICATION OF AMINO ACIDS A N D PEPTIDES BY DISPLACEMENT CHROMATOGRAPHY III. PURIFICATION OF PROTEINS BY DISPLACEMENT CHROMATOGRAPHY A. Ion Exchange B. Stationary Phases Other than Ion Exchange IV. ALTERNATIVE MODES OF DISPLACEMENT CHROMATOGRAPHY A. Sample Displacement Chromatography B. Selective Displacement Chromatography C. Displacement Using Retained pH Gradients V. METHODS DEVELOPMENT FOR DISPLACEMENT CHROMATOGRAPHY A. Estimation of the SMA Parameters B. The Dynamic Affinity Plot C. Affinity Ranking Plot D. Operating Regime Plot E. Developing Displacement Separations VI. DISPLACEMENT CHROMATORGRAPHY FOR THE PURIFICATION OF BIOMOLECULES: INDUSTRIAL CASE STUDIES A. Purification of an Antigenic Vaccine Protein by Selective Displacement Chromatography B. Purification of Recombinant Proteins from Variants C. Purification of Antisense Oligonucleotides by Displacement Chromatography VII. DESIGN OF L O W MOLECULAR WEIGHT DISPLACERS VIII. CONCLUSIONS REFERENCES Separation Science and Technology,

Volume 2

Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

379

380

ABHINAV A. SHUKLA AND STEVEN M. CRAMER

I. INTRODUCTION The biopharmaceutical industry has grown rapidly in the past decade with over 75 biotechnology drugs approved for sale in the United States alone.^ In addition there are over 500 biopharmaceutical candidates in various phases of clinical trials. In contrast to most of the earHer biotechnology therapeutics which were produced in relatively small scale (kilograms per year), many of the recent products are expected to have production scales of the order of hundreds of kilograms per year. In addition, many biopharmaceuticals are making the transition to generic drugs with more than one manufacturer competing for market share. Thus there is an urgent need for the development of efficient, large-scale purification processes in the biotechnology industry. Since preparative chromatography is the single most widely used unit operation for process-scale purification of biologicals,^'^ the development of more efficient chromatographic operations is assuming increasing importance. Displacement chromatography is an efficient mode of preparative chromatography. Operationally, displacement chromatography is performed in a manner similar to step-gradient chromatography in which the column is subjected to sequential step changes in the inlet conditions (Fig. 1). The column is initially equilibrated with a carrier buffer in which the feed solutes exhibit a relatively high retention on the chromatographic stationary phase (e.g., low ionic strength in ion exchange, high salt concentrations in hydrophobic interaction chromatography, and low mobile phase modifier concentrations in reversed-phase chromatography). Following the equilibration step, the feed mixture is introduced into the column, which is then followed by a constant infusion of the displacer solution. The displacer is selected such that it has a higher affinity for the stationary phase than any of the feed components. Under appropriate conditions, the displacer induces the feed components to develop into adjacent "square-wave" zones of highly concentrated pure material. After the breakthrough of the displacer, the column is regenerated and is reequilibrated with the carrier buffer. The displacer, having a higher affinity than any of the feed components, competes effectively, under nonlinear conditions, for the adsorption sites on the stationary phase. An important distinction between displacement and gradient chromatography is that the displacer front always remains behind the adjacent feed zones in the displacement train whereas desorbents (e.g., salts in ion exchange, organic modifiers in reversed-phase chromatography) move through the feed zones. The displacement mode of chromatography takes advantage of the thermodynamic characteristics of the chromatographic system to overcome many of the shortcomings of preparative elution chromatography. In linear elution chromatography, the amount of feed is kept deliberately low so that the peak profiles are approximately symmetrical and essentially Gaussian. Scaling up the separation by linear elution chromatography is straightforward but results in an economically less favorable process. Upon increasing the feed size, the throughput can only be increased at the cost of diminishing resolution and lower purity. In contrast, in displacement chromatography the

381

BIOSEPARATIONS BY DISPLACEMENT CHROMATOGRAPHY

Carrier Feed

Displacer development WKSSK^^M

Product train

Regeneration

Carrier FIGURE

I

Schematic of the operation of displacement chromatography.

displacer suppresses the adsorption of the feed components in the displacer zone and thus prevents the tailing of the most strongly retained feed component. In a fully developed displacement train, each of the components displaces the component ahead of it, leading to a suppression of tailing in all the solute zones. This characteristic of displacement makes it less sensitive to the feed loads than the elution modes of operation, thus enabling it to deliver higher process throughputs. Another advantage is the high resolution that displacement can deliver as compared to elution processes. Displacement chromatography exploits the nonlinear, multicomponent competition among the components to be separated, resulting in higher resolution, particularly among closely related species. In contrast, in elution processes (e.g., linear gradient, step gradient), the separation takes place under relatively v^eaker binding conditions (which are essential to get the solutes off the column). The separation factors among solutes are thus lower in elution than in displacement, leading to poorer resolution in the elution modes of operation. Other advantages of displacement include a better control over the product concentrations and the emergence of the product in relatively low concentrations of the mobile phase modifier. The combination of high throughputs and high resolutions in a single process makes displacement an attractive mode of operation for preparative separations."^

382

ABHINAV A. SHUKLA AND STEVEN M. CRAMER

The advent of displacement chromatography can be attributed to Tisehus, v^ho in 1943 first classified the modes of chromatography as frontal, elution, and displacement/ How^ever, the years that follow^ed saw^ an increase in the use of chromatography as an analytical tool and linear elution chromatography eclipsed the overloaded modes of chromatography in importance. Indeed, many of the preparative separations that are carried out in the biotechnology industry are still carried out at relatively lovv^, linear loading conditions, largely due to unfamiliarity w^ith nonlinear modes of operation. With the birth of the biotechnology industry in the early 1980s, there has been an increasing interest in the field of displacement chromatography. Recent advances in the field have included the advent of low^ molecular weight displacers, a focus on the design of high-affinity displacer molecules, the extension of displacement chromatography to systems other than ion exchange and the application of displacement chromatography to several challenging problems from the biotechnology industry. The aim of this review^ is to summarize the recent developments in this field and to place in perspective the role that displacement chromatography could play in preparative bioseparations in the years to come.

II. PURIFICATION OF AMINO ACIDS AND PEPTIDES BY DISPUCEMENT CHROMATOGRAPHY Corticosteroids^ and polymyxin antibiotics'^ v^ere purified by displacement chromatography on reversed-phase supports. This w^ork v^as later extended to the purification of short peptides w^ith chain lengths of tw^o to three amino acids on silicaceous cation-exchange supports.^ Peptides synthesized w^ith immobilized carboxypeptidase Y w^ere purified on a C-18 reversed-phase stationary phases using butoxyethoxyethanol, cetramide, and dodecyltrimethyl ammonium bromide as the displacers.^ The tendency of these displacers to form micelles v^as controlled by temperature and solvent programming and by the introduction of a w^ash step betw^een the introduction of the feed and the displacer. Mellitin, a 26 amino acid peptide was displaced on micropellicular C-18 reversed-phase stationary phases.^^ Figure 2 show^s the purification of crude synthetic P14A mellitin from its variants using benzyl dimethylhexadecyl ammonium chloride as the displacer. More recently, a theoretical study of the displacement of tv^o interconverting species, such as the cis and the trans forms of peptides containing petidyl-proline residues has been carried out.^^ The displacement of single amino acids on cation-exchange materials was investigated by Bendall et al}-^ The authors separated glycine and creatinine on a cation-exchanger using NaOH as the displacer. Mixtures of a-amino butyric acid (ABA) and isoleucine have been separated by cation-exchange displacement chromatography on a Dowex resin with NaOH as the displacer.^^ Finally, the D and L forms of tryptophan have been separated by displacement on chiral cyclobond stationary phases using mandelic acid as the displacer.^"^

383

BIOSEPARATIONS BY DISPLACEMENT CHROMATOGRAPHY

£ E

CD O

c o O

20

30

40

50

Time (min) F I G U R E 2 Purification of crude synthetic PI4A mellitin by displacement. Column: Hy-Tach micropellicular Cjg silica, 105 X 4.6 mm; mobile phase: 10% ( v / v ) acetonitrlle in water, 0 . 1 % TFA; flow rate: 0.2 m L / m i n ; temperature: 23°C; displacer: 25 m M benzyl dimethylhexadecyl ammonium chloride; feed: 10 mg PI4A mellitin (from Kalghatgi et o/.,'° with permission from Elsevier Science).

III. PURIFICATION OF PROTEINS BY DISPLACEMENT CHROMATOGRAPHY A. Ion Exchange Displacement chromatography has been widely investigated for the preparative purification of proteins on both anion- and cation-exchange stationary phases. Initial v^ork in this area employed relatively large polyelectrolytes as the displacers. Carboxymethyldextrans have been employed as "spacer displacers" for the purification of serum proteins, /3-lactoglobulins, and albumin on a DEAE cellulose anion-exchange stationary phase.^^"^^ This technique involved the use of polyelectrolytes spanning a range of affinities for the stationary phase. The resulting bands of the proteins formed by this displacement w^ere separated between the fronts formed by the polyelectrolytes. Displacement chromatography was employed for the purification of j8lactoglobulins^^'^^ and j8-galactosidase^^ using chondroitin sulfate as the displacer on an anion-exchange stationary. Subramanian et al}^ demonstrated that proteins could be successfully displaced in cation-exchange systems using PEI-600 and Nalcolyte 7105. Several other polyelectrolyte displacers were subsequently identified for cation-exchange chromatography including protamine sulfate^^ and DEAE-dextrans.^"^ Additional displacers employed for anion-exchange systems have included heparin,^^ dextran sulfate,^^ pentosan polysulfate,^^ and block methacrylic polyampholytes.^^ Ghose and Mattiason^^ have examined the purification of lactate dehydrogenase using a carboxymethyl starch displacer. Jen and Pinto have employed polyvinyl sulfonic acid^^ and dextran sulfate^^ to separate a mixture of moderately

384

ABHINAV A. SHUKLA AND STEVEN M. CRAMER

retained proteins, conalbumin and ovalbumin, and to separate j8-lactoglobulins A and B.^^ Displacement separation of j8-lactoglobulins A and B has been carried out at elevated flov^ rates on a perfusive chromatographic support using heparin as the displacer.^^ As shown in Fig. 3, this resulted in an effective separation even at velocities of 1440 cm/hr. The elevated flow rates possible with perfusive supports are expected to enhance the productivities in displacement chromatography for this class of stationary phase materials.^^ Whey proteins have been separated on a monolithic stationary phase using polyacrylic acid as the displacer.^"^ Several of these high molecular weight polyelectrolytes employed for the displacement of proteins are listed in Table 1.^^-^^ A significant development in the field of displacement chromatography of proteins has been the use of low molecular mass displacers,"^^'"^^ in contrast to the high molecular weight polyelectrolytes described earlier. Low molecular weight displacers have significant operational advantages as compared to large polyelectrolyte displacers and have generated significant interest from the biotechnology industry. First and foremost, if there is any overlap between the displacer and the protein of interest, these low molecular weight materials can be readily separated from the purified protein during subsequent downstream processing involving size-based purification methods."^^ The relatively low cost of synthesizing low molecular weight displacers can be expected to significantly improve the economics of displacement chromatography. Furthermore, the salt-dependent adsorption behavior of these low molecular weight displacers greatly facilitates column regeneration. The use of low molecular weight displacers also enables the development of selective displacements^^ in which the displacer selectively displaces the bioproduct of interest while desorbing and inducing the elution of the higher and lower affinity impurities, respectively. Selective displacement chromatography will be described in Section IV. Table 2"^^'^^ lists several of the low

35

beta-Lactoglobulin A

30

beta-Lactoglobulin B

1.25 c

20

CO

Sc 15

o o c o O

Heparin

10

2.8

3.3

3.8

4.3

4.8

5.3

5.8

Elution volume (ml) F I G U R E 3 Displacement chromatography of jS-lactoglobulins at 1440 c m / h r . Column: 100 X 4.6 mm POROS 10 H Q (10 / i particles); mobile phase: 10 m M Tris-HCI, pH 7.0; displacer: 12 m g / m L heparin in carrier; feed: 60 mg j8-lactoglobulin crude mixture; fraction volume: 200 fA. (from Gerstner et o/.,^^ with permission from Elsevier Science).

385

BIOSEPARATIONS BY DISPLACEMENT CHROMATOGRAPHY

^ ^ H T A B L E I High Molecular W e i g h t Displacers Employed for the Ion-Exchange Displacement Chromatography of Proteins Stationary phase

Displacer

Proteins

Reference

DEAE Sephadex A50 and DEAE Sephacel (weak anion exchange); DEAE cellulose CM cellulose

Carboxymethyld extrans

Ovalbumin, j8-lactoglobulins A and B; serum proteins; RNA polymerase II Human serum albumin, a-fetoprotein j8-lactoglobulins A and B

15-17

Ampholine (pH 4 - 6 )

TSK DEAE 5PW (weak anion exchange, 5 /x, particles) TSK-DEAE-5PW(weak anion exchange, 5 /JL particles)

Chondroitin sulfate and polygalactouronic acid Carboxymethyl dextrans

TSK DEAE 5-PW(weak anion exchange, 5 fi particles) RG Bio PSM 300 (weak cation exchange, 5 /JL particles) and strong cation exchange

Chondroitin sulfate

TSK DEAE-5PW(weak anion exchange, 5 fi particles) Matrex PAE-300 (silica based, polyethyleneimine coated weak anion exchange, 10 iJL particles) Tris Acryl DEAE M (weak anion exchange) Matrex PAE-300 (silica based, polyethyleneimine coated weak anion exchange, 10 IJL particles) Fractogel DEAE 650S, DEAE cellulose (weak anion exchange) Protein-Pak SP-8HR (strong cation exchange, 8 /JL particles) Protein-Pak Q-8HR (strong anion exchange, 8 /JL particles) Waters SP-8HR (strong cation exchange, 8 ^t particles) Waters Q-8HR (strong anion exchange, 8 fi particles) and DEAE-8HR(weak anion exchange, 8 /it particles) Protein-Pak Q-8HR (strong anion exchange, 8 JJL particles) POROS lOHQ, POROS H Q / M (strong anion exchange, 10 and 20 ^^ particles) POROS H S / M POROS HS50 (strong cation exchange, 20 and 50 /A particles) Uno Q l and Q6 (strong anion exchange, monolithic stationary phase)

35 19

Alkaline phosphatase from E. coli periplasmic space proteins jS-lactoglobulins A and B

36

Cytochrome c and lysozyme; RNAse, a-chymotrypsinogen A, and lysozyme j8-galactosidase

22, 37

Polyvinyl sulfonic acid (2kD)

Ovalbumin, conalbumin

29

Carboxymethyl starch

Lactate dehydrogenase (LDH) from beef heart j8-lactoglobulins A and B

28

Guinea pig serum proteins, mouse liver cytosol proteins a-chymotrypsinogen A, cytochrome c and lysozyme j8-lactoglobulins

18

Nalcolyte 7105, PEI 600

Chondroitin sulfate

dextran sulfate (MW 5-500 kD), polyvinyl sulfonic acid (MW 2kD) carboxymethyld extrans

Protamine sulfate

Heparin DEAE-dextrans

20

21

30,31

23

25

a-chymotrypsinogen A, cytochrome c j8-lactoglobulins A and B, a-lactalbumin, soybean trypsin inhibitor (STI)

24

j8-lactoglobulins A and B

27

j8-lactoglobulins A and B

32

Protamine sulfate

rHu-BDNF (variants removal)

39

Polyacrylic acid

Whey proteins ( a and j3-lactalbumins)

34

Pentosan polysulfate. dextran sulfate

Block methacrylic polyampholytes Heparin

38

386

ABHINAV A. SHUKLA AND STEVEN M. CRAMER

^ H i T A B L E 2 Low Molecular W e i g h t DIsplacers ( < 2 kD) for the Ion-Exchange Displacement Chromatography of Proteins Stationary phase

Displacer

Proteins

Reference

Waters SP-8HR (strong cation exchange, 8 /JL particles)

Low molecular weight dendrimers (PETMA4,12, 36) Protected amino acids (BAEE, CLME) Antibiotics (neomycin sulfate, streptomycin sulfate) EGTA, IDA (iminodiacetic acid)

a-chymotrypsinogen A and cyctochrome c

44

Cytochrome c. a-chymotrypsinogen A Lysozyme, cytochrome c

45

a-lactalbumin. j8-lactoglobulins A andB /3-lactoglobulins A andB

48

Waters SP-8HR (strong cation exchange, 8 /i particles) Waters SP-8HR (strong cation exchange, 8 /A particles) Super Q (strong anion exchange, 35 /A particles) Waters Q-8HR (strong anion exchange, 8 fi particles) and Waters Q-40HR (strong anion exchange, 40 /I particles) Waters Q-8HR (strong anion exchange, 8 fi particles)

Waters SP-8HR (strong cation exchange, 8 fji particles)

Waters SP-8HR (strong cation exchange, 8 /JL particles) Pharmacia DEAE Fast Flow Sepharose (weak anion exchange, 90 /JL particles) Waters SP-8HR (strong cation exchange, 8 JJL particles)

Waters SP-8HR (strong cation exchange, 8 JJL particles)

Waters Q-8HR (strong anion exchange, 8 JJL particles)

Sucrose octasulfate

Low molecular weight aromatic sulfonates (e.g., p-toluene sulfonic acid) Streptomycin sulfate, BAEE

Protected amino acid (BAEE) Low molecular weight aromatic sulfonates (e.g., p-toluene sulfonic acid) Linear amines (spermine, spermidine, triethylene tetramine, diethylene triamine, tetraethylene pentamine); low molecular weight branched aromatic and nonaromatic dendrimers (e.g., PETMA4, DPE-TMA6, PE-DMABzCl4) Low molecular weight branched, hydrophobic (PE-DMABzC14, PhTMA6, PETMA4) Low molecular weight aromatic sulfonates (e.g., p-toluene sulfonic acid, napthalene disulfonic acid)

46,47

42

jS-lactoglobulins A and B by selective displacement

43

Ribonuclease B, cytochrome c, lysozyme by selective displacement Bovine and horse heart cytochrome c Antigenic vaccine protein from complex feed stream by selective displacement rHu-BDNF (recombinant human brain-derived neurotrophic factor)

43

lysozyme, a-chymotrypsinogen A dendrimers

52

j8-lactoglobulins A

53

andB

49 50

51

BIOSEPARATIONS BY DISPLACEMENT CHROMATOGRAPHY

387

molecular weight displacers that have been employed for protein purification in ion-exchange systems. Jayaraman et al."^"^firstdemonstrated that low molecular weight displacers (less than 2 kD) could be successfully employed for protein separations using pentaerythritol based dendritic polyelectrolytes ranging from 480 to 1500 Da. Subsequently, protected amino acids"^^ and antibiotics'^^ were identified as useful low molecular weight displacers for cation-exchange chromatography. The sulfonic acid salts of aromatic compounds^^ and sulfonated sugars such as sucrose octasulfate"*^ have been employed as low molecular weight displacers for anion-exchange systems. More recently, there has been significant effort directed toward the design and synthesis of high-affinity, low molecular weight displacers for cation-exchange chromatography.^^'^^'^"^ These displacers have included linear amines such as spermine and spermidine and branched molecules based on the early generation dendrimeric molecules described earlier. High affinity low molecular weight displacers will be covered in depth in Section VII. One of the major advantages of operation in the displacement mode is the potentially higher resolution that can be achieved. The separation factors between most solutes tend to decrease under higher salt conditions resulting in lower resolutions in elution chromatography. On the other hand, since displacement chromatography is carried out under relatively low salt conditions (i.e., stronger binding) it can often result in higher resolutions for difficult problems. The higher resolving power of displacement chromatography has been exploited for the separation of cytochrome c from two sources, bovine and horse heart."^^ These proteins differ from each other in just three amino acids and their respective adsorption isotherms are shown in Fig. 4A. As seen in Fig. 4B, displacement chromatography in a cation-exchange system using BAEE as the displacer resulted in an excellent separation with high yields and purities. This example is significant in that it illustrates the capability of displacement chromatography to resolve very closely related species in a single process step. Displacement chromatography has been recently employed for the purification of proteins from complex industrial mixtures.^^'^^ These industrial examples will be described in greater detail in Section VI.

B. Stationary Phases Other than Ion Exchange While, displacement chromatography of proteins has, for the most part, been carried out on ion-exchange stationary phases, several investigations have been carried out on stationary phases involving other modes of adsorption. Displacement of moderately retained proteins (e.g., lactoferrin and RNase A) has been carried out in immobilized metal affinity (IMAC) stationary phases using myoglobin as the displacer,^^'^^ While, imidazole was employed as the mobile phase modifier in these studies it was subsequently established that it possessed sufficient affinity to act as a displacer for proteins in IMAC systems.^^'^^ Figure 5 shows the separation of RnAse and myoglobin using imidazole as a displacer on a Cu^"^ charged Sepharose column. To date.

388

ABHINAV A. SHUKLA AND STEVEN M. CRAMER

Bovine @ 90 mM Bovine @ 125 mM

0.2

0.1

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Mobile phase c o n e , C(mM)

B 0.5 r

i''•-"-"'.

0.4 i

pi-

s

J

1o °-^1r

L! ^

Imp

o

•

o c

•

en

'

I

,11

+-

I

1

1

6

8

J160 I |50

1

>> •

"> O

0.1 L

80

[70 _

^%^-ki^_|.-

1

01

1 :

^^ 0.3 K co 1 o

90

...Salt

o

OOOG

j i BAEE

^ o

140:2 CO 1 w ^

130^ 120^

L_i

1110

1

10

12

0 14

Effluent volume (ml) F I G U R E 4 (A) Adsorption isotherms of bovine and horse heart cytochrome c each at tv/o different salt concentrations (90 and 125 m M ) . Column: 50 X 5 mm i.d. strong cation exchanger (8 jLim); flow rate: 0.2 mL/min. (Kundu et al^^) (B) Displacement separation of cytochrome c's from bovine and horse heart using BAEE as the displacer. Column: 105 X 5 mm i.d. strong cation exchanger (8 /im); mobile phase: 50 mAi sodium phosphate buffer, pH 6.0; feed: 1.6 mL of 0.52 mM each of bovine and horse heart cytochrome c In carrier; displacer concentration: 40 m M N-a-benzoyI arginine ethyl ester (BAEE) in the carrier; flow rate: 0.2 mL / min; fraction size: 200 m L

Studies on MADC (metal affinity displacement chromatography) have been limited to the separation of model protein mixtures. Displacement chromatography has also been carried out in dye affinity chromatographic systems for the purification of lactate dehydrogenase (LDH)/^ In that study, polyethyleneimine (PEI) was employed as a displacer on dye affinity matrices prepared by immobilizing Cibacron Blue 3GA or Procion Red HE-3B. Whey proteins have been purified on a hydroxyapatite support using EGTA (ethylene glycol bis( j8-aminoethylether)-N, N,N',N' tetraacetic acid) and PGA (polyglutamic acid) as displacers."*^

BIOSEPARATIONS BY DISPLACEMENT CHROMATOGRAPHY

389

3 4 5 Elution volume (ml) F I G U R E 5 Displacement chromatography of proteins on IMAC using imidazole as the displacer. Column: 50 X 5 mm i.d. Cu^"*" charged metal chelate Sepharose (10 /im); mobile phase: I M NaCI, 25 mA^ phosphate buffer, pH 7.0; feed: l.8mL of 0.89 m M ribonuclease A and 0.69 m M myoglobin in the carrier; displacer: 10 m M imidazole in the carrier; flow rate: 0.1 m L / m i n ; fraction size: 100 /iL fractions. (Vunnum et o/.^^)

Both reversed-phase liquid chromatography (RPLC) and hydrophobic interaction chromatography (HIC) are widely employed in preparative-scale protein purification because of their unique selectivity. Both techniques are based on the solvophobic effect^^'^^ and separate biomolecules based on their relative hydrophobicities. Hydrophobic displacement chromatography offers the possibility of combining the unique selectivities of HIC and RPLC stationary phases w^ith the high loadings and high resolutions possible in the displacement mode of operation. In addition, the displacement mode of operation in reversed-phase systems w^ould have the additional advantage of low^ering the solvent consumption in these systems. Displacement of peptides has been carried out on RPLC supports.^'^^'^^ In HIC systems, higher retained proteins such as a-chymotrypsinogen A have been employed as displacers for proteins writh lower retention such as BSA and lysozyme.^^ More recently, a series of methacrylic block copolymers have been employed as displacers for proteins in HIC.^"* Pancreatic trypsin and a-chymotrypsin were separated on an octyl-Sepharose HIC column using block methacrylates of varying hydrophobicities. Recent results in our laboratory have demonstrated the possibility of performing displacement chromatography of proteins on both HIC and RPLC stationary phases using small molecules as displacers.^^ Figure 6 shows the separation of lysozyme and conalbumin on a Phenyl 650 M HIC stationary phase using benzyl tributyl ammonium chloride (BTBAC) as the displacer. Further investigations in our laboratory have demonstrated that displacement chromatography in reversed-phase systems can result in high resolution of very difficult bioseparation problems.

390

ABHINAV A. SHUKLA AND STEVEN M. CRAMER

30

25 Conalbumin ^

I

20

25

lysozyme -BTBAC

6 c

20

£

15

o o

10

o CO o

0

8 c 10 E

Q-

CO

Q

(D

H5

5 15 Volume (ml)

20

25

30

F I G U R E 6 Displacement of conalbumin and lysozyme using BTBAC as a displacer. Column: 10 mm D X 95 mmL Phenyl 650 M (TosoHaas) HIC resin; mobile phase: 1.5 molal ammonium sulfate in 100 m M , pH 7 phosphate buffer; displacer: 25 m M BTBAC; loading: 100 mg lysozyme + 35 mg conalbumin; flow rate: 0.2 m L / m i n . (Shukia et o/.^^)

lY. ALTERNATIVE MODES OF DISPLACEMENT CHROMATOGRAPHY A. Sample Displacement Chromatography In overloaded chromatography, significant rectification of the mixture may occur during the column-loading step itself. In this situation, the highest retained component, if present in sufficiently large amounts, may itself act as a displacer for the lesser retained components. Thus, the separation in sample displacement chromatography is produced by competitive binding occuring during frontal development. While sample displacement will result in a similar resolution for the lesser retained components as in conventional displacement chromatography, the component with the highest retention will tail significantly, as in other elution processes and may result in a loss of resolution from other minor components with slightly higher retentions. The principal advantage of sample self-displacement over conventional displacement is the lack of need for an extraneous displacer compound. Sample displacement has been utilized for the preparative purification of peptides,^^ proteins, and for oligonucleotides and nucleic acids 68 B. Selective Displacement Chromatography In contrast to high molecular weight polyelectrolyte displacers, the efficacy of low molecular weight displacers is dependent on both mobile phase salt and displacer concentrations. This sensitivity to the operating conditions opens up the possibility of carrying out selective displacement where the products of interest can be selectively displaced, while the low affinity impurities can be desorbed in the induced salt gradient ahead of the displacement train and the high-affinity impurities either retained or desorbed in the displacer zone."^^ A

391

BIOSEPARATIONS BY DISPLACEMENT CHROMATOGRAPHY

1.0

160

09

Salt

140

0.8

120

0.7

Bioproduct

Eluted protein

0.6 0.5 0.4 Q.

\

Displacer

100 ^ o 80

Desorbed protein

Imp

40

0.2

Q.

b

20

0.1

FIGURE 7

8

60 «w

0.3

n

^

1

1

1

2

1

3

1

4 5 6 Effluent volume (ml)

1

1

7

8

i\

n

10

A schematic of a selective displacement process. (Kundu et o/.^')

schematic of this mode of operation is shown in Fig. 7. Selective displacement chromatography results in baseline resolution of the bioproduct of interest from the low- and high-affinity impurities at high loadings and facilitates on-line monitoring of the displacement process. This latter issue is a matter of concern when deciding where to make cuts while pooling the product in displacement chromatography. In selective displacement, the displacer acts as a "spacer" between the bioproduct of interest and the higher retained impurities and serves to reduce tailing of the bioproduct into the zone of the higher retained impurities, as is observed in overloaded step elution processes. This can enable higher loadings in selective displacement as compared to step elution processes. Kundu et al^^ have shown the application of selective displacement for separating a mixture of ribonuclease B, cytochrome c, and lysozyme on a cation-exchange column using streptomycin sulfate as a displacer (Fig. 8A). Use of the on-line UV profile to make cuts between components in this process was also demonstrated (Fig. 8B). They have also shown the selective displacement of a mixture of whey proteins on anion exchange in which /3-lactoglobulin B is displaced while eluting a-lactalbumin A in the induced salt gradient and leaving j8-lactoglobulin A to be desorbed in the zone of the displacer (p-toluene sulfonic acid) (see Fig. I I B in Section V.D). Selective displacement chromatography has also been employed for the purification of an antigenic vaccine protein from an industrial process stream.^^ This example is described in detail in Section VI.A. Selective displacement chromatography appears to be the method of choice for separations in the early part of the downstream process sequence where the levels of impurities are higher. In such a scenario, selective displacement will result in baseline resolution from impurities with markedly different retentions, while making use of the high resolving power of displacement for the separation of closely related impurities. Furthemore, the process will result in significant concentration of the desired bioproduct.

392

ABHINAV A. SHUKLA AND STEVEN M. CRAMER

100

Effluent volume (ml)

B 2.b

Ribo-B

2.0

-

1.5

-

1.0

-

I-ysozyme

3yt-c

J

o

00 0)

o c cd o CO

> 3

0.5

n

-f U '-^

\

/

1

\

1

1

1

10

15

20

Effluent volume (ml) F I G U R E 8 Displacement histogram and UV detector trace for a selective displacement process. (A) Displacement separation of a three-component protein mixture using streptomycin sulfate A as a displacer. Column: 100 X 5 mm i.d. strong cation exchange (8 m); carrier: 30 m M sodium phosphate buffer, pH 6.0; feed: 1.6 mL of 0.392 m M ribonuclease A, 0.42 m M horse cytochrome c and 0.34 m M lysozyme in the carrier. Total column loading: 12.7 m g / m L column; displacer: 25 m M streptomycin sulfate A; flow rate: 0.2 m L / m i n ; fraction size: 200 fxL. (Kundu et o/."*^) (B) UV detector trace monitored at 280 nm for the displacement separation shown below.

393

BIOSEPARATIONS BY DISPLACEMENT CHROMATOGRAPHY 100

I

7

PHV

6 £

1

pH 5

H50 g

i^N

/

1

LacB— J

4

1

1

1

2

Q.

— Lac A

y^

J-l

3

\ 0

u

8

10

Dimensionless time [t/(L/Vf|uJd)] F I G U R E 9 Displacement chromatography on a retained pH gradient. The presaturation and elution buffers are formed by titrating 0.05 M NaOH with MOPS and acetic acid, respectively. Adsorbent: TosoHaas TSK Q-5PW (5 /JL particles); flow rate: 0.1 mL / min; proteins: j8-lactoglobulins A and B (from Narahari et o/.,^' with permission from Elsevier Science).

C. Displacement Using Retained pH Gradients The advantages associated with the use of low molecular weight displacers in contrast to the conventional high molecular weight polyelectrolytes have greatly increased the interest in the biotechnology industry regarding displacement chromatography. Another recent discovery that could prove important for the implementation of displacement chromatography has been a process termed "displacerless displacement."^^ The use of retained pH gradients using simple buffering species has been shown to result in displacementlike profiles during the preparative separation of j8-lactoglobulins A and B (Fig. 9). This technique makes use of a retained pH transition in place of a displacer compound to displace proteins from ion-exchange supports. While several questions about this method remain unanswered, including the tendency of high concentrations of proteins to precipitate at a pH close to their pis, this technology could be significant in the years to come.

Y. METHODS DEVELOPMENT FOR DISPLACEMENT CHROMATOGRAPHY Methods development for displacement chromatography has been previously described for a Langmuirian system using a graphical approach which employs the adsorption isotherms for the displacer and the various components

394

ABHINAV A. SHUKLA AND STEVEN M. CRAMER

in the feed mixture in concert with a displacer operating line (drawn from the origin to the point on the displacer isotherm corresponding to the inlet displacer concentration^^'^^ However, this theory does not account for the induced salt gradient which is produced by the displacer front in ion-exchange systems. Furthermore, according to this approach, a compound can act as a displacer only if its adsorption isotherm lies above the isotherms of the proteins."^^ The steric mass action (SMA) model has been shown to successfully predict nonlinear, multicomponent behavior in ion-exchange systems over a range of mobile phase salt concentrations.""^"^^ It has also been widely employed as a methods development tool for displacement separations."^^'"^^'^^ In this section, we will describe several graphical techniques derived this theory which can facilitate methods development in ion-exchange displacement systems.

A. Estimation of the SMA Parameters The SMA adsorption isotherm involves three parameters: the characteristic charge (v), which is the average number of interaction sites of a molecule with the ion-exchange surface; the steric factor ( a ) , which is the average number of sites on the surface which are sterically shielded by the adsorbed molecule and which are thus unavailable for interaction with other macromolecules in solution; and the equiUbrium constant (K) for the exchange reaction between the salt counterions and the solute. The SMA isotherm for a single component is given by^^:

^°(f)(A-(';.)Qr

<')

where C and Q are the solute concentrations in the mobile and stationary phases, respectively; C^ is the background salt concentration; A is the total ionic bed capacity; and v, cr, and K are the SMA parameters. Isocratic elution under linear conditions at various mobile phase salt concentrations can be employed to determine v and X^^ by the following expression: log^' = log(/3KA'')-vlogC,

(2)

where a plot of log k' vs log Q yields a straight line with a slope oi — v and an intercept of log( jSiCA^). Here k' is the dimensionless retention time (also called the capacity factor) for a solute under a specific mobile phase salt concentration, j8 is the phase ratio, A is the ionic capacity, and K and v are the linear SMA parameters. Linear gradient experiments can also be employed to determine K and v,^'^ Equation (3) relates a solute retention volume (V^) to initial and final carrier salt concentrations (x^, x^), the gradient volume (V^), the column

BIOSEPARATIONS BY DISPLACEMENT CHROMATOGRAPHY

39^

dead volume (V^) and the SMA linear parameters (v and K): V =

xr' +

(3)

Vr.

This expression can be employed with two or more gradient experiments to determine v and K (using a least-squares fit). The gradient technique is better suited for simultaneously determining parameters for several components of a complex mixture. The parameter a can be determined by nonlinear frontal experiment(s).^^ Once the SMA parameters have been obtained, they can be employed to predict the nonlinear behavior of solutes under any mobile phase salt concentration. B. The Dynamic Affinity Plot It has been shown by a stability analysis^^ that the measure of relative affinities in a displacement separation is the dynamic affinity of a species which is given by

[^

'=ij

(4)

where A is the slope of the displacer operating line and can be calculated from the expression: A= ^

(5)

where Q^ and Q are the stationary phase and mobile phase concentrations of the displacer, respectively. Taking the logarithm of both sides of Eq. (4) yields logi<:> log(A) -h log(A)i/

(6)

Thus, a plot of log K vs v (dynamic affinity plot) defines two regions demarcated by a line with slope log(A) and intercept log(A). The line originates at the point A on the ordinate axis and passes through the point defined by the parameters K and v of the species. The region above the affinity line includes all solutes which will displace the solute when traveling at a velocity characterized by A. Conversely, the solutes in the region below the affinity line will be displaced by the solute under these conditions. A dynamic affinity plot is presented in Fig. 10 for the proteins bovine and horse heart cytochrome c on a cation-exchange column. As one moves in a counterclockwise direction, the dynamic affinity of the molecule increases. The dynamic affinity plot provides an easy method of determining the order of elution in a displacement experiment as is shown in Fig. 4B.

396

ABHINAV A. SHUKLA AND STEVEN M. CRAMER 100

Direction of increasing dynamic affinity c o o E 3 3

Horse Cyt-c

00.1

000, 3

4

Characteristic charge F I G U R E 10 Dynamic affinity plot for cytochrome c's from bovine and horse heart and BAEE under the condition described in Fig. 4B. (Kundu et al.^^)

C. Affinity Ranking Plot While the dynamic affinity plot provides a simple graphical representation of the order of elution in a displacement, that order is restricted to the particular operating conditions chosen for that experiment (i.e., for a specified value of A). In contrast, the affinity ranking plot can shov^ the variation of the dynamic affinity of a molecule over a range of A values. To enable this comparison, the dynamic affinity A (w^hich is the criterion for displacement) can be plotted against the displacer partition ratio A (which is the operating variable for displacement). The equation for such a plot can be determined by rearrangement of Eq. (6) as 1 1 log(A) = - l o g K - - l o g ( A )

(7)

For a given A value, the higher the A value, the better the displacer under those operating conditions. As expected, an increase in the A value as one moves along the x axis (v^hich corresponds to lovsrer displacer concentrations or lowrer salt concentrations) results in a decrease in the value of the dynamic affinity (A). This plot has been employed to rank the relative efficacy of displacers^^'^"^ and to provide insight into the important structural features of low molecular weight displacers (Section VII). D. Operating Regime Plot While the displacer ranking plot described in the preceding section is useful for the identification of displacers possessing sufficient affinity for a given separation problem, it does not directly give the actual operating conditions. Operating conditions can be selected by an operating regime plot (plot of displacer concentration vs salt concentration"^^). This plot has separate curves

BIOSEPARATIONS BY DISPLACEMENT CHROMATOGRAPHY

397

for the displacement and elution of solutes. For a given solute to be displaced by a given displacer, the displacer concentration should be above the minimum value required to displace the solute. The operating line for displacement is given by

w^here C^ is the mobile phase displacer concentration, Q^i^ is the mobile phase salt concentration in the displacer zone, A is the ionic bed capacity, K^ is the SMA equilibrium constant for the displacer, v^ is the displacer characteristic charge, and a^ is the steric factor for the displacer. The A value for Eq. (8) is the minimum value of A at which the displacer can displace the protein and is given by VJ

log A =

^

log K^ - v^ log ^ ^

KJ

^

(9)

v^here the subscript p refers to the protein and the subscript d refers to the displacer. In addition to the requirement for a minimum displacer concentration to effect a displacement, there is also a constraint on the magnitude of the salt gradient induced by the displacement front. If the induced salt gradient is very large, elution of the protein ahead of the displacer breakthrough may occur. The equation for the elution curve is given by K d\

Qalt

K,

r—^'<—r *""

Thus, for a given salt concentration (Q^it), Eq. (10) can be used to obtain the corresponding value of the displacer stationary phase concentration (Q^). Substituting the value of Q j thus obtained into the equation for the SMA isotherm (Eq. 1), the corresponding value of C^ can be obtained. This set of implicit equations can be readily employed to generate the elution curve for that specific displacer and protein. The elution curve (Fig. 11 A), maps the minimum displacer concentration v^hich will produce an induced salt gradient that will result in elution of the protein. Thus, for a given combination of mobile phase salt and displacer concentrations, if the point lies in the elution region (above the elution curve), the protein will not be displaced by the displacer but will elute in the induced salt gradient ahead of the displacer front. On the other hand, if it lies in the displacement region (below the elution curve), the protein will be displaced.

398

ABHINAV A. SHUKLA AND STEVEN M. CRAMER

100

Region A: displacement of a-lact A, p-lact B, p-lact A Region B: displacement of p-lact B, p-lact A Region C: displacement of a-lact A, p-lact B Region D: displacement of p-lact B Region E: displacement of a-lact A Region F: no displacement

90 80

\>^^^EIution curve for a-lact A

70 o c o

60

A ^ ^ ^ \

B

C

50

Displacement line for p-lact A

^

D

CD

CO

40

Q

30 h

Displacement line for (3-lact B

E

20

^ V

F

10 0 10

1

1

20

30

1

1

n

1

1

40

50

60

70

80

Salt cone. (mM)

B

Effluent volume (nnl) F I G U R E I I (A) Operating regimes plot for a system of milk proteins and p-toluene sulfonic acid (PTS) as the displacer on a Waters strong anion-exchange column, pH 7.5 (B) Selective displacement separation of three milk proteins with PTS as the displacer. Column: 100 X 5 mm i.d. strong anion-exchanger (8 fju); carrier: 50 m M Tris buffer, pH 7.5; feed: 1.8 mL of 0.36 m M a-lactalbumin and 0.335 m M of a mixture of /3-lactoglobulin A and B in the carrier; total column loading: 15.7 m g / m L column; displacer concentration: 46 m M ; flow rate: 0.2 m L / m i n ; fraction size: 200 fxL (Kundu et

An operating regimes plot has been employed to develop a selective displacement process for a system of whey proteins."^^ Figure l l A shoves the displacement lines for the proteins j8-lactoglobulin A and B and the elution curve for a-lactalbumin A using p-toluene sulfonic acid (PTS) as the displacer. To displace j8-lactoglobulin B, desorb /3-lactoglobulin A, and elute a-lactalbumin A in the induced salt gradient one must operate: (1) above the

BIOSEPARATIONS BY DISPLACEMENT CHROMATOGRAPHY

399

displacement line for /3-lactoglobulin B (2) below the displacement line for /3-lactoglobulin A, and (3) above the elution curve of a-lactalbumin A. Thus, one must operate in region D of the operating regimes plot. A displacement experiment carried out w^ith operating conditions in region D resulted in selective displacement chromatography (Fig. IIB). Thus, the operating regime plot can be a useful tool for methods development, particularly in the case of selective displacement chromatography."^^'^^ E. Developing Displacement Separations A schematic for developing displacement separations is shown in Fig. 12. The first step consists of selecting a stationary phase and mobile phase conditions which result in the greatest selectivity for the separation problem at hand. The SMA parameters of the principal components of the mixture should then be determined as described earlier. The next issue at hand is the choice of the displacer. Foremost among the criteria to be considered is the affinity of the displacer. Use may be made of the displacer ranking plot to examine displacer affinity relative to the components of the mixture over a range of operating conditions. As shown in Section VII, displacer affinity can be highly dependant on the stationary phase chemistry. In general, the highest affinity displacer should be selected as it will provide the greatest flexibility in terms of operating conditions. However, apart from affinity, some of the other factors that must be

Stationary phase and buffer selection

Estinnation of SMA parameters for principal feed components

Choice of displacer i) Affinity: use of affinity ranking and dynamic affinity plots ii) Other issues: non interaction with feed components, cost, availability toxicity, disposal, availability of accurate assay protocols

Choice of operating conditions i) Operating regimes and affinity ranking plots

Optimization Experimental/numerical FIGURE

12

Flow sheet for methods development in displacement chromatography.

400

ABHINAV A. SHUKLA AND STEVEN M. CRAMER

considered while selecting the displacer include cost, availability, toxicity, the presence of accurate assay protocols, noninteraction w^ith any of the feed components (especially the product), and disposal. In practice, many of these factors may dominate the choice of the displacer. An operating regime plot can then be employed to provide further insight into the choice of operating conditions for a displacement process. Use of this plot is particularly important for a selective displacement process vs^hich typically has narrower operating regimes for successful operation. The development of displacement separations has historically been an empirical process and even though chromatographic theory may guide the selection of operating conditions the final stage must involve experimental validation. Typically, several experiments will be carried out at or near the conditions determined by the theory. The final stage in the procedure is either experimental or numerical optimization of the displacement process to produce optimal yields, purities and productivities. At this point, the relative efficacy of selective and conventional displacement chromatography can also be evaluated.

VI. DISPUCEMENT CHROMATOGRAPHY FOR THE PURIFICATION OF BIOMOLECULES: INDUSTRIAL CASE STUDIES A. Purification of an Antigenic Vaccine Protein by Selective Displacement Chromatography Selective displacement chromatography, as discussed in Section IV.B is a technique that is v^ell suited for the early stages of a dow^nstream process sequence. Selective displacement chromatography has been employed for the purification of an antigenic vaccine protein (AVP) from an industrial process stream.^^ An analytical chromatogram of the feed mixture on anion exchange is shown in Fig. 13A. As seen in the figure, most of the impurities have a higher affinity than the AVP, while a few impurities with lower retention are also present. The operating conditions for a selective displacement process were selected using an operating regime plot. A selective displacement experiment was then carried out resulting in the separation shown in Fig. 13B. The pooled AVP was assayed by a variety of techniques including size-exclusion chromatography (SEC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 13C), and isoelectric focusing (IFF) (Fig. 13D) and was shown to be of comparable purity to the product obtained from the conventional step gradient process. Importantly, the selective displacement process had the advantage of six- to sevenfold higher column loading than the gradient process. Techniques such as N-terminal sequencing and amino acid composition analysis were also employed to establish the identity of the AVP. This example provides an illustration of the efficacy of selective displacement chromatography for purifying a protein from a complex industrial mixture.

401

BIOSEPARATIONS BY DISPLACEMENT CHROMATOGRAPHY

I.U

0.9

-

High affinity/ impurity /

0.8

\ \

AVP

0.7

11

8 0.6 S 0.5

11 A

8 0.4 ^ 0.3 0.2 0.1 0 -0.1 L

10

20

30

40

50

60

Time (min)

0.9

I 40

r-|

0.8 ^0.7

_

L^

to.6hQro.5 -5 0.4

-

[\n

^' PTS

?

other impurities

0.2

\

0

J

'

25' 20 ^ o

15

CO I

10

\^^--L

0.11-

0L

30

-

\

AVP

o O

35

5 1

1

10

20

1

.••••'

30

! • • ••1

40

• • •

1

50

••

60

Volume (ml) F I G U R E 13 Purification of an antigenic vaccine protein (AVP) by selective displacement chromatography. (Shukia et o/.^°) (A) Analytical chromatogram of the AVP feed stock on anion exchange. Column: Waters Q-BHR, 5 X 100 mm; linear gradient: 20 t o 1000 m M C I " in 60 min; flow rate: 0.2 mL / min; mobile phase: pH 7.0, Tris buffer; detection: UV at 280 nm. (B) Selective displacement of the AVP. Displacer: 35 m M PTS; mobile phase: 20 m M Tris buffer; column: 10 X 290 mm DEAE Fast Flow Sepharose; loading: ~ 6.5 mL AVP feedstock per milliliter column volume; flow rate: I m L / m i n ; fraction size: I m L (C) SDS-PAGE of AVP samples from (B). Lanes I and 4: molecular weight standards; lane 2: AVP purified by displacement chromatography; lane 3: AVP purified by step gradient process (D) lEF of AVP samples from (B) performed in the pH range 3 - 1 0 . Lanes I and 4: standards; lane 2: AVP purified by displacement chromatography; lane 3: AVP purified by step gradient chromatography.

402

ABHINAV A. SHUKLA AND STEVEN M. CRAMER

F I G U R E 13

(Continued)

B. Purification of Recombinant Proteins from Variants The expression of proteins in Escherichia coli often results in the formation of protein variants that have only slight differences differ from the product. Common modifications include mistranslation of a few amino acid residues or oxidation or deamidation products. The removal of these protein variants on a preparative scale is one of the most challenging dow^nstream process steps in biomolecule purification. Displacement chromatography on a cationexchange stationary phase has been show^n to result in the effective removal of variants from recombinant human brain derived neurotrophic factor (rHu-BDNF).^^ rHu-BDNF is expressed in E. coli and harvested as an inclusion body. An analytical high-temperature reversed-phase assay of the column feedstock show^ed the presence of several peaks close to that of the native protein (Fig. 14A). These variants have been chemically characterized and have been found to be single amino acid changes to the main protein comprising mistranslations of methionine to norleucine or oxidations of methionine.^^ A room temperature analytical chromatogram on a POROS H S - M cation-exchange column did not exhibit separate peaks for the variants emphasizing the difficult nature of this separation problem. Displacements of the protein were carried out on POROS H S - M (Fig. 14B) and POROS HS50 stationary phases using protamine as a displacer. The pooled

BIOSEPARATIONS BY DISPLACEMENT CHROMATOGRAPHY

FIGURE

13

403

(Continued)

product obtained by displacement on the POROS H S - M (20 JJL particle size) had a purity of 93.6% with a yield of 7 3 % as compared to the feedstock which was 85% pure. Similar results (78% yield at 93.4% purity) were obtained on the POROS HS50 (50 JUL particle size) material by using a lower displacer concentration which had the concomitant effect of widening the zone of the displaced protein. Analytical chromatograms of the fractions from the beginning, middle, and end of the displacement train are shown in Fig. 14C. The variants which elute before the main peak on the high-temperature reversed-phase assay are present in higher levels in the first few fractions, while the higher retained impurities on the reversed-phase assay appear at higher levels in the end fractions of the zone. Thus, the order of elution of the variants appears to be the same on RPLC and cation exchange which may point to nonspecific interactions as the basis for selectivity. Productivity comparisons between displacement and linear gradient modes of operation was also carried out and showed a four to eightfold enhancement in the productivity with displacement chromatography. This example illustrates the utility of displacement chromatography for high-resolution protein separations from complex industrial feed streams.

404

ABHINAV A. SHUKLA AND STEVEN M. CRAMER

1.40 1.20 -rHuBDNF

1.00 0.80 0.60 Variants

Host cell proteins

Variants

0.40 0.20 0.00 0.00 -0.20 L_

n

10.00

1

20.00

I

T-

40.00

50.00

""—I

30.00

60.00

70, 00

Time (min)

B 14

12

r

10

8 c B o

16

r

14 12 Protamine

rHuBDNF Total protein

10

E

8

S o

6 6

QI

Q.

w

b

H4 2

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

Volume (ml) F I G U R E 14 Purification of r H u - B D N F from its variants by displacement chromatography. (Barnthouse et o/.^') (A) Analytical chromatography of feed stock by high temperature reversed-phase acetonitrile gradient. Column: 4.6 X 250 mm Vydac C4; sample: 50 [xg injection of rHu-BNDF. (B) Displacement of r H u - B D N F by protamine. Column: 4.6 X 500 mm, POROS H S / M ; loading: 20 m g / m L column volume; mobile phase: 50 m M phosphate buffer, pH 7.0, 500 m M NaCI; displacer: 2 m M protamine sulfate. (C) High-temperature reversed-phase assay of three displacement fractions. Overlay of fractions from the early (fraction 16), intermediate (fraction 18), and late (fraction 3!) parts of the displacement train.

405

BIOSEPARATIONS BY DISPLACEMENT CHROMATOGRAPHY

4.00

1

3.50 F 3.00

1 Fraction #18 (95.07% purity)

2.50

S < ^

2.001

]

1 Fraction #31 (83.21% purity)

1.50 1.00

KA

r Jv

1

if

1

9

Fraction #6 (77.85% purity)

0.50 1 0.00 0.00

-U

1

10.00

20.00

j ^

1 js.^^^

30.00 Time (min)

1

40.00

1

50.00

=i 60.00

FIGURE 14 (Continued)

C. Purification of Antisense Oligonucleotides by Displacement Chromatography Antisense oligonucleotides are a class of molecules that are rapidly gaining importance as a new class of therapeutics/^'^^ This class of compounds presents an especially difficult challenge to the separations engineer for several reasons. First, oligonucleotides exhibit an extremely high binding to anion exchange resins as compared to the molecules typically encountered in downstream processing. Second, the existence of multiple closely related failure sequences formed during chemical synthesis of these oligonucleotides presents a complex separation problem. Depending on what base is deleted in an "n - 1" failure sequence, its retention could be very similar to that of the "^''-length product. This makes resolution of the product from the failure sequences particularly challenging. The purification of a 24 mer phosphorothioate oligonucleotide contaminated with 1-12 base deletions was investigated using displacement chromatography.^^ A POROS H Q - M (20 fx particle size, perfusive particles) anion-exchange stationary phase was employed and dextran sulfate with a molecular weight of 40 kDa was used as a displacer. The displacement histogram is shown in Fig. 15. The displacement resulted in a 70% yield of the n-length product at a 96.4% purity with 1.2 g of product loaded on a 50 mmD X 100 mmL column. High mobile phase salt concentrations were utilized to improve the kinetics of adsorption and desorption in an attempt to obtain higher purities for the oligonucleotide. However, this did not improve the separation. Use of a higher temperature for the process

406

ABHINAV A. SHUKLA AND STEVEN M. CRAMER

8

100

7

90 80^

6|-

70^ o 60 o O 50 Q-

^ 5

£4 d § 3 O

70% n-length yield @ 96.4% purity

3.1

2 1 410

20 '^ 10 0

Fraction 31

460

510

560

610

660

Volume (ml) Oligo

— - Displacer

• n-length purity

F I G U R E 15 Preparative displacement chromatography of a 24 mer phosphorothioate oligonucleotide. (Gerstner et al.P with permission from Oxford University Press.) Load: 1.2 g (6 m g / m L CV); column: 50 mmD X 100 mmL POROS H Q - M ; flow rate: 150 c m / h r (50 mL/min); mobile phase: 100 m M NaCI, 10 m M NaOH, 5% methanol; displacer: 800 mL of 7 m g / m L dextran sulfate; fraction size: 14 m L

also did not improve the selectivity of the system. A lov^ concentration of organic modifier w^as also added to the mobile phase. While this did not improve the separation, this did result in better mass recoveries. The authors concluded that many of the "n — 1" impurities are similar to the product and hence it is difficult to improve the separation beyond the extent show^n in their results. This example shoves the efficacy of displacement chromatography for another class of bioseparation problems v^here high resolutions are required.

Yll. DESIGN OF LOW MOLECUUR WEIGHT DISPUCERS While there has been significant research in displacement chromatography for identifying new^ displacers and in applying displacement for the purification of biomolecules, not much effort has been focused to date on understanding the mechanisms of affinity in ion-exchange displacement systems. In addition to the fundamental aspects, the lov^ molecular v^eight displacers employed to date have possessed moderate affinities and have been unable to displace highly retained biomolecules. Thus, there is a need for high-affinity, low^ molecular w^eight displacers that w^ould enable a v^ide range of displacement separations. Several of the important structural features of low^ molecular weight displacers have been identified on a polymethacrylate based cation exchange support (8 )Lt particle size^O. This study made use of the displacer ranking plot described in Section V.C to rank homologous series of molecules which differ from each other in predominantly one structural characteristic. The

BIOSEPARATIONS BY DISPLACEMENT CHROMATOGRAPHY

407

order of ranking of these homologous series of molecules can provide insight into the relative importance of various structural features of the displacers and aid in the rational design of high affinity displacers. One of the important structural features studied w^as the geometry of the displacer molecule. Figure 16A shoves a comparison of linear, branched, and cyclic structures, each with four charges. As seen in the figure, the linear structure is superior to the branched and cyclic structures. This increased affinity is probably due to the greater flexibility associated v^ith a linear chain, resulting in a better orientation w^ith the stationary phase surface. Another important feature studied was the role of the number of charges and the spacing between these charges on linear amines. Figure 16B shows a comparison of several linear molecules. As seen in the figure, increasing the number of charges (e.g., tetraethylene pentamine > triethylene tetramine > diethylene triamine, or spermine > spermidine) improves the efficacy of these linear displacers. Another interesting effect is that of the spacing between the charges. It turns out that the greater the spacing between the charges, the higher the affinity of the displacers. For example, while spermine and triethylene tetramine both have four charges, spermine has a greater spacing between the charges and a correspondingly higher affinity. Similarly, spermidine was shown to have higher affinity than diethylene triamine. This spacing effect could be attributed to the increased flexibility of the molecule and its ability to align itself with the charges on the stationary phase surface. Figure 16C shows a comparison of branched displacer structures including PETMA4, DPE-TMA6, PhTMA6, and PE-DMABzC14 (structures given in the figure). Thus the order of affinity of these displacers is: PETMA4 < DPETMA 6 < PhTMA6 < PE-DMABzC14 within the practical operating range of 2.6 < A < 13. The increase in affinity from PETMA4 to DPE-TMA6 is primarily due to an increase in the number of charges. The higher affinity of PhTMA6 relative to DPE-TMA6 was attributed to the presence of the benzene ring in the core of the molecule, which increased the hydrophobicity of the molecule. The PE-DMABzCl4 displacer had a dramatically higher affinity than the other molecules even though it has only four charged moieties. This result was ascribed to an increase in the overall hydrophobicity of the molecule, as well as due to the placement of benzyl moieties at the charged termini where they have the greatest possibility of interaction with the stationary phase surface. These results emphasize the importance of nonspecific interactions in determining the affinity of displacers in ion exchange chromatography. In fact, the high affinity displacers resulting from this study were shown to be able to displace rHu-BDNF which is very highly retained in cation-exchange systems.^^ The displacer ranking plot has also been employed to compare the affinity of low molecular weight displacers on various classes of ion-exchange materials.^"^ This study was carried out on three different stationary phases: a polymethacrylate (PMA)-based stationary phase (Waters SP-8HR), an agarose-based material (Pharmacia High Performance SP Sepharose), and a hydrophilized PS-DVB support (POROS HS50). These phases are representative of different classes of stationary phases typically employed for protein chromatography. The displacers employed in this study were based on the

408

ABHINAV A. SHUKLA AND STEVEN M. CRAMER

10 Triethylene tetramine

3^ c

% o

CO

c

•D

0.1 100

10 A

B 10 Spermine NHg^ Tetraethylene pentamine

NH\

^-^

NHv. ^^^

NHv

c

% o £

CO

Spermidine

'^•

Diethylene triamine

NH ^•

0.1 1

10 A

100

F I G U R E 16 Structural characteristics of low molecular weight cationic displacers on a Waters SP-8HR (8 /A) strong cation-exchange column. (Shukia et 0/.^') (A) Effect of molecular geometry on cation-exchange displacers, linear, branched and cyclic structures. Parameters: triethylene tetramine (v = 4, K = 20); pentaerythrityl tetramine (J^ = 4, K = 4.12); 1,4,8,11 tetrazacyclotetradecane (v = 3.9, K = 0.58). (B) Relative ranking of extended cation-exchange displacer structures. Parameters: spermine (v = 4, K = 36.3); spermidine (v=3, K= 12.6); tetraethylene pentamine (z^ = 5, K = 40.3); triethylene tetramine (J^ = 4, K = 20); diethylene triamine (v=3, K = 4). (C) Relative ranking of branched cation-exchange displacer structures. Parameters: PETMA4 ( j / = 2 . 6 , K = 1.52); PE-DMABzCI4 (v = 3.12, K = 70.2); DPE-TMA6 (v = 4.47, K = 0.79); PhTMA6 (v = 5.65, K = 3.47).

409

BIOSEPARATIONS BY DISPLACEMENT CHROMATOGRAPHY

PETMA4 FIGURE

16

(Continued)

PETMA4 structure with the R group on the quaternary ammonium group consisting of either a benzyl (PE-DMABzCl4), a cyclohexyl (PE-DMACyI4), a heptyl (PE-DMAHepI4), or a methyl unit (PETMA4). The cyclohexyl group has comparable hydrophobicity as the benzyl functionality, but lacks aromaticity. As seen in Fig. 17 there is a dramatic difference in the relative affinities of these homologous displacers on the three stationary phases. The trends on the Waters SP-8HR material (Fig. 17A) indicate an affinity ranking of PE-DMABzC14 > PE-DMAHepI4 > PE-DMACyI4 > PETMA4. This trend indicates that all of the molecules with hydrophobic R groups have higher affinity than the PETMA4. In addition, the results indicate that aromaticity plays an important role in the affinity of these molecules (e.g., PE-DMABzCl4 > PE-DMACyI4). The trends on the POROS HS50 resin (Fig. 17c) shows an affinity ranking of PE-DMAHepI4 > PE-DMABzCl4 = PEDMACyI4 > PETMA4. Again, the incorporation of hydrophobic R groups results in an elevated affinity. In contrast to the results on the Waters material, however, the PE-DMABzCl4 had comparable affinity to PEDMACyI4. Thus, aromaticity does not appear to play as important a role on this material. In sharp contrast to the results on the Waters and the POROS materials, there is minimal difference in the affinities of these homologous displacers on the SP-Sepharose stationary phase (Fig. 17B). The lack of hydrophobic contributions to the retention on the SP-Sepharose material is due to the highly hydrophilic nature of the polysaccharide support. These results are quite compelling in that they clearly demonstrate the marked difference in the affinity of different displacers on various stationary phase materials. Further, they highlight the effect that interactions with the stationary phase backbone can have on displacer affinity. Clearly, the choice of

410

ABHINAV A. SHUKLA AND STEVEN M. CRAMER

100

B

100 F I G U R E 17 Comparison of the effect of nonspecific interactions on displacer affinity in three classes of stationary phases. (Shul
dispiacers for a given separation problem v^ill be dependent on the stationary phase being employed for the separation.

VIII. CONCLUSIONS Preparative chromatography has achieved a unique position in the purification of biotechnology products on an industrial scale. As an increasing number of biotechnology products are approved by the Food and Drug Administration (FDA) and v^ith the rapid increases in the scales at w^hich these products are produced, there is a grow^ing need for the design of dov^nstream processes v^ith few^er, more efficient steps. While elution chro-

411

BIOSEPARATIONS BY DISPLACEMENT CHROMATOGRAPHY

100 PE-DMABZCI4

PE-DMAHepl4

PE-DMACyl4

PETMA4 PE-DMABzCI4 PETMA4 PE-DMACyl4 PE-DMAHepl4

FIGURE 17

(Continued)

matography in the linear and step gradient modes has thus far been the method of choice, these processes are often Hmited with respect to column loading, yield, or purity. Thus, there is an urgent need for preparative processes with greater efficiencies which can potentially reduce the number of unit operations in the downstream process. Displacement chromatography offers an attractive alternative to the elution mode of operation for preparative purification. As shown in this review, significant advances have occurred in the state of the art of displacement chromatography over the past decade. Several applications have been demonstrated for complex biological mixtures, novel low molecular weight displacers have been identified, and theoretical techniques have been developed to facilitate methods development. Nevertheless, there are several issues that remain to be addressed before the industry will fully adopt this technology. Further investigations must be carried out aimed to identify more cost-effective, nontoxic, and readily detectable displacers which are commercially available to the biotechnology industries. Displacers with high affinities on a range of commercially available stationary phases must be identified to facilitate the development of displacement steps on these materials. This will require significant advances in our understanding of the nature of affinity in these systems. The disposal or reuse of the displacer molecules is another issue that may need to be considered for very large scale operations. In

4 I2

ABHINAV A. SHUKLA AND STEVEN M. CRAMER

addition, for process scale operation of displacement processes the decision of where to make the cuts for pooling the product will be important. Lot to lot variability in the relative levels of the impurities and the main product may make it difficult to predetermine where the cuts should be made. On-line detection and/or rapid off-line chromatographic analysis of the fractions should be investigated to enable automation of the displacement process. On a more theoretical level, chromatographic modeling will continue to provide further insights into the development of optimal displacement separations for a range of chromatographic stationary phase materials. Finally, while displacement chromatography is currently being considered for a number of drug candidates in various stages of clinical trials, it will be very important in the long run to have an FDA approved process which successfully employs displacement chromatography.

REFERENCES 1. Dibner, M. D. (1997). Biotechnology and pharmaceuticals: 10 years later. Biopharmacology 10(9), 24-30. 2. Wheelwright, S. M. (1991). "Protein Purification: Design and Scale-up of Downstream Processing." Hanser, Munich. 3. Sofer, G., and Hagel, L. (1997). "Handbook of Process Chromatography: A Guide to Optimization, Scale-up and Validation." Academic Press, San Diego, CA. 4. Guiochon, G., Shirazi, S. G., and Katti, A. M. (1994). "Fundamentals of Preparative and Nonlinear Chromatography." Academic Press, Boston. 5. Tiselius, A. (1943). Studies uber adsorptionanalyse I. Kolloid-Z. 105, 101-111. 6. Kalasz, H., and Horvath, Cs. (1982). High Performance Displacement Chromatography of corticosteroids, / . Chromatogr. 239, 423-438. 7. Kalasz, H., and Horvath, Cs. (1981). Preparative scale separation of polymyxins by high performance displacement chromatography. / . Chromatogr. 215, 295-302. 8. Horvath, Cs., Frenz, J., and El Rassi, Z. (1983). Operating parameters in high performance displacement chromatography. / . Chromatogr. 255, 273-293. 9. Cramer, S. M., and Horvath, Cs. (1988). Displacement chromatography in peptide purification. Prep. Chromatogr. 1, 29-49. 10. Kalghatgi, K., Fellegvari, I., and Horvath, Cs. (1992). Rapid displacement chromatography of melittin on micropellicular octadecyl silica. / . Chromatogr. 604, 4 7 - 5 3 . 11. Rathore, A. S., and Horvath, Cs. (1997). Displacement chromatography with on-column isomerization. / . Chromatogr. 7S7, 1-12. 12. Bendall, J. R., Partridge, S. M., and Westall, R. G. (1947). Displacement chromatography on cation-exchange materials. Nature (London) 160, 374-375. 13. Carta, G., and Dinerman, A. (1994). Displacement chromatography of amino acids: Effects of selectivity reversal. AIChEJ. 40, 1618-1628. 14. Quintero, G., Vo, M., Farkas, G., and Vigh, G. (1995). Series of homologous displacers for preparative chiral displacement chromatographic separations on Cyclobond II columns. / . Chromatogr. 693, 1-5. 15. Peterson, E. A. (1978). Ion exchange displacement chromatography of serum proteins using carboxymethyldextrans as displacers. Anal. Biochem. 90, 767-784. 16. Torres, A. R., and Peterson, E. A. (1979). Concentration of proteins using carboxymethyldextran. Anal. Biochem. 98, 353-357. 17. Peterson, E. A., and Torres, A. R. (1983). Ion-exchange displacement chromatography of proteins using narrow range carboxymethyldextrans. Anal. Biochem. 130, 271-282.

BIOSEPARATIONS BY DISPLACEMENT CHROMATOGRAPHY

4 I 3

18. Torres, E. A., and Peterson, E. A. (1992). Purification of complex protein mixtures by ion-exchange displacement chromatography using spacer displacers. / . Chromatogr. 604, 39-46. 19. Liao, A. W., El Rassi, Z., LeMaster, D. M., and Horvath, Cs. (1987). High performance displacement chromatography of proteins: Separation of j8-lactoglobulins A and B. Chromatographia. 24, 881-885. 20. Lee, A. L., Liao, A. W., and Horvath, Cs. (1988). Tandem separation schemes for preparative high performance liquid chromatography of proteins. / . Chromatogr. 443, 3 1 - 4 3 . 21. Liao, A. W., and Horvath, Cs. (1990). Purification of j8-galactosidase by combined frontal and displacement chromatography. Ann. N. Y. Acad. Set. 589, 182-191. 22. Subramanian, G., Phillips, M. W., and Cramer, S. M. (1988). Displacement chromatography of proteins. / . Chromatogr. 439, 341-351. 23. Gerstner, J. A., and Cramer, S. M. (1992). Cation exchange displacement chromatography of proteins v^ith protamine displacers: Effect of induced salt gradients. Biotechnol. Frog. 8, 540-545. 24. Jayaraman, G., Gadam, S. D., and Cramer, S. M. (1993). Ion-exchange displacement chromatography of proteins: Dextran based polyelectrolytes as high affinity displacers. / . Chromatogr. 630, 53-68. 25. Gerstner, J. A., and Cramer, S. M. (1992). Heparin as a non-toxic displacer for anionexchange displacement chromatography of proteins. Biopharmacology, 5, 4 2 - 4 5 . 26. Gadam, S. D., Jayaraman, G., and Cramer, S. M. (1993). Characterization of nonlinear adsorption properties of dextran-based polyelectrolyte displacers in ion-exchange systems. / . Chromatogr. 630, 37-52. 27. Patrickios, C. J., Gadam, S. D., Cramer, S. M., Hertler, W. R., and Hatton, T. A. (1995). Novel acryhc block copolymeric displacers for ion-exchange separation of proteins. Biotechnol. Prog. 11, 3 3 - 3 8 . 28. Ghose, S., and Mattiason, B. J. (1991). Evaluation of displacement chromatography for the recovery of lactate dehydrogenase from beef heart under scale-up conditions. / . Chromatogr. 547, 145-153. 29. Jen, S. C. D., and Pinto, N. G. (1990). Use of the sodium salt of poly(vinyl) sulfonic acid as a lov^ molecular w^eight displacer for protein separations by ion-exchange displacement chromatography. / . Chromatogr. 519, 87-98. 30. Jen, S. C. D., and Pinto, N. G. (1991). Dextran sulfate as a displacer for the displacement chromatography of pharmaceutical proteins. / . Chromatogr. Sci. 29, 478-484. 31. Jen, S. C. D., and Pinto, N. G. (1995). Nonlinear chromatography of b-lactoglobulins A and B, non-langmuirian behavior. Ind. Eng. Chem. Res. 34, 2685-2691. 32. Gerstner, J. A., Morris, J., Hunt, T., Hamilton, T., and Afeyan, N. B. (1995). Rapid ion-exchange displacement chromatography of proteins on perfusive chromatographic supports. / . Chromatogr. 695, 195-204. 33. Gerstner, J. A., Londo, T., Hunt, T., Morris, J., Pedroso, P., and Hamilton, R. (1995). Take another look at displacement chromatography. Chemtech, November, pp. 27-32. 34. Vogt, S., and Freitag, R. (1998). Displacement chromatography using the UNO continuous bed column as a stationary phase. Biotechnol. Prog. 14, 742-748. 35. Peterson, E. A., and Torres, A. R. (1984). Displacement chromatography of proteins. In "Methods in Enzymology" (W. B. Jakoby, ed.). Vol. 104, pp. 113-133. Academic Press, New York. 36. Dunn, B. E., Edberg, S. C , and Torres, A. R. (1988). Purification of E. cofi alkaline phosphatase on an ion-exchange HPLC column using carboxymethyl dextrans. Anal. Biochem. 168, 25-30. 37. Subramanian, G., and Cramer, S. M. (1989). Displacement chromatography of proteins under elevated flov^ rate and crossing isotherm conditions. Biotechnol. Prog. 5, 92. 38. Gadam, S. D., and Cramer, S. M. (1994). Salt effects in anion exchange displacement chromatography: Comparison of pentosan polysulfate and dextran sulfate as displacers. Chromatographia 39, 409-418. 39. Barnthouse, K. A., Trompeter, W., Jones, R., Inampudi, P., Rupp, R., and Cramer, S. M. (1998). Cation exchange displacement chromatography for the purification of recombinant protein therapeutics from variants. / . Biotechnol. 66, 125-136.

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40. Cramer, S. M., Moore, J. A., Kundu, A., Li, Y., and Jayaraman, G. (1995). Displacement chromatography of proteins using low molecular weight displacers. U. S. Pat. 5,478,924. 41. Cramer, S. M., Moore, J. A., Kundu, A., Li, Y., and Jayaraman, G. (1997). Displacement chromatography of proteins using low molecular weight anionic displacers. U.S. Pat. 5,606,033. 42. Kundu, A., Shukla, A. A., Barnthouse, K. A., Moore, J., and Cramer, S. M. (1997). Displacement chromatography of proteins using sucrose octasulfate. Biopharmacology 10(5), 64-68. 43. Kundu, A., Barnthouse, K. A., and Cramer, S. M. (1997). Selective displacement chromatography of proteins. Biotechnol. Bioeng. 56, 119-129. 44. Jayaraman, G., Li, Y. F., Moore, J. A., and Cramer, S. M. (1995). Ion-exchange displacement chromatography of proteins: dendritic polymers as novel displacers. / . Chromatogr. 702, 143-155. 45. Kundu, A., Vunnum, S., and Cramer, S. M. (1995). Protected amino acids as novel low molecular mass displacers in ion exchange displacement chromatography. Biotechnol. Bioeng. 48, 452-460. 46. Kundu, A., Vunnum, S., and Cramer, S. M. (1995). Antibiotics as low molecular mass displacers in ion exchange displacement chromatography. / . Chromatogr. 707, 57-67. 47. Gallant, S. R., and Cramer, S. M. (1997). Productivity and operating regimes in protein chromatography using low molecular weight displacers. / . Chromatogr. 771, 9-22. 48. Vogt, S., and Freitag, R. (1997). Comparison of anion-exchange and hydroxyapatite displacement chromatography for the isolation of whey proteins. / . Chromatogr. 760, 125-137. 49. Kundu, A., and Cramer, S. M. (1997). Low molecular weight displacers for high resolution protein separations. Anal. Biochem. 248, 111-116. 50. Shukla, A. A., Hopfer, R. L., Chakravarti, D. N., Bortell, E., and Cramer, S. M. (1998). Purification of an antigenic vaccine protein by selective displacement chromatography. Biotechnol. Prog. 14, 9 2 - 1 0 1 . 51. Shukla, A. A., Barnthouse, K. A., Bae, S. S., Moore, J. A., and Cramer, S. M. (1998). Structural characteristics of low molecular mass displacers for cation exchange chromatography. / . Chromatogr. 814, 83-95. 52. Shukla, A. A., Bae, S. S., Barnthouse, K. A., Moore, J. A., and Cramer, S. M. (1998). Synthesis and characterization of low molecular mass displacers for cation exchange chromatography. Ind. Eng. Chem. Res. 37, 4090-4098. 53. Kundu, A., Vunnum, S., and Cramer, S. M. (1998). Displacement chromatography of proteins using low molecular weight anionic displacers. Adsorption 4, 373-381. 54. Shukla, A. A., Bae, S. S., Moore, J. A., and Cramer, S. M. (1998). Structural characteristics of low molecular mass displacers for cation-exchange chromatography II: Role of the stationary phase. / . Chromatogr. 827(2), 295-310. 55. Kim, Y. J., and Cramer, S. M. (1991). Metal affinity displacement chromatography of proteins. / . Chromatogr. 549, 89-99. 56. Kim, Y. J., and Cramer, S. M. (1994). Experimental studies in metal affinity displacement chromatography of proteins. / . Chromatogr. 686, 193-203. 57. Vunnum, S., Gallant, S., Kim, Y. J. and Cramer, S. M. (1995). Immobilized Metal Affinity Chromatography: Modeling of nonlinear multicomponent equilibrium. Chem. Eng. Sci. 50, 1785-1803. 58. Vunnum, S., Gallant, S., and Cramer, S. M. (1996). Immobilized Metal Affinity Chromatography: Displacer characteristics of traditional mobile phase modifiers. Biotechnol. Prog. 12, 84-91. 59. Galaev, I. Y., Arvidsson, P., and Mattiason, B. (1995). Polymer displacement in dye affinity chromatography. / . Chromatogr. 710, 259-266. 60. Horvath, Cs., Melander, W., and Molnar, I. (1976). Solvophobic interactions in liquid chromatography with nonpolar stationary phases. / . Chromatogr. 125, 129-156. 61. Melander, W., and Horvath, Cs. (1977). Salt effects on hydrophobic interactions in precipitation and chromatography of proteins: An interpretation of the lyotropic series. Arch. Biochem. Biophys. 183, 200-215. 62. Frenz, J., and Horvath, Cs. (1985). High Performance Displacement Chromatography: Calculation and experimental verification of zone development. AIChE / . 31, 400-409.

BIOSEPARATIONS BY DISPLACEMENT CHROMATOGRAPHY

4 I 5

63. Antia, F. D., Fellegvari, L, and Horvath, Cs. (1995). Displacement of proteins in hydrophobic interaction chromatography. Ind. Eng. Chem. Res. 34, 2796-2804. 64. Ruaan, R. C , Hsu, D., Chen, W. Y., Chen, H., and Lin, M. S. (1998). Protein separation by hydrophobic interaction chromatography using methacryhc copolymers as displacers. / . Chromatogr. 824, 3 5 - 4 3 . 65. Shukla, A. A., Sunasara, K., and Cramer, S. M. (2000). Hydrophobic displacement chromatography of proteins. Biotechnol. Bioeng. (in press). 66. Hodges, R. S., Burke, T. W. L., and Mant, C. T. (1988). Preparative purification of peptides by reversed phase chromatography—sample displacement versus gradient elution modes. / . Chromatogr. 444, 349-362. 67. Veeraraghavan, K., Bernier, A., and Braendli, E. (1991). Sample displacement mode chromatography: Purification of proteins by use of a high performance anion exchange column. / . Chromatogr. 541, 207-220. 68. Deshmukh, R., Leitch, W. E., and Cole, D. L. (1998). Application of sample displacement techniques to the purification of synthetic oligonucleotides and nucleic acids: A mini-review with experimental results. / . Chromatogr. 806, 77-92. 69. Narahari, C , Strong, J., and Frey, D. D. (1998). Displacement chromatography of proteins using a self sharpening pH front formed by adsorbed buffering species as the displacer. / . Chromatogr. 825, 115-126. 70. Horvath, Cs., Nahum, A., and Frenz, J. (1981). High performance displacement chromatography. / . Chromatogr. 218, 365. 71. Brooks, C. A., and Cramer, S. M. (1992). Steric Mass Action Ion-exchange: Displacement profiles and induced sah gradients. AIChEJ. 38, 1969-1978. 72. Gallant, S., Kundu, A., and Cramer, S. M. (1995). Optimization of step gradient separations —Consideration of nonlinear adsorption. Biotechnol. Bioeng. 47, 355-372. 73. Gallant, S., Kundu, A., and Cramer, S. M., (1995). Modeling nonlinear elution of proteins in ion-exchange chromatography, / . Chromatogr. 702, 125-142. 74. Gallant, S., Vunnum, S., and Cramer, S. M. (1996). Modeling gradient elution of proteins in ion exchange chromatography. AIChE J. 42, 2511-2520. 75. Gallant, S., Vunnum, S., and Cramer, S. M. (1996). Optimization of preparative ionexchange chromatography of proteins-finear gradient separations. / . Chromatogr. 725, 295-314. 76. Sunasara, K. M., Cramer, S. M., Hauer, C. R., Rupp, R. G., and Shoup, V. A. (1999). Characterization of recombinant human brain-derived neurotrophic factor variants. Arch. Biochem. Biophys. 372(2), 248-260. 77. Crooke, S. T., and Bennett, C. F. (1996). Progress in antisense oligonucleotide therapeutics. Annu. Rev. Pharmacol. Toxicol. 36, 107-129. 78. Stein, C. A., and Cheng, Y. C. (1993). Antisense oligonucleotides as therapeutic agents—is the bullet really magical? Science 261, 1004-1012. 79. Gerstner, J. A., Pedroso, P., Morris, J., and Bergot, B. J. (1995). Gram scale purification of phosphorothioate oligonucleotides using ion-exchange displacement chromatography. Nucleic Acids Res. 23, 2292-2299.

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PHYSICOCHEMICAL BASIS OF EXPANDED-BED ADSORPTION FOR PROTEIN PURIFICATION B. M A T T I A S S O N A N D M. P. N A N D A K U M A R Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, Lund, Sweden

I. INTRODUCTION II. TYPICAL PROCEDURE T O OPERATE A N EXPANDED-BED CHROMATOGRAPHIC SYSTEM A. Expansion Behavior B. Washing C. Support Material D. Dense Particles E. Sanitation F. Viscosity III. LIGAND SELECTION IV. APPLICATIONS A. Recovery of Protein B. Analytical Applications V. CONCLUSION REFERENCES

I. INTRODUCTION When purifying biomolecules from a complex mixture, a sequence of unit operations is normally needed. Each of these steps involves losses of substance and will add to the overall cost of the process. One v^ay to simplify the situation has been through process integration. This may either be by combining a bioconversion step v^ith one or two steps involving separation. However, one can also combine two or more steps in downstream processing, thereby reducing the number of different processing steps that are needed. Still another way may be to design processing steps that eliminate the needs for a certain treatment (see page 426). This was the case when the concept of affinity-mediated separation came about; since then the use of biospecificity in the interaction replaced several different steps that earlier had to be used.^ Separation Science and Technology, Volume 2 Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

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B. MATTIASSON AND M. P. NANDAKUMAR

A key problem that per se does not involve difficult theoretical challenges, but rather technical and economical ones, is the need to remove particulate matter before any substantial chromatography can be applied. The problem is that particulate matter causes clogging of the column and thereby destroys the separation powder. Another option is to use a batch procedure in which an adsorbent is added directly to the feedstock in a stirred tank. The advantage of this method is that the product is captured directly from the unclarified feedstock; how^ever, the disadvantage is that the stirred tank acts as one theoretical plate in a separation process, which leads to a long process time due to poor contacting efficiency.^ One way to circumvent such problems is to use fluidized-bed adsorption instead of the packed-bed mode of operation. If just the adsorption-desorption of one single entity is wanted, then a fluidized bed may be sufficient. However, there is a constant mixing and thus extreme band broadening upon passage of a pulse of liquid through such a reactor. The concept of expanded-bed chromatography combines the advantages of good distances between the chromatographic beads when operated in the expanded mode and the adsorption power of the adsorbent particles without severe backmixing. The particles tend to be sorted spontaneously with regard to size and density, such that the smaller and lighter particles will be found in the top fraction of an expanded bed that has been operating until equilibrium is estabUshed. The larger and denser particles are found in the bottom section of the bed. It has furthermore elegantly been proven that a fraction from the top will be found in the top next time the column is in operation.^ It is therefore realistic to state that an expanded-bed column is stable with regard to particle size-particle density distribution. Therefore, backmixing must be less than in a fluidized bed. It has, in fact, been shown that expanded beds show a relatively low dispersion, and thus such beds would be useful for separation purposes. The idea is to use particles with adsorption ability, either nonspecific or specific, so that the target molecules are trapped while cell debris or cells and other particulate matter passes by.

II. TYPICAL PROCEDURE TO OPERATE AN EXPANDED-BED CHROMATOGRAPHIC SYSTEM The expanded-bed process is operated in a manner similar to that of a packed-bed process. The main difference is that the direction of liquid flow is upward during the expansion, feedstock application, and washing, while elution of the target protein can be done either in the expanded-bed mode or in the packed-bed form. The experimental setup is shown in Fig. 1. The following steps are involved: 1. The adsorbent in the bed is expanded to create a stable bed by passing buffer through the bed. 2. The upper flow adapter must be placed a bit above the upper level of the bed in the expanded mode, since when viscous material such as crude homogenate is added, the expansion may increase even further.

419

EXPANDED-BED ADSORPTION FOR PROTEIN PURIFICATION

V4 •^l<-

V1

V5 T1

T2

^

v3

V2

T

>i<-

w F I G U R E I Expanded-bed affinity chromatography (EBAC) setup: T l and T2, tanks for homogenate, washing, elution, and reequilibration buffers; P, peristaltic pump; C, expanded-bed column; W , wastes collector; E, eluent collector; V I , V2, V3, and V4, three-way valves; V5, four-way valve; K, knitted silicone tubing used to reduce flow pulsation.

3. The crude feedstock containing particulate matter is applied to the expanded bed until the capacity with respect to the target protein(s) is exceeded. 4. Particulate matters are removed from the bed by a washing procedure. 5. Elution of the target protein is carried out with the bed returned to the packed-bed configuration (or it can be done in expanded mode). 6. If necessary, the bed can be subject to a cleanup procedure before the next cycle of operation is initiated. The first step is to obtain a stable expanded bed. This can be done by selecting a good adsorbent and a well-defined column. There are a number of special features-challenges using expanded-bed chromatography as compared to conventional packed-bed operation. These are discussed individually in the subsequent pages, and then toward the end, some applications are presented. A. Expansion Behavior An important feature when operating expanded-bed chromatography is that the liquid is evenly distributed over the whole cross section of the column. To achieve this, certain arrangements are made. The design of proper flow distributors is an essential task. At the top it is less demanding, but one must still be secure that no mixing takes place. It is important to keep the column in a vertical mode, otherwise band-broadening may appear."^ When no flow goes through the bed, a packed bed is achieved. On increasing the flow, initially the flow will pass through the bed of sedimented

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B. MATTIASSON AND M. P. NANDAKUMAR

beads. At a certain linear velocity, the beads will start to be moved by the flow whereby the bed expands. In characterizing the behavior of a bed, one usually plots the height of the expanded bed vs the flow rate. The ideal fluidization velocity is one at which the terminal velocity of the particulate matter of the crude extract is exceeded, but that of the adsorbent particles is not. As a result, the latter are retained while the former pass out of the column with the liquid flow.^ The increase in the external voidage of the adsorbent beds enables most particulate matter present in the crude extract to pass unhindered through the column. An important feature of the expanded bed compared with a conventional fluidized bed is its low backmixing, achieved by the proper design of the adsorbent and the column. The particle size distribution of the adsorbent gives a segregated or stratified bed in expanded-bed mode. This means that the larger adsorbent particles are found in the lower part of the bed and the smaller adsorbent particles are in the upper part. Usually, adsorbents for expanded beds have particle sizes ranging from 50-400 /im. Smaller particles result in overexpansion at low flow velocities and larger particles require very high flow velocities to expand the bed sufficiently. In the first case, the overall productivity of the process will be low, and in the second case, protein binding is impaired due to the restricted diffusion into the adsorbent particles. Densities in the range of 1.1-1.3 g/cm^ are required to make it possible to operate the expanded bed at flow velocities that result in high productivities. When increasing the linear velocity in the expanded bed, the bed expansion increases and consequently also the axial dispersion. This observation was reported by Draeger and Chase^ who found that a fourfold increase in flow rate increased the axial dispersion coefficient seven times. Therefore, expansion of the bed must be restricted to maintain plug flow and multiplate process. The expansion behavior should follow the Richardson-Zaki equation. Figure 2 shows the flow behavior for a commercial STREAMLINE DEAE together with two preparations measured in the lab. One is the STREAMLINE DEAE and the other is the same polymer structure treated with a cationic polymer carrying the ligands Cibacron blue-Eudragit S 100 conjugate, thereby transferring the ion-exchanger into an affinity support. Expansion behavior according to the Richardson-Zaki equation provides reassurance that the particles behave properly. The next factor to determine is dispersion. A pulse of a tracer is passed through the bed and monitored after leaving the expanded bed. The dispersion is calculated by comparing the shape of the elution profile with that of the original plug of liquid. Often acetone or phenol is used as a tracer and is monitored using UV. Knowledge of the hydrodynamics of liquid flow and particle movement are required for scale-up and optimization of expanded-bed processes. Residence time distribution (RTD) analysis i.e., a plot of the dimensionless tracer concentration in the effluent stream versus the dimensionless time, can determine whether the liquid flow in the expanded bed is plug flow or well mixed. Using the method described by Levenspiel,^ values of mean residence time in the expanded bed (t), the dimensionless variance of the RTD curve.

421

EXPANDED-BED ADSORPTION FOR PROTEIN PURIFICATION

o *3) c

a

X CD O O

k.

D) O Q

500

Flow rate, cm/h F I G U R E 2 Bed expansion of STREAMLINE^^ DEAE reported ( • ) , measured ( O ) , and STREAMLINE"^^ DEAE loaded with Cibacron blue - Eudragit SI 00 conjugate ( • ) . (From Lall et al}^ with permission.)

and the axial dispersion coefficient (D^^) can be calculated. These values are important for characterizing the performance of the expanded bed. B. Washing When exposing a chromatographic material to a crude homogenate, obvious risks for nonspecific adsorption and fouling prevail. Washing therefore becomes even more important than it is under conventional conditions. Cell debris may be "sticky" and attach to chromatographic material, thereby changing both its surface properties and its specific density. Such fouling can lead to impaired chromatographic behavior. A further risk with such fouling is leakage of components during the washing and elution processes, which may result in contamination of the product. In conventional affinity chromatography, washing can be carried out using high ionic strength a n d / o r a shift in pH (often lowered pH is needed). Only under exceptional circumstances does one use surfactants or hydrogen bond interfering compounds (urea, guanidinium chloride, and the like). When dealing with expanded bed being in contact with cell debris, this must become a much more frequent treatment. The examples discussed later in this chapter make it clear that this is the case. C. Support Material

Most studies have been carried out with spherical agarose beads containing pieces of quartz to give it the proper density. The initial studies were done using regular Sepharose, but then the linear velocity had to be kept very low

422

B. MATTIASSON AND M. P. NANDAKUMAR

in order not to wash the beads out of the system. There is a need for a difference in density between the bed material and the liquid passing through it. Additions of quartz to the agarose solution before bead formation resulted in preparations with better flow properties. Streamline® is commercially available and is the most frequently used material. Denser materials of larger average particle diameter have been developed especially for use in expanded-bed procedures at higher linear velocities (300-1000 cm/hr).""'^ Other materials made of a composite such as dextransilica are used for expanded-bed purification.^'^^ These materials are commercially available in various particle sizes and different pore radii to enable the user to adapt the adsorbent to the product needs. The glass surface consists of silanol groups that are easily derivatized by silanizating reagents, offering a wide variety of possible reactive groups to which ligands may be attached. Before the binding capacity and resolution of the matrix is characterized, the matrix must be characterized with respect to fluidization properties. A description of the fluidizer bed behavior of several standard chromatography matrices was provided by Dasari et al}^ Liquid perfluorocarbon emulsions have also been shown to have excellent fluidization properties.^^'^^ D. Dense Particles A critical issue when dealing with expanded-bed chromatography is linear flow rate vs degree of expansion. To a certain limit, it is desirable to increase the linear flow rate, thereby improving productivity in the process. With very high density particles, flow could be so fast that the contact time for diffusion and binding to take place would be too short. There is thus an optimum when designing beads for expanded-bed behavior. Besides the beads containing quartz, particles with denser materials such as zirconium, titanium, and even hafnium have also been studied.^"^ It is important to have filling materials that will not cause nonspecific binding to the medium passing through the column. When using very dense beads, smaller particles can be used, which contributes to reduced diffusion restrictions and thereby to a facilitated binding of target molecules. E. Sanitation When reusing an expanded bed one must have guarantees that it is clean and sterile. Therefore, proper sanitation is required. In most cases, treatment with 0.5 M NaOH is recommended. After a treatment for 1 hr it is regarded that any cells present have been killed. The sanitation is often carried out as a cleaning-in-place (CIP) procedure. To meet the challenges that such treatment exerts on the system, support and attached ligands as well as the coupling link must be stable to the treatment. The most common support for expanded-bed chromatography so far is Streamline. The material consists of crosslinked agarose with entrapped quartz crystals. The experiences of cleaning processes are clear: Streamline stands up very well to the CIP treatment. In addition, ion-exchange and

EXPANDED-BED ADSORPTION FOR PROTEIN PURIFICATION

423

hydrophobic derivatives of the support material have been shov^n to be stable. When it comes to affinity matrices involving protein structures as ligands, hov^ever, the situation is more complex. Most proteins w^ill denature under the conditions offered. However, the z-fragment from protein A seems stable to relatively alkaline conditions and even more resistant forms have been created by protein engineering. Furthermore it has been demonstrated that by modifying some of the amino acid residues at the immunoglobulin (IgG)binding domain, one gets a library of proteins with different binding properties, out of which selective binders for a range of proteins were selected.^^ It is thus likely that both support and protein ligands may show good resistance to the harsh conditions of CIP treatment. F. Viscosity

The flow behavior of an expanded-bed column is very much a function of the properties of the liquid fed and the flow rate. When adding a dense homogenate to the feed, viscosity will change drastically and thereby also change the expansion behavior of the bed. The presence of nucleic acids contributes substantially to these changes. The addition of nucleases prior to chromatography substantially improves the situation. It is furthermore clear that when dealing with ion-exchange particles, low molecular weight nucleic acid fragments are preferred over long native structures. The long molecules may bind to more than one chromatographic particle, thereby crosslinking them. This phenomenon will eventually lead to channelling or formation of backwaters, i.e., it will ruin the expansion behavior of the bed. The addition of benzoase, a broad spectrum nuclease, helps to improve the situation. The general rules for expanded-bed chromatography are outlined in the preceding sections. Provided these, or at least some of them, are met, separation involving expanded-bed chromatography will be an interesting processing alternative. Next examples of separations that have been carried out are given. The focus is on the type of raw materials treated rather than target proteins isolated.

III. LIGAND SELECTION

Expanded-bed affinity chromatography is a capturing step early in the purification process. This is attractive, since thus high resolution can be used early and high resolving power can be applied early in the purification process. However, it also leads to some restrictions as compared to the situation where affinity chromatography is used as a polishing step. The rather crude material that the affinity sorbent is exposed to makes it recommendable to avoid using ligands of protein nature such as antibodies or lectins. These will soon be exposed to proteolytic attack by proteases in the homogenate. Furthermore, the risks of nonspecific binding to the support material is enhanced when working with crude preparations, and therefore it is often

424

B. MATTIASSON AND M. P. NANDAKUMAR

recommended to use cleaning-in-place conditions. These often involve washing the chromatographic column with 0.5 M NaOH, and therefore only stable ligands should be used. The trend has been toward the use of textile dyes, hydrophobic residues, and other stable molecules with at least group selectivity. The application of an early affinity step has led to a more common use of group-selective ligands, and this leads to less selective separation processes. In most cases, however, it is fully satisfying to apply a group-selective ligand in the first step, since several advantages are gained. The volume will decrease dramatically and this will make further processing less costly, and furthermore, many of the disturbing compounds such as proteases will be eliminated. By redesigning the ligands that were group specific it is possible to increase selectivity. The concept of "designer dyes" involves specific synthesis of ligands tailored to fit the binding site of the target protein. The use of shielding polymers to prevent nonspecific interactions from taking place with the ligand has also been demonstrated as a powerful tool to improve the selectivity.^^'^"^ Another alternative has been to use combinatorial chemistry to raise libraries of structures with potential binding abilities and then to screen for selectivity. This has been done in many different chemistries as well as in dye chemistry.^^ The area is developing rapidly and a forecast is that this type of ligand will increase in importance in the future. In recent years there has also been a trend toward designing stable protein ligands for CIP conditions. One such example is the ligand library based on ZZ-fragments of protein A.^^

lY. APPLICATIONS Expanded-bed adsorption has been used in a number of processes mainly for the recovery of protein from feedstocks containing cells or cell debris. It has been reviewed recently.^^ However, expanded-bed processing has also gained attention in other areas of biotechnology such as bioremediation. Immobilized expanded bed technology can also be applied in analytical applications. Note that so far the expanded bed mainly has been used with rather uncomplicated systems such as for purification of monoclonal antibodies from culture broth, isolation of extracellular substances from microbial cultivations, harvesting of fusion proteins from Escherichia coli cell homogenates, affinity isolation of certain enzymes from microbial homogenates, and separation of serum proteins from serum. To our knowledge there are very few reports on the isolation from homogenates of mammalian tissues or plant material. In a muscle homogenate, actin-myosin was found to have formed complexes that were able to settle on the chromatographic material. Moreover, the actin-myosine complexes bound various glycolytic enzymes.^^ An elution profile resulting when lactate dehydrogenase was being purified from muscle homogenate is shown in Fig. 3. Plant material is well known for its contents of tannins and various phenolic compounds that may polymerize-precipitate on the surface. It is

EXPANDED-BED ADSORPTION FOR PROTEIN PURIFICATION

425

F I G U R E 3 Breakthrough and elution profile for total protein ( # ) and L-lactate dehydrogenase activity ( O ) on an expanded-bed affinity column. (From Lali et al}^ with permission.)

well documented that some of these plant-derived materials may even react covalently with proteins, either the target or a protein ligand. The presence of polymeric carbohydrates that cause severe problems in other separation systems can be foreseen to also cause problems here. When dealing with feeds that also contain larger particles or clumps, it may be advisable to avoid liquid application units involving membranessieves. The concept of upfront chromatography^^ involving a stirred zone at the bottom provides certain advantages here. The feed is dispersed among the beads and is transported upward through the expanded bed. As long as elution is carried out in the expanded mode, there ought to be no restrictions concerning the column height used. A. Recovery of Protein The expanded-bed adsorption procedure was successfully applied for the recovery of both intracellular and extracellular protein from crude feedstock containing particulate matter. The technology has been applied by many industries both at the laboratory scale and at the pilot plant level for the recovery of several different recombinant proteins. Feedstocks of yeast cell suspensions are relatively common starting material, as the yeast cell usually exports the expressed recombinant protein efficiently in the culture medium. A process was developed for the recovery of human serum albumin produced in Pichia pastoris using a 5 cm diameter column run in expanded-bed mode and scaled up directly to a 100 cm diameter column using 150 liters of cationic exchangers. Using this size of column, it was possible to apply a 1900 liter cell suspension. The yield of human serum albumin varied from 83 to 9 1 % over four different runs.^^

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B. MATTIASSON AND M. P. NANDAKUMAR

Affinity adsorbents with immobilized biomimetic dyes have been used to purify the enzyme phosphofructokinase from nonclarified homogenates of Saccharomyces cerevisiaeP Expanded-bed affinity chromatography using a dye-hgand perfluoropolymer support was used to recover dehydrogenases from Baker's yeast with 85-94% yield. Dehydrogenase enzymes were also purified based on polymer-shielded dye ligand affinity chromatography in an expanded bed system from crude feed of porcine muscle.^"^ Recombinant proteins expressed in £. coli are successfully recovered by an expanded bed adsorption procedure. The proteins may be of cytoplasmic or periplasmic origin. In such cases, the starting material for the expanded-bed process is a cell homogenae or a cell containing periplasmic extract. Barnfield-Frej et al}^ described the recovery of annexin V from an £. coli homogenate using an ion-exchange adsorbent. At pilot scale the process was capable of treating 26 liters of cell homogenate in less than 2.5 hr in a 20 cm diameter expanded-bed column. Proteins were also recovered from E. coli and yeast cell homogenates with significantly higher purification factor.^^'^^ An example of an expanded bed process for recovery of a Pseudomonas exotoxin from an £. coli system has been recently reported in pilot plant scale.^^ A single-step recovery of a secreted recombinant protein has been carried out in expanded-bed mode directly from the fermentation broth without prior cell removal. The fusion protein was designed to have relatively low isoelectric point to enable anionic exchange adsorption at pH 5,5 where most of the E, coli host proteins are not adsorbed. The gene product was secreted to the culture medium of E. coli in high yield and the recovery of the protein was 90% in one step.^^ Feedstocks of mammalian cell cultures which are highly sensitive to changes in their environment and to shear forces have been used directly for purification of soluble proteins. Monoclonal antibodies were purified using immobilized protein A in expanded-bed mode.^^ It was possible to capture 2 g of pure mouse IgG 2a per cycle from an unclarified hybridoma cell culture, by using a 5 cm diameter expanded-bed column. Recently recombinant proteins have been recovered from milk of transgenic live stock in a one-step expanded-bed adsorption process.^^ Other applications of expanded bed purification of proteins are reviewed recently.^^ Table 2^^~^^ lists some relevant applications

T A B L E I Means to Combine Affinity Interactions with Other Separation Steps to Reduce the Total Number of Unit Operations in a Separation Process Extraction in aqueous two-phase combined with precipitation yielding a removal of cell debris in the extraction step, and elimination of most impurities in the affinity precipitation step Partitioning of affinity adsorbent particles in aqueous two-phase systems with a subsequent elution in a conventional chromatographic mode combined the advantages of the two-phase system in dealing with a particulate matter with the resolution of the chromatography. Expanded bed chromatography for removal of cell debris with concomitant capture of target molecule.

427

EXPANDED-BED ADSORPTION FOR PROTEIN PURIFICATION

• I H T A B L E 2 Applications W h e r e Expanded-Bed Chromatography has Played an Important Role

Source - material E. £. £. £.

coli coli coli coli

cell suspension homogenate homogenate homogenate

£. coli periplasmic extract Bacillus hrevis homogenate Yeast cell suspension Mammalian cell culture Plant tissue Porcine muscle

Feed volume (L)

Purification factor

Yield

(%)

Reference

NA 2 NM NM

90 95 93 50-60

29 15 32 33

2

79

28

NM

80

34

NA 30

87 83

22 30

Phenyl or butyl, Recombinant human NA STREAMLINE lysosomal enzyme Cibacrone blue Lactate-dehydrogenase 4.1

90

35

78

24

Adsorbent

Protein

8 26 NM NM

DEAE DEAE DEAE DEAE

180

DEAE

ZZ-M5 Annexin V Sialidase Human fibronectinC-274 Exotoxin

30

Cation-exchanger Human epidermal growth factor SP rHSA Protein A Monoclonal IgG

2000 60 NA NA

where expanded-bed chromatography has played an important role in the downstream processing. B. Analytical Applications A new concept of using an expanded-bed column instead of a packed-bed column in flow injection analysis was reported.^^ A miniaturized version of the expanded-bed column to an inner diameter of 0.5 cm and a length of 5-10 cm was used for the determination of lactate and glucose during fermentation without a sample pretreatment such as cell removal, which is a bottle neck in all the other analytical techniques.^^ In another set of experiments, both extracellular and intracellular proteins were determined using a setup having on-line cell lysis and capture of target protein in an antibody-based miniaturized expanded-bed column. The system was successfully used to monitor human serum albumin and intracellular j8-galactosidase^^'^^ (Fig. 4). Future applications of miniaturized expanded-bed columns could be the detection of fusion proteins from crude fermentation broth, which gives an alternative to the conventional analytical techniques which require a series of sample cleanup procedure prior to analysis.

Y. CONCLUSION Expanded-bed chromatography is a young technique, but it has already proven itself a powerful and versatile tool in downstream processing work. It is likely that the technology will develop rapidly in the next few years.

428

B. MATTIASSON AND M. P. NANDAKUMAR

40

X

g 30

X

X X

^

0

O O

S

•

o

(A

2 S P O

IS 20 (A

O Lactose I

10

1 ixx

0

g.

io OO . • •guOO 200

400 600 800 1000 cultivation time (min)

0 1200

F I G U R E 4 Monitoring of a shake-flask experiment. On-line ( • ) and off-line ( O ) /3-galactosidase activity and o.d.(x) measurements. The assay was set up using an expanded bed with immobilized lysozyme coupled to an on-line ultrasound treatment for disrupting the cells, in sequence arranged with an expanded-bed with Streamline with immobilized antibodies against j8-galactosidase. The assay was set up as a binding assay and after binding had taken place, the substrate was added to quantify the amount of trapped enzyme. A t the arrow, the inducer lactose was added. (From Tocaj et o/.^® with permission.)

ACKKOWLEDGMENTS This work was supported by the Swedish Center for Bioseparation and the Swedish Agency for Research Cooperation with Developing Countries (SAREC).

REFERENCES 1. Mattiasson, B., Olsson, U., Senstad, C , Kaul, R., and Ling, T. G. I. (1987). Affinity interactions in free solution as an initial step in down stream processing. Proc. Eur. Congr. BiotechnoL, 4th, Amsterdam, The Netherlands, 1987, Vol. 4, pp. 667-676. 2. Slater, M. J. (1991). Axial mixing and flow abnormalities. In "The Principles of Ion Exchange Technology" (M. J. Slater, ed.), pp. 41-49. Butterworth-Heinemann, Oxford. 3. Chase, H. A. (1994). Purification of protein by adsorption chromatography in expanded beds. Trends Biotechnol. 12, 296-306. 4. Bruce, L. J., and Chase, H. A. (1998). The effect of column alignment on separation efficiency in scaled down expanded bed adsorption. Int. Conf. Expanded Bed Adsorption, 2nd, Napa Valley, CA, 1998, Abstr. P7:3. 5. Draeger, M. N., and Chase, H. A. (1990). Liquid fluidized bed for protein purification. / . Chem. Eng. Symp. Ser. 118, 12.1-12.2. 6. Levenspiel, O. (1977). "Chemical Reaction Engineering." Wiley, New York. 7. Hedman, P., and Barnfield-Frej, A. K. (1992). In "Harnessing Biotechnology for the 21st Century" (M. Ladisch and Bose, eds.), pp. 271-274. Am. Chem. Soc, Washington, DC. 8. Gibson, A., and Lyddiatt, A. (1993). In "Cellulosics; Materials for Selective Separations and Other Technologies" Q. F. Kennedy, M. G. O. Philippos, and P. A. Williams, eds.), pp. 52-62. Ellis Horwood, Chichester, England.

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9. Morton, P. H., and Lyddiatt, A. (1992). In "Ion Exchange Advances" (M. J. Slater, ed.), pp. 237-244. Elsevier, Amsterdam. 10. Gilchrist, G. R., Burns, M. T., and Lyddiatt, A. (1994). SoHd phases for protein adsorption in Hquid fluidised bed. In "Separations for Biotechnology" (D. L. Pyle, ed.). Vol. 3, pp. 329-335. Royal Society of Chemistry, Cambridge. 11. Dasari, G., Prince, I., and Hearn, M. T. W. (1993). High performance Hquid chromatography of amino acids, peptides and proteins. CXXIV. Physical characterization of fluidized bed behaviour of chromatographic packing materials / . Chromatogr. 631, 115-124. 12. McCreath, G. E., Chase, H. A., Purvis, D. R., and Lowe, C. R. (1992). Novel affinity separations based on perfluorocarbon emulsions. / . Chromatogr. 597, 189-196. 13. McCreath, G. E., Chase, H. A., and Lowe, C. R. (1992). Novel affinity separations based on perfluorocarbon emulsion—use of perfluorocarbon affinity emulsions for the direct extraction of glucose-6-phosphate dehydrogenase from homogenized Baker's yeast. / . Chromatogr. 659, 275-287. 14. Voute, N., and Boscetti, E. (1998). Highly dense beaded sorbents suitable for fluid bed applications. Int. Conf. Expanded Bed Adsorption, 2nd, Napa Valley, CA, 1998, Abstr. O.IO. 15. Nord, K., Gunneriusson, E., Ringdahl, J., Stahl, S., Uhlen, M., and Nygeren, P. A. (1997). Binding protein selected from combinatorial libraries of an alpha-helical bacterial receptor domain. Nature Biotech. 15(8), 71-77. 16. Galaev, L Yu., Garg, N., and Mattiasson, B. (1994). Cibacron blue interaction with polymers: Implications for polymer-shielded dye-affinity chromatography of phosphofructokinase from Baker's yeast. / . Chromatogr. 684, 45-54. 17. Galaev, I. Yu., and Mattiasson, B. (1994). Competitive binding on the affinity matrix improves selectivity. Bio /Technology 12, 1086. 18. Li, R., Dowd, V., Stewart, D. J., Burton, S. J., and Lowe, C. R. (1998). Design, synthesis and application of a protein A minetic. Nat. Biotechnol. 6, 190-195. 19. Hjorth, R. (1997). Expanded bed adsorption in industrial bioprocessing: Recent developments. Trends Biotechnol. 15, 230-235. 20. LaU, A., Kaul, R., Galaev, I. Yu., and Mattiasson, B. (1997). Purification of L-lactate dehydrogenase from crude homogenate of porcine muscle by expanded bed affinity chromatography (EBAC). Iso. Purif. 2, 289-300. 21. Zafirakos, E., and Lihme, A. (1998). Upfront expanded bed columns—performance and scale up. Int. Conf. Expanded Bed adsorption, 2nd, Napa Valley, CA, 1998, Abstr. 0 . 1 2 . 22. Noda, M., Sumi, A., Ohmura, T., and Yokoyama, K. (1996). Eur. Pa. Appl. EP 069 968 7A2. 23. Chase, H. A., and Draeger, M. N. (1992). Expanded bed adsorption of proteins using ion-exchangers. Sep. Sci. Technol. 17, 2021-2039. 24. Garg, N., Galaev, I. Yu., and Mattiasson, B. (1996). Polymer-shielded dye-ligand chromatography of lactate dehydrogenase from porcine muscle in an expanded bed system. Bioseparation 6, 193-199. 25. Barnfield-Frej, A. K., Hjorth, R., and Hammarstrom, A. (1994). Pilot scale recovery of recombinant Annexin V from unclarified Escherichia coli homogenate using expanded bed adsorption. Biotechnol. Bioeng. 44, 922-929. 26. Chang, Y. K., and Chase, H. A. (1996). Development of operating conditions for protein purification using expanded bed techniques; The effect of degree of bed expansion on adsorption performance. Biotechnol. Bioeng. 49, 204-216. 27. McDonald, J. R., Ong, M., Shen, C , Parandoosh, Z., Sosnowski, B., Bussel, S., and Houston, L. L. (1996). Large scale purification and characterization of recombinant fibroblast growth factor-saporin mitotoxin. Protein Express. Purif. 8, 97-108. 28. Johansson, H. J., Jagersten, C , and Shiloach, J. (1996). Large scale recovery and purification of periplasmic recombinant protein from £. coli using expanded bed adsorption chromatography followed by new ion exchange media. / . Biotechnol. 48, 9-14. 29. Hansson, M., Stahl, S., Hjorth, R., Uhlen, M., and Moks, T. (1994). Single step recovery of secreted recombinant protein by expanded bed adsorption. Bio/Technology 12, 285-288.

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B. MATTIASSON AND M. P. NANDAKUMAR 30. Thommes, J., Bader, A., Halfar, M., Karau, A., and Kula, M. R. (1996). Isolation of monoclonal antibodies from cell containing hybridoma broth using a protein A coated adsorbent in expanded beds / . Chromatogr, A 752, 111-122. 31. Degener, A., Belew, M., and Velander, W. H. (1996). Expanded bed purification of a recombinant protein from the milk of transgenic livestock. 211th Am. Chem. Soc. Nat. Meet., New Orleans, LA, 1996, Abstr., p 87.9. 32. Chang, Y. K., and Chein, C. H. (1998). Simple two step procedure for purification of cloned small sialidase from unclaridied £. coli feedstocks. Int. Conf. Expanded Bed Adsorption, 2nd, Napa Valley, CA, 1998, Abstr., p. 8.2. 33. Chono, H. (1998). Purification of recombinant proteins from £. coli using Streamline. Int. Conf. Expanded Bed Adsorption, Napa Valley, CA, 1998, Abstr., p. 9.3. 34. Miyauchi, A. (1998). A commercial application of EBA in a veterinary pharmaceutical production. Development of recombinant epidermal growth factor production process using Bacillus brevis. Int. Conf. Expanded Bed Adsorption, 2nd, Napa Valley, CA, 1998, Abstr. 0.03. 35. Cameron, T. L, Liten, A. D., Lawrence, J., Farenmark, J., and Turpen, T. H. (1998). Recovery of two recombinant human lysozomal enzymes from plant tissue by expanded bed adsorption with hydrophobic resins. Int. Conf. Expanded Bed Adsorption, 2nd, Napa Valley, CA, 1998, Abstr., p. 10.2. 36. Nandakumar, M. P., Lali, A., and Mattiasson, B. (1999). On-line monitoring of glucose a n d / o r lactate in a fermentation process using an expanded micro-bed flow injection analyser. Bioseparation 8, 229-235. 37. Nandakumar, M. P., and Mattiasson, B. (1999). Binding assays in heterogeneous media using a flow injection system with an expanded micro-bed adsorption column. Bioseparation 8, 237-245. 38. Tocaj, A., Nandakumar, M. P., Hoist, O., and Mattiasson, B. (1999). Flow injection analysis of intracellular j8-galactosidase in Escherichia coli cultivations, using an on-line system including cell disruption, debris separation and immunochemical quantification. Bioseparation 8, 255-267.

EXPANDED-BED ADSORPTION PROCESS FOR PROTEIN CAPTURE JOSEPH SHILOACH Biotechnology Unit, National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, Bethesda, Maryland 20892

ROBERT M. K E N N E D Y Separations Group, Amersham Pharmacia Biotech, Piscataway, New Jersey 088SS

I. II. III. IV. V. VI.

INTRODUCTION PRINCIPLES OF EXPANDED-BED OPERATION EXPERIMENTAL STRATEGY INSTRUMENTATION MATRICES APPUCATIONS A. Capturing Extracellular Proteins B. Capture of Intracellular Proteins VII. DISCUSSION AND CONCLUSIONS REFERENCES

INTRODUCTION

The complete process for obtaining pure proteins can be divided into three main steps: capture, purification, and poHshing. The first step is the immobihzation of the target protein onto some adsorptive surface, and it can be viewed as a combination of clarification, concentration, stabilization and initial purification. Because the starting protein solution (feedstock) is usually crude, it is essential to clarify the solution. The traditional or conventional approach involves centrifugation, microfiltration, ultrafiltration, or diafiltration before the target protein solution can be loaded on an adsorbing material, utilizing packed-bed chromatography. The clarification step is a demanding operation, and is particularly difficult v^hen processing large quantities of microorganisms, especially disrupted microorganisms. Highspeed, large-scale centrifugation and microfiltration are the most common processes used to obtain protein solutions that are suitable for packed-bed chromatography; therefore, it is obvious that an approach that eliminates the Separation Science and Technology, Volume 2 Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

43 I

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JOSEPH SHILOACH AND ROBERT M. KENNEDY

clarification step can significantly simplify and improve the overall purification process. Direct adsorption of the protein not only eliminates the clarification step, but also produces a concentrated and partially purified product ready for the next purification step (Fig. 1). Several protein capture procedures, such as batch adsorption, solvent extraction, and expanded-bed adsorption, are protein capture procedures that do not require centrifugation and filtration. In this chapter wt describe the expanded-bed adsorption approach for capturing target proteins. In the expanded-bed mode, the starting protein solution is pumped upw^ard through a bed of adsorbent beads that are constrained by a flow^ adaptor.^ As a result of the upward flow and the properties of the beads, the bed expands as spaces open up between the beads. If the physical properties of the beads are significantly different from those of the particles in the feedstock, the particles can pass freely through the bed without being trapped.

Extracellular

Fermentation

Intracellular

Cell separation ppt (Cells)

Sup. Clarification

Suspension

Concentration

Cell disruption

Dialysis

Clarification

Column chromatography

Concentration

f

' Dialysis

Column chromatography

.--

F I G U R E I General purification schemes of extracellular and intracellular proteins using traditional and expanded-bed approaches. The solid lines indicate the traditional approach and the broken lines the expanded-bed approach. Using the expanded bed, when the product is extracellular, the fermentation broth Is pumped directly on the column; when the product is intracellular, the suspension of disrupted cells is pumped directly on the column.

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EXPANDED-BED ADSORPTION

An effective process, which means the formation of a stable expanded bed, depends on parameters such as the viscosity, ionic strength, sohds content, and pH of the feedstock as well as the linear flow rate. Recently, the expanded-bed approach has been successfully used to combine clarification, concentration, and initial purification. The references cited in two recent reviews provide a good summary of these applications.^'^

II. PRINCIPLES OF EXPANDED-BED OPERATION The expanded bed is, in principle, similar to a fluidized bed, a common unit-operation in the chemical industry.^ However, in the expanded-bed method, mixing of the adsorbent material is minimal during the operation, whereas a fluidized bed is turbulent. This means that an expanded bed is more effective for adsorption and elution processes than the fluidized bed. A complete review of all engineering aspects, comparing expanded beds and fluidized beds, can be found in Thommes."^ The expanded-bed adsorption operation is illustrated in Fig. 2. The overall operation is comprised of several phases. In the first phase, the adsorbent material is expanded and equiUbrated by applying an upward liquid flow to the column. To allow for sufficient contact time and efficient binding of the target molecule, the expansion should be three times that of the sedimented bed ("expansion ratio"), to a height of approximately 50 cm. A stable bed is formed when the adsorbent particles achieve equilibrium between particle sedimentation velocity and upward liquid flow velocity. In the second phase, the sample is applied to the expanded adsorbent. The crude, unclarified protein solution of intact or disrupted biomass is pumped upward on the column. In a well-defined process, the expanded adsorbent will remain stable and will not change its expansion ratio. However, if the

Before start-up sedimented adsorbent FIGURE 2

Expansion and equilibration of the adsorbent

Application of feed, followed by washing

Schematic presentation of the steps of expanded-bed adsorption.

Elution in packed bed

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JOSEPH SHILOACH AND ROBERT M. KENNEDY

process is not defined, there is a chance that the expanded adsorbent will not remain stable and will collapse or expand to the top of the column. More details are provided later in Section VI, "Application." During loading, the target proteins are bound to the adsorbent matrix, while cell debris and contaminants pass through the bed and the upper flow adaptor. The third phase is the washing of the expanded adsorbent (in an upward flow) using a buffer, most often the loading buffer. During this step, weakly bound materials, such as residual cells, cell debris, and other types of particles, are washed out. Following this step, the flow through the column is stopped and the adsorbent material settles. The upper flow adaptor is lowered to the surface of the settled adsorbent and the flow is reversed, going downward. The captured proteins are eluted from the sedimented bed, using appropriate elution solutions. The final phase, as in other adsorption processes, is the washing and regeneration of the adsorbing material. Detailed information is provided by the manufacturer.^ It is possible to use the adsorbent for a large number of runs without losing its functionality. As indicated, expanded-bed adsorption is based on controlled, stable fluidization, thus combining the hydrodynamic properties of a fluidized bed with the chromatographic properties of a packed bed. Stable fluidization with minimal backmixing results in a performance that is similar to that of a traditional plug flow column. The stability of the bed arises from the special design of the distributor plate at the base of the column and from two properties of the adsorbent: particle size distribution and particle density distribution. The distributor plate generates the required pressure drop and directs the flow only in a vertical direction, thus eliminating any radial flow that might create turbulence through the column. The size distribution and the density distribution of the beads ensure minimal local mixing. Table 1 summarizes the work of Hanson^ and illustrates the distribution of particle size and density in the bed. Particle distribution is described from the top of the column down: the top 30 mL contain 12% of the particles with an average size of 144 ^tm and an average density of 1.15 gm/mL, while the bottom 104 mL, 4 1 % of the expanded bed volume, contain particles with an average size of 238 fim and an average density of 1.19 g/mL. The polydispersity of the beads positions them at specific heights in the expanded bed; the smaller, lighter particles move to positions at the top and the larger.

TABLE I

Distribution of Beads in an Expanded Bed°

Sedimented gel volume (mL)

%

Average particle size ()Jim)

Density (gm / mL)

30 49 72 104

12 19 28 41

144 164 186 238

1.15 1.16 1.17 1.19

Degree of expansion: 2.5 folds in water, at 300 cm/hr, in a 5.0 cm diameter column.

435

EXPANDED-BED ADSORPTION

o o

o

o o o

Upper adapter o o o o O

no o

o , '^o

Qp^ O O ^

Lower adapter

booOocn I Flow

FIGURE 3

Packed-bed (a) and expanded-bed (b) columns.

heavier particles move to the bottom, resulting in a stable expansion (Fig. 3). In other v^ords, the beads find their ideal position in the column, v^hich is the reason for the lowr axial dispersion in the expanded-bed operation. The high density of the beads is necessary in order to be able to run the expanded bed at high flow velocities, thus achieving higher productivity.

III. EXPERIMENTAL STRATEGY As with other purification procedures, to develop an expanded-bed procedure, one must first follow an experimental strategy composed of method scouting, method optimization, process verification, and production. The purpose of method scouting is to define the most suitable adsorbent and the optimal conditions for binding and elution. The adsorbent of choice should be the one showing the strongest binding of the target protein, while hardly binding contaminating proteins, demonstrating the highest selectivity a n d / o r the highest capacity for the protein of interest. These initial determinations are done using packed-bed chromatography with clarified feedstock. The conditions must be verified and adjusted using the expanded bed with unclarified feedstock. The feedstock flows through the expanded bed at the relatively high flow rate of 300 to 400 cm/hr; therefore, it is important to ensure that the binding parameters of the expanded bed with the unclarified feedstock are similar to those determined using the packed bed process. Figure 4 shows breakthrough capacity curves of three different columns, one packed-bed and two expanded-bed that represent laboratory and pilot scale productions. Increasing amounts of BSA were loaded into the columns and the fraction of the unbound BSA was monitored. Please note the similar

436

JOSEPH SHILOACH AND ROBERT M. KENNEDY

C/Co Packed XK16

1.0

Streamline® 50 Streamline 200

0.8

0.6

h

0.4

0.2

0.0 00

20

40

60

Applied BSA (mg/ml adsorbent) FIGURE 4 Breakthrough curves for BSA on STREAMLINE DEAE. The breakthrough capacity is determined using frontal analysis.

shapes of the breakthrough curves for the packed-bed and expanded-bed modes, as well as for the different sizes of expanded-bed columns with internal diameters of 50 and 200 mm. This similarity demonstrates that the adsorption performance is the same in expanded-bed and packed-bed modes. The special expanded-bed column (STREAMLINE, Amersham Pharmacia Biotech, Sweden) can be scaled up while maintaining the same performance. Once a suitable maximum loading capacity is defined, it is suggested that a safety margin of 75% of the breakthrough capacity be applied, to compensate for sample variability that can affect the binding capacity. The translation of the packed-bed conditions to expanded-bed operation is done with crude, unclarified feedstock and an expanded-bed column with a diameter of 2.5 or 5 cm. Since proper operation of the expanded bed depends on stable expanded adsorbent, and the stability of the adsorbent depends on the viscosity, the ionic strength and the flow velocity of the loading solution, these parameters are adjusted.

437

EXPANDED-BED ADSORPTION

Cleaning-in-place (CIP) procedures^ are important for expanded-bed operations due to the high debris load. Most other chromatographic matrices are never exposed to such levels of contamination. It is also important to verify that bed characteristics have not been altered after the CIP. After establishing process parameters, pilot-scale work may begin with a pilot-scale column (e.g., 20 cm in diameter). For some applications, this column may be suitable for the final production, since such a column has a capacity for 4.7 liters of matrix. Further scale-up is accomplished by increasing the column diameter while maintaining the sedimented bed height, the flow velocity, and the expanded bed height. Larger columns currently in use have a diameter of 1 m.

IV. INSTRUMENTATION The basic configuration and optional setup of the expanded-bed operation is shown in Fig. 5. In addition to the special column, there is a need for two pumps and a set of valves to control the flow to and from the column. Columns for expanded-bed adsorption are commercially available from vari-

Hydraulic liquid

Waste F I G U R E 5 Basic configuration of expanded-bed column operation. Pump 2 pushes down the flow adaptor, pump I controls the upward flow of the sample and the various buffers. Valve VI is a four-way valve directing the flow to and from the column.

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JOSEPH SHILOACH AND ROBERT M. KENNEDY

ous sources. The two main features that characterize the STREAMLINE columns are the unique design of the liquid distribution system and a moveable adaptor. Columns range in size, from 2.5 cm in diameter used for method development, 5 and 10 cm used for process development, and up to 1200 cm used for production purposes. Columns ranging from 2.5 to 200 cm are built from glass, while the larger ones are made of stainless steel. Another company, Upfront (Denmark), offers an expanded-bed column with a special mixing device at its base. As mentioned, the expanded-bed operation requires two pumps: one for pumping the feedstock, the wash, and the elution solutions on the column, and the other for lowering the flow adaptor. To control flow direction, four-way valves and two-way valves are needed. Manual and automated control systems are available for operating an expanded-bed column.

V. MATRICES

Gel matrices for expanded-bed adsorption are commercially available, and their fluidization properties are reviewed by Thommes. 4 The adsorbent (e.g., STREAMLINE DEAE) exhibits a Gaussian-like distribution of particle sizes and particle densities. As explained previously, this particle polydispersity is an important factor that contributes to the stability of the expanded bed. The mean particle size of STREAMLINE beads is 200 ~ m compared to approximately 90 ~ m for matrices used in packed-bed chromatography, and the density is about 1.2 g/mL. The various matrices available under the STREAMLINE name are summarized in Tables 2 and 3. The ion-exchangers are based on highly crosslinked 6% agarose modified by including an inert quartz core to give the desired density. In the XL versions of the STREAMLINE matrices, long molecules of dextran are coupled to agarose, and the ion-exchange groups are then attached to these dextran chains. The STREAMLINE protein A is based on highly crosslinked 4% agarose modified by including an inert metal alloy core. Not mentioned in the tables is STREAMLINE phenyl used for hydrophobic interaction chromatography.

Vl. APPLICATIONS

Sections I-V addressed the principles of expanded-bed operation, the instrumentation, and the special matrices. This section focuses on applications of the expanded bed, describing actual processes and providing operational guidelines. The expanded-bed adsorption process replaces the centrifugation, clarification, and concentration steps and allows the direct adsorption of the

439

EXPANDED-BED ADSORPTION

T A B L E 2 S T R E A M L I N E Matrices Available for Ion-Exchange Expanded-Bed Adsorption Matrix

SP

SPXL

DEAE

QXL

Type of ion-exchanger Ionic capacity (mmol/mL) Particle size ({xm) Mean particle size (/xm) Approx. mean density (gm/mL) Expansion at 300 c m / h r pH stability^ Long term Short term Working velocity (cm/hr) Capacity (mg/mL) Lysozyme BSA

Strong cation 0.17-0.24 100-300 200 1.2 2-3

Strong cation 0.18-0.24 100-300 200 1.2 2-3

Weak anion 0.13-0.21 100-300 200 1.2 2-3

Strong anion 0.23-0.33 100-300 200 1.2 2-3

4-13 3-14 300

4-13 3-14 300-500

2-13 2-14 300

2-12 2-14 300-500

>60 n.d.

>140 n.d.

n.d. >40

n.d. >110

Total ionic capacity by titration. Long term refers to the pH interval v^here the gel is stable over a long period of time w^ithout adverse effects on its chromatographic performance. Short term refers to the pH interval suitable for regeneration and cleaning. For the SP and the DEAE matrices, breakthrough capacity v^as determined in a STREAMLINE 50 column at a How velocity of 300 c m / h r using a 2.0 m g / m L protein solution in 50 m M sodium phosphate buffer pH 7.5 (lysozyme) and 50 m M Tris-HCl buffer pH 7.5 (BSA). Sedimented bed height w^as 15 cm. For the SPXL and QXL matrices, breakthrough capacity v^as determined in a packed bed, 4.4 mL, at a flov^ velocity of 300 c m / h r using a 2.0 m g / m L solution of protein in 50 m M glycine-HCl buffer pH 9.0 (lysozyme) and 50 m M Tris-HCl buffer pH 7.5 (BSA). Bed height was 10 cm.

protein on a proper matrix. The conditions of a specific process depend on the source and the properties of a particular protein and on its location in the microorganism. The protein can be obtained from prokaryotic and eukaryotic microorganisms (native or recombinant), from mammalian cells and tissues, insect cells, plant tissues, and other sources. In some cases, the protein can be intracellular and in others extracellular. This application section is, therefore, divided into two parts: one covering the capture of extracellular proteins and the other covering the capture of intracellular proteins. In each case, general considerations are given, and several examples of different matrices and different protein sources are described. It is important to remember that the capture process is comprised of seven major steps: (1) loading the suitable matrix into an expanded-bed column, (2) expanding the adsorbent matrix in the proper buffer, (3) upward loading of the protein sample while the matrix is in the expansion mode, (4) washing the expanded matrix in an upward direction, (5) packing the adsorbent matrix, (6) washing the packed matrix in a downward direction, and (7) eluting the protein from the packed column.

440

JOSEPH SHILOACH AND ROBERT M. KENNEDY

T A B L E 3 S T R E A M L I N E Matrices Available for Affinity Expanded-Bed Adsorption Matrix

Chelating

Heparin

r-ProteIn A

Ligand Ligand density Particle size (fjum) Mean particle size (/im) Approx. mean density (gr/mL) Expansion at 300 c m / h r pH stability Long term Short term Working velocity (cm/hr) Capacity (mg/mL) Total Dynamic

Iminodiacetic 40 fimol Cu^+/mL 100-300 200 1.2 2-3

Heparin 4 mg/ml 100-300 200 1.2 2-3

r-Protein A 6 mg/ml 8-165 120 1.3 2-3

3-13 2-14 300

4-12 4-13 300

3-10 2-11 300-500 50 m g / m L 20 m g / m L

Heparin is therapeutic grade, derived from porcine sources. Recombinant protein A expressed in Escherichia coli. Long term refers to the pH interval where the gel is stable over a long period of time without adverse effects on its chromatographic performance. Short term refers to the pH interval suitable for regeneration and cleaning. Breakthrough capacity for STREAMLINE r-Protein A in packed bed and expanded bed, at a flow velocity of 300 cm/hr. Sedimented bed height is 15 cm. Capacity for STREAMLINE Chelating and STREAMLINE Heparin is comparable ( + 1 0 % ) to the Fast Flow versions of these matrices.

A. Capturing Extracellular Proteins i. General Considerations This category includes proteins that are produced and secreted by microorganisms, prokaryotes and eukaryotes, and by mammaUan cells grown in suspension in bioreactors. The starting solution contains the targeted protein, other proteins, intact producing cells, and media components such as carbon sources, nitrogen sources, phosphate, various metals, amino acids, and antifoam. The producing cells can be bacteria, which are approximately 1 ixm in size and have a cell wall; they can be yeast, which are of the order of 10 /xm and also have a cell wall; or they can be mammaUan cells, which are also around 10 ^tm but do not have a cell wall. Mammalian cells can be easily disrupted, thus adding intracellular components such as nucleic acids to the solution, possibly interfering in the expanded-bed process. The biomass concentration of the loading solution should be between 5 and 8% (50 to 80 g packed cells per liter) or less, and the viscosity should be below 10 mPa. The loading linear flow rate should be at least 300 cm/hr. In addition, it is important to consider the binding conditions of the targeted protein to the matrix. For example, in the case of an ion-exchanger, it is important to adjust the pH and the ionic strength of the solution, sometimes requiring a dilution

441

EXPANDED-BED ADSORPTION

that can significantly reduce the protein concentration and increase the volume. As was indicated in the introduction section, a stable expanded bed is critical to the overall expanded-bed operation. However, when dealing with a solution of intact cells, cell solid concentration and ionic strength have a minimal effect on bed stability, and therefore, the entire solution can be pumped directly on the expanded matrix. It is important to mix the loading solution continuously throughout the loading process to ensure homogeneous loading. Sometimes, depending on the cell type and media components, the upper flow adaptor can be clogged, likely due to cell aggregation. When clogging occurs, it is essential to reverse the direction of the flow for a few seconds. Various expanded-bed processes differ in the protein source, the composition of the protein solution, the type of adsorbent material, and the various buffers used for the process. ii. Practical Examples

This segment includes a detailed example of capture and recovery of extracellular mutant diphtheria toxin from Cory neb acterium diphtheria^ and some key parameters and conditions for obtaining other products. a. Niutant Diphtheria Toxin from Corynebacterium diphtheriae

CRM 9 is a mutant diphtheria toxin with a molecular weight of 60,000 secreted extracellularly from C. diphtheriae under certain growth conditions.^ Traditionally, this protein is captured on an anion-exchanger only after centrifugation, clarification, and extensive diafiltration, since the ionic strength of the medium is too high for direct adsorption (Fig. 6). When using the

Fermentation ]

^

ppt -*

, 401

Centrifugation

,, Sup ^

ppt -

Clarification

,, 401 Ultrafiltration

' 30 liters 20mM Tris PH=7.4 (NH4)2 SO4

(60 OD 600nm)

2-31

Diafiltration

'

21

(sharplessAS-16P) 1 hour (sharplessAS-16P) 1 hour (10ft2 10000 MWCo) 3-4 hours (10ft2 10,000 MWCo) 2 hours

Ammonium sulfate precipitation F I G U R E 6 Flow diagram of the traditional capture process for CRM 9. (Reprinted from Shiloach and Kaufman^ by courtesy of Marcel Dekker, Inc.)

442

JOSEPH SHILOACH AND ROBERT M. KENNEDY

10rFlow rate: Loading 400 cm/hr Elution 100 cm/hr Protein loading: 40mg/ml packed bed

o

00

CVJ

<

280

140

420

liters FIGURE 7 Chromatography of C diphtheria fermentation broth on expanded bed. Sixty liters of bacterial culture, diluted six times, were applied on 4 liters STREAMLINE DEAE in a STREAMLINE 200 column. After loading, the CRM 9 was eluted using 0.175 M KCI.

expanded-bed approach, it is possible to adsorb the protein directly from the bacterial suspension only after its proper dilution (six- to eightfold).^ The expanded-bed procedure, using STREAMLINE DEAE as the adsorbent, is performed as follows: At the end of the fermentation, the culture's conductivity is adjusted to 3.5 mS/cm by diluting the culture with 20 m M Tris buffer pH 9.0 (approximately sixfold). The culture is then pumped upwards on equilibrated STREAMLINE DEAE expanded in a STREAMLINE column at a ratio of 1 liter original culture to 80-100 mL packed resin at a flow rate of 300-400 cm/hr. After all the culture is applied to the column, it is washed with the same Tris buffer until the eluent OD (optical density) at 280 nm returns to baseline. At this point, the upward flow is stopped, the bed is

T A B L E 4 Capture-Step Comparison for Processing 40 Liters of C diphtheria Culture for Production of C R M 9 Diphtheria Toxin "Traditional" process CRM 9 yield (g) Filtration area (m^) Column volume (liters) Processing time (hr) Product volume (liters)

3.3 0.97 NA 8.0 2.5

Expanded-bed process 3.0 NA 4.8 4.0 6.0

443

EXPANDED-BED ADSORPTION

Fermentation Tris buffer 40mM PH=9.0

••

''

Dilution

loading 125 I/hour [400 cm/hr] 3 hours Washing 60! 1/2 hour

40 1 (60 OD 600nm)

'

3201

*

Washing Elution 0.175M

4.81 Expanded bed

31 |/hour [100 cm/hr.] KCI. 30 min.

ii

6.0 Liters (NH4)2 SC>4 Ammonium sulfate precipitation F I G U R E 8 Flow diagram of the expanded-bed capture process for CRM 9. (Reprinted from Shiloach and Kaufman® by courtesy of Marcel Dekker, Inc.)

allowed to settle and the flow adaptor is lowered to the surface of the packed bed. The column is then washed with two column volumes of the 20 m M Tris buffer in a downward mode at a rate of 100 cm/hr, and the CRM 9 diphtheria toxin is eluted with 20 m M Tris buffer, pH 7.4 containing 0.175 M KCI. The OD peak at 280 nm is collected (Fig. 7) and the protein is precipitated with ammonium sulfate. The overall operation can be seen in Fig. 8. A comparison between the traditional process and the expanded-bed process can be seen in Table 4. b. Aprotonin from Hansenula polymorpha^

Adsorbent: STREAMLINE SP in 20 m M sodium acetate pH 3.5 Capture conditions: cell concentration 5 %, conductivity 25 mS/cm, pH3.5 Flow rate: 300 c m / h r Wash: 20 m M Sodium acetate pH 3.5 Elution: contaminants in 0.5 M NaCl in the sodium acetate buffer; aprotinin in 0.9 M NaCl. c. Recombinant Human Serum Albumin from Pichia pastoris^^

Adsorbent: STREAMLINE SP in 50 m M acetate buffer pH 4.5, containing 50 m M NaCl Capture conditions: heat-treated yeast culture, diluted 1:2, pH 4.5, information on percent solids is not available (the estimated concentration is around 20%)

444

JOSEPH SHILOACH AND ROBERT M. KENNEDY

Flow rate: 100-250 cm / h r Wash: 50 m M acetate buffer pH 4.5, containing 50 m M NaCl Elation: 100 m M phosphate buffer pH 9.0, containing 300 m M NaCl downward at 100 c m / h r d. Recombinant Human Nerve Growth Factor (rhNGF) from Cells^^

CHO

Absorbent: STREAMLINE SP in 25 m M MES/NaMES, 0.3 M sodium acetate pH 6.0 Capture conditions: temp. 37°C. Flow rate: 375 c m / h r Wash: 25 m M MES-NaMES, 0.3 M sodium acetate pH 6.0 Elution: 25 m M MES-NaMES 1 M sodium acetate pH 6.0. e. Recombinant Monoclonal Antibody from CHO Cell Culture^^

Adsorbent: STREAMLINE SP, 25 m M MES buffer pH 5.4 Capture conditions: the cell culture is kept for 2 to 3 days to settle the cells, then the supernatant, containing 5 X 10"^ cells/mL, is diluted 3 times and the pH adjusted to 5.4 Flow rate: 135-144 c m / h r Wash: 24 volumes of 25 m M MES buffer pH 5.4 Elution: linear gradient from 40 to 400 m M NaCl in 25 m M MES, pH 5.4 f. Humanized lgG4 Monoclonal Antibody from Myeloma Cell Cu/ture'^

Adsorbent: STREAMLINE Protein A in 50 m M glycine glycinate pH 8.0, containing 250 m M NaCl Capture conditions: the cell culture suspension is applied directly at 37°C. Flow rate: 300 c m / h r Wash: 50 m M glycine glycinate pH 8.0, containing 250 m M NaCl, and additional 10 column volumes, after the OD comes back to baseline Elution: 0.1 M glycine pH 3.0. iii. Discussion

As indicated in the previous section, the expanded-bed capture process for extracellular proteins from various sources is quite straightforward. Extracellular proteins from bacteria, mammalian cells and yeast can be captured, using different types of adsorbent materials, by directly pumping the cell suspension on the expanded-bed column. Preparations prior to the adsorbing process are minimal. For example, in the case of the mutant diphtheria toxin, culture dilution was needed; in the case of recombinant human serum albumin from P. pastor is ^ the culture was heat-treated; and in the case of monoclonal antibody from CHO cell, the culture was allowed to settle for 2 - 3 days before pumping it on to the anion-exchanger. Compared with the traditional approach (Fig. 1), the expanded-bed method is shorter and requires fewer steps (Table 4). The elimination of centrifugation, filtration, and ultrafiltration is likely to reduce processing time and capital ex-

EXPANDED-BED ADSORPTION

1 ^ 1

445

T A B L E 5 Economic Considerations for Obtaining C R M 9 Diphtheria Toxin"

Equipment Consumables Time (h)

Traditional

Expanded-bed adsorption

$90,000-$120,000^ $3,000-$4,300 (filters) 15 (set up and cleaning 10 hr, operation 5 hr)

$20,000'' $3,600 (resin) 14 (set up and cleaning 6 hr. operation 5 - 7 hr)

The comparison is for processing 70 liters of bacterial culture. Equipment needed: continuous centrifuge and tangential flow filtration system. Equipment needed: expanded bed column for 4.5 liter resin.

penses. Table 5 summarizes the economical considerations of the two approaches when mutant diphtheria toxin is recovered. B. Capture of Intracellular Proteins i. General Considerations

This category includes proteins that are produced inside microorganisms or mammaUan cells. To release these proteins, the biomass is first concentrated by conventional continuous centrifugation, and then it is disrupted. Therefore, the starting protein solution contains the targeted protein, other proteins, cell contents such as cell wall fragments and DNA, but it does not contain any media components. Unlike the solution that contains extracellular proteins, this solution is not homogenous, possibly affecting the bed stability; therefore, it is more difficult to achieve a stable expanded bed when dealing with intracellular proteins than when dealing with extracellular proteins. As previously stated, the bed stability is affected by the solution's ionic strength, pH, solids concentration and viscosity, and, in this case, also by the method of cell disruption. The disrupted cell suspension, after proper adjustment, is pumped directly on the expanded bed. The solid concentration in the suspension should be between 50 and 80 g per liter, the viscosity below 10 mPa, and the loading linear flow rate not less than 300 cm/hr. Unlike the extracellular protein case that deals with intact cells, in the disrupted cells case, the ionic strength has an effect on column stability. It is important to mix the loading solution continuously throughout the loading process to ensure homogeneous loading, and to adjust the binding conditions based on the properties of the targeted protein and the choice of matrix. ii. Practical Examples

This segment includes a detailed example of capture and recovery of intracellular protein (recombinant exotoxin A from E. coli) and key parameters and conditions for obtaining other products. a. Recombinant Pseudomonas aeruginosa Exotoxin A from £. co/i'^' '^

The modified P. aeruginosa exotoxin A is overproduced in £. coli BL21 (ADE3). The modified toxin, which lacks the enzymatic activity but retains the binding activity, is missing glutamic acid in position 553. The expression

446

JOSEPH SHILOACH AND ROBERT M. KENNEDY

Sucrose20 mM Buffer-

4.5 kg Bacteria 90 I Suspension Centrifugation

2-3 h

Clarification

2-311

DEAE Sepharose fast flow 25.5 1 30 cm X 36 cm

loading 1h elution 2h .(100 cm h""")

I

36 1 F I G U R E 9 Flow diagram of the traditional capture process of recombinant exotoxin A from £ coli. (Reprinted from Johansson et o/.'^ with permission from Elsevier Science.)

of the modified toxin is controlled by the T7 promoter and the protein accumulates in the periplasmic space following induction with IPTG. When applying the traditional recovery process (Fig. 9), at the end of the fermentation, the cells are collected and suspended in two volumes of 20% sucrose in 20 m M Tris buffer pH 7.4 containing 1 m M EDTA. After the cells are well dispersed, they are mixed for an additional 10 min and then diluted with 18 volumes of 20 m M Tris buffer pH 7.4. The cell suspension is centrifuged and microfiltered through a 0.45 jubm filter. The clarified supernatant is applied on DEAE Sepharose Fast Flow at a flow rate of 100 c m / h r and a ratio of 40 OD280 nm per 1 mL. column. The elution is performed with a linear gradient of eight column volumes from 0 to 0.5 M NaCl in the Tris buffer. In contrast, when applying the expanded-bed process (Fig. 10), the collected cells are suspended in 2 volumes of 20% sucrose in 50 m M Tris buffer pH 7.4 containing 1 m M EDTA. After the cells are well dispersed.

Bacteria 4.5 kg E Sucrose50 mM buffer-

.

.

(400 cm h ') 2 1 min ^ loading-1801 washing - 40 1 2h

•

180ISu spension

i Streamline DEAE 4.71 20 cm X 15 cm

• 0.5 NaCI 0.5 I min""" (100 cm h-'')

t ,I \

13 51

F I G U R E 10 Flow diagram of the expanded-bed capture process of recombinant exotoxin A from £. coli. (Reprinted from Johansson et 0/.'^ with permission from Elsevier Science.)

EXPANDED-BED ADSORPTION m

447

T A B L E 6 Capture Step Comparison for Processing 4.5 Kg £. Co/f Cells for Production of Exotoxin A 553D a

Packed-bed DEAE Sepharose FF Specific activity after the step (mg toxin/mg protein) Recovery (%) Processing time (hr) Column volume (liters) Eluent volume (liters)

0.1 73.0 8.0 25.5 36.0

Expanded-bed STREAMLINE DEAE 0.06 79.0 2.5 4.7 13.5

Reprinted from Johansson et al}^ W\t\v permission from Elsevier Science. Packed column numbers are extrapolated values.

they are mixed for an additional 10 min and then are diluted with 18 volumes of 50 m M Tris buffer, pH 7.4. The mixture is mixed for an additional 10 min, endonuclease (Benzonase) is added at a ratio of 7S units per gram cells, and the suspension is diluted with 18 volumes of the Tris buffer. The cell suspension is then pumped upward on an equilibrated and expanded STREAMLINE DEAE at a ratio of 1 g cell extract per 1 mL packed column at a flow rate of 400 cm/hr. After all the cell suspension is applied on the column, the column is washed with 50 m M Tris buffer pH 7.4 until the OD at 280 m M of the eluent is back to baseline. At this point, the upward flow is stopped, the bed is allowed to settle, and the flow adaptor is lowered to the surface of the packed bed. After washing in a downward mode with 2 column volumes at a rate of 100 cm/hr, the protein is eluted with 20 m M Tris buffer pH 7.4 containing 0.5 M NaCl. The OD280 P^ak is collected for the next purification step. Successful adsorption procedure is achieved using the following conditions: concentration of the dry cell mass is 6 g/liter, the buffer concentration is 50 m M and the endonuclease content is 75 units per gram cells. The comparison between the traditional process and the expended-bed process is summarized in Table 6. b. Recombinant

Diphtheria

Toxin from £. c o / i ' *

Method of cell disruption: £. colt cells are suspended in the binding buffer (0.25 gram cells per mL), and the suspension is passed twice through a homogenizer (Manton Gaulin) at 9000 psi. Absorbent: STREAMLINE Chelating, saturated with NiS04 and equiUbrated in 20 m M Tris, pH 8.0 containing 0.5 M NaCl and 5 m M imidazole. (The protein is expressed with His-tag). Capturing conditions: broken cell suspension at a concentration of 0.13 g / m L in binding buffer. Flow rate: 200 m L / h Wash: 20 m M Tris pH 7.2 containing 0.5 M NaCl and 30 m M imidazole.

448

JOSEPH SHILOACH AND ROBERT M. KENNEDY

Elution: 20 m M Tris pH 7.2, containing 0.5 M NaCl and 250 m M imidazole c. Recombinant Annexin V from £. coli^^

Method of cell disruption: cell suspension is passed three times through a high-pressure homogenizer 10,500-13,500 psig. Adsorbent: STREAMLINE DEAE equilibrated in 30 m M ammonium acetate pH 5.5 Capturing conditions: broken cell suspension at a concentration of 3.6% solids in 30 m M ammonium acetate pH 5.5; the suspension contains 1% detergent Triton X-100 Flow rate: 300 c m / h r Wash: 30 m M ammonium acetate pH 5.5 Elution: 30 m M ammonium acetate pH 5.5 containing 250 m M NaCl 100 c m / h r d. Recombinant Anti-HIV Fab-Fragment from £. co/i'®

Method of cell disruption: cell suspension passed three times through high-pressure homogenizer at 10,000 psig Adsorbent: STREAMLINE SP in 50 m M sodium acetate pH 5.0 Capturing conditions: dry cell concentration, 1.4% suspended in 50 m M sodium acetate pH 5.0, containing endonuclease (benzonase) at a ratio of 10 fiL per 60 g cells (dry weight). Flow rate: 300 c m / h r Washing: 50 m M sodium acetate pH 5.0 Elution: 50 m M sodium acetate pH 5.0, containing 1 M NaCl, at 100 cm/hr e. Recombinant lnterluldn-8 from £. co/i Inclusion Bodies

Method of cell disruption: cell pellet suspended in three volumes of 30 m M sodium phosphate pH 6.5 containing 6 M guanidine hydrochloride. Suspension is mixed for 3 hr and diluted with six volumes of water in two steps: three volumes in the first step followed by mixing for 30 min, and three volumes in the second step followed by mixing overnight. Adsorbent: STREAMLINE SP in 30 m M sodium phosphate pH 6.5 Capturing conditions: 1% (dry weight) cell suspension in 30 m M sodium phosphate containing 6 M guanidine hydrochloride Flow rate: 300 c m / h r Washing: 30 m M sodium phosphate containing 6 M guanidine hydrochloride Elution: 30 m M sodium phosphate containing 6 M guanidine hydrochloride, and 0.5 M NaCl at a flow rate of 100 c m / h r f. Glucose 6 Phosphate Dehydrogenase (G6PDH) from S. cerevisiae^^

Method of cell disruption: yeast suspension 50% wet weight is passed through a bead mill using 0.5 mm glass beads

449

EXPANDED-BED ADSORPTION

Adsorbent: STREAMLINE DEAE in 50 m M sodium phosphate pH 6.0 Capturing conditions: 6.5% (dry weight) yeast suspension in 50 m M sodium phosphate pH 6.0, viscosity 5.0 mPa Flow rate: 200 c m / h r Washing: 2 5 % glycerol in 50 m M sodium phosphate pH 6.0 in an expanded mode, followed by 50 m M NaCl in 50 m M sodium phosphate pH 6.0 in a packed mode Elution: 150 m M NaCl in 50 m M sodium phosphate pH 6.0 iii. Discussion

Capturing intracellular proteins on an expanded bed involves preliminary steps that are not required for the capture of extracellular proteins. These steps are the initial cell concentration, usually by filtration or centrifugation, and cell disruption, using a high-pressure homogenizer, a bead mill, or osmotic shock. To prepare a disrupted cell suspension for direct adsorption on an expanded bed, suspension viscosity and ionic strength can be adjusted by adding detergent, endonuclease, and salt. It is also important to adjust the concentration of solids in the broken cell suspension because it affects the stability of the expanded matrix. As seen in the example of capturing recombinant Interlukin-8 from £. coli, the expanded-bed process is also suitable for capturing a protein extracted from inclusion bodies using guanidine hydrochloride. Since concentration of the cells is an essential preliminary step when processing intracellular proteins, and instrumentation for this unit operation is required, a comparson of the economical considerations between the described approach and the traditional method is different from the case of the extracellular proteins (Table 7).

Yll. DISCUSSION AND CONCLUSIONS The production examples described here, and the information published since this chapter was written, clearly indicate that the expanded-bed operation offers an efficient alternative to the conventional protein capturing process.

T A B L E 7 Economic Considerations for Obtaining Recombinant Exotoxin A (533D)°

Equipment Consumables Time (hr)

Traditional

Expanded-bed adsorption

$103,000 $1,500 (filters) $10,000 (resin) 20 (set up and cleaning 11 hr. operation 8-9 hr)

$90,000"" $3,600 (resin) 12.5 (set up and cleaning 6.5 hr. operation 6 hr)

The comparison is for processing 4.5 kg of bacterial biomass. Equipment needed: continuous centrifuge and chromatography column. Equipment needed: continuous centrifuged and expanded-bed column for 4.5 liter

450

JOSEPH SHILOACH AND ROBERT M. KENNEDY

The latter requires that the starting product solution be centrifuged, clarified, and at times, dialyzed, before it can be adsorbed on a packed column. In contrast, capturing the desired product by the expanded-bed method is simpler because the crude starting solution is pumped directly on the adsorbent. To achieve a successful expanded-bed process operation, the adsorbent must be stable in its expanded mode, requiring that careful evaluation of the loading conditions take place during the process development stage. The follow^ing points should be considered: establishing optimal loading conditions for intracellular proteins was found to be more complicated than for extracellular proteins; starting solutions with high viscosity and solid content often must be diluted; and when the loading solution is very crude, the upper flow adaptor may get clogged, requiring that the flow direction be changed frequently. Note that the expanded-bed approach is not a viable option if the binding of the desired product to the adsorbent is possible only after initial partial purification. In summary, when evaluating the suitability of the expanded-bed approach for capturing a particular protein, one must consider whether the product is from bacteria, yeast, or mammalian cells; whether it is intracellular or extracellular; and the specific activity of the final product, its overall yield, the processing time and capital, labor, and maintenance costs.

REFERENCES 1. Chase, H. A. (1994). Purification of proteins by adsorption chromatography in expanded beds. Trends Biotechnol. 12, 296-303. 2. Hjorth, R. (1997). Expanded bed adsorption in industrial bioprocessing: Recent developments. Trends Biotechnol. 15, 230-235. 3. Gailliot, E. P., Gleason, C , Wilson, J., and Zwarick, J. (1990). Fluidized bed adsorption for whole broth extraction. Biotechnol. Prog. 6, 370-375. 4. Thommes, J. (1997). Fluidized bed adsorption as a primary recovery step in protein purification. Adv. Biochem. Eng. Biotechnol. 58, 185-230. 5. Amersham Pharmacia (1995). "Application Note: Cleaning in Place," Publ. No. 18-1115-27. 6. Hanson, K. A. (1995). Physical chemical properties of STREAMLINE Ion exchangers. Poster Presentation, Eur. Congr. Biotechnol., 7th, Nice, France. 7. Fass, R., Bahar, S., Kaufman, J., and Shiloach, J. (1995). High yield production of diphtheria toxin mutants by high density culture of C7(6)*'^^^ strains grov^n in a non deferrated-media. Appl. Microbiol. Biotechnol. 43, 83-88. 8. Shiloach, J., and Kaufman, J. B. (1999). The combined use of expanded bed adsorption and gradient elution for capture and partial purification of mutant diphtheria toxin (CRM 9) from Cory neb acterium diphtheriae. Sep. Sci. Technol. 34, 29-40. 9. Zurek, C , Kubis, E., Keup, P., Hoerlein, D., Beunink, J., Thoemmes, J., Kula, M. R., Hollenberg, C. P., and Gellissen, G. (1996). Production of two Aprotonin variants in Hansenula plymorpha. Process Biochem. 31, 679-689. 10. Noda, M., Sumi, A., Ohumura, T., and Yokoyama, K. (1966). Process for purifying recombinant human serum albumin. Eur. Pat. Appl. EP 0 699 687 A2. 11. Beck, J., Liten, A., Viswanathan, S., Emery, C , and Builder, S. (1996). Direct capture of Nerve Grov^th Factor from CHO cell culture by EBA. Presentation, Recovery Biol. Prod., 8th, Tucson, AZ. 12. Batt, B. v., Yabannavar, V. M., and Singh V. (1995). Expanded bed adsorption process for protein recovery from whole mammalian cell culture broth. Bioseparation 5, 4 2 - 5 3 .

EXPANDED-BED ADSORPTION

45 I

13. Jagerstern, C , Johansson, S., Bonnerjea, J., and Pardon, R. (1996), Capture of a humanized IgG4 directly from the fermentor using STREAMLINE r-protein A. Presentation, Recovery of Biol. Prod., 8th, Tucson, AZ. 14. Fass, R., Van de Walk, M., Shiloach, A., Joslyn, A., Kaufman, J., and Shiloach, J. (1991). Use of high-density cultures of Escherichia coli for high level production of recombinant Pseudomonas aeruginosa exotoxin A. Appl. Microbiol. Biotechnol. 36, 65-69. 15. Johansson, H. J., Jagersten, C , and Shiloach, J. (1996). Large-scale recovery and purification of periplasmic recombinant protein from E. coli using expanded bed adsorption chromatography follow^ed by new ion exchange media. / . Biotechnol. 48, 9-14. 16. Noronha, S., Kaufman, J., and Shiloach, J. (1999). Use of STREAMLINE Chelating for capture and purification of poly His Tagged recombinant proteins. Bioseparation 8, 145-151. 17. Barnfield Frej, A. K., Hjorth, R., and Hammarstsrom A. (1994). Pilot scale recovery of recombinant Annexin V from unclarified £. coli homogenate using expanded bed adsorption. Biotechnol. Bioeng. 44, 922-929. 18. Jagerstern, C. (1994). Purification of recombinant Anti-HIV Fab-fragment expressed in £. coli. Presentation, Recovery Biol. Prod., 7th San Diego, CA. 19. Chang, Y. K., and Chase, H. A. (1966). Ion-exchange purification of G6PDH from unclarified yeast cell homogenate using expanded bed adsorption. Biotechnol. Bioeng. 49, 204-216.

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ADSORPTIVE MEMBRANES FOR BIOSEPARATIONS RANJIT R. DESHMUKH* Manufacturing Process Development, his Pharmaceuticals, Inc., Carlsbad, California 92008

TIMOTHY N. WARNER+ Sartorius Corporation, Edgewood, New York 11717

I. INTRODUCTION II. COMPARISON OF MEMBRANE CHROMATOGRAPHY T O TRADITIONAL CHROMATOGRAPHY A. Velocity B. Capacity C. Resolution D. Cleanability and Sterilization IN. SCALE-UP OF CHROMATOGRAPHY MEMBRANES A. General Considerations B. Scale-Up: Multimode Systems for Pilot-Scale Purification IV. APPLICATIONS OF MA TO PREPARATIVE BIOSEPARATIONS A. Purification of Proteins on MA: Case Study of Recombinant Vaccine Protein B. Negative Chromatography: Removal of Contaminants C. Case Study: Purification of Oligonucleotides V. CONCLUSIONS REFERENCES

Membrane chromatography is gaining wider interest and acceptance in the process bioseparation industry. The understanding of membrane materials, large-scale availability, and identification of applications have promoted this new technology. The focus of this chapter is on aspects of membrane adsorbers (MAs) that have the greatest impact on large-scale preparative chromatography applications in the bioindustry. We will also address a few novel technologies such as thin columns, monolithic matrices, and innovative media, which seem to cross the classical definition of chromatography media. The general technology has been reviewed,^'^ and transport related disper"" Current address: Wydeth Lederle Vaccines, One Great Valley Parkway, Malvern, Pennsylvania 19355. Current address: BioSecure, 511 Dov^ling Forge Rd., Dov^ningtown, Pennsylvania 19335 Separation Science and Technology,

Volume 2

Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

453

454

RANJIT R. DESHMUKH AND TIMOTHY N. WARNER

sion in such media has been discussed elsewhere.^"^ This chapter aims to provide a state-of-the-art look at the various MA separation modes, discuss commercially available technology, and provide guidelines to develop largescale applications. INTRODUCTION In general, membrane adsorbers are membranes with chemically functionalized surface sites for chromatography. The appeal of the membrane-based chromatographic surface stems from the fact that it is an ideal monolithic support. The functional groups are uniformly distributed over the membrane surface and convection enhanced separation is possible. On the practical side, these can be modular in format, enabling easy and convenient use. The scale-up for MA builds on the knowledge gained in scale-up of both technologies: chromatography and membrane filtration. To rival traditional chromatography, MA technology must address the age-old chromatography problems. The ligand chemistry must have high dynamic binding capacity, as well as very low nonspecific binding. The development of membrane housings must have appropriate fluid distribution to take advantage of the high resolution. The ultimate challenge is to develop scaleable membrane devices capable of good chromatography. A viable alternative to bead-based chromatographic supports should be able to deliver consistent results after repeated cleaning cycles, and should be able to fulfill all FDA regulatory guidelines. Table 1 lists the commercially manufactured modules, their available formats, and their chemistries. The geometries reflect the widely used formats in membrane filtration. These are flat sheets in filter holders, sheets wound in spiral or cylindrical configuration, and hollow-fiber membranes. Syringe filters are popular housings containing flat sheets of MA. These enable easy low-pressure chromatography with low cost, disposability, and easy connectivity of units for series operation. In a manner similar to small columns, these syringe-type MAs may be connected to chromatographic workstations for quick method development. Figure 1 shows a photograph of three syringe filters containing three (15 cm^) modules in series connected to a low-pressure workstation. The scale-up of this concept is accomplished with larger disks of flat membranes. Multiple stacks of these membranes may be placed into a stainless steel filter holder. A photograph of this configuration is shown in Fig. 2. The maximum size for such a configuration is reported to be approximately 22,000 cm^. Larger surface area can be achieved by rolling a flat sheet MA and operating it in a radial flow manner as shown in the schematic in Fig. 3. This design is discussed in detail in Section III. Hollow fibers are another popular membrane filtration configuration used at large scales. There are various forms of hollow-fiber-based MA, some embed chromatographic beads, while others are derivatized membranes.^"^^ A schematic of a small dead-end hollow-fiber-based MA is shown in Fig. 4A, and a diagram of its flow path is presented in Fig. 4B. The hollow-fiber-based MA units may enable the use of particulate-laden feed streams. Both the hollow-fiber-based and spiral wound microporous filter configurations are

455

ADSORPTIVE MEMBRANES FOR BIOSEPARATIONS

TABLE I

Summary of Commercially Available Membrane Adsorbers

Trade name

Manufacturers

Module type

Membrane material

Ligand chemistry

References

Biodyne Fractoflow

Pall Corp., East Hills, NY Merck KGaA., Darmstadt, Germany Millipore, then PE Biosystems, Framingham, MA Sartorius, Goettingen, Germany

Flat sheet Hollow fiber

Nylon Polyamide

lEX lEX

24 25

Syringe, disk

Regenerated cellulose

lEX; AF

19, 26-30

(MemSep) '

Sartobind

(ZetaAffinity)^ ZetaPlus (ZetaPrep)

Cuno Life, Meriden, CT Cuno Life, Meriden, CT LKB, Bromma, Sweden

Flat sheet Regenerated cross- lEX; AF; (syringe, disk); linked cellulose epoxy; cylindrical aldehyde; IMAC AF Disk Depth filter Charged filter aid lEX lEX Disk

31

32 33 34-38

Abbreviations for type of application: lEX: Ion Exchange, HIC: Hydrophobic interaction chromatography, AF: Affinity chromatography, RP: Reverse phase, IMAC: Immobilized metal affinity chromatography. Product under development. Product names in parentheses are currently not commercially available. Product line sold to PE BioSystems, and now discontinued.

FIGURE

I

Photograph of syringe type membrane adsorber devices. (Courtesy of Sartorius Corp.)

F I G U R E 2 Photo of stack of flat sheet disk membrane adsorbers in a filter holder. (Courtesy of Sartorius Corp.)

456

RANJIT R. DESHMUKH AND TIMOTHY N. WARNER

F I G U R E 3 Schematic of a cylindrical membrane adsorber module, operated in the radial flow mode. (Courtesy of Sartorius Corp.)

typically scaled up by connecting modules in series or in parallel banks. MA applications can be scaled up in a similar manner. Pilot-scale prototype modules with 8 m^ membrane area are now available. Commercial-scale operations of 4 m^ modules have been used in pharmaceutical manufacturing. There are other novel media with characteristics similar to functionalized membranes. Some of the commercially available media are listed in Table 2. These materials in many cases are at the cross-lines of definitions and are frequently compared in the MA literature. Organic separations in the reserve-phase (RP) and hydrophobic interaction chromatography (HIC) mode are not very common on filtration-based MA materials. However, the methacrylate copolymers can be used for this purpose. Also rodlike monolithic materials enable greater flexibility in these types of chemistry.^^'^"^ The method of Tennikova and Svec^^"^^ has been used to commercialize a novel disk type separation media, called CIM (Convective interaction media, BIA, Ljubljana, Slovenia).^^"^^ Analytical-scale separations can be performed on

457

ADSORPTIVE MEMBRANES FOR BIOSEPARATIONS

A

outlet white

inlet colour code

Flow

™3v™

embedding

hollow fiber membrane

housing lumen

B

F I G U R E 4 (A) Schematic of the Fractoflow^'^ hollow-fiber membrane adsorber module and (B) flow path through the hollow fibers is indicated. (Schematics reproduced with permission of Merck KGaA, Darmstadt, Germany.)

such disks in a very short time scale. This material is currently being scaled up for small-scale preparative separations with a radial-flow cartridge. UNO material from Biorad Corp, is a recently commercialized monolithic material formed in-situ.^^ It can be operated at high linear velocities without significant back pressure. Both these materials have promise for large scales, however, demonstrations beyond a few milliliters have not yet been developed. A functionalized sponge material, Seprasorb (Sepragen Corp.) is another intriguing variation of MA available in anion exchange (AX) chemistries.^^

TABLE 2

Novel Membrane-like Commercial Chromatographic Media

Trade name

Manufacturers

Description

References

CIM disk (convective interaction media) (Quick disk)^

BIA, Ljubljana, Slovenia

Disk/monolith formed by copolymer Product discontinued; CIM disk is the present version of the product Derivatized sponge in a radial-flov^^ module Monolithic polymer rod of polyacryl-amide

19-21

Seprasorb

Saulentechnik Knauer, Berlin, Germany Sepragen, Hawthorne, CA

UNO

Biorad, Hercules, CA

Cross-linked poly(glycidyl)methacrylate-co-ethyleneglycoldimethacrylate. Presently not available commercially.

19 23 22

458

RANJIT R. DESHMUKH AND TIMOTHY N. WARNER

II. COMPARISON OF MEMBRANE CHROMATOGRAPHY TO TRADITIONAL CHROMATOGRAPHY

A. Velocity As discussed earlier, the hallmark of membrane chromatography is in its speed. Membranes are capable of flow rates 10 to 100-fold faster than classical chromatography. Single membrane modules capable of flowing 7 liters per minute are now available. The rate-limiting step in the mass transfer is the diffusion of the solute to the functional groups. In traditional chromatography, the majority of the functional groups are located within pores. Interaction within the pores are severely limited by diffusional forces. Membranes have convective flow through their pores, which enables a faster interaction with the functional groups. Diffusion is still a limiting factor for mass transfer within membranes. Diffusional effects may be reduced by one to two orders of magnitude by the reduction of diffusional distances (10- to 100-fold). It has been shown that a pore size of 1-10 jum is the ideal size for rapid mass transfer with reasonably fast flow rates.^^ A second factor which may influence mass transfer is turbulent flow. The flow through a membrane is a tortuous path; thereby creating turbulent eddies. These eddies may serve to speed contact with functional groups. Therefore, both shorter diffusional distances and turbulent flow within the membrane may increase mass transfer.

B. Capacity It appears that membranes and bead matrices have similar capacities on a volumetric basis. If the membrane is approximately 200 fjum thick, then approximately 50 cm^ of the membrane will be equivalent to 1 mL of traditional media."^^ Although the ionic capacity, i.e., the small molecule capacity, is often measured, it may not be directly related to the protein or DNA capacity. The accessibility of functional groups may explain these differences in binding capacity. The functional group may not be accessible to all sizes of molecules. Some chromatographic companies have grafted large polymers onto beads or membranes in an attempt to alleviate problematic size exclusion. These polymers contain a large number of charged groups and thereby increase the matrix capacity. The highest capacity membranes have approximately 1 mg of protein binding per square centimeter.^^ The most important measurement of capacity is the "dynamic capacity," which is the assessment of the actual functional capacity at a specified flow rate. The dynamic capacity of any chromatographic media is usually lower than the static capacity. Generally, as the flux increases the dynamic capacity decreases. However, MA capacities are less affected by flow rate than are traditional chromatography matrices. Figure 5 shows identical breakthrough curves for cytochrome c at flow rates of 5 mL/min (60 cm/hr) and 50 mL/min (600 cm/hr) flow rates. This shows that the dynamic capacity is the same for both flow rates.

ADSORPTIVE MEMBRANES FOR BIOSEPARATIONS

459

Flow rate: 5 ml/min (A) 50 ml/min (B)

o 00 CN

60

mg protein

FIGURE 5 Effect of fluid velocity on MA performance: Comparison of breakthrough curves at 5 mL/min (60 cm/hr) and 50 mL/min (600 cm/hr). Membrane QI5. (Courtesy of Sartorius Corp.)

C. Resolution Even if membrane chromatography has superior speed and equivalent capacity, it must have good resolution to find applicability to chromatography. Fortunately, the resolution of membrane adsorbers appears to be very good. The resolution of traditional chromatographic beads is inversely proportional to the size of the chromatographic beads. In membrane chromatography, the size of the pores and the uniformity of the membrane are important factors for resolution. Experiments by Jungbauer on membrane devices with varying layers of 5 cm^ disks suggests that very high resolution may be achieved."^^ Very good resolution is obtained with even three layer modules and the resolution increases with the increase in number of membrane layers. Largescale experiments are even more illustrative of superior resolution feasible with the MA. Experiments by Demmer and Nussbaumer"^^ with 15, 30, and 60 layers of 1 m^ overall area in each cyclendrical module shows excellent resolution of bovine hemoglobin and hen egg white lysozyme (HEWL) Fig. 6. This also shows that as number of layers increase the resolution is increased. The impurities in HEWL are best resolved in the 60 layer module. D. Cleanability and Sterilization Membranes composed of the correct materials may have excellent chemical compatibility. A highly cross-linked regenerated cellulose (e.g., Hydrosart, Sartorius Corp.) is stable to a wide variety of chemical agents. It is stable in 40°C I M NaOH, and 1 M H2SO4, and a variety of organic solvents. The extreme stability enables a thorough cleaning and depyrogenation. In a

460

RANJIT R. DESHMUKH AND TIMOTHY N. WARNER

Volume (Ular) F I G U R E 6 Increase in efficiency with increase in number of MA layers. All modules are Sartobind S membranes with 10,000 cm^ overall membrane surface area. Module I has 60 layers, length 6 cm; module 2 has 30 layers with 12 cm length; and module 3 has 15 layers with 25 cm length. Sample I g each of purified bovine hemoglobin and hen egg white lysozyme. (Reprinted from Journal of Chromatography, Vol. 852, W . Demmer and D. Nussbaumer, Large-scale membrane adsorbers, 7 3 - 8 1 . Copyright 1999 with permission from Elsevier Science.)

cleaning study, the elution patterns of a recombinant protein were reproducible after 40 repeated cleaning and purification cycles."^^ An overlay of chromatograms after 1, 10, 20, 30, and 40 purification/cleaning cycles is shown in Fig. 7. Sterilization of MA products may be more practical than conventional chromatographic products. The same technology employed to sterilize standard 0.2 and 0.45 /xm membranes for the past decades can be utilized for chromatography membranes. Laboratory- and process-scale membrane devices capable of withstanding 50 autoclave cycles are now commercially available. Thus, membrane chromatography offers a higher degree of control over sterility. This may be advantageous for pharmaceutical purifications.

III. SCALE-UP OF CHROMATOGRAPHY MEMBRANES A. General Considerations Capacity is basically a linear function of the membrane area. Thus, if one wished to scale up 10-fold, one would simply use 10-fold the area of the membrane. The usual considerations of column height and diameter do not

ADSORPTIVE MEMBRANES FOR BIOSEPARATIONS

461

E2.5

o

00 CN

Run#1 — Run#10 —•Run#20 --Run#30 — Run#40

@2 CD O

c

CD 1.5

O CO

Time (minutes) F I G U R E 7 Overlay of chromatograms after repeated cleaning-purification cycles. The purification performance shows consistency after I, 10, 20, 30, and 40 purification and cleaning cycles. (Courtesy of R. McMaster, Aventis Pasteur.)

influence the success of the membrane scale-up. Other usual considerations such as the compaction of bed, or the "wall-effect," are not critical. In fact, as pressure is increased the multiple layers will be forced together, and approach a monolithic layer. The number of membrane layers determine efficiency in flat sheet MA. Therefore, in scale-up the number of layers should remain constant, if the same degree of resolution and dynamic capacity is desired. The number of layers can be varied in method development to select the appropriate compromise between efficiency and flow rate for a particular application. As one scales up, the flow rate per square centimeter should be held constant. The deviation in flux will not dramatically change capacity nor resolution in MA. It is prudent to hold the linear velocity constant when scaling up. Pressure has little effect on the scale-up. It is a consideration only in achieving the desired flux. Fluid distribution is perhaps the most critical component in scaling membrane adsorbers, just as it is in conventional chromatography. There may be a change in performance due to a different geometry at large scales. Variations in flow path and the filter housing can affect loading and elution. The most common impact of this change is that the dynamic capacity may be lowered and housing induced dispersion may increase. Coffman et al? have summarized some of the dispersion related issues of these modules. One strives to achieve a uniform distribution across the entire membrane surface. Again, like traditional chromatography, the dead volume must be minimized. The design of the standard "dead-end" filtration does not minimize the hold-up volumes in the inlet or outlet channels. The commonly available Luer-Lok connector for ease of use is one of the biggest contributors to housing related dispersion in the small syringe-type devices since the gap

462

RANJIT R. DESHMUKH AND TIMOTHY N. WARNER

between the connectors acts as a small mixing chamber."^^ Extramembrane effects destroy the resolution just as the extracolumn effects in chromatography. A good design for MA device is extremely low dead volumes in both the loading channels (inlet) and the elution collection channels (outlet). Low dead volume designs avoid the unnecessary mixing and resulting destruction of resolution. A schematic of one such design is shown in Fig. 3 in Section I. This is a cylindrical module where the membrane is rolled around a stainless steel core. The flow of the fluid is from the inside to the outside in a radial manner. The resistance of multiple layers creates a uniform distribution across the entire length of the thin annular 3 mm channel. Another critical factor affecting efficiency is the entrapment of air in the modules. This is a common problem for all sizes of modules. Pockets of air can affect fluid distribution as well as significantly lower dynamic capacity by reducing the accessible membrane area. The removal of entrapped air is usually a manual operation. Preequilibration, by flushing both in the forward and reverse directions, in conjunction with injection of well sparged buffer should remove any entrapped air. Unlike beaded chromatography columns, the air entrapment does not affect the integrity of the chromatography bed. Some large scale modules (e.g., Sartobind 0.1-8 m^ module) have air vents (Fig. 3) which are initially used to remove trapped air. Flowever, once the membrane is wetted completely, the module can be stored in solution within its housing thereby eliminating further air-related problems. MA systems can easily add a module to increase membrane capacity or resolution. Alternatively, the series addition of a traditional chromatography column for improvement of capacity or resolution is seldom practiced. Thus, membrane chromatography offers the chromatographer a new flexibility of easy expansion of inadequate systems. Alternatively, the easy connectibility also enables a simple way to couple multiple units of different chemistries for a mixed-mode separation."*^ The next section describes these configurations in greater details. B. Scale-Up: Multimodule Systems for Pilot-Scale Purification The connection of modules in series or parallel arrangements will affect the overall performance of a chromatographic system. The difference in modular configuration directly affects both flux and efficiency. For example, if cartridges are connected in a parallel arrangement, the flux of each module remains unchanged. The intermodule piping would increase the dead volume, thereby decreasing resolution. Parallel connection will also highlight any heterogeneity in the membrane modules. If, on the other hand, modules were connected in series, the dead volumes would be minimized, while the resolution and dynamic capacity would be maximized. At a constant pressure, however, the flux would decrease in a series arrangement; unless the number of membrane layers (resistance) is decreased. For this reason, there are practical limitations to the number of modules that can be connected in series. Scale-up to manufacturing levels will require an optimization between series and parallel connections. Pioneering work in the area of modular configurations has been done by Demmer and Nussbaumer."*^ Here, they compared a simple system consisting

463

ADSORPTIVE MEMBRANES FOR BIOSEPARATIONS

of two 8 m^ modules connected in parallel to a compound system. The compound system consisted of three stages. The first stage consisted of the two 8 m^ modules in parallel. The effluent of the first stage was routed to a secondary 4 m^ stage by a serial connection. The effluent of the secondary stage was directed to the third (final) 1 m^ stage by serial connection (Fig. 8). The interesting aspect of this three-stage system was that each sequential stage has half the number of layers and length of the previous stage. This configuration achieved similar fluxes within each stage. The performance of the two systems was compared for the purification of hemoglobin. In Fig. 9A the dynamic capacity of the two 8 m^ modules in parallel is shown to be approximately 100 g. The addition of the serially connected 4 m^ and 1 m^ modules gives a tremendous improvement in performance. There is a dramatic increase in the dynamic capacity to 140 g hemoglobin (Fig. 9B). Demmer and Nussbaumer"^^ showed that this compound system could purify 60 g hemoglobin every 7 min. Therefore, with cycling this system can

Upper end plate Single colunn three stage Sartobind plant (21 n"2) Extension core 60 layers

Outer shell, double length Fi rst stage. 16 n"2

Base core 60 layers

Serial connection nenber Outer shell, unit length Base core 30 layers Second stage, 4 n^2 Serial connection nenber Outer shell, half length Thi rd stage. 1 n"2

Core 15 layers, half length lotton end plate

F I G U R E 8 Arrangement of multiple radial flow modules to increase capacity and flow rate. Two 8 m^ modules were connected In parallel, with one 4 m^ module in series, in conjunction with a final I m^ module in series. Total membrane area is 21 m^. (Adapted from Journal of Chromatography, Vol. 852, W . Demmer and D. Nussbaumer, Large-scale membrane adsorbers, 7 3 - 8 1 . Copyright 1999 with permission from Elsevier Science.)

464

RANJIT R. DESHMUKH AND TIMOTHY N. WARNER

BC Hemoglobin 2* S-80k-60-50 parallel

0 B

20

40

60

80 100 protein [g]

120

140

160

180

BC Hemoglobin 2* S-80k-60-50 par + S-40k-30-50 ser. + S-1 Ok-15-25 ser 2T

20

40

60

80 100 protein [g]

120

140

160

180

F I G U R E 9 Shows the dynamic capacity curves for hemoglobin adsorption on two membrane module setups: (A) two 8 m^ modules in parallel show capacity of 100 g and (B) addition of 4 m^ and I m^ modules in series (as shown in Fig. 8) increased the dynamic capacity to 140 g of hemoglobin.

produce 514 g of hemoglobin per hour. They calculated that automation of this small three-stage system (21 m^ of membrane) could produce 2 metric tons of hemoglobin per year.

lY. APPLICATIONS OF MA TO PREPARATIVE BIOSEPARATIONS MAs have been applied to a variety of applications.^'^"^ There are numerous examples in the literature for the use of MAs for affinity purification using a variety of ligands.^'^^'"^^""^^ Ion exchange is also well documented in the MA literature. Hydrophobic interaction and reverse phase separations are rela-

ADSORPTIVE MEMBRANES FOR BIOSEPARATIONS ^ ^ 1

TABLE 3

465

Chromatographic Modes in M A Applications

MA separation mode

References

Affinity chromatography IMAC Ion exchange HIC/reversed phase

9-11, 20, 32, 43, 50-56 12,57 15, 16, 21, 35, 36, 43, 50, 51, 54, 56, 58-61 8, 15, 16, 61

tively fewer.^^"^^'^^'^^'"^^ The different modes of chromatography reported on MA is briefly summarized in Table 3/^"^^ Several practical bioseparations are discussed next. These examples show the use of different sizes and geometries of MA. The first example shows protein purification with the modules larger than 4 m^. The second example of viral clearance uses membranes up to the 1 m^ range. The third oligonucleotide example shows purification on 2 m^ pilot scale module.

A. Purification of Proteins on MA: Case Study of Recombinant Vaccine Protein The desired 40 IcDa recombinant vaccine protein was expressed in Escherichia coli^^ Cells were lysed by high-pressure disruption and the resulting lysate was filtered through a 2 /^m filter. This crude lysate contained very high levels of both DNA and endotoxin requiring 6-10 log reductions to meet pharmaceutical standards. The lysate was first passed through a Qmembrane (Sartorius Corp.) in low salt buffer at pH 8.1. The initial method development was performed on a small 15 cm^ module to optimize the salt gradient. A stepwise elution with NaCl gradient in 100 m M increments of 5 min durations showed that the vaccine product eluted at 200 m M NaCl (Fig. 10). This purification was further optimized such that a wash of 50 m M salt removed the contaminants. The target molecule was eluted by a pH step to minimize diafiltration or dilution step. Complete elution was accomplished by 15 m M citrate, pH 6.0. Due to the high DNA and endotoxin levels a second Q-step was added as a safety net. The pH was adjusted to 4.7, such that the vaccine product would flow through; while the DNA and endotoxins would be retained. The low pH allowed sequential adsorption onto a carboxyl (C) membrane (Sartorius Corp.). A stepwise citrate pH gradient showed elution of the product at pH 5.5 (Fig. 11). The DNA and endotoxin levels were monitored throughout the purification process (Fig. 12). The final optimized process was robust and able to handle variable levels of these contaminants. The process achieved a high level of purity (99%) with reasonable yields (68%). The pH shifts used in elution eliminated the need for time-consuming diafiltration steps. The entire purification was completed within approximately 75 min. It was found that this process could easily be modified to accommodate other similar recombinant proteins. This process could easily be scaled up.

466

RANJIT R. DESHMUKH AND TIMOTHY N. WARNER

280 nm Isocratic gradient (conductivity)

F I G U R E 10 Optimization of elution salt step for a recombinant vaccine protein purification. A step elution ladder with increments of 100 m M salt for 5 min durations, from 0 t o I M was used: MA, Sartobind Q-15 (anionic 100 cm^). Load conditions: double load, 15 m M Tris, pH 8.1; flow rate: 5 m L / m i n . Most of the vaccine protein elutes in the 200 m/Vl salt step. FT indicates the flow through peaks. (Courtesy of R. McMaster, Aventis Pasteur.)

pH 8.0 Isocratic pH gradient pH 6.5

2.0

pH6.0 280 nm

pH5.5

1.0 pH4.5 P H ^

o.ofy"^ 0

25 min

50

F I G U R E I I Optimization of pH elution steps for recombinant vaccine protein purification. Incremental pH step gradients 5.0, 5.5, 6.0, 6.5, and 8.0 are shown. Membrane 100 cm^ C (Sartobind CI00): Load conditions: 15 m M citrate, pH 4.75, flow rate 7 mL/min. The product elutes at pH shift of 5.5. (Courtesy of R. McMaster, Aventis Pasteur.)

467

ADSORPTIVE MEMBRANES FOR BIOSEPARATIONS

10000 1000 2 100 Q. 10 0 1

6224

c

-I—•

32

1

0.1 0.01 LU 0.001

B

g m 0) -«—• o

0.02 0.002

—(—

First Anion

Sterile Crude

1000

1

Anion/Cation

Sterile Filt.

0.08

n r\R

Anion/Cation

Sterile Filt.

180

100

30

0)

10 =1.

1

1

0.1 + Q.

0.01

1

Sterile Crude

•^•^•^•••^^^^^•^•^•^•^•^^<^-^<-&^>^

First Anion

F I G U R E 12 (A) Endotoxin and (B) D N A reduction monitored during purification steps for vaccine protein purification. (Courtesy of R. McMaster, Aventis Pasteur.)

The purification process was scaled up 10-foid from 100 to 1000 cm^ of stacked 142 mm disks. The scale-up exhibited a good separation with a 7 log reduction in endotoxin levels. The yield was 98% with a final purity of 98.2%. This process was again scaled up with the modular configuration described previously (Fig. 3). Two 30-layer 4 m^ Q modules were used in conjunction with a 1 m^ C module. The chromatograms appeared very similar to the 1000 cm^ purification. The product purity was found to be 99.7% with < 0.002 endotoxin units per microgram of product and 0.003 pg DNA per microgram of product. The final product exceeded the clinical pharmaceutical specifications for this product. B. Negative Chromatography: Removal of Contaminants i. Viral DNA and Endotoxin Reduction under GMP Conditions The high throughput of the membrane adsorbers is perhaps best utilized in a passthrough separation, where large volumes are problematic with traditional chromatography. Reduction of DNA contamination in monoclonal antibody purification is one such application. Ruth^^ has demonstrated the application of a Q-type flat sheet membrane in this application. There was validated reduction of over 3 to 5 logs of contaminating DNA in a GMP application. The separation was developed on small syringe filters (Q-5

468

RANJIT R. DESHMUKH AND TIMOTHY N. WARNER

^ B l

T A B L E 4 Effect of Flow Rate on Efficiency of D N A Removal (Single layer) Flow rate (cm / hr)

Logs DNA removal

125 250 500 1000

2.8 2.6 2.4 2.2

Data courtesy of Dr. Mary Ruth and IDEC Pharmaceutical Corp., San Diego.

and Q15, Sartorius), then scaled up to 11,000 cm^ cassettes of flat sheet membrane. The effect of number of layers of membrane on efficiency of DNA capture was studied. Table 4 shows the DNA removal as a function of the flux. Linear flow rates of 125 to 1000 c m / h r showed only modest reduction in efficiency with an increase in flow rate. The throughput was much higher than an equivalent bead-based system, and the initial startup costs for MA based system was found to be favorable. Belanich et al.^^ reported the removal of endotoxins from protein mixtures. Endotoxins in a bacterial extract containing a protein photolyase was passed through a stack of 10 disk membranes (Q-type, Sartorius). LAL assay was used to monitor the endotoxin levels after each pass. There was over 5 log reduction in endotoxin content after three passes through the membranes. The protein content was reduced during this process, however, the enzyme's specific activity was increased 35-fold. This study also determined that the binding capacity of the membrane was greater than 2.25 million EU/cm^ of membrane area. ii. Monoclonal Antibody Purification with Virus Removal

Another application developed for monoclonal antibody (MAb) purification was effective in virus reduction. Levine^"^ developed a two-part membrane adsorber process to purify HeFi-1, a murine IgGl monoclonal antibody. The product was captured onto a cationic membrane (S15x) at pH 4.0; and eluted by 150 m M NaCl. This partially purified monoclonal antibody was pooled, diluted, and pH adjusted to 7,5. This was then applied on an anionic membrane (Q15x) and eluted by a salt gradient. The resulting product was found to be > 95% pure by SDS gel analysis. The purification regime was assessed for its abflity to remove spiked virus loads. Three viruses-polio, herpes, and murine leukemia—were analyzed for log reduction in viral titers. As seen in Table 5, both the cationic and anionic steps partially removed the indicator viruses. There was an 8.2 log removal of herpesvirus; a 7.5 log reduction of murine leukemia and a 3.2 log reduction of polio virus. Although the purification was optimized for MAb production, it was also effective in removal of a variety of viruses. Karger et al.^^ have used cationic exchange membranes for purification of alpha-herpes viruses from cell culture supernatents. This method was found to be more convenient and faster than a sucrose density gradient

ADSORPTIVE MEMBRANES FOR BIOSEPARATIONS

469

TABLE 5 Virus Removal Log Reduction

S-15 cartridge Q-15 cartridge Total removal

Polio

Herpes

MuLV

1.52 1.64 3.16

3.36 4.81 8.17

2.69 4.82 7.51

method. The yield, purity, and specific infectivity were superior with the MA method. C. Case Study: Purification of Oligonucleotides Ohgonucleotide use has increased dramatically due to the low cost and wider availability of synthetic oligonucleotides for biochemical and genetic research. Large-scale quantities are required for the therapeutic applications and techniques are being developed to purify oligonucleotides at the kilogram scale.^^'^^ The first antisense drug has recently been approved by the U.S. Food and Drug Administration (FDA) for treatment of CMV retinitis (fomivirsen sodium, discovered and manufactured by Isis Pharmaceutical, Inc.). Although reverse-phase and anion-exchange methods work well for the purification of oligonucleotides, Q-type membrane adsorbers were used to demonstrate their potential use at a large scale. This application demonstrates the use of very large scale modules, 2 to 4 m^, and shows that large-scale high-resolution purifications are possible. The details of the oligonucleotide purification are given elsewhere^'' (this volume, chapter 14.) The feasibility of oligonucleotide purification on MA was demonstrated on a small 15 cm^ Q syringe filter. An analytical scale purification of a 20 mer phosphorothioate oligonucleotide crude is shown in Fig. 13. The buffer was 20 m M NaOH with NaCl as the eluent. A linear gradient of 0-100% over 10 min was used. A similar separation was developed using step gradients on the Q-15 module. This purification was then scaled up to a 2 m^ strong anion exchange (Q-type) module. Buffers were the same as those used at small scale, but elution was carried out manually. Salt steps of required ionic strengths were premixed and the step changes were performed manually during the purification. The output of the module was monitored by a side stream to a chromatographic skid (BioCad 60, PE Biosystems, Framingham, MA). In this manner, it was possible to monitor the conductivity, pH, and absorbance of the elution stream. This chromatographic setup can be improved by using an automated low-pressure skid with appropriately high flow rates. The chromatographic output is shown in Fig. 14. There is excellent separation of the impurities and the product containing peak as analyzed by high-performance liquid chromatography (HPLC). Analysis by analytical anion exchange shows that an impurity-rich feed of 34% purity could be increased to 75%, with a yield of 76% of the main product. Experiments were also performed with crudes more representative of routine synthesis, with full-length purity of 65%. A step-

470

RANJIT R. DESHMUKH AND TIMOTHY N. WARNER

F I G U R E 13 Small-scale separation of antisense oligonucleotides on Q modules. MA: Q I 5 (Sartobind 15 cm^); flow rate: 5 m L / m i n ; buffer A: 20 m M NaOH; buffer B: 20 m M NaOH plus 2.5 M NaCI; sample: 20 mer phosphorothioate crude, ISIS 2302; gradient: 0 - 100% B in 10 min.

gradient purification of this crude resulted in a product purity greater than 90% full length at a yield of S7%, This purification performance is comparable to conventional anion-exchange chromatography, and can be carried out at much higher flow rates. It is possible to carry out the entire purification under sanitary and good manufacturing practice (GMP) conditions.

Y. CONCLUSIONS Membrane adsorber technology has recently advanced to a new level of sophistication. This offers a valid alternative to chromatographic bead technology. There is considerable pubHshed literature to enable one to develop a specific separation methods. The scale-up technology has also expanded the scope of operation to the multiple square meter range, enabling GMP processing at a reasonable cost. The membrane-based adsorption systems may not be advantageous in every application. There are several niche applications where fast adsorption kinetics is beneficial, e.g., DNA and endotoxin reduction, capture of dilute product, sequential anionic and cationic

471

ADSORPTIVE MEMBRANES FOR BIOSEPARATIONS

100

o o

Q. C

o

o" 3

15

20

25

30 Time, min

F I G U R E 14 Large-scale separation of antisense oligonucleotides on 2 m^ Q MA. Sample: ISIS 2302 DMT-off crude (20 mer phosphorothioate oligonucleotide); load amount: I g; elution: equilibration and wash; 20 m M NaOH, eluents step gradients of various strengths with 2.5 M NaCI stock solution; flow rate: 1.25 liters / min; monitoring: side streak directed through BioCad 60 conductivity and absorbance flow cells (266 and 300 nm).

purifications. Here membrane adsorber chromatography can offer an order of magnitude advantage in throughput, cost, and operating convenience.

ACKNOWLEDGMENTS The authors thank their colleagues for help in this work, especially Frederick Hutchison, Michael Murphy, Dr. Abdul Weiss, Dr. Stefan Fischer-Fruhholz, Dr. Wolfgang Demmer, and Dr. Nussbaumer at Sartorious Corporation, and William E. Leitch II, Patricia De Leon, Dr. Susan Srivatsa, Dr. Yogesh S. Sanghvi, and Dr. Douglas L. Cole at Isis Pharmaceuticals.

REFERENCES 1. Roper, D. K., and Lightfoot, E. N. (1995). Separation of biomolecules using adsorptive membranes. / . Chromatogr. A 702, 3-26. 2. Thoemmes, J., and Kula, M.-R. (1995). Membrane chromatography—An integrative concept in the downstream processing of proteins. Biotechnol. Prog. 11, 357-367. 3. Coffman, J. L., Roper, D. K., and Lightfoot, E. N. (1994). High-resolution chromatography of proteins in short columns and adsorptive membranes. Bioseparation 4, 183-200. 4. Frey, D. D., Van de Water, R., and Zhang, B. (1992). Dispersion in stacked-membrane chromatography. / . Chromatogr. 603, 43-47. 5. Roper, D. K., and Lightfoot, E. N. (1995). Estimating plate heights in stacked-membrane chromatography by flow^-reversal. / . Chromatogr. A 702, 69-80. 6. Suen, S.-Y., and Etzel, M. R. (1992). A mathematical analysis of affinity membrane bioseparations. Chem. Eng. Sci. 47, 1355-1364.

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7. Suen, S.-Y., Caracotsios, M., and Etzel, M. R. (1993). Sorption kinetics and axial diffusion in binary-solute affinity-membrane bioseparations. Chem. Eng. Set. 48, 1801-1812. 8. Ding, H., Yang, M . - C , Schisla, D., and Cussler, E. L. (1989). Hollow-fiber liquid chromatography. AIChEJ. 35, 814-820. 9. Kim, M., Saito, K., Furusaki, S., Sato, T., Sugo, T., and Ishigaki, I. (1991). Adsorption and elution of bovine y-globulin using an affinity membrane containing hydrophobic amino acids as ligands. / . Chromatogr. 585, 4 5 - 5 1 . 10. Nachman, M., Azad, A. R. M., and Bailon, P. (1992). Membrane-based receptor affinity chromatography. / . Chromatogr. 597, 155-166. 11. Brandt, S., Goffe, R. A., Kessler, S. B., O'Connor, J. L., and Zale, S. E. (1988). Membranebased affinity technology for commercial scale purifications. Bio/Technology 6, 779-782. 12. Serafica, G. C., Pimbley, J., and Belfort, G. (1994). Protein fractionation using fast flow immobilized metal chelate affinity membranes. Biotechnol. Bioeng. 43, 21-36. 13. Wang, Q. C., Svec, P., and Frechet, J. M. J. (1993). Macroporous polymeric stationery-phase rod as continuous separation medium for reversed-phase chromatography. Anal. Chem. 65, 2243-2248. 14. Wang, Q. C., Svec, F., and Frechet, J. M. J. (1994). Reversed-phase chromatography of small molecules and peptides on a continuous rod of macroporous poly(styrene-co-divinylbenzene). / . Chromatogr. A 669, 230-235. 15. Tennikova, T. B., Belenkii, B. G., and Svec, F. (1990). High-performance membrane chromatography. A novel method of protein separation. / . Liq. Chromatogr. 13, 63-70. 16. Tennikova, T. B., Bleha, M., Svec, F., Almazova, T. V., and Belenkii, B. G. (1991). High-performance membrane chromatography of proteins, a novel method of protein separation. / . Chromatogr. 555, 97-107. 17. Tennikova, T. B., and Svec, F. (1993). High-performance membrane chromatography: Highly efficient separation method for proteins in ion-exchange, hydrophobic interaction and reverse-phase modes. / . Chromatogr. 646, 279-288. 18. Strancar, A. (1997). Separation of biopolymers with different techniques of Hquid chromatography. Ph.D. Dissertation, University of Ljubljana, Ljublijana, Slovenia. 19. Josic, D., Zeilinger, K., Lim, Y.-P., Raps, M., Hofmann, W., and Reutter, W. (1989). Preparative isolation of glycoproteins from plasma membranes of different rat organs. / . Chromatogr. 484, 327-335. 20. Abou-Rebeyeh, H., Korber, F., Schubert-Rehberg, K., Reusch, J., and Josic, D. (1991). Carrier membrane as a stationery phase for affinity chromatography and kinetic studies of membrane-bound enzymes. / . Chromatogr. 566, 341-350. 21. Josic, D., Reusch, J., Loster, K., Baum, O., and Reutter, W. (1992). High-performance membrane chromatography of serum and plasma membrane proteins. / . Chromatogr. 590, 59-76. 22. BioRad Corp. (1997). "UNO Ion Exchange Columns," Bull. No. 2116. BioRad Corp., Hercules, CA. 23. Sepragen Corp. (1998). "Seprasorb Cartridges," Appl. note. Sepragen Corp., Hayward, CA. 24. Pall Gelman Sciences (1998). "Biodyne Nylon 6,6 Membranes," Prod, data sheet. Pall Gelman Sciences, East Hills, NY. 25. E. Merck (1998). "Fractoflow," Appl. note. E. Merck, Darmstadt, Germany. 26. Millipore Corp. (1992). "MemSep Chromatography Cartridges," Rep. No. AN106. Millipore Corp., Bedford, MA. 27. Gerstner, J. A., Hamilton, R., and Cramer, S. M. (1992). Membrane chromatographic systems for high-throughput protein separations. / . Chromatogr. 596, 173-180. 28. Sackett, D. L. (1995). Rapid purification of tubulin from tissue and tissue culture cells using solid-phase ion exchange. Anal. Biochem. 228, 343-348. 29. Van Huynh, N., Motte, J. C , Pilette, J. F., Deleire, M., and Colson, C. (1993). Sequential elution of denatured proteins, hydrolyzed RNA and plasmid DNA of bacterial lysates adsorbed onto stacked DEAE-Cellulose membranes. Anal. Biochem. 211, 61-65. 30. Weinbrenner, W. F., and Etzel, M. R. (1994). Competitive adsorption of a-lactalbumin and bovine serum albumin to sulfopropyl ion-exchange membrane. / . Chromatogr. A 662, 414-419.

ADSORPTIVE MEMBRANES FOR BIOSEPARATIONS

473

31. Sartorius Corp. (1997). "Membrane Separation Products for Enhanced Productivity in the Biotech Laboratory," Appl. No. K003. Sartorius Corp., Edgewood, NY. 32. Mandaro, R. M., Roy, S., and Hou, K. C. (1987). Fihration supports for affinity separation. Bio/Technology 5, 928-932. 33. Cuno Life (1998). "Product Catalogue," Appl. note. Cuno Life, Meriden, CT. 34. Jungbauer, A., Unterluggauer, P., Uhl, K., Buchaner, A., Steindl, F., Pettauer, D., and Wenisch, E. (1988). Scaleup of monoclonal antibody purification using radial streaming ion exchange chromatography. Biotechnol. Bioeng. 32, 326-333. 35. McGregor, W. C , Szesko, D. P., Mandaro, R. M., and Rai, V. R. (1986). High performance isolation of a recombinant protein on composite ion exchange media. Bio/Technology 4, 526-527. 36. Menozi, F. D., Vanderpoorten, P., Dejaiffe, C , and Miller, A. O. A. (1987). One-step purification of mouse monoclonal antibodies by mass ion-exchange chromatography on Zetaprep. / . Immunol. Methods 99, 229-233. 37. Tan, L. U. L., Yu, E. K. C , Louis-Seize, G. W., and Saddler, J. N. (1987). Inexpensive, rapid procedure for bulk purification of cellulase-free /3-1,4-D-Xylanase of high specific activity. Biotechnol Bioeng. 30, 96-100. 38. Upshall, A., Kumar, A. A., Bailey, M. C , Parker, M. D., Favreau, M. A., Lev^ison, K. P., Joseph, M. L., Maraganore, J. M., and McKnight, G. L. (1987). Secretion of active human tissue plasminogen activator from the filamentous fungus aspergillus nidulans. Bio/Technology. 5, 1301-1304. 39. Etzel, M. (1997). Affinity and ion exchange chromatography on membranes. Purdue Chromatogr. Workshop: Sorbents, Membr. Sep. Strategies Scale-up, W. Lafayette, IN., 1997. 40. Demmer, W., and Nussbaumer, D. (1999). Large-scale membrane adsorbers, / . Chromatogr. A 852, 7 3 - 8 1 . 41. Jungbauer, A. (1998). Personal communication. 42. McMaster, R., Kruk, J., Christianson, G., Gomez, P., Warner, T., Demmer, W., and Nussbaumer, D. (1998). Purification of clinical vaccine proteins by high performance membrane chromatography. Proceedings of 18th Int. Symp. on Sep. and Anal, of Prot., Pep. and Polynucleotides (ISPPP'98), p. 29, abstract 114, Vienna. 43. Freitag, R., Splitt, H., and Reif, O.-W. (1996). Controlled mixed-mode interaction chromatography on membrane adsorbers. / . Chromatogr. A 728, 129-137. 44. Wang, W. K., Lei, S.-P., Monbouquette, and McGregor, W. C. (1995). Membrane adsorber process development for the isolation of a recombinant immunofusion protein. BioPharm. 8, 52-59. 45. Ritter, K. (1991). Affinity purification of antibodies from sera using polyvinylidenedifluoride (PVDF) membranes as coupling matrices for antigens presented by autoantibodies to triosephosphate isomerase. / . Immunol. Methods 137, 209-215. 46. Krause, S., Kroner, K. H., and Deckw^er, W.-D. (1991). Comparison of affinity membranes and conventional affinity metrices with regard to protein purification. Biotechnol. Tech. 5, 199-204. 47. Arnold, F. H., Blanch, H. W., and Wilke, C. R. (1985). Analysis of affinity separations. I: Predicting the performance of affinity adsorbers. Chem. Eng. J. 30, B9-B23. 48. Planques, Y., Pora, H., and Menozzi, F. D. (1991). Affinity purification of plasminogen by radial-flov^ affinity chromatography. / . Chromatogr. 539, 531-533. 49. Simmonds, R. J., and Yon, R. J. (1976). Protein chromatography on adsorbents with hydrophobic and ionic groups. Biochem. J. 157, 153-159. 50. Langlotz, P., and Kroner, K. H. (1992). Surface-modified membranes as a matrix for protein purification. / . Chromatogr. 591, 107-113. 51. Briefs, K.-G., and Kula, M.-R. (1992). Fast protein chromatography on analytical and preparative scale using modified microporous membranes. Chem. Eng. Sci. 47, 141-149. 52. Champluvier, B., and Kula, M.-R. (1991). Microfiltration membranes as pseudo-affinity adsorbents: Modification and comparison with gel beads. / . Chromatogr. 539, 315-325. 53. Champluvier, B., and Kula, M.-R. (1992). Dye-ligand membranes as selective adsorbents for rapid purification of enzymes; A case study. Biotechnol. Bioeng. 40, 33-40.

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54. Lutkemeyer, D., Bretschneider, M., Buntemeyer, H., and Lehmann, J. (1993). Membrane chromatography for rapid purification of recombinant antithrombin III and monoclonal antibodies from cell culture supernatant. / . Chromatogr. 639, 57-66. 55. Unarska, M., Davies, P. A., Esnouf, M. P., and Bellhouse, B. J. (1990). Comparative study of reaction kinetics in membrane and agarose bead affinity systems. / . Chromatogr. 519, 53-67. 56. Reif, O.-W., and Freitag, R. (1994). Comparison of membrane adsorber (MA) based purification schemes for the down-stream processing of recombinant h-AT III. Bioseparation 4, 369-3981. 57. Reif, O.-W., Nier, V., Bahr, U., and Freitag, R. (1994). Immobilized metal affinity membrane adsorbers as stationary phases for metal interaction protein separation. / . Chromatogr. A 664, 13-25. 58. Church, F. C , and Whinna, H. C. (1986). Rapid sulfopropyl-disk chromatographic purification of bovine and human thrombin. Anal. Biochem. 157, 77-83. 59. Splitt, Fl., Mackenstedt, I., and Freitag, R. (1996). Preparative membrane adsorber chromatography for the isolation of cov\^ milk components. / . Chromatogr. A 729, 87-97. 60. Chaudhary, A., Mehrotra, B., and Prestv^ich, G. D. (1997). Rapid purification of reporter group-tagged inositol hexakisphosphate on ion-exchange membrane adsorbers. BioTechniques 23, 427-430. 61. Luksa, J., Menart, V., Milicic, S., and Kus, B. (1994). Purification of human tumor necrosis factor by membrane chromatography. / . Chromatogr. A 661, 161-168. 62. Ruth, M. E. (1996). Use of a Q-type membrane adsorber for the removal of DNA during the purification of a monoclonal antibody. PrepTech Conf., East Rutherford, NJ, 1996. 63. Belanich, M., Cummings, B., Grob, D,, Klein, J., O'Konner, A., and Yarosh, D. (1996). Reduction of endotoxin in a protein mixture using strong anion-exchange membrane absorption. Pharm. Technol. 20(3), 142-150. 64. Levine, H. (1995). The use of membrane adsorbers for the purification of monoclonal antibodies. BioWest'95. 65. Karger, A., Bettin, B., Granzow, H., and Mettenleter, T. C. (1998). Simple and rapid purification of alphaherpesviruses by chromatography on a cation exchange membrane. / . Virol. Methods 70, 219-224. 66. Deshmukh, R. R., and Sanghvi, Y. S. (1997). Recent trends in large-scale purification of antisense oligonucleotides. IBC Conf.: Large Scale oligonucleotide Synth., San Diego, CA, 1997. 61. Deshmukh, R. R., Leitch, W. E., II, and Cole, D. L. (1998). Application of sample displacement techniques to the purification of synthetic oligonucleotides and nucleic acids: A mini-reviev^ vs^ith experimental results. / . Chromatogr. A 806, 77-92.

SIMULATED MOVING-BED CHROMATOGRAPHY FOR BIOMOLECULES R. M. NICOUD NouaSep, Vandoeuvre-les-Nancy, France

I. INTRODUCTION II. BASIC PRINCIPLE III. OPERATING CONDITIONS A. Step I: Determine the Required Experimental Data B. Step 2: Calculate the TMB C. Step 3: Calculate the SMB D. Step 4: Practical Implementation and Fine-Tuning IV. MAIN APPLICATIONS A N D DEVELOPMENTS A. Separation of Sugars B. Desalting C. Purification of Proteins and Complex Molecules D. Separation of Ionic Molecules E. Separation in Organic Molecules F. Separation of Optical Isomers V. PRACTICAL APPLICATION: SEPARATION OF SUGARS A. Step I: Determination of the Required Experimental Data B. Step 2: Calculation of the TMB C. Step 3: Calculation of the SMB D. Step 4: Practical Implementation and Fine-Tuning VI. CONCLUSION REFERENCES

I. INTRODUCTION Many different products are now purified by chromatographic processes, from the laboratory scale (a few grams) up to the industrial pharmaceutical scale (a few tons per year) or even up to the "petrochemical scale" (100,000 tons per year). Among the possible technologies, the elution high-performance Hquid chromatography (HPLC) technology (sometimes with recycle) has taken a very important part of the small-scale (10 tons per year) market Separation Science and Technology,

Volume 2

Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

475

476

R. M. NICOUD

during the previous decade, meanwhile the simulated moving-bed (SMB) technology has been extensively used for the very large scale fractionation of sugars and xylenes for the last 30 years.^'^ Currently, there is considerable interest in the preparative applications of liquid chromatography, even though it is often considered expensive. To make the chromatographic process more attractive, attention is focused on the choice of the operating mode^ to minimize the eluent consumption and to maximize the productivity which is of key importance when expensive packings are used. Among the alternatives to the classical process (elution chromatography), much attention is paid to the SMB. Even though it is known as a process able to maximize productivity and to minimize eluent consumption, SMB has been ignored in the pharmaceutical and fine chemical industries during the last 30 years. The reason was probably due to the patent situation and the complexity of the concept. Only a relatively short time ago, separations of pharmaceutical compounds began to be performed using the SMB technology"^'^ and other application areas are now open: fine chemistry, cosmetics, and the perfumes industry.^ SMB is now considered as a real production tool (for instance, the Belgium pharmaceutical company U.C.B. Pharma recently announced the use of SMB for performing multi-ton-scale separation of optical isomers) and in addition to the huge commercial plants offered for petrochemistry (IFP, Rueil-Malmaison, France; UOP, Des Plaines, IL), smaller plants are now commercially available for fine chemistry, the pharmaceutical industry, or biotechnologies. As presented in the following sections, even if SMB is still not extensively used in biotechnologies, it could quickly lead to very attractive applications.

II. BASIC PRINCIPLE The basic idea of a moving-bed system is to promote a countercurrent contact between the solid and the liquid phases, as described in Fig. 1. The solid phase goes down in the column as a result of gravity; when it exits the system (zone I), it does not contain adsorbed products and is thus recycled at the top of the system (zone IV). The liquid (eluent) stream follows exactly the opposite direction: It goes up and is recycled from zone IV to zone I. Feed, containing components A and B, is injected at the middle of the column, and the fresh eluent at the bottom. Provided that the affinity of A and B for the solid are different (B being more retained than A), it is possible to choose flow rates to cause A to move upward and B to move downward, leading thus to a spatial separation. This system requires two inlet lines (one for the feed and one for the eluent) and two outlet lines (one for the raffinate A and one for the extract B). The classical moving bed is made of four different zones,* in which different constraints must be fulfilled^: Zone I (between the eluent makeup and the extract points): The more retained product (B) must be completely desorbed. * Note that the zone definitions vary according to the authors

477

SIMULATED MOVING-BED CHROMATOGRAPHY

Liquid

SoUd

1L Zone IV

bate (A)

Zone III Feed ^ A+B Zone II Extraict (B) ix^ Zone I ly Eluent make-up X— FIGURE

I

Principle of a true moving bed.

Zone II (between the extract and the feed points): The less retained product (A) must be completely desorbed. Zone III (between the feed and the raffinate points): The more retained product (B) must be completely adsorbed. Zone IV (between the raffinate and the eluent makeup points): The less retained product (A) must be completely adsorbed. All the internal flow rates are related to the inlet-outlet flow rates by simple mass balances: Qll = Ql - QExt

QUI = Qll + Qpeed

Qiv = QUI - QRaff

Qi = Qiv + Q E .

(1)

And the inlet-outlet flow rates are related by QEXC + QRaff = Qpeed + QEI

(2)

Because of the difficulty in operating a true moving bed (TMB) (due to the solid circulation), this interesting idea must be implemented in a different way. As will be shown, most of the benefit of a true countercurrent operation can be achieved by using several fixed-bed columns in series and an appropriate shift of the injection and collection points: this is the SMB concept (Fig. 2). The solid does not actually move, its flow is only simulated by shifting inlet and outlet lines. In fact, this simulated solid flow rate downward is directly linked to the shift period. The key is the proper selection of the flow rates: they must be chosen to stabilize the B front between zones I and II and the A front between zones III and IV and to allow separation between zones II and III. The adequate choice of the flow rates requires a minimum knowledge of the physicochemical

4/8

R. M. NICOUD

Raffinate (

^* Extract (B) FIGURE 2

Principle of the simulated moving bed.

properties of the system (adsorption isotherms, plate number, etc.) as presented later. Although the most widely used scheme among simulated moving-bed separation processes is the four-zone one described in the first part of this section, there are alternative schemes that are more suited to very particular cases.^'^ Those cases are related to binary to ternary separations. This subject is discussed more extensively in Ref. 10. The simplest SMB system is made of three zones, for which zone number IV does not exist, and the flux exiting zone III is sent to concentration unit. The three-zone SMB process (Fig. 3) is an economical alternative to the four-zone system when the eluent consumption is not an important concern a n d / o r when the raffinate is a zero- or low-value by-product. A simple mass balance shows that the raffinate obtained with the three-zone process is more diluted than the raffinate obtained with the four-zone process: Qy^f (four-zone) Q,,(three-zone) = 2^^^(fo,,.,,„e) + Q,,(four-zone) ' C.af(four-zone)

(3) Actually this dilution phenomenon is partially avoidable in three-zone SMB separation processes when a simulated countercurrent is employed instead of a true countercurrent; when port motion occurs, a fully rinsed column (thus full of eluent) is added to the end of zone 3. As a matter of fact, during a first part of the switching period, pure eluent will exit from the raffinate outlet. Of course, this can be discarded or recycled to the eluent tank. During the

SIMULATED MOVING-BED CHROMATOGRAPHY

Solid

479

Raffinate (A)

Zone III Fei

A+B Zone II Extract £x^r (B) Zone I luent ^l!!! FIGURE 3

Equivalent true countercurrent (three-zone) systenn.

second part of the switching period, the component A diluted by eluent will be recovered. Nevertheless there are cases where the advantages of this three-zone scheme (i.e., decreasing the overall length of the system, avoiding a recycle pump operated with four different flow rates) overcome the additional eluent consumption: for example, when the eluent is water (and thus cheap) or when the packing is really expensive (saving one zone can lead to significant cost savings). These situations can happen in real cases, for example, removing salts from biological products or sucrose from molasses. A more complex implementation of the SMB involves a five-zone system. There are basically three main reasons for adding a fifth zone in an SMB: 1. Recovery of a third fraction: The addition of a fifth zone enables us to obtain three fractions, two being pure and one containing two products. For instance, starting from a feed containing three products A, B, and C (A less retained than B, B less retained than C), the flow sheet given in Fig. 4 (option 1) would enable us to obtain: two raffinate lines, one containing pure A and the second one a mixture A + B, one extract line containing pure C. 2. Removal of strongly retained products: By adding a fifth zone which could be eluted by a second eluent, the process described in Fig. 4 (option 2) enables us to perform simultaneously the separation between A and B and the removal of the strongly retained impurities. A similar process has been described by Ganetesos and Barker.^^ 3. Improvement of one outlet purity (usually the extract): Even if there is theoretically no need to add a fifth zone for binary separation, in practice, we must deal with real systems and two effects may prevent the system from reaching extreme purities (i.e., greater than 99.5%). The first effect is related to competitive adsorption: In some cases, for low concentration of one of the two species compared to the other, the selectivity disappears or may even be reversed; this is equivalent to a "pinch" or an azeotrope in distillation. The

480

R. M. NICOUD

Solid

Solid

Liquid

ZoneV

Raffinate (A)

Zone IV 1^1ffinate (A+B)

ri.

Solid

F^d

Liquid

rit

Zone IV Raffinate (A)

ik+B+c

Zone i n

Liquid

Zone III

ZoneV

F^d |A+B

Zone II

Rafrinate (A)

•>4

Zone IV

Zone III

Feed Extract (B)

lA+B+C Zone I

Zone II Extract (C) [^ Zone I

I

1^

L—Jr

FIGURE 4

Zone II Extract (B)

I Eluent 1 ZoneO

Eluent

Int.r|nux (B)

L—f

Strongly adsorbed products

Zone I

Eluent 2

—(-

m&mmsmS

^ Eluent

Different 5-zones schemes: left, option I; middle, option 2; and right; option 3.

second effect is tied to the technology of the SMB separation equivalent, and for instance, possible very small pollution due to connection at the valves level. Discussions regarding the advantages of this implementation can be found in Refs. 2, 8, and 10. The different equivalent moving beds associated v^ith the previous objectives are presented in Fig. 4. In conclusion, the four-zone SMB process scheme is generally the best choice and must be investigated first for binary separations. In several instances, how^ever, alternative schemes offer a better technical solution. The three-zone SMB should be considered v^hen the eluent consumption is not a limiting factor. The five-zone SMB is interesting for multicomponent separation or v^hen an extreme purity of one of the tv^^o effluents is wished w^ith a maximum unit throughput. A simulated moving bed (w^hatever the number of zones) consists of a given number of columns (usually betw^een 4 and 24), pumps (usually 3 to 5), and valves, enabling us to connect the different fluxes betw^een the columns. In the foUov^ing, we will restrict our analysis to the classical four-zone SMB. They are different ways to connect columns in order to build a SMB. One important option is linked to the presence or absence of a recycling pump, as explained in Ref. 10. We will restrict our analysis to the most classical (and preferred) way to implement an SMB process in which a recycling pump is located between two columns (for instance columns 12 and 1), as explained in Fig. 5. For instance, let us assume that at a given time a SMB system made of 12 columns is in the following configuration: the feed is injected at position 9 and the eluent at position 3, and the extract recovered at position 6 and the raffinate at position 11. For this configuration, the recycling pump (fixed between columns 12 and 1) is located in zone IV. After a few shifts, the pump

481

SIMULATED MOVING-BED CHROMATOGRAPHY

Extract

FIGURE 5

R a ffin ate

Classical SMB implementation.

will be located in zone III, then II, then I, and again IV. With the position of the recycling pump varying according to the zones, its flow rate can not be constant: The pump must deliver the flow rate required in the zone in which it is located. As the flow rates in the different zones are different, the recycling pump flow rate varies from cycle to cycle. Note that this constraint is perfectly mastered and that this design is relatively simple and is used with small differences in all the large-scale units. A more serious limit to this implementation is due to the volume of the recycling pump and associated equipment (flowmeter, pressure sensors, etc.): as the pump moves with respect to the zones, its volume leads to a asymetry which can lead to a decrease in purities. This decrease, which is especially important for short columns a n d / o r low retention, can be significant, but it can be perfectly and easily controlled, for example, by using a shorter column or an asynchronous shift of the inlets-outlets.^^ The last solution is extremely efficient and does not induce extra costs because it is a purely software solution. The principle has been explained in Nicoud.^^ They are many different technical options enabling us to implement a SMB process. The main features of the different elements are discussed in the following. Columns: These should be able to deliver a reasonable efficiency at a low pressure drop. Note that the pressure drop constraint is more stringent for the SMB than for classical elution mode (simply because several columns are connected in series). Moreover, the columns must obviously be identical. Pumps: According to the fluids, the size of equipment, and the pressure drop requirements, membrane, piston, or centrifugal pumps can be selected. However, they should absolutely deliver a reliable flow rate, which is a key for a successful operation of SMB. Valves: Among the different possible valves (rotary multiposition with one or several inlets associated to several outlets, or two-way valves), we recommend the use of two-way piston valves because of a possibly enhanced rehability and the easiness of troubleshooting if it becomes necessary. Fittings, dead volume: Everything must be made to minimize dead volume in the internal circulation loop.

482

R. M. NICOUD

The technical conception of an SMB is not easy, as this technology requires high precision for all the flow rates ( usually better than 1%), and great care must be taken with all the connections between the different lines to minimize dead volumes. Moreover, all the columns should be nearly identical, and very stable. This can be achieved even with soft gels, provided that an adapted procedure is used.^^ Note that if rough solutions are implemented, dreams can be transformed into nightmares. Finally, as SMB is now proposed for biochemical applications, great care must be taken for all the good manufacturing practice (GMP) issues (cleaning, reproducibility, software validation, etc.). Moreover, as SMB is probably always linked with a device enabling product recovery in the extract-raffinate streams and/or solvent recycling, these solvent-handling devices should be considered as part of the SMB. III. OPERATING CONDITIONS The steps to be followed when designing an SMB which enable it to process a given amount of feed per unit time have been described in detail in Ref. 14. The procedure described is based on the modeling of nonlinear chromatography, an experimental procedure being likely to fail unless the adsorption isotherms are linear (which is very uncommon, in fact, or requires working at very low concentrations). This procedure is a rigorous, versatile, and general procedure which mainly requires the determination of competitive adsorption isotherms, which is not as tedious and does not require as a large an amount of work as sometimes claimed. A few competitive adsorption data, measured using the mixture to be separated (the pure components are not necessary), are usually enough to determine the operating conditions of a SMB.^"^ A. Step I: Determine the Required Experimental Data Once the packing and the eluent have been selected, some important physicochemical parameters must be determined at the laboratory scale. All that is required at this stage, is a simple laboratory procedure, and that there is no need to use an SMB system. i. Initial Slope of the Adsorption Isotherms In the case of a single-component system, the adsorption isotherm gives the concentration in the stationary phase C (in moles per liter of bead or grams per liter of bead) versus the mobile phase concentration C when equilibrium is reached (in the same units as for C), at a given temperature. For small concentration, the adsorption isotherms are linear and can be written: C, = K , - C ,

(4)

Notice that the initial slope of the adsorption isotherm can be easily obtained from the knowledge of the retention time (mean position of the peak rather

SIMULATED MOVING-BED CHROMATOGRAPHY

483

than position of the maximum) associated with a small injection performed on a column, as this retention time is given by

t^ = to-il

+ ^-K]

(5)

v^here tQ = e- V/Q is the "zero-retention time" based on the external bed porosity e (commonly, e is about 0.36-0.4). Equation (5) is valid under only linear conditions; it is mandatory to check that the test has been performed under linear condition. To accomplish this, it is best to perform tw^o injections of different concentration; the obtained retention time should not be affected by the concentration. ii. Complete Adsorption Isotherms

Even if it can sometimes be linear in a vv^ide concentration range, the relation C vs C is usually not linear. Moreover, in the case of a multicomponent mixture, there is usually a competition betw^een the various compounds to access to the adsorption sites. Consequently, Q depends not only on Q but on all liquid phase concentrations. Each component adsorption isotherm is a relation of the follov^ing type: C, = / ^ , ( C i , C „ . . . )

(6)

Many different isotherm equations have been described in the literature.^^ Even though it has been challenged because it does not agree w^ith the Gibbs adsorption isotherm unless all saturation capacities are identical,^^ the competitive Langmuir isotherm is very often used:

v^here N is the saturation capacity assumed here to be equal for all components and K^ is a numerical coefficient quantifying the affinity of the solute toward the solid. In fact, the Langmuir isotherm often fails to fit experimental data, and more complex isotherms, such as modified competitive Langmuir^^ or a bi-Langmuir,^^ are more suitable. This is especially true with systems exhibiting concentration dependent apparent selectivities. (cy/Cy)/(c^/c^). A good compromise between simplicity and accuracy is relatively often obtained by adding a linear term to the classical competitive Langmuir (modified Langmuir isotherm):

C. = A;C, +

^ L

(8)

484

R. M. NICOUD

Note that the physicochemical mechanisms that enables us to perform the chromatographic bioseparations are not always "adsorption-like" but can involve ion exchange, ion exclusion, or size exclusion. Even if it is generally possible to fit experimental data with a mathematical function derived from the adsorption theory, it is strongly advisable to refer to the proper physicochemical process before modeling the separation. For instance, ion exchange can be modeled with selectivity coefficients (derived from the mass action law) that can be constant or not,^^'^^ ion-exclusion can be modeled thanks to theories based on the Donnan exclusion, etc. The determination of adsorption isotherms is less tedious than is often considered, and in some case, a simple first approximation based on a few experimental results enables us to immediately acquire interesting first process designs. Among the different methods presented,^^ the frontal method is of general applicability when the investigation of the evolution of maxima of retention peaks with respect to the injected quantities is attractive when high-efficiency columns are available. a. Hydrodynamics—Mass

Transfer Limitation

Several models are available that enable us to model as precisely as possible the respective influence of hydrodynamics and the mass transfer limitation on the chromatographic behavior. According to our experience, the simple "plate" model offers an adequate compromise between simplicity and accuracy. The height equivalent to a theoretical plate H is defined as L H = —

(9)

where L is the column length. This value can be related to the experimental system parameters through the Van-Deemter^^ or Knox^^ equations, which especially give H as a function of the interstitial mobile phase velocity u. In the case of preparative chromatography, where relatively high velocities are used, these equations can very often be simplified into a linear relation^^'^^: H = a-^b'U

(10)

The a and b parameters are related to the diffusion coefficient, porosity, mass transfer parameters, etc. If internal diffusion is the main resistance to mass transfer (which is the usual case in preparative chromatography), the dependence on the particle diameter dp is given by^"^ a = A-dp

(11)

b = B-dl

(12)

Experience shows that A is usually about 2 - 3 . From a practical point of view, parameters A and B are obtained from the determination of a few plate counts associated with chromatograms obtained at different velocities.

485

SIMULATED MOVING-BED CHROMATOGRAPHY

b. Modeling of Pressure Drop

The Kozeny-Carman equation is suitable for the laminar flows met in chromatography: AP

36

/l-6\^

cp

where h^ is the Kozeny coefficient (close to 4.5), (p = hj^ - 36 - (1 — e/eY • /i, and /I the eluent viscosity. From a practical point of view, the parameter O is obtained from the determination of few experimental pressure drops at different fluid velocities. At this level, all the required information for a first process calculation is available.

B. Step 2: Calculate the TMB For a given feed concentration, the design of the TMB relies mainly on the adequate choice of the different flow rates: recycle, feed, eluent, extract, raffinate, and solid (equivalent to a shift period in SMB). In a first step, the idea is to calculate adequate flow rates for a given feed concentration (and purities specifications). The calculation of the required flow rates is made by writing some constraints that the different products must fulfill in the different zones (for instance, the liquid flow rate in zone I must be strong enough to desorb completely the most retained product). In the particular case of linear adsorption isotherms when two species are considered, one has CA

= ^A * Q

Q = Xg • Cg

(14)

and a good separation between products is achieved with the following possible set of flow rates^:

The system as Eq. (15) enables us to choose all the TMB flowrates for a given feed flow rate and for a given value of the /3 parameter, which is a safety factor: If j8 is close to 1, the SMB is operated under its maximum possibilities, and it becomes very sensitive with respect to the number of plates and to the flow rates. If the j8 value is increased, the system is less productive but more robust. Practically, typical values of j8 are located between 1.00 and

486

R. M. NICOUD

1.05. Noted that according to the system of Eq. (15), this parameter must fulfill the constraint

l<^
(16)

We stress the fact that these relations are valid only for linear adsorption isotherms [sugars on cationic resins, diluted feed on silica (usually less than 10 g/liter)]. In preparative chromatography, high feed concentrations are suitable and lead to nonlinear adsorption behaviors. The nonlinear (and related competitive) effects must absolutely be taken into account when evaluating the flow rates. This issue has barely been addressed in the literature.^^"^^ For adsorption isotherms similar to the Langmuirian isotherms, Morbidelli and coworkers^^ have pubhshed a solution associated to the equihbrium theory in a simple way. The first step consists in solving the characteristic equation: (l + iC^ • C l - ^ + K, • C r ^ ) . co' + [ N . K , . (1 + K^ • C l - ^ ) + N . K^ • (1 + K , • C r ^ ) ] . co' + N'K^'N'KB

= 0

(17)

which has two roots (OQ and cop. The flow rates enabling us to get 100% pure products for a system equivalent to an infinity of plates are given by mi = -^^ = N'

KQ

Q

'

Qi.

K^

Q

KB

nt:, = ^=^ = Q

N'K^{N'K^-cop)

(18)

niA =

Q

12

-}- ^ 3 -h Kg • C | • {m^ - m2)\

_

^

- 4 - N - Kg • m

The preceding set of equation is valid only for Langmuir adsorption isotherms, and numerical simulation must be used to obtain the flow rates for other adsorption isotherm shapes or for multicomponent mixtures.

487

SIMULATED MOVING-BED CHROMATOGRAPHY

At this level, the flow rates required by an "ideal TMB" (which mainly means that kinetic and hydrodynamic dispersive effects are assumed to be negligible) to get 100% pure products for a feed of a given concentration are known. The final flow rates will be extremely close. This procedure is sensible because it has been proven that a TMB or SMB performance is only slightly sensitive to the number of plates.^^ In most cases, the required number of plates can easily be achieved and optimum flow rates are then available. The feed concentration has a strong influence on the SMB performance and must be well chosen. The productivity and the eluent consumption are two main economic criteria involved in chromatographic processes.^^ Their variations versus the feed concentrations can be checked in order to choose an appropriate feed composition. This study can be quickly carried out for an "ideal" TMB, as mentioned in the previous section. It has been reported^"^ that the productivity increases and the eluent consumption decreases when the feed concentration increases: The variations are usually rather steep in the low concentration range and very smooth in the high concentration range. As a consequence, low injection concentrations will have to be avoided. However, even if achievable, very high concentrations will not be suitable because • As soon as concentrations are high enough, the performance of the TMB (or SMB) is almost constant. • Very high concentrations can lead to a very low feed flow rate, which might be difficult to control. The flow rates given previously lead to 100% purities in the case of an "ideal" TMB, equivalent to an infinite number of plates. The approach used here is to keep these flow rates and to seek the minimum number of plates N^ required to reach the required purities as high as 99%. To do so, we must use a model of the TMB that enables us to focus the influence of the system efficiency on purities. Many different models have been applied to the modeling of chromatographic processes.^^ The equilibrium stage model has been proven to be suitable under the usual conditions of high-performance preparative chromatography^^ and can also be applied to TMB.^^ The countercurrent column is considered as a cascade of cells in series. The adsorption equilibrium is assumed to be reached in each cell or equilibrium stage or plate. The broadening effects, linked to the mass transfer kinetics and to the hydrodynamics, are lumped together and are quantified by the number of theoretical plates N , which can be derived from an "analytical" pulse injection. The mass balance equation of a component / over a plate k when steady state is reached is

Q

-

Q

Q,k ~

fi(^ik'>'"-f^i^k^-")

488

R. M. NICOUD

where Q and Q are the fluid and sohd flow rates. Solving equation (19) for each plate in each zone (with the proper liquid flow rate) together with the proper boundary conditions enables us to calculate the internal concentration profiles, and thus the extract and raffinate purities. The steady state of a TMB is calculated for different numbers of theoretical plates, where an identical number of plates in each zone is assumed. The extract and raffinate purities are derived from each numerical simulation. At this stage, everything that can be obtained from the simple TMB representation has been obtained. In order to get more information, numerical work must not be performed on a real SMB model.

C. Step 3: Calculate the SMB The TMB and SMB concepts are similar. In fact, it has been shown that an SMB and its hypothetical corresponding TMB have very close performances.^^ Knowing that optimum operating conditions can be found directly for a TMB and that simulating this kind of process leads to much shorter computation times, it is in our interest to take advantage of this similitude when designing a SMB. Consequently, a study of an hypothetical TMB is performed first. Table 1 gives the relation between a SMB and its corresponding TMB.^"^ In the table V^ is the volume of one SMB column and the equation given in Table 1 enables us to calculate the SMB that would behave closely to its corresponding TMB. The inlet-outlet flow rates of an SMB and its corresponding TMB are identical. An SMB does not exactly work in steady state but in periodic steady state: During a given period, the internal concentration profiles vary, but the internal profiles examined at the same time in two successive periods are identical (except for a one-column translation). The rules used to calculate the SMB flow rates from the TMB flow rates mean just that the velocity of the liquid relative to the solid is kept constant. i. Determination of Column Number, Diameter, and Length

The only way to estimate the number of columns per zone N^ is to perform numerical simulations of an SMB, including the shift of the injection and collection points at regular time intervals. Such calculations can be performed using dummy values of the column volume and TMB solid flow rate (to estimate the period At). In the case of fixed-bed operations (elution chromatography, SMB, etc.), if the column is simulated to a series of mixed cells, the mass balance equation of a component / over a plate k is dC: L

1 - e

dC: L

c,,.-. = c,,, + v - ^ + v ^ - - ^

(20)

where ^Q is the mean residence time of the mobile phase in a plate, and 6 is the external porosity, which is usually in the range 0.35-0.45. The system in Eq. (20) is a system of first-order ordinary differential equations and can be solved with classical numerical methods^^ when associated with a set of boundary and initial conditions. The solution of the system of Eq. (20)

489

SIMULATED MOVING-BED CHROMATOGRAPHY

j ^ ^ H TABLE I

Relation between an SMB and Its Corresponding TMB

TMB

SMB

Steady state Solid flow rate: Q

Periodic steady sate Periodic shift of the injection-collection lines: A T = -^

Internal flow rates: Qj^^k

= I, II, III, or IV

=-

Internal flow rates: Q | ^ ^ = QJ^^

Eluent, extract, feed, and raffinate flow rates: WEI ? WfEx 5 y ? 5 and (^R^f

+ —— •

Eluent, extract, feed, and raffinate flow rates: yEi ? S^EX ^ ^ F ? and ^R^f

enables us to simulate the complete SMB behavior and thus to select precisely the required configuration. In a first approximation, the previous complex calculation can be avoided by assuming that the minimum number of plates required to obtain adequate purities is identical to the number of plates that was required for the TMB. Taking into account a pressure drop constraint, from Eq. (10) and (13), one can write a system of equations giving the system total length and mean velocity by AP. =

^'U^

(21) N^.n

1 a + b 'U„

If we know L and u^, the column length and diameter are easily calculated by i^col =

L/K

ficol= '^^-0,01/4=

where Q'^^ is the SMB average internal flow rate, and Cl is the column section. The optimum number of columns cannot be determined without the complete process calculation. However, experience shows that systems of eight columns are able to solve most of the problems without too much technical complexity. Step 4: Practical Implementation and Fine-Tuning At this level, most of what can be done with numerical tools and simple laboratory equipment has been done. We must now check this design on a laboratory pilot plant. Experience shows that the numerical prediction enables us to get a set of flow rates which are very close to the adequate ones.

490

R. M. NICOUD

Usually, the simple determination of an internal profile enables us to identify simply which flow rate(s) must be modified. Determining an internal profile consists of taking small samples of fluid between columns in order to analyze them and to plot, at a given time, the concentration of the product versus the position in the system. The profile obtained is equivalent to the steady-state internal profile obtained in the TMB. The shape of the profile tells a lot regarding the system behavior, as shown in the example presented at the end of this chapter.

lY. MAIN APPLICATIONS AND DEVELOPMENTS There are many bioseparations problems for which SMB can be used. Most of them are reviewed in the following and can be classified as • • • • • •

Separation of sugars Desalting Purification of proteins and complex molecules Separation of ionic molecules Separation in organic molecules Optical isomers separation

A. Separation of Sugars This application is the most known, and the separation of fructose and glucose is one of the largest applications of chromatography separation. Since the pioneering work of Barker,^"^ this separation has been investigated by many workers.^^"^^ A review of the work done by the Barker's team is given in Ref. 11. This separation is performed on ion-exchange resins, using warm water as the eluent. The preferred implementation consists of using polystyrene cation-exchange resins in the calcium form: The fructose forms a complex with the calcium ions and is retarded; the glucose and other oligosaccharides are eluted with the eluent. To improve the productivity, some work has been done with zeolites (calcium form) instead of resins.^^ Another possibility consists of using anion-exchange resins in the bisulfite form, where the glucose is retarded by complexing with the bisulfite, and the other oligosaccharides are eluted first. This option is not used at the production scale because of the lower stability of anion-exchange resins. The Sarex process has been reported^^ to be used to separate continuously a 500 g per liter inverted carbohydrate syrup containing 42% fructose, giving 90 to 94% pure fructose at recovery of over 90%. The glucose-rich fraction is about 80%, and both product concentration were about 200 g per liter. This separation can be implemented on columns of a few meters internal diameter. SMB packed with cationic resins in the calcium form have also been used to obtain other monosaccharides such as xylose or arabinose. ^ Experiments have been performed using a feed mixture containing 21 g per liter of glucose, 155 g per liter of xylose, and 20 g per liter of arabinose, the

SIMULATED MOVING-BED CHROMATOGRAPHY

49 I

retention order being glucose then xylose then arabinose. The goal was to obtain a raffinate with no detectable traces of arabinose with a maximum recovery of xylose. The goal was reached as the following extract and raffinate composition were obtained: Raffinate: glucose: 8.26 g per liter; xylose: 73.69 g per liter; arabinose: below detection limit Raffinate: glucose: 0.09 g per liter; xylose: 0.47 g per liter; arabinose: 14.16g per liter This example shows perfectly that SMB is a binary separator: It enables us to split a mixture in two fractions even if this mixture contains more than two products. Another interesting application is devoted to the separation between mono- and disaccharides or between disaccharides. For instance, Kishihara et al^^ studied the separation between palatinose and trehalulose and results obtained by NOVA SEP for the separation between fructose and trehalulose are given in the following paragraphs. The separation has been performed on Dowex 99 monosphere (350 /xm) in calcium form Ca form using water (65°C) as eluent. Under these conditions, the retention factors of the two sugars are: i^^xreha = 0.17 and K^^^^^ = 0.4. The separation has been performed on a SMB made of 12 columns of 2.6 cm i.d. and 1 m length, working with a maximum pressure drop of 5 bars and at a temperature of 65°C. The feed containing 120 g per liter fructose and 120 g per liter of trehalulose was injected at a flow rate Qp = 10.83 mL/min leading to a productivity of 3750 g of feed per day on this small pilot plant. Excellent results were obtained: both extract (position 3) and raffinate (position 9) were recovered at 98% purity; an internal profile (internal concentrations normalized by the feed concentration) is given in Fig. 6. Moreover, note that the pure fractions are recovered at a very significant concentration: 79% of the feed concentration for the fructose and 65% for the trehalulose. The SMB technology has also been used for performing the fractionation of dextran (polyglucoside mainly used as a blood plasma volume expender) by size exclusion.^ The columns were packed with Spherosil XOB075 of 200 to 400 /mm porous silica beads, and the technology has been proven to be efficient allowing to obtain, according to the flowrates very different fractions (from 10,000 to 125,000 Dal).

B. Desalting Desalting is a second simple and interesting application of SMB in biotechnologies. Different mechanisms can be used, for example, ion exclusion, hydrophobic interaction, size exclusion, or the ion-retardant effect.^^ Glucose and NaCl have been separated on Retardion 11 A-8,'^^ very high purity products have been reported for feed mixtures containing 3 mol per liter of glucose and 3 mol per liter of NaCl. In that case, the adsorption isotherms are favorable (Langmuirian type).

492

R. M. NICOUD

- Fructose -Trehalulose

1

2

3

4

5

6

7

8

9

10 11 12

Column Number FIGURE 6 Separation between fructose and trehalulose. Internal profile obtained on a LICOSEPLab (NOVASEP).

A similar work was performed to separate NaCl from glycerol on Amberlite HFS-471X (8% DVB) which is a strongly acid cation-exchange resin of the sodium form. In this case, the mechanism is ion exclusion (the glycerol can enter the internal porosity of the resin when the salts are excluded). The adsorption isotherm of glycerol is linear (as expected because glycerol has no interaction with the resin: it just enters in the internal porosity) when the adsorption isotherm of the salt is non favorable (anti-Langmuir) as expected from an ion exclusion process (Donnan behavior). Instead of ion exclusion, the size exclusion process has been used to perform the separation of NH4SO4 and a protein."*^ In this case, the adsorption isotherms were simply linear. Finally, a hydrophobic interaction was used by Hashimoto"^^ to perform the separation of phenylalanine and NaCl. In this case, NaCl, having almost no interaction with the packing, had a linear adsorption isotherm when the phenylalanine exhibited a classical Langmuirian adsorption isotherm. C. Purification of Proteins and Complex Molecules Only a few references regarding the use of SMB to perform the separation of proteins have been reported. The first attempt can probably credited to Huang et al!^^ who performed the purification of trypsin from extract of porcine pancreas. They successfully used an SMB consisting only six affinity columns, which shows that SMB with a very limited number of columns can be attractive. Even if the four-zone process had fairly high performances in separation efficiency, the addition of a fifth zone used as a washing section (cf. Fig. 4, option 2) was advised. A recent proposal consists of performing the purification of human serum albumin (HSA)"^^ on two SMBs connected in series: The first one was used to remove the less strongly retained components, and the second one to remove the more strongly retained components.

493

SIMULATED MOVING-BED CHROMATOGRAPHY

Finally, some results concerning the separation of myoglobin and lysosyme have been presented."*^ This separation can be performed on a support such as ACA 54 (Biosepra, France) with an eluent containing NaCl 0.15 M in vvrater (L. Guerrier, personal communication). On an SMB made of eight columns of 2.6 cm i.d. and 0.1 m length, very pure extract ( > 98%) and raffinate ( > 98%) can be obtained from a 50-50 mixture at 2 g per liter. An internal profile is given in Fig. 7. One of the most difficult biochemical separations to perform is associated with obtaining cyclosporine from fermentation broth. Some options using SMB have been described in by Kinkel'^'^ and in a patent application."^^ The problem is to purify cyclosporine a (Fig. 8) from a mixture containing various isomers in order to get a quality corresponding to USP XXIII and European Pharmacopoeia, 2.Ed. 1995. The minimum required purity is 98.5%. This purification is tough because of the number of products closely related to cyclosporine a and because of significant kinetics limitations (due to the size of the molecule) constraining the columns efficiencies. Due to the complexity of the problem, some attempts have been made to use different chromatographic processes. For instance, a batch system packed with silica could lead to two prepurified fractions containing enriched cyclosporine a. The first fraction contains cyclosporine a and impurities that elute before it, and the second fraction contains cyclosporine a and impurities elute after it. These two fractions can then be processed in batch systems or in SMB systems packed with reversed-phase material or with normal silica (Fig. 9). Using this coupling between batch and SMB systems and because of the choice of the stationary phase, the retention orders as well as the composition of the prepurified fractions enables cyclosporine a to be obtained at the raffinate. Both SMB steps have been performed on a LICOSEP 8 X 50 (NOVASEP) equipped with eight axial compression columns (100 m length X 50 mm i.d.). - Myoglobin - Lysozyme

3

4

5

Column Number FIGURE 7 Separation of myoglobin and lysozyme: internal profile on an eight-column SMB for an ACA 54 (Biosepra).

494

R. M. NICOUD

Cyclosporine A

CM,

HaC^ ^CH3

CH3

r ^ ' Cja

HjC-N

CH3 CH3

HjC^

FIGURE 8

CH3

Formula for cyclosporine and analytical chromatogram of the feed mixture.

For step 1, the mobile phase is ethyl acetate and the solid phase normal silica. The feed concentration is fixed to 5.8 g per liter, the feed flow rate is 5.3 mL/min, and the recycling flow to 151 mL/min. For step 2, the mobile phase is acetonitrile-H20 and the solid phase is reversed-phase chromatography. The feed concentration is fixed to 1 g per liter, the feed flow rate is 12.7 mL/min, and the recycling flow is 151 mL/min. The results are summarized in Tables 2 and 3. A comparison of the batch and the SMB modes proved to greatly favor SMB: The productivity and the eluent consumption was improved by three at least times using SMB.

s

Cyclosporine

Cyclosporine

I

C.B.L.UAG.D

C.B.L.UAG.D

Cyclosporine J—'

Stat, phase: Silica Mob. phase: ethyl acetate. Cyclosporine

Stat, phase : RP18

Stat, phase : Silica

Mob. phase ACN/water

m Mob. phase : ethyj W acetate

Cyclosporine Cyclosporine

FIGURE 9

Mob. phase: ethyl acetate. Cyclosporine

C,B,L,U,A

A.G

A

Cyclosporine

Stat, phase : Silica

Raffinate:

Raffinate:

Cyclosporine

Cyclosporine

A

A

A

Purification of cyclosporine a batch - batch and batch - S M B methods.

SIMULATED MOVING-BED CHROMATOGRAPHY

H H

495

T A B L E 2 Results Obtained on a Licosep 1 2 - 5 0 for the Purification of the First Fraction Obtained by the Batch System (Fig. 9)

Type of cyclosporlne

Feed composition (%)

c b I u a g d Yield of a

2.3 6.9 0.7 1.2 86.2 1.0 0.1

RafFinate composition (%)

0.2 0.4 0.0 0.4 97.4 1.1 0.1 >95%

Note that other schemes involving SMB can also be considered. For instance, the feed can be processed in an SMB working in the reversed mode in order to get a fraction containing only cyclosporine /, w, and a. This fraction is then purified in a second SMB w^orking also in the reversed mode to get the final cyclosporine. The first step can also be performed using a SMB packed v^ith silica (see Fig. 10). D. Separation of Ionic Molecules The SMB technology has also been used to perform the purification of different ionic molecules. For instance, large-scale production of lysine can be performed by SMB."*^ In addition, pure betaine can be obtained from molasses via a process involving tvv^o chromatographic steps^^: In step 1, a fraction containing a mixture of betaine and glycerol is separated from the rest of the feed (mineral, carboxylic acids) using ion exclusion. Then, in step 2, glycerol is separated from betaine in a second ion-exclusion step. The tripeptide L-glutathione^^ produced by yeast fermentation is used in cases of liver diseases. High purity L-glutathione (99%) is required in the final

T A B L E 3 Results Obtained on a Licosep 1 2 - 5 0 for the Purification of the Second Fraction Obtained by the Batch System (Fig. 9)

Type cyclosporine

Feed composition (%)

c b I u a g d Yield of a

— — 0.3 0.8 92.5 4.1 1.6

RafFinate composition (%)

0.2 0.5 99.1 0.0 0.0 >95%

496

R. M. NICOUD

Cyclosporine C.B.L.U.A,G.D

Cyclosporine C,B.L.U.A.G,D

Raffinate: Cyclosporine L.U.A

Extract:

Raffinate:

Cyclosporine

Cyclosporine

A

A

F I G U R E 10 Two possible processes enabling the acquisition of pure cyclosporine using SMB. Left: first SMB packed with reversed phase, and right: first SMB packed with silica.

crystallization step. Obtaining this highly pure glutathione is difficult, especially because of the presence of amino acids, and one of the limiting species is glutamic acid. The separation of the L-glutathione and glutamic acid is performed on a cation-exchange resin (Rohm & Haas, Amberlite IR200C, 350-590 jLtm). The separation was implemented in an SMB composed of 16 columns of 1 cm i.d. and 10 or 20 cm length. The SMB parameters were determined via a procedure which is similar in essence to the procedure given in this chapter (determination of the internal flow rates using knowledge of the adsorption isotherms). Operating at 0.05 mol per liter of HCl (the pH value obviously controlling the adsorption of both products on the resin) the glutathione was obtained in the raffinate stream at 1.62 X 10"^ mol per liter at 99% purity with 99% yield. The productivity was 4.52 X 10""^ mol per liter of adsorbent per minute. Moreover, in this case, the steady state was reached in about 4 hr which is a very reasonable time. E. Separation of Organic Molecules Many separations of organic molecules have been performed with the SMB technology, but according to our knowledge, only two of them belong to the biotechnology area. 1. Some work has been done to perform the separation of fatty acids. The first results were published by Szepy^^ and were associated with the separation of C^^ to C22 methyl esters. An interesting patent has been granted to Pronova^^ which associates classical (batch) chromatography with an SMB with two inlets to obtain pure EPA(C2o) and pure DHA(C22) from

SIMULATED MOVING-BED CHROMATOGRAPHY

FIGURE I I

497

Eight = column LICOSEP 8-200.

fish oil. This example shows nicely the need to associate different technologies to optimized a production process. 2. The separation of the stereoisomers of phytol (3, 7, 1, 15-tetramethyl2-hexadecen-l-ol, C20H40O) has been described^ at a relatively large scale (about 20 kg per day). The synthetic phytol is a mixture of cis (33%) and trans (67%) isomers, the latter being used in perfumery. This separation is presented in the foUow^ing. The separation of the phytol isomers is performed on classical silica (Lichroprep Si 60, 25-40 /xm from Merck KGaA, Darmstadt) w^ith an eluent made of heptane-ethyl acetate (75-25 v/v) at 27°C. This separation is implemented on a LICOSEP 8-200 (NOVASE?) v^hich includes eight axial compression columns of 200 mm i.d. and up to 400 mm long. For this particular case, the columns w^ere packed w^ith 3 kg of silica, leading to a column length of 17.7 cm (±0.6%). The LICOSEP 8-200 (Fig. 11) is built according to the flow sheet given in Fig. 5.

498

R. M. NICOUD

Each outlet (extract and raffinate) was processed using a falling film evaporator consisting of one to twelve 3 m tubes of 10 mm i.d. For this separation, the pressure was set to 180 mbar, and the oil temperature to 80°C. The concentrated extract and raffinate were recovered in 50 liter glass vessels. For a feed concentration of 105 g per liter the system was operated night and day under the following conditions: Q R , , = 226 liter/hr Q,^^, = 7.8 liter/hr Q^,^,^, = 36.0 liter/hr QExtract = 32.4 litcr/hr QR,ff = 11.4 liter/hr leading to an extract purity of 98.4% and to a raffinate purity of 99.4% Note that the pressure drop being only 26 bar under these conditions, the productivity could have been increased of about 52%. Finally, note that when products are soluble in organic solvents, the use of supercritical fluid as eluent can be of key interest. Moreover, substituing liquid by supercritical fluids enables us to introduce a pressure gradient and thus an eluent strength modulation in the system.^"^'^^

F. Separation of Optical Isomers The interests in SMB to perform large-scale separations of optical isomers^^ are now recognized (very short development time, extremely high probability of success, attractive purification cost, etc.). An increasing number of pubhshed results are available;^^'^^ among them: prazinquatel,^^ j8-blockers,^° chiral epoxide,"^ Thiadiazin EMD5398,^'^ Hetrazepine.^ The Belgium company UCB-Pharma has announced their decision to purchase a large-scale SMB from NOV A SEP to perform optical isomer separation at several tons per year. Almost all of these separations are performed on cellulose-based packings using organic eluent. The two first references were published in 1992 by Negawa on phenylethyl alcohol^^ and Fuchs on threonine.^^ The second one is described in the following. Among all the examples cited previously, the threonine example is not by far the most productive. It is presented here because it involves amino acids and a ligand exchange chiral chromatography which is seldom used at preparative scale and is thus probably more "bio" than the others. The separation of the two optical isomers of threonine has been performed on a SMB made of 12 columns of 2.6 cm i.d. and 1 m length. These columns were packed with Chirosolve L-Proline (200 jjum) from J.P.S. Chime (Bevaix, CH2022). These support involves ligand exchange chromatography which was introduced by Davankov in 1968. The mechanism is based on the formation of complexes between the grafted proline, copper, and the optical isomers contained in the solution. The eluent used is a mixture of aqueous acetic acid (0.05 M) and aqueous copper acetate (0.000125 M ) and the operating temperature was 25°C.

SIMULATED MOVING-BED CHROMATOGRAPHY

499

The racemic mixture was injected continuously at 5 g per liter, the operating parameters being given in the following. Q R , , = 22 mL/min Qfeed = 4.2mL/min Qmuent = 6.2 mL/min QExtract = "7.4 mL/miu QR,ff = 3.0 mL/min AT = 43 min Under these conditions, about 30 g of feed were processed per day, and very pure extract (99%) and raffinate (99%) are obtained. An internal profile obtained with this set of parameters is provided in Fig. 12. This profile has a very interesting property which demonstrates what can be done when one is able to play with the nonlinear behavior of the system: The extract is recovered at a concentration which is 60% what is already an excellent result, but the raffinate is recovered in a pure form at a concentration which is 2 5 % greater than the feed concentration.

Y. PRACTICAL APPLICATION: SEPARATION OF SUGARS Note that this example is built as a pedagogical example. The results given in the following are not all experimentally obtained, but are very typical of what is obtained with sugars. The methodology proposed enables us to get a precise understanding of the method that makes it possible to calculate an SMB. If a very precise design is required, however, some simple hypothesis should be relaxed and more sophisticated model used. The problem consists in performing the separation of a monosaccharide (mono) and a disaccharide (di), A preliminary screening has shown that these sugars can be separated on a cationic ion-exchange resin (250 fim) in calcium form Ca using water (65°C) as eluent. The problem is to design a

F I G U R E 12 Separation of the isomers of threonine. Internal profiles obtained on a LICOSEP 12-26 (NOVA SEP).

500

R. M. NICOUD

SMB able to process a flow rate of 50 liters/hr of a solution containing 100 g per liter of each sugar. Each stream (extract and raffinate) should contain products at a minimum purity of 97%. A. Step I: Determination of the Required Experimental Data All the tests were performed on a column of 75 X 1 cm (the laboratory column) packed with the cation-exchange resin to be used in the SMB; the temperature is fixed to 65°C. A small injection (Vj^j = 0.5 mL) of a mixture containing 10 g per liter of monosaccharides and 10 g per liter of disaccharides is performed on the laboratory column eluted by a flow rate of 5 mL/min of water. The retention times of the peaks are: tj^imono) = 5.7 min and tj^idi) = 7.54 min. Assuming an external porosity of 0.4 for the laboratory column, the zero retention time associated to the pulse test is ^0 =

V,^i 0.4 X 58.9 ::— = 1 = 4.71 min

From Eq. (5) we obtain tj,{mono)-to Xmono =

X I. = ^^

e,

5.7-4.71

* ^0 t^{di)-t, ^0

= 1 - ^.

*

e, 1 - ^e

=

0.4 *

4.71 7.54-4.71 4.71

= 0.14 1 - 0.4

*

0.4 1 - 0.4

= 0.40

The initial slopes of the adsorption isotherms are thus known. The adsorption isotherms are measured via the frontal chromatography method. Because the interaction between the products was expected to be weak, both products were simultaneously present in the injected feed. Injections were performed on the laboratory column, at a fixed flow rate of 5 mL/min. Samples were taken at the outlet of the columns every 30 s and analyzed. Obtained results are given in the following Table 4. From the results of the Table 4, it appears that the retention time of the breakthrough curves of the mono-and disaccharides are not affected by the feed concentration: They are close to 5.7 min for the monosaccharides and to 7.4 min for the disaccharides. Having shown that the mean position of the breakthrough curves are independent of the concentration and identical to the retention time obtained for an elution peak, we can state that the isotherms are linear, with a slope given by K^^^^ = 0.14 for the monosaccharides and K^^ = 0.40 for the disaccharides. The investigation of the efficiency of the system was performed by injecting small amounts of mono-and disaccharides (V^^- = 0.5 mL of a mixture containing 10 g per liter of monosaccharides and 10 g per liter of disaccharides) on the laboratory column at different flowrates. An example of

501

SIMULATED MOVING-BED CHROMATOGRAPHY

TABLE 4 Frontal Chromatography: Experimental Breakthrough Curves in G r a m s Per L i t e r O b t a i n e d f o r t h e M i x t u r e s o f M o n o - and Disaccharides of Different Concentrations Feed: 2.5 + 2.5 g per liter Feed: 25 + 25 g per liter Feed: 100 + 100 g per liter Time (min)

Mono

Dl

Mono

Dl

Mono

Dl

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10

0 0 0 0 0 0 0 0 0.03 0.15 0.49 1.05 1.65 2.1 2.35 2.45 2.49 2.5 2.5 2.5 2.5

0 0 0 0 0 0 0 0 0 0 0.01 0.05 0.16 0.41 0.8 1.26 1.7 2.05 2.27 2.4 2.46

0 0 0 0 0 0 0 0.02 0.25 1.44 4.73 10.24 16.35 21.13 23.94 25.22 25.62 25.57 25.3 24.94 24.59

0 0 0 0 0 0 0 0 0 0.01 0.1 0.49 1.7 4.22 8.14 12.84 17.34 20.85 23.14 24.42 25.04

0 0 0 0 0 0 0 0.08 1.01 5.91 18.34 40.07 64.22 86.45 93.11 101.1 103.9 103.6 101.7 99.21 96.85

0 0 0 0 0 0 0 0 0 0.05 0.39 2 6.98 16.4 33.5 51.8 67.3 85.7 90 95.8 99.9

2,500 -r 2,000 1,500 1,000 0,500 0,000 0,0

20.0

40,0

60,0

Time (min) FIGURE

13

Injection disaccharides on the laboratory column. Flow rate: I m L / m i n .

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R. M. NICOUD

TABLE 5 Influence of the Fluid Velocity on the Efficiency of the Analytical Column (Most Retained Connponent) Flowrate (mL/min)

1

5

10

20

Fluid velocity (m/min) Number of plates on the column Number of plates per meter

0.0127 375 500

0.0637 127 170

0.127 79 105

0.254 37 50

obtained chromatogram is given in Fig. 13. In chromatograms such as that shown in Fig. 13 one can determine the number of plates associated with each peak. The resuhs obtained are summarized in Table 5. The results obtained can be adequately fitted by

N/L = H = l/{a -\- b ^ u) = 1/(0.001 + 0.075 * u)m~^ where u is given in meters per minute. In this simple example the most retained product is considered because it gives the smallest number of plates; it will enable us to create a conservative design of the SMB. The pressure drop was determined according to the fluid velocity on the laboratory column: one obtained 1 bar at 5 mL/min and 5 bar at 20 mL/min. To get an average value of the viscosity that can be expected in the SMB, a solution containing 50 g per liter of each sugar was used for the tests. The results are presented in Table 6. Consequently, the pressure drop in the system is fitted by: AP —— = 22 * u b a r / m At this point, all the experimental information required to perform the calculation of the SMB is available.

B. Step 2: Calculation of the TMB The flowrates of the TMB are calculated thanks to the equation given on system (15). For a feed flow rate of 50 liter/h, and if one selects j8 to 1.02

T A B L E 6 Pressure Drop Obtained in the Laboratory Column Flowrate ( m L / m i n )

5

20

Pressure drop (bar) Pressure drop (bar/m) Fluid velocity (m/min)

1.1 1.48 0.0637

4.2 5.60 0.254

SIMULATED MOVING-BED CHROMATOGRAPHY

H ^ l

503

T A B L E 7 Influence of the Equivalent Number of the T M B on the Extract and Rafflnate Purities Number of plates

100

150

200

250

Extract purity Raffinate purity

92.07 96.77

98.10 95.10

98.76 96.70

99.14 97.68

one obtains (in 1/h): 50 = 200.5 0.4/1 .02 - 0.14 * 1.02

^'

0 ^

K,/p-K^'P

Q™^ = Q™^ =

P-M-KB

=

1.02*200.5 * 0 . 4 = 81.8

QE^, = Q • (Kg - K ^ ) • /3 = 200.5 * (0.40 - 0.14)/1.02 = 53.17 QRaf = Q{KBQEIU

=

QEXC

KA)/P

= 200.5 * (0.40 - 0.14)/1.02 = 51.1

+ QRaf - Qpeed = 53.17 + 51.1 - 50 = 54.27

This set of flow rates is then used in a model solving the set of Eq. (19) to get the internal profiles and thus the extract and raffinate purities. The influence of the total number of plates on the purities of a system composed of four identical zones is given in Table 7. The targeted purities ( > 97%) are reached with a system containing about 200 plates. Note that in this case, the highly purified extract is easier to get than highly purified raffinate. This result is not at all general. To optimize the system completely, other values of the parameter j8 could be selected, leading to different flow rates and plate requirements. The optimum j8 is then selected introducing economical criteria. C. Step 3: Calculation of the SMB From Table 1, we know that the SMB internal flow rates are related to the TMB internal flow rates by QSMB ^ gTMB + ^

•Q

which for zone I (recycling flow rate) gives Qf^^ = 81.8 +

0.4 • 200.5 = 215.47

The global column size is estimated by taking into account the pressure drop and the number of plates constraint. For instance, if we decide to fix a maximum pressure drop of 20 bar for the complete system and assuming that the SMB must have 200 plates (as in the TMB), the global length and fluid

504

R. M. NICOUD

velocities must be fulfilled (as given by Eq. 21): —J— = ^u^ N™„

thus Y 2 2 * u„ 1

a + b'u^ thus

200

1

L

0.001 + 0.0075 * w.

leading to = 0 . 2 4 m / m i n L = 3.8 m The column section can be calculated as the ratio of the mean internal flow rate to mean fluid velocity. To keep a safety margin, one can replace the mean internal flow rate by the flow rate in zone I, which is greatest. Consequently, one obtains

iicoi =

215.47/60/1000 ^J^ = 0.015 m^

which leads to a column diameter of 13.7 cm. Now, assuming as a first guess that the system is made of eight columns, the length of each column should be 0.475 m (in order to get a total length of 3.8 m). The column volume is thus 0.0071 m^ (7.1 liters) and the periodic shift of the injection-collection lines is given by (1 - e) • V, (1 - 0.4) * 7.1 AT = -^^ ^ = -^ = 0.0213 hr M 200.5 All the parameters (flow rates, columns size, and shift period) defining a first SMB are thus known. This SMB is probably able to supply adequate purities with an interesting productivity, but is not fully optimized. A complete optimization of the process would require an intensive use of numerical tools to investigate the precise influence of the number of columns, number of columns per zone, column length, etc. on an defined economical function. D. Step 4: Practical Implementation and Fine-Tuning The separation was performed in a pilot test equipped with columns of 2.6 cm i.d. The first test was performed after a simple extrapolation of the parameters obtained for the calculation of the production scale unit (keeping the fluid velocity constant. Table 8). To check the quality of the columns of the SMB pilot plant, a pulse test is performed on all the columns connected in series, the eluent flow rate being fixed to 4 liters/h. The total bed volume of the pilot plant being 2.016 liters, assuming an external porosity of 0.4, the zero retention time associated to

505

SIMULATED MOVING-BED CHROMATOGRAPHY

H H T A B L E 8 Comparison of Key Parameters and the Pilot Plant and the Expected Production Unit Feed (liter/hr) Pilot plant (column i.d.: 2.6 cm and 47.5 cm length) Designed production unit (column i.d.: 13.7 cm and 47.5 cm length)

Raffinate (liter/hr) 1.84

1.80 50

Recycle (liter/hr)

Switch (hr)

1.91

7.76

0.052

53.17

215.47

0.052

Extract (liter/hr)

51.1

this test is 0.4 *2016 +

^0

—

A

0.2 h r = 12 min

The expected retention times are t^{mono) t^{di)

/ 1-0.4 \ = 12 * 1 + ——-— * 0.14 = 14.52 min / 1-0.4 = 12 * 1 +

\ * 0.4

= 19.2 min

In fact, the experimental retention times are tj^imono) = 15.3 min and t^{di) = 21.0 min, and are thus 5 to 10% greater than the expected times. This discrepancy can be due to differences in packing procedures between the laboratory column and the columns used in the SMB or differences between the lots of resins. The SMB was started with the set of flow rates presented in Table 8, and the results are given in Fig. 14. The associated extract and raffinate purities are, respectively, 81.6 and 72.6%. Looking at the profiles given on Fig. 14, one can easily see that the internal flow rates are not sufficient enough to stabilize the fronts at the right position. The fronts are shifted to the left, leading to a pollution of the raffinate by products recycled from zone I to zone IV (on the hypothetical solid flow rate) and to a pollution of the extract by mono coming from zone III. This behavior agrees with the fact that from the pulse test on the SMB pilot plant, retention times are greater than anticipated; with the products being more retained one must push them more strongly. Another test was performed on the pilot plant after increasing the recycling flow rate from 7.76 to 8.35 liters/h. The results presented in Fig. 15 show now well positioned profiles, the extract and raffinate purities are respectively 96.3 and 95.5%. The achieved purities are now acceptable even though they are slightly below the target. In this case, they can be increased by about 1% by decreasing all the internal flow rates by about 20%, which leads to an increase of the number of theoretical plates for the system. Consequently, we were successful in designing an SMB able to perform the separation of the

506

R. M. NICOUD 100

F I G U R E 14 Internal profiles obtained on the pilot plant with the set of parameters presented in Table 8 (column 9 is column I).

saccharides. A precise process optimization would now be required, starting from this first design by adjusting the key parameters (flow rates and columns number) and checking the result.

Yl. CONCLUSION Simulated moving beds have been successfully used during almost 30 years at a very large scale in petrochemistry. It appears clearly that this technology has a great potential for fine chemistry and the pharmaceutical industry. More and more applications are described for the biochemical field (leading sometimes to 10 times lower eluent consumption compared to the usual chromatography). Because small-scale units are already available, SMBs can

1,00E+02 ^

8.00E+01

g

6,00EK)1

I

4.00EH)1

3

1 o c 8

2,00EK)1 O.OO&OO

column

F I G U R E 15 Internal profiles obtained on the pilot plant with the set of parameters presented in Table 8 except that the recycle flow rate has been set to 8.35 liters/hr (column 9 is column I).

SIMULATED MOVING-BED CHROMATOGRAPHY

507

be operated for small-scale production ( < 1 kg) as well as for very large production (100,000 tons per year) for very different products: enzymes or sugars, desalting or optical isomers separations, etc. The SMB is basically a binary separator that presents three main advantages over batch chromatography. 1. It enables us to save significant amounts of eluent. According to our experience, the previous result is general provided that binary separations are involved. Regarding multicomponent systems, the situation is more complex. SMB is basically a binary separator. If the product to be recovered is the first or the last eluted one in a multicomponent mixture, SMB can be used without technical modifications (only the method of determining the flow rates must be changed). In other situations, two SMB systems must usually be used in series. 2. It enables us to maximize productivity. The value of SMBs with respect to batch chromatography is maximized for low selectivity problems or low efficiency systems. 3. It is a continuous process that simplifies the operation and especially the connection to associated equipments (evaporation for instance). However, SMB utilization requires a strict procedure, an efficient simulation software and is less versatile than batch chromatography. In fact these two modes are more complementary than competitive: SMB is really a production tool, when batch chromatography, thanks to its flexibility, has some interesting advantages during the first stages of development.

Notation a, b, A, B

H K„N L Q

K N

OTMB Qpeed QExt QRaf QEI

P ^ max

t to u

oSMB

Coefficients relating the height equivalent to a theo retical plate to the mobile phase velocity Mobile and soUd phases concentrations Kozeny coefficient Height equivalent to a theoretical plate Isotherm parameters Column length Solid flow rate Number of columns per zone Number of theoretical plates Minimum number of plates required TMB and SMB internal flowratesin zone k. Feed flow rate Extract flow rate Raffinate flow rate Internal flow rate in zone k Maximum pressure acceptable Time Zero retention time Linear mobile phase velocity

508

R. M. NICOUD

u^ V^ AP AT cp, 3> phase velocity e ^t fl

SMB average mobile phase velocity SMB column volume Pressure drop Period Parameter relating the pressure drop to the mobile External porosity Mobile phase viscosity Column section

REFERENCES 1. Nicoud, R. M. (1992). LC-GC Int. 5 43. 2. Balannec, B., and Hotier, G. (1993). In ''Preparative and Production Scale Chromatography'" (G. Ganetsos and P. E. Baker, ed.), Dekker, New York, pp. 301-357. 3. Nicoud, R. M., and Bailly, M. (1992). Proc. Symp. Prep. Ind. Chromatogr., Prep '92, 9th; INPL, Nancy, France, pp. 205-220. 4. Nicoud, R. M., Fuchs, G., Adam, P., Bailly, M., Kiisters, E., Antia, F. D., Reuille, R., and Schmid, E. (1993). Chirality 5, 267. 5. Nicoud, R. M., Bailly, M., Kinkel, J. N., Devant, R., Hampe, T., Kiisters, E. (1993). In ''Simulated Moving Bed: Basics and Applications'' (R. M. Nicoud, ed.), pp. 6 5 - 8 8 . INPL, Nancy, France. 6. Blehaut, J., Charton, F., and Nicoud, R. M. (1996). LC-GC Int. 9 (4), 228-238. 7. Ruthven, D. M., and Ching, C. B. (1989). Chem. Eng. Sc. 44, 1011. 8. Hotier, G. (1993), In "Simulated Moving Bed: Basics and Applications" (R. M. Nicoud, ed.), pp. 95-117. INPL, Nancy, France. 9. Ganetsos, G., and Barker, P. E., ed. (1993). "Preparative and Production Scale Chromatography." Dekker, New York. 10. Nicoud, R. M. (1998). In "Bioseparation and Bioprocessing" (G. Subramanian, ed.). Wiley-VCH, New York, pp. 3-39. 11. Ganetsos, G., and Barker P. E. (1993). In "Preparative and Production Scale Chromatography" (G. Ganetsos and P. E. Barker, ed.), Dekker, New York, pp. 233-255. 12. Hotier G. et al., (1996). U.S. Pat. 5,578,216. 13. Nicoud, R. M. (1993). LC-GC Int. 6, No. 10, pp. 636-637. 14. Charton, F., and Nicoud, R. M. (1995). / . Chromatogr. A 702, 97-112. 15. Nicoud, R. M., and Seidel-Morgenstern, A. (1996). Isol. Purif. 2, 165-200. 16. Levan, M. D., and Vermeulen, T. / . Phys. Chem. 85, 3247. 17. Jacobson, S., Golshan-Shirazi, S., and Guiochon, G. (1990). / . Am. Chem. Soc. 112, 6492. 18. Nicoud, R. M., and Schweich, D. (1989). Water Resour. Res. 25(6), 1071-1082. 19. Dye et al., Ind. Eng. Chem. Res. 29, 849-857. 20. Van Deemter, J. J., Zuiderweg, F. J., and Klinkenberg, A. (1956). Chem. Eng. Sci. 5, 271. 21. Knox, J. H. (1977). / . Chromatogr. Sci. 15, 352. 22. Horvath, Cs., and Lin, H. J. (1978). / . Chromatogr. 149, 43. 23. Endele, R., Halasz, I., and Unger, K. (1974). / . Chromatogr. 99, 377. 24. Villermaux, J. (1981). In "Percolation Processes: Theory and Applications" (A. E. Rodrigues and D. Tondeur, ed.). Sijthoff & Noordhoff, Alphen aan den Rijin, The Netherlands. 25. Rhee, H., Aris, R., and Amundson, N. R. (1971). Philos. Trans. R. Soc. London 269, 187. 26. Ching, C. B., Ho, C , and Ruthven, D. M. (1988). Chem. Eng. Sci. 43, 703. 27. Storti, G., Masi, M., Carra, S., and Morbidelh, M. (1988). Prep. Chromatogr. 1(1), 1-27. 28. Mazzotti, M., Storti, G., and Morbidelh, M. (1997). / . Chromatogr. A 769, 3-24. 29. Tondeur, D., and Bailly, M. (1993). In "Simulated Moving Bed: Basics and Applications" (R. M. Nicoud, ed.). pp. 95-117. INPL, Nancy, France. 30. Nicoud, R. M., and Colin, H. (1990). LC-GC Int. 3, No. 2, pp. 28-36. 31. Golshan-Shirazi, S., and Guiochon, G. (1994). / . Chromatogr. A 658, 149.

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32. Ernst, U. P., and Hsu, J. T. (1989). Ind. Eng. Chem. Res. 28, 1211. 33. Finlayson, B. A. (1980). "Non-linear Analysis in Chemical Engineering." McGraw-Hill, New York. 34. Barker, P. E., and Critcher, (1960). Chem. Eng. Set. 13, 82. 35. Hashimoto, K., Adashi, S., Noujima, H., and Maruyama, H. (1983). / . Chem. Eng. Jpn. 16, 400. 36. Ching, C. B., and Ruthven, D. M. (1985). Chem. Eng. Set. 40, 877. 37. Ching, C. B., Ruthven, D. M., and Hidajat, K. (1985). Chem. Eng. Set. 40, 1411. 38. Hashimoto, K., Adachi, S., Shirai, Y., and Moritshita, M. (1992). In "Preparative and Production Scale Chromatography" (G. Ganetsos and P. E. Barker, eds.), Dekker, New York, pp. 273-300. 39. Blezer, H. J., and De Rosset, A. J. (1977). Staerke 29, 393. 40. Kishihara, S. et al., (1989). / . Chem. Eng. Jpn. 22(4), 434. 41. Maki, H., Fukuda, H., and Morikawa, H. (1987). / . Ferment. Technol. 65, 1. 42. Hashimoto, K., Adashi, S., and Shirai, S. (1988). Agric. Biol. Chem. 52, 2161. 43. Hashimoto, K., Yamada, M., Adashi, S., and Shirai, Y. (1989). Chem. Eng. Jpn. 22, 432. 44. Huang, S. Y., Lin, C. K., Chang, W. H., and Lee, W. S. (1986). Chem. Eng. Commun. 456, 291. 45. Houwing, J., Van der Wielen, C. A. N., and Luyben, K. (1996). Froc. Eur. Symp. Biochem. Eng. Set. 1st, Dublin, 1996. 46. Nicoud, R. M. (1996). Recovery of Biol. Prod., 8th, Tuscon, AZ 1996. 47. Kinkel, J. N. (1999). Personal communication. 48. A.W.D. (1997). Pat. Appl. P C T / D E 9 / 0 0 5 2 5 . 49. Van Walsem, H. J., and Thompson, M. C. (1996). Eur. Symp. Biochem. Eng. Sci. 1st, Dublin, 1996, p. 27. 50. Kampen, W. H. Eur. Pat. Appl. 90307701.4. 51. Maki, H., (1992). In "Preparative and Production Scale Chromatography" (G. Ganetsos and P. E. Barker, eds.), Dekker, New York, pp. 359-371. 52. Szpepy et al., (1975). / . Chromtogr. 108, 285-297. 53. (1996). Eur. Pat. Appl. 697034. 54. Nicoud, R. M. (1998). In "Practical Supercritical Fluid Chromatography and Extraction" (M. Caude and D. Thiebaut, eds.). Harwood Academic Publishers, Chur, Switzerland. 55. Nicoud, R. M., and Perrut, M. (1992). Fr. Pat. 9,205,304. 56. Nicoud, R. M., Pharm. Technol. 11(3), 36-44; and Pharm. Technol. 11(4), 28-34. 57. Nicoud, R. M. (1996). Proc. Chiral Eur. '96, Strasbourg, 1996. 58. Kinkel, J. N. (1995). Proc. Chiral Eur. '95, London, 1995. 59. Ching, C. B., Lim, B. G., Lee, E. J. D., and Ng, S. C. (1993) / . Chromatagr. 634, 215-219. 60. Ikeda, H., and Murata, K. (1993). Symp. Chiral Discrimination, 4th, 1993. 61. Negawa, M., and Shoji,. F. (1992). / . Chromatogr. 590, 113-117. 62. Fuchs, G., Nicoud, R. M., and Bailly, M. (1992). Proc. Symp. Prep. Ind. Chromatogr. Prep '92, 9th, INPL, Nancy, France, pp. 205-220.

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LARGE-SCALE CHROMATOGRAPHIC PURIFICATION OF OLIGONUCLEOTIDES RANJIT R. DESHMUKH* Manufacturing Process Development, Isis Pharmaceuticals, Inc., Carlsbad, California 92008

WILLIAM E. LEITCH, II Argyll Associates, Palm Desert, California 92210

YOGESH S. SANGHVI Manufacturing Process Development, Isis Pharmaceuticals, Inc., Carlsbad, California 92008

D O U G L A S L. C O L E Development Chemistry and Pharmaceutical Development, Isis Pharmaceuticals, Inc., Carlsbad, California 92008

I. INTRODUCTION A. Antisense Oligonucleotides B. Large-Scale Production of Therapeutic Antisense Oligonucleotides C. Composition and Analysis of Crude Oligonucleotides II. GENERAL PURIFICATION STRATEGIES FOR OLIGONUCLEOTIDES III. LARGE-SCALE PURIFICATION OF THERAPEUTIC OLIGONUCLEOTIDES A. Reversed-Phase Purification of DMT-on Antisense Oligonucleotides B. Hydrophobic Interaction Chromatography of DMT-on Antisense Oligonucleotides C. Anion Exchange Chromatography D. Displacement Chromatography and Sample Self-Displacement Chromatography * Current address: Wydeth Lederle Vaccines, One Great Valley Parkway, Malvern, Pennsylvania 19355.

Separation Science and Technology, Volume 2 Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

•• • i 511

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RANJIT R. DESHMUKH et al.

IV. PURIFICATION OF RELATED MOLECULES — DNA FRAGMENTS, PLASMIDS, RIBOZYMES, AND RNA V. ECONOMICS OF OLIGONUCLEOTIDE PURIFICATION VI. SUMMARY REFERENCES

I. INTRODUCTION Production of synthetic oligonucleotides has become increasingly important with recent developments in biochemistry, diagnostics, genetic engineering, genomics, and very significantly, antisense therapeutics. The purification challenge differs depending on the amount of synthetic oligonucleotide product required. At one extreme, large amounts of relatively fev^ compounds must be purified to therapeutic quality, and at the other, a large number of compounds must be purified at high throughput in small quantities. The applications of separation techniques are generally similar, however. Sanghvi et al. report over 12 antisense oligonucleotides in human clinical trials.^ Fomivirsen sodium, discovered and manufactured by Isis Pharmaceuticals, became the first antisense drug approved for market by the U.S. Food and Drug Administration (FDA). The success of antisense drugs is spurring innovation and process development in all facets of oligonucleotide manufacture and purification. This chapter will focus on two of these aspects: (i) application of various separation modes to purification of these compounds and (ii) state-of-the-art large-scale technologies for purification of therapeutic antisense oligonucleotides. A. Antisense Oligonucleotides In general, antisense oligonucleotides are short, single-strand DNA or RNA analogues. The antisense drugs in current clinical trials are typically 20 nucleotides in length. Many of these compounds are phosphorothioate-linked oligomers, wherein a nonbridging phosphate oxygen is formally replaced by sulfur, as shown in Fig. 1. This modification increases stability of the oligonucleotide toward degradation by cellular nuclease. There are many other chemical modifications of DNA noted in the literature, under development as potential therapeutics, and in human clinical trials.^ This chapter will focus on phosphorothioate-modified DNA and RNA antisense oligonucleotide drugs because of their progress through clinical trials to the NDA (new drug application) and approval for distribution and sale. B. Large-Scale Production of Therapeutic Antisense Oligonucleotides In recent years, DNA synthesis technology has advanced significantly under pressure of the need for large-scale antisense oligonucleotide manufacture for human clinical trials. The early gene machines which made ^ 1 mg of oligonucleotide per run have been supplanted by advanced synthesizers producing almost a kilogram of oligonucleotide per run. Solid-phase oligonucleotide synthesis technology has progressed further than solution-phase

5 I 3

J^RGE-SCALE CHROMATOGRAPHIC PURIFICATION

Base

Phosphodlester FIGURE

I

Phosphorothloate

Chemical structure of phosphorothloate oligonucleotides.

synthesis. Packed-bed reactors are particularly far advanced. Various reactor column geometries and fluid contact mechanisms have been tested, but axial flow columns have been most successful and are used at the largest scales. Reagents are delivered under precise control of programmed computer protocols. A packed-column reactor affords optimal solvent efficiency and enables minimal amidite excess, and its near-continuous flow enables ready real-time process control. The largest such synthesizer available at present is the OligoProcess^^ (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) which can synthesize 20-mer oligonucleotides at the 150 mmol level, producing roughly 900 g of crude material per 10 hr synthesis cycle. Such syntheses are done routinely under good manufacturing practice (GMP) conditions at the Isis Pharmaceuticals facilities in Carlsbad, California, including those for fomivirsen sodium, the first antisense drug to reach market. Briefly, oligonucleotides are typically synthesized using phosphoramidite coupling chemistry.^ A simplified schematic of the production process is shown in Fig. 2. A detailed account of the chemistry is given elsewhere.^ Oligonucleotides are synthesized from 3' to 5', with the starting nucleoside attached to the solid-phase support via an ester linkage. The 5' protecting group 4,4'-dimethoxytrityl (DMT) group is acid-cleaved to prepare for second nucleotide coupling. The nucleoside 3'-0-amidite is activated with IHtetrazole and then coupled to the free 5'-OH of the first residue. The resulting phosphite triester linkage is converted to a phosphorothioate using a sulfurizing reagent. Subsequent acylation blocks any unreacted 5'-OH groups to prevent participation in further chain elongation. Acetonitrile washes are performed between steps to remove unreacted reagents. The cycle is then repeated for the each nucleotide addition until an oligonucleotide of desired length and sequence has been synthesized. At this stage, the terminal 5'-DMT group may be retained or removed, depending on choice of subsequent purification method. The crude product is

514

RANJIT R. DESHMUKH et al.

"^Step 1 A - Amidite

Amidite

Automated solidphase synthesizer appx. 10 h for a 20-mer 20 mer oligonucleotide attached to solid support

^ Step 2. Cleavage of oligonucleotide from solid support and removal of P, and Base protecting groups with Ammonia at 55° C

i

Step 3. Remove ammonia (concentration)

J

Crude oligonucleotide (Feed for purification) FIGURE 2

Schematic for the production of oligonucleotides.

treated with concentrated ammonium hydroxide at 55°C for 8-12 hr to cleave the oUgonucleotide from the support and remove the backbone and the base protecting groups. The ammonia is evaporated and the crude product is ready for purification. There are tv^o general protocols available for postsynthesis purification, and downstream processes depend on which one is selected. These are described in detail later. In one approach, the hydrophobic 5'-DMT protecting group is left on the oligonucleotide and used as an aid to reversed-phase (RP) purification. The DMT group is removed under acidic conditions after purification, and the resultant oligonucleotide product is precipitated from solution, isolated, and lyophilized to obtain the dry active pharmaceutical ingredient (API). In another approach, the DMT group is removed on the solid support and the resulting crude solution is purified by anion-exchange (AX) chromatography. The postpurification product pool is desalted, then lyophilized. Oligonucleotides for smaller scale applications are generally produced in bench-top automated synthesizers. These systems often use small gas-sparged

LARGE-SCALE CHROMATOGRAPHIC PURIFICATION

5 I 5

glass reactors and controlled pore glass as solid support. When appropriately operated, more than 6000 sequences can be synthesized daily at a single site. Robotics may be used to automate synthesis, purification, and in some cases quality control functions.^ Synthesis time per oligonucleotide tray can be under 5 hr for such high-throughput operations.

C. Composition and Analysis of Crude Oligonucleotides Crude 20 mer phosphorothioate synthesis mixtures made by phosphoramidite chemistry typically contain about 75% full-length oligonucleotide, because average coupling efficiency is high (approximately 98.5%). The major impurities are internal deletion and truncated trityl-off sequences of length {n — 1),(w — 2 ) , . . . ,(w — :v) arising via coupling failure.* Since a deletion can occur at any point in the chain, 20 mer crudes contain 19 individual (n — 1) mer impurities."^ Partial phosphodiesters are also present, predominately as monophosphodiester linked (P = 0 ) i oligomers with a single random diester linkage. There are low levels oi (n -\- x) length species produced mainly through double-coupling side reactions.^'^ These phosphorothioate oligonucleotide process-related species are determined by two complementary techniques. For most oligonucleotides in the 10 to 30 mer range, AX-high-performance liquid chromatography (HPLC) is used to determine phosphodiester components and quantitative capillary gel electrophoresis (QCGE) is used to analyze and quantify length based impurities. Srivatsa et al.^'^ have studied the applicable selectivity of these techniques for characterization of phosphorothioate oligonucleotide drug substances. Representative AX and QCGE analysis traces for a crude phosphorothioate are shown in Figs. 3A & 3B. Analytical AX-HPLC does not require sample pretreatment, but some QCGE samples may require prior desalting. For analysis of preparative AX HPLC fractions by QCGE, for example, extensive desalting is often necessary. Our preference is to use Centricon (Amicon, Division of Millipore, Beverly, MA) centrifuge cartridges in three or more cycles to desalt and concentrate the fractions. Desalted material is diluted to approximately 0.01 m g / m L concentration for loading into the QCGE capillary. The n mer (i.e., a full-length oligonucleotide with n nucleotides) and (n — 1) mer components of phosphodiester oligonucleotide products may be determined by analytical AX-HPLC. While selectivity is likely similar, AX-HPLC peak capacity is generally inadequate for quantitative determination of the corresponding all-phosphorothioate n mer and (n — 1) mers, because of the added width of the diastereomer-containing peaks. Protocols for analysis by AX-HPLC may be found in published literature.^ There are also literature indications that for all-phosphodiester oligonucleotides, AX-HPLC sequence-based selectivity may be achieved among oligomers of the same length.^ *Note (n — x) indicates a shorter oligonucleotide with x less nucleotides than n mer. Note (n + x) indicates a longer oligonucleotide.

5t6

RANJIT R. DESHMUKH et al.

2.60CD

2.40-

II

i

2.202.001.801.60^ ^ 1.40^ 1.201.00^

1

o II

0.80-

Is

0.60-^

CO 6

0.400.20-^ 0.000.00

/ ^

^

r W ^ y y /^

--—. ,

1

20.00

i

1

,

J

1

1

Cu

\

V:yi~,^ 1

40.00 Minutes

.

]

\

60.00

F I G U R E 3 (A) Representative analytical anion exchange (AX) trace for crude 20 mer phosphorothioate and (B) representative QCGE trace for the same oligonucleotide. Conditions for the analytical AX: Column, resource Q I mL column (6.4 X 30 mm); buffer A, 20 m M NaOH; buffer B, A + 2.5 M NaCI; gradient, 0 - 100% B in 50 min, temperature 70°C; sample, ISIS 2302 crude, a 20 mer phosphorothioate. Conditions for analytical QCGE: column; eCap gel-filled capillary (Beckman-Coulter, Fullerton, CA), 14.1 kV run voltage, 40°C, electrokinetic injection 13 s at 10 kV; system, Beckman P/Ace 5000; sample: ISIS 2302 crude sample at concentration of 0.01 m g / m L

II. GENERAL PURIFICATION STRATEGIES FOR OLIGONUCLEOTIDES Synthetic oligonucleotide purification strategies applicable at a small scale are discussed first, followed by a discussion of scaleable technologies, described in detail in Section III. Several properties of the oligonucleotides have been exploited for their purification.^^"^^ Some of the key methods are summarized in Table 1.^^"^"^ The net negative charge of an oligonucleotide is dependent on length and backbone structure, but for a given synthesis product, AX chromatography is

517

LARGE-SCALE CHROMATOGRAPHIC PURIFICATION

B 0.024 i

0.000 20 Minutes FIGURE 3

(Continued)

selective for length-variant process-related impurities and can be thus used for purification. The original use of AX LC for nucleotide and oligonucleotide separations is generally attributed to Cohn.^^ There is now extensive literature data regarding application of AX to oligonucleotides.^'^^~^^'^^~^^ Samelength oligonucleotides differing in base composition exhibit unique hydrophobic properties, so reversed-phase and hydrophobic interaction chromatography (HIC) separations are useful. Oligomer charge and hydrophobic character can also be exploited through mixed-mode strategies, including use of ion-pairing RP chromatography (IP-RP),^^ stationary phases such as RPC-5 that combine anion-exchange and reversed-phase characteristics,^^'^"^ and hydroxyapatite media.^^'^^ Mobile-phase ion-pairing regents such as tetraalkyl ammonium compounds can dramatically improve chromatographic

5 I8

RANJIT R. DESHMUKH et al. m

T A B L E I Application of Various Chromatographic Techniques for Purification of Oligonucleotides and Nucleic Acids Technique

Property used

References

Ion exchange Reversed phase Hydrophobic interaction Affinity Gel permeation Mixed mode Ion-paired RP Hydroxyapatite Slalom chromatography RPC-5 media

Charge on phosphate backbone Hydrophobicity of bases and DMT protecting group Hydrophobicity of base and DMT protecting group Watson-Crick base pairing Size of the molecule

8,13-20 8,13-: 1,20 21 22--24 25

Hydrophobicity of bases and charge interaction Charge and phosphate groups Size and hydrophobicity of large nucleic acids Charge interaction and hydrophobicity

12, 2626-29 30, 31 32 33, 34

resolution of oligonucleotides at small scales,^^'^^ but ion-pairing reagents are not always useful in preparative applications. IP-RP chromatography effectively purifies synthetic oligonucleotides and can function as chromatographic front end for on-line LC-ES-MS analyses."^^'"^^ RPC-5 is an AX medium that has reversed-phase properties because of the absorbed C30 quaternary ammonium compound comprising its stationary phase. RPC-5 exhibits excellent utility for separation of structurally similar isoaccepting t-RNAs, restriction fragments, large DNAs, and plasmids. Unfortunately, the RPC-5 solid-phase support material, Plaskon® (Allied-Signal Corp.), is presently unavailable. Paired-ion chromatography (PIC) is a term used to describe a preparative separation mode similar to IP-RP."^^ The media and columns, which can perform similarly to RPC-5 in some applications, are commercially available from PureSyn, Inc. (Malvern, PA).^^ It is also possible to obtain the performance of RPC-5 media with other commercial AX or RP media. Hydroxyapatite has a specific interaction with oligonucleotide backbone phosphate groups and at the same time exhibits mixed-mode interactions with oligomers through their charged and hydrophobic moieties. Affinity chromatography has been used as the basis for oligonucleotide column chromatographic purification media functioning through WatsonCrick base pairing. Thus, oligomers complementary to the targeted oligonucleotide have been used as covalently bound ligands on chromatographic stationary phases.^^"^"^ Other moieties have been attached at oligonucleotide termini to enable their purification by non-Watson-Crick affinity chromatography. Examples include biotinylated end groups, PEG-coupling for aqueous two-phase separations,'*^ synthesis of oligos with poly-His chains for immobilized metal affinity chromatography,'^'* and affinity-aided specific precipitations."*^ Molecular size is an important separation parameter, but it is most often exploited for chromatographic separation of oligonucleotides shorter than 10 nucleotides or for isolation of very large DNA fragments. Kasai^^ has reviewed techniques useful for size-based purification of nucleic acids. Gel

LARGE-SCALE CHROMATOGRAPHIC PURIFICATION

5 I 9

electrophoresis can be use for a wide range of sizes and is routinely used for small-scale separations. Gross size-based separations are also possible by gel permeation chromatography, selective precipitation,"^^ ultracentrifugation, and slalom chromatography.^^ The next section discusses application of large-scale separation techniques to the purification of synthetic oligonucleotides.

III. LARGE-SCALE PURIFICATION OF THERAPEUTIC OLIGONUCLEOTIDES Antisense oligonucleotide drugs are currently manufactured at larger scale than any other class of synthetic nucleic acids. Demand for GMP antisense oHgonucleotides is forecast to reach ton scale within the next few years."^"" Therefore, only those purification techniques that have been demonstrated at multigram scale and are likely to be scaled to hundred-kilogram to metric ton levels are reviewed here. Because of the highly regulated nature of therapeutic agents, their manufacturing processes must be robust, validated, and consistent with current GMPs (cGMPs). They also must be scaleable. Thus, only RP-HPLC, HIC, and AX chromatographies are generally suitable for GMP use, as documented in published reports showing feasibility of these methods at hundred-gram levels.^'^^'^^'^^ Crude trityl-on solid phase synthesis products are typically purified by RP-HPLC^ or HIC chromatography.^^'"^^ For AX purification, the oligomer is typically detritylated before loading on the column, though the DMT group may be removed on the synthesis support followed directly by AX-HPLC. In general, three methods are used for large-scale synthetic oligonucleotide purification, as described next.

A. Reversed-Phase Purification of DMT-on Antisense Oligonucleotides Reversed-phase (RP) purification has been the most extensively used method for synthetic oligonucleotide purification^'^^ and has been scaled up by Isis under cGMP conditions for the manufacture of clinical trial and marketed antisense oligonucleotide drugs. For reversed-phase purification, 5' DMT group is left intact at the end of synthesis. The DMT function imparts hydrophobicity to the oligonucleotide and the product peak can thus be easily separated from non-DMT failure sequences. While this method depends for its efficacy on the high efficiency of both the DMT protection and deprotection processes, results with the technique are quite good. Purities achieved are excellent, with very high product recovery. Although final purity depends on the sequence synthesized, relatively similar results have been obtained across a large number of oligonucleotide sequences and structural modifications. The RP method works well for synthetic phosphodiester DNA molecules and phosphorothioate-modified oligonucleotides, as well as synthetic RNAs, DNA-RNA chimeras, and ribozymes. There are occasional chromatographic bandwidth challenges with 2'-modified oligonucleotides, but the method remains effective for these oligomers. The basic framework for a quick and reliable purification method is shown in Fig. 4 in the form of

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Automated solid phase synthesis (5' DMT protecting group intact)

Cleavage from solid support removal of P and Base protecting groups

IL

Reverse Phase HPLC

IL

Detritylation Ethanol precipitation

^ Lyophilization FIGURE 4

Schematic for RP purification of oligonucleotides.

a schematic of the now-classic RP purification protocol and its associated postpurification operations. Silicate or organic polymer C^g'^erivatized columns are the simplest stationary phase options for RP purification. Weakly buffered RP eluents, typically sodium- or ammonium acetate-based, are preferred. The DMT-on oligonucleotide crude product is loaded at low or zero mobile phase organic content, then eluted by a gradient of increasing organic content. Acetonitrile and methanol are frequently used as the organic eluent component, but methanol is often preferred at production scale for economic reasons, as minimal selectivity differences are observed between the two eluents. Oligonucleotides exhibit good solubility in these strongly aqueous solvent mixtures. Phosphate buffers, however, are typically avoided due to potential for salt precipitation in aqueous organic solution. RP-HPLC is most widely used for small scale oligonucleotide purifications where purity in the range of 85-92% is needed. Optimized methods at large scale could give much better performance compared to the off-the-shelf methods. At production scales, column compression is preferred for the purification. We have utilized the Biotage (division of Dyax Corp., Cambridge, MA) radial compression columns for the production scale, since these columns give nearly identical performance over a wide range of scales up to 20 liters. Among various chromatographic media, silica-based media offer a cheap alternative with good binding capacity and resolution. Polymeric supports, on the other hand, are robust and offer the flexibility of using high pH buffers that helps avoid secondary structure related contributions during purification of G-rich sequences. Pore size of the media is important as well. Wide-pore particles were used very early in nucleic acid separations and were found to be useful. However, newer chromatographic media can fine-tune the selectivity and capacity without resorting to very large pore sizes such as the Oligo R3 media (PE BioSystems, Framingham, MA).

521

LARGE-SCALE CHROMATOGRAPHIC PURIFICATION

E c in

Time (min) F I G U R E 5 RP separation of DMT-on phosphorothioate oligonucleotides. A 21 nner phosphorothioate, DMT-on crude was separated on a Cjg nnedia (Oligo R3, PE BioSystems). The column was 10 X 100 mm length, and 160 mg of sample was loaded (approx. 20 mg / mL column volume). Buffer A: 200 m M NaOAc in delonized (Dl) water, pH 7.2; and buffer B: 200 m M NaOAc in MeOH, pH 7.2. The gradient was 0 - 8 0 % B in 50 min, at a flow rate of 15 m L / m i n ( I I 4 5 c m / h r ) . The experiment was performed on the BioCad 60 workstation and monitored at A = 265 nm. Peaks (a) and (b) DMT-off failures, (c) DMT-on full length product pool, and (d) DMT-on failures.

Figure 5 shows a representative RP purification of a DMT-on phosphorothioate crude. The sample was DMT-on 21 mer crude. About 20 mg of sample per milliliter of packed column volume was loaded on the polymeric column. This loading is generally considered as the standard loading in most state-of-the-art RP media for oligonucleotides. About four peak clusters are seen. The first blow through contains very small failures and some protecting groups. The second broad set of peaks represents the DMT-off failure peaks. The main product peak is flanked by failures at the beginning of the peak and DMT-on deletions are the tail of the peak. The final peaks may or may not be prominent in purification of every synthesis. As seen in this figure, the separation of the DMT-on product pool from the DMT-off failures is quite good. The optimization efforts therefore focus on maximizing purity by separating the DMT-on impurities from the product pools by manipulating the gradient, load, and proper fractionation. Very consistent purification

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profiles can be obtained using this basic RP method. This method can be scaled up easily by using a larger column and matching the linear flow and sample loading. A similar method on different column media and column are routinely run at 10 liter scale in our manufacturing unit under GMP conditions. Reproducible results are obtained at this high scale for a number of different sequences. After purification the DMT group from the oligonucleotide is removed by acid treatment, such as, acetic acid or some other milder acid in aqueous solution. After final detritylation, the salts if excessive are removed via precipitation, and then the product is lyophilized. Several process improvements have been tried and implemented to enable processing at the kilogram scale in our GMP facilities.^'"^^ The final aqueous detritylation is a complicated step that requires careful process optimization, such as control of pH, oligo and salt concentrations etc. "^^ After detritylation, the oligonucleotide is precipitated quantitatively from the acidic DMT cation containing solution under optimized conditions. This step can be labor intensive at the large scale, and may be inconvenient for the high-throughput small-scale synthesis. One v^ay to circumvent the problem is to use on column detritylation, where the RP and detritylation steps are combined in one chromatographic operation. Since the acid can leach the silica based columns, this is more useful on polymeric supports. Figure 6 shows the example of this method. A 21 mer phosphorothioate crude is on-column detritylated and purified on the polymeric Oligo R3 media (PE BioSystems). The sample was loaded on the column and then in the first part of the separation the DMT-off failures were eluted off the column, keeping the DMT-on product containing peak bound to the column. A dilute acidic solution, in this case 2% TFA, was then pumped into the column for a specific duration. The acid was then washed off with the equilibration buffer and the detritylated bound material was then eluted with increasing organic content. Peak b in Fig. 6 shows the main peak, and peak c is the material that was not detritylated. In this case, the acid exposure was insufficient for complete detritylation. However, this method inherently separates the undetritylated portion from the desired product pool. This method was very fast (under 30 min) and was run on automated workstation. The disadvantages are that there is a small yield loss due to incomplete detritylation. Another disadvantage is that smaller sample loadings than normal RP purification are used. It is more difficult to make the process more robust at high sample loadings; however, it is a very reliable and convenient method for small sample sizes. In our lab, we generally use these methods for total sample size of < 1 g and sample loading of < 5 mg/mL of column volume. Yet another way to detritylate synthetic oligonucleotide intermediates is on the AX purification column itself, since AX-HPLC is at times used as an orthogonal purification step in oligonucleotide manufacture.^^'^^ In our hands, it has been most straightforward to optimize purification of a new oligonucleotide drug by RP-HPLC than to proceed directly to on-column detritylation on and AX column as the initial step, even though resulting purities can be equivalent.

523

LARGE-SCALE CHROMATOGRAPHIC PURIFICATION

E c

lO CD CVJ

0.0-b^ 15 Time (min) F I G U R E 6 Example of on-column detritylation of phosphorothioates. A 21 mer phosphorothioate, DMT-on crude was detritylated on a polymeric Cjg media (Oligo R3, PE BioSystems). The column was 10 X 100 mm in length, and 160 mg of sample was loaded ( ~ 20 mg / mL column volume). Buffer A: 200 m M NaOAc in Dl water, pH 7.2; buffer B: 200 m M NaOAc in MeOH, pH 7.2; and buffer C: 2% trifluoroacetic acid (TFA) in Dl. Gradient in the percentage of buffer B is as indicated in the figure. In the detritylation step, 100% buffer C was pumped for 5 min, and then washed with the equilibrium buffer A. The experiment was performed on the BioCad 60 workstation and monitored at A = 260 and 290 nm. Peaks (a) DMT-off failures, (b) DMT-on full length product pool, and (c) DMT-on failures.

Small-scale solution phase detritylation can be tedious and often compromises yield. In such cases, the on-column detritylation method is an alternative with potential for automation. For oligonucleotide production beyond the 10 g scale, on-column detritylation techniques, though possible, may not prove robust enough for routine use. In particular, sample loading may have to be less than the chromatographically allov^ed maximum to achieve robust detritylation efficiency. We have developed tv^o additional variants that are more robust. These approaches use two columns or use the same column twice. In the first step, the DMT-on product pool is eluted from the column. This fraction pool is loaded on the second column, all the absorbed material is detritylated without elution and then oligonucleotide is eluted. RP is favored as the first step followed by on-column detritylation on either another RP column or AX column. In an orthogonal two-column purification scheme, using RP as first step and AX as second step enhances final product

524

RANJIT R. DESHMUKH et o/.

purity because DMT-off failure removal is separation from the detritylation operation, leading to more consistent overall results. B. Hydrophobic Interaction Chromatography of DMT-on Antisense Oligonucleotides Hydrophobic interaction chromatography has also been used at large scale for separation of DMT-on crude oligonucleotides from DMT-off failure sequences.^^'^^ An advantage of this method is direct loading of the ammoniacal solution from succinate cleavage on the chromatographic column w^ithout prior ammonia stripping. The resulting eluate is in low^ organic-content solution, thus avoiding a concentration step if AX-HPLC is the next step. Puma^^ reports use of Phenyl Sepharose Fast Flow^ media (Amersham Pharmacia Biotech) in the HIC step. The column v^as equilibrated in ammonium acetate buffer and the product eluted in water for injection. The DMT-on pool was detritylated in solution as described in Section III.A then subjected to orthogonal purification on DEAE-5PW (TosoHaas Corp., Montgomeryville, PA) AX resin. This method is reported to result in > 95% (P = S) purity as determined by analytical AX-HPLC area percent. Both steps were carried out under GMP conditions and shown scaleable to the 100 g scale. While both RP and HIC media achieve equivalent results, limited pubHshed work indicates that capacity and cost advantages may favor RP media at the present time. C. Anion Exchange Chromatography Since oligonucleotides bear multiple negative charge, AX chromatography is a logical separation and purification method. At large industrial scale, several modes of AX chromatography have been used to purify synthetic oligonucleotides. In some cases, orthogonal RP and AX approaches have been used to produce high purity oligonucleotides. For example, DMT-on oligonucleotides may be RP-purified as (Section III.A) and following detritylation of the product pool the oligonucleotide may be further purified by AX chromatography. This process is used in the manufacture of Vitravene^^* fomivirsen sodium, the first antisense drug approved by FDA for market. In this case, RP purification is followed by AX chromatography resulting in > 96% pure oligonucleotide. Previous sections likewise describe orthogonal AX following HIC purification.^^ We and others have also shown that starting with DMT-off crude product, high-purity oligonucleotide can be obtained using a single AX chromatography step. A schematic for this strategy is shown in Fig. 7. DMT-off crude, after cleavage and deprotection, was chromatographed on an AX column, followed by desalting and lyophilization of the product pool. Advantages of this approach include avoidance of postpurification detritylation and concomitant oligomer precipitation, compatibility with low-pressure LC pumps and columns, elimination of organic solvent, and availability of *Vitravene is a trademark of Novartis AG.

525

LARGE-SCALE CHROMATOGRAPHIC PURIFICATION

Automated solid phase synthesis (5' DMT protecting group removed)

Cleavage from solid support removal of P and Base protecting groups

O Strong Anion Exchange Chromatography

^ Desalting

^ Lyophilization FIGURE 7

Schematic for A X purification of oligonucleotides.

several suitable chromatographic media. Typical protocols are discussed in the following text. Figure 8 shows the reconstructed histogram from an AX purification of DMT-off crude ISIS 2302, a 20 mer phosphorothioate oligonucleotide. A strong AX resin, in this case POROS H Q / 5 0 (PE BioSystems), was used. Sample loading in related experiments varied from 5 to 10 g. High pH eluents prepared with NaOH were used to minimize secondary structure

3000

2000

o

B < 1000

Fraction number F I G U R E 8 A X Purification of DMT-off oligonucleotide crude. A 20 mer phosphorothioate, DMT-off crude was purified on a A X column (POROS HQ / 50, PE BioSystems). A 200 mL column (50 X 200 mm length) was used and approximately 10 g of sample was loaded on the column ( ~ 50 mg/ mL column volume). Buffer A: 20 m M NaOH; Buffer B: 20 m M NaOH + 2.5 M NaCI. Optimized gradient was used, with a flow rate of 35 m L / m i n (107 c m / h r ) . The experiment was performed on the BioCad 60 workstation and monitored at A = 260 and 290 nm.

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RANJIT R. DESHMUKH et al.

contributions to chromatographic band width. Buffer A was 20 m M NaOH and the eluent was 2.5 M NaCl. The initial method was developed using linear gradients, then optimized to use step gradients to enable reducing the number of fractions collected. As the chromatogram shows, most processrelated oligomers are well separated from full-length product-containing fractions, but (n — 1) species are very similar in properties to full-length oligonucleotides so that an optimal product cut must be made to balance product purity and yield. Fractions 6-8 were pooled for this separation and the product pool was analyzed for area percent purity, as shown in Table 2. The 85% pure crude yielded > 95% full-length purity final product. Partial phosphodiester-related components were reduced as reflected by analytical AX-HPLC area-percent analyses of 76% for the column feed and 99% for the product. Yield of full-length oligonucleotide based on column feed full-length content was 72%. While purity obtained compares well with that from optimized RP purification, isolate yield is somewhat lower than from RP purification. Some yield increase can be realized by recycling side fractions. This method uses conventional anion-exchange hardware typical of industrial bioseparations, and the stationary phase and buffers used are suitable for production scale use. Additional results from AX purification of DMT-off synthetic oligonucleotides are discussed in the following section, based on the principles of displacement and sample self-displacement chromatography.

D. Displacement Chromatography and Sample Self-Displacement Chromatography The major deletion sequence impurities in crude synthetic oligonucleotides are very similar in properties to the full-length desired product, varying slightly in molecular weight to chemical charge ratio and base sequence. The former characteristic inherently suits the crude product to self-displacement chromatographic purification. These techniques were recently reviewed for their application to oligonucleotides and nucleic acids.^^ Column displacement chromatography is a method in which a compound with greater binding avidity than the desired product is used to elute desired compounds from the column. For example, Gerstner et al.^^ have demonstrated feasibility and scaled-up use of dextran sulfate displacer for separation of synthetic oligonucleotides on AX resin, implying that a well-optimized method could

TABLE 2

Summary of Analysis for A X Purification of ISIS 2302 CGE area percent purity

Feed Product

n mer

(n - 1)

85.0 95.8

3.4 2.7

Other impurities [sunn(n - x)^>, + (n+x)] 11.6 1.5

(P=S)

(P=0),

75.6 98.5

6.5 1.5

Sum(P=0),>, 17.9 0.0

527

LARGE-SCALE CHROMATOGRAPHIC PURIFICATION

be fine tuned and made useful for a variety of sequences. A major limitation of this technique is that displacer contamination of the product must be minimized and may require a follow-on orthogonal chromatography to ensure complete removal. Nevertheless, good oligonucleotide purity can be achieved at high yield by displacement chromatography. In the sample self-displacement approach, the components of the sample itself are used to effect chromatographic displacement. Since the desired oligomers in crude synthetic oligonucleotide mixtures have relatively higher affinity than most process-related impurities, they displace lov^er affinity shortmers w^ith lov^er net charge, and they in turn displace even shorter oligomers. Thus, under optimal conditions of sample load and other operating conditions, the sample self-displacement phenomenon can displace impurities to the leading portion of the eluted material band to produce a high purity product pool. The purity and yield obtained in this case can be very similar to those obtained through true displacement chromatography, and the displacer contamination issue is avoided. Sample self-displacement is highly scalable, as with other AX chromatography processes. Figure 9 shows an example of sample self-displacement chromatographic purification of DMT-off crude phosphorothioate oligonucleotide ISIS 2302

TlOO

-(p=o) -oligorUV

S 60 +

> •§

40 +

+ 40 g.

20 +

+ 20

o Z

45

F I G U R E 9 Sample self-displacement of oligonucleotides. A 20 mer phosphorothioate, DMT-off crude was purified on an A X column (Fractogel TMAE Hi Capacity, Merck KGaA, Darmstadt, Germany). A 7.8 mL column (10 X 100 mm length) was used and approximately 235 mg of sample was loaded on the column [ ^ 30 m g / mL column volume (CV)]. Buffer A: 50 m M NaOH; buffer B: 50 m M NaOH + 2.5 M NaCI. Optimized gradient was used, with a flow rate of 3 mL / min (230 cm / hr). Gradient: Step to 20% B, 20% B to 40% B in 15 CV, 100% B for 2 CV. Experiment was performed on the BioCad 60 workstation and monitored at A = 260 and 290 nm.

528

RANJIT R. DESHMUKH et al.

on a strong anion exchanger. Sample load and gradient profile were optimized so that deletion sequence impurities eluted in the leading edge of the chromatogram. Elution profiles for partial phosphodiester components (P = 0)„ and (n — 1) mer deletion sequences are shown in the figure. Following the initial peak there is a zone of very high purity product as indicated by pooling tics on the chromatogram. By optimizing sample load, gradient, and the flow rate, product purity can be maximized. In this case the product purity was 90% with 76% yield, and the starting purity was 62%. Isolated yield is lower than for underloaded linear chromatography, but can be maximized by recycling mixed-zone fractions. Despite the need for recycling, this is a very convenient method for synthetic oligonucleotide purification at the gram scale. Purification of an 8 mer phosphorothioate oligonucleotide is illustrated in Fig. 10. This nonantisense oligomer spontaneously forms a parallel-chain tetrad structure that determines its specific antiviral activity. For purification of the synthesis product as the single-chain "monad," a tetrad-denaturing eluent at high NaOH concentration was used. Fluent A was 50 m M NaOH and eluent B was 50 m M NaOH plus 2.5 M NaCl. This eluent converted all material to the monad form. Q Hyper D F (BioSepra) strong AX media was used for the purification. Purity of the starting crude product was 71 area percent by analytical AX. Since this is a relatively short oligonucleotide, impurities are more readily resolved than from a longer crude oligonucleotide, impurities are more readily resolved than from a longer crude oligonucleotide. In the first cycle of purification, a simple linear gradient was used. This enabled separation of (n — 2) mer and shorter process-related impurities and some partial phosphodiester components and (n — 1) mer impurities. The product pool from first purification cycles was pooled and combined and run again on the AX column. For the second step, conditions were optimized such that sample self-displacement effects were emphasized, yielding high-purity full-length product. Again, some side fractions were selectively recycled to increase yield. This powerful approach increased initial purity of 7 1 % to > 99 area percent purity by analytical AX, and > 98 area percent purity by CGE. A similar approach was used for a 20 mer phosphorothioate oligonucleotide. The buffers and columns were same as in the preceding example, but, the column feed was prepurified by reversed phase and detritylated in solution. The chromatograms in Fig. 11 show results of the subsequent orthogonal AX steps. In the first cycle of purification, the bulk of the shorter deletion sequences were removed and the product rich cut was taken as shown in the chromatogram. Similar product cuts from several runs were combined, conductivity was adjusted by dilution, and a second series of runs were performed. In both separations, conditions were adjusted so that sample self-displacement enhanced purification by displacing impurities toward the leading edge of the chromatogram. The latest eluting regions showed greatest product purity. Pools from the second series were combined to obtain the purified product. The starting purity of 85% was increased to 96% full-length purity.

529

LARGE-SCALE CHROMATOGRAPHIC PURIFICATION step 1: Pre-purification

Step 2: Sample displacement

Waste T Recycle | Feed Step 1 (70.7% purity by SAX)

.1. i l

him

Purified pool after Step 2 (99.5% purity by SAX)

U

,

Pool 2

1" '

.. 1 r^1\

\

F I G U R E 10 Purification of a 8 mer phosphorothioate on A X with sample self-displacement chromatography. A 8 mer phosphorothioate, DMT-off crude was purified on an A X column (Q Hyper D F, BioSepra Corp., Marlborough, MA). Buffer A: 50 m M NaOH; buffer B: 50 m M NaOH + 2.5 M KCI; column: 50 X 100 mm Waters AP-5 column; flow rate: 35 m L / m i n (107 c m / h r ) . Gradients for the two purification rounds are shown in the chromatograms. A pool for the first purification rounds was collected, the conductivity reduced by dilution with water and injected for the second purification. Feed purity by CGE: 77%. Purity of the product pool after second cycle: 98% by CGE. Purity by AX: feed = 7 1 % , product pool after second cycle = 99.5%.

lY. PURIFICATION OF RELATED MOLECULES—DNA FRAGMENTS, PLASMIDS, RIBOZYMES, AND RNA Techniques similar to those discussed can be used for purification of a wide variety of nucleic acid oligomers and analogues. However, depending on product quality criteria and required purification scale, critical parameters

530

RANJIT R. DESHMUKH et o).

II

\

L/J

\

Pool 2

F I G U R E I I Purification of a 20 mer phosphorothioate on A X with sample self displacement chromatography. The DMT-on crude was first separated on a RP column (Oligo R3, PE BioSystems) with a on-column detritylation procedure. The chromatograms I and II shown are for the orthogonal A X steps. Product pool from step I was loaded onto the same column for step II after appropriate conductivity adjustment. Buffer A: 50 m M NaOH; buffer B: 50 mM NaOH + 2.5 M NaCI; gradient: load in no salt, elute step gradient 44% B for 10 CV, clean 100%. The conductivity trace is superimposed on the chromatograms. Flow rate 40 m L / m i n (122 cm / hr). Media: Q Hyper D F (BioSepra Corp.). Feed purity for A X steps 85% and purity after purification: 96% by CGE.

may vary. Thompson has extensively reviev^ed isolation techniques for supercoiled plasmid DNA and restriction fragments/"^'^^ Huber has reviewed purification of ssDNA on micropellicular chromatographic media.^^ AX chromatography has been compared to ion-paired RP in such cases, and the literature show^s a general trend to best results with AX for larger oligomers such as plamids/^ For short RNA segments and ribozymes, the methods developed for antisense oligonucleotides are good starting points for the development of specific methods. Particular handling care is generally required for RNA molecules due to potential for ribonuclease degradation. Reversed-phase purification has been used at large and small scales for high-throughput ribozyme purification.^'^~^^

V. ECONOMICS OF OLIGONUCLEOTIDE PURIFICATION Raw material costs for production of antisense oligonucleotide drug substances have been reduced more than 97% since 1991 through process optimization, scale-up economies, and reduced raw material costs,^' ^^ while the manufacturing and control associated labor costs have fallen even more precipitously. Key changes have converted the process from use of costly

LARGE-SCALE CHROMATOGRAPHIC PURIFICATION

53 I

commercial reagents to cheaper proprietary reagents, such as phenylacetyl disulfide (PADS) for sulfurization instead of Beaucage reagent.^^ The economic aspects of oligonucleotide purification have been discussed earlier for displacement chromatography and large-scale AX chromatography/^'^^ As prices have fallen, the percentage costs of raw materials and reagents have remained relatively constant. Currently the solid support, protected nucleoside phosphoramidites and the sulfurizing reagent constitute about 80% of the total cost of raw materials. The cost of purification reagents and stationary phases is less than 5% of total raw material costs at present. The high efficiency with which current chromatographic purification methods recover full-length oligonucleotide from crude synthesis products is a tribute to progress in the area. Further increases in isolation yield will result from ongoing research in oligonucleotide purification and will directly contribute to further lowering cost of the broad new class of antisense oligonucleotide therapeutic agents. VI. SUMMARY There are two robust single-step methods for purification of synthetic oligonucleotides at any scale, as shown in this chapter. RP purification is the simpler of the two and has been widely used in large-scale production and high-throughput small-scale applications. AX chromatography may also be suited for production-scale use, but has not yet been scaled to the levels achieved with reversed-phase chromatography in commercial manufacture. While selectivity enhancements with modified versions of these methods appear promising at small scale, at the present time the two demonstrably robust techniques offer the most direct routes to scale-up of oligonucleotide purification. REFERENCES 1. Sanghvi, Y. S., Andrade, M., Deshmukh, R. R., Holmberg, L., Scozzari, A. N., and Cole, D. L. (1999). Chemical synthesis and purification of phosphorothioate antisense oligonucleotides. In "Manual of Antisense Methodology" (G. Hartman and S. Endres, eds.), pp. 3-23. Kluwer Academic Publishers, New York. 2. Sanghvi, Y. S. (1999). DNA with altered backbone in antisense applications. In "DNA and Aspects of Molecular Biology" (E. T. Kool, ed.). Vol. 7, pp. 285-311. Pergamon, New York. 3. Poddenin, B. (1997). The industrial challenge—Custom oligonucleotide synthesis. IBC Conf.i Large Scale Oligonucleotide Synth.^ San Diego, CA, 1997. 4. Chen, D., Yan, Z., Cole, D. L., and Srivatsa, G. S. (1999). Analysis of internal (« - 1)deletion sequences in synthetic oligonucleotides by hybridization to an immobilized probe array. Nucleic Acids Res. 27, 388-395. 5. Krotz, A. H., Klopchin, P. G., Walker, K. L., Srivatsa, G. S., Cole, D. L., and Ravikumar, V. T. (1997). On the formation of longmers in phosphorothioate oligodeoxyribonucleotide synthesis. Tetrahedron Lett. 38, 3875-3878. 6. Srivatsa, G. S., Batt, M., Schuette, J., Carlson, R., Fitchett, J., Lee, C , and Cole, D. L. (1994). Quantitative Capillary Gel Electrophoresis (QCGE) assay of phosphorothioate oligonucleotide in pharmaceutical formulations. / . Chromatogr. A 680, 469-477.

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7. Srivatsa, G. S., Klopchin, P., Batt, M., Feldman, M., Carlson, R. H., and Cole, D. L. (1997). Selectivity of anion exchange chromatography and capillary gel electrophoresis for the analysis of phosphorothioate oligonucleotides. / . Pharm. Biomed. Anal. 16, 619-630. 8. Thayer, J. R., McCormick, R. M., and Avdalovic, N. (1996). High resolution liquid chromatographic nucleic acid separations. In "Methods in Enzymology" (B. L. Karger and W. S. Hancock, eds.). Vol. 271, pp. 147-174 Academic Press, San Diego, CA. 9. Alazard, D., Tran, L., Allen, J. R., Weisburg, W. G., and Russell, J. (1998). Ion chromatography analysis of oligonucleotide mixtures in the medical diagnostics industry. Int. Symp. High Performance Liq. Phase Sep. Related Tech., HPLC98, 22nd, St. Louis, MO, 1998. 10. Haupt, W., and Pingoud, A. (1983). Comparison of several high-performance liquid chromatography techniques for the separation of ohgodeoxynucleotides according to their chain lengths. / . Chromatogr. 260, 419-427. 11. Zon, G. (1990). Purification of synthetic oligodeoxyribonucleotides. In "High-performance Liquid Chromatography in Biotechnology" (W. Hancock, ed.), pp. 301-397. Wiley, New^ York. 12. Zon, G., and Thompson, J. A. (1986). A reviev^ of high-performance hquid chromatography in nucleic acids research. IL Isolation, purification and analysis of oligodeoxyribonucleotides. Bio Chromatography 1, 22-32. 13. Ausserer, W. A., and Biros, M. L. (1995). High-resolution analysis and purification of synthetic oligonucleotides w^ith strong anion-exchange HPLC. BioTechniques 19, 136-139. 14. Bergot, B. J., and Egan, W. (1992). Separation of synthetic phosphorothioate ohgodeoxynucleotides from their oxygenated (phosphodiester) defect species by strong-anion-exchange high-performance liquid chromatography. / . Chromatogr. 599, 35-42. 15. Bergot, B. J. (1993). Method for chromatographic separation of synthetic phosphorothioate oligonucleotides. U. S. Pat. 5,183,885. 16. Drager, R. R., and Regnier, F. E. (1985). High-performance anion-exchange chromatography of oligonucleotides. Anal. Biochem. 145, 47-56. 17. Metelev, V., and Agrawal, S. (1992). Ion-exchange high-performance liquid chromatography analysis of oligodeoxyribonucleotide phosphorothioates. Anal. Biochem. 200, 342-346. 18. Deshmukh, R. R., Leitch, W. E., II, and Cole, D. L. (1998). Application of sample displacement techniques to the purification of synthetic oligonucleotides and nucleic acids: A mini-review with experimental results. / . Chromatogr. A 806, 77-92. 19. Liautard, J., Ferraz, C , Widada, J. S., Capony, J. P., and Liautard, J. P. (1989). Purification of synthetic oligonucleotides on a weak ion-exchange column. / . Chromatogr. 476, 439-443. 20. Warren, W. J., and Vella, G. (1994). Analysis and purification of synthetic oligonucleotides by high-performance liquid chromatography. In "Protocols for Oligonucleotide Conjugates" (S. Agrawal, ed.), pp. 233-265. Humana Press, Totowa, NJ. 21. Puma, P. (1997). Chromatography in process development. In "HPLC: Practical and Industrial Applications" (J. Swadesh, ed.), pp. 81-110. CRC Press, Boca Raton, FL. 22. Agrawal, S., and Zamecnik, P. C. (1990). Method of separating oligonucleotides from a mixture. PCT Int. Appl. WO 90/09393. 23. Orum, H., Nielsen, P. E., Jorgensen, M., Larsson, C , Stanley, C , and Koch, T. (1995). Sequence-specific purification of nucleic acids by PNA-controUed hybrid selection. BioTechniques 19, 472-480. 24. Schott, H., Schrade, H., and Watzlawick, H. (1984). Isolation of oligonucleotides from partial hydrolysates of DNA using template chromatography (in German). / . Chromatogr. 285, 343-363. 25. Kasai, K.-I. (1993). Size-dependent chromatographic separation of nucleic acids. / . Chromatogr. 618, 203-221. 26. Oefner, P. J., Huber, C. G., Umlauft, F., Berti, G.-N., Stimpfl, E., and Bonn, G. K. (1994). High-resolution liquid chromatography of fluorescent dye-labled nucleic acids. Anal. Biochem. 223, 39-46. 27. Green, A. P., Burzynski, J., Helveston, N. M., Prior, G. M., Wunner, W. H., and Thompson, J. A. (1995). HPLC purification of synthetic oligodeoxyribonucleotides containing base- and backbone-modified sequences. BioTechniques 19, 836-841. 28. Huber, C , Oefner, P., and Bonn, G. (1992). High-resolution liquid chromatography of oligonucleotides on non-porous alkylated-styrene-divinyl benzene copolymers. Anal. Biochem. 212, 351-358.

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29. Huber, C. G. (1998). Micropellicular stationary phases for high-performance Uquid chromatography of double stranded DNA. / . Chromatogr. A 806, 3-30. 30. Yamasaki, Y., Yokoyama, A., Ohnaka, A., Kato, Y., Murotsu, T., and Matsubara, K.-I. (1989). High-performance hydroxyapatite chromatography of nucleic acids. / . Chromatogr. 467, 299-303. 31. Bernardi, G. (1971). Chromatography of nucleic acids on hydroxyapatite colummns. In "Methods in Enzymology" (L. Grossman and K. Moldave, eds.). Vol. 2 1 , pp. 92-140. Academic Press, New York. 32. Hirabayashi, J., and Kasai, K.-I. (1996). Applied slalom chromatography improved DNA separation by the use of columns developed for reversed-phase chromatography. / . Chromatogr. A 722, 135-142. 33. Pearson, R. L., Weiss, J. F., and Kelmers, A. D. (1971). Improved separation of transfer RNA's on polychlorotrifluoroethylene-supported reversed-phase chromatography columns. Biochim. Biophys. Acta 228, 770-774. 34. Wells, R. D., Hardies, S. C., Horn, G. T., Klein, B., Larson, J. E., Neuendorf, S. K., Panayatatos, N., Patient, R. K., and Selsign, E. (1980). RPC-5 chromatography for the isolation of DNA fragments. In "Methods in Enzymology" (L. Grossman and K. Moldave, eds.). Vol. 65, pp. 327-347. Academic Press, New York. 35. Cohn, W. E. (1949). The separation of purine and pyrimidine bases and of nucleotides by ion exchange. Science 109, 377-378. 36. Li, Y., and Spencer, H. G. (1992). Selective protein separations using formed-in-place anion exchange membranes. / . Biotechnol. 26, 203-211. 37. Deshmukh, R. R., and Sanghvi, Y. S. (1997). Recent trends in large-scale purification of antisense oligonucleotides. IBC Conf.: Large Scale Oligonucleotide Synth., San Diego, CA, 1997. 38. Baba, Y., and Ito, M. K. (1989). Optimization of gradients in anion-exchange separations of oligonucleotides using computer assisted retention prediction and a high-performance liquid chromatographic simulation system. / . Chromatogr. 485, 647-655. 39. Baba, Y. (1993). Prediction of the behaviour of oligonucleotide in high-performance liquid chromatography and capillary electrophoresis. / . Chromatogr. 618, 41-55. 40. Griffey, R. H., Greig, M. J., Gaus, H. J., Liu, K., Monteith, D., Winniman, M., and Cummins, L. L. (1997). Characterization of oligonucleotide metabolism in vivo via liquid chromatography/electrospray tandem mass spectrometry with a quadrupole ion trap mass spectrometer. / . Mass Spectrom. 32, 305-313. 41. Apffel, A., Chakel, J. A., Fisher, S., Lichtenwalter, K., and Hancock, W. S. (1997). Analysis of oligonucleotides by HPLC-electrospray ionization mass spectrometry. Anal. Chem. 69, 1320-1325. 42. Thompson, J. A. (1986). A review of high-performance liquid chromatography in nucleic acids research. I. Historical perspectives. Bio/Chromatography 1, 16-20. 43. Jaeske, A., Fuertste, J. P., Erdmann, V. A., and Cech, D. (1994). Hybridization-based affinity partitioning of nucleic acids using PEG-coupled oligonucleotides. Nucleic Acids Res. 22, 1880-1884. 44. Min, C , and Verdine, G. L. (1996). Immobilized metal affinity chromatography of DNA. Nucleic Acids Res. 24, 3806-3810. 45. Agrawal, S., Habus, I., and Kandimalla, E. (1998). Affinity-based purification of oligonucleotides using soluble multimeric oligonucleotide. PCT Int. Appl. WO 9 8 / 0 4 5 7 1 . 46. Jost, J.-P., Jiricny, J., and Saluz, H. (1989). Quantitative precipitation of short oligonucleotides with low concentrations of cetyltrimethylammonium bromide. Nucleic Acids Res. 17, 2143. 47. Cole, D. L. (1999). Keynote address—GMP manufacturing of antisense oligonucleotides at Isis Pharmaceuticals for clinical trials and the marketplace: Yesterday, today and tomorrow. IBC Conf. Oligonucleotide Pept. Manuf. Strategies, San Diego, CA, 1999. 48. Puma, P. (1997). Increasing large scale production limits. IBC Conf.: Large Scale Oligonucleotide Synth., San Diego, CA, 1997. 49. Krotz, A. (1999). In preparation. 50. Johansson, H. J., and Svensson, M. A. (1995). A novel method for large-scale purification of oligonucleotides. Nucleic-acid Based Thera., San Diego, CA., 1995.

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51. Sofer, G. K., and Hagel, L. (1997). "Handbook of Process Chromatography: A Guide to Optimization, Scale up, and Vahdation." Academic Press, San Diego, CA. 52. Zhang, Z., and Tang, J.-Y. (1998). Recent advances in scale-up of modified oligodeoxynucleotides. Curr, Opin. Drug Discovery Dev. 1, 304-313. 53. Gerstner, J. A., Pedroso, P., Morris, J., and Bergot, B. J. (1995). Gram-scale purification of phosphorothioate oligonucleotides using ion-exchange displacement chromatography. Nucleic Acids Res. 23, 2292-2299. 54. Thompson, J. A. (1986). A review of high-performance liquid chromatography in nucleic acids research. III. Isolation, purification, and analysis of supercoiled plasmid DNA. Bio/Chromatography 1, 74-80. 55. Thompson, J. A. (1987). A review of high-performance liquid chromatography in nucleic acids research. IV. Isolation, purification, and analysis of DNA restriction fragments. Bio/Chromatography 2, 4 - 1 8 . 56. Prazeres, D. M. F., Schuluep, T., and Cooney, C. (1998). Preparative purification of supercoiled plasmid DNA using anion-exchange chromatography. / . Chromatogr. A 806, 31-45. 57. Wincott, F. E. (1997). High-throughput ribozyme production. IBC Conf.: Large Scale Oligonucleotide Synth., San Diego, CA, 1997. 58. Wincott, F. E., DiRenzo, A., Shaffer, C , Grimm, S., Tracz, D., Workman, C , Sweedler, D., Gonzalez, C , Scaringe, S., and Usman, N. (1995). Improvements in synthesis, deprotection, analysis and purification of RNA and analogues. Nucleic Acids Res. 23, 2677-2684. 59. Bellon, L. (1998). Accelerating the discovery of drugs. BioPharm, 26. 60. Andrade, M., Scozzari, A. S., Cole, D. L., and Ravikumar, V. T. (1997). Efforts toward synthesis of oligonucleotides for commercialization. Nucleosides Nucleotides 16, 1617-1620. 61. Cheruvallath, Z., Wheeler, P. D., Cole, D. L., and Ravikumar, V. T. (1999). Use of phenylacetyl disulfide (PADS) in the synthesis of oligonucleotide phosphorothioates. Nucleosides Nucleotides 18, 485-492. 62. Gerstner, J. A. (1996). Economics of displacement chromatography—A case study. BioPharm 9, 30-35.

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY EGISTO BOSCHETTI Life Technologies-BioSepra, 95804 Cergy Saint Christophe, France

ALOIS JUNGBAUER Institute of Applied Microbiology, University of Agriculture, A-1190 Vienna, Austria

I. INTRODUCTION A. Milestones B. General Considerations of Antibody Purification II. ANTIBODIES: A N OVERVIEW A. Structural Properties B. Chimeric Antibodies C. Antibodies from Phage Display Libraries D. Common Properties Regarding Purification III. BIOLOGICAL STARTING MATERIAL A. General Considerations B. Plasma as a Source of Antibodies C. Cell Culture as a Source of Antibodies D. Transgenic Animals as a Source of Antibodies E. Antibodies from Transgenic Plants F. Egg Yolk as a Source of Antibodies IV. PREPURIFICATION A. Stability of the Culture Supernatant B. Harvest and Clarification of Culture Supernatant C. Concentration of Culture Supernatant by Ultrafiltration D. Concentration by Precipitation V. PURIFICATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY A. Packed Beds B. Fluidized Beds C. Role of Ion-Exchange Chromatography D. Role of Hydrophobic Interaction Chromatography E. Role of Hydroxyapatite Chromatography F. Role of Affinity Chromatography G. Role and Place of Gel Filtration H. Conclusions Concerning Single Chromatographic Techniques I. Combined Chromatographic Separation Processes

Separation Science and Technology, Volume 2 Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

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VI. REGULATORY CONSIDERATIONS A. Risks Related to Initial Raw Materials B. Virus Detection and Clearance C. Risks Associated with Leached Material from Affinity Columns D. Sanitization of Columns: General Considerations VII. GENERAL CONCLUSION O N ANTIBODY SEPARATION TECHNOLOGIES REFERENCES

INTRODUCTION A. Milestones In the past decades monoclonal antibodies and recombinant antibodies became the largest class of proteins that are currently in clinical trials and having received Food and Drug Administration (FDA) approval as therapeutics and diagnostics. This situation is the result of large intellectual and capital efforts. Significant progress in understanding antibody function, hostdefense mechanisms, the role of antibodies in cancer, and substantial improvement of production and purification technology have been achieved. The development of protein free culture media, continuous production of animal cells in perfusion culture, genetically engineered "humanized" antibodies,^ development of single-chain antibodies, phage display,^'^ and cellsurface display libraries have been important steps in this dynamic discipline. The design of antibodies according to the special needs for therapy, diagnosis, and purification technology is now possible. Specific properties for in vivo behavior such as defined pharmacokinetics and tumor targeting"^ are simply achieved by combining various fragments with desired properties. These highlights are only a few examples demonstrating the progress. Antibodies are expressed by hybridoma cells formed by cell-fusion of sensitized animal or human B lymphocytes with myeloma cells or they are generated by EBV (Epstein-Barr virus) transformation of sensitized B lymphocytes. Other heterologous expression systems such as bacteria,^ yeast,^ insect cells,"^ and mammalian cells^ have also been used for the expression of antibodies and fragments thereof. Due to renaturation problems, glycosylation, and expression levels, however, mammalian cells are mostly used for the expression of monoclonal antibodies. More recently technologies have been extensively developed for the expression of antibodies in transgenic animals^ and transgenic plants.^^ Intact antibodies with the biologically active glycosylation profile, which is crucial for the effector functions, require eukaryotic expression {in vitro or in vivo). These circumstances inspired a lot of scientists to find effective methods for production as well as methods for the selection of the best extraction-purification methods.

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Purity, safety, potency, and cost effectiveness are the main points to consider when designing the expression method and more importantly when defining purification processes. Purification of antibodies is a story that commenced with the separation of proteins, mainly paraproteins, several decades ago. A plethora of protocols have been described involving precipitation with a variety of chemical agents, electrophoretic separation, membrane methodologies, and fiquid chromatography. The latter probably represents the most popular technique as a result of the easiness of implementation, of the capability to play on the selectivity, and finally because of the level of purity that can be achieved. Specific hquid chromatography methodologies and resins have been especially developed for the purpose; this is a pecufiar situation in the discipline of downstream bioprocessing. Today monoclonal antibodies and more largely immunoglobulins with all their derivatives represent by far the largest class of produced and purified protein in number and mass.

B. General Considerations of Antibody Purification Although purification of antibodies can be performed by a combination of different technologies such as precipitation, membrane technology, and separation by electrophoretic techniques, it is the scope of this chapter to focus on description of antibody purification by chromatographic means. Numerous sorbents have been developed for protein separation, and they are based on a variety of adsorption-desorption principles; in the following description, only materials and principles that are suited for the separation of antibodies are selected. Selection of such materials and principles depend first on the properties of the particular immunoglobulins to be separated and on the composition of the impurities that constitute the feedstock. Therefore the antibody structure and function will be described first and then a short overview of the production technology will be given. Antibodies are very diverse in molecular properties, chemical characteristics, and biological activity. Purification strategies are therefore very diverse, since they are based on a large variety of molecular interactions. Antibodies have, however, several common properties that are frequently exploited for their capture from the initial feedstock. It is known in fact that several physicochemical and structural similarities are shared between antibody molecules, and that is why they are considered as an homogeneous group of proteins. As is valid for any protein separation problem, the knowledge of the nature and concentration of the impurities is the key for success. Therefore the initial composition of feedstock is of importance when designing the separation process. Today expression systems can be selected to simplify the extraction-purification procedures. In the following pages all described methodologies are discussed with the purpose of separating antibodies at bench scale as well at large scale.

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II. ANTIBODIES: AN OVERVIEW A. Structural Properties Antibodies or immunoglobulins are serum glycoconjugates expressed in all mammals. They are induced by the lymphoid system if and when a foreign immunogenic antigen is recognized. During the maturation of the immunologic reaction polyspecific primary antibodies are somatically mutated to increase avidity of the antibody-antigen complex. Therefore immunoglobulins are part of the adaptive immune system. Five immunoglobulin classes can be distinguished (IgG, IgA, IgM, IgD, and IgE), which differ in size, amino acid, and carbohydrate composition of the heavy chain. IgG is a tetrameric protein representing the main part of the antibody pool. Immunoglobulins of the IgG class have a typical structure composed of four polypeptide chains separated into two groups: two light chains and two heavy chains. They are tightly linked together by disulfide bonds to form the entire construct as illustrated in Fig. l}^ The conjunction of light- and heavy-chain extremities constitutes the active site (hypervariable region) where the antigen is recognized. The tail formed by the association of two extremities of heavy chains constitutes a constant region where most of the glycosylation of the protein takes place. The molecular weight of a whole immunoglobulin G is relatively constant and is in the range of 160 Kd. This is a high molecular mass that can be advantageously used for their separation from other proteins that are generally smaller in size. IgM antibodies (much larger structures than IgG) are expressed first by lymphocytes defending a new antigen. They show a pentameric structure (Fig. 2) stabilized by a peptide-linker called the joining

disulfide bond antigen binding site heavy chain 450 residues

light chain 212 residues FIGURE

I

The basic structure of IgG. (Reprinted from Roitt et a/.")

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F I G U R E 2 Pentameric polypeptide chain structure of human IgM. The IgM heavy chain have five domains with disulfide bonds crosslinking adjacent CjLt3 and CfiA domains of different units. Also shown are the carbohydrate side chains and possible location of the J chain. (Reprinted from Roitt et 0/.")

chain (J chain). IgA are dimeric structures and constitute an immunological barrier in seromucosal secretion where they are mostly present. IgD antibodies have some relevance in antigen-triggered lymphocyte differentiation, and finally IgE antibodies are displayed on the surface of membrane of basophilic and mast cells; they are often associated with hypersensitivity. i. Structure and Function of Constant Region

Immunoglobulins consist of two identical heavy chain-light chain heterodimers. Heavy chains have a molecular weight of about 50 kD; they possess a constant region that determines the class contribution. Light chains also called CA and CK are composed of a carboxyl-terminal moiety characterized by a constant region CL (constant light chain) except for certain allotypic and isotypic variations, whereas the amino-terminal half shows a high sequence variability and is known as VL (variable light chain). Each light chain is linked to one heavy chain via a disulfide bond formed by a cystein on the C-terminal end of CL and the end of CHI region on the heavy chain. Every light chain has two intrachain disulfide bonds, one in the variable and one in the constant region. The molecular weight of light chain is of about 25 kD. The constant parts of the heavy chain y, a, and 8 can be divided into three domains, each generating an intradomain disulfide bond spanning about 60-70 amino acids. Thus, the heavy chain displays four sections, VH,

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C H I , CH2, and CH3, defined by homologies in the secondary and tertiary structure through similar loops. In fi and e heavy chains, an additional domain after CHI is present so that C^t3 is homologous to C/x2. ii. Variable Regions of Antibodies

Variable regions of heavy and light chain together form the antigen-binding domain of the immunoglobulin. This three-dimensional structure is generated by the three a-helical domains of the complementary regions (CDRs) on both heavy and light chain. These CDRs are stabilized by relatively invariant framev^ork segments v^ith j8-sheet structure, building the variable regions backbone. The binding of the antigen takes place by multiple noncovalent bonds, v^hereby the association forces depend on the distance betv^een the interacting groups and their number. This determines the antibody affinity. To ensure optimal fit of the humoral immunity to a maximum number of antigens, the number of potential antibodies in man must be very large. This is accompanied by a separate diversification mechanism existing for heavy and light chains coded on different chromosomes. Recombination of gene segments for heavy- and light-chain variable regions during B-cell maturation enables that specific antibodies for numerous antigens can be produced from a limited pool of genes. Light chain kappa and lambda are located on tv^o different chromosomes (2 and 22) in humans. During B-cell differentiation one of the V genes recombines with the J segment to form a V-J combination. Heavy V, D, and J segments are located on chromosome 14 in humans, leading to a greater variability through differential splicing of the segments. Additional steps of the immune system in generating a maximum of antibody diversity-are-the recombinatorial accuracy, the somatic point mutations, and the assorting of heavy and light chains. These different mechanisms taken together are able to generate 10^ different antibodies, able to recognize most of non-self antigens invading the body. Due to incomplete splicing in hybridoma cells, antibodies with aberrant structure are also observed. Either they contain an additional CH or CL fragment or one of these fragments is missing. These antibodies have a different molecular weight, although they are still recognized by the various immunological techniques such as Western blots, enzyme-linked immunosorbent assay (ELISA), and dot blot. These antibodies do also recognize the antigen, and they may also have effector function activity. Incomplete IgM antibodies due to incorrect splicing have also been observed. As long as the domains responsible for interacting with ligands in affinity chromatography are not affected, these antibodies with aberrant structure can still be purified with the same methods as applied for normal antibodies. iii. Effector Functions of Antibodies

The effector function of an antibody is defined as the mediation of other host-defense mechanism besides antigen recognition. It is highly determined by the carbohydrate structure of the heavy chain. The primary duty of antibodies is to bind antigens. In some cases the penetration of cells by bacterial toxins or viral infections can be combat by generating such antigen-antibody complexes. Most of the time, an effector

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54 I

function mediated by the Fc-specific receptors of other cells of the immune system is necessary to defend invasing agents.^^ The most important effector function is the activation of the complement system, mediating inflammatory reactions. The initiating step after antigen fixation is the binding of antibody domains C/x, C y l , or CyS to the protein of the complement system clq. Partial cleavage of different proteins activates the complement cascade and three major steps can be initiated: activation of macrophages, cytolysis of target cells, or phagocytosis of the antigen. Another effector function is the cell-mediated cytotoxicity. Infected cells that v^ere recognized and opsonized by specific antibodies can be lysed by natural killer cells v^hich are the classical K cells. These properties are highly determined by the Fc part of antibodies. IgGl and IgG3 have the highest capacity for cell-mediated cytotoxicity. The effector function of an antibody is frequently used as a mean of antibody recognition. This is especially the case w^ith bacterial proteins such as protein A and protein G that specifically interact w^ith the same domain. Therefore purification can interfere with the antibody effector function. iv. The Microheterogenic Nature of Antibodies

Although a monoclonal antibody is considered to consist of a single molecule, in reality this is not the most frequent case. Due to posttranslatorial modification, especially glycosylation,^^'^^ a family of molecules with identical aminoacid backbone, but different carbohydrate composition, is generated.^"^'^^ The possible structure of N-linked oligosaccharides of the complex types is shown in Fig. 3. Usually, immunoglobulins are N-glycosylated at the Fc part; in some cases, terminal neuraminic acid may be missing. This leads to slightly different isoelectric points of the various members in the family called isoforms. Therefore, an IgG preparation which exhibits a single band in sodium dodecyl sulfate (SDS) electrophoresis exhibits a characteristic band pattern in isoelectric focusing (Fig. 4). As a consequence for purification, antibodies separated by chromatographic methods acting through electrostatic forces (e.g., ion-exchange chromatography) or biorecognition of the Fc part may influence the isoform composition of separated antibodies.^^ This is not of great importance, as long the immunoglobulin is not used as therapeutic agent. Conversely, as the

(7) (6) (5) (4) ±Siala2->6±Gaipi ^4±GlcNAcpi ->2Mana1

i 6 (3) (2) (1) ±GlcNAcp1->4Manp1->4GlcNAcpi-^4GlcNAcpi->Asn 3

t

±Siala2->6±Gaipi->4±GlcNAcpi->2Mana1 (7') (6') (5') (4') F I G U R E 3 Structure of an N-linked oligosaccharide of the complex type occurring in human IgG. The bold typed saccharides constitute the conservative structure, all others are optional.

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Mm 'MMm^Mmmmm&*&mmnmimmm0rm':AW^r. .^/smm^^Mmm^mmm^^^mma 7i:rm::rmm. .^ ^' mm:0S^:%mmm.ii'aikimmfi'':.::m-':w- nm • n,. •. m: m ; ;:•-,. mm.t»s : ^mwmmst-mmtmm %•;: %: I: m:M:mk:msmmmmm-mzm^^^^^ F I G U R E 4 Microheterogeneity of a human monoclonal antibody determined by isoelectric focusing. Lane I, marker proteins; lane 2, purified antibody; and lanes 3 - 8 , separated isoproteins according to the method of Kaltenbrunner et o/.^'

effector functions and the pharmacokinetics are determined by the Fc part, attention must be paid on the microheterogeneity during purification of therapeutic antibodies.^^ Microheterogeneity may also change with fermentation conditions.^^ Protein purifiers must be aware that the biological activity can vary with the starting material which may vary when production scale-up is performed. Therefore if a fermentation mode or device is changed it may be accompanied with some reoptimizations of chromatographic conditions. It has been reported that the pharmacokinetics have drastically declined when antibodies produced by airlift fermenter instead of in vivo by the ascites technology. ^^ The authors mention that these findings cannot be generalized, since they have investigated two other clones which were less sensitive to fermentation conditions. Secreted proteases and glycosidases may also contribute to a modification of the antibody structure; this effect generally happens extracellularly. Karl et al}^ described an acidic protease in cell-free culture supernatant of a hybridoma culture with a similar activity of a lysosomal protease. This enzymatic activity could be inhibited by pepstatin A. Investigations of cell-free

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SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

culture supernatants of various insect cell cultures showed also significant exoglycosidase activity.^^

B. Chimeric Antibodies Chimeric antibodies is a definition that appUes to antibodies which are composed of at least two different structural elements originated from immunoglobulins of another species or class. An overview of the most common types of chimeric antibodies is given in Fig. 5. Chimeric antibodies with changes in the variable domain or in the constant region were established mainly because of the need for humanized antibodies.^^ Rodent variable domains for antigen binding were fused with human constant regions, so that the resulting molecule is largely human, but binds with the specificity of the parental monoclonal. Chimersization enhances effector functions, but a significant part of the molecule is still of rodent origin and recent human trials have shown that over a half human mount, generates an antimouse response after receiving a chimeric antibody. In CDR-grafted antibodies, the six hypervariable loops of aminoacids (CDRs) are grafted on human framework regions to improve the therapeutic benefit.^^ Another variant of antibodies are bispecific molecules, which can be created conventionally by fusing to hybridoma lines or chemically, by crosslinking two halves of two different antibodies. Those bispecific mono-

] v (rodent)

single domain antibody (Fv)

e.g. human antibody

rodent antibody antibody fusions with other proteins

F I G U R E 5 Restructured versions of antibodies, antibody fragments and fusion proteins available due to recombinant D N A technology. (Reprinted from Sachaffner et al}^^ by kind permission of Walter de Gruyter GmbH & Co., Berlin.)

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EGISTO BOSCHETTI AND ALOIS JUNGBAUER

clonal antibodies have been used to bring together cytotoxic effector cells and targets, that they would otherwise not recognize, e.g., tumor cells. Chimerization of an antibody can also be accounted when designing a separation process based on immunoaffinity recognition. C. Antibodies from Phage Display Libraries The intact humoral immune system is essentially the largest library of antibodies. After challenge with an antigen the most suitable antibodies are selected amplified and affinity maturated. If this whole process could be reproduced in vitro^ isolation of high-affinity human monoclonal antibodies might be greatly simplified. A single phage library consists of at least 108 clones that each express one antibody on the surface and can be selected from the library. Afterward low-affinity antibodies from such libraries can be randomly mutated by polymerase chain reaction (PCR) technology under imperfect conditions and higher affinity variants can then be selected from this library, leading to in vitro affinity maturation.^'^'^^ The structural properties of such antibodies are not different from other monoclonal antibodies and the same separation principles can be applied for. D. Common Properties Regarding Purification Still in the domain in similarities between antibodies is the glycosylation moiety. It is mostly restricted in the Fc fragment and can be used as a recognition region for the chromatographic separation. Fc fragment is relatively constant and is, as such, used very largely to standardize the separation. Specific portions of Fc fragment are recognized by some microbial proteins such as protein A from Staphylococcus aureus^^ (designated also as SpA); this was at the basis of the extraordinary development of protein A resins used extensively today for antibody separation at any scale. Fc fragments contain interacting zones for metal ions and amino acid sequences that confer structures able to interact with aromatic or heterocyclic chemicals. Both characteristics are used as properties to develop generic methods for antibody separation described below. Fragmentation of antibody is a regular way to prevent nonspecific binding to cells such as macrophages and monocytes with receptors for Fc fragments. Cleavage of IgG by papain, a nonspecific thiol protease, is one of the best-known methods (Fig. 6). Digestion is stopped by an irreversible alkylation reaction using an excess of iodoacetamide. Separation of Fc from Fab produced fragments is accomplished by either the specific adsorption on a protein A column (see Section V.F.ii), or anion exchange chromatography (see Section V.C), since Fab fragments are significantly more alkaline than the Fc and emerge in the flowthrough of the column. Papain is also used for partial digestion of antibodies. F(ab02 are obtained when papain is free of cysteine. The interest of these fragments is their higher antigen avidity when compared to Fab fragments. Those F(ab')2

545

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY -F(ab')2

low mol. wt. - * - - p F c ' — ^ peptides

CiDDfi I I — gOOQ

333

I

II . ^ I

secondaiy papain cleavage points

-Fab F I G U R E 6 Enzymatic cleavage of human IgGI. Pepsin cleaves the heavy chain at the amino acid residues 234 and 333 to yield F(ab')2 and pFc' fragments. Further action reduces the central fragment to low molecular weight peptides. Papain splits the molecule in the hinge region yielding two Fab fragments and the Fc fragment. Secondary action on the Fc fragment at residues 431 and 433 gives rise to Fc'. (Reprinted from Roitt et a/.")

fragments can then be chemically conjugated to a variety of substances including plant and bacterial toxins, enzymes, radionuclides, and cytotoxic drugs. In most cases, such modifications are useful tools to image tumorogenic cells, recognizing surface marker proteins. IgM can also be fragmented enzymatically, but information on proteolytic breakdown is limited and the mechanism of cleavage not entirely knov^n. Enzymes are used alone or in association with urea to enhance the dissociation and fragments obtained are of different molecular weight. They appear to be constituted of two light and two heavy chains by molecular rearrangement. Other fragments are also identified as constituted of one light chain and one heavy chain.

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Table 1 shows a list of antibody characteristics utilized for their separation from crude mixtures.

III. BIOLOGICAL STARTING MATERIAL A. General Considerations Originally antibodies were obtained by injecting an antigen to an animal to induce an immune response leading to the secretion of specific antibody molecules. This is still an important method for generating antibodies. When antibodies are intentionally induced in humans by vaccination and the blood is collected from volunteer donors, then these sera are called hyperimmune sera. Each additional immunization and each individual animal creates a different pattern of specific antibodies. This peculiar situation may limit the continuity of long-term studies using only polyclonal antibodies as diagnostic tools. On the other hand, polyclonal antibodies are almost not susceptible to single mutations of antigens, and therefore they are more proper in all cases when a more broadly specificity is desired. In practice the question is not if monoclonal antibodies are better than polyclonal antibodies. Requirements and problems in diagnosis of livestock diseases are individually different and it will be advantageous and helpful if both types of antibodies are available and combinatorial use is possible. Compared with the generation, such as the laborious steps in screening, subcloning, and the in vitro production, monoclonal antibodies require more technology and equipment and therefore cause higher costs and consume more time. H H T A B L E I Molecular Properties of Antibodies and Related Solid-Phase Separation Methods Main properties of antibodies

Related chromatographic separation

Amphoteric molecule Large molecular size Presence of hydrophobic clusters Presence of charged clusters Recognition of some microbial proteins Presence of sugars

Ion exchange Gel filtration Hydrophobic interaction chromatography Hydroxyapatite chromatography Bioaffinity

Ability to make metal complexes Antigen specific recognition

Metal affinity chromatography Bioaffinity

Antigenic structure

Immunoaffinity

Bioaffinity

Type of Interaction on solid phase Ionic Exclusion from small pores Association with hydrophobic regions of resin Mediated by phosphate-rich regions Complementarity of Fc region with protein A and protein G Complementarity of sugars with lectins and boronic acid Formation of a metal complex with histidyl clusters Association of immobilized antigen on binding site Association with an immobilized specific antibody

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547

Monoclonal antibodies developed by Kohler and Milstein^^ imply the selection of the right B lymphocyte responsible for the secretion of the selected antibody and its fusion with a myeloma cell to assure longevity of secretion. Monoclonal and recombinant antibodies are broadly accepted for the diagnosis of various diseases, therapeutic applications, and as a general biochemical tool. EBV transformation is another widely used way for establishing cell lines for antibody production in the past.^^'^^ Cell lines transformed with EBV preferably produce IgM.^^ Due to some instability of the cell lines, however, hybridoma technology based on cell fusion is preferred. In vivo production of hybridoma cells is accomplished by injecting them into the intraperiton. They grow and an ascite is formed which contains large amounts of monoclonal antibodies where the concentration can reach 10 mg/mL and more. This production technology has been banned in several European countries for animal welfare reasons. Moreover, ascites fluid also contains serum proteins and fats that make the separation of antibodies more complex. Protein impurities are less numerous than in serum, but the lipids are removed for better longevity in chromatographic columns. Modern in vitro fermentation technologies can compete with in vivo production of antibodies regarding concentration and purity of the produced material. In fact, a number of more sophisticated methods of antibody expression have been developed in the last decade; the most important are cell culture in protein-free media and the expression in the milk of transgenic animals, in transgenic plants, and in egg yolk. Each expression system is characterized by the quality of the expressed antibody, by the antibody concentration, and in particular by the impurities from the culture medium. The choice is mostly dictated by the amount of antibodies to be expressed, its intended application (therapy, diagnosis, fine chemical, or research), the investment, and the available infrastructure to produce and to purify the antibody. Recombinant technologies enable us to produce bispecific antibodies, chimeric antibodies, and antibody fragments. The feedstock from which the antibodies are to be extracted dictates the choice of the separation tools and to some extent the sequence of the chromatographic columns (see Section V.I). B. Plasma as a Source of Antibodies Plasma is still the biggest source of antibodies. Hyperimmune sera as well as normal purified IgG from serum or plasma have broad medical applications. Although fractionation of plasma proteins with the goal of antibody purification has been accomplished without chromatography for years, increasing requirements on the quality (purity, consistency, safety, and potency) of the product have led to the introduction of fiquid chromatography. Besides heat treatment and treatment with TnBP in association with detergents, chromatographic techniques also exhibit a highly efficient virus clearance (see Section VLB), introducing thus an improved level of product safety.

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Mammalian plasma from which polyclonal antibodies are extracted contain several hundreds of proteins, the most important in mass being albumin. Impurities constitute a difficult obstacle if classical separation methods are used; however, affinity separation are efficient enough to eliminate most of foreign proteins. In plasma, polyclonal antibodies are present at relatively high concentration (5 to 12 mg/mL of polyclonal IgG); they are among a large number of other biological compounds relatively difficult to separate even by fiquid chromatography. C. Cell Culture as a Source of Antibodies Hybridomas and other animal cells can be cultivated either in small Roux flasks, in spinner flasks, in hollow fibers, or in fermenters.^^ At a small scale, hybridomas, and other host cells are cultivated batchwise, thus the antibody concentration depends on the harvesting time, inocculum density, and qualj^y 30-33 gjj^^g production of inocculum is very tedious at large scale, continuous production can alternatively be used.^"^ Continuous production can be carried out either in stirred vessels without retention of cells or in a perfusion mode, where cells are retained in the cultivation vessel.^^'^^ Cells are either growing in suspension or on a solid phase. Even hybridoma cells, which are suspension cells, show a tendency to adhere on solid phases, therefore specific microcarriers can also be applied for hybridoma production.^^'^"" As a benefit of the perfusion culture, the harvested culture supernatant does not contain a lot of cells and debris. Such suspensions can be easily clarified by dead-end filtration or easily processed by expanded-bed technology (see Section V.B) or packed-bed technology using large beads. It is the nature of continuous fermentation that the harvest is continuously delivered. So far the continuous process is cut down after harvesting and even clarification is carried out batchwise. A significant advantage of continuous production is the homogeneity of the harvested product, conversely the stability of cell line must be checked aU along the process for possible drifts. A certain problem in a large-scale continuous cell culture is, in fact, ageing of retained cells. Antibody composition may slightly change in a long-term production run. Validation of consistent antibody composition (purity, aminoacid composition, glycosylation, isoforms, peptide mapping, etc.) throughout the whole production cycle is a critical issue. This is one of the reasons why antibody producers very frequently rely on traditional batch production strategies. These methods are, however, more and more sophisticated by, for instance, continuous addition of defined biologicals or chemicals to buffer deficiencies aU along the batch process to increase the expression of the antibody.^^'^^ A summary of the situation is presented in Table 2. Cell culture supernatants containing fetal bovine serum (from 1 to 10%) are characterized by their content of serum proteins and their low concentration of monoclonal antibodies (50-500 mg/mL). A perequisite for a good separation is to eliminate cells and cell debris first; in some cases, the concentration of the feedstock is also a requirement. Special care must be taken for the choice of the fetal bovine serum so as it does not carry any trace

549

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TABLE 2

Main Hybridoma Cell Culture Processes

Culture method

Product yield

Process manipulation

Batch Repeated batch Perfusion Fed batch Chemostat

Low Low Medium High Low

Low Medium High Medium Medium

Cost (capital investment and labor)

Throughput

Low Medium Medium Low Low

Low Medium High Low Low

of bovine polyclonal antibody that will be difficult to separate from the expressed monoclonal antibody. Up to 1% bovine IgG has been reported in final purified monoclonal antibody preparations from cell culture supernatant containing fetal bovine serum."^^'"^^ This can be a problem, since current existing affinity chromatography matrices cannot discriminate between the monoclonal antibody and other irrelevant IgGs from fetal bovine serum present in the tissue culture supernatant. Therefore, when present bovine IgG must be removed from fetal bovine serum by, for instance, the use of an affinity chromatography matrix before being used as part of standard tissue culture medium."*^ The essential benefit of abundant serum proteins in the cell culture is related to possible damages of expressed antibodies due to proteolytic or glycolytic breakdown. Serum contains enzymatic inhibitors and therefore degradation processes are better downregulated. Fetal bovine serum can be effectively replaced by a few known proteins such as albumin, transferrin, and insulin as supplement to basal cell culture medium. In specific cases protein-free cell culture media are advantageously used at large scales."*^ The specific role of proteins is progressively taken over by known natural and synthetic molecules."^"^ Resulting cell culture supernatants are thus characterized by a low concentration of foreign proteins, which is of considerable importance for the effectiveness of the separation technologies to isolate antibodies. The concentration of the antibody in such a cell culture supernatant can, however, be as low as few dozen of milligrams per milliliter. With optimized protein free-cell culture media, expressed antibody can reach concentrations as high 500 /xg/mL. The actual choice of cell culture medium depends on a variety of parameters connected to the host cell, since not all of them require foreign proteins to grow and not all of them secrete degradation enzymes in a significant level. D. Transgenic Animals as a Source of Antibodies The best-known process that uses transgenic animals to produce antibodies is expression in the milk.^ Production of recombinant antibodies in the milk of transgenic animals often presents the advantage of an overexpression level that reaches values as high as several milligrams per milliliters of milk. This is

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about 10-100 times higher than in recombinant cell culture systems. Another advantage of expression in transgenic animals is that the glycosylation of the immunoglobulin may be closer to the human antibody, depending on the choice of the animal. The cost and availability of feedstock are also important points to consider for better economics. The counterpart of this kind of expression for chromatographic purification is that the milk contains a large amount of proteins (30-40 mg/mL) and a variety of fats and casein micelles that must be removed. Microfiltration and or centrifugation are required to obtain a clear feedstock. This is also beneficial to reduce the high lactose content together v^ith low molecular w^ater-soluble species (salts, vitamins, amino acids, etc.). Milk can also contain lov^ levels of autologous animal immunoglobulins that must be removed from the expressed antibody. Specific strategies are put in place for this kind of separation such as the selection of the right animal in relation to separation methodologies. The use of transgenic goats, for instance, enables the use of protein A resins since they adsorb the expressed antibody and do not recognize goat immunoglobulins (see the specificity of protein A in section V.F.ii and Table 8 in that section). Separation of other proteins such as lactoglobulins and lactalbumin, the most representative proteins in milk, are as easy as the separation of proteins from serum. The same situation can be encountered v^ith the separation of global antibodies from hyperimmune animals treated for the expression in their milk for specific diagnostic or therapeutic uses. E. Antibodies from Transgenic Plants The production of antibodies in transgenic plants v^as introduced in 1989 by Hiatt, Cafferkey, and Bow^dish."^^ One of the important justifications of the choice of this host organism is the lov^ cost of plant maintenance. Another advantage over other recombinant expression systems is the ability to assemble full-length heavy chains with light chains to form full-length antibody efficiently.^^'^^ In transgenic plants, the expression of heterologous proteins can be targeted in different organs, whereas in animals the biological fiquids where antibodies can be expressed and then separated are blood and milk. Accumulation in different cellular compartments such as the cytosol, the apoplastic space, and the endoplasmic reticulum are well-described locations where antibodies are expressed."^^ A high accumulation of active antibodies was found when targeting the apoplastic space. This has been demonstrated in tobacco seeds and potato tubers."*^ The amount of expressed protein compared to the total extractable proteins is also an important information for the purification of antibodies. In corn seeds, for instance, the expressed proteins represent up to about 5% of total extractable proteins."*^ Plant extracts from which expressed proteins are separated are very different from animal biological liquids. Impurities include not only foreign proteins but also peculiar lipids and polysaccharides, depending on the plant organ. The presence of polyphenols must also be considered for separation by chromatography. One of questionable characteristics of antibodies expressed

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

55 I

in corn seeds is the absence of glycosylation that may impact the potency and the stabihty of the molecule.

F. Egg Yolk as a Source of Antibodies It has long been known that specific antibodies can be extracted from the eggs of immunized chickens. The particular specificity of avian antibodies is that they appear phylogenetically different from mammalian and are also structurally different. The advantages of egg yolk antibodies with respect to the welfare of animals and to scientific and economic considerations are described in a recent review.^^ Although antibodies issued from this source are mostly used in laboratories and for diagnostics, they must be extracted and purified from very complex mixtures. The presence of massive amount of insoluble material and lipids is the most important problem. The collected egg yolk must first be clarified and the lipid removed for a consistent chromatographic separation. Table 3 summarizes the most important characteristics of expression systems for antibody preparation with regards to the chromatographic separation.

lY. PREPURIFICATION A. Stability of the Culture Supernatant Usually, except when sera and ascitic fluids are used as a raw materials, the concentration of the antibody in the feeds stock is very low. The actual titer in a cell culture broth may be from 50 ^lg/mL (often less) up to a few hundred; in average it varies from 50 to 200 ^ig/mL. This dilute antibodycontaining solution is usually stable at 4°C. Harvesting should be immediately effected when batch fermentation is completed or on a daily basis when monoclonal antibodies are continuously produced. Meanwhile, the broth should be cooled down to 4°C. Cells should be removed as soon as possible, since they may be lysed and deliver a number of proteolytic enzymes which may partially degrade the antibody. This can happen either by unspecific proteolytic cleavage or by a highly selective cleavage forming Fab fragments. Such a selective degradation depends on individual antibodies and cell lines. When fetal bovine serum is used as a cell culture supplement, proteolytic cleavage is reduced by the presence of inhibitors such as al-anti-trypsin, a2-macroglobulin, and others protease inhibitors are naturally present in the feed stock.

B. Harvest and Clarification of Culture Supernatant I. Centrifugation Cell harvesting can be carried out by centrifugation; disk, bowl, tube, or basket centrifuges are the most popular. The sedimentation process is de-

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^ I H T A B L E 3 S u m m a r y of Characteristics and Advantages of Various Expression Systems Used for t h e Production of A n t i b o d i e s

Expression system

Amount of antibody (mg/mL)

Amount of foreign proteins (mg/mL)

Specific operations

Advantages ~ drawbacks

Animal sera Ascites

5-12 1-10

30-40 20-25

None Lipid removal

Hybridoma culture with FBS Serum-free culture

0.05-0.5

3-10

Cell removal

0.01-0.5

0.1-1

Hyperimmune milk

0.1-20

20-35

Transgenic milk

1-10

30-40

Transgenic plants

0.1-0.3

10-15

Egg yolk

2-8

20-40

Cell removal; concentration Removal of casein and lipids Removal of casein and lipids Protein extraction; lipid and pigments removal Dilution; removal of lipids

Large amount of foreign proteins High concentration of antibodies; small volume of feed stock per animal Consistency, automation; possible contamination of FBS Very clean material; low^ expression level Low cost; heavy pretreatments Low cost; heavy pretreatments Very low cost; heavy pretreatments High concentration, low cost; heavy pretreatments

Expressed as total amount of weight (%) for corn seeds.

scribed by Stoke's law w^rAo18/1

(1)

where ^^ is the sedimentation velocity, (o is the angular velocity, Acr is the density difference between medium and particle, jx is the dynamic viscosity, and r is the radial distance of the particle from the axis of rotation. For the removal of cell debris, the angular velocity or the residence time must be increased. Note that o) and r are limited by the stability of the construction material of the centrifuge. Tube centrifuges are better suited for the removal of cell debris than disk centrifuges; higher g forces are achievable. For comparison of different centrifuges, the static settling area is generally used; however, when scaling up, the nozzle capacity must also be taken into consideration. Since the amount of separable solids is very low in a mammalian cell culture, decanters are never used. In fact, it is essential to operate the centrifuge above a minimum g force due to the low difference of density of cells compared to the cell culture supernatant. In practice, animal cell culture centrifuges were developed in a manner which enables a gentle separation of the cells from the liquid to maintain the viability of the cells. In downstream bioprocessing, very often cell viability is not an important issue; a clear supernatant is the essential aim of the operation. In the case when the centrifugation is performed in an insufficient manner, nucleosomes^^ may remain in the supernatant, and these proteins, after having formed large complexes, may interfere in the subsequent purification steps.

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ii. Microfiltration Microfiltration is a unit operation for the separation of small particles. The separation limits are between 0.02 and 10 fim particle dimensions. Microfiltration can be carried out in a dead-end mode and a cross-flow mode. In downstream processing, the cross-flow filtration is carried out continuously or discontinuously. The most important parameters that determine the productivity of cross-flow microfiltration are transmembrane pressure, velocity, particle size and surface, viscosity of the liquid and additives such as surfactants, and changing the surface and surface tension. Many models have been published to calculate the microfiltration process. One important factor is the concentration polarization, which represents the most important limiting physical obstacle. At high particle concentration and with time, a layer is formed on the membrane. This layer is responsible for the flux reduction. A comprehensive overview on this technique is given by Ripperger^^ and Staude.^^ Often similar or identical module types are used in microfiltration and ultrafiltration. iii. Delipidation Working with ascite fluids, removal of lipids is frequently a requirement. This can be done by selective adsorption or by filtering throughout membranes having a high affinity for lipids. The downside is that these membranes may also bind a significant amount of immunoglobulins. The addition of sorbents such as silicon dioxide powder (e.g., Carb-O-Sil, a trademark of Union Carbide, Danbury, CN) may be helpful. A minimum time is required for the complete reaction of the lipophilic silicon dioxide powders with free lipids. Then the material can be easily decanted or removed by centrifugation. Separation of lipids by very high speed centrifugation is also known, but it is applicable only at laboratory scale. C. Concentration of Culture Supernatant by Ultrafiltration As already mentioned, cell culture supernatants are very dilute antibody solutions. Therefore a concentration step is necessary, unless the culture supernatant is directly processed by expanded-bed adsorption.^"^'^^ Ultrafiltration (UF) and diafiltration (DF) are very popular methods for conditioning the clarified culture broth for chromatography. As general rule, dilute animal cell culture supernatants are concentrated up to a factor of 20 and even more prior to fractionation. Ultrafiltration is a process in which a solution containing macromolecules is passed across a functional membrane, while macromolecules are retained by the membrane. The process is driven by the pressure drop across the membrane also called transmembrane pressure (Pj) expressed as PT

- ^ - ^

- Pp

(2)

where Pj is the inlet pressure, PQ is the outlet pressure, and Pp is the permeate back pressure of the membrane.

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The difference between conventional dead-end filtration and cross-flow filtration is the configuration of the system. For large-scale operations, only cross-flow filtration will be used. The membranes for miocrofiltration as well as ultrafiltration are commonly utilized in a variety of filtration devices. There are three basic types of tangential flow filtration devices: plate and frame, hollow fiber, and spiral wound membranes. The differences cannot be easily explained. The selection rules are often heavily influenced by economical considerations alone. The technological parameters are flux, recirculation rate, chemical compatibility, pore size or molecular weight cutoffs, pressure and temperature limitations, and cleaneability. Once the feasibility has been established, the engineering design and scale-up come into play. The additives (antifoam or stabilizers) to the culture medium may strongly interfere the UF process by reduction of the flux.^^ UF-DF at a large scale is always carried out in cross-flow mode, discontinuously or continuously or in dynamic mode to reduce fouling of membranes.^^ A simple relationship between flux ( / ) and time (t) is the empirical relationship

J=Jot"

(3)

where /Q is the initial flux, and n is an exponent less than zero^^ also called fouling rate constant. This constant must be experimentally determined. Equation (3) shows that the flux is decreasing with time, because a layer is built up at the membrane surface. Membranes of appropriate quality and characteristics are available to cover the prepurification problems in processing of antibodies. A detailed description would exceed the scope of this chapter. D. Concentration by Precipitation Three chemical agents are commonly used for the precipitation of antibodies: ammonium sulfate, caprylic acid, and polyethyleneglycol (PEG). Precipitation of immunoglobulins by ethanol is applied only for blood plasma fractionation at the industrial scale, and it would exceed the scope of this chapter to review 50 years experience of this technology.^^ Ethanol precipitation does not play a role for purification of monoclonal or recombinant antibodies. Protein fractionation on the basis of solubility in aqueous salt buffers or organic solvents is a well-accepted and documented method for pre-purification of proteins. The major drawback of precipitation is the aggregation of proteins during the process. As a result, bioactivity is frequently lost.^^ Another disadvantage is the handling of wastes, especially when dealing with ammonium sulfate. i. Ammonium Sulfate Precipitation Ammonium sulfate precipitation follows the principle that at high concentrations the protein solubility decreases. This behavior is called salting out, is common to all nonelectrolyte solutes, and appears to be due to "dehydration" of the protein molecule.

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Isolation of monoclonal antibodies from supernatant by ammonium sulfate precipitation is widely used. A short protocol using saturated (NH4)2S04 solutions is given by Jonak/^ The disadvantage of this procedure is the at least 1:1 dilution. The addition of solid ammonium sulfate is preferred, hov^ever, one must take into consideration that it low^ers the pH of the supernatant. Therefore a good buffered supernatant must be used. The combination of ammonium sulfate precipitation with hydrophobic interaction chromatography (HIC) or thiophilic adsorption is an elegant way to shortcut purification processes (see Sections V. D and V. F.V). The two steps are connected together without any intermediate treatment. The purification sequence consists of following phases: clarification, ammonium sulfate precipitation, washing of the precipitate, redissolution of IgG, clarification, and hydrophobic interaction chromatography or thiophillic adsorption. IgG binds to HIC matrices at very high ammonium sulfate concentrations ( > 50% saturation), where IgG already precipitates. A repetitive injection of IgG dissolved in a low salt buffer followed by a solution containing high concentrations of ammonium sulfate circumvents this problem. Direct HIC purification of monoclonal antibodies from a serum containing supernatant is not recommended, since albumin binds very strongly to hydrophobic matrices and reduces the binding capacity for IgG. ii. Caprylic Acid

Caprylic acid (also called octanoic acid) can also be used for the precipitation of antibodies from serum, ascites, or hybridoma culture supernatant.^^ ~^^ In terms of antibody purity, precipitation with caprylic acid is significantly less efficient than the chromatographic methods. Note also that precipitation with caprylic acid is associated with a reduction in the affinity properties of some antibodies and is not suitable to purify murine IgA and IgG3. The extraction and purification of serum-derived immunoglobulin fraction in hen egg yolk by the combined treatment of the raw egg yolk with caprylic acid and ammonium sulfate has been reported.^"^ This simple two-step method proved to be rapid, reproducible, and suitable for batch processing of pooled egg yolks. The extraction procedure had no adverse effects on antibody titer. Caprylic acid is, in fact, a natural substance; the concentration of autologous caprylic acid in serum is in the range of 40 ppm.^^ No adverse effect of caprylic acid should be expected because of traces present in a pharmaceutical solution. Caprylic acid has a low toxicity for vertebrates but acts as a bacteriostatic agent. iii. Organic Solvents

Cohn's ethanol precipitation is still used for blood protein fractionation.^^ The advantage of the method is the good recovery and the high efficiency in virus inactivation. The greatest disadvantage of the method is the necessity to cool the mixture below the freezing point of water to protect the proteins from inactivation. The whole precipitation procedure is very sensitive to changes in pH and ionic strength.

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Polyethylenglycol precipitation is a very gentle purification procedure. The precipitation principles are related to ethanol fractionation. PEG is often considered as a polymerized organic solvent. Neoh et a\.^^ described PEG precipitation for monoclonal antibodies from mouse ascites fluid. These conditions could also be applied to cell culture supernatants produced from hybridoma grow^th in a dialysis reactor.^^ A short protocol for monoclonal antibodies (concentrations between 2 and 4 mg/mL) is as follow^s: polyethylenglycol 6000 (PEG) is dissolved to a concentration of 30% (w^/v); this solution is added to the cell culture supernatant up to a final concentration of 15%, gently mixed, incubated at least overnight at lov^ temperature, and centrifuged. The sediment is v^ashed v^ith a 15% (v^/v) aqueous solution of PEG 6000 and then the precipitate containing immunoglobulins is redissolved in a physiological buffer. To this antibody solution an additional ammonium sulfate precipitation can also be added.^^ The removal of PEG from the precipitated protein may causes problems in ultrafiltration and gel filtration. iv. Isoelectric Precipitation

Isoelectric precipitation and acid precipitation are also used to separate antibodies. Isoelectric precipitation (also called euglobulin precipitation) uses the solubility properties of a protein near its isoelectric point.^^ When a concentrated protein solution in a low^ ionic strength buffer is titrated to its isoelectric point, it precipitates very slov^ly. Table 4 shoves a balance of a process for purification of murine monoclonal IgM antibodies.'^^ The success of precipitation can be follov^ed by the cumulative IgM content in the centrifugate and the sediment. The procedure is very gentle, but very sensitive to environmental conditions.

Y. PURIFICATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY Liquid chromatography is very often the method of choice for adsorption, separation, and purification of antibodies. Chromatographic separations are based on the differential adsorption and migration speed of components of a protein mixture through a column filled v^ith small particles called chromatographic sorbent or solid phase."^^ At the level of adsorption, solid phases are TABLE 4 Yields and Purification Factors during Purification of Monoclonal IgM Purification step

IgM ( m g ml -

Culture supernatant Ultrafiltration Precipitation Gel chromatography

0.039 15.5 43.84 4.7

I)

IgM / Protein (%)

Kp

K^

1.42 9.8 95.3 99.0

1 6.8 66.8

1 398 1124 115

(i^.S

Yield (%)

100 95 (^(,.S

40.3

The starting volume is 150 liters hybridoma culture supernatant; Kp and K^ are the purification factors related to protein or volume respectively according to Steindl et al/^

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

557

mostly used in packed bed, however, they can be used in batch as well as in fludized bed mode. The literature on chromatographic separation of proteins is abundant and describes a variety of theoretical as well as practical aspects for analytical purposes and for preparative applications. It is beyond the scope of this chapter to give details of the technology for the separation of antibodies. Instead, first some basis is presented and then how liquid chromatography is usable for the adsorption, separation, and purification of antibodies is explained. Method development phases such as, for instance, resin screening, optimization of running conditions, flow sheeting, operational parameters for productivity optimization, and the choice of system components, will not be detailed here. For general reviews, see Refs. 71 and 72. All individual methodologies described in the following, such as ion exchange, hydrophobic interaction, and affinity chromatography, are potentially usable in packed-bed mode as well as in fluid-bed mode if the solid phase sorbent meets the specific requirements of density and particle size. The only exception today is gel filtration, which requires a large number of plates for an acceptable resolution that the fluid bed cannot guarantee.

A. Packed Beds

Packed beds are constituted of chromatographic columns filled with sofid phase media which are sedimented inside the column tube over the bottom filter. The column tube is then closed with an upper adaptor and are generally used downward. The solid phase packed between the bottom filter and the upper piston does not move, while the fiquid phase composed of aqueous buffers or solutions of proteins is pushed across the column bed. During this passage the proteins come directly into contact with the solid phase, with which they interact. Difference in migration speed of the proteins constituting the mixture being separated depends on the strength of their interaction with the soUd phase and on the partition between the particles (the solid phase) and the liquid (the mobile phase). This distribution depends on various interrelated physicochemical factors that are involved in the interaction of the solute molecules with the particles and the solvent (see later). Preparative liquid chromatography (in opposition to analytical chromatography) introduces specific requirements, which are mostly related to the separation selectivity and the loadability of a sofid phase rather than the separation efficiency or peak resolution. While analytical chromatography separations may use complex profiles of elution gradients, in most of the cases, preparative chromatography utilizes linear or step gradients or a combination of both. A large variety of liquid chromatographic separation techniques have been described to date, and it can be said that practically all of them have been tried for the separation or purification of antibodies. Only liquid chromatographic separation involving organic solvents is not usable for the separation of large proteins such as immunoglobulins.

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Although in a number of cases chromatography is used in packed-bed mode, there are an increasing number of examples of the use of solid phases in fluidized-bed mode. This does not change the adsorption phenomenon based on the complementarity of the solute for the solid phase. Fluidized-bed columns are essentially used to resolve specific problems related to the feedstock, as detailed later. B. Fluidized Beds Fludized-bed or expanded-bed processes have been described for the separation of biologicals from crude feedstocks^^"^^ and they offer significant advantages over packed beds in specific cases. Contrary to classical packed beds, fluidization of beads provides a practical option to process very crude material containing particles in suspension such as protein aggregates, cells, or cell debris. In this separation mode, microbeads are lifted inside a column by an upv^ard liquid stream generated by buffers and sample solutions. The particles leave larger empty zones betv^een beads w^here the feedstock and particles in suspension pass through. Important criteria in the design of a fluidized-bed process are numerous and the most important are sorbent bead size and density, ligand accessibility for a given residence time, and stability of the solid phase. Upv^ard speed is also critical and, w^ith beads of a given density and particle size, is the result of a compromise betw^een the maximum loading speed and the bed expansion factor. Theoretical values of the terminal velocity (u^) of the beads can be calculated by the Stokes equation:

u, =

dJiPp -

Pi)'g

(4)

w^here p^ is the particle density, p/ is the liquid density, and /x, is the viscosity. The expansion of a bed can be described by the Richard-Zaki equation^^: (5) w^here e is the bed voidage, and n is an empirical exponent. The exponent has been found in the range of 3-5 for Sepharose Fast Flov^.*'^^ The size and the geometrical shape of the sorbent beads influence the dynamic fluid bed performance to a significant extent.^^ Considerations about fluid beds v^ith small particles of high density to improve the capture of large macromolecules at high speed have been extensively described.^^"^^ This technology has been described for the capture of monoclonal antibodies from crude feedstocks containing cells using dense solid phases. In a first example, soluble antibodies from mammalian * Sepharose is a trademark of Amersham Pharacia Biotech, Uppsala, Sweden.

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559

cell culture broth were adsorbed on a cation exchange resin/"^ Adsorption was performed upward with a device to minimize backmixing and to create a multiplate adsorption process. After an extensive washing phase to eliminate cells and cell debris, elution was performed in packed-bed mode with a reversed flow. Recovery of antibodies was 7 0 - 8 5 % , and antibody concentration was 39 times higher than in the broth. Purification factor was about 7. Most generally during elution, the liquid flow is reversed and the resin bed is therefore packed. In contrast to conventional complex initial feedstock treatments, fluidized-bed processes combine clarification, expressed product specific capture, and concentration into a single step. Residence time distribution analysis showed a small degree of axial dispersion and the generation of a few dozen theoretical plates that are enough for a good efficiency of the capture step. The efficiency of the separation is, however, dependent on the particle size of the solid phase material. At the large industrial scale the reverse flow step for elution is, however, a relatively heavy operation. It first requires one to stop the flow and wait for solid phase settlement and then to adjust the plunger on the top of packed bed. Additionally, when initiating another step the bed must be reexpanded again, requiring some time. Difficulties are related to the expansion of resins after several cycles; they form aggregates and time needed for the perfect bed expansion equilibrium becomes longer. Affinity capture in fluid-bed mode was also described. This was accompUshed using dense sorbents carrying protein A.^^ Untreated effluents from a continuous hybridoma culture was injected into a fluidized bed containing the affinity sorbent. After clarification, IgGla antibodies were desorbed by decreasing the pH to 4. The binding capacity of the resin under normal conditions was about 14 mg of antibodies per milliliter; a 50-fold concentration effect was obtained with good purity of antibodies. It has been reported that with protein A resins designed for fluidized-bed utilization all the processes for antibody capture from recombinant CHO cells were performed in expanded-bed mode.^^ Equilibration, capture, washing, elution, and regeneration were performed without changing the flow direction. This saved a significant amount of time without greatly compromising the dilution of the collected IgG fraction. Yields compared to packed beds, however, expanded-bed technology provided antibodies with a larger amount of host cell contaminant proteins (four fold) and of DNA material (20-fold). Although most of the principles of antibody capture in packed bed mode are applicable to fluidized-bed technology, today applications are hindered by the very limited availability of sorbents specifically designed for fluidized beds. For instance, the potential applicability of a protein A affinity capture in a fluid bed seems very attractive and may become a useful operation with appropriate dense sofid phases. Collected fractions rich in antibody obtained at the issue of a capture step in fluidized-bed mode can be further repurified or polished by other types of packed-bed chromatography, as described in Section V.I.

560

EGISTO BOSCHETTI AND ALOIS JUNGBAUER

C. Role of Ion-Exchange Chromatography i. General Principles Ion exchange is the most widely used chromatographic method at the large scale and is extensively applied to protein separation. Its separation efficiency depends on a number of factors which have been studied in depth at theoretical and practical levels.^^'^^ Ion-exchangers are composed of a solid porous structure carrying functional groups, that are charged and vary in sign. A wide variety of functional groups have been described. Resins for different applications. In different particle sizes, and with different ligand densities and pore sizes are easily available.^"^ Ion-exchangers are classified into four categories according to their electrical charge and the shape of the titration curve; weak and strong cation and weak and strong anion exchangers. Concepts and mechanisms of action of modern ion-exchangers have been extensively reviewed.^^'^^ For isocratic conditions, Kopaciewicz et a\}'^ developed a protein retention model for ion-exchange chromatography based on an earlier treatment of polyelectrolyte retention. Here fe', the relative retention (or capacity factor), is defined as k' = -

^

(6)

where tj^ is the retention time and t^ is the retention time of an unretained solute. The influence of mobile phase modifier on k' can also be approximated by k'=al-^

(7)

which can be written in logarithmic form as log)^' = blog{l/I)

+loga

(8)

where / is the mobile phase modifier concentration, and a and b are constants. For the monovalent counter ion NaCl, the authors found the following relationship:

where z reflects the number of charges interacting with the sorbent, and K^ reflects the electroneutrality conditions. Note that z and K^ must be determined experimentally. The log of the k' value is plotted versus the log of the reciprocal mobile phase modifier concentration. The slope of this graph represents z. The intercept K^ which is defined as K, = K{iye

(10)

561

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

where K is the equiUbrium reaction constant of the ion-exchange process, and 6 is the phase ratio defined as

e

1 - 6

(11)

where e is the void fraction the column. The dependence of the distribution coefficient of the protein on an ion-exchanger with regard to protein and salt concentration [ X ( C ^ , J)] can be empirically described by the following equation assuming a Langmuir-type adsorption behavior for the protein:

K{C„,I)

=

[i + ii/b)Y-ii

+ d-cj'

(12)

where K(C^I) is the distribution coefficient depending on salt and protein: C^ and I are the protein and salt concentrations, respectively; and a,fc,c, and d are empirical parameters which have different meaning than in Eq. (8) Equation (12) shows that the distribution coefficient is inversely proportional to the salt concentration ( / ) to the power of c. A plot of the distribution coefficient of a model protein in equilibrium with an anion-exchanger (QCeramic HyperD*) is shown in Fig. 7. Although the stoichiometric displacement model does not describe the physical situation rigorously enough, it has been widely used and corrected for some shortcomings. Whitley et al.^^'^^ have corrected the model since not all charges are accessible for the protein. They have introduced a correction *HyperD is a trademark of BioSepra-Life Technologies Inc., Rockville, MD.

o^-.*-^'-^' 1 0 ^

r

o^^'-^"'^ \P«

F I G U R E 7 Plot of distribution coefficient (K) versus feed concentration (C) and salt concentration of bovine serum albumin (/) on Q-HyperD. Data have been fitted according t o Equation (10).

562

EGISTO BOSCHETTI AND ALOIS JUNGBAUER

term compensating for this effect. Gallant et al.^^ have introduced a correction term that compensates for the shielding of charges. Proteins, when adsorbed onto an ion-exchanger, may shield charges, and these charges are not available for the adsorption of another protein. Both mentioned correction factors even encounter for shielding and accessibility. The beauty of the stoichiometric displacement model is that log k' can be plotted against the salt concentration of the running buffer (see Eq. 9). A rough estimate can be extracted for the number of interacting charged groups on the protein. Plots of log k' versus I have been frequently reported, including those for immunoglobulins. For the adsorption of antibodies, virtually all ion-exchangers compatible w^ith proteins can be used. Anion-exchangers have been used extensively for the isolation of polyclonal IgG from human plasma and from that of different mammals in a single step with a high degree of purity in a single pass, as shown in Fig. 8. They have also been used to isolate isoforms of monoclonal IgG after optimization of adsorption and elution conditions to fit with isoelectric point properties of the antibody.^^ The key parameter for the development of successful separation conditions is the isoelectric point. Antibodies are actually very diverse in their isoelectric point that can be between 4 and 9. An antibody should adsorb on an anion exchanger at pH value above its pi and to a cation exchanger below its pi. This rule is, however, valid only at low conductivity conditions, because the presence of ions in the running buffer prevents adsorption. Nevertheless, the net charge of an antibody at a given pH does not always reflect its ability to bind to an ion-exchanger. Negative and positive charges are not randomly distributed at the surface of a protein. They may be

F I G U R E 8 Separation of rabbit polyclonal antibodies by ion-exchange chromatography on DEAE Trisacryl M. Column dimensions: 16 mm i.d. X 100 mm; initial buffer 50 m M T r i s - H C I , 0.035 M sodium chloride, pH 8.8; load: 5 mL of rabbit serum previously precipitated with ammonium sulfate at 50% saturation and redissolved in column buffer; flow rate: 50 m L / h r ; elution of adsorbed protein performed using I M sodium chloride solution in the initial Tris buffer. The first peak represents IgG; the second peak is composed of all other serum proteins precipitated by ammonium sulfate. The straight line is absorbance at 280 nm, and the broken line represents the variation of ionic strength of the buffer. The purity of IgG estimated by gel electrophoresis was over 98% and the calculated yield was over 90%.

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

563

clustered in certain domains and then they may bind in an oriented manner. This effect may influence the expected behavior. Sufficient adsorption of antibodies on cation-exchange resins is generally reached in running buffers with low ionic strength, which is equivalent to a conductivity of 10 mS/cm or lower at a pH between 4 and 6. At a pH above 8 and low ionic strength, sufficient adsorption is reached for an anionexchanger. Desorption at the laboratory scale is effected by a linear sodium chloride gradient, a pH gradient, or both. The peak position in the linear gradient elution can be used to determine the required salt concentration for stepwise elution, which is preferred at preparative pilot and industrial scales. Ion-exchange chromatography can be applied to virtually all monoclonal antibody separation from different classes, species, and feedstocks, however, in each case, adsorption and elution conditions must be empirically determined. Until now it has not been possible to predict the adsorption behavior in ion-exchangers from the amino acid sequence. An estimate can be obtained for the number of charged groups at a certain pH. Limiting cases are monoclonal antibodies with isoelectric points that require an extreme pH for adsorption or for elution, that may contribute to the denaturation of their biological activity. A pH higher than 9 or lower than 4 is generally to be avoided. ii. Capture of Antibodies by Ion-Exchangers

Feedstocks containing monoclonal antibodies are physiological liquids characterized by a pH close to neutrality and a conductivity above 14 mS/cm. These properties of the crude feed stock are not compatible with a direct adsorption on ion-exchangers, therefore the ionic strength must be lowered to a conductivity of 10 mS/cm or below and the pH must be adjusted. In practice, the actual salt concentration for adsorption must be determined experimentally; it depends on the net charge of the antibody and the charge density of the ion-exchanger. To this end, most of the time the feedstock is diluted with appropriate buffered solutions. A diafiltration followed by a concentration step, whenever appropriate, are additional operations to adjust the properties of the feedstock to be suited for a proper adsorption on an ion-exchange resin.^^ The dilution of crude feedstock can, however, be very critical when the antibody is present in low amount, it increases the loading volume of the column and consequently the loading time; it also decreases the ability of the column to capture effectively the antibody due to unfavorable conditions for a proper ion exchange interaction. To circumvent this inconvenience, special cation-exchangers with high charge densities have been developed especially for antibody capture to avoid dilution of crude feed stocks. Representatives of highly substituted ion exchangers are, CM HyperD and Sepharose XL. They carry 300-400 /xmol of carboxyl groups per miUiliter of packed resin. With these resins, specific studies^^ demonstrated the possibility to adsorb efficiently antibodies directly from crude feed stream with conductivity that can be as high as 22 mS/cm (Fig. 9).

564

EGISTO BOSCHETTI AND ALOIS JUNGBAUER

F I G U R E 9 Interdependence of ionic strength (I.S.) and pH for the adsorption of hunnanized mouse monoclonal antibodies on a highly substituted cation exchanger CM HyperD F. Open circles represent the behavior of lgG2; close circles are IgG I. Adsorption essentially occurs when the pH is below 4.7 with ionic strengths close or above physiological buffers ( 1 2 - 2 5 mS/cm).

The binding capacities of such sorbents are higher than 60 m g / m L in physiological conditions. The pH for adsorption, however, must be adjusted according to the individual property of the antibody, but pH should be aWays in the range betw^een 4.2 and 4.8. This conditioning procedure may cause slight precipitation of proteins that can be avoided by titrating the crude feedstock with concentrated citric acid. Phosphoric acid, lactic acid, acetic acid, hydrochloric acid, and acetic acid may not be recommended when proteins precipitate. The pH adjustment must be done in the cold and the conditioned feedstock must be stored at 10°-12°C. Figure 10 represents a capture example of monoclonal IgG 2b from a cell culture supematant on CM HyperD at a pH of 4.7 and conductivity of 22 mS/cm. The binding capacity of highly substituted cation exchangers can be very high resulting into relatively large elution volumes. The elution volume naturally correlates with column saturation, as shown in Fig. 11. By selective modification of desorption conditions, the elution volume can be modulated. Buffer conditions must be found that enable a simultaneous change of salt concentration and pFl value in the elution buffer. When combining a salt gradient with a pH gradient in such a way that the antibody changes the sign of its net charge, the elution volume can be reduced by a factor of 2. Reduced elution volume is also observed with other buffers. Examples are shown in Table 5. Borate buffers must be cautiously used, since they form complexes with polyols, which are present in immunoglobulins at the level of the sugar moieties. The choice of the elution buffer is an interesting method to consider to achieve a good compatibility with the following chromatographic operation (see Section V.I) and biocompatibility with proteins. The purity of antibodies that can be reached by ion-exchange chromatography in a single pass is 4 0 - 9 8 % . This depends on the choice of the ion-exchanger and on the

565

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

F I G U R E 10 Direct capture of an lgG2b monoclonal antibody by cation exchange chromatography on CM HyperD F. Column: 10 mm i.d.X 100 mm; binding buffer. 50 m M sodium acetate pH 4.5; sample: 200 mL of protein free-cell culture supernatant adjusted at pH 4.5 by addition of 2 M citric acid; final ionic strength of the sample was 22 mS / cm. Elution (first arrow from the left) was obtained by raising the ionic strength with sodium chloride up to I M in the initial buffer. Regeneration was performed with 0.5 M sodium hydroxide (second arrow from the left). Flow rate was 300 cm / hr. The insert represents H P L C - G P C analysis of the initial sample (a), of the flowthrough (b), and of the eluted antibodies (c). The purity of collected antibody was estimated above 95%.

nature of feedstock carrying protein impurities. For instance, Mono Q and MonoS* column resins have been widely used for purification of antibodies from cell culture supernatants and ascites fluid.^"^"^^^ Usually they yield purities between 70 and 90% have been reported. Often these ion-exchangers have been connected to a gel filtration polishing columns. M o n o Q and Mono S resins are used for small preparative research purposes and have not been appHed for the purification of clinical grade material. In spite of the very small particles of these resins (10 /xm), their extremely narrow bead size distribution yields column pressures within acceptable ranges ( < 20 bar for *Mono Q and Mono S are trademarks of Amersham Pharmacia Biotech, Uppsala, Sweden.

4

1

F I G U R E I I IgG elution volume from a cation-exchange column as a function of the column saturation; load was (a) 10% of column saturation or 6 mg per milliliter of resin, (b) 20% saturation, (c) 50% saturation, and (d) 100% saturation or 60 mg per milliliter of resin. As resin CM HyperD F was used, the loading conditions and elutions were as described in Fig. 10.

566

EGISTO BOSCHETTI AND ALOIS JUNGBAUER

^ ^ H j TABLE 5 Effect of Buffer Composition on the Elution Volume of IgG Fraction from C M HyperD F Volume needed for elution (CV) (depending on flow rate)

Buffer 50 m M acetate, 1 M NaCl, pH 4.5

7-9

200 m M borate, pH 8.6

14-16

200 m M Tris-HCl, pH 8.6

10-12

200 m M Tris-HCl, 1 M NaCl, pH 8.6 155 m M phosphate, pH 8.6

3-4 6-8

0.8 M phosphate, pH 7 50 m M phosphate, 0.8 M (NH4)2S04, pH 7

2-3 5-6

Compatibilities columns Needs pH a n d / o r ionic strength adjustment, gel filtration Anion-exchangers, HCIC, IMAC, Protein A, gel filtration Anion-exchangers, HCIC, IMAC, Protein A, gel filtration HCIC, immunoaffinity Hydroxyapatite, anion exchange, dye affinity Hydroxyapatite after dilution Hydrophobic interaction, thiophilic

columns of 10 cm in length). This material is resistant to relatively harsh chemical conditions, which enables efficient regeneration of fouled columns. An example is the treatment with saw-tooth gradients made from trifluoracetic acid and acetonitrile. These treatments are occasionally necessary since a known drawback of these resins is their relative high hydrophobicity, which leads to lower yield compared to the natural and synthetic hydrogel-based supports. Ion-exchange chromatography can be used as a capture step at the initial stage of antibody separation and can then be followed be one or two additional chromatography steps to remove other protein impurities. Note that proteolysis in conjunction with ion-exchangers may be used in special cases. The pH used with anion exchangers (typically around 8) may induce the activation of proteases such as trypsin-like proteases as well as plasmin and kallikrein when traces are present (these come from animal sera). In this case DEAE and Q, ion-exchangers should therefore be avoided as the first separation chromatographic column. D. Role of Hydrophobic interaction Chromatography i. General Theoretical Considerations Hydrophobic interaction chromatography (HIC) is based on the tendency of apolar molecules to associate in aqueous solutions. The association of apolar groups is characterized by a high entropy contribution to the free energy of the whole aggregation; the entalpy contribution, however, is low or even negative. Hydrophobic associations of molecules are attributed to the mobilization of ordered water molecules structurally organized around the apolar molecule and apolar groups of sorbents. In the case of proteins and especially antibodies, HIC acts through molecular association involving two hydrophobic moieties in the presence of

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

567

high concentrations of salts of strong lyotropic effect. This effect is at the basis of the interaction on hydrophilic resins that have clusters of hydrophobic sites. Several mechanistic retention modes in isocratic conditions are described. The effect of salt concentration on HIC at sufficient high ionic strength (I) can be described as logife' =log)^'o + A^

(13)

where A is an empirical parameter that is related to the retention strength and is similar to the salting-out constant,^^"* and k' and k^ are the capacity factors of a protein (relative retention time) at a particular salt concentration (I) and at zero salt concentration, respectively. Staby and MoUerup^^^ pubHshed a more rigorous model describing the retention of a protein and the salt concentration in the mobile phase. The capacity factor k' or the relative retention can be further defined as k' = (tj, - tj/t^

= tjt^

= njn^

(14)

where t^ is the retention time of a solute in the stationary phase (t^ ^ tj^ — f^), and n^ and n^ denote the number of moles of solute in the stationary and mobile phases, respectively. The activity coefficient y, which accounts for the deviation from ideality, is defined as the ratio of the activity and the mole fraction x. y = a/x

(15)

If the phase ratio in the column is constant, then v is the molar volume of the phase. At equilibrium, the chemical potential of the solute on the stationary phase and mobile phase are equal. The capacity factor can thus be calculated as ym

yS

jjtn

lnfe'=lnn + l n — - l n - + l n — 7o To ^0

(16)

The first term on the right side of this equation is the capacity factor, at zero ionic strength. The ratio of activity coefficients in the second term is the ratio of the solute activity coefficients at finite ionic strength over the solute activity coefficient at zero ionic strength. The activity coefficients of the mobile phase modifier are modeled by the Deye-Hiickel theory and can be approximated by 7^

1.5

{I

where / is the ionic strength, and a is the relationship between the mole fraction and the activity coefficient. The activity coefficient of the active sites

568

EGISTO BOSCHETTI AND ALOIS JUNGBAUER

in the stationary phase have been described by an empirical equation:

I n — = W - cP

(18)

70

where b and c are empirical parameters. The latter model predicts a minimum in the retention time that is different from k^. That means, that it is not necessary to reduce the ionic strength in the elution buffer to zero. The capacity factor at zero ionic strength is assumed to be independent from the active group of the supporting material, and ^Q i^ assumed to be only a function of the mobile phase pH. ii. Optimization and Selected Examples

Ammonium sulfate is frequently used as lyotropic salt to promote hydrophobic interaction. It is also known that in the presence of this salt, antibodies tend to precipitate (see Section IV.D.i) and share their water content with the solid phase with consequent association. This is a dual mechanism where each phenomenon contributes for the adsorption of the antibody on an hydrophobic resin and has been frequently used for purification of antibodies from different sources.^^^"^^^ Antibodies are proteins well adapted for hydrophobic interaction chromatography because they have specific conserved zones with high density of hydrophobic aminoacids (see Fig. 1). It has been shown that IgG have 18 highly conserved hydrophobic sites, most of them located close to C y 2 and CyS domains, and are rich in valine, leucine, isoleucine, and alanine.^^^'^^^ These sites are also contributing to the interaction of antibodies with protein A from S. aureus (see Section V.F). Solid-phase matrices used for hydrophobic interaction chromatography are composed of hydrophilic structures such as agarose, dextran, and hydrophilic polymers. Hydrophobic sites such as methyl, butyl, octyl, dodecyl, and phenyl groups are chemically attached by means of activation reactions. The degree of hydrophobicity results not only from the type of ligand, but also from its density on the hydrophilic matrix.^^^'^^^ Weakly hydrophobic sorbents require a very high concentration of salts to have the adsorption of antibody happening, while lower salt concentrations are required with strong hydrophobic sorbents. The recovery of antibodies is, however, proportional to the decreased level of hydrophobicity of the solid phase. With strong hydrophobic resins the reduction of lyophilic salts is not always adequate to recover antibodies: the addition of nondenaturing organic solvents, such as ethanol or ethylene glycol, may be required. The selection of a hydrophobic sorbent implies we must perform an individual screening operation since the type of hydrophobic ligand is not a reliable indicator for hydrophobicity of an HIC sorbent. Rational applicability of HIC is actually relatively difficult. Contrary to ion-exchange interaction where the net change of a protein can be calculated, in HIC interaction, there are no defined rules for how to measure the hydrophobicity degree of a protein.

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

I^B

569

TABLE 6 Lyotropic Effect of Ions on Hydrophobic Association between Molecules N a t u r e of ions

High lyotropic effect

Low lyotropic effect

Cations Anions

P O | - ; S O | - ; C O Q - ; Cl" N H ^ ^ ; Rb+; Na+; Cs+

SCN"; CIO4-; NOJ; Br" Ba^ + ; Ca^ + ; Mg^ + ; Li+

Hydrophobicity is a very relative parameter that induces problems not only at the level of the determination of optimized operational conditions, but also at the level of the consistency of sohd phase sorbents. In spite of the frequent use of ammonium sulfate, other hydrophobic association promoting salts can be useful at neutral pH. In that v^ay, it is possible to select the degree of lyotropic property adapted to the antibody to purify (see Table 6). When the volume of the feedstock exceeds 10% of the total column volume, it is advisable to add lyotropic salts to a concentration similar to the ones used in the running buffer. Diafiltration of the feedstock against the adsorption buffer before loading the column is another possibility, but this consumes high amounts of sulfate buffers. This buffer is inexpensive, but the disposal is difficult at large scale. The amount and the type of salts must be chosen in such a v^ay to avoid IgG precipitation. When ammonium sulfate is used at a concentration higher than 0.5 M a precipitation may occur, leading to a substantial decrease in yield, and even preventing chromatographic separation. Elution of adsorbed antibodies is achieved by low^ering salt concentration with linear gradients of ammonium sulfate at neutral pH or with ethylene glycol. Hydrophobic interaction chromatography on a phenyl resin was used as a single step to purify a bispecific monoclonal antibodies from continuous hollow fiber hybrid-hybridoma cell culture.^^"^ Adsorption was performed in the presence of 1 M ammonium sulfate at pH 7 and IgG elution by lowering the concentration of the salt to zero. Monoclonal antibodies were desorbed in pure state when the concentration of ammonium sulfate was close to 0.02 M. Binding capacity reported was of several mg per ml of resin; high purity of monoclonal IgG2a was demonstrated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) in reducing conditions. Figure 12 presents an example of separation of antibody by hydrophobic interaction chromatography. The binding capacity of hydrophobic interaction chromatography sorbents is in the range of 5-20 mg of antibody per milliliter of resin depending on chosen conditions; mass balance depends on conditions of elution but can be as high as 90%. Biological activity recovery can be, however, a problem associated with the risk of denaturation with excessively hydrophobic sorbents which induce conformational changes.^^^'^^^ Effector functions of antibodies located in Fc fragment supporting most of hydrophobic sites, may be somewhat modified.

570

EGISTO BOSCHETTI AND ALOIS JUNGBAUER

Time (mjn) F I G U R E 12 Purification of a murine monoclonal antibody against IgE form hybridoma culture supernatant on a phenyl Sepharose HP. The feedstock has been added with ammonium sulfate to a final concentration of 0.5 M. The sample volume was 130 m l . The column was operated at 100 cm / hr. As equilibrium buffer a 20 m M potassium phosphate buffer, pH 7.0, supplemented with 0.5 N\ ammonium sulfate was used. Elution was performed by a linear gradient with a 20 m M potassium phosphate buffer, pH 7.0. The gradient volume was equal to 10 column volumes.

E. Role of Hydroxyapatite Chromatography Hydroxyapatite is a well-known and useful mineral sorbent for protein purification.^^^ It is composed of a complex hydrated form of calcium phosphate. It is comprosed of a positively charged pair of calcium ions and negatively charged clusters associated with phosphate sites. Interaction with proteins happens on the surface of crystals of calcium phosphate resulting into a relatively complex phenomenon. Cationic groups from the protein structure interact weakly with phosphate clusters and carboxyl sites adsorb quite tightly on calcium ions. Although interactions appear mostly ionic, they are not based on the classical ion-exchange effect only, since binding capacity for acidic proteins diminishes when pH increases and, for proteins with low isoelectric points, the presence of sodium chloride does not induce desorp118 tion Hydroxyapatite has been described in a number of protocols for the separation of polyclonal and monoclonal antibodies.^^^'^^^ Binding of antibodies is stronger under slightly acidic pH, due to the increase of positive charge on the protein structure; however, the pH is always close to neutrality. Phosphate buffers are generally used for the chromatography; their initial concentration is close to 1 m M and the pH, between 6.5 and 6,9^ remains constant. Elution of antibodies is obtained by phosphates, citrates, and fluorides. The most common way to desorb is, in fact, to apply a gradient of phosphate buffer at constant pH. When an antibody with a high isoelectric point is applied, the resolution power of phosphate may not be sufficient enough to separate albumin from the antibody.^^^ It is interesting to know that while the interaction between acidic proteins and hydroxyapatite is not sensitive to sodium chloride gradients, cationic proteins such as most of the antibodies, are eluted by a salt gradient. This is an advantage for their purification because all acidic contaminants

571

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

such as DNA, endotoxins and other acidic proteins will remain adsorbed on the resin. Hydroxyapatite has been used for the separation of polyclonal as well as monoclonal antibodies in a variety of cases, alone and in associations with other resins.^^^ An example of monoclonal separation of a IgGl monoclonal antibody is represented in Fig. 13. The binding capacity of hydroxyapatite for antibodies of the IgG and IgM classes is relatively low and in the range of few milligrams per milliliter of resin. Antibody purity from ascite fluids in a single pass is typically in the range of 55 to 75%. Hydroxyapatite columns are mostly recommended as polishing steps after a capture performed for instance with a cation-exchange resin (see Section V.I.VIII). The most striking advantage of hydroxyapatite chromatography is its high selectivity for DNA. Extremely high DNA clearance factors, higher than 6 log steps, have been reported.^^^ Whenever DNA impurities cause problems in antibody purification processes, an additional hydroxyapatite step may be the key for success. Column cleaning is performed with high concentrations of phosphate buffer, with nonionic detergents, urea, and sodium hydroxide to restore the complex structure of calcium phosphate. Three forms of hydroxyapatite sorbents are currently commercially available. The most classical is under crystalline form of variable particle size from few to several hundred of micrometers. This presentation shows generally good sorption capacity, especially when the particle size is small, but is very difficult to use in a packed column because the formation of fines dramati-

[POi"l

*r-

05-

•;-

50

150

250

ml

F I G U R E 13 Separation of mouse IgG I monoclonal antibody from ascitic fluid on a hydroxyapatite column (HA Ultrogel). Column: I.I i.d.X 10 cm height; initial buffer; 10 m M phosphate, pH 6.8; sample 16 mL of ascitic fluid previously filtered; elutions: 50 m M phosphate buffer pH 6.8 (a), 100 m M phosphate buffer, pH 6.8 (b), 200 m M phosphate buffer, pH 6.8 (c). Flow rate 20 cm / hr; regeneration I M sodium hydroxide. Antibodies are collected in buffer b; purity by SDS-PAGE is 83%, yield is 11%.

572

EGISTO BOSCHETTI AND ALOIS JUNGBAUER

cally reduces the flow rate. Preparative columns are not recommended with such a material. Hydroxyapatite is also available under spheroidal shape with better flow properties once packed into a chromatographic column; formation of fines under manipulation and possible collapse when in large columns are, however, technical drawbacks that must be carefully considered. Spheroidally shaped hydroxyapatite resulting from crystal agglomeration is available in different particle sizes. This material can be considered for large-scale processes. Microcrystals of hydroxyapatite trapped within crosslinked agarose beads is the third presentation of commercially available hydroxyapatite. It does not create fines or problems of flow rate once in small and large columns, however, binding capacity is slightly diminished because of the presence of agarose matrix. Hydroxyapatite is not stable in the presence of chelating agents and at pH below 5: Both phenomena contribute to the degradation of the mineral structure and to partial or total dissolution of the crystals. F. Role of Affinity Chromatography I. Theory and Principles of Affinity Chromatography Affinity chromatography, first described by Cuatrecasas et al.^^^ utilizes the ability of a protein or another biopolymer to recognize a natural or synthetic ligand. The affinity chromatography sorbent consists of a porous matrix itself on which a ligand is chemically immobilized directly or by means of a spacer arm. The art of designing a perfect affinity sorbent is based on the selection of the appropriate support, the length and type of spacer, and the appropriate ligand combined with the immobilization chemistry. The support material must provide functional groups for attaching the spacer or the ligand, it must be chemically inert, and it should not contain any ionic or hydrophobic characteristics. The support material and activation principles for the covalent linkage of a ligand were comprehensively reviewed by Clonis,^^^ Narayanan and Crane,^^^ and Carlson et alP'^ Affinity chromatography uses the highest selectivity of the protein ligand interaction. The specificity of this interaction, which is a probabilistic term, can be described by the simple law of mass action; therefore the association of two molecular species is described by: A -h B - ^ AB

(19)

and the increase of the complexes can be written as

4 AB] dt

KA^Wn

(20)

where k^^^ is the association rate constant with the dimension [M~^s~^]. The association rate constant k^^^ indicates how fast the concentration of AB complexes increase when the concentrations of A and B are [ A] and [B].

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

573

Values for k^^^ typically range from 10^ to 10'^ M~^ • s~^ The opposite reaction, the decrease of AB complexes over time, A B ^

A-^B

(21)

= k,,ss[AB]

(22)

can be written as d[AB] dt

where k^^^^ is the dissociation rate constant with the dimension of inverse seconds. The dissociation rate constant k^^^^ indicates the fraction of AB complexes that dissociate per second. Values for k^^^^ typically range from 10"^ to 10~^ s~^, corresponding to half-life of protein complexes from a few minutes to several hours. A protein-ligand complex with a k^^^^ value of 10"^ is very stable. According to the law of mass action the position of the equilibrium is dependent on the concentration and the reaction constants (^aiss? ^ass) ^^^ can be expressed as association equilibrium constant K^: [AB]

k,,,

with the dimension [M~^]. The dissociation equilibrium constant Kj^ is the reciprocal unit of the equilibrium association constant [M[B]

^diss ^ass

with the dimension [ M ] . In affinity chromatography, a high association rate during loading and washing and a high dissociation rate during elution, are required. High dissociation constant rates can be achieved by the addition either of chaotropic agents or deforming agents into the elution buffer or even by selective elution such as EDTA, for Ca^"^ binding proteins. Competition with a free ligand is also a mode to force the protein to go into the mobile phase as for example addition of sugars for the dissociation of lectin-glycorpotein complexes (Section V.F.xii). An excellent ligand for catching the targeted protein does not mean that the ligand will also release the protein under mild conditions. A rule of thumb for the characterization of the ligand and the ligand density is given by Lowe and Dean.^^^ The equation of the equilibrium dissociation constant Kj^ (Eq. 24) can be then extended as ^

[P][L]

^°" ULT

[Po-PL][L,-PL]

~

[ET]

^^^^

where pQ and LQ are the initial concentrations of the proteins and the immobilized ligand respectively, PL is the formed complex. Assuming LQ :»

574

EGISTO BOSCHETTI AND ALOIS JUNGBAUER

PQ, the equation can be simplified to

The chromatographic distribution coefficients K is defined as bound protein-free protein and can be modified to bound protein free protein

I

PL

\

\PQ — PL j

L-0 K^

(27)

The retention of a protein V, = Vo + K-iV,-Vo)

(28)

and the relationship between iigand concentration and affinity can be estimated from an aforementioned equation (Eq. 27). The knowledge of these parameters enables a rough calculation of the elution behavior: Vc

Vo

Lo = 1 + -^

(29)

KD

From Eq. (28) one can estimate the required reduction of the equilibrium constants between the binding and the elution step to obtain a reasonable elution volume. All these equations, however, do not tell anything about the selectivity of the interaction. In affinity chromatography, high selectivity is presumed; this means that only one molecule species binds to the Iigand with high affinity in the presence of a large number and mass of other molecules. The specificity of the affinity chromatography is based on selective biorecognition. The selectivity is a statistical term, defined as the number of molecules that bind in the presence of other molecules. The simplest representation of the adsorption of a protein (C) with a Iigand (L) assuming single component, monovalent association, is represented by Eq. (30) and (32). When the concentrations of the protein in the mobile phase and stationary phase is Q and C^ and the maximum binding capacity C^^^^^ the rate expression is

^

= k^cf'""'^^'^' - k_,C,,

(30)

The equilibrium expression for the dissociation constant is therefore transformed into Eq. (29): J.

_

^-1

_

Cm(Cs,max ~

Q)

,

.

575

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

which is equivalent to the Langmuir type isotherm: (32) KD +

C„

li Kj^ <^ C^, this isotherm reduces to the rectangular or irreversible isotherm, Q :^ Q max-^^"^ For many natural and synthetic systems, the equilibrium dissociation constant is in the range of 10~^ and 10"^^. For the dissociation of strong protein-ligand complexes, destructuring agents such as chaotropic agents and detergents have to be used to facilitate elution within a reasonable elution volume. Harsh desorption conditions is one of the general obstacles of affinity chromatography, since the protein or the ligand may be partially destroyed. A variety of affinity separation techniques have been described for the isolation of immunoglobulins. They are based on biorecognition properties of these molecules and have been designed with the purpose of making a molecular recognition as selective as possible. Although biorecognition between the immobilized ligand and the immunoglobulin should be a highly selective process, the obtained purity of collected antibodies is the result of both molecular recognition and the choice of the most appropriate desorption conditions, that may eliminate impurities that are also coadsorbed with the antibody. Table 7 summarizes the principles of most known affinity related mechanisms applied to antibody separation. ii. Protein A Affinity Chromatography a. General Considerations

The best characterized natural ligand for antibody interaction is protein A (SpA), a cell wall constituent of S, aureus. This ligand gained an increasing importance as a tool in both quantitative and qualitative immunological techniques because of its ability to interact very selectively with immunoglobulins, mainly IgG, from mammalian species.^^^ Due to its high specificity. ^ ^ H T A B L E 7 Ligand Selection for Antibody Adsorption by Affinity Related Mechanisms Properties of antibodies

involved ligand

Type of affinity separation

Fc constant region Kappa chain composition Adsorption of an antigen Induction of antibodies Glycoprotein

Proteins A and G Protein L Antigen Antibody Boronic acid and lectins Chelating agents

Complex with hydrophobic effect Stereospecific complex effect Molecular recognition Immunoaffinity Sugar lectin

Ability to make metal complexes Composed of aromatic aminoacids Complex aminoacid sequences structure

Active dye ligands Thiophilic like ligands

Immobilized metal chelate adsorption Aromatic ionic interactions Hydrophobic charge transfer effect

576

EGISTO BOSCHETTI AND ALOIS JUNGBAUER

SpA rapidly became the IgG separation method of choice after its first publication in early 1970s.^^^ SpA is constituted of a single polypeptide chain with a C-terminus related to a transmembrane domain and five homologus IgG-binding domains of about 6.6 kDa in size.^^^ Purified cell-wall-bound SpA has a markedly extended shape with large hydrodynamic volume. It has a relative molecular mass of 42.000, whereas the molecular mass of extracellular SpA is 41.000; its isoelectric point is as low as 5. The secondary structure of SpA has been estimated to be constituted of 3 1 % a-helix, 13% /3-structure, and 56% random coil. The SpA molecule may be divided into two structurally and functionally different regions: an N-terminally located region consisting of four globular, highly homologus immunoglobulin binding units, and one C-terminally located nonimmunoglobulin binding region of extended shape covalently linked to the cell wall.^^^ Protein A has been chemically attached to soUd porous chromatographic phases such as agarose beads, dextran beads, silica, and controlled porous glass, and has been used for the specific adsorption of immunoglobulins G. The primary binding site of protein A is on the Fc fragment of IgG at the junctures of the CH2 and CH3 domains. Its specificity is very high, but is restricted to a limited number of antibodies, as shown in Table 8. Association of protein A with Fc fragment of IgG affects somewhat the local structure of the antibody destabilizing the structure of carbohydrate moiety with consequent altered susceptibility to proteolytic attack. This interaction can also partially alter antibody effector functions. b. Structure and Function of Immunoglobulin Binding Domain

A large number of staphylococcal and streptococcal strains express on their cell surface proteins that bind mammalian immunoglobulins. It was found at an early date that SpA interacts with human, rabbit and guinea-pig IgG primarily via the Fc part of the immunogloubin. This binding site for SpA has been shown to be located both on the CH2 and CH3 domains on the Fc part of human IgG. Immunoglobulins of 65 different mammalian species so far tested, interacted with SpA in immunodiffusion assays, although quantitative and qualitative differences between species have been noted. The relative affinities between SpA and IgG in different mammalian species have been compared and found to differ more than 1000-fold. Subclass restriction in the bidding of SpA have been reported, namely, that it binds to human IgG4, IgG2, and IgGl but to only a fraction of IgG3, has its counterpart in IgG of many other mammals. These differences in the affinity for SpA can be utilized in separating the different subclasses of IgG. Furthermore, SpA is not highly specific for IgG, as first thought, since fractions of human IgM, IgA, and IgE have been found to interact with SpA. This property of SpA is not restricted to human immunoglobulins, but is true for the most species tested except rabbits, guinea pigs and horses. An additional site for binding of SpA on the immunoglobulin molecules has been identified in the Fab portion. This property can easily be exploited for the separation of Fc fragments after proteolytic breakdown with papain (see Fig. 16 on page 581). This site has been demonstrated to be present in all

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

577

T A B L E 8 Affinity of Protein A for Antibodies from Different Species Animal species

Antibody subclass

Affinity for protein A

Human

IgGl IgG2 IgG3 IgG4 IgA IgM IgGl IgG2a IgGlb IgG3 IgM IgGl IgG2a IgG2b IgG2c IgM IgGl IgG2 IgM IgGl IgG2 IgM IgG IgM IgG IgG IgY

High High Low High Low Low Low High High High No interaction Low No interaction No interaction High No interaction Low High No interaction Low High No interaction High No interaction High High No interaction

Mouse

Rat

Goat

Sheep

Rabbit Pig Dog Chicken

IgG of different species tested, except for rabbit. It is different from the antigen-binding site on the Fab portion and consequently it does not represent an ordinary antigen-antibody reaction. Furthermore, the Fab binding site on the SpA molecule seems to be distinct from the classical Fc binding site. There is evidence that the binding of human IgM, IgA, and IgE to SpA is mediated exclusively via the Fab portion, however, the molecular structure of the SpA-Fab interaction is not completely elucidated. Experiments with fragmented proteins suggest that weak interactions with the Fc parts of IgM and IgA may occur. To locate the precise region responsible for the binding between SpA and IgG different approaches such as nuclear magnetic resonance (NMR) studies,^^^ protein engineering,^"*^ or site specific mutagenesis^"^^ may be employed. As already mentioned, the extracellular part of SpA contains a repetitive structure of five highly homologous Fc-binding domains designated as E, D, A, B, and C, each of them comprises about 60 amino acid residues. The domains are on average about 80% homologus at the amino acid level. The C-terminal part is a cell wall binding domain designated as X, which does not bind to the Fc portion and comprises approximately 180 amino acid residues.

578

EGISTO BOSCHETTI AND ALOIS JUNGBAUER

c. Interaction between SpA-Derived Domains and IgG, IgA, IgM, and F(ab '^2

SpA-derived proteins containing different number and composition of domains have been produced by recombinant methods. The binding properties of intact SpA and some SpA-derived protein fragments to IgG, IgA, IgM, and FCabOi have been analyzed. The results suggest that the binding is affected both by the number of immunoglobulin binding domains and by the composition of the domains. In contrast to intact SpA and derived domain VI, v^hich are natural products from specific strains of S. aureus, Z, ZZ, Z-V, and EB domains are products obtained by recombinant methods. The EB domain consists of two IgG-binding domains, Z is derived from domain B, and Z-V and ZZ are multiply of domain Z (Fig. 14). The binding properties of SpA, VI, Z-V, ZZ, EB, and Z to IgG, IgA, IgM, and FCabOi have been investigated in saturation assays and in competitive inhibition assays. Determination of the affinity constants indicates that the number of IgG-binding domains greatly affects binding strength. The five-domain proteins SpA and Z-V have relatively high binding constants when compared to the shorter proteins. The number of accessible binding sites on the immunoglobulins for the various proteins have been determined. The results show a discrepancy in specificity between SpA and VI on one side and Z-V, ZZ, EB, and Z on the other side. Whereas SpA and VI bind to all immunoglobullins tested, Z-V, ZZ, EB, and Z bind poorly to IgA, IgM, and FCabOi-

SpA

E

r^ n^

B

Tc

X

1

Protdn Engineenng

VI

EB

E

ID

1 A

1 E

B

Duplicadon

ZZ Polymerizatioii

Z-V

z

z

z

z

z

F I G U R E 14 Schematic structures of protein A Fc-binding domains and recombinant fusion proteins. Intact SpA and Z-V contain five IgG binding domains with different compositions. Protein VI contains two intact IgG-binding domains (E and D) and one truncated (A' domain).

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

579

The most characterized nonimmune interaction between protein and immunoglobuHn is the binding of SpA to the Fc portion of the IgG molecule. An additional binding site for SpA on IgG has been demonstrated in the Fab part of the immunoglobulin and it has been suggested that the interaction between SpA and IgG and IgM is mediated via a Fab binding. The relative binding properties of SpA-derived proteins to isolated FCabOi fragments are similar to the binding properties to IgA and IgM. This implies that intact SpA and VI are able to react with the immunoglobulins via the Fab portion in addition to Fc. However, the other SpA-derived proteins bind to Fc, but the occupancy to IgA, and IgM, and F(ab02 is significantly lower. Since SpA and Z-V both contain five IgG-binding regions, the discrepancy cannot be explained by differences in number of IgG-binding domains. Studies on the five separate domains E, D, A, B, and C have shown that all of them can interact with human polyclonal IgG. Domains Z-V, ZZ, and Z, all based on region B of intact SpA, show low binding occupancy to the FCabOi fragment of IgG. This suggests that A, D, a n d / o r E regions contain a structural motif, which is not present in Z, and which is necessary for the Fab binding. This suggestions is supported by the fact that VI, containing D, E, and a truncated A region, shows a binding activity to FCabOi- Since EB binds only poorly to IgA, IgM, or FCabOa this would imply that the addition of domain E to domain B is not sufficient for full Fab binding. Therefore, it has been proposed that domain D possesses that ability of SpA to bind to Fab, since it possesses an interesting amino acid insertion compared to the other protein A domains, and this significant difference between this domain and the others could also be responsible for the interaction with Fab. d. Chromatographic Conditions

Binding of IgG onto protein A resins takes place mostly in physiological conditions, however, the presence of polyethylene glycols, glycine buffers, at high pHs and high ionic strength enhances the interaction. Interaction is complex, but one of dominant contribution is the hydrophobic association. Elution of antibodies from protein A can be accomplished using acidic pH in the region of 2 . 5 - 3 . When the association is performed at alkaline pH and high ionic strength, for instance when adsorbing mouse IgGl, the elution can be obtained by just lowering the ionic strength and pH close to 5-6. Resins carrying protein A as ligand show binding capacities for pure polyclonal human IgG that can reach 30-40 mg/mL. A large variety of resins are available, differences being in their mechanical and chemical stability, the level of leakage and the amount of protein A attached to the matrix. The amount of immobilized protein A per unit volume of resin governs the IgG binding capacity when the pores of the matrix does not hinder the diffusion of antibodies. Figure 15 presents an example of purification of a mouse monoclonal IgG3 antibody from an ascite fluid. Antibody purity reached in a single pass can be as high as 98%, but usually is in the range of 90%. Recovery is also as high as 80-90%.'^^''^^' ^^^' ^"^^ These numbers are obtained in adequate conditions that are not necessarily the same for all IgG subclasses. For example, human and guinea pig IgG

580

EGISTO BOSCHETTI AND ALOIS JUNGBAUER

F I G U R E 15 Separation of a mouse lgG3 monoclonal antibody by affinity chromatography on a column of protein A Ceramic HyperD. 0.7 mL sample of ascitic fluid previously diluted with an equal volume of phosphate buffered physiological saline was directly injected into the chromatographic column (dimensions 4.3 mm i.d. X 100 mm). After washing to eliminate all non adsorbed proteins, IgGB were desorbed by 0.1 M acetic acid (arrow I). The column was finally regenerated using a 0.1 M solution of sodium hydroxide (arrow 2). Final purity of the antibody as attested by capillary electrophoresis (insert) was of about 98%. Insert: Analytical capillary electrophoresis of crude sample (a) and eluted fraction (b).

adsorb directly without dilution (except hIgG3); mouse IgG2a, IgGlb, and igG3 can bind in physiological conditions, but the capacity is improved if ionic strength and pH are increased. Mouse IgGl always requires high pH and high ionic strength for a good interaction. Due to discrimination from species to species, protein A resins can be used to separate monoclonal antibodies from transgenic milk. Autologous antibodies are not adsorbed if the animal is well chosen. For instance, monoclonal humanized IgG expressed in goat milk can be separated. Protein A itself is very stable in a large variety of physiochemical conditions such as alkaline pH, temperature, chaotropic agents, and detergents. If it is properly attached it can be easily cleaned with different solutions including sodium hydroxide and mixtures containing 0.5M acetic acid and 60% ethanol. It is, however, susceptible to proteases. The property of SpA to bind antibodies at Fc domain level allows the separation and purification of Fab fragments. In a first time whole antibodies are purified and, after molecule breakdown by pepsin, Fc fragments are adsorbed on a protein A column while Fab are found in the flowthrough. An example of separation of Fc fragments from Fab is given in Fig. 16. e. Ligand Leakage

Ligand leakage may become a severe problem when SpA is used as an affinity ligand for therapeutic antibodies. Free released ligand, protein-ligand complexes that are present in the collected antibody fraction must be separated by further chromatography procedures, which may reduce yield and add cost. Fliigistaller^"^^ reported that the immobilization chemistry influence the stability of the protein A ligand. Immobilization chemistry for commercial available SpA affinity matrices are cyanogen bromide, triazine.

581

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

igc

50 kO

vvA F I G U R E 16 Affinity separation of Fc fragments on a protein A column; 100 mg of pure human IgG in 10 mL of 0.1 M phosphate buffer, pH 7.5, containing 100 m M cysteine and 2 m M EDTA were first added with 2 mg of pure papain and the mixture incubated for 2 hr at 37°C. Proteolysis reaction was then stopped by addition of I mL of 20 m M iodoacetamide; 3 mL of IgG hydrolyzate were injected into a protein A column (chromatogram on the left) previously equilibrated with a 10 m/Vt Tris - HCI buffer, pH 8. After washing to eliminate all unbound material the elution of Fc containing fraction was performed with 0.1 M acetic acid (arrow). Collected acidic peak was immediately neutralized and analyzed. Left: Gel filtration analysis on Ultrogel AcA 34 of collected fractions: (A) Initial human IgG; (B) flowthrough fraction from protein A column (contains traces of IgG, fragments of a molecular weight close to 50 kD; first main peak and degradation products with a molecular weight below 7 kDa; (C) acid eluted fraction containing Fc fragments (main peak) and other proteins of higher molecular weight. Main peak (Fc) showed a positive reaction with protein A by double diffusion on agarose gel.

aldehyde-Schiff's base formation, and a number of not disclosed chemistries. Peng et al}"^"^ reported findings for proteins, that the most stable binding for immobilization to a matrix was via amine or amide bonds in contrast to cyanogen bromide linking which slowly degrades. This observation was further supported by Fliiglstaller^"^^ who found on different SpA matrices that ligand leakage was lowest for those employing alkalamine or ether-immobilization linkages. Furthermore, activation agents such as cyanogen bromide and triazine are highly toxic and may leach out together with purified antibodies, suggesting that these immobilization chemistries are inappropriate for the purification of therapeutic antibodies. Godfrey et al}"^^ have compared eight different commercial available SpA-matrices. Ligand leakage was in the 10 ppm range, except for one matrix which was of the order of 100 ppm. Testing of leaked SpA is very cumbersome. SpA complexes must be disassociated prior to immunological detection. Quantitative Western blots and ELISA have been used for this purpose. Furthermore, it is very difficult to obtain antibodies which react with the antigen-binding site to SpA.^"^^ The working range of this assay is between 0.5 and 10.0 n g / m L (coefficient of variation less than 5%), with a lower limit of detection of 0.2 ng/mL

582

EGISTO BOSCHETTI AND ALOIS JUNGBAUER

(coefficient of variation less than 10%) in presence of 1 mg immunoglobulin per ml. iii. Protein G Affinity Chromatography a. General Considerations

Another microbial protein well known for the separation of antibodies is protein G.^"^^ It is also composed of a single polypeptide chain with binding domains for IgG. It contains also other domains that diminish the specificity when used as ligand for antibody separation. Recombinant versions of protein G, however, have been prepared and contain only the IgG binding domains. Although the specificity of IgG binding sites is the same for both protein A and protein G, they have distinct aminoacid sequences. Optimum pH for binding is rather acid for protein G and it binds human IgG3 which is not adsorbed on protein A resins. Typically, purity of polyclonal IgG can be as high as 90% in a single pass; in the case of monoclonal antibodies, purity is most currently around 70 to 85%. Recovery is similar to protein A resins except for human polyclonal antibodies. b. /nteractfon between Streptococcal Protein G and IgG

Protein G is a large multidomain cell surface protein of groups. A, G, and C streptococci, which exhibits a broader spectrum of binding to IgG subclasses than protein A. It binds to all four subclasses as well as to a variety of mouse and rat monoclonal antibodies.^"^^ Protein G contains repeats of two or more IgG-binding domains, each comprising 55 residues and at least two albumin binding domains. Recombinant protein G has been depleted from the albumin binding regions as well as from the cell membrane spanning domain and from the anchoring domain. These engineered proteins are used as ligands for affinity chromatography. Like protein A, protein G binds primarily and tightly to the Fc region of IgG, apparently interacting with the same site on the antibody. In addition a weak binding to Fab region has been observed. The crystal structure of a molecular complex between one of the five homologous Fc-binding domains of protein A and a human Fc fragment demonstrated that the binding site for the protein A fragment is located at the junction between the CH2 and CHS domains of Fc. The IgG binding domain of protein A is an all-helical structure with two helices involved in the interaction. Interestingly, protein A and protein G exhibit neither sequence nor structural homology in their IgG-binding domains, although they appear to bind to the same site. A high-resolution structure has been determined for the BI IgG-binding domain of protein G. The structure comprises of four stranded j8-sheet made up of two antiparallel j8-hairpins connected by an a-helix. The two central strands of the sheet are parallel and comprise the N- and C-terminal residues. Comparison of the protein A and protein G IgG-binding domain architectures reveals no immediately obvious region that could take the place of the two interacting helices of protein A and protein G complex.

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

^ B i

TABLE 9

583

Main Bacterial Proteins and Their Affinity for Antibodies

Microbial protein

Origin

Specificity

Main antibody interaction

Protein Protein Protein Protein Protein

Staphylococcus aureus C and G streptococci Peptococcus magnum A streptococci Clostridium perfringens

Fc Fc Kappa chains Fc Kappa chains

IgG IgG IgM and IgG IgA IgM

A G L ARP P

iv. Other Bacterial Immunoglobulln-Binding Proteins

Several other proteins have been reported w^ith binding properties for antibodies (Table 9). One of them is protein L from the bacterial species Peptostreptococcus magnus, which binds specifically to the variable domain of immunoglobulin light chain without interfering with the antigen binding site^"^^ it binds all classes of human antibodies constituted of kappa light chains. Immunoglobulin-binding repeating units were employed for the purification of immunoglobulins from various sources. Thus, IgG, IgM, and IgA could be purified from human and mouse serum in a single step using protein L-agarose affinity chromatography. Moreover, human and mouse monoclonal IgG, IgM, IgA, and human IgG Fab fragments, as well as mousehuman chimeric recombinant antibody, could be purified from cultures of hybridoma cells or antibody-producing bacterial cells, with protein L-agarose. This was also the case with a humanized mouse antibody, in which mouse hypervariable antigen-binding regions had been introduced into a protein L-binding kappa subtype III human IgG. These experiments demonstrate that it is possible to engineer antibodies and antibody fragments (Fab, Fv) with protein L-binding framework regions, which can then be utilized in a protein L-based purification protocol. Protein LG^^^'^^^ is a 50 kDa hybrid molecule containing four immunoglobulin light-chain-binding domains from protein L of Peptostreptococcus megnus and two IgG-Fc-binding repeats from streptococcal protein G. Protein LG was shown to bind human IgG of all subclasses and other immunoglobulin classes that carry kappa chains. The binding to human IgG was only marginally influenced by changes in temperature (4°-37°C) or salt concentration (0-1.6M), and was stable over a wide pH range (pH 4-10). Protein LG binds to immunoglobulin from 11 of 12 mammalian species, including those of rabbit, mouse, and rat. The affinity constants obtained for the interactions between protein LG and polyclonal IgG from rabbit (4.0 X 10^ M - ^ , mouse (1.7 X 10^ M ' O , and rat (1.3 X 10^ M ' ^ ) were similar to the value previously reported for the interaction between the hybrid protein and human polyclonal IgG (5.9 X 10^ M " ^ . The interaction between protein LG and a mouse IgG monoclonal antibody was not influenced by the presence of the specific protein antigen, nor was the binding of this antibody to its ligand affected by protein LG. Protein LG selectively absorbed 85-90% of the total immunoglobulin present in human and rabbit sera and 75-80% of the immunoglobulins in sera from mouse and rat.^^^

584

EGISTO BOSCHETTI AND ALOIS JUNGBAUER

V. Thiophilic Chromatography

Thiophiiic adsorption chromatography was introduced by Porath et al. In 1985.^^^ Structurally these sorbents result from the activation of agarose beads by divinylsulfone and coupled with 2-mercapto-ethanol. The ligand contains two sulfur atoms. These derivatives showed good selective binding of immunoglobulins in the presence of high concentration of structure-forming salts such as ammonium sulfate or sodium sulfate (also called lyotropic salts). For this behavior, thiophilic adsorption chromatography resembles to hydrophobic interaction chromatography. Adsorption is promoted by highly concentrated salts and elution occurs when salt concentration is lowered. The type of salt also influences the strength of the adsorption. Sulfates and phosphates are mostly used in thiophilic mediated chromatography, while the presence of sodium chloride even at high concentrations does not promote adsorption of proteins. No exhaustive explanations are given about the mechanism of adsorption of thiophilic sorbents; thioethylsulfone structures do not possess in fact pronounced hydrophobicity and do not contain ionic charges. Thiophilic adsorption chromatography has been described for the purification of murine monoclonal antibodies from hybridoma cell culture containing fetal bovine serum.^^"^ Due to the very low concentration of immunoglobulins in cell culture supernatants, binding capacity remains modest in spite of the presence of 0.5 to 1 M potassium sulfate. Further developments of this technology described by Nopper et al}^^ indicated that thiophilic sorbents could be modified in their structure to increase the specificity and the binding capacity. Linear ligand structures with three,^^^ four, five, and six sulfur atoms were described. Affinity constants were determined and all derivatives evaluated for their ability to separate IgG. All these structure required relatively large amounts or sodium sulfate to promote IgG adsorption; pH for adsorption was between 5 and 9. Elution occurred at pH 3. The binding capacity obtained with this material was between 18 and 28 mg of IgG per milliliter of silica as matrix. When agarose beads were used, binding capacity was of about 15 mg/mL. It has been, however, indicated that with cell culture supernatants containing phenol red as pH indicator, binding capacity decreased to few milligrams of IgG per milliliter of resin. Thiophihc chromatography for the separation of antibody was also applied to the extraction of IgG from sweet cheese whey. The purity of immunoglobulin G was about 75% after a single separation and required only the addition of sodium or potassium sulfate to the initial feedstock.^^^ Heterocyclic structures acting as thiophilic sorbents for the separation of immunoglobulins have also been described. In 1988 Porath and Oscarsson^^^ described several structures involving thiophilic electron-donor-acceptor mechanism. Pyridyl sulfide and phenyl sulfide molecules were attached to agarose beads and then used for the separation of plasma proteins. All these ligands adsorbed preferably immunoglobulins. Mercaptopyridine derivatized agarose beads were used as thiophilic adsorbents for antibodies in radioimmunoassay (RIA) and ELISA procedures.

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

585

All these sorbents required analogously as observed for the linear thiophilic ligands relatively high concentrations of potassium sulfate. This salt promoted also the adsorption of immune complexes formed in solutions, suggesting that the adsorption sites are present in the Fc fragment of immunoglobulin molecules. Human, mouse, goat, and rabbit IgG were adsorbed on these thiophilic resins. In 1992 Knudsen et al}^^ described sulfone aromatic ligands for the purification of human and mouse immunoglobulins. Agarose beads were first derivatized with divinylsulfone and then various aromatic structures were immobilized. Among them 2-hydroxypyridine, 3-hydroxypyridine, 4-hydroxypyridine, 2-aminopyridine, imidazole, 4-aminobenzoic acid, and 4-methoxyphenol were tested for the separation of IgG from human serum. These ligands exhibited remarkable binding capacity, but still required the presence of lyotropic salts. This result was attributed to an increase of the binding constant between the ligand and IgG. Adsorption of immunoglobulins was performed using 1.2 M ammonium sulfate in 50 m M acetate buffer, pH 5.2, followed by a washing with a solution of similar composition where concentration of ammonium sulfate was decreased to 0.3 M. Elution of IgG was performed using 50 m M Tris buffer, pH 9. In all cases collected IgG were still contaminated by other serum proteins. However, purity could be improved by an additional washing step with a buffer containing polyethylene glycol. Monoclonal IgGl were purified from 680 mL of cell culture supernatant using 7 mL of 2-hydroxypyridine containing agarose with a yield of 7 1 % and a purity of 90%. Binding capacity for IgG in these conditions was in the range of 30-60 mg per milliliter of resin. In 1995 Schwarz et al}^^ described novel heterocyclic structures able to adsorb selectively immunoglobulins. The structures contained sulfur and nitrogen. Reported ligands were 2-mercapto-pyridine, 2-mercapto-pyrimidine, and mercapto-thiazoline. These structures were chemically immobilized on silica and agarose beads using epoxy-activated matrices. Binding capacities for silica based material were about 25 mg of IgG per milliliter of resin, while for agarose beads it was about 18 mg/mL. Subsequently five membered mercaptoheterocyclic compounds have been described as affinity chromatography ligands for the separation of immunoglobulins.^^^ IgG were separated from ascitic fluid using these resins; adsorption was performed in the presence of 350 m M sodium sulfate at pH 7.4 and elution obtained with 10 m M HEPES buffer, pH 7.4, or 10 m M Tris-HCl, pH 7.4. Reactive antibody recovery was between 65 to 96% according to the nature of the ligand while the purity measured by poly aery 1amide gel electrophoresis was estimated by the authors close to 90%. Thiophilic sorbents have also been described for the isolation of immunoglobulins from egg yolk.i^^'^^i More recently thiophilic heterocyclic ligands have been claimed as improved compounds for the adsorption of antibodies in a salt independent manner.^^^'^^^ Table 10^^"^ assembles some thiophilic structures described in the literature used for the separation of antibody. All studies on thiophilic adsorption of immunoglobulins make evident that the adsorption is improved when sulfur atoms are present in the ligand. The specificity for IgG is not extremely high, but can be modulated by the

=

TABLE 10

Main Thiophilic Ligand Structures Described for the Separation of Antibody Separated immunoglobulin

Reference

Polyclonal IgG monoclonal Various Various Various

153 155 155 155 155

Polyclonals; IgGl

158

Polyclonals; IgGl

158

-0-CH2-CH2-SO2-CHz-CH2-0-

Polyclonals; IgGl

158

-0-CH2-CHOH-CHz-S

Polyclonal

164

Monoclonal

159

Chemical structures

0 u

-0-CHz-CH2-SO2-CH2-CH2-NH

-COOH

N -0-CH2-CHOH-CH2-S<

@

N

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

587

presence of aromatic or heterocyclic structures. A major drawback of thiophilic chromatography for immunoglobuHns is the required high sah to promote the adsorption. Feedstock-conditioning and severe environmental issues related to the elimination of large amounts of buffer containing sulfates have prevented thiophilic adsorption to become a dominating method for immunoglobulin purification, although it is extremely simple. vi. Hydrophobic Charge-Induction Chromatography

Hydrophobic charge-induction chromatography (HCIC) is a pubUshed method for protein separation based on the use of special ligands.^^^"^^'^ HCIC ligands can induce mild hydrophobic interaction effect in given conditions and, when environmental pH is changed, the ligand becomes ionically charged inducing thus an effect of repulsion of the adsorbed protein. Ligands for HCIC are composed of a hydrophobic tail and of an ionizable head. They are solidly immobilized on matrices through their tail by means of ether or thioether bonds. The density of the ligand of the matrix is high so as the binding capacity is industrially exploitable. One of the most important properties of HCIC is their hydrophobic effect without the presence of salts and at neutral pH. Adsorption is therefore performed in physiological conditions at a pH between 6 and 8.5, depending on the nature of proteins. This method of separation has been applied to monoclonal antibody purification (A. Schwarz, personal communication). The selectivity for the antibodies is played by the choice of the ligand while the HCIC effect is still the one described above. Ligands selected for HCIC appUed to purification of antibodies, exhibit a mild hydrophobic effect, a thiophilic effect and an ionic charge at the ligand head. Table 11 shows some ligands selected for the separation of immunoglobulins by HCIC. HCIC demonstrated its effectiveness to adsorb immunoglobulins of different classes. IgG, IgM were adsorbed on the matrix in similar conditions as per protein A and protein A mimetic ligands, respectively, described in Sections V.F.ii and V.F.x. Contrary to thiophilic chromatography, HCIC does not require the presence of salts during the adsorption phase, thus immunoglobulin capture can be effected in physiological conditions and pH close to neutrality; no feedstock pretreatment is required. Figure 17 shows no effect of ionic T A B L E 11 Composition of Main Ligands for H C I C Applicable to Antibody Separation Ligand head

Ligand tail

Ionic charge

Mercapto-pyrimidine Mercapto-methyl-thiazoline Mercapto-methyl-imidazole 4-Mercapto-pyridine 2-Mercapto-pyridine Mercapto-thiadiazole

Allylglycidylether Allylglycidylether Allylbromide Allylbromide Allylglycidylether Butanedioldiglycidylether

Mild cation Mild cation Cationic effect Cationic effect Cationic effect No charge effect

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EGISTO BOSCHETTI AND ALOIS JUNGBAUER

20

10H

10 PH

0.2

0.6

1 [NaCl]

F I G U R E 17 Dynamic binding capacity (DBC) for human poiyclonal IgG of a HCIC column as a function of the pH (A) and of the ionic strength (B) of the medium. Determinations were performed by frontal analysis and calculations done at 10% breakthrough. IgG concentration was I mg / mL of initial solution; linear flow rate was 75 cm / hr.

Strength for the adsorption capacity of IgG and a mild pH effect when it goes below 7. At pH 6 the adsorption of IgG is still effective and strong but binding capacity is lower. In all cases binding capacity at 10% breakthrough with pure human IgG is above 30 per milliliter of resin. HCIC is currently used for the separation of a number of immunoglobulins such as monoclonal antibodies from ascites fluid and cell culture supernatants, from bovine colostrum, cheese whey, and egg yolk. Typically adsorption for cell culture supernatants is made without any pH or ionic strength adjustment; the column is then washed with a 50 m M Tris buffer, pH 8. Alternative possible washings can be effected at pH 5 with a 50 m M acetate buffer containing 0.5 M sodium chloride. Antibodies are always desorbed by using a 20-50 m M acetate buffer, pH 4-4.5. In the case where large amounts of albumin are present in the feedstock, after the adsorption phase the column is washed with a low ionic strength buffer at pH 8-8.5 such as 10 m M Tris-HCl, pH 8.5, to weaken the hydrophobic interaction responsible for albumin adsorption. Antibodies are then eluted using a 20-50 m M acetate buffer, pH 4 - 4 . 5 . Variations of these conditions can be made during adsorption phase to prevent capture of undesired impurities; for instance, traces of albumin still present on the column and that can coelute with antibodies are washed out with diluted solutions of caprylic acid at neutral pH. Elutions can also be modulated by using different buffers at acidic pH such as acetate or citrate buffers. Higher the ionic strength of the buffer lower the pH for a complete elution of antibodies. Figure 18 shows a separation of IgGl from a cell culture supernatant. As shown by electrophoresis, the purity of monoclonal antibodies in a single pass is of about 8 0 - 9 5 % . This method represents the most advanced separation method for immunoglobulin for its good compromise between selectivity, binding capacity, and cost. Selectivity is close to most effective affinity ligands without issues associated with leachables and instability caused by strong caustic treatments. The cost of this material is significantly lower than any protein A resins.

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F I G U R E 18 Separation of an lgG2b monoclonal antibody from a cell culture supernatant containing 5% of fetal bovine serum on a HCIC column. Two hundred and ten milliliters of cell culture supernatant were directly injected into the column (10 mm i.d. X 100 mm height) without any preliminary treatment at a flow rate of 75 c m / h r . The column was then washed with a 50 m M Tris-HCI buffer, pH 8, followed by a washing with 25 m M sodium caprylate in the same buffer (arrow I) and then by a water wash (arrow 2). Elution of antibodies was accomplished by using a 50 m M sodium acetate buffer, pH 4 (arrow 3). Collected antibodies were analyzed by polyacrylamide gel electrophoresis in the presence of SDS and 2-mercapto-ethanol. Results showed in the insert demonstrated that the eluted fraction contained mainly antibodies as attested by the presence of light and heavy chains. Purity is estimated at about 65 - 70% with a purification factor of about 80. (a) pure standard IgG, (b) crude extract, (c) flowthrough fraction, and (d) Desorbed antibody fraction at pH 4 with evidenced heavy (H) and light chains (L).

HCIC can be advantageously used for the production of monoclonal antibodies in association with an ion-, cation-, or anion-exchange column or even hydroxyapatite (see Section V.I). vii. Immobilized Boronic Acid Ligands

Organic boronic acids are known to reversibly esterify carbohydrates containing 1,2-ds-diol structures. This interaction occurs regardless the nature and the position of the sugar. Immobilized boronic acid structures are therefore resins for the adsorption of nucleic acids, glycoproteins and carbohydrates.^^^ Specificity for glycoproteins is so high that it has been used for the measurement of glycosylated forms of proteins.^^^ Although the most widely used ligand is 3-aminophenylboronic acid, a variety of other boronate ligands have been described to identify modulation in their specificity for glycoproteins and to favor the esterification with ds-diols at a neutral pH. Attempts to lower pKa of boronate ligands involved bromo derivatives^^^ aminoethyl derivatives^'^^ and (N-methyl)-carboxyamido groups on the phenyl ring.^^^ More recently catecol esters have been proposed to improve the conditions of adsorption-elution of glycoproteins without denaturation.^^^ Specific applications have been described for the separation of immunoglobulins.^^"^ The ligand used and immobilized on a solid phase carriers was m- or /?-aminophenyl boronic acid. Antibodies were adsorbed from feed

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Stock equilibrated with 50 m M HEPES buffer, pH 8.5, and elution was obtained by a solution of 0.2 M sorbitol in 25 m M ammonium acetate buffer, pH 8.5. Starting from blood serum immunoglobulins G and A as well as complement factors C3 and C4 were adsorbed selectively. Upon addition of 20 m M magnesium chloride in the adsorption buffer the selectivity for immunoglobulins was increased. Tightly adsorbed protein on boronate resins are desorbed by 6 M urea or 30% ethylene glycol prior reuse of the columns. Proteins adsorbed on boronate resins are similar to those adsorbed on thiophilic adsorbents, however, some differences can appear for some plasma proteins. For instance boronates have a significant lower affinity for a^macroglobulin. While boronic acid-glycoconjugates interaction is due to ds-diol complexation, in the case of immunoglobulins it seems that the aromatic structure of the ligand also contributes for binding. That is why the specificity of the interaction may be affected by aromatic compounds. Immunoglobulin recovery data are not reported, but purity estimated by electrophoresis analysis can be as high as 7 0 - 8 5 % starting from whole human blood serum. Boronate affinity chromatography can be useful during a separation or polishing phase. The selectivity of boronate affinity chromatography is not high enough for initial capture steps from crude feed stocks. viii. Dye Interaction Chromatography

Reactive dyes are common ligands in affinity chromatography for years.^^^ They have been used for the separation of a number of proteins, the most common being albumin^^^ and dehydrogenases.^^^ Dyes are relatively complex organic structures inducing different concomitant interactions. They contains most of the time sulfonate groups with a strong ionic effect; they are constituted of multiaromatic rings with donor-acceptor effect described as major contribution in chromatography; hydrophobic interactions are also promoted by their chemical structure. Although antibodies can easily be adsorbed when the pH is rather acidic and elution produced when sodium chloride concentration increases, only few papers have been published on the matter. One of them relates the separation capability of a sorbent on which Cibacron Blue F3GA dye molecules and DEAE groups are both chemically attached to the same agarose matrix.^''^"^^^ Adsorption of proteins was performed in 20 m M phosphate buffer, pH 8.0, and 0.02% sodium azide. The IgG-specific fraction was eluted with two volumes of equilibration buffer. Analysis of this fraction on SDS-PAGE in reducing conditions showed only two bands of, respectively, 45K and 22K representing the heavy and light chain of IgG. In another paper Remazol Yellow GGL was described as a ligand for the separation of IgG from plasma.^^^ This dye is characterized by the presence of sulfur atom located in the linker and by the presence of heterocyclic groups involving nitrogen atoms. The overall character of the dye is anionic with a car boxy 1 group and a sulfonate group. IgG were applied in the presence of 20 m M phosphate buffer, pH 7.4, and desorbed by increasing the ionic strength with 1 M sodium chloride. Recovery was 40 to 60% reflecting differences in composition. With this ligand it has been evidenced

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59 I

the interaction did not occur with hght chains. Experimental data suggested the interaction happens in the N-terminal half of the heavy chain of IgG. Among contaminants adsorbed on the dye was the thyroxin-binding globulin. More recently Berg and Scouten^^^ described the screening of a large number of dye ligands for their ability to adsorb selectively human IgG. Two dyes were identifies as good candidates Drimarene Blue K-R and Rubine R/K5-BL. Both had a strong affinity for immunoglobulins, but no satisfactory procedure was found for their proper recovery in a native state. This preliminary work was then developed by Cochet et al}^^ with the use of Rubine R/K5-BL. This dye contains copper and therefore was tried as native and after copper depletion with EDTA. For IgG elution a large variety of eluents have been tried such as phosphorylated compounds, nucleosides, nucleotides, aromatic aminoacids, and their derivatives, imidazole, glucosamine, benzylamine. Although these agents modulated the elution properties of the buffer, none of them was really effective to obtain pure IgG. Nevertheless the sorbent demonstrated its ability to separate subclasses of immunoglobulins. The elimination of copper, that contributed to increase the affinity of IgG, was beneficial for a better elution. A special procedure was defined to separate human IgG2 subclass from other IgG using an AMP elution gradient. Immobilized Rubine R/K5-BL can be used for the separation of egg yolk antibodies. The yolk is first diluted with four volume of phosphate buffered saline and added with 3 % of PEG 6000 final concentration. The precipitate is removed and the supernatant filtered prior column loading. The column is then washed with a MES buffer, pH 6, containing 0.3 M sodium chloride and antibodies desorbed by either increasing the ionic strength or displacing with imidazol or by increasing the pH of the buffer. Obtained IgY are of good purity and can be totally isolated by adding a precipitation step using 15% final concentration of sodium sulfate to the collected chromatographic fraction. The same dye can be used for the separation of IgGl from milk whey or from colostrum. The material is loaded onto the column and antibodies eluted by increasing sodium chloride concentration to 2 M. Collected fraction contains IgG, IgA, and lactoferrin. Procion Red HE-3B was also found effective to separate mouse monoclonal antibodies in given conditions. Adsorption of filtered ascitic fluid is followed by a first washing with a 50 m M Tris-HCl buffer, pH 8.5, and a second wash with the same buffer containing 0.2 M sodium chloride to eliminate impurities. Elution is then performed by increasing the concentration of sodium chloride up to 1 M. Figure 19 shows results of adsorption-elution of mouse IgGl monoclonal antibodies on immobilized Procion Red HE-3B. Purity of antibodies is of about 8 0 - 8 5 % , few contaminants being still present in the eluted fraction. This separation recipe works also with IgG2 monoclonal antibodies from mouse, however, sodium chloride concentration must be defined case by case. ix. Metal Chelate Affinity Chromatography

This type of affinity separation is also known under the name of immobilized metal affinity chromatography (IMAC) and is based on the ion-mediated interaction with proteins. Metal ions are adsorbed first on a chelating

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EGISTO BOSCHETTI AND ALOIS JUNGBAUER

Dilution 1092 F I G U R E 19 ELISA assay of chromatographic fractions obtained from a column of Procion Red HE-3B-agarose. The sample was an ascitic fluid dialyzed against the column adsorption buffer before injection. Fractions were collected from the column previously equilibrated with a 50 m M T r i s - H C I buffer, pH 8.5. A first washing was performed using a solution of 0.2 M sodium chloride in the adsorption buffer and antibodies desorbed by increasing the concentration of sodium chloride to I M in the same buffer. Open triangles represent the nonadsorbed fraction; close circles represent the fraction obtained by a washing with 0.2 M sodium chloride; open circles represent the antibody activity of eluted fraction using a I M sodium chloride solution.

resin (iminodiacetic acid or nitrilotriacetic acid chemically immobilized on a solid matrix) and the solid phase complex is then used for protein adsorption. Interaction generally occurs between the metal ions and histidyl residues of the protein. A variety of proteins have been purified by metal chelate affinity chromatography. ^ ^"^ Although exposed hystidyl residues of the antibody are mostly responsible for the interaction v^ith the metal ions other residues such as tryptophane, phenylalanine and arginine contribute for the phenomenon. Metal chelate affinity chromatography is influenced by several parameters such as the nature of immobilized chelating group, the immobilized metal ion, the pH environment as well as the ionic strength. El Rassi and Horvath^^^ could fit the logarithmic retention factor of proteins on metal-chelate sorbents by a three-parameter equation: log k' = a -\- b log I + cl

(33)

where / is the molarity of salt in the mobile phase. The parameters a and b are negative for the investigated proteins (cytochrome c, lysozyme, j3-lactoglobulin A, a-chymotrypsin A). The empirical parameters b and c have some physical meaning; b is the electrostatic interaction parameter; it also measures the effect of the salt on the magnitude of the electrostatic interaction between the protein and the stationary phase. Parameter c is the hydrophobic interaction parameter because it expresses the magnitude of the corresponding salt-mediated hydrophobic interaction. Immunoglobulins G are a peculiar group of metal-interacting proteins as a result of the presence in their structure of a highly conserved histidyl cluster at the junctures of CH2 and CH3 domains of Fc fragment,^^^ and therefore they are easily adsorbed on metal chelating resins.^^^

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Antibody interaction generally occurs with nickel ions^^^ or copper ions.^^^ Polyclonal IgG were efficiently recovered from low concentration cheese whey at high binding capacity and purity. In terms of being competitive, IgG was found to be able to displace less tightly bound proteins such as /3-lactoglobulin, a-lactalbumin, serum albumin, and lactoperoxidase, during whey loading. In practice alkaline pH favors the selectivity of adsorption of antibodies on chelated metal ions on solid phases. To achieve high levels of antibody purity a wash with 0.5-1 M sodium chloride can be helpful to desorb weakly interacting proteins.^^^ Elution is achieved by a variety of options. The first method is based on pH lowering from 8 to 4; competitive imidazol, histidine, or histidine analogs can also be used; chelating agents such as EDTA and iminodiacetic acid are also very effective agents to desorb proteins since they capture specifically all immobilized metal ions. Purification of humanized monoclonal antibodies has also been published using nickel affinity chromatography.^^^ Directly after filtration of feed stock, antibodies were adsorbed and elution was achieved by a descending pH gradient from 7.5 to 4.25. Purity obtained was about 90% and the fraction did not contain free albumin nor free light chains. Recovery was also reported as greater than 9Q% from ascitic fluids as well as from cell culture supernatants. Purification performance of metal chelate affinity chromatography for antibodies can be very attractive as very high purity levels in a single pass have been reported.^^^ Known contaminants of purified antibodies using metal chelate affinity chromatography include transferrin, and albumin traces. Eluted antibodies can also be contaminated by significant amounts of metal ions that must be removed for the following separation or before the final formulation is performed. X. Immobilized Ligand-Derived Combinatorial Chemistry

In the last few years the identification of new drugs is performed by making large libraries of molecules in combinatorial ways starting from preselected scaffolds.^^^"^^"^ A variety of strategies have been developed for the preparation of combinatorial ligand libraries such as peptide libraries by chemical synthesis from known amino acids^^^ or by phage display technique,^^^ combinatorial molecules from nucleotides,^^'^ and pure synthetic chemical libraries.^^^ By all of these methods is possible to make at least 10^ combinations covering all possible molecular aspects for a better molecular interaction. This drug screening procedure can be also applied for discovery of new affinity chromatography ligands. Until now, however, only rarely here combinatorial library of molecules been described as a source for new affinity chromatography ligands.^^^'^^^ In the area of immunoglobulin purification, ligands from combinatorial approaches have also been applied. Li et aL describe the design of a protein A mimetic ligand based on a rational approach in understanding the specific recognition parameters between the B domain of protein A and the Fc fragment of IgG. A combination of com-

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EGISTO BOSCHETTI AND ALOIS JUNGBAUER

puter-aided molecular structures and organic synthesis enabled them to prepare several ligand candidates with affinity for immunoglobulins G.^^^ Since the interaction appeared to be predominantly hydrophobic and supported by some hydrogen-bonding effects, the structures studied where around phenylalanine, tyrosine, and isoleucine residues. These aminoacids were attached to a trichlorotriazine ring in different proportions and the various obtained structures were studied for their affinity for IgG. Constructs were then immobilized on agarose beads by means of a spacer arm. Measured affinity constants obtained with these ligands were in the range of 2.7 X 10^ and 1.0 X 10^ compared to 1.4 X 10^ for protein A. Purity of IgG from human plasma evaluated by SDS-PAGE were reported to be in the range of 50 to 98%. The mechanism of action being similar to the one described for protein A, adsorption was performed in physiological conditions and elution by lowering the pH to 3.8 and below. Contrary to protein A, the described mimetic ligand adsorbed also IgA and IgM; binding capacity was reported to be in the range of 4 to 25 mg per milliliter of resin. Another peptide was also described for its ability to interact with immunoglobulins and was used as an affinity chromatography ligand.^^^ This peptide structure resulted from a combinational peptide library screening and was identified by its ability to make complexes with IgG that were displaced by the addition of protein A. The structure of this ligand is pecuhar in the sense that it is composed of four parallel chains of a tripeptide linked together by three lysine molecules linkers. After immobilization onto the chromatography matrix, it showed good properties for the separation of antibodies from ascites fluid and from cell culture supernatants. As for the previous described peptidomimetic ligand, it does not distinguish classes of antibodies. Binding capacity was reported to be close to 15 mg per milliliter of resin. The separation of immunoglobulins was performed by adsorbing the material in 100 m M phosphate buffer and after a washing step immunoglobulins are desorbed by increasing the pH to 8-8.5 with carbonate or borate buffer. Similar results have also been recently published with a synthetic ligand able to mimic protein A in the recognition of Fc portion of antibodies.^^^ Ligand specificity is broader than SpA since Ig, IgA, IgM, IgY, and IgE were all of them interacting with no specific discrimination. Elution was described as effective either at an acidic pH (e.g., acetic acid) or using a sodium bicarbonate buffer, pH 9. Purity checked by SDS-PAGE was higher than 90% in described conditions with a binding capacity that could reach up to 25 m g / m L of resin. Another protein A mimetic molecule of nonpeptide nature was recently described.^^^ The nature of the ligand was not disclosed but was described as able to bind a large variety of immunoglobulin G and M from different animal species. In typical experiments the capacity described was in the range of 1.2 to 11.5 mg per milliliter of resin; purity of immunoglobulins was in the range of 35 to 99% and the recovery of 9 0 - 9 8 % . With this resin the adsorption was performed at pH 5.1 after adjustment of the feedstock, in the presence of sodium lauroyl sarcosinate. A washing

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was then performed with 10 m M citrate buffer to ehminate nonspecifically adsorbed material and the elution achieved by increasing the pH between 7 and 9 with phosphate or carbonate or Tris buffers. As per previous reported protein A mimetic Ugands, this resin adsorbed also IgM. xi. Immunoaffinity Chromatography

Immunoaffinity chromatography covers the real aspect of biological affinity purification. In the context of this review two distinct parts are considered: (i) the immobilization of the antigen and (ii) the immobilization of specific antibodies both intended for the adsorption of the antibody to purify. Typical molecules that are specifically recognized by antibodies are antigens. Therefore the immobilization of the antigen on a chromatographic matrix is also a means to purify selectively the antibody. To that end, the first requirement is the availability of the antigen for its chemical immobilization on a solid phase. If it is available, an effective affinity sorbent can be prepared by its chemical immobilization on a solid matrix. For small-size antigen molecules, a spacer arm may be required for a good accessibility to the active site of the antibody.^^"^ Interaction on the solid phase involves the hypervariable region of the antibody and sorption efficiency is related to the affinity constant. When this latter is very high and requires elution conditions that can have a deleterious effect on the integrity of the antibody, it may be necessary to chemically modify the structure of the antigen to decrease the affinity constant. Adsorption of the antibody on antigen ligands attached on a solid phase is performed in physiological conditions while the desorption of the antibody is achieved using deforming buffers or chemicals. An acidic pH may be used as well as high concentrations of magnesium chloride or even 3 M sodium isothiocyanates. Highly concentrated urea or guanidine hydrochloride solutions, though very efficient to dissociate the antigen-antibody complex, are not always recommended for their strong denaturing effect on antibodies. As an example immobilized antigens were used for the purification of monospecific polyclonal antibodies from hyperimmune sweet whey.^^^ Immunoaffinity separation chromatography can also be performed using immobilized specific antibodies to separate immunoglobulins. Actually immunoglobulins are antigenic molecules and, as such, they can produce specific antibodies in animals. The use of these specific antibodies as immobilized affinity ligands is the second aspect of immunoaffinity chromatography and constitutes an effective specific way to purify monoclonal and polyclonal antibodies. Anti-immunoglobulins are first chemically coupled on a solid phase chromatographic matrix and used as an affinity sorbent (see Fig. 20). An advantage of this approach is that the antibody ligand can be chosen as a function of its affinity and specificity so elution conditions remain mild for a minimum denaturation of the antibody to be separated. Immunoaffinity chromatography with antibody ligands is, however, so specific for the target antibody that it is necessary to design a new column each time a separation of a novel antibody is wished. This implies the

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F I G U R E 2 0 Separation of mouse monoclonal IgG, by immunoaffinity chromatography on rat antimouse IgG antibodies chemically immobilized on agarose beads (7.1 mg per milliliter of resin). Column, 11 mm i.d. X 100 mm. Load of I mL of whole mouse ascitic fluid previously filtered containing 8.8 mg of IgG, among 32 mg of other proteins. Buffer: phosphate buffered saline, pH 7.2. Wash performed using I M sodium chloride solution in phosphate buffer. Elution obtained by lowering the pH to 2.8 with 0.1 M glycine - HCI buffer, containing 0.15 M sodium chloride. The flowthrough and the wash peak did not contain IgG, while elution peak contained about 7.4 mg of antibody. Purity was over 70% and the yield of 84%.

development of the specific capture antibody ligands, which is a relatively expensive operation. Immobilized anti-light or anti-heavy-chain-specific antibodies could conversely be appUed more broadly as they can copurify antibodies of the same group.206.207

In immunoaffinity chromatography adsorption phase is performed in physiological conditions followed by a wash with high ionic strength buffers to eliminate nonspecifically adsorbed proteins. Elution of human IgG is obtained by a deforming buffer such as a 0.2 M glycine-HCl, pH 2.7. Binding capacities achievable with this type of chromatographic separation can be as high as 5-8 m g / m L of resin. Recovery is generally in the range of 8 5 - 9 5 % with medium affinity antibodies; however, acidic pHs may be a problem as they may induce partial denaturation of the separated antibody. A typical example of immunoaffinity chromatography is the separation of rat monoclonal IgM using an anti-^t-chain antibody, as illustrated in Fig. 2 1 . Purity of the separated antibody in a single pass was about 95%. The ideal column of immunoaffinity chromatography would be made using monoclonal anti-immunoglobulin antibodies. These might be selected for their appropriate epitope affinity, specificity, and association constant to release the monoclonal antibody under defined gentle, nondenaturing conditions. Electroelution has also been reported as a method to avoid denaturation in immunoaffinity chromatography.^^ ^ A specific aspect of immunoaffinity chromatography is the so called epitope affinity. Actually antibodies are either directed against a continuous aminoacid sequence, the continuous epitopes, or a domain which is located on various sites in the aminoacid chain, discontinuous epitopes, or conformational epitopes. Continuous epitopes have served as affinity ligands for antibody

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(1 UA. F I G U R E 21 Separation of mouse monoclonal IgM by immunoaffinity chromatography on rat anti-/Lt-chain antibodies chemically immobilized on agarose beads (4.2 mg per milliliter of resin). Column: 10 mm i.d. X 45 mm. Load of 5 mL of whole mouse ascitic fluid previously filtered. Buffer: phosphate buffered saline, pH 7.2. Elution by lowering the pH to 2,8 with 0,2 M glycine - HCI buffer. The first peak did not contain IgM while eluted peak (arrow) contained about 7,8 mg of IgM total.

purification. Peptides have been synthesized and immobihzed to agarose and dextran derivatives.^^^ Purification required in some cases very harsh conditions, since the equihbrium binding constant of the epitope-antibody complex w^as remarkably high. xii. Lectin Affinity Separation

Lectins are proteins with different amino acid sequence derived from a variety of biological sources w^ith a common property: They bind carbohydrates. Structural requirements for interaction of lectins with glycoproteins have been elucitated. Their specificity is based on the recognition of sugar structures found on the surface of glycoproteins. Differences in binding specificity of lectins is considered due to either the difference in the structure of the glycoconjugate or to the nature of the terminal sugar moiety of the glycan exposed on the surface of the protein. Besides a large number of protocols for the separation of glycoproteins, lectins have also been studied for antibody purification. Concanavalin A, a frequently used lectin, has been described for the purification of IgM. The specificity for IgM is not exclusive, however, when IgG, a: 2-macroglobulin, and a 1-antitrypsin are not present in the initial feed stock, this is the case with serum free-cell culture supernatants, high levels of purity can be reached in a single step. Figure 22 shows the purification of a monoclonal IgM using Concanavalin A as ligand. Different classes of antibody have been separated with lectins according to their specificity, as illustrated on Table 12. For instance, IgA antibodies have been purified using immobilized Jacalin.^^^ IgM have been separated using Galanhtus nivalis agglutinin^^^ and using mannan-binding protein (MBP) from rabbit serum.^^^ Lentil lectin as well as phytohemagglutinin and ricin have been used to separate IgG and Griffonia simplicifolia lectin (GS-1) has been described for the separation the purification of IgD with a overall yield of nearly 95%.^^^

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L_kJiL

^

F I G U R E 2 2 Separation of a murine IgM monoclonal antibody by affinity chromatography on Concanavalin A. Two milliliters of cell culture supernatant in low protein Ig-free medium (Ultroser HY, Ultroser is a trademark of Biosepra-Life Technologies Inc., Rockville, MD) was first precipitated with ammonium sulfate at 50% saturation and redissolved and dialyzed against 50 m M T r i s - H C I buffer, pH 7.4, containing I m M MnCl2 and CaClj before column loading. The amount of IgM loaded was 90 jjbg total. Column 11 mm i.d. X 90 mm; first wash t o eliminate nonspecifically adsorbed proteins was done using I M sodium chloride in the initial buffer (first arrow) and elution of IgM was performed with the use of 300 m M a-methyl-mannose (M-M) in the initial buffer containing I M sodium chloride (second arrow). Flow rate was 20 m L / h r . The right part of the figure represents the content in micrograms of IgM found in the flowthrough and in the eluted fraction determined by a classical ELISA test. Overall yield was 93%.

As a general rule, adsorption of antibodies is performed in physiological conditions and desorption of glycoconjugates by using competitive sugars. In spite of their excellent specificity for glycoconjugates, immobilized lectins suffer form their relatively low binding capacity and their instability to agents currently used for column cleaning. Moreover they are sensitive to proteases as are other immobilized proteins mentioned in Sections V.F.ii, V.F.iii, and V.F.iv. xiii. Other Affinity-Like Techniques Among affinity related methods described for the separation of antibodies there are fev^ minor others that are difficult to classify but that deserve to be mentioned for their effectiveness. One example is given by sorbents carrying aza-arenophilic ligands.^^"^ They are prepared by first reacting 3,5-dichloro-2,4,6-trifluoropyridinewith agarose beads follow^ed by the attachment 4-dimethylaminopyridine and an endcapping w^ith 2-mercaptoethanol.^^^ The

TABLE

12

Main Lectins Usable for Their Interactions

with Immunoglobulins Lectin name

Source

Sugar specificity

Interacting antibody

Concanavalin A GSI-B4 Jacalin PNA RCA-2 MBP LCA PSA GNA

Concanavalia ensiformis Griffonia simplicifolia Artocarpus heterophyllus Arachis hypogea Ricinus communis Mammalian serum Lens culinaris Pisum sativum Galanthus nivalis

Mannose, glucose Galactose N-acetyl-galactose Galactose N-acetyl-glucose Mannan Glucose, mannose Glucose, mannose Mannose

IgM,IgG,IgA IgD IgA,IgE IgG IgD IgM IgG, IgM IgG IgM

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599

adsorption of antibody from whole serum onto the column is obtained by diluting the sample with about four volumes of a physiological phosphate buffered saline solution prior loading. The column is then washed with the same buffer used for serum dilution and immunoglobulins eluted using a 50 m M acetate buffer, pH 2.8. Purity of antibodies in the collected fraction is about 95% by SDS polyacrylamide gel electrophoresis. This method can be considered as a complementary step to affinity chromatography with protein A and protein G ligands. This sorbent can be used for the separation of other immunoglobulins such as IgY from egg chicken and IgM.^^^ Immobilized histidine on agarose beads by means of aminohexyl spacer arm has also been utilized to purify polyclonaP^^ and monoclonal antibodies.^^^ Adsorption of IgG on histidine-agarose occurs in the presence of 25 m M Tris-HCl buffer, pH 7.4, and elution is achieved using a solution of 0.2 M sodium chloride in the same buffer. In described conditions the binding capacity of antibodies was about 11 m g / m L of resin and purity of separated antibody was estimated by the authors at around 98% when combined with ethanol precipitation. Dissociation constant was also determined and reported between 2.4 X 10"^ and 4.6 X 10~^ M. Abx sorbents are also well known in the separation of antibodies. They are based on a mix mode interaction involving weak cation and anion exchange as well as mild hydrophobic interactions. Affinity-like antibody binding is complex and may vary according to pH and ionic strength of the initial buffer.^^^ Abx sorbents are also able to resolve multiple forms of immunoglobulins such as subclasses, conjugates, and fragments.^^^ Binding capacity of the resin depends on buffer composition and purity of antibodies is typically above 70%. One of the major disadvantages of Abx resins is that other proteins such as albumin, trasnferrin, a2-macroglobulin, proteases, and lipopolysaccharides are adsorbed and may be co-purified. DNA binds to the resin to some extent. For several antibodies a high purity has been reported when purified using Abx sorbents. High purity as well as endotoxin removal can be achieved by adding an anion exchange chromatography column.^^^ The achieved high purity depends, however, on the individual antibody. Therefore Abx cannot be considered as a generic selective resin such as solid phases carrying proteins A, G, L, or LG as ligands.

G. Role and Place of Gel Filtration Gel filtration, also called size-exclusion chromatography and first described by Porath and Flodin,^^^ is a chromatographic method that separates proteins according to their size. Under ideal conditions, surface binding does not occur between proteins and the solid phase. Gel filtration is frequently used for desalting and for the separation of aggregates or degradation products. Proteins penetrate into the pores of the hydrogel network. According to the size of the protein and the pore size distribution of the gel network, the proteins can be separated, since the small ones are more retarded that the larger ones.

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EGISTO BOSCHETTI AND ALOIS JUNGBAUER

The simple relationship between the distribution coefficient and the elution volume can be expressed as -^ = 1 + (1-6)^3,

(34)

where V^ is the elution volume, VQ is the void volume, K^^ is the available distribution coefficient, and e is the fraction of the void volume related to the total column volume.^^^ Furthermore, an empirical relationship between the K^^ and the molecular mass (M^) of a macromolecule has been found and was expressed by a logistic equation: ^av =

h

(35)

where b and c are constants that depend on the particular separation conditions.^^"^ Both equations show that the proteins are eluted in the order of their molecular mass or Stokes radii, if interactions other than diffusion into the pores are prevented. Gel filtration chromatography can be carried out in two different modes: desalting and resolution mode. In the desalting mode, the K^^ value of the target protein is close to zero and does not enter the pores of the gel chromatography medium. The protein is eluted with the void volume and the contaminants or salts, which are substantially smaller than the product, are retained. In that case the dilution of the product is very limited or even negligible. In the resolution mode, the K^^ value is different from zero, therefore proteins are able to enter the pores and are delayed according to the inverse of their molecular weight. High molecular mass contaminants can be removed by this technique because they elute earlier than the target product while small species are eluted later. In the resolution mode, the product is diluted between 5- and 10-fold. In general, a gel filtration chromatography step is preferred as a final step in an antibody purification sequence. Optimization of gel filtration is much simpler than the other types of chromatography. The problem of fingering when a very viscous sample applied onto the column, is the major obstacle in this type of chromatography. Gel filtration is restricted by small feed volume, low feed concentration, and low feed viscosity. Limited flow rates are required since separation is achieved by selective diffusion of the various feed components into the pores. This separation mode, where several classes of proteins are separated according to their size, is considered a laboratory technique. When used at industrial scale, its role is usually limited to elimination of small amount of impurities. The main role of gel filtration in preparative antibody purification is desalting, which may precede another chromatography step or may be the final formulating step. Purification of IgM is often performed by gel filtration after a preconcentration step.^^'^^'^^'^^^ The process is carried out in long columns (typically 60 to 100 cm long) at low linear flow rates (5 to 20 cm/hr) and the feed

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SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

H H T A B L E 13 Selection of Gel Filtration Media (GF) for the Separation of Antibodies and Related Molecules Antibodies and related nnolecule to separate

Molecular size (average)

IgG (poly or monoclonal) Secretory IgA IgM Fc fragment Fab fragment F(ab)2 fragment Immunoconj ugates

160 320 960 60 50 100 >200

Exclusion llnnit of GF medium 300 500 1200 90 90 160 500

Fractionation range

Example of GF medium

20-300 40-500 100-1200 5-90 5-90 10-160 40-500

Polyacrylamide agarose Polyacrylamide agarose Polyallyldextran Polyacrylamide agarose Polyacrylamide agarose Polyacrylamide agarose Polyallyldextran

b

All numbers are in kilodaltons. Polyacrylamide agarose is known under the trade name Ultrogel AcA (BioSepra-Life Technologies, Inc., Rockville, MD); polyallyldextran is known under the trade name Sephacryl (Amersham Pharmacia Biotech, Uppsala, Sweden).

volume does not exceed a volume corresponding to 5% of the total column volume. Gel filtration media are characterized by their exclusion limit and their fractionation range for macromolecules. Therefore the most important selection criterion to choose among gel filtration media is the range of molecular mass of the proteins that have to be separated. For IgG pohshing, the solid phase of choice should have an exclusion limit of 300 KDa; for IgM the exclusion limit should be of about 1200 KDa and for Fab fragments it should be of 80 KDa. Table 13 provides a simple guideline for choosing the appropriate gel filtration medium for various immunoglobulins and their fragments. Salt concentration and feed composition are not limited in contrast to other chromatographic techniques. Virtually any ionic strength and pH compatible with the separation medium can be used; how^ever, feed volume must be reduced when a protein solution with a high salt concentration is loaded. Furthermore, it is essential to note that diffusion may be partially hindered at high salt concentration. Yields are generally high; no risk of denaturation is associated with gel filtration. H. Conclusions Concerning Single Chromatographic Techniques i. Applicability and Limitations The principles of separation of antibodies by chromatography are based on specific interactions on solid phase surfaces. Beyond their biological properties and role, antibodies possess a variety of common characteristics that can be exploited to develop a generic separation procedure. In spite of a large spectrum of existing separation techniques for antibodies, development of novel approaches is still an attractive task. Available methodologies are not applicable to all antibodies. Furthermore requirements on therapeutic antibodies are continuously increasing.

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Nevertheless with the right combination of existing techniques it is possible to prepare pure antibodies for research, diagnostic, and human therapeutic use. Chromatographic methods are not the sole separation methods for antibody purification, however, they play a dominant role especially when they are combined to complementary methodologies such as precipitation by chemical agents or membrane-based separations. Existing chromatographic methods can be very effective in special circumstances, but all of them have critical limitations related to the nature of the antibody or the source of raw material. The choice is, in most of the cases, a trade-off between advantages and drawbacks, each case constituting a specific compromise. Table 14 presents a general guide describing the principle of several chromatographic separations and limitations of the technology. Usually, the first choice of a chromatographic technique for antibody separation is dictated by the selectivity. Protein A and protein G sorbents are the first choice for most of IgG antibody separation, whatever the feedstock containing the antibodies. No specific loading conditions must be defined, such as adjustment to a specific ionic strength or pH or addition of selected chemical agents. Protein A solid phases are, however, not always practical due to their very high cost and, sometimes, the lack of specificity. In the next section, specific antibody purification recipes are suggested involving combinations of orthogonal columns.

j ^ ^ H T A B L E 14 Summary of Chromatographic Separation Methods for Antibodies and Related Molecules Type of chromatography Gel filtration Ion exchange Hydrophobic interaction Hydroxyapatite Protein A and G affinity Protein L affinity Dye affinity Immunoaffinity IMAC adsorption Thiophilic separation Boronate affinity Hydrophobic charge induction Combinatorial ligands Antigen affinity

Exploited parameter

Applicability

Technical limitations

Molecular size Net ionic charge Water repelling associations Dual ionic interaction Bioaffinity Bioaffinity Mix mode Bioaffinity

All antibodies All antibodies All antibodies

Metal binding properties Complex mix mode Sugar complex Mix mode

IgG

Low selectivity; poor flow rate Activation of proteases Denaturation; high amount of salts Low binding capacity Toxic leakage Toxic leakage Limited specificity Availability of antibodies and poor stability Metal ions leakage

Complementary structures Bioaffinity

Separate classes

IgM mainly IgG /c-light chains Various Single antibody

All antibodies All antibodies All antibodies

Single antibody

High amount of salts Low specificity Definition of intermediate washings Cost; stability; specificity; binding capacity Availability of antigen; affinity constant

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

603

ii. Separation Methods as a Function of Scale

As underlined in the introduction, all described methodologies for the purification of antibodies address preparative purposes at bench and industrial scales. They are diverse in the principle of adsorption-desorption, in their productivity aspect, in the purity of antibody that they are able to yield and, finally, in the exploitation cost. The latter depends not only on the initial cost of a solid phase sorbent, but also on run-to-run stability and consistency to withstand dozen or hundred or even thousand of cycles. Although a rational sequence is not really critical for small or medium preparative scale, it becomes important to make the right choice when the scale of the separation involves large amounts of feedstock with the consequent necessity to enhance the recovery and reduce the production cost. The preparation of pure antibody for human therapeutic use is a long process that commences by in vitro use and last for several years of intensive work at various clinical stages. Risks related to the noneffectiveness of the antibody for the target application are relatively high at the early stage of the project. In this context, it would be irrational to search at the beginning for an optimized sophisticated separation and purification process to make the antibody at a large scale. Therefore initially the most logical approach is to use a specific classical method that is easy to use although it may be expensive. Protein A resins are one of the tools of choice because of the specificity and the predictability in terms of separation results. A gel filtration column can follow to polish the antibody for preliminary studies. Such an approach is not the best way to start for the scale-up of the process because of the high price of protein A resins, their limited usability (that also impacts the cost per gram of produced antibody), and the risk of leachables. Additionally, a gel filtration is not really a productive large-scale method for reasons related to its very low loadability and low speed (see Section V.G). Most generally, a first laboratory process is kept for the early clinical stages; however, as soon as the clinical results are promising and there is evidence that the process would have to be scaled up, a novel phase in the design of an efficient and cost effective process should start. The principle is to have about three steps: the first with specific properties to capture the antibody from the crude feedstock, the second to separate impurities, and the third to play a polishing role with properties to remove traces of peculiar agents such as nucleic acids and endotoxins, and able to add virus clearance. The choice of the type of resin is a compromise between its cost, binding capacity, and longevity in harsh cleaning conditions. Three main situations can distinguished. The first is the preparation of antibodies for diagnostic use in vitro ^ the second is related to the therapeutic use for relatively modest amounts of antibodies, and the third addresses very large amounts of antibodies for both therapeutic and prophylactic applications. In the second and third situations, although antibodies would meet similar requirements of safety, purity, and potency, the issue of market must be addressed. The production cost resulting from both the method of expression and the purification process must be considered.

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For diagnostic applications, most generally, one single column is used. Protein A resins are used very frequently. High cost as well as problems related to possible leakage are not really large issues, as long as they do not impact on the result of the diagnostic test. The separation process is easy, and does not always justify the development of sophisticated processes for the production of such antibodies. For therapeutic use, the question is very different. At large scale it would be beneficial to stay away from protein A whenever possible to save money, and to avoid the risks related to the possible leakage of protein A and its fragments. Substituents of protein A sorbents can be column sequences that are orthogonal in adsorption so as to purify the antibody by their different properties. In this respect, concepts for large amount of monoclonal antibody purification processes have changed significantly in the past ten years. Although many were already predicted, the envisioned purification approaches were still very labor intensive^^^ with a low level of productivity. For instance, feedstock pretreatments, expensive affinity purifications, and lowspeed gel filtration are no longer considered for very large scale future monoclonal productions. For very large scale, current expression methods of interest are transgenic animals and transgenic plants. These both represent a challenge for extraction and purification processes. Low cost and specificity seem to be the key success factors. In this respect, thiophilic-like materials, such as the one identified in this review as HCIC, which are without the need to add salts or to modify physicochemical conditions, should have a great impact on the definition of the capture phase. From these very crude raw materials, an important issue will undoubtedly be the ability to clean resins with harsh agents such as strong alkafis, acids, oxidizing agents, and detergents without degrading the resins. I. Combined Chromatographic Separation Processes Antibody purification processes are rational combinations of two or more chromatographic separations techniques with the aim of removing all relevant impurities from the antibody preparation according to the demands of the application. It is known that none of the already described methods for antibody separation provides sufficient pure, homogeneous products in a single chromatographic step. For higher purity, as required in therapeutic applications, the combination of two or more chromatographic procedures is mandatory. As with any other classical approach in downstream processing of proteins, the process is split into three steps: a capture step, followed by a separation step, and finally by a polishing step to remove residual impurities originating from leached compounds of the sorbent a n d / o r fragments and aggregates. ^^ A desirable attribute of a capture step is the high selectivity for the antibody, to eliminate bulk impurities during the early phase and, moreover, to reduce water content, resulting in a substantial concentration of the product. The requirement of such a sorbent to capture the antibody at high capacity even from feedstocks where the antibody is present at very low

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

605

concentration, is first of all a rectangular adsorption isotherm. Furthermore for antibody capture, the sorbent must withstand harsh cleaning conditions, which are a consequence of the contact with crude feedstocks. Capture is best operated with affinity-based material because of their selectivity for the target antibody. Cost issues play also an important role for the selection of the appropriate material. Fractionation is less specific and essentially requires separation efficiency. This is generally performed with ion-exchangers and hydrophobic interaction chromatography, which enable efficient separation by salt or pH gradients. Hydroxyapatite is also a good tool for fractionation where proteins can be separated by both phosphate gradients and sodium chloride gradients. Finally, gel filtration is most often considered an appropriate polishing method when the target antibody is already pure and the only impurities are foreign protein traces or fragments or aggregates that must be eliminated. In this context, separation processes for antibody purification are logical orthogonal combinations of methods. The literature is very rich in describing combinations of chromatographic methods. Flow-sheet tools for designing the most suitable combination are not available to date; however, Osterlund^^^ has developed a simple computer program called an antibody-purifier. This program is a training tool for setting up combinations based on the most common methods. This chapter discusses only the most relevant suggestions and Table 15 presents a summary of possible powerful combinations. i. Protein A Affinity, Ion Exchange, and Gel Filtration

This is the most-documented classical way to purify antibodies.^^^ Crude feedstock is first adsorbed on a column of immobilized protein A under physiological conditions (with some weakly interacting antibodies a high ionic strength, high pH buffer is required) and eluted with an acidic buffer. Collected IgG are of good purity and yield can be as high as 70-90%.^^^ The eluted antibody fraction is immediately adjusted to a pH of about 5 and fractionated by cation exchange chromatography. The separated IgG peak is

TABLE [ 5

Possible Combinations of Methods for Antibody Purification

Capture

Fractionation

Polishing (optional)

Protein A

Ion exchange

Gel

filtration

Ni-IMAC

Cation exchange

Gel

filtration

Thiophilic Cationic exchange HCIC Anion exchange Cation exchange

Ion exchange Hydroxyapatite

Hydroxyapatite —

Protein A HCIC

Cation exchange Anion exchange

Cation exchange Cation exchange Gel filtration

Connnnents Good selectivity; very low productivity of gel filtration Risks of nickel release; lov^ productivity of gel filtration Large amounts of salt needed; good selectivity Good capture; good selectivity; low^ binding capacity Good selectivity of capture; high binding capacity Poor selectivity; usable w^ith protein-free feedstocks Good productivity first; poor productivity w^ith gel filtration Good selectivity; good binding capacity; high cost Good selectivity; high binding capacity; low^ cost

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EGISTO BOSCHETTI AND ALOIS JUNGBAUER

then injected into a gel filtration column for the separation of possible aggregates and other small molecular weight impurities. This includes possible protein A fragments resulting from the presence of proteases in the initial feedstock and degradation by chemical processes, especially the harsh elution conditions. A final gel filtration column is also a good means to desalt and formulate the antibody preparation. ii. I M A C , Cation Exchange, and Gel Filtration

As described in Section V.F.ix, immobilized nickel affinity resins are selective sorbents for antibodies. Desorption is achieved by relatively acidic pH values, enabling a direct linkage with a cation-exchange chromatography as a second step. Ionic strength as well as pH must be adjusted according to the nature of the antibody and the charge density of the ion-exchanger to meet the required conditions for adsorption. Eluates from ion-exchange resin yields antibody fractions that can easily be desalted by gel filtration independently on the composition of the elution buffer. The combination of cationexchange chromatography and gel filtration after an IMAC is necessary for the separation of nickel ion traces present in the eluate. These will primarily be adsorbed by the cation-exchangers, and, in case traces of metal ions are still present, they will be separated by gel filtration. iii. Thiophilic, ion Exchange, and Hydroxyapatite Chromatography

This sequence is chosen for the complementarity of separation techniques that increase the probability of obtaining highly pure antibodies. Thiophilic chromatography requires high salt concentration during loading. Therefore the feedstock must be added with ammonium or potassium sulfate up to a concentration of 1 M in a neutral 50-100 m M phosphate buffer. Once the antibody is adsorbed, it can be dissociated after washing off the unbound material by lowering the salt concentration in the same phosphate buffer. Depending on the salt concentration where the antibody elutes, the collected fraction can sometimes be directly loaded onto an anion-exchange column. If the ionic strength is too high, the antibody fraction must be diluted to enable impurities to bind on the ion-exchanger. The antibodies pass the ionexchange column in the flowthrough and can then be directly loaded onto a hydroxyapatite column for further purification, as indicated in Section V.E. The advantage of this approach is that all processes are strictly performed at neutral pH in a phosphate buffer. Danger of antibody denaturation in therefore excluded. This separation process can be advantageously applied to the separation of IgM antibodies, as they are sensitive to denaturation. A disadvantage of the method is the relatively low binding capacity of hydroxyapatite. iv. Hydrophobic Charge Induction and Cation-Exchange Chromatography

In Section V.F.vi, hydrophobic charge induction chromatography was presented as a good alternative to protein A affinity chromatography for the capture of antibodies. It can bind antibodies under physiological conditions with no changes in pH and ionic strength and can be eluted by decreasing the pH. Purity is not as high as that obtained with protein A columns, therefore a

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

607

further column (which is used also in the case of protein A affinity chromatography) will achieve the purity of the target antibody. As second column, an S or CM ion-exchanger can be easily used. No adjustment of ionic strength and pH are necessary. The advantage of this approach is its simplicity, the high binding capacity of both columns, and the ability to clean them with harsh cleaning agents to preserve the effectiveness of the adsorption and of separation for a long period. Contrary to protein A affinity sorbents, columns used in this process are not sensitive to proteases or to strong alkaline or to dissociating agents. Figure 23 illustrates an example of antibody purification by the combination of a HCIC column and a S Ceramic HyperD column. The process in performed in relatively mild conditions of ionic strength and pH throughout separation stages. V. Anion Exchange Followed by Cation Exchange

Although both steps utilize ion-exchange effects, cation- and anion-exchange chromatography can be used in series in both combinations with good results. As reported by Schwartz et al.,^'^^ antibodies can easily be isolated from serum. First the feedstock is precipitated by ammonium sulfate, and after diafiltration, the sample is loaded onto an anion-exchange column. Collected immunoglobulins from the flowthrough are then pH adjusted and loaded onto a cation-exchange column. The purity of antibodies are satisfactory, as attested by isoelectric focusing. This approach can easily be applied for the separation of monoclonal antibodies from cell culture supermatants when direct capture by the ion-exchanger does not induce protein precipita-

l g C 1 R.R

JIUI

L

igCI

\A

F I G U R E 2 3 Two steps purification of a mouse monoclonal IgGI from an ascitic fluid using hydrophobic charge-induction chromatography followed by a cation-exchange column (S Ceramic HyperD). Eight milliliters of filtered ascitic fluid were directly injected into a phosphate-buffered saline equilibrated HCIC column (A). The column (I.I cm i.d.X 10 cm) was first washed with a 50 m M acetate buffer, pH 5, containing 0.5 M sodium chloride (arrow I); a second wash was performed using the same salted buffer at pH 4.7 (arrow 2). Elution was then obtained with a 50 m M sodium acetate buffer, pH 4.5 (IgG containing peak). Eluted IgG I -rich fraction (IgG I R.F.) was then directly injected into a column ( I . I cm i.d X 5 cm) of S Cermaic HyperD, previously equilibrated with a 50 m M sodium acetate buffer, pH 5.4 (B). The column was then washed with the same buffer t o eliminate all nonbound proteins. Elution of pure IgGI (arrow 3) was achieved using a 50 m M sodium acetate buffer, pH 5.4, containing I M sodium chloride. Nonspecifically adsorbed material was then eliminated by cleaning the column with a solution of 0.2 M sodium hydroxide (arrow 4). Flow rates for both columns were 75 m L / h r . The intermediate purity of IgGI from the first column was estimated close to 70%; the final purity from the second column (IgGI) was estimated as close to 97% by PAGE in reduced and nonreduced form.

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EGISTO BOSCHETTI AND ALOIS JUNGBAUER

tion. It is, in fact, known that in some cases acidification of the feedstock prior to column loading generates protein precipitation, somewhat reducing the overall antibody recovery. In this case, an anion-exchange column used as first separation resolves the problem.^^^ Anion-exchange columns are first used to adsorb many foreign proteins while the antibodies are collected in the flowthrough. The collected fraction is then acidified at a pH of 4-5.5 and directly injected into a cation-exchange column. For more details see Fig. 24. This method is very simple, and takes advantage of sorbents of high binding capacity and of low cost compared to affinity resins. vi. Hydrophobic Charge Induction Chromatography Followed by Hydroxyapatite

The specificity advantages of HCIC for antibodies are detailed in Section V.F.vi. This sorbent captures relatively specifically antibodies from any feed-

Steps

Separation conditions

Filtered serum free feed stock

pH adjustment to 6.5

50 mM Tris-acetate pH 6.5. Separation on Q Ceramic HyperD F

IgG in the flowthrough. Regeneration with 1 M NaCI

GPC analytical results of 1 IgG fractions

J ll

J k.

Adsorption at pH 5 Purification on CM HyperD F

Eiution of adsorbed IgG with sodium chloride gradient

F I G U R E 2 4 Schematic representation of a monoclonal separation process involving two ion-exchangers: anion (Q Ceramic HyperD) followed by a cation-exchanger (CM HyperD). Chromatographic separations on the right represent analytical gel filtration profiles from the crude feedstock to isolated fractions from Q Ceramic HyperD and CM HyperD. The final purity of the isolated monoclonal antibody was estimated close to 90%. The initial material was a cell culture supernatant. The volume of processed sample was 650 mL containing 100 [xg of antibody per milliliter. The column dimensions were 10 mm i.d. X 100 mm for both ion-exchangers. Flow rates: 500 cm / hr.

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SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

lgGlR.F.

i m V^

400

ml

80 ml

F I G U R E 25 Two-step purification of a mouse monoclonal IgGI from a cell culture supernatant containing 5% of fetal bovine serum using hydrophobic charge-induction chromatography followed by a column of hydroxyapatite. Three hundred milliliters of cell culture supernatant were directly injected into a HCIC column (A). The column (I.I cm i.d.X 10 cm) was first washed with a 50 m M tris buffer, pH 8 (arrow I), then by one column volume of 25 m M sodium caprylate In the previous buffer (arrow 2), and finally with distilled water (arrow 3). Elutlon of IgG was then obtained using a 50 m M sodium acetate buffer, pH 4 (arrow 4). Eluted IgG-rich fraction (IgGI R.F) was first adjusted at pH 6.8 by adding solid dipotassium phosphate. The obtained solution was then injected into a column of hydroxyapatitie (B) (HA Ultrogel) (6.6 mm i.d.X 100 mm) previously equilibrated with a 10 m M phosphate buffer, pH 6.8. The hydroxyapatite column was first washed with a 10 m M phosphate buffer, pH 6.8, to eliminate impurities and then antibodies were desorbed using a 0.5 M potassium chloride in 10 m M phosphate buffer, pH 6.8 (arrow a). Other bound proteins were finally desorbed by using 0.1 M posphate buffer pH 6.8 (arrow b). Flow rates for both columns was 90 m L / h r . The intermediate purity of IgG 1 from the first HCIC column was estimated close to 70%. The final purity from the second column (IgGI) was estimated above 97% by PAGE under reducing and nonreducing conditions and high-performance GPC.

Stock without the need to adjust the ionic strength and pH. Loading is therefore direct and the nonadsorbed proteins are pushed out by a 50 m M Tris buffer, pH 8. If the initial feedstock contains albumin (cell culture in the presence of fetal bovine serum or in the presence of pure albumin as supplements), the column has to be washed with a 25 m M sodium caprylate in tris buffer and elution follows by using a 50 m M acetate buffer, pH 4, or by a solution of 0.1 M citric acid at pH 3. Antibody fraction from an HCIC column can be then adjusted at pH 8 and loaded into a column of hydroxyapatitte, previously equilibrated with 10 m M phosphate buffer, pH about 7. Impurities are found in the flow through and pure antibodies are desorbed by increasing the ionic strength by a phosphate-buffered solution of potassium chloride. The main advantage of this method is its ability to eliminate not only impurities such as albumin, but also nucleic acids and possibly endotoxins. This methodology could also be used for the separation of IgM after a few adjustments and method optimization. Figure 25 represents an example of purification of a monoclonal IgGI. vii. Cation Exchange Followed by Gel Filtration

Frequently antibodies are produced by cell culture in protein-free media. Although the expression is low, their purification becomes a simple task. When classical cation-exchangers with S groups are used for the capture step, the ionic strength of the cell culture supernatant must be decreased to at least 5 mS/cm and the pH adjusted to 5.2-5.5. Note, however, that the required

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EGISTO BOSCHETTI AND ALOIS JUNGBAUER

ionic strength highly depends on the individual resin and antibody.^^^ The feedstock can be diluted by the addition of distilled water or can be diafiltered to maintain the antibody in the same starting concentration. With the dilution of the feedstock, the column loading will be very long and yield may be endangered by denaturation. With the the use of special highly substituted CM cation exchangers (see Fig. 26), the feedstock is not required to be adjusted in ionic strength, whereas pH is to be lowered to 4.2-4.8. The collected fraction is then loaded into a gel filtration column. This operation is achieved by adding concentrated solutions of citric acid. The use of other acids may induce precipitation of some proteins comprising the feedstock. Loading is then direct and elution can be performed by increasing ionic strength or by raising the pH while increasing the ionic strength. Although the fraction collected from the cation-exchange column is relatively pure, for further purification it can be directly loaded into a gel filtration column. A preliminary concentration may be necessary before the gel filtration column step. viii. Protein A Affinity- and Ion-Exchange Chromatography

IgG purification by protein A affinity chromatography produces usually relatively pure antibody in one step. Due to the high probability of finding traces of protein A fragments in the IgG preparation and other impurity traces, it is necessary, however, to use a second polishing column. Protein A-leached material from the affinity column has an acidic isoelectric point and, as such, it does not interact with cation-exchangers and can leave the column in the flowthrough, while immunoglobulins G in appropriate buffers

Gel filtration

F I G U R E 2 6 Two steps purification of a humanized monoclonal IgG I from a serum-free cell culture supernatant using a cation exchanger CM HyperD (a) followed by a gel filtration (b). Forty milliliters of filtered cell culture supernatant were added with 2 M citric acid so as to decrease the pH to 4.6, ionic strength was 18.8 mS • c m ~ '. The resulting solution was then directly injected into a 3 mm i.d.X 100 mm column at a flow rate of 300 c m / h r . The column was washed with a 50 m M sodium acetate buffer, pH 4.6, and elution of IgG I (arrow) accomplished using 50 m M T r i s - H C I buffer, pH 8, containing I M sodium chloride. Eluted IgG I fraction was directly injected into a gel filtration column (b) (4.6 mm i.d. X 600 mm in length) previously equilibrated with a 50 m M Tris - HCI buffer, pH 8, containing I M sodium chloride. Flow rate was 60 mL / hr. The purity of IgG I from the first column was estimated close to 9 0 - 9 5 % ; the final purity from the second column (IgGI) was estimated close to 99% by capillary electrophoresis in reduced and nonreduced form. Gel filtration removed large molecules of proteins identified as antibody aggregates and a small amount of very small molecules (third peak).

61

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

are adsorbed by ion exchange. Under normal conditions, traces of protein A are, however, associated with IgG by biological affinity and cannot be separated by regular ion-exchange chromatography. Dissociation agents are required to have protein A adsorbed and IgG released separately. Ethylene glycol and urea are agents that contribute to dissociate protein A from IgG molecules; they do not change the ionic environment and ion-exchange separation can be performed normally. Figure 27 shows the separation of an antibody of IgG class using protein A followed by a polishing step using a cation-exchange column at a pH where antibodies are adsorbed on the solid phase while impurities are found in the flowthrough. The separation by ion exchange can also be done using cationic sorbents. In this case and in the presence of mild dissociating agents (dilute buffered solutions of ethylene glycol and or urea), antibodies will be found in the flowthrough and impurity traces as well as traces of protein A will be adsorbed by ion exchange. ix. Cation-Exchange and Hydroxyapatite Chromatography

This separation process applies independently to antibodies of the IgG and IgM class. The feedstock is loaded onto a strong cation-exchanger after lowering the conductivity to less than 5 mS/cm and the pH adjusted to 5.0-5.5. Impurities can be partially removed by repeated washing using different buffers with different ionic strengths and pHs in such a way that the antibody still remains adsorbed on the cation-exchanger. Elution is then performed by using a phosphate buffer of pH between 7 and 7.5 at a concentration of 100-150 m M . Collected antibodies are already in a suited buffer, which enables loading on a hydroxyapatite column without further modification of pH and ionic strength. After several washing steps, antibodies are desorbed from hydroxyapatite column by a linear gradient or by a step gradient of phosphate buffer. This process is exemplified on Fig. 28. Mild

igCi

L F I G U R E 2 7 Purification of mouse IgG! monoclonal antibodies in t w o steps using an affinity column (protein A Ceramic HyperD) followed by a cation-exchanger (CM HyperD). A six milliliter sample of ascitic fluid previously diluted with an equal volume of phosphate-buffered physiological saline was directly injected into the chromatographic column (dimensions 6.2 mm i.d. X 120 mm), (A). After washing t o eliminate all nonadsorbed proteins, IgG I were desorbed by 0.1 m acetic acid (arrow). The collected fraction was then directly injected into the ion-exchanger at pH 4.5 (B). Column dimensions: 3 mm i.d. X 100 mm. Antibodies were finally desorbed by raising the ionic strength with sodium chloride up to \ M (arrow). Flow rates: 75 cm / hr.

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SP Trlsacryl M

HA Ultrogel

.''Ab

|>o.i 0.05

F I G U R E 2 8 Two-step purification of an IgGI monoclonal antibody from an ascitic fluid using a cation-exchange column (SP Trisacryl M, Trisacryl is a trademark of BioSepra-Life Technologies Inc., Rockville, MD) followed by a hydroxyapatite column (HA Ultrogel, HA Ultrogel is a trademark of BioSepra, Inc.). SP Trisacryl column: Column dimensions: 25 mm i.d. X 120 mm (60 mL of sorbent): loading buffer: 50 mM sodium acetate, pH 5.5; elution by addition of 0.2 M sodium chloride to the initial buffer; flow rate 40 m L / h r . The starting material was I mL of filtered ascitic fluid previously diluted with 9 volumes of adsorption buffer. The initial purity of IgG I in the feedstock was 28%. Eluted peak containing IgGI (Fl) showed a purity estimated by electrophoresis of 53%. This collected fraction was added with 15 m M of sodium phosphate up to pH 6.8. HA, Ultrogel column: Column dimensions: 11 mm i.d.X 100 mm (10 mL of sorbent); adsorption buffer: 10 m M phosphate buffer, pH 6.8; elution buffers were 10, 50, 100, 200, and 500 m M phosphate buffers pH 6.8; flow rate: 10 m L / h r . Collected peak at 100 m M phosphate buffer contained the IgGI antibody; its purity estimated by PAGE was 92%.

conditions used throughout the separation process are the most important advantage of the method.^^^ Binding capacity of hydroxyapatite is, however, modest. X. Hydrophobic Charge Induction Followed by Anion-Exchange Chromatography From a column of hydrophobic charge-induction chromatography the antibody fraction can be easily polished by an anion-exchange column. Due to the high binding capacity and good specificity of the first column, the amount of impurities present in the IgG fraction is lov^. The second column, being designed to adsorb impurities, will consequently have a small size. Impurities are dominantly composed of albumin especially with initial feedstocks that naturally contain albumin and cell culture supernatants supplemented with fetal bovine serum. This is also the case with ascite fluids. With anion-exchangers, albumin is tightly adsorbed and antibodies are found in the flowthrough (see also Fig. 8). One of the main advantages of this column sequence is the reduction of nucleic acids possibly present on the antibody fraction. DNA molecules as well as endotoxins are tightly adsorbed on the anion-exchange column while antibodies are in the flowthrough. Figure 29 shows an example of anion-exchange purification of antibodies captured by hydrophobic charge-induction chromatography.

VI. REGULATORY CONSIDERATIONS The production of therapeutic antibodies is guided by the same principles as are applied for recombinant proteins or plasma proteins. A common rule is to check for potency, consistency, and purity at each level of the production

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

A

jgC R.F.

613

B

pure IgG

F I G U R E 2 9 Two-step purification of a mouse monoclonal IgG I from an ascitic fluid using hydrophobic charge induction chromatography (A) followed by an anion exchange column (DEAE Ceramic HyperD) (B). Eighteen milliliters of filtered ascite fluid were directly injected into a phosphate buffered saline equilibrated HCIC column (A). The column (I.I cm i.d.X 10 cm) was first washed with a 50 m M T r i s - HCI buffer, pH 8; a second wash was performed using 25 m M sodium caprylate in the same buffer (arrow I) followed by a water wash (arrow 2). These washing steps were designed to eliminate the maximum amount of albumin. Elution was then obtained with a 50 m M sodium acetate buffer, pH 4.0. Eluted IgG I -rich fraction (IgG I R.F.) was added with Tris base up to pH 8.8 and ionic strength 7.4 mS / cm. It was then injected into a column of DEAE Ceramic HyperD (6.6 mm i.d. X 100 mm) previously equilibrated with a 50 m M Tris - HCI buffer, pH 8.8 (B). The column was then washed with the same buffer to collect all nonbound proteins corresponding to pure antibodies. Elution of adsorbed impurities (arrow 4) was achieved using a 50 m M T r i s - H C I buffer, pH 8.8, containing I M sodium chloride. The flow rates were 75 m L / hr for the first column and 160 cm / hr for the second column. The intermediate purity of IgG I from HCIC column was estimated as about 65 - 75%; the final purity from the second column (IgG!) was estimated close to 98% by PAGE in reduced and nonreduced form.

process. These three quahty criteria are the cornerstones on which all regulatory principles are founded. In general, the regulations of the different countries do not differ, although they are quite different when it comes to detailed protocols and tracing of the production process. In parallel, a laboratory information management system must be established to ensure the quality required for testing in process and the intermediate or final products. Especially the problem connected with the removal of biologically active contaminants will be discussed here. Detailed guidelines have been released from various U.S., European, and Japanese authorities. The most important guidelines can be downloaded from the Internet. The respective Internet addresses are Hsted in Table 16. These guidelines are rapidly developing and frequently revised, therefore it is recommended to check the latest information from the Internet sites. In 1995 a new European system for the authorization of medicinal products came into effect. After 10 years of cooperation between national registration authorities at the European Union (EU) level and 4 years of negotiations, in June 1993 the Council of the EU adopted three directives and a regulation, which together form the legal basis of the system. Since 1995, two new registration procedures for human and veterinary medicinal products have become available throughout the European Union: The centralized procedure is compulsory for medicinal products derived from biotechnology

=

TABLE I 6 Important Authorities Releasing Detailed Instructionsfor Manufacturingof Therapeutic Antibodies; the Listed U.S. Authorities Are Suborganizations of the Food and Drug Administration Country

Authority

United States

Center for Biologic Evaluation and Research (CBER)

Europe

International

Field of regulation

Releases “points to consider” for manufacturing of monoclonal antibodies and other biologics including test guidelines Center for Drug Evaluation Releases guidelines concerning pharmaceutical production (nonbiologics); some guidelines are also relevant for and Research (CDER) biologics Releases guidelines for approval of drugs, import licenses, Office of Regulatory Affairs and withdrawal of released products ( O W Releases guidelines for radiolabeled antibodies for Center for Devices and Radiological Health (CDRH) in uivo imaging Releases guideline for manufacture and distribution of Center for veterinary drugs and feed additives intended for animals Medicine (CVM) European Agency for the Regulatory authority of the European Commission regulation of human products and of veterinary products Evaluation of Medicinal Products (EMEA) Advisory body for EMEA: releases general guidelines Commitee for Proprietory regulating pharmaceutical production Midicinial Products (CPMP) Biotechnology Working Party Advisory body for EMEA: releases general guidelines (BW) regulating biotechnological production Advisory body for EMEA: releases general guidelines Blood Products Working regulating blood plasma products Group (BPWG) International Conference on Releases guidelines for quality control, safety testing, Harmonization (ICH) clinical studies, and common terminology

Internet address

www.fda.gov/cber/points.htm

www.fda.gov/cder/ www.fda.gov/ora/ www.fda.gov/cdrh/ www.fda.gov/cvm/ www.eudra.org/en-home.htm www.eudra.org/humandocs/about.htm www.eudra.org/vetdocs/a bout. htm Same as EMEA

Same as EMEA Same as EMEA www.pharmweb.nt/pwmirror/ pw9/ichS.htm

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

6 I 5

and is available at the request of companies for other innovative new products. The decentralized procedure, applying to a majority of conventional medicinal products, is based on the principle of mutual recognition of national authorizations. It provides for the extension of a marketing authorization granted by one member state to one or more other member states identified by the applicant. The production of proteins is associated with risks of either introducing substances with adverse effects, or failing to achieve total removal of impurity, or both. Potential contamination with adventitious agents is actually one of the main concerns for the product safety of biopharmaceuticals. This situation is extendible to antibodies, whether they are monoclonal or polyclonal, since they are isolated from various biological liquids (see Section III), which are themselves considered as potential sources of risk. The biological starting material contains other proteins, DNA and RNA, either from the medium or secreted by the host cell, and endotoxins. Endogenous and exogenous viruses, mycoplasma, bacteria, and proteins responsible for transmissible degenerative encephalopathies (TDE) must also to be considered as serious contaminants present in biological starting material. Existing regulations concerning biologicals for therapeutic applications demonstrate the high concern to define quality standards. With this concept, product purity and identity are assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectric focusing, aminoacid analysis, enzymatic breakdown combined with HPLC and mass spectrometry, and biological activity. Additionally, stringent DNA^^^ and virus removal validations are to be considered as well as leached chemicals from solid phase matrices used for chromatography. A regulatory consideration review for recombinant products has been published by Kozak et alP"^ In monoclonal antibody purification, biological risks are primarily related to the host animal cells, but also to animal supplements for culture medium such as fetal bovine serum or pure proteins (e.g., bovine albumin, insulin, and transferrin). A special risk associated with production of antibodies with rodent cell lines is their high load of C-type particles. These particles are considered as incomplete retroviruses. The danger regarding infecting humans is not clear. Thus, the efficient separation of these particles must be guaranteed. These particles are quantified either by immunological techniques or electron microscopy. A. Risks Related to Initial Raw Materials As detailed in Section III, initial raw materials for the production of antibodies are very different. Animal sera, transgenic milk, bovine colostrum, cell culture (hybridomas or other recombinant cells), ascites fluid, and egg yolk are the best known. Each raw material represents a potential carrier for adventitious agents. All of them can contain viruses. More specifically, animal sera used as source of antibodies, or as a cell culture medium supplement, can additionally carry mycoplasma and pyrogens depending on the method of collection.

6 I6

EGISTO BOSCHETTI AND ALOIS JUNGBAUER

Bovine colostrum as well as milk whey contain essentially bacteria and pyrogenic substances and may contain viruses. Humanized or specifically reconstructed antibodies from transgenic animals can contain traces of endogenous antibodies from the host animal that are very difficult to separate from the newly expressed molecules. When antibodies are from bovine and ovine origin, the possibility of having them contaminated by slow and latent agents such as TDE should be considered (e.g., BSE from bovine extracts, or scrapie from sheep or CJD in man). Possible biological contamination of antibodies expressed from plants are not yet clearly reported because this technology is not yet well developed. With the advent of this kind of production, attention must be paid to possible presence of plant agents of biological and biochemical origin such as alkaloids and related substances. B. Virus Detection and Clearance Contamination of biologicals by viruses is not just a theoretical possibility. This has been documented throughout the history of biologicals production. Human growth hormone, blood-derived products, and monoclonal antibodies are typical examples. The risk associated with the presence of viruses justified clearance studies that are one of the important places of safety documentation for final pure antibodies. These studies are referred to as "viral validation" and are based on clearance effect of extraction-purification steps and on virus removalinactivation steps. A comprehensive rational approach to guaranteeing a minimum risk has been reported by Berthold et alP^ Virus clearance studies are performed at the laboratory scale and are designed is such a way that the process is scaled down while all process operations are the same. Scaling down processing must be carefully validated to ensure it represents the regular purification scale process. Spiking with high titers of appropriate agents is a way to determine the clearance effect of a purification step. Virus reduction is then expressed as log^o reduction value (LRV) from one step to another or for the total process.^^^ Virus inactivation is another way to eliminate the infectivity. This operation is generally done before or after chromatography with well-known physical or chemical procedures. However, during a chromatographic process, virus inactivation can occur to a certain extent when inactivating buffers are used. Low pH buffers, urea-containing buffers, solvents, and detergents are examples usable in chromatography.^^ ^ For such vaUdation studies, one of the critical factors is the choice of viruses. This will unavoidably be dependent on the starting raw material; in the case of monoclonal antibody purification, which is frequently done from rodent cell line cultures, retroviruses are of particular concern. Terms used for virus classification related to the biological of interest and the manufacturing processing are "relevant viruses," "specific model viruses," and "nonspecific model viruses." The first refers to a virus that is likely to be present in the initial crude starting biological material, the second is a model

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

I B I

6 I 7

T A B L E 17 Removal of Viruses by Chromatographic Procedures Given in log,o Units Enveloped viruses Type of chromatography Affinity Protein G Cation exchange Sulfonate Type 1 Sulfonate Type 2 Anion exchange Diethyl aminoethyl Trimethyl amino ethyl Quaternary aminomethyl Gel filtration 3-70 KD-fraction

X-MuLV

E-MulV

>5.7

6.5

n.d. <1.0

PI-3

Nonenveloped viruses

vsv

Reo-3

SV-40

Polio

7.0

3.0

2.0

3.3

3.0

>6.3 n.d.

>6.3 <1.0

n.d. n.d.

2.8 <1.0

1.7 1.7

n.d. n.d.

4.3 n.d. 4.1

n.d. >6.3 >5J

6.0 3.1 >6.0

n.d. n.d. 9.1

6.0 >6.8 4.1

5.9 3.6 >7.0

n.d. n.d. <1.0

3.2

3.3

3.0

n.d.

2.6

2.6

n.d.

Adapted from Walter and Allgaier."^

but of the same family as the virus that could be present in the initial biological material, and the third is a model that is chosen on the basis of physical characteristics. The latter are used, for instance, to cover the risk of possible presence of unknown viruses. Models v^ith variations of nucleic acid content (RNA, DNA viruses), size (large and small), and external structure (enveloped, nonenveloped) are used. As an example, poliovirus, adenovirus, and herpesvirus are nonspecific models.^^^ Commonly used methods for virus detection are cell culture assays, in vivo inoculation into small animals, electron microscopy, and PCR.^^^ An example of virus clearance factors in chromatographic processes frequently used for purification of antibodies is given in Table 17. Lower clearance factors for protein A affinity chromatography have been found by Mariani and Tarditi^^^ when compared to results found with protein G by Walter and Allgaier.^^'' An explanation of this fact can be found in the fact that protein G requires harsher elution conditions than protein A. The probability of detecting a small number of virus particles must, however, be always taken into consideration.^^^ For instance, virus concentrations as low as 10-1000 particles per liter may not be detected, resulting in a wrong analytical conclusion. C. Risks Associated with Leached Material from Affinity Columns One of the aspects of resin deterioration is the leakage of ligands attached to the resin matrix by chemical links. Although ligand leakage has been particularly described for affinity sorbents, it also occurs for any kind of packing material where adsorption sites are available. Therefore ionizable chemical groups in ion-exchangers as well as hydrocarbon chains in hydrophobic sorbents can be the source of ligand deterioration and leakage. This phenomenon is important enough to be considered because it can be the origin of

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EGISTO BOSCHETTI AND ALOIS JUNGBAUER

the contamination of final pure monoclonal antibodies with possible adverse effects. Mechanisms resulting in the release of entire or partial ligand into the mobile phase during the chromatographic separation cycle are classified as a function of their origin. Breakage of the attachment point, ligand leaching because partial matrix hydrolysis, release of physically entrapped material within the matrix network, dissociation of adsorbed but not covalently bound ligand, subunit dissociation of ligands, and chemical or biochemical hydrolysis of the ligand itself are the major reasons for ligand release. These phenomena are well known and described extensively; they are, however, somewhat neglected because they do not significantly impact the behavior of the column. When dealing with biological liquids such as those described in Section III, proteases can be present in active or inactive form. They can both degrade the protein to be purified and hydrolyze the immobilized ligand if it is of a proteinaceous nature. Partial hydrolysis or subunit dissociation has been actually shown with macromolecular ligands such as proteins. Dissociation of Concanavalin A used as a ligand in individual subunits in the absence of calcium ions and in the presence of chaotropic agents can result into the loss of partial structure that may contaminate the purified antibody. All multimeric ligands involving subparts in their structure that may reversibly be dissociated (e.g., LDH and antibodies when used as ligands) represent a potential danger of leakage. In the specific case of affinity chromatography with immobilized protein A and protein G, very popularly used in the purification of antibodies, it has been shown that there is some level of ligand leakage due proteolytic action. Released peptides can still be associated with the purified antibody and are very difficult to evidence and to separate. Special process steps addressing this specific point are indicated elsewhere.

D. Sanitization of Columns: General Considerations Disinfection, sanitization, germ reduction, and sterilization are very important topics in the production of injectable pharmaceuticals and fine chemicals for in vivo use. Depending on the step in a production process, there are different requirements for the germ content of the environment which is in direct contact with the product to be purified. Most frequently, germ inactivation is required, but pyrogen removal and virus clearance are often mandatory for therapeutic protein purification. At the beginning of the process low germ counts can be accepted, but at later stages complete sterility and apyrogenicity of the product are required. Generally, for complete aseptic column chromatography processes, the resins must be sterilized, although the complete success of these operations are still the dream of many process engineers. A highly effective way of destroying germs by dry or wet heat is limited by the temperature sensitivity of the resins or the process equipment. Treatment with radiation may be hazardous and is often not applicable in industrial environments. Germ reduction with gaseous agents such as beta propiolactone or ethylene oxide is

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

6 I 9

not appropriate because of their chemical reactivity and explosion hazards. Formaldehyde solutions should also be avoided because of their claimed carcinogenic effect, or at least because they can be potent allergens. The germ reduction of chromatographic resins is conventionally carried out by chemical treatments before or after packing a column. The choice of the sanitizing agent is largely dependent on the sensitivity of the resin. i. Ethanol Mixtures

A very commonly used disinfectant is ethanol-water in neutral or, preferably, in acidic conditions. Aqueous ethanol displays its best germicidal efficiency at a concentration of 60 to 70%. However, the most commonly used concentration in industry is about 20%, because higher concentrations require specific explosion-proof facilities. At 20%, ethanol has no sporicidal effect, its effect on viral inactivation is only partial and it does not destroy pyrogens (it only tends to destabilize large molecular aggregates of lipopolysaccharide molecules). For these reasons, 20% ethanol can only be considered as a bacteriostatic agent. Mixtures of ethanol with bases or acids are somewhat more sporicidal, but are not sufficient to provide sterilization at short incubation times and low temperatures. ii. Sodium Hydroxide

Sodium hydroxide is widely used in the biotech industry to both regenerate and sanitize chromatography resins to a certain extent. The practice of using sodium hydroxide as a cleaning agent varies a lot in terms of concentration (0.1 to 1 N ) , incubation times (hours to several days), temperature, and column volumes passed through the column (1 to more than 10). Sodium hydroxide, even at concentrations of 0.5 N and for several hours incubation, is not effective in totally inactivating sporulated germs of Bacillus subtilis used as a model recommended by the Pharmacopoeia and the American Microbial Society (Fig. 30). Tens of hours are necessary to reduce significantly the germ concentration of spores of Bacillus subtilis using sodium hydroxide.^"^^ Sodium hydroxide at concentrations at concentrations of 0.1 to 0.2 N for 30 min at room temperature can sometimes destroy pyrogens generated on a chromatographic column. Pyrogens are practically unaffected by ethanol, but are very sensitive to alkaline hydrolysis. iii. Oxidizing Agents

The oxidizing agent sodium hypochlorite is a well-known sanitizer. Its toxicity toward microbes is well documented. Despite its excellent germicidal properties it has the advantage of being harmless to human skin and mucoses. Its use as an agent for sanitizing chromatographic resins has been limited by the fear of generating toxic chlorinated by-products during the procedure. Other hypohalogenites, based on F, Br, or I are limited to special applications because of corrosive and hazardous effects as well as their high cost. Among strong oxidizing agents with bacterial, sporicidal, and virucidal effects with a low toxicity level, peracetic acid has been described with a special interest.^^^"^"^^

620

EGISTO BOSCHETTI A N D ALOIS JUNGBAUER

1E7

^4*C

1EB •

3.7E-K)5

TrsE^os

1E8

y0.6C»(>»

164 4.7&KH

LOE^OS

I I 11 I 11 111 I 11 11 I I I I 11 i I I I 11 I I i 1111 i I

1.0E^02

tliiM (iilin]

1E7

i25X

AZO&KB 168 1ES

-

. 3,1Et06 • 1.9&K»

B

#6,^-^

164

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•3.5&KB 31,06*02

|6.0EtO1 _46400

1.06^1

I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I M M I

1,06*00 0

1000

2000

3000 4000 tiOM (mln]

6000

6000

F I G U R E 3 0 Inactivation kinetics of spores of Bacillus subtilis affected by 0.5N sodium hydroxide at 4°C (A) and at 25°C (B) according to Jungbauer and Lettner.^"*^

VII. GENERAL CONCLUSION ON ANTIBODY SEPARATION TECHNOLOGIES In spite of the large amount of resources spent at the research level for a number of years, disappointing therapeutic results prevented the commercial development of therapeutic antibodies. Because of their high specificity, they therefore have heavily supported the diagnostic development with numerous applications. A number of positive clinical data based on different antibody constructions generated a considerable opportunity of development for w^ell-defined applications. The question of their possible use for w^ell-targeted human diseases does not require repeating. Presently, antibodies along v^ith all their derivatives represent the largest class of biodrugs in development v^ith a promising emerging antibody therapeutics. Extraction, separation, and purification of antibodies w^ill undoubtedly follow^ and evolve in parallel v^ith the evolution of the productions and applications. Today biochemists have a complete set of recipes and tool boxes to obtain very pure antibodies. The use of what exists in a rational and effective

SEPARATION OF ANTIBODIES BY LIQUID CHROMATOGRAPHY

62 I

manner is something that must be considered according to the nature and the source of the antibody in question. The design of an antibody purification process must first take into consideration the final utifization of the molecule. The degree of purity will not actually be the same if the product is to be used for research purposes or for long-term therapeutical application. The design should also consider the scale of the final process to meet economic requirements. One of these is related to the use of water. Liquid chromatography involves a large use of aqueous buffers and a way to save part of this element is to adjust conditions between columns so as to use first similar buffers and decrease the column volumes. This can be achieved by choosing high-binding-capacity resins that are easily cleanable with standard cleaning solutions composed of chemical agents. Finally, since specific impurities must be eliminated for regulatory reasons, the process design configuration must be part of a global approach. REFERENCES 1. Neuberger, M. S., Williams, G. T., Mitchell, E. B., Jouhal, S. S., Flanagan, J. G., and Rabbitts, T. H. (1985). A hapten-specific chimaeric IgE antibody with human physiological effector function. Nature, (London) 314, 268-270. 2. Winter, G., Griffiths, A. D., Hawkins, R. E., and Hoogenboom, H. R. (1994). Making antibodies by phage display technology. Annu. Rev. Immunol. 12, 433-455. 3. Winter, G. (1998). Synthetic human antibodies and a strategy for protein engineering. FEES Lett. 430, 92-94. 4. FitzGerald, K., Holliger, P., and Winter, G. (1997). Improved tumour targeting by disulphide stabilized diabodies expressed in Pichia pastoris. Protein Eng. 10, 1221-1225. 5. Skerra, A., and Pluckthun, A. (1988). Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science 240, 1038-1041. 6. Horwitz, A. H., Chang, C. P., Better, M., Hellstrom, K. E., and Robinson, R. R. (1988). Secretion of functional antibody and Fab fragment from yeast cells. Proc. Natl. Acad. Sci. U.S.A. 85, 8678-8682. 7. Heseman, C. A., and Capra, D. J. (1990). High level production of a functional immunoglobulin heterodimer in a baculovirus expression system. Proc. Natl. Acad. Sci. U.S.A. 87, 3942-3946. 8. Weidle, U. H., Borgya, A., Mattes, R., Lenz, H., and Buckel, P. (1987). Reconstitution of functionally active antibody directed against creatine-kinase from separately expressed heavy and light chains in non-lymphoid cells. Gene 51, 131-141. 9. Fulton, S. (1999). Production of antibodies in transgenic milk. Proc. Antibody Prod. Downstream Process., San Diego, CA, 1999. 10. Russell, D. A., (1999). Plant-based production of monoclonal antibodies: From field to clinic. Proc. Antibody Prod. Downstream Process., San Diego, CA, 1999. 11. Roitt, I., Brostoff, J., and Male, D. (1986). "Immunology." Gower Medical Pubfishing, London and New York. 12. Nose, M., and Wigzell, H. (1983). Biological significance of carbohydrate chains on monoclonal antibodies. Proc. Natl. Acad. Sci. U.S. A. 80, 6632-6636. 13. Silverton, E. W., Navia, M. A., and Davies, D. R. (1977). Three-dimensional structure of an intact human immunoglobulin. Proc. Natl. Acad. Sci. U.S.A. 74, 5140-5144. 14. Wenisch, E., Jungbauer, A., Tauer, C , Reiter, M., Gruber, G., Steindl, F., and Katinger, H. (1989). Isolation of human monoclonal antibody isoproteins by preparative isoelectric focusing in immobilized pH gradients. / . Biochem. Biophys. Methods 18, 309-322. 15. Hamilton, R. G., Reimer, C. B., and Rodkey, L. S. (1987). Quality control of murine monoclonal antibodies using isoelectric focusing affinity immunoblot analysis. Hybridoma 6, 205-217.

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16. Bond, A., Jones, M. G., and Hay, F. C. (1993). Human IgG preparations isolated by ion-exchange or protein G affinity chromatography differ in their glycosylation profiles. / . Immunol. Methods 166, 2 7 - 3 3 . 17. Patel, T. P., Parekh, R. B., Moellering, B. J., and Prior, C. P. (1992). Different culture methods lead to differences in glycosylation of a murine IgG monoclonal antibody. Biochem. } . 285, 839-845. 18. Maiorella, B. L., Winkelhake, J., Young, J., Moyer, B., Bauer, R., Hora, M., Andya, J., Thomson, J., Patel, T., and Parekh, R. (1993). Effect of culture conditions on IgM antibody structure, pharmacokinetics and activity. Bio/Technology 11, 387-392. 19. Karl, D. W., Donovan, M., and Flickinger, M. C. (1990). A novel acid proteinase released by hybridoma cells. Cytotechnology 3, 157-169. 20. Licari, P. J., Jarvis, D. L., and Bailey, J. E. (1993). Insect cell hosts for baculovirus expression vectors contain endogenous exoglycosidase activity. Biotechnol. Prog. 9, 146-152. 21. Morrison, S. L., Johnson, M. J., Herzenberg, L. A., and Oi, V. T. (1984). Chimeric human antibody molecules: Mouse antigen-binding domains vv^ith human constant region domains. Proc. Natl. Acad. Sci. U.S.A. 81, 6851-6855. 22. Orlandi, R., Gussow^, D. H., Jones, P. T., and Winter, G. (1989). Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. Proc. Natl. Acad. Sci. U.S.A. 86,3833-3837. 23. McCafferty, J., Griffiths, A. D., Winter, G., and Chiswell, D. J. (1990). Phage antibodies: Filamentous phage displaying antibody variable domains. Nature (London) 348, 552-554. 24. Forsgren, A., and Sjoquist, J. (1966). "Protein A" from S. aureus. I. Pseudo-immune reaction with human gamma-globulin. / . Immunol. 97, 822-827. 25. Kohler, G., and Milstein, C. (1975). Continuous cultures and fused cells secreting antibodies of predefined specificity. Nature (London) 256, 495. 26. Casah, P., Inghirami, G., Nakamura, M., Davies, T. F., and Notkins, A. L. (1986). Human monoclonals from antigen-specific selection of B lymphocytes and transformation by EBV. Science 234, 476-479. 27. Buchacher, A., Predl, R., Strutzenberger, K., Steinfellner, W., Trkola, A., Purtscher, M., Gruber, G., Tauer, C , Steindl, F., and Jungbauer, A. (1994). Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for peripheral blood lymphocyte immortalization. AIDS Res. Hum. Retroviruses 10, 359-369. 28. Unteriuggauer, F., Doblhoff-Dier, O., Tauer, C , Jungbauer, A., Gaida, T., Reiter, M., Schmatz, C , Zach, N., and Katinger, H. (1994). Stable, continuous large-scale production of human monoclonal HIV-1 antibody using a computer-controlled pilot plant. BioTechniques 16, 140-147. 29. Samoilovich, S. R., Dugan, C. B., and Macario, A. J. (1987). Hybridoma technology: New^ developments of practical interest / . Immunol. Methods 3, 153-170. 30. Ozturk, S. S., and Palsson, B. O. (1990). Chemical decomposition of glutamine in cell culture media: Effect of media type, pH, and serum concentration. Biotechnol. Prog. 6, 121-128. 31. Ozturk, S. S., and Palsson, B. O. (1990). Loss of antibody productivity during long-term cultivation of a hybridoma cell line in low^ serum and serum free media. Hybridoma 9, 167-175. 32. Ozturk, S. S., and Palsson, B. O. (1990). Effect of initial cell density on hybridoma growth, metabolism, and monoclonal antibody production. / . Biotechnol. 16, 259-278. 33. Kundu, P. K., Prasad, N. S., and Datta, D. (1998). Monoclonal antibodies: High density culture of hybridoma cells and downstream processing for IgG recovery. Indian J. Exp. Biol. 36, 125-135. 34. Cherlet, M., and Marc, A. (1998). Intracellular pH monitoring as a tool for the study of hybridoma cell behavior in batch and continuous bioreactor cultures. Biotechnol. Prog. 14, 626-638. 35. Bohmann, A., Portner, R., and Markl, H. (1995). Performance of a membrane-dialysis bioreactor with a radial-flow fixed bed for cultivation of a hybridoma cell line. Appl. Microbiol. Biotechnol. 43, 772-780.

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199. Buettner, J. A., Dadd, C. A., Baumbach, G. A., Masecar, B. L., and Hammond, D. J. (1996). Chemically derived peptide libraries: A new resin and methodology for lead identification. Int. J. Pept. Protein Res. 47, 70-83. 200. Li, R., Dowd, V., Stewart, D. J., Burton, S. J., and Lowe, C. R. (1998). Design, synthesis and application of a protein A mimetic, Nat. Biotechnol. 16, 190-195. 201. Guerrier, L., Flayeux, L, Schwarz, A., Fassina, G., and Boschetti, E. (1998). IRIS 97: An innovating Protein A-peptidomimetic solid phase media for antibody purification. / . MoL Recognition, 11, 1-3. 202. Fassina, G., Verdoliva, A., Palombo, G., Ruvo, M., and Cassani, G. (1988). Immunoglobulin specificity of TG19318: A novel synthetic ligand for antibody affinity purification. / . Mol. Recognition 11, 128-233. 203. Lihme, A., and Bendix Hansen, M. (1997). Protein A mimetic for large scale monoclonal antibody purification. Biotechnol. Lab. 15, 3 0 - 3 1 . 204. Mohan, S. B., and Lyddiatt, A. (1997). Recent developments in affinity separation technologies. In "Affinity Separations" (P. Matejtschuk, ed.), pp. 1-38. IRL Press, Oxford. 205. Konecny, P., Brown, R. J., and Scouten, W. H. (1996). Purification of monospecific polyclonal antibodies from hyperimmune bovine whey using immunoaffinity chromatography. Prep. Biochem. Biotechnol. 26, 229-243. 206. Bazin, H., Xhurdebise L. M., Burtonboy, G., Lebacq, A. M., De Clercq, L., and Cormont, F. (1984). Rat monoclonal antibodies. I. Rapid purification from in vitro culture supernatants. / . Immunol. Methods 66, 261-269. 207. Bazin, H., Cormont, F., and De Clercq, L. (1984). Rat monoclonal antibodies. II. A rapid and efficient method of purification from ascitic fluid or serum. / . Immunol. Methods 71, 9-16. 208. Williamson, K. C , Duffy, P. E., and Kaslow, D. C. (1992). Immunoaffinity chromatography using electroelution. Anal Biochem. 206, 359-362. 209. Murray, A., Sekowski, M., Spencer, D. I., Denton, G., and Price, M. R. (1997). Purification of monoclonal antibodies by epitope and mimotope affinity chromatography. / . Chromatogr A 782, 49-54. 210. Kondoh, H., Kobayashi, K., and Hagiwara, K. (1987). A simple procedure for the isolation of human secretory IgA of IgAl subclass by a jackfruit, lectin, jacalin, affinity chromatography. Mol. Immunol. 24, 1219-1222. 211. Shibuya, N., Berry, J. £., and Glodstein, I. J., (1988). One-step purification of murine IgM and human a2-macroglobulin by affinity chromatography on immobilized snowdrop bulb lectin. Arch. Biochem. Biophys. 267, 676-680. 212. Nevens, J. R., Mallia, A. K., Wendt, M. W., and Smith, P. K. (1992). Affinity chromatographic purification of immunoglobulin M antibodies utilizing immobilized mannan binding protein. / . Chromatogr. 597, 247-256. 213. Oppenheim, J. D., Amin, A. R., and Thorbecke, G. J. (1990). A rapid one step purification procedure for murine IgD based on the specific affinity of Bandeiraea (Griffonia) simplificifolia-1 for N-linked carbohydrates on IgD. / . Immunol. Methods 130, 243-250. 214. Ngo, T. T. (1994). Rapid purification of immunoglobulin G using aza-arenophilic chromatography: Novel mode of protein-solid phase interaction. / . Chromatogr. 662, 351-356. 215. Ngo, T. T., Khatter, N., and Avid, A. L. (1992). A synthetic ligand affinity gel mimicking immobilized bacterial antibody receptor for purification of immunoglobulin G. / . Chromatogr. 597, 101-109. 216. Khatter, N., Matson, R. S., and Ngo, T. (1992). Utilization of a synthetic affinity ligand for immunoglobulin purification. Int. Chomtogr. Lab. 11, 14-18. 217. el-Kak, A., Manjiny, S., and Vijayalakshmi, M. A. (1992). Interaction of immunoglobulin G (IgG) with immobilized histidine: Mechanistic and kinetic aspects. / . Chromatogr 604, 29-37. 218. Mbida, A. D., Kanoun, S., and Vijayalakshmi, M. A. (1989). Purification of IgGl subclass from human placenta by pseudoaffinity chromatography. In "Biotechnology of Plasma Proteins," Vol. 175, pp. 237-243. INSERM, Paris. 219. Nau, D. R. (1986). A unique chromatographic matrix for rapid antibody purification. Biochromatography 1, 82-94.

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220. Ross, A. H., Herlyn, D., and Koprowski, H. (1987). Purification of monoclonal antibodies using Abx. / . Immunol. Methods, 102, 227-232. 221. Neidhardt, E. A., Luther, M. A., and Recny, M. A. (1992). Rapid, two-step purification process for the preparation of pyrogen-free murine immunoglobulin G^ monoclonal antobodies. / . Chromatogr. 590, 255-261. 222. Porath, J., and Flodin, P. (1959). Gel filtration: A method for desalting and group separation. Nature (London), 4676, 1657-1659. 223. Laurent, T. C., and Kilander, J. (1964). A theory of gelfiltration and its experimental verification, / . Chromatogr. 14, 317-330. 224. Rodbard, D. (1976). "Methods of Protein Separation" (N. Catsimpolas, ed.). Plenum, New York. 225. Schmidt, C. (1989). The purification of large amounts of monoclonal antibodies. / . Biotechnol. 11, 235-252. 226. Osterlund, B. (1995). "Mab Assistant." Pharmacia Biotech AB, Uppsala. 227. Duffy, S., Moellering, B. J., Prior, G. M., Doyle, K. R., and Prior, C. P. (1989). Recovery of therapeutic-grade of antibodies: Protein A and ion exchange chromatography. BioPharm, June, pp. 35-47. 228. Ostlund, C., Borwell, P., and Malm, B. (1987). Process-scale purification from cell culture supernatants: Monoclonal antibodies. Deu. Biol. Stand. 66, 367-375. 229. Schwartz, W., Clark, F., and Sabran, I. B. (1986). Process scale isolation and purification of immunoglobulin G. LC-GC 5 310-316. 230. Necina, R., Amatschek, K., and Jungbauer, A. (1998). Capture of human monoclonal antibodies from cell culture supernatant by ion exchange media exhibiting high charge density. Biotechnol. Bioeng. 60, 689-698. 231. Carlsson, M., Hedin, A., Inganas, M., Harfast, B., and Blomberg, F. (1985). Purification of in vitro produced mouse monoclonal antibodies. A two-step procedure utilizing cation exchange chromatography and gel filtration. / . Immunol. Methods. 79 89-98. 232. Sene, C , Santambien, P., Pouradier Dutail, X., and Boschetti, F. (1990). Purification of IgG monoclonal antibodies. A simple and adaptable method. Chim. Oggi, 8, 1 5 - 2 1 . 233. Tauer, C , Buchacher, A., and Jungbauer, A. (1995). DNA clearance in protein A affinity chromatography. / . Biochem. Biophys. Methods. 30, 75-78. 234. Kozak, R. W., Durfor, C. N., and Scribner, C. L. (1992). Regulatory considerations when developing biological products. Report of the center for biologies evaluation and research. Cytotechnology, 9, 203-210. 235. Berthold, W., Walter, J., and Werz, W. (1992). Experimental approaches to guarantee minimal risk of potential virus in purified monoclonal antibodies. Cytotechnology, 9, 189-201. 236. Josic, D., Schulz, P., Biesert, L., Hoffer, L, Schwinn, H., Kordis-Krapez, M., and Strancar, A. (1997). Issues in the development of medical products based on human plasma. / . Chromatogr. B 694, 253-269. 237. Walter, J., and AUgaier, H. (1997). Validation of downstream processes. In "Mammalian Cell Biotechnology" (H. Hauser and R. Wagner, eds.), pp. 453-482. de Gruyter, Berfin. 238. Irving, J. M., Chang, L. W., and Castillo, F. J. (1993). A reverse transcriptase-polymerase chain reaction assay for the detection and quantitation of murine retroviruses. Bio Technology 11, 1042-1046. 239. Lower, J. (1996). Risk assessment and limitations of evaluation studies. Dev. Biol. Stand. 88, 109-117. 240. Whitehouse, R. L., and Clegg, C. F. L. (1963). Destruction of JB. Substilis spores with solutions of sodium hydroxide. / . Dairy Res. 30, 315-321. 241. Jungbauer, A., Lettner, H. P., Guerrier, L., and Boschetti, E. (1974). Chemical sanitization in process chromatography. Part 2: In situ treatment of packed columns and long-term stability of resins. Bio-Pharm. 7, 37-42. 242. Jungbauer, A., and Lettner, H. (1994). Chemical disinfection of chromatographic resins. Part 1: Preliminary studies and microbial kinetics. BioPharm 6, 46-56. 243. Schaffner, A., Kaluza, B., and Weidle, U. H. (1997). Genetic engineering of antibodies and derivatives from mammalian cells. In "Mammalian Cell Biotechnology in Protein Production." de Gruyter, Berlin.

PROCESSING PLANTS AND EQUIPMENT P. BOWLES Kvaerner Process (UK), Ltd., Whiteley, Hants, United Kingdom

I. INTRODUCTION II. INDUSTRIES USING BIOSEPARATIONS A. Pharmaceuticals and Biopharmaceuticals B. Food and Beverage C Waste Water Treatment D. Chemicals and Fuels III. PROCESS-SCALE BIOSEPARATIONS A. Selection Criteria B. Biomass Separation and Primary Recovery C. Product Purification and Final Isolation IV. PROCESS-SCALE CONSIDERATIONS A. Materials of Construction and Mechanical Design B. Automation C. Safety and Biosafety D. Location E. GMP and Validation F. Hygienic Design V. SUMMARY REFERENCES

INTRODUCTION The diversity of industries which involve bioseparations has led to the development of a wide range of techniques and unit operations for the efficient processing of biological materials. The objective of this chapter is to aid the scientist or engineer in selecting a method of bioseparation which will be suited to the particular requirements of the process and the product at a commercial scale of operation. The complexity of biological processes generally requires many stages to produce a final, purified product from a particular composition of raw materials. Although a typical bioprocess consists of two main parts: upstream fermentation and downstream product recovery, it is not unusual to have between 10 and 20 steps in the overall process. This reflects the complex Separation Science and Technology,

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nature of a typical fermentation broth, which will consist of an aqueous mixture of cells, intracellular or extracellular products, unreacted substrates, and by-products of the fermentation process. From this mixture, the desired product must be isolated at a given purity and specification, and all of the unwanted contaminating materials must be removed. The choice of a bioseparation technique will depend on a number of factors, including the initial location of the product inside or outside the cell as well as the product size, charge, solubility, chemical, or physical affinity to other materials, and so on. Economic factors also come into play, including the value of the product, the regulatory environment in which the product is manufactured, and the balance between the capital cost of the bioseparation equipment and the operating cost of running it. In moving from laboratory- or pilot-scale processing to full-scale manufacture, it can be difficult to scale up certain types of bioseparation equipment easily, for example high g centrifuges are available as bench mounted units (using test tubes), but an equivalent industrial machine with a similar g force is unlikely to be a cost effective solution, even if it is possible to build a suitable unit. It would not be realistic to consider 10 or 100 identical units as a realistic alternative. Compromises are therefore required as a process is commercialized to ensure that it remains technically and economically feasible. In this chapter, guidance is provided concerning the choice of industrial bioseparation equipment which is available, and the issues which must be taken into account when selecting a suitable system to meet both technical and economic objectives. II. INDUSTRIES USING BIOSEPARATIONS In this section, the wide range of industries using bioseparation techniques are briefly reviewed. A. Pharmaceuticals and Blopharmaceuticals Bioseparations are essential in the manufacture of high-volume, low-value bulk pharmaceuticals and nutraceuticals such as antibiotics and vitamins, where economies of scale are used to ensure commercial competitiveness. At the opposite end of the pharmaceutical product spectrum, genetically engineered therapeutic proteins of extremely high value are produced at very small scale. These different products share a common requirement for a large number of bioseparation stages to isolate the product at an acceptable level of purity. This is particularly important in the pharmaceutical industry where product manufacturing is closely regulated and controlled at all stages to ensure that the medicines produced are effective and safe to use. Product consistency between batches must also be achieved. Manufacturers are obliged to make great efforts to demonstrate these requirements while developing and operating pharmaceutical manufacturing processes, and a later section in this

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chapter discusses validation and good manufacturing practice (GMP) in more detail. B. Food and Beverage The majority of food and beverage products are based on biological materials, although not all are produced using fermentation techniques. Dairy products such as yoghurt and cheese; beverages including beer, fruit juices, and v^ine; and single-cell proteins for human or animal consumption all involve bioseparations. As with pharmaceuticals, the food industry is highly regulated and food hygiene considerations are paramount in any manufacturing process. Many food production processes are based on batch rather than continuous manufacturing, with the need to dismantle and clean equipment between batches. Cleaning in place, or CIP, is increasingly important in pharmaceutical and food processes to reduce labor requirements for manual cleaning and to improve levels of cleanliness, in particular, the repeatability of cleaning between different batches. The competitive nature of the food and beverage industry and the need for continued improvements in cost-effective manufacturing have provided an impetus for companies to develop and use new bioseparation techniques at very large scales, for example, freeze-drying in coffee production and continuous centrifugation in brewing. Many food industry innovations are now slowly being adopted by pharmaceutical manufacturers as they also come under increasing pressure to help reduce health care costs. C. Waste Water Treatment Many waste water treatment processes involve biological processes to reduce the concentration of a wide range of contaminants including organic materials, nitrogen, and phosphates. Activated sludge, aerobic and anaerobic digestion processes are used for industrial and municipal effluent treatment. Generally these processes require subsequent bioseparation of the microorganisms from the treated waste water. Examples of commonly used bioseparations include sedimentation, coagulation, and filtration. The scale of operation for such bioseparation processes is considerable, because of the volumes of effluent which are processed and the throughputs required. Proprietary aerobic digesters such as the Deep Shaft process may use centrifugation to recover biomass from the treated effluent for recycle as an inoculum for the digester or to reduce the quantity of water before sending the solid material either to incineration or land fill. D. Chemicals and Fuels There is an increasing trend toward the production of fine and commodity chemicals on a very large scale using fermentation processes followed by downstream bioseparation and purification. This trend is being driven by the

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availability of renewable feedstocks for such processes, with a positive effect on the environment, the possibility of processing at near ambient conditions, compared with the high temperatures and pressures required for chemically synthesised equivalents, and the isolation and commercialization of more efficient microbial strains which can convert raw materials to useful products. Citric acid and vitamin C are examples of very large scale fermentation processes where the subsequent product isolation involves several bioseparations, including filtration, precipitation, evaporation, crystaUization, and drying methods. The scale of operation requires careful choice of equipment which is robust, efficient in separating product from unwanted by-products, and cost effective to be competitive. There are continuing efforts to develop cost-effective processes for fuel alcohol production, although the economics are often dependent on the availability of subsidized feedstocks to compete with traditional fuels derived from oil. The pretreatment and fermentation of such feedstocks, derived from corn, sugar cane, and even municipal waste, yields a dilute aqueous solution of ethanol which must be separated from a complex mixture of waste materials and then concentrated by distillation to remove water. Both batch and continuous production processes have been developed, with the requirement for effective bioseparations during both the pretreatment and ethanol recovery parts of the process. The development of genetically engineered plants offers the prospect of pharmaceutical production from crops as well as improved yields for cereals, vegetables and other agricultural products. The challenge will then be to find suitable bioseparations to enable the efficient isolation of such products.

III. PROCESS-SCALE BIOSEPARATIONS The industries described are diverse but all require bioseparations at various scales. Although not all such manufacturing processes involve fermentation, it is possible to identify common types of bioseparations which are required at particular stages. A typical process will include the following bioseparation steps: Biomass separation of insoluble from soluble material, with either phase being retained depending on the location of the product as intracellular or extracellular material. Examples of unit operations commonly used include centrifugation, filtration, and sedimentation. Primary recovery of the active ingredient from the solid or liquid phase to remove large quantities of unwanted waste materials, which may themselves be processed further. Suitable techniques include solvent extraction, precipitation by chemical or physical changes to the product-containing solution, and ultrafiltration or microfiltration to separate products above a particular size. Work done on combined biomass separation-primary product recovery processes such as expanded-bed adsorption are now being commercialized in the pharmaceutical industry.

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Purification of the product and removal of specific impurities by suitable methods, based on the size, charge, or solubility of the materials being separated. Chromatographic techniques are widely used for these types of purification steps, as well as adsorption and crystallization. Final product isolation in a form suitable for further processing into the final dose form of the pharmaceutical, e.g., as a tablet or an injectable solution. Secondary production of this type is sometimes done in a separate facility, with the raw material referred to as the bulk product or, more recently, the active pharmaceutical ingredient. Examples of unit operations at this stage of processing include lyophilization, precipitation, or crystallization followed by solid isolation using filtration and drying techniques. In some cases, the final product must be produced in a sterile form, which introduces additional compHcations when selecting suitable process equipment.

A. Selection Criteria

For all of the bioseparation types last referred to, there are a number of selection criteria to be considered when developing a commercial scale process. [. Location and Nature of Product

The product may be located inside a microorganism (intracellular) or outside in the growth medium (extracellular), or alternatively, the product could be the whole cell material. The nature of the product may be solid or dissolved in the aqueous phase. For example, the product is found in the aqueous phase for a fuel ethanol fermentation, within the cell for a therapeutic protein, while the product is the whole cell in the case of single cell protein. The location of the product influences the choice of a bioseparation method which may favor the efficient recovery of either the solid or liquid phase. The relative difficulty of separating intracellular products from other unwanted insoluble materials may influence the subsequent processing steps once the solids phase has been recovered from the fermentation broth. The cell line from which the product is derived will also play a part in the decision-making process, because bioseparation techniques are likely to be needed depending on whether the fermentation is based on mammalian, microbial, fungal, or yeast cells. Different fermentation broths demonstrate varying characteristics of viscosity, density, particle size, and charge which may enable exploitation of a difference in that characteristic between the phases to be separated. ii. Production Scale, Quality, and Regulatory Environment

In the industries using bioseparations described above, there is a great variation in terms of production scale and product quality between waste water treatment and pharmaceutical production. This will obviously affect the choice of equipment for the process, although in many cases the principle on which bioseparation is based will be common. For example, centrifuga-

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tion techniques are widely used in both industries, ahhough the size, design of the equipment and type of centrifugation method are different. Where small-scale bioseparations have been developed, particularly in the biopharmaceutical industry, there has been a tendency to retain laboratory type equipment even if this results in more labour and capital intensive processing. The reason for this is often to avoid the need for extended periods of process development work with new equipment designs, which might delay the launch of a product where competitors are not far behind. Manufacturers are also wary of adopting new bioseparation techniques for processes if there is any risk that regulators such as the U.S. Food and Drug Administration (FDA) will require more evidence that the equipment is fit for the purpose. This conservative tendency is understandable and may influence the choice of bioseparation equipment for pharmaceutical manufacturing in particular. The required product quality and therefore the value of the product to the consumer will also influence the choice of bioseparation technique. Usually a more efficient or specific bioseparation technique will have higher cost, and so it would obviously be uneconomic to consider a series of chromatography columns to treat a very low value waste water stream to remove some specific impurities. High- and medium-value products such as pharmaceuticals and foods are manufactured within a regulated environment which imposes various legislation and guidelines on manufacturers. These regulatory constraints will also influence the choice of bioseparation equipment. For example, to maintain appropriate levels of cleanliness or sterility for certain products requires specialized equipment at a premium cost. Apart from regulations aimed at product quality, there are also issues concerned with the safe operation of certain processes, for example, where genetically modified or pathogenic microorganisms are being handled. In such cases, the bioseparation process is normally contained; in other words, the potential for release of hazardous material is minimized by various methods. Many bioseparations also involve the use of solvents which must be handled in appropriately designed equipment and facilities with proper explosion protection. Again there are cost implications associated with these types of processes which must be identified at the outset of the development phase. iii. Waste Production and Disposal

In most bioseparations, a waste stream will be generated which has no commercial value. Depending on the nature of this waste stream, it may not be possible to dispose of the material easily without further processing. For example, solvent-rich mother filtrates in antibiotics production are usually distilled to separate the solvent and aqueous phases so that the aqueous phase composition is acceptable for discharge to the sewer and the solvent phase can be reused or incinerated as a smaller volume of material. Biologically hazardous material must always be inactivated before it can be disposed of, or even removed from the production facility. This will normally require a validated heat or chemical deactivation system for aque-

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ous materials, or autoclaving for solid materials, to ensure that no live organisms remain after treatment. It is important to identify the relevant environmental and regulatory constraints affecting the disposal of material from bioseparations, so that any additional steps are allowed for within the overall manufacturing process. iv. Cost and Program Issues All process-scale bioseparations will have implications for project cost and program when developing a new process. It is important to consider both the capital costs associated with designing, purchasing, and installing a piece of bioseparation equipment and the operating costs of maintenance; utilities such as electricity, steam, and compressed air; labor; and any raw materials. In most cases, there will be a trade-off between capital and operating cost which may favor a particular type of equipment, depending on the desired initial capital investment and the economics of the process. The complexity of many industrial bioseparation equipment items means that the design and construction can be time consuming, particularly if process development is required to test the equipment on a typical product to see if it will work at the larger scale. It is not unusual to have 6 to 9 month delivery periods for this type of equipment, and even when delivered, it will be necessary to install, commission, and validate it. Therefore, the project program must recognize the long duration for introducing commercial bioseparation equipment. B. Biomass Separation and Primary Recovery In a bioprocess the desired end product may be present as whole cells or intracellular or extracellular material at the end of a fermentation. Therefore in this first bioseparation stage, it may be necessary to recover either the solid or aqueous phase, with as much of the unwanted phase removed as possible, and with minimal loss of the desired material to maximize product yield. After biomass removal has been achieved if appropriate, the main objective of the primary recovery stages is to isolate the product from significant impurities which will generally be in the same phase. At this stage of bioseparation, it is necessary to exploit some difference between the product and impurities such as solubility (in water or an organic solvent), particle size, surface affinity, charge and so on. Since many unit operations are common to cell separation and primary recovery, the possible options for process-scale equipment are discussed in the following. i. Pretreatment Depending on the nature of the product, pretreatment of the feed material may be desirable to improve the separation characteristics. Possible techniques are based on chemical or physical treatment and include thickening, flocculation, and coagulation. A simple heat treatment process where the temperature of the broth is elevated and held for a period of time can reduce

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the broth viscosity by breaking down the microbial structure or promoting cell lysis (breakage). The addition of chemicals including polymeric flocculants or filter aids can increase the particle size prior to the bioseparation stage or reduce the resistance of the solids phase (usually biomass) on the filter medium. An example of the latter pretreatment is in antibiotics manufacture where rotary vacuum filtration of fermentation broths, where the filter is precoated to improve the separation of biomass from the clarified filtrate containing the crude product. ii. Sedimentation

In sedimentation processes, the major driving force for separation is the difference in specific gravity between the liquid and solid phases. This can be enhanced by increasing the g force, by using a centrifuge device, or by increasing the size and density of particles by flocculation. Gravity sedimentation systems are relatively cheap at high throughputs and are well suited to low-labor, continuous operation. Even if sedimentation does not achieve complete phase separation, it can be a useful first stage of bioseparation process to reduce the quantity of material to be handled in subsequent processing stages. Settling time is a key parameter for choice of sedimentation equipment, together with the feed slurry solids concentration. Gravity sedimentation techniques are used commonly in effluent treatment processes for separation of activated sludge from aqueous solutions, in fuel ethanol production for recovery of yeast cells from aqueous ethanol solutions for recycle to the fermenter, and in the pharmaceutical industry for separation of solvent and aqueous phases in product recovery and isolation of impurities. Equipment types range from simple circular tanks, equipped with rake arms for large thickeners used in effluent treatment, through lamella type thickeners, which are fitted with inclined plates to increase the solids handling capacity, to flotation tanks where particles are caused to rise to the surface of the tank through natural low density or the use of gas bubbles or chemical flocculating agents. iii. Filtration Processes a. Pressure Filtration

Conventional pressure or vacuum filtration techniques are widespread in industry for separating cells and other biological materials from a liquid phase which can be solvent based or aqueous. A pressure differential between the dirty and clean sides of the filter, created with over pressure or vacuum, provides a driving force for the liquid to be forced through the filter material which retains solids above a particular size. This type of filter is often used in conjunction with a precoat material on the filter to improve the separation characteristics. Solids in the range 0.1 to 10 /xm are typically removed by this type of bioseparation, which is generally used where the sofid material retained by the filter is the unwanted by-product, and the desired product is dissolved in the liquid phase.

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b. Gravity Filter

Gravity filters are seldom used in the process industries because they offer low filtration rates, however, simple Nutsch filters are sometimes found in the pharmaceutical industry at pilot scale. The Nutsch filter is a tank with a perforated base on which a filter cloth can be supported. The feed slurry liquid filters through the cake and cloth under its own weight. Although these units are low cost, they are labor intensive to operate, cannot be contained for protection of the product or the operator, and are slow. c. Pressure Filter

Pressure filters are operated above atmospheric pressure at the slurry surface using a slurry pump or compressed gas, typically between 0.5 and 4.0 bar gauge. Usually they are suitable for batch operation only. Pressure filters have several advantages including faster filtration rates, a large filter area for a given floor space, and the flexibility to operate at different stages within a batch manufacturing process. However, they are labor intensive and expensive to operate in a contained manner. Examples of pressure filters include the plate and frame filter press, which gives a variable filter area depending on the number of plates installed on the frame, improving flexibility. Cake washing is also possible with this type of filter. High-pressure contained-plate and frame filters are now available where separations are more difficult or where solvents are being handled. The Rosenmund filter is a specialized type of pressure filter in the form of an agitated vessel with a flat base and a perforated false bottom on which a filter cloth can be mounted. The agitator can be used to spread the filter cake evenly, carry out reslurrying, and assist with cake washing and delumping. A rotary screw is used to discharge the solids from the filter. It is possible to combine filtration and drying operations within the Rosenmund filter to contain filtration, washing, and drying operations within one single piece of equipment. Minimal liquid hold-up and very efficient cake washing can also be achieved, and these filters are commonly used for final recovery of bulk pharmaceutical products following crystallization or precipitation. The Funda filter is another type of pressure filter with a stack of rotating circular horizontal leaves within a cylindrical vessel, with the filter medium on the upper side of each leaf. Filtrate is drawn through the leaves and removed while solids are deposited on the upper surface of the leaf. It is possible to blow compressed gas through the filter to help dewater and dry the filter cake; cake washing is also feasible. SoUds are discharged by rotating the filter at a sufficient speed to throw them off the filter medium, such that the solids fall to the bottom of the housing and can be removed through a slide valve. d. Vacuum Filtration

In this type of filter, the pressure differential across the filter is achieved by imposing a vacuum on the downstream side, rather than pressure on the upstream side. Both batch and continuous vacuum filters are widely used in

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the process industries, for example, in pharmaceuticals manufacture and waste water treatment. e. Rotary Drum Filter

This equipment consists of a horizontal axis revolving drum. The surface of the drum is segmented, with compartments fitted with drainage pipes leading inside the drum to a common manifold and a rotary valve located at one end of the drum. A filter medium is attached to the external surface of the drum. When the drum is mounted with part of its surface in a tank of slurry, the application of a vacuum behind the filter medium pulls the filtrate through. The solid material is retained on the filter surface and, as the drum turns slowly past a knife, is dislodged into a collection hopper for disposal or further treatment. The filter medium may be precoated by placing the precoat solution in the immersion tank before being replaced by the process slurry. Generally precoated filters must operate on a semicontinuous basis because after a period of time the precoat layer will deteriorate and must be replaced. Filters without a precoat can be operated continuously. Rotary vacuum belt filters are a similar type of filter with the added advantage of being able to continuously filter, wash and dry the filter cake as it travels along an endless belt. Precoat is not needed on this type of filter. Although the belt filter is in many ways superior in performance to the drum filter, it does rely on an easily filterable cake being used and requires a large area within a production building. Containment of solvents requires a complex enclosure design to minimize fume losses. f. Centrifugal Filtration

Centrifugal force, rather than pressure differential, is used to throw the solid phase against the filter medium and force liquid through the filter medium and cake. The filter medium may be wire mesh, perforated metal or cloth and is not usually used in a precoat manner. Centrifugal filters can be either batch or continuous operation. Higher gravitational force centrifuges are not considered in this section since they do not involve any filtration, but are described in detail below. g. Basket Centrifuge

The basket centrifuge is operated in batch mode and comprises a rotating basket within a housing, onto which the drive unit is fixed and driven from the top or bottom of the unit. Solids discharge may be from the top or bottom of the machine. Typically, these machines are operated at speeds of 400 to 600 rpm for slurry feeding and washing, but up to 1100 rpm for cake dewatering. A special sofids plough is used to push the cake off the filter medium. They are very flexible machines capable of handling a wide range of solids, but are relatively labor intensive to operate. h. Continuous Centrifuge Filters

Continuous centrifugal filters are available as truly continuous or batchcontinuous units. Conical screen centrifugal filters consist of a perforated

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conical screen which rotates in either the vertical or horizontal plane. Feed slurry is introduced at the cone angle end, and liquid passes through the screen, while the solids slide down the cone and are thrown off at the end. The cone angle is important for the correct operation of this type of filter. They are best suited to high solids concentrations and uniform particle size so that liquid will drain easily while avoiding screen blockage. Pusher (reciprocating) centrifugal filters enable filtration, cake washing, and drying as sequential operations, hence they are batch-continuous units. Cake formed on the screen is pushed sideways by an advancing annular ring and thrown off the edge of the spinning basket. Multistage baskets can be used. Pusher centrifugal filters can be automated to a high degree. Peeler centrifuges are also batch-continuous machines where the cake is removed by a plough. Different solids content feedstocks can therefore be handled in this type of unit, provided that they do not clog up the filter medium. A high degree of automation can be achieved with this type of unit. I. Cartridge Filtration

These are housings of metal or plastic containing one or more replaceable and renewable cartridges which contain the active filter element, usually based on a polymeric filter medium or in some cases, sintered stainless steel. They are useful as polishing filters where the level of solids to be removed is relatively low, to prevent the filter from blocking up. A most important application of cartridge filters is in sterile (absolute) filtration of gases and liquids either as part of a fermentation process or during final purification stages for a bulk or sterile pharmaceutical liquid product. In such processes, the filter and housing will usually be sterilized in an autoclave or in place using steam before filtration starts. Integrity testing of filters is important to demonstrate that the ability of the filter to remove all particles above a certain cut off point has not been compromised, to ensure that sterile solutions do not contain harmful contaminating microorganisms or viruses which could affect the product and ultimately the patient. /. Cross-Flow Filtration

Although this technique is not limited to the initial cell recovery stages of a downstream process, cross-flow filtration is commonly used for product recovery operations, particularly in lower volume processes where stringent hygiene requirements apply, as in the pharmaceutical and food industries. Cross-flow filtration is also referred to as tangential flow filtration or microfiltration, but all three terms refer to a process by which membranes are used to separate components in a Hquid solution (or suspension) on the basis of their size. The development of robust membranes in polymeric and ceramic materials has provided a powerful new technology for bioseparations, which is already widespread in the process industries as well as for water treatment processes. The principle of operation for a cross-flow filtration system is to recirculate a hquid solution or suspension, usually using a positive displacement pump, through the membrane module, which may be arranged as multiple tubes, a spiral wound sheet or in a plate and frame configuration. The use of

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several membrane modules enables flexibility in designing a cross-flow filtration system to suit a particular application. The pressure imposed on the feed side of the membrane creates a driving force which forces material of smaller size than the membrane through to the clean side. This is referred to as the transmembrane pressure, the clean material passing through the membrane as permeate, and the remaining material not passing through the membrane as retentate. The transmembrane pressure tends to be higher as the pore size decreases. Cross-flow filter performance is often characterized by a flux rate, which equates to the permeate flow rate per unit area of membrane surface. The flux rate in most biological separations is reduced by a fouling phenomenon called gel polarization, which tends to concentrate material at the surface of membrane to impose an additional resistance to transmembrane flow. The deterioration in flux rate must be well characterized for a commercial bioseparation process to ensure the correct size for the cross-flow filtration unit and avoid hold-ups at this processing stage. Cross-flow filters can be operated in three different modes according to the nature of the product to be recovered and the stage in the processing. Concentration mode enables a product in either soluble or insoluble form to be reduced in volume prior to further processing, provided that the product size is greater than the membrane pore size, so that the material cannot pass through the membrane. This is useful when there is a large volume of aqueous material to be removed as waste in order to reduce the batch size for downstream purification operations. Typical, a concentration stage can be used immediately after fermentation, especially for mammalian cell cultures, or following an elution stage from a chromatography step which has increased the product solution volume. Diafiltration (see later) can be used to wash further soluble impurities from the concentrated product fraction if desired. Clarification mode enables a product in soluble form to be separated from larger sized solid or dissolved impurities, by passing it through a suitable size membrane and collecting the filtered liquid as permeate. There will usually be a limit on the volume reduction which is possible before the membrane surface becomes badly fouled. To avoid losing a significant proportion of product in the retentate fraction, diafiltration can be used (see later). Diafiltration is essentially a washing step which can be used either to remove more impurities as part of a concentration process, or to increase yield by recovering more product as permeate in a clarification process. The feed volume is maintained at a constant level by adding a suitable solvent to the feed tank as permeate is removed through the membrane. Several volumes of feed solution can be added as diafiltrate according to the processing requirements. There is usually a point beyond which diafiltration becomes uneconomical, due to the marginal reduction in impurities or increase in product recovery achieved for a given volume of diafiltrate. Cross-flow filtration systems are suitable for aqueous and solvent based solutions and suspensions, provided that electrical equipment is appropriately specified to meet relevant hazardous area classifications. Most systems are

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suitable for cleaning in place and, with a suitable membrane material, for sterilization in place (SIP) using saturated steam. If SIP is not possible, a chemical solution, typically sodium hydroxide, is commonly used to sanitize the membrane system and minimize any microbial growth. They are therefore well suited to the hygienic and sterile applications found in the food and pharmaceutical industry, as well as for contained operation if hazardous materials are being processed. Commercial cross-flow filtration units are often supplied as packaged units where the membrane modules, feed pumps, feed and permeate tanks, heat exchangers, pipes, and controls are all arranged on a frame, or skid, which is preassembled and tested in the fabricator's workshop. It is possible to provide these units with a high level of automation and control, depending on the application. Cross-flow filtration systems are attractive when a high-quality filtrate or permeate is required, where washing is needed, and if contained operation is desirable for safety or hygiene reasons. It is also a useful technique in heat-sensitive processes where dewatering is required, and where other techniques such as evaporation could damage the product. They may be less attractive where the feed solution is highly fouling, or where an acceptable product recovery requires a large amount of diafiltration, thus diluting the product concentration significantly. It is not possible to use these units to recover dried products, but they may be useful in reducing the quantity of material to be handled in a downstream bioseparation process, for example, a spray dryer or an adsorption process. iv. Centrifugation

In centrifugal separation, the sedimentation rate of a particle is increased many thousands of times compared with gravitational forces, to enable efficient separation of particles with relatively small differences in density or size. Centrifugal bioseparations are an important group of available industrial techniques and are found in most of the industries described earlier for concentration and washing of solid phase material, countercurrent extraction of soluble products, and removal of solid phase impurities to clarify a product in solution. They are most commonly found in the early cell recovery and primary product separation stages of a bioprocess and are capable of handling relatively high liquid throughputs, solids concentrations, and a wide range of particle sizes. However, the choice of industrial centrifuge will be influenced by these parameters, as well as the need for sterility, containment, batch or continuous operation, and the nature of the product as solid or liquid phase material. In most cases, it will be necessary to carry out centrifuge trials using a typical feed material and pilot-scale version of the candidate machine so that the effectiveness of the chosen type can be verified. a. Tubular Centrifuge

Tubular centrifuges are small diameter, vertical axis machines running at high g forces up to 15,000 rpm and feed rates between 0.4 and 4.0 m^/hr.

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They are best suited to liquid-liquid separations and clarification duties, but not high solids concentrations due to the limited solids hold-up capacity. They can handle small-density difference separations and best suited to batch processes. They are difficult to clean, and as large-scale laboratory units, best avoided unless another type of centrifuge design will not work. b. Solid Bowl Centrifuge

Solid bowl centrifuges are similar to tubular centrifuges, but with a larger diameter bowl and running at slower speeds. Feed rates can be as high as 10 m^/hr, provided that the solids concentration in the feed is not too high, as the solids hold-up is again limited. c. Disk Stack Centrifuge

These versatile machines are used for liquid clarification, concentration of light slurries, and liquid-liquid separation. They are capable of continuous operation even with high soHds concentrations, since the solids phase can be discharged periodically if desired. They can also be arranged as hermetically sealed units for handling hazardous materials or in sterile applications. The centrifuge contains a stack of conical disks spaced between 0.5 and 2 mm apart. The feed is supplied through a pipe to the center of the bowl at the base of the machine and is distributed into many thin layers. The different phases therefore have only a very short distance to travel to free themselves from each other. Once the solids have contacted the underside of the disks, the settling process is over and the solids move to the periphery of the disks, where they are thrown off the edge onto the bowl wall. Feed rates of up to 250 m^/hr can be handled by this type of centrifuge, with g forces in the range 5000 to 10,000 rpm. If the solids phase is fairly small, then disk stack centrifuges can be configured as the solids retaining type. If necessary, bowl liners can be fitted to assist with product removal and cleaning. Alternatively, solids discharging types are available in two configurations, either the nozzle type, which ejects solids continuously, or the solids ejecting type, where intermittent discharge through valves on the nozzles avoids excessive liquid loss or dilution of the slurry. This type of centrifuge is well suited to CIP and some models can also be sterilized in place as part of a hygienic or contained bioseparation process. d. Decanter Centrifuge

The decanter centrifuge is a horizontal axis machine comprising a long rotating cylindrical bowl which contains a screw conveyor rotating at a slightly different speed in the same direction. Typically, the bowl rotates at 5000 to 8000 rpm, and the screw rotates 50 rpm more slowly. If the bowl is tapered at one end, dewatering of the solids on a "beach" is possible before discharge. The angle of the tapered section, the relative length of the beach and the remainder of the bowl, and the differential speed between the bowl and the scroll are all important parameters which must be fine-tuned to a particular process.

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Decanter centrifuges are well suited to high solids concentrations at feed rates between 1 and 100 m^/hr. They cannot achieve such high standards of separation as a disc stack machine, and are more difficult to engineer as truly hygienic or contained units. They are suitable for continuous operation and can discharge solids continuously. V. Sorption Processes

Adsorption and ion-exchange processes involve contacting a solution with a rigid and durable particulate phase which is able to selectively take up either the product or one or more specific contaminants. Generally, after the column has been loaded with the product or an impurity, another solvent is used to elute the material back into solution, possibly with an intermediate washing stage if some impurities are likely to be taken up along with the product. The particulate phase must then be regenerated and possibly sanitized using suitable solutions. Bioseparations using sorption processes have a number of advantages over alternative methods, including mild operating conditions of temperature and pressure, low energy consumption, high specificity, simple equipment, and the possibly for separation and recovery of the product essentially in the same physical and chemical state. However, sorption processes are usually run in batch mode with consequently high labor requirements. They tend to have a relatively slow "reaction" rate and the product hold-up per unit volume of packing is low, also large volumes of effluent are generated in the washing and regeneration stages. The attachment of particular solute molecules to the surface of the particulate sorption packing material can be achieved by a number of different methods, which are outside the scope of this paper. There are a large number of different sorption materials and complex physical and chemical interactions which must be considered. The most common sorption materials are activated carbon, silica gel, activated alumina, molecular sieves, and ion exchange resins. This chapter deals with the industrial aspects of handling these materials and operating process-scale equipment, but does not look at the choice of sorption material for a particular process. vi. Packed-Bed Systems

In most cases, apart from the expanded-bed systems discussed earlier, sorption processes tend to be based on packed-bed configurations where the material is loaded into a suitable column and remains static during the sorption process. Operation can be batch, with one column or several in series, or continuous, with two or more columns in parallel. When designing sorption columns, important process parameters include the pressure drop across the packing, especially if fouling might occur, and velocity and flow through the bed to achieve the correct residence time for adsorption. Large columns often have problems of poor flow distribution, since preferential channeling via the path of least resistance can reduce the ability of the sorption material to take up the product. Some packing materials will expand by up to 20% when wetted and this must be accounted for in the column sizing. The handling of different solution types and

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quantities is also important: How will the solutions be made up at the correct volume, how will they be disposed of, and what operating conditions are required? The loading and unloading of particulate material from the column must also be considered: Can the sorption material be regenerated, how many times before change is needed, and how will this operation be done when several tonnes of material could be involved? Backwashing of some packed beds is practiced to remove impurities and to regenerate the sorption material. It may also be necessary to chemically sanitise the column to minimise the build up of microorganisms. Examples of sorption processes using packed beds include recovery of crude antibiotics from fermentation broth filtrate, heavy metal removal from product streams, and separation of amino acids from solutions. vii. Expanded-Bed Adsorption

This is a relatively new technique which is now becoming commercialized. It combines the cell separation and primary product separation stages in a single unit operation, to reduce equipment costs, improve product yields and reduce processing times. The principle of operation is a column of suitable adsorptive material showing affinity toward the product. Due to the fouling nature of most fermentation broths, which contain a large amount of solid phase material, the adsorption bed would quickly become fouled if operated in a classical packed-bed mode. To get around this problem, the bed is fluidized using the liquid flow, so that the particles expand and move away from each other, enabling debris and cell material to pass through the bed without causing blockage, while the product can still be adsorbed onto the packing. A clean product solution can then be eluted from the adsorption bed, for further processing. The advantage of this technique is that it can avoid the need for two separate process stages to first separate the product from cells, perhaps using a centrifuge, and then a second adsorption stage using a packed bed or chromatography column to isolate the crude product. However, it is best suited where the product is in the aqueous phase as dissolved material, and requires careful control of the expanded bed to give good product recovery while maximizing the removal of impurities. viii. Cell Disruption

Cell disruption techniques are used to recover materials produced within the cell, for example, industrial enzymes and some pharmaceutical proteins. Generally this stage of bioseparation will follow cell recovery, for example, by centrifugation, and precede the isolation of the desired product from the cell debris which is also produced during the disruption process. There are a range of physical and chemical methods available at laboratory scale for cell disruption which involve the use of reagents or temperature and pressure changes to break the cell wall to release the desired products. However, at an industrial scale it is more common to use a mechanical disruption technique, and a number of companies have developed efficient

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cell disruption equipment suitable for yeast and bacterial cells. Mammalian cells with fragile cell walls do not generally require this type of treatment. Since cell disruption is a relatively specialized technique with specific difficulties depending on the organism being handled, it is worth obtaining assistance from the equipment manufacturer at an early stage, and possibly using a small-scale version of the apparatus to carry out pilot scale tests. When carrying out cell disruption operations it is often necessary to provide cooling of the cell concentrate due to the high pressures developed in the equipment. An additional consequence of high-pressure operation is that cell disruption equipment can generate aerosols which may be undesirable, particularly for biologically hazardous organisms. In these cases, the ability to steam sterilize the equipment is required, for decontamination, and some type of secondary containment may also be required, such as an isolator or a contained area within a facility to which access is controlled. Mechanical cell disruption techniques are based on high shear effects as fluid is forced through a narrow orifice, or a chamber containing rotating disks and glass beads to break up the cellular material. Both techniques enable some control over the extent of cell disruption. C. Product Purification and Final Isolation Following the initial stages of product recovery from a fermentation broth, a number of purification stages will be required in all but the simplest industrial processes. In the case of high-purity pharmaceutical products, a large number of separation stages are usually required to remove all impurities from the desired final product. By identifying some difference between the product and its impurities, either physical or chemical, the desired bioseparation can be achieved. i. Chromatographic Separation Chromatography is an effective bioseparation technique suitable for low-volume, high-value products such as pharmaceutical proteins. In chromatographic separations, an aqueous or organic solution containing the product is passed through a packed column containing a separation matrix. Differences in chemical or physical properties between the product and its impurities are exploited to achieve the separation. In most industrial purification processes, several stages of chromatography are required to achieve the required product purity, generally with a concentration step using ultrafiltration to reduce the volumes of liquid handled from stage to stage. At industrial scale, various chromatographic techniques are available: adsorption chromatography, which uses physical binding effects which are dependent on pH or salt concentration; affinity chromatography, where a specific binding between a molecule and the matrix is achieved; and partition chromatography, where product and impurities move through the bed at different rates. Typical stages in a chromatographic separation process are first loading of the column with the product while impurities pass through, various

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washing stages to further purify the product, then elution where some change to the column operating conditions removes the product from the matrix into the solvent phase. After the product has been removed, the column is cleaned, sanitized, and regenerated. All of these stages involve the passage of buffer solutions though the column, this term being a general description for any aqueous or solvent-based solution usually containing organic or inorganic salts. The scale-up of a chromatographic process to industrial scale can be difficult to achieve while maintaining an acceptable throughput and yield of product. Problems may occur which are not met at the laboratory scale, for example, the flow distribution pattern through a large-diameter column, excessive pressure drop in a longer column due to compression of some matrices, and the need to maintain equipment cleanliness over an extended number of purification cycles. Since most chromatographic stages are found toward the end of the production process, and because very few matrices are suitable for steam sterilization, product protection must be ensured by locating the equipment within a clean room environment. Often, the temperature of the process must be controlled, typically at 4°C, and so the entire clean room may be cooled to avoid the need for local process cooling. At an industrial scale, the preparation of buffer solutions and the loading of the column with a matrix is also more difficult. Large volumes of solutions must be prepared ready for use, requiring vessels for dissolution, mixing, and storage. The larger volumes of matrix required in a large-diameter column require special equipment for packing to avoid channeling and consequential poor flow distribution. These two requirements may create the need for dedicated clean rooms where buffer preparation and column packing can be carried out. As a general rule, the use of organic solvents in chromatographic processes should be minimized, because of the requirement for specialized flameproof equipment which can be extremely costly compared with the equivalent item for a "safe" area. Where unavoidable, a separate flameproof room for handling solvent-based materials is recommended, with the appropriate specification for mechanical and electrical equipment, controls and room fittings including lights, switches, and telephone systems. ii. Crystallization Crystallization is an important unit operation at industrial scale found commonly in both the food and pharmaceutical industries. It is capable of producing highly pure solid materials in a form suitable for sale. For example, bulk antibiotics and sugar are two biologically derived products which are crystallized at the end of their manufacturing process. The crystallization step enables the removal of unwanted impurities by a convenient route and, coupled with subsequent washing of the crystal slurry on a filter, produces a highly pure end-product. As a widely used unit operation throughout the process industries, there is a wealth of scientific and engineering literature which underpins this important separation technique, beyond the scope of this section. The com-

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plex heat and mass transfer problems associated with crystaUization have meant that such processes have sometimes been considered a "black art." Also, the need to recover the soHds from a liquid slurry by filtration and subsequent drying requires a more complex train of process equipment at an industrial scale. For foods and pharmaceutical products, the handling of slurries and solids at industrial scale presents problems where the product is exposed on filters and in dryers, rather than being enclosed within a pipe or vessel as a liquid. Contamination of the crystalline material can occur, and the product itself or the solvent phase it must be removed from might be toxic. Various types of equipment have therefore been developed to enable wet crystalline "cakes" and dry powders to be handled in a contained manner, using specially designed booths, isolators, and pack off systems to protect the product from its environment (and vice versa for highly potent materials). Both batch and continuous crystallization equipment are available at industrial scale, although batch operation is normally favored for pharmaceutical products where batch integrity must be maintained for quality control reasons. Continuous crystallization is suited to higher throughputs and enables more energy efficient operation. Batch crystallization vessels can be agitated or unagitated, and may use either cooling or evaporation to cause crystallization, sometimes with "seeding" required. In some cases, very smooth surfaces are required to enable the crystals to form in the desired form, and this can be achieved using purposebuilt glass-lined vessels and stirrers. iii. Thermal Separations

Thermal separations are commonly required for biological products to reduce or remove unwanted solvents, most commonly water, from a liquid or wet soHd material. Evaporation produces a more concentrated liquid while drying results in a product with lower moisture content. Such processes are energy intensive at industrial scale and so it is important to investigate possible alternatives such as reverse osmosis for concentration of a liquid, or preliminary filtration by mechanical means before final product drying, so that more liquid is removed from the feed material. Dried products are generally more stable and suited to storage for extended periods, compared with liquids or wet solids where there may be potential for microbial growth to occur especially for nonsterile products. Drying conditions for bioseparations may be milder than for other types of products, but still require the removal of liquid from between the interstices of the solid and as droplets on the solid surface, as well as from within the bulk of the particle either in pores or encapsulated within the particle surface. a. Thermal Dryers

Industrial drying equipment is available in many different forms for both batch and continuous duties, using hot air or a combination of indirect heating and vacuum to remove the liquid.

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Tray dryers, the simplest type of dryer, are commonly used for batch drying of biological materials, where the wet solids are placed on trays which are then transferred into a chamber. The chamber may have a heating jacket, heated tray supports, or a hot air supply. Vacuum may be applied to reduce the temperature at which the liquid evaporates, preserving heat labile products. These are well suited to low-volume products or flexible plants where a number of different products with different characteristics must be dried. They are relatively inefficient to operate, difficult to clean, and labor intensive to operate. The product is exposed when being loaded and unloaded, so the dryer may need to be located in a clean room or area for pharmaceutical products. Various types of agitated dryers are available, again operating in batch mode, with vacuum and indirect heating to increase the drying rate. The dryer chamber may be a horizontal cylinder, a pan, or a vertical cone. These units are more efficient and offer faster drying rates due to the constant turnover of solids within the chamber. They can be loaded and unloaded in a contained manner where product or operator protection is required. However, they are still relatively difficult to clean and operate on a batch basis with a relatively low possible throughput. For vacuum dryers, a solids filter between the drying chamber and the vacuum pump is required to collect any "fines" from the dried product. Combined filter-dryer units are available in a number of different configurations including the Funda filter, Titus-Nutsche filter dryer, Rosenmund filter, and Seitz-Wega filter. These equipment types enable filtration and drying in a single piece of equipment to reduce the amount of product exposure and the necessity to transfer wet cake from one plant to another. However, the time required to process a batch of material through filtration and drying stages means that this equipment may create a bottleneck to production unless several units operate in parallel, at a higher capital cost. Hence a balance must be made between contained processing needs and plant capacity and flexibility. b. Spray Dryers

Spray dryers are operated continuously and commonly used for foods, enzymes, and pharmaceutical intermediates and products. Most if not all of the solvent phase can be removed provided that the feed slurry is converted to a fine spray to maximize the surface area for heat and mass transfer. Spray dryers have a relatively large space requirement in a plant and have a high energy consumption. They can be difficult to clean effectively but are well suited to single products where continuous operation is desirable. Again dust can be a problem and contained pack off systems are recommended to maintain a clean environment around the spray dryer unit. c. Freeze-Dryers

Freeze-drying is a specialized process by which moisture is removed from a wet solid, usually placed on trays or in small containers, by sublimation under high-vacuum conditions. This is an energy-intensive procedure even compared with conventional drying processes but is used in a number of industries where heat causes damage to the product, reduces its yield, or

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spoils the presentation of the product. Examples of freeze-dried biological materials include instant coffee granules and high-value pharmaceutical proteins such as growth hormones. Freeze-dryers are large and expensive items which require a large amount of supporting equipment for heating, vacuum production, and condensation.

lY. PROCESS-SCALE CONSIDERATIONS A. Materials of Construction and Mechanical Design At industrial scale, careful consideration of the materials of construction for the bioseparation equipment is vital to ensure that the product does not become contaminated, by rust, for example, and also to assure long plant life with good reliability to maximize throughput. Materials that were suitable on a laboratory or pilot scale may no longer be appropriate, where the process and mechanical demands on the equipment may be greater. For example, the plant could be located outside where there are greater extremes of temperature in summer and winter, or equipment may need to be steam sterilized in situ rather than being autoclaved. Mechanical design conditions refer to the range of temperatures and pressures that are encountered during bioprocessing. The equipment supplier will add a margin to the stated design conditions to ensure that failure will not occur in normal operation, and test the equipment before delivery under the most stringent conditions. Where high pressures or vacuum conditions are encountered, a recognized pressure vessel code such as ASME VIII in the United States or BS 5500 in the United Kingdom will be followed, and particular construction techniques and testing requirements will be either optional or mandatory, according to these codes to suit a particular application. The chemical and physical nature of the products being processed will also be important, especially high or low pH and chloride content, which can mean that even some grades of stainless steel could corrode. For example, pharmaceutical chromatographic separations involve aqueous buffer solutions containing sodium chloride as a sanitizing agent, and under certain conditions of concentration and temperature, chloride-induced stress corrosion cracking may occur. Cleaning chemicals can also attack certain materials and so any requirement for cleaning, sanitizing, decontaminating, or flushing the equipment with chemicals other than those used in the processing operation must be clearly defined and checked for compatibility. It is therefore important to draw on the experience of the equipment supplier and to fully define all anticipated operating conditions for the bioseparation equipment. It is also vital that any other components such as gaskets, O-ring seals, instruments, and other parts are checked for compatibility with the products to avoid failure in service and possible product contamination or equipment downtime for repairs to be made.

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B. Automation To increase production capacity, to give more repeatable process conditions, or to reduce labor costs, it may be advantageous to automate certain bioseparation processes rather than relying on manual operation. For example, a decanter centrifuge could be controlled from a local PLC (programmable logic controller) so that sterilization, separation, and cleaning operations are automatically monitored and controlled using on-off and modulating control valves and appropriate instrumentation. Automated plant is also useful in semicontinuous and continuous processes to make the control of the plant more stable. Hov^ever, there are several disadvantages with automated bioseparation systems, including cost, development time, and the need for complex validation activities if the equipment is being supplied as part of a regulated production facility, for example, for pharmaceuticals production. In such cases, specific guidelines have been developed for computer system validation known as GAMP (good automated manufacturing practice). A further disadvantage is that suppliers may offer only one specific type of hardware a n d / o r software, which may be incompatible with a company's standards or with other equipment in the plant. The operation of a particular bioseparation step often involves unique features which require specialized software to be written rather than an "off-the-shelf" system, with consequent delays for testing and implementation. It is recommended that a careful analysis of costs is made before making the decision to automate a particular process, looking at capital and operating costs (such as man power), and to compare the process with existing plants or competitors to see if there is an industry benchmark. C. Safety and Biosafety In all industrial processes, the safety of operators and staff, as well as the general public in surrounding areas, is of paramount importance. Every effort should be made during design and construction to ensure that the bioseparation plant is safe to operate with all risks identified and minimized through appropriate precautions. Hazardous features to be considered will include pressure relief; handling of hazardous materials such as acids and alkalis; protection from steam and other high-temperature fluids; and electrical classification for handling solvents or protection from water ingress, high speed rotating machinery, and noise levels. A unique feature in biological separations in the potential presence of biologically hazardous materials, in particular pathogenic or genetically modified microorganisms. Where these are being handled, specific safety guidelines are mandatory both in Europe and the United States, depending on the level of hazard presented by the microorganism. Formal safety reviews such as Hazops (Hazard and Operability reviews) may be required during the design of a new facility or the modification of an existing plant. It is recommended that specialist help be sought in carry-

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ing out such reviews to ensure that all potential hazard are identified at an early stage in the project where the appropriate protective measures can be specified. D. Location Equipment and plant location can vary from an outdoor, exposed site, as in the case of an effluent treatment plant, to a clean room with controlled climate and air quality, for example, in biopharmaceuticals manufacture. The specification for bioseparation equipment will depend on its location, for both mechanical and electrical components. The need to weatherproof a piece of equipment and protect it from rain, wind, and temperature extremes will require specific provision to be made. Equipment located indoors will not necessarily require such features, although cleaning down of plant areas with hoses, as is common in the food industry, requires some degree of protection against water ingress for electrical items such as motor drives, control panels, and instruments. E. GMP and Validation For pharmaceuticals and foods, the safety and efficacy of the product is strictly regulated to ensure that consumers are protected. Both the European Union (EU) and the United States have regulatory bodies who are responsible for food and drug safety. The U.S. Food and Drug Administration and their European counterparts provide high-level regulations and directives for manufacturers of foods and pharmaceuticals. Good manufacturing practice, or GMP, encompasses this legislation plus more detailed guidelines which have been prepared to help the industries operate to a consistent standard. It is important to identify the markets where the product may be sold and to understand the prevailing GMP regulations and guidelines which will apply. Even if a manufacturing facility is outside the United States, the FDA must inspect the facility and license its products for sale if products are to be sold within the country. During the earliest stages of product and process development, facility design, and planning, due consideration must be given to ensure that GMP requirements are satisfied and the product or products will be approved. Validation is the process by which a facility is demonstrated to be compliant with GMP and fit for the manufacture of a particular product. Validation within the pharmaceutical industry normally falls into three or four parts: Design qualification (DQ) is the validation of a design to ensure that all GMP requirements are met at an early stage in the life cycle of the project, while there is still an opportunity to make changes relatively easily. Installation qualification (IQ) is where the construction of the facility and equipment is checked against the design specifications to ensure compliance with the original design intent.

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operational qualification (OQ) then enables the functionahty of the faciUty and equipment to be tested against the design specifications. Finally, performance qualification (PQ) ensures that the actual product can be manufactured within the required specification and quality parameters which are claimed. Usually, three batches of product must be produced and carefully documented to demonstrate the repeatability of the process and the consistent specification of the product. The cost and complexity of vaUdation is often underestimated and can cause delays in bringing the product to market. Validation requirements for a project should be identified as early as possible in the project life cycle to ensure that adequate time and cost is allowed to complete them successfully. It is particularly important to establish the documentation required from suppliers and contractors working on the project, and the extent to which they will be required to play a part in the validation process. For example, if material certification is required for equipment components, these must be ordered from the supplier's own stockholders with the raw materials, and cannot be obtained retrospectively. It is particularly important to put in place quality control procedures for the development of hardware and software for plant automation. Good automated manufacturing practice provides a systematic and structured approach to the development of these systems including change control and validation methods. F. Hygienic Design For the pharmaceutical and food industries, surface finish is very important to enable effective cleaning and sterilization or sanitization. Equipment should be specified with a polished internal finish, possibly with electropolishing for critical applications, and designed with a minimum of crevices or dead spaces where dirt can collect. Welds must be finished to the same standard as the plates and ground flush with the internal surface and must be pinhole and crevice free. External surface finish may also be important for visual reasons and to enable cleaning down for surface decontamination in clean room locations. Where steam sterilization is required, the equipment must be self-venting and draining so that condensate drains to a single low point where it can be removed, and air which is trapped at high points can be displaced by steam.

V. SUMMARY In this chapter, a brief overview of the approach required when selecting and specifying industrial bioseparation equipment has been provided. Because of the range of industries involving bioseparations, it has been possible to give only general guidelines and advice. Each industry and product type will have particular requirements which will determine the choice of suitable processing plant and equipment.

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A holistic approach to the selection of bioseparation equipment is vital, so that the unit operation is not considered in isolation, but in relation to the whole process, the facility or site where it will be located, the nature of the product, and the operating and capital costs. Only then can an informed decision be made to find the right balance between product quality and yield, processing costs and capital investment. REFERENCES Atkinson, B., and Mavituna, F. (1983). "Biochemical Engineering & Biotechnology Handbook." Macmillan, New York. Bailey, J. E., and OUis, D. F. (1986). "Biochemical Engineering Fundamentals," 2nd ed. McGraw-Hill, New York. Lydersen, B. K., D'Elia, N. A., and Nelson, K. M., eds. (1994). "Bioprocess Engineering Systems Equipment and Facilities." Wiley, New York. Perry, R. H, and Green, D. (1984). "Perry's Chemical Engineer's Handbook," 6th ed. McGrawHill, New York.

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17ENGINEERING PROCESS CONTROL OF BIOSEPARATION PROCESSES RANDEL M. PRICE AND AJIT SADANA Department of Chemical Engineering, University of Mississippi, University, Mississippi 38677

I. NEED FOR PROCESS CONTROL IN BIOSEPARATIONS II. BRIEF OVERVIEW OF CURRENT CONTROL METHODS A. Bioreactor Control B. Batch Process Control C. Control in Food Processing III. APPLICATION EXAMPLES A. Chromatography B. Electrophoresis C. Extraction IV. OPPORTUNITIES FOR CONTINUING DEVELOPMENT REFERENCES

This chapter addresses the engineering process control of bioseparations. "Bioseparation" will be defined as process steps used to purify the products from bioreactors (such as fermentors): "downstream" processing steps which may include extraction, precipitation, electrophoresis, and chromatography. It has been suggested that these separation and purification steps comprise 4 0 - 8 0 % of the total cost of a bioprocess (Mattiason and Hakanson,^ p. 221). An example of the scale of the bioseparation portion of a processing sequence is the production of human insulin using fermentation and recombinant DNA technology.^ After the fermentor stage, this process requires cell harvesting and disruption, recovery and solubilization, enzymatic conversion, refolding, sulfitolysis, cleavage, purification, and crystallization. These steps involve the unit processes of centrifugation, reaction, adsorption, filtering, chromatography, and crystallization. Clearly, any steps to improve the efficiency of the process must address the downstream steps. "Engineering process control" involves measurement of a product property and comparison to a desired value. The process operation is then immediately adjusted to reduce the deviation from the specification. This feedback procedure adjusts the process whenever the product deviates from setpoint and is used to change operating points and to reject the effect of outside disturbances. Separation Science and Technology, Volume 2 Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

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Engineering process control is not generally the same as "quality control" or "statistical" process control; engineering control is intended to continuously reject and attenuate errors which enter while the process operates, while most statistical control techniques seek to identify sources of error so that they may be eliminated by subsequent process or operation revisions. At times the two approaches are in conflict, for by correcting or compensating for a disturbance an engineering control system may distort the statistical parameters used in quality control analysis.

NEED FOR PROCESS CONTROL IN BIOSEPARATIONS The objectives of an engineering process control system apply equally to bioseparation processes and traditional chemical systems. These include quality control; guiding operating transitions; providing effective turndown management; minimizing inventory, utility requirements, and required operator attention; and production scheduling (Buckley,^ pp. 11-16). Adler,"^ addressing control in the bulk pharmaceutical industry, Hsts both tangible benefits— reduced personnel requirements, yield enhancement, reduced lot variation, and increased capacity—and intangible benefits—improved product quality, increased operator morale, regulatory compliance—of process control systems. An effective control system provides detection and rejection of unanticipated disturbances which might cause deterioration of the product, continuous monitoring of process state and product quality, and improved and reproducibility (Mattiason and Hakanson,^ p. 221), all of which lead to a more efficient, productive process system. Process control structures include three major operations—measurement of a process variable, calculation of the required adjustment, and manipulation of the process to implement the correction. The measurement step may be the most routine, since almost all bioseparation systems, regardless of scale, function, or constraints are usually equipped with instruments for monitoring the process. Monitoring instrumentation is generally well understood and is documented in discussions of particular bioseparation processes and implementations. Far less common in the open literature are examples of the calculation and manipulation steps. Most published bioseparation examples seem to be small enough in scale that "manual" operation, where the human operator provides both of those actions and so functions as both controller and actuator. In small-scale systems, manual operation is probably adequate, but the large scale required for viable commercialization will probably require automatic control. Automatic control would seem to be ideally suited to commercial bioseparations to comply with the stringent quality control and validation demands of these systems. The regulatory requirements of such bioseparations products as protein therapeutics are of major concern.^ Because most modern control systems are capable of monitoring extensive data records of process

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operation and because automatic systems are more consistent and repeatable than human operators, it would seem that they are ideally suited to the requirements of process validation. To be suitable for use in validated processes, the control system itself would require validation, but procedures for doing this have been reported.^' ^ Process control system instruments do not necessarily need to be extremely accurate. For effective control, repeatability is generally more important than absolute accuracy; consequently, automating a bioseparation process should not require duplication of highly accurate instruments. In addition, it should be possible to use the most accurate instruments only at the most critical stages of the operation. For example, when batch processing is employed, only the final state of the product must meet product quality specifications. It is not required that these specifications be met during the entire production sequence.^ A similar situation exists in continuous processing, where the product state may be more conveniently monitored by tracking levels of impurities or contaminants.

II. BRIEF OVERVIEW OF CURRENT CONTROL METHODS A. Bioreactor Control Bioseparation practitioners can probably benefit from the control experience of bioreactor operation. Several extensive articles on the instrumentation of bioreactors discuss issues and approaches to measuring temperature, pressure, flow, and volume; pH, dissolved oxygen and CO2, redox potentials, and specific ions or chemicals.^"^^ Control of bioreactors has been a fertile research area for a number of years, particularly with respect to fermentor control.^^ A key problem in bioreactor control is the difficulty in obtaining reliable sensors and consequently of refiable on-line process information. Demands for product consistency and process productivity produce requirements for more process information.^^ Especially in the case of fermentors, rapid, accurate on-line measurement of process variables is often a complex task. As a result, much research effort has focused on methods for quantitatively estimating compositions within reactors and on using model-based control techniques. An "estimator" (or more specifically an "optimal state estimator") in this usage is an algorithm for obtaining approximate values of process variables which cannot be directly measured. It does this by using knowledge of the system and measurement dynamics, assumed statistics of measurement noise, and initial condition information to deduce a minimum error state estimate. The basic algorithm is usually some version of the Kalman filter. ^"^ In extremely simple terms, a stochastic process model is compared to known process measurements, the difference is minimized in a least-squares sense, and then the model values are used for unmeasurable quantities. Estimators have been tested on a variety of processes, including mycelial fermentation and fed-batch penicillin production,^^ and baker's yeast fermentation.^^ The

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effectiveness of an estimator is strictly limited by the model accuracy, leading to the recent interest in the use of artificial neural networks for estimator models. A second approach to the problem of difficult to obtain measurements is "knowledge-based" or "model-based" control. "Knowledge-based" systems attempt to use various types of knowledge of the biological process (rules etc.) to supplement traditional mathematical control approaches.^^ "Expert systems" are one type of knowledge-based control. "Model-based" control systems use a model of the process as part of the control algorithm; their reliability depends on the accuracy of the model. These complex approaches may not be necessary in bioseparation applications—assuming that the components of interest can be readily measured —since processes such as chromatography and extraction operate under more manageable conditions. B. Batch Process Control Control practitioners have devoted a great deal of effort to a better understanding of batch process optimization and control. Interest has primarily been focused on specialty chemicals, but the characteristics of the systems— varied products made in relatively small quantities, multiple products made using the same equipment^"^—would also seem applicable to bioprocesses. ISA Standard SP88, Batch Automation in the Process Industries, defines a structured, modular approach devised to provide a tool for process and performance qualification, that provides a framework for documenting and vahdating batch processes independent of recipe.^^'^^ In particular, the standard allows the modular design of control software modules; a procedure which has been demonstrated in the biotech manufacturing environment.^^ C. Control in Food Processing Lessons may also be learned from applications of control systems in the food processing industries. These applications must satisfy hygiene requirements (including periodic cleaning and sterilization), time constraints imposed by product perishability, and requirements for accurate records of sources and operation histories of materials.^^ The industry also experiences slim profit margins, short production runs, and frequent product changeovers—characteristics shared with many industrial bioprocesses.

III. APPLICATION EXAMPLES In most published applications of monitoring and control of bioseparation systems, the goal seems to be monitoring and maintaining operating parameters of the separation device—temperature, flow, pressure, etc.—rather than product specifications. The separation may be governed by a "recipe" requiring a predetermined temperature and the control system tasked with maintaining the device operating temperature at that value. The performance of

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the process and any adjustments required to improve product quality are then judged by off-Une measurements. In most continuous bulk chemical plants, the product quality would be directly measured and used to set the temperature requirements, providing compensation for changes in the feed makeup, ambient conditions, etc. This "cascade" control arrangement (where product quality measurements are used to adjust the setpoints of temperature and flow controllers) might be a simple and effective tool to transfer to bioseparations.

A. Chromatography Chromatography appears to be one of the more common bioseparation techniques, finding application in such areas as protein therapeutics. It is, though, not suitable for very large scale production.^ In operation, chromatography requires three sets of information^^: operating data (pressure, flow, temperature, etc.), system status data (valve positions, pump status, etc.), and product data for quality assessment. Most chromatographic systems employ process control of operating parameters. These may well be built into the instrument. Temperature control is particularly important, especially for contemporary techniques such as chiral recognition and protein interaction.^^ In liquid chromatography, for instance, temperature directly effects retention, separation efficiency, and selectivity. Stability of temperature is thus extremely important, since variations of more than 1°C can lead to noticeable effects.^"^ Monitoring and control of system status and operation has also been reported. Automation has been used in analytical chromatography to speed certain extremely time consuming techniques, such as selection and reinjection of cuts by means of computer-controlled valves.^^ One would anticipate that such methods might also find application in production chromatography.

B. Electrophoresis Capillary zone electrophoresis is another technique which has been used to separate products such as organic acids.^^ Separation is based on differences in the mobility of analytes exposed to an electric field. Resolution and separation time in such systems depends on factors including electroosmotic flow (EOF), and a number of approaches for adjusting the EOF have been examined. While some of the approaches (pretreatment of capillaries) are not useful as means of process control, adjusting buffer pH and the electric field^^ seem to be possible handles for true feedback control of the separation, although closed-loop operation does not seem to have been attempted. Temperature programming has also been used in electrophoresis, using a thermostatically regulated circulating water jacket to maintain constant values.^^ Temperature effects convective flow through the system, ionization of the analyte, and the viscosity and pH of the buffer solutions.^^

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C. Extraction The bioseparation technique which is probably the most readily adapted to modern process control techniques is extraction. Liquid-liquid extraction is a mature unit process with application in industrial-scale protein separation.^^ Control techniques used on similar systems in other industrial applications should be readily adaptable to bioprocessing, the primary difficulty being the lack of data on the partitioning and related behavior of the product. IV. OPPORTUNITIES FOR CONTINUING DEVELOPMENT For effective process control of bioseparations, it is necessary to have reliable, repeatable, on-line measurements. The delays caused by off-line analysis can be prohibitive. Offline approaches also tend to result in increased labor and costs.^^ Development of such sensors appears to offer promise as an area of research. Biosensors may be one solution, but the characteristics of a typical biosensor do not align well with those required for control. Tools to enable the coupling of "quality control" techniques with those required for "process control" are another interesting area for continued investigation. If the two tasks can be harmonized, less instrumentation may be needed. REFERENCES 1. Mattiasson, B., and Hakanson, H. (1991). Measurement and control in down-stream processing. In "Measurement and Control in Bioprocessing" (K. G. Carr-Brion, ed.), Chapter 8, pp. 221-250. Elsevier Applied Science. 2. Petrides, D., Sapidov, E., and Calandranis, J. (1995). Computer-aided process analysis and economic evaluation for biosynthetic human insulin production—A case study. BiotechnoL Bioeng. 48, 529-541. 3. Buckley, P. S. (1992). "Process Control Strategy and Profitability." Instrument Society of America, Research Triangle Park, NC. 4. Adler, D. J. (1998). Instrumentation and process control system strategy. In "Automation and Validation of Information in Pharmaceutical Processing" (J. F. DeSpautz, ed.), pp. 59-68. Dekker, Nev^ York. 5. Chapman, G. E., Rott, J., More, J. E., Feldman, P. A., Matejtschole, P. (1994). Chromatographic purification of protein therapeutics: An industrial perspective. / . Chem. Tech. BiotechnoL 59, 108. 6. Coady, P. J., and deClima, A. P. (1995). Best practice engineering for vahdation of process control systems. Pharm. Eng. 15(4), 18-30. 7. Lopez, O. (1997). Automated process control systems verification and validation. Pharm. Technol. 21(9), 100-108. 8. Liibbert, A., and Simutis, R. (1994). Using measurement data in bioprocess modelling and control. Trends BiotechnoL 12, 304-311. 9. Armiger, W. B. (1985). Instrumentation for monitoring and controlling bioreactors. Compr. BiotechnoL, 2, 133-148. 10. Bull, D. N. (1985). Instrumentation for fermentation process control. Comp. BiotechnoL 2, 149-163. 11. Hartnett, T. (1994). Instrumentation and control of bioprocesses. In "Bioprocess Engineering: Systems, Equipment, and Facilities" (B. K. Lydersen, N. A. D'Elia, and K. L. Nelson, eds.), pp. 413-468. Wiley, Nev^ York.

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12. Rolf, M. J., and Lim, H. C. (1985). Systems for fermentation process control. Compr. Biotechnol. 2, 165-174. 13. DiMassimo, C , Lant, P. A., and Saunders, A. (1992). Bioprocess applications of model-based estimation techniques. / . Chem. Tech. Biotechnol. 53, 265-277. 14. Gelb, A. ed. (1989). "Applied Optimal Estimation." MIT Press, Cambridge, MA. 15. Beluhan, D., Gosak, D., Pavlovic, N., and Vampola, M. (1995). Biomass estimation and optimal control of the Baker's yeast fermentation process. Comput. Chem. Eng. 19 (SuppL), S387-S392. 16. Konstantinov, K. B., and Yoshida, T. (1992). Knowledge-based control of fermentation processes. Biotechnol. Bioeng., 39, 479-486. 17. Davidson, R. S. (1989). Using process information to control a multipurpose batch chemical reactor. Computs Chem. Eng., 13(1/2), 83-84. 18. Haxthausen, N. (1995). The painkiller for batch control headaches. Chem. Eng. 102(10), 118-124. 19. Nelson, P. R., and Shull, R. S. (1997). How to organize for S88 batch control. InTech 44(7), 44-47. 20. Growl, T. E. (1998). S88.01 batch standard's concepts -I- "cloning" = big dollar savings. InTech 45(8), 4 2 - 4 3 . 21. Macchietto, S. (1997). Batch food processing: The proof is in the eating. AIChE Symp. Ser. 316, 7 1 - 8 1 . 22. Kelly, M. (1991). Measurement and control in process scale liquid chromatography. In "Measurement and Control in Bioprocessing" (K. G. Carr-Brion, ed.). Chapter 7, pp. 191-219. Elsevier Applied Science, Amsterdam. 23. Ooms, B. (1996). Temperature control in high performance liquid chromatography. LC-GC 14(4), 306-324. 24. Baba, Y., Yoza, N., and Ohashi, S. (1985). Effect of column temperature on high performance liquid chromatographic behavior of inorganic polyphosphates. / . Chromatogr. 350, 119-125. 25. Tomlinson, M. J., and Wilkins, C. L. (1989). Evaluation of a semi-automatic multidimensional gas chromatography-infrared-mass spectrometry system for irritant analysis. / . High Resolut. Chromatogr. 2 1 , 347-354. 26. Hsieh, M. M., and Chang, H. T. (1998). Dynamic control for the separation of organic acids in capillary electrophoresis. / . Chromatogr. A 793, 145-152. 27. Wu, C. T., Lee, C. S., and Miller, C. J. (1992). Ionized air for applying potential gradient in capillary electrophoresis. Anal. Chem. 64, 2310-2311. 28. Whang, C. W., and Yeung, E. S. (1992). Temperature programming in capillary zone electrophoresis. Anal. Chem. 646, 502-506. 29. Chang, H. T., and Yeung, E. S. (1993). Voltage programming in capillary zone electrophoresis. / . Chromatogr. 632, 149-155. 30. Asenjo, J. A. (1994). Industrial prospects of aqueous two-phase processes. / . Chem. Tech. Biotechnol. 59, 109. 31. Scheper, T., Plotz, P., Miiller, C , and Hitzman, B. (1994) Sensors as components of integrated analytical systems. Trends Biotechnol. 12, 4 2 - 4 6 .

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ECONOMICS OF BIOSEPARATION PROCESSES ANAND RAMAKRISHNAN AND AJIT SADANA Chemical Engineering Department, University of Mississippi, University, Mississippi 38677

I. INTRODUCTION A. Example I: Role and Use of Outsourcing Companies during the Development of a Process II. DRUGS MARKET A N D SALES A. Example 2: Increased Profitability and Market for Drugs (Erythropoietin) B. Example 3: Some Data on the Existing Market for Thrombolytic Drugs III. APPLICATIONS OF MODELS AND FLOW SHEETS IN BIOSEPARATION ECONOMICS A. Example 4: A Model for Process Economics B. Example 5: An Economic Analysis of Biosynthetic Human Insulin Production C. Example 6: An Economic Analysis for Heparinase I Production from £. coli D. Example 7: A Process and Economic Evaluation of j8-Glucuronidase (rGUS) Production from Transgenic Corn REFERENCES

INTRODUCTION Valuable products are being produced increasingly by biotechnological methods. Burrill and Roberts^ estimate that by the year 2000 the worldwide sales of these biotechnological products will be $100 billion. The western hemisphere countries, particularly the United States, Germany, France, and England, are the leading players. For example, diabetic and thrombolytic drugs have a very large market throughout the world, hence it would be desirable to review the sales of these classes of drugs. In the United States, the estimated market for diabetic drugs is $1.8 billion which includes $800 million for insulin.^ The estimated market for thrombolytic drugs is $355 million.^ Biotechnological products include not only pharmaceutical drugs but also other biological macromolecules of interest. One needs to separate the biological macromolecule of interest; and herein lies a very significant Separation Science and Technology,

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cost of the entire process. For different processes, the fraction of the entire cost of the process required for bioseparation will vary. Ronsohoff et aL"^ indicated that the purification and recovery costs may be as high as 80% of the total manufacturing costs. These costs may be higher if ultrahigh purity of DNA-involved products are manufactured. Recognize that during processing one may have to purify products at 99.9% levels v^ith virtual complete removal of DNA, viruses, and endotoxins. The key to cutting production costs is to emphasize improvements in downstream processing. Traditionally all the steps occurring in the fermentor which result in the production of the desired biological macromolecule can be considered as upstream processes and all the other processes occuring after the fermentation and which result in the separation, purification, concentration, and conversion of the biomolecule to a form suitable for its intended final use can be classified under downstream processes. Thus, it is helpful to better analyze and understand the different facets involved during downstream processing. Better physical insights are required and are continuously being obtained in downstream processes, and these will eventually lead to a more efficient and economical process. Note that upstream processes are already well understood. Even though further improvements in upstream processes are possible, they do not have the potential of making as significant an impact on production costs as improvements in downstream processes. Also, one should not or may not treat the upstream and downstream processes separately, but one should integrate the downstream processes with the upstream processes. For instance, any change or improvement envisaged in an upstream process should also consider the possible effect of this change on the downstream process. Minor changes in upstream conditions may have a significant impact on downstream processes. Thus, early in the design of processes, one must consider the impact of upstream processes on downstream processing. One technique where different "what-if" possible scenarios can be analyzed is with the development of an appropriate model for the process. Ernst et al.^ emphasize the importance of computer-aided process simulation, and the early necessity of providing a workable process flow sheet. They emphasize the need for this during the early stages of process development. This is an important tool to help optimize the process expeditiously. Considering the high stakes that are involved in getting a drug to the market, and the fierce competition involved, it seems appropriate to get as much useful information of a process as early as is possible during the development process. This also explains the extreme secrecy involved in the research and development of key steps in the bioprocessing of a highly marketable and valuable product. It does take $100 milHon^ to $300 miUion^ to bring a pharmaceutical drug to the market. Also, a minimum of 7 to 10 years are required. The Biotechnology Industry Organization indicates that it takes 10 to 12 years to move a product from the bench to the bedside. This is twice as long as it took 20-25 years ago. Stone^ estimates that the cost of a drug has increased by a factor of 5 to $360 million, in the period between the early 1970s to the mid-1990s. Another estimate by Hassler^ is $350 to $400 miUion to develop a drug. These figures are a few years old, and it is presumably reasonable to

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assume that it now takes $400 to $450 million to develop a drug considering the ever increasing and stricter Food and Drug Administration (FDA) regulations. Another phenomenon that is occurring, and that which is not only restricted to this industry, is the alliance of two or more companies to provide a synergistic effect which makes it more and more competitive in a particular area. More often than not, the alliance occurs between a small company that has a novel idea, but does not have the resources to economically bring it to the market or take the financial risk involved, since a considerable amount of a variety of resources are required to do so, and one or more large companies with the needed resources. According to a report appearing in BioWorld Today, in 1993, there were 69 aUiances accounting to a total collaboration value of $1.3 billion, in 1997 the number grew to 227, and the total value of these collaborations to $4.3 billion. These multifaceted requirements are available only with the more established and well-known pharmaceutical companies. In essence, the small company essentially discovers a new technique or drug; and the larger pharmaceutical corporations then effectively bring it to the market after forming a collaboration with the smaller company. In fact it may be predicted, and there are indications that due to the high cost involved, eventually there will be one to three fully integrated pharmaceutical companies, and presumably 50-100 successful novel idea-generating companies. Everybody else will be doing something else in some other capacity. Another phenomenon that has come about is the emergence of outsourcing companies. Remarkably, some of these have done very well. In the list of "Molecular Millionaires" published by Genetic Engineering News in its 1997 edition, two of the top three on the list owned considerable stock in Quintiles Transnational Corporation, a leading contract research organization catering to biotechnology and pharmaceutical companies. One of the reasons for the continued success of these companies is the increasing resources required to bring a drug to the market; and most companies do not have either the resources or the many-faceted knowledge requirements necessary to bring a drug economically to the market. Scarlett^^ emphasizes that scientifically competent and service-based outsourcing companies are a boon to the biotechnology and pharmaceutical companies. They provide a muchneeded flexibility to the entire manufacturing process. This is true, however, not only for the pharmaceutical companies but also for the other industries as well. A. Example I: Role and Use of Outsourcing Companies during the Development of a Process'^ Considerable flexibility is provided to biotechnology and biopharmaceutical companies by scientifically competent, financially secure, and service-based outsourcing companies. During the selection process, a potential client should carefully check the technical capacity of the outsourcing organization, its facility, and its ability to provide economic production of the desired product. Scarlett^^ indicates the high risk involved in releasing a recombinant-based

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product in the market. The author emphasizes that an organization needs to commit about $40-75 milhon for building a faciUty at about the time when the product is ready for cHnical trials. This is because it takes 30-36 months to design, construct, and vaHdate a licensed current good manufacturing practice (cGMP) facility. Besides, one requires about another year to get the first conformance lot. Another two additional lots are required even before one may apply for a license. Thus, the financial risks are very high. Needless to say, many companies are very reluctant to commit these vast resources to a single project. Product failure in phase III clinical trials is not unknown, and this can have a devastating effect on not only the company morale but also its stock price. Most small companies just do not have these resources, and the ability to absorb this kind of failure at this late stage. This may perhaps also be true for some of the larger corporations. Scarlett^^ indicates that another point to consider is that a corporation may not be running its facility at its full capacity; nevertheless it will still have to bear the capital cost, the cost of the personnel, and the utilities. Most corporations just do not have the depth of products required to fully utilize its expensive facility. Sometimes, the facility may be running at even about 30% of its capacity. The outsourcing organization can overcome this drawback by carefully selecting multiple contracts. This enables it to enhance its capacity utilization of its facility. Considering the expense required to maintain an in-house facility, this author suggests that it is worthwhile outsourcing in some cases, even when the profit margin of the outsourcing organization is taken into account. Finally, this (recent) phenomenon speaks for itself considering the high capitalization of some of these outsourcing companies who cater to the needs of the biotechnological corporations. II. DRUGS MARKET AND SALES Different issues about getting a biotechnological product ready for the market have been proposed.^ These include the product conformation to FDA standards, its safety, its purity, and its potency. Other considerations include shelf life and product stability in injectable form. However, if a corporation successfully gets a product to the market after crossing the seemingly never-ending hurdles, the profitability even for a single drug is quite substantial, especially if one has captured a large part of the market share, or has the only drug in the market for a particular ailment. A. Example 2: Increased Profitability and Market for Drugs (Erythropoietin)" Erythropoietin is a drug having a very large worldwide market. Furst^^ has recently analyzed the sales of erythopoietin (EPO) made by Amgen. During the years 1992, 1993, 1994, 1995, and 1996, the sales of this drug were (in milhons of dollars) 506, 587, 721, 883, and 1072, respectively. This is a blood cell stimulant, and Amgen is keen on maintaining its current supremacy in this market. Its next generation erythropoietin is in phase III trials. This drug is known as novel erythopoeisis-stimulating protein (NESP). This is a

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preparation of one or more of several highly active isoforms of erythropoietin. Amgen is keen on maintaining its lead position in this area. It does have competition from Boehringer v^hich has an erythropoietin annual sales of $400 milUon. It would be of interest for corporations to obtain a predictive function (general, if possible) for drugs v^ith respect to the sales figures for every year. The sales figures for EPO for Amgen are available for five years, as already mentioned. A linear regression v^as performed on the data mentioned above for the sales of EPO between the years 1992 and 1996. The year 1992 was the first year for which the data are available, and that is taken as year = 0. Sigmaplot^^ may be utilized to model the sales number for EPO using an equation of the form Sales of EPO, in million $ = a (time in years, with 1992 = 0)

(1)

Here a and b are coefficients that are to be determined by a regression analysis using Sigmaplot.^^ Figure lA shows the fit of the predictive equation: Sales of EPO, in million $ = (602.64 ± 34.61) (time in years, with 1992 = of'"^^^-^'^^^^ (2)

1100

1 2 3 year starting with 1992 (= 0)

0

2

4

year starting with 1992 (=0)

6

F I G U R E I Profit functions for EPO: (A) power-law model and (B) second-order polynomial. (Data taken from Furst")

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The fit is reasonable, and the availabiUty of more points (sales figures for different years) would more firmly establish this equation. Obviously, this equation applies only after t > 1 year(s). For year 0, the number was directly used. It would be of interest to note if a similar form of this predictive equation applies to the sales figures of other drugs also. Thus, the necessity of developing and utilizing a general form of the equation to predict sales figures for future years for different drugs. There is some scatter in the data. Since we are talking about millions of dollars it would be of interest to also be able to model the sales figures as closely as is possible even at the cost of some rigor in the analysis. Figure IB shows the curve obtained using a second-order polynomial. This equation is given by sales of EPO, in million $ = 17A29t^

+ 38.229^ + 447.4

(3)

Here t is time in years. This equation provides a perfect fit for the sales figures for EPO as a function of time in years. Once again, ^ = 0 here indicates the base year, 1992, for which the sales numbers were first available. For practical or predictive purposes Eq. (2) may be better to use than Eq. (3) since it is a power-law type of equation and has some physical basis, even though Eq. (3) provides more accurate estimates than Eq. (2). Secondorder polynomial fits can of course be applied to a wide variety of sales curves for different drugs with considerable accuracy. In other words, if one were to attempt to predict future sales of EPO, one should (a) obviously use caution, (b) extrapolate only for a short period (since one cannot predict market forces), and (c) use the powerlaw model as compared to the secondorder polynomial model (Eq. 3). Considering the high level of profit that is possible in the manufacture of drugs (if one is successful in bringing it to the market) it is not unexpected that the level of competition will be intense. More often than not, alternative drugs for performing the same corrective action for a medical ailment will be manufactured by two or more companies. Let us get a better idea of this competition by analyzing an example. B. Example 3: Some Data on the Existing Market for Thrombolytic Drugs^ The thrombolytic drug market is estimated to be $355 million.^ Genentech's tPA (tissue plasminogen activator) is a drug frequently used to clear the arteries of blood clots. Genentech's tPA (alteplase) has recently been challenged by Boehringer Mannheim's [reteplase (RPA)]. Recent studies indicate that heart-attack patients have about the same chance of survival whether they were treated either by alteplase or by reteplase. A study was made on patient survival 30 days after a heart attack when they were given one of these two drugs. Note that 7.22% of alteplase patients and 7.43% of reteplase patients died. This is a statistically insignificant difference, and one is unable to say which drug is better. Davidson^ indicates that researchers could not agree if the drugs are equivalent. No cost figures were given for either alteplase or reteplase. Perhaps economics or the severity of side effects

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for survival patients may be able to help us decide which of these tv^o drugs, if any one, is better than the other. The market for diabetes drugs is $1.8 billion and the market in United States including insulin is $800 million.^ It is estimated that 40-50 million people take insulin worldv^ide. Note that for 90% people, insulin is only partially effective in controlling sugar. A better drug introduced in the market by Warner Lambert is Rezulin. This drug not only lowers blood sugar level but it also help control type II diabetes. Rezulin or troglitazone acts by decreasing insulin resistance, which is the most common indicator of type II diabetes. This is done by facilitating the uptake of the insulin in the skeletal muscle, and by decreasing glucose production in the liver. The actual mechanism of action is not quite clear.^ However, it is suggested that rezulin binds to nuclear receptors that regulate the transcription of insulin response genes. Warner Lambert indicates that Rezulin is headed for Europe. In clinical trials Rezulin effectively brought down glucose levels such that 15% of the users stopped insulin altogether, whereas others cut down their insulin doses. In animal studies, it was noted that very high doses cause tumors and gave rise to enlarged hearts in some rats. An unexpected benefit from using Rezulin is that it has the effect of lowering free fatty acids in the blood. Apparently, this drug may have a host of beneficial side effects.

III. APPLICATIONS OF MODELS AND FLOW SHEETS IN BIOSEPARATION ECONOMICS Models provide physical insights into the economics of industrial processes, are useful for comparative purposes, and if they posses enough detail they can be applied to a variety of cases with only a few appropriate modifications.^^ A detailed process flow sheet combined with good engineering judgment can be utilized to obtain general conclusions about the manufacturing cost of biopharmaceuticals, or in general of any other chemical of interest. A. Example 4: A Model for Process Economics'^ Models can be very helpful in analyzing and understanding a particular process. Datar^^ has proposed a model to analyze the costs of an Escherichia coli based fermentation system. Though the analysis is based on the production of an intracellular enzyme (j8-galactosidase), the author indicates that with only minor modifications, the analysis may be extended to include recombinant products such as human growth hormone or interferon. This flexibility in the model presented is a welcome addition. At the outset, this author indicates that the actual production costs may differ from that predicted by the model due to differences between the processes involved, raw material costs, labor, inefficient or efficient utilization of resources, etc. In any case, the model does provide physical insights into the cost structure of the process, and where improvements and significant effort should be expended to help minimize the process cost effectively.

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Datar^^ indicates that the total manufacturing cost of a bioprocess may be given by: total manufacturing cost = [ a R M + jSFCI + yOL + 8E]/Z

(4)

The total manufacturing cost includes administration, research and development, distribution, and selling. Here RM represents the costs of the raw materials; FCI is the fixed capital investment; OL is the cost of operating labor; E is energy costs; Z is the step or overall yield; and a, j8, y, and 8 are coefficients that must be determined for a specific situation. In the example analyzed, the authors noted the following distribution in costs: raw materials 20%, labor 36%, and capital related charges 28%. The capital-related costs include taxes, insurance, and depreciation. The labor costs include operating, maintenance, and overhead. The remaining 16% of the costs may be assigned to energy. The coefficients a, j8, y, and 8 were determined by the method suggested by Bridgewater.^"^ Datar^^ indicates that these coefficients were determined during detailed pilot-plant trials. The author does point out that the type and quality of product required for the high-resolution fractionation stages does significantly determine the sequence of the primary separation stages. The analysis helped the author to suggest the modifications to help improve process economics at different stages. Increase the fermentation yields by genetically engineered microorganisms. However, one should ensure that these organisms continue to be productive at high levels; make every effort to optimize existing downstream processes, especially the different unit operations, such as centrifugation and ultrafiltration; and integrate existing unit operations thereby minimizing product loss as well as increasing the overall efficiency of operations; and finally attempt to attain maximum utilization of recovery equipment. Every aspect of the process must be analyzed within an economic framework. Sometimes, the choice of a unit operations for a primary separation between centrifugation, membrane filtration, and aqueous polymer extraction is difficult to determine on a cost structure basis. In that case, one should resort to other factors such as reliability, ease of scale-up, maintenance, yield, etc. In any case, the initial model suggested by Datar^^ is very valuable since it correctly breaks down the cost structure of the different components during the different stages, and helps provide guidelines where significant improvements may be made. It is a flexible model, and depending on one's particular situation, one may make modifications at appropriate places, especially after identifying critical separation steps. However, while making modifications one should always keep in mind the stability, purity, and yield of the biological product. One's choices may often be constrained by these variables, especially if these constraints are imposed by some external controlling agency, such as the Food and Drug Administration. The development of further models such as these, and the sensitivity analysis that one can perform with them should go a long way to help make processes more and more profitable. In fact, in this excessive competitive market and age, it is difficult to envisage doing without them.

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The next two examples pursue this Hne of thought further wherein we analyze a computer-aided process of human insulin production. ^^ This is then followed by an analysis of process simulation of heparinase I production from E. coli.^ B. Example 5: An Economic Analysis of Biosynthetic Human Insulin Production'^ As mentioned earlier, insulin accounts for about 50% of the worldwide market for diabetic drugs. The metabolism of carbohydrates is facilitated by insulin. Diabetes mellitus results if insulin is not properly produced in the body. Barfoed^^ indicates that diabetes is the third largest cause of death after cardiovascular diseases and cancer. Petrides et al}^ indicate that insulin is a two-chain polypeptide that consists of 51 amino acids. The A chain contains 21 amino acids, and the B chain contains 30 amino acids. These chains are connected by disulfide bonds. One method of making biosynthetic human insulin is to produce each chain as a j8-galactosidase fusion protein in E. colt. The two chains are purified and then combined together to yield the human insulin. Another method to produce insulin eliminates the step that involves the combination of the two steps. In this method, proinsulin is produced by fermentation as inclusion bodies. Cyanogen bromide (CNBr) is then used to cleave the methionine linker. Petrides et al}^ indicate that the proinsulin chain is placed in a folding environment wherein the disulfide bonds are formed. The C peptide is then cleaved to yield the human insulin using enzymes. These authors proposed a flow sheet for the production of biosynthetic human insulin (BHI). They used information available in the patent and open technical literature, and combined it with their engineering experience and judgment. The basis utilized by Petrides et al}^ is 1500 kg of purified BHI per year. They indicate that this represents 1 0 - 1 5 % of the world demand.^'^ In essence, the following downstream steps are involved in sequence during synthetic BHI production. The fermentation step (not a downstream step) is also included to provide some continuity. The steps are fermentation, cell harvesting, cell disruption, inclusion body recovery, inclusion body solubilization, enzymatic conversion, refolding, sulfitolysis, CNBr cleavage, final purification steps, and crystallization. In the flow chart provided by Petrides et al}^^ a surge tank separates the upstream from the downstream processes. This tank is in between the fermentor and the downstream processing steps. A disk-stack centrifuge is used for cell harvesting. A high-pressure homogenizer is utilized to break the cells and release the inclusion bodies. These inclusion bodies are recovered in another disk-stack centrifuge. The inclusion bodies are then solubilized in a well-mixed reactor with urea. This chaotropic agent dissolves the denatured protein. A filter is used to remove the fine particles such as biomass, debris, and inclusion bodies. The chimeric protein is then cleaved with CNBr into Trp-LE'-Met (which contains 121 amino acids) and the denatured proinsulin (82 amino acids). This denatured proinsulin must be converted to the active insulin form. Sulfitolysis is carried out under alkaline conditions wherein the proinsulin

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molecule is unfolded, and the SO3 moiety is added to all sulfur residues on the cysteines. In this reaction a denaturant (such as guanidine-HCl) is added to minimize refolding and cross-folding of the protein molecules. Folding of the proinsulin (S-SO3-) and disulfide formation then occurs in a reactor. Mercaptoethanol is added to facilitate the disulfide interchange reaction. Thereafter, trypsin and carboxypeptidase are added to remove the C peptide from human insulin. Trypsin is added to cleave the proinsulin at the carboxy terminal of the internal lysine and arginine residues. Carboxypeptidase removes the terminal acids. Petrides et a\P indicate that the final purification steps involve multimodal chromatography.^^ Differences in hydrophobicity, molecular charge, and size are typically utilized to separate the human insulin. An ion-exchange chromatographic step is follov^ed by a reversed-phase high-pressure liquid chromatography (RP-HPLC) step. Care must be taken to remove structurally similar insulin-like compounds. Minor modifications in the insulin molecule are carefully removed. They have a higher retention time than the actual insulin molecule. Filtration is employed at different stages, and the process is completed w^ith a gel-filtration step. Finally, zinc crystallization is performed by initiating with small amounts of zinc chloride. The crystals are recovered by a basket centrifuge and freeze dried. Petrides et a\}^ utilized a BioPro Designer to assist in the process design and economic analysis. They indicate that this may be used in the design and analysis of biochemical, pharmaceutical, and in the food processes. Their analysis is based on 1994 prices. For a plant of base case (1500 kg per year of BHI) the total equipment cost is estimated to be $18.1 miUion. The fixed capital investment is estimated to be $141 million. A total of $13.1 million is required for the rav^ material costs. The consumables cost is $8.23 million, v^ith the chromatographic resins contributing about $8.02 million to this cost. A pie chart clearly broke down and summarized the operating costs (Fig. 2). Over 50% of the total operating cost may be allocated to directfixed-capital (DFC)-dependent cost. Raw materials is the next most expensive category accounting for 23.3% of the total operating cost. This is followed by the cost of different consumables which is 14.6% of the operating cost.

Administration (2.00%) Waste Treatment/Disposal (6.60%) Utilities (0.30%) Consumables (14.60%)- ^^^^^^^^^^^^^^^^^^^^ -DFC (48.20%)

Raw Materials (23.30%) Labor (5.00%) FIGURE 2

Breakdown of operating costs.

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Another way of looking at the cost structure is to note that about 97% of the cost is required in the product recovery and purification steps. Chromatographic separation is a very expensive item, and accounts for almost 70% of the total operating cost. Petrides et al}^ indicate that due to the very high purity required in the biopharmaceutical industries, this high fraction of the total cost for the chromatographic steps is not unusual. These authors were careful to include a cost for waste minimization, treatment and disposal. Their cost accounted for G.(i% of the total cost. This is a small though important factor in the cost structure, since as environmental standards get stricter in the future (which they will), this waste disposal cost will rise very quickly. Note that as one removes the last few percentages of waste from a solution, the costs rise very quickly (almost in an exponential or similar function fashion). Petrides et alP indicate that the BioPro Designer is ideally situated to ask "what if" type questions. It facilitates a sensitivity analysis of the influence of key variables on the production cost and the profitability of the project. The effect of three variables were analyzed. Their analysis indicates that product cost is quite sensitive to a biomass content of 20% and less. The product cost does not change significantly for a biomass content of greater than 20%. This is because most of the production cost is associated with the product recovery and purification costs. They emphasize that this sort of information may be utilized to prioritize future work. Petrides et a\}^ also analyzed the product cost as a function of plant throughput process scheduling. The base case analyzed involves a plant batch time of 180 hr, with 100 batches per year. The limiting step is the fermentation step, and that required 30 hr. In other words, if one wants one can start a new batch every 30 hr, and thus one may run 240 batches a year. Their analysis indicates that about a 2 5 % cost reduction is possible if one were to increase the number of batches from 100 to 200. If one were to operate only 50 batches per year, then the product cost increases by about Sl% from $37 per gram to $58 per gram. Furthermore, their analysis indicates that the production cost is rather sensitive to production rates less than 1000 kg per year. The production costs are quite insensitive to production rates greater than 3000 kg per year. The authors indicate that for production rates greater than 3000 kg per year, all expensive equipment are operating in parallel, and cost savings is difficult in this situation. Finally, Petrides et al}^ analyzed the influence of up-front research on the internal rate of return. They emphasize that the production and commercialization of biopharmaceuticals is a high-risk (due to the high cost involved) endeavor. The high cost must to be amortized into the life of a product, and this expense must be accounted for in the profitability function. A selling price of $110 per gram of insulin for their base case would yield them an internal rate of return of 30%. The authors indicate that this number is high for the standards of commodity chemicals, but average or low for high-risk products. They plotted the influence of up-front research expenditures on the internal rate of return for the insulin plant profitability. It was noted that an up-front research expenditures of $75 million would decrease the insulin profitability (plant operation for 1500 kg per year) to around the levels of

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commodity chemicals (15%). A gradual increase in the up-front research expenditures led to a gradual decrease in the internal rate of return. The analysis of Petrides et al}^ is useful in quite a few ways. It shows clearly, though not unexpectedly, that the major cost in manufacturing high-valued products lies in the recovery and purification steps. This is due to the very high purity involved. The sensitivity analysis presented by these authors is of interest since it enables one to not only enhance the economics of the manufacturing process but also to select the optimum range of conditions from a myriad of optimum operating parameter ranges determined by these sensitivity analysis. This is particularly useful, since sometimes there are constraints that prevent one from attaining a particular set of operating parameters. Furthermore, in a dynamic and competitive environment, the sensitivity analysis quickly helps one to select the right choice of operating parameters which are most beneficial for a particular organization under a specified set of circumstances (that may be influenced by external as well as internal constraints). Finally, more such studies must be carried out on real-life examples and made available in the literature. No doubt such studies are available in the different pharmaceutical industries, but due to economic and obvious reasons, they are restricted to only in-house circulation. This is an unfortunate aspect of the economics of bioseparation of different processes. Hopefully, people in the universities, at least, will pay more attention to this aspect, and fill this critical gap in the complete analysis of bioseparation processes. Considering the importance of this technique it is worthwhile analyzing another example wherein cost estimates have been made along with sensitivity analysis using process simulation. Ernst et al.^ indicate that computer-aided process simulation is widely used to help design and optimize the performance of chemical processes. These ideas have been extended to bioprocesses.^^'^^'^^ Ernst et al,^ indicate that process simulation involves initially delineating the process flow sheet. This is followed by describing and solving for the mass and energy balances. Cost estimates are made for the different steps, and after an overall picture is obtained, sensitivity analysis may be undertaken. C. Example 6: An Economic Analysis for Heparinase I Production from £. COU"^ Ernst et al.^ indicate that heparinase I (EC 4.2.2.7) production from Flavobacterium heparinum is an interesting choice for economic analysis. Heparinase I is an acidic polysaccharide that degrades heparin, which is used as an anticoagulant drug.^^ Ernst et al.^ indicate that heparinase has wide clinical applications and may also be used in the treatment of angiogenesisdependent diseases.^^ Due to its potential clinical applications there is a significant demand for high-purity heparinase I. These authors indicate that a manufacturing process for the production of heparinase I from £. coli is in the initial stages of development. It would be worthwhile exploring a method to design and to evaluate economically a manufacturing process for heparinase I. They designed a process based on data made available from laboratory-scale experiments.

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Ernst et al,^ estimated the demand per year for the chnical apphcations of heparinase I to be 1.8 kg. This include appHcations for heart surgery patients, for immobihzed heparinase I reactors for use in acute dialysis procedures, and for analytical purposes. They designed for a base process to manufacture 3 kg of heparinase per year. They proposed two flow sheets. One flow sheet is for the production of recombinant heparinase I by insoluble expression. Another flow sheet involves production by soluble expression of heparinase I. The flow sheet for the insoluble expression case involved the following steps: fermentation, centrifugation, homogenization, centrifugation, unfolding, filtration, refolding, filtration, affinity chromatography, diafiltration, thrombin cleavage, diafiltration, affinity chromatography, and finally, gel filtration. The flow sheet for the soluble expression case involved the following steps: fermentation, centrifugation, homogenization, centrifugation, filtration, affinity chromatography, diafiltration, thrombin cleavage, diafiltration, affinity chromatography, and finally, gel filtration. Ernst et al.^ indicate that the SuperPro Designer contains models to help estimate the direct fixed capital cost based on 1995 dollars. These numbers may be suitably updated using the current CPI or other suitable index. Maintenance cost were set at 2 5 % of the purchase cost of the equipment per year. Laboratory costs were allocated to capital costs. Labor costs were carefully calculated based on number of operator hours per equipment hour. This was done after analyzing scheduling of the entire process as well as noting that the operator time on each equipment was reasonable. i. Insoluble Expression

This process was designed to run in campaigns of 24 batches per year. The fermentation volume is 2.9 m^, and the largest volume occurs after dilution for refolding (12 m^). The authors indicate that the capital investment is not dominated by any single piece of equipment, and the total capital cost of producing heparinase at this level is $12.5 miUion. The manufacturing cost (that includes the cost of capital depreciation) is $560,000 per kilogram. Capital cost is the highest expense category that includes: $92,000 for fermentation, $94,800 for harvesting and refolding, $58,800 for affinity chromatography 1, $23,100 for filtration and cleavage, $39,200 for affinity chromatography 2, and finally, gel filtration. The capital cost represents 55% of the total cost of production. The next expensive category is raw materials and consumables which represents 19% of the total cost. This is followed by labor and maintenance which are 17 and 9%, respectively, of the total cost. Fermentation (actually an upstream cost) (26%) and primary affinity chromatography (23%) represent the most expensive individual steps. These authors emphasize that since they were able to design a manufacturing process based on laboratory scale data they were able to obtain an idea about the profitability of the process. Furthermore, an additional advantage was the sensitivity analysis possible due to the detafled flow sheet made available. This enabled the authors to examine and to analyze the profitability for different possible scenarios, and thus they were able to tailor make a possible flow sheet most suitable for their application. This, in our opinion, is a powerful advantage, and the sooner this type of information is made

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available during the design of a process, the more beneficial it will be for a particular organization. It behooves an organization to obtain as much information as is possible as early as is possible from all sources and view points, so that they may be able to effectively design a economic process. Also, possible pitfalls are avoided, and any advantage, no matter how trivial, is fully exploited. ii. Soluble Expression

Ernst et al.^ indicate that for this case, 24 batches are required to produce the 3 kg/year (base case). The fermentation volume is 12 m^, and eventually results in 94 liters of pure product at 1.3 mg/mL. The most expensive pieces of equipment in this case are the two centrifuges for processing the cell lysate ($225,000 and $350,000 each). The fermentor is estimated to cost $388,000, $24.1 miUion is required as direct fixed capital cost, and $1,024 million is required for the manufacturing cost. This number includes the depreciation cost. In this case, the fermentation (42%) and the harvesting (36%) are the two most expensive process costs. Also, the capital cost is 59% of the total cost. Ernst et al}^ emphasize that the soluble expression method is 80% more expensive than the insoluble expression method. This is because of the low level of expression of heparinase I. This leads to large process volumes during fermentation and harvesting. This then shows up in higher capital and raw material costs. Ernst et aL^ compared the costs involved in the insoluble and soluble expression methods. At the outset, it appeared that the insoluble method is more cost effective than the soluble expression method. However, these authors cautioned that if the values of certain key parameters were to change then this might significantly change the order of preference. For example, they analyzed the influence of (a) the change in the expression level of soluble heparinase I and of (b) a change in the protein concentration during refolding. Their preliminary calculations indicated that if the expression level of soluble heparinase could be increased to 100 mg/mL as compared to 129 m g / m L (for insoluble heparinase I expression), the two techniques are equally cost effective. Also, if the refolding process could be carried out only at low protein concentrations ( < 5 0 mg/mL), then the process based on soluble heparinase I expression would be more economical than that of the insoluble heparinase I expression even at the present expression level. These are interesting pieces of information, and if future research or research improvements permit the application of these conditions, then this may have a direct impact on the choice of the method selected for the production process. Another technique that Ernst et al.^ utilized was to reduce the temperature of the fermentor to 15°C during the induction phase. This reduced the inclusion body formation, and achieved soluble heparinase I at a level of 4.1 m g / g dry cell weight. Their calculations indicated that the manufacturing cost in this case would be $653,000 per kilogram. This is only 16% higher than the cost obtained for the insoluble heparinase I expression method. Ernst et al.^ also indicate that process development in the bioprocess industry is a compromise between optimization and time to market launch.

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Thus, one is often keen on obtaining improvements in the process in the shortest interval of time. These authors developed a mathematical formulation to assist them in doing this. Their formulation was based on two simple principles. The first is to estimate the improvement in the cost structure by enhancing the performance by a certain fraction. Also, one should have a fair assessment of the probability of success of attaining this enhancement of performance. They emphasize that this enhancement of performance is often subjective, however, do try to make it as quantitative as is possible. Using the preceding technique, the authors identified that they should focus on improving the fermentation step, and the affinity chromatography recovery steps. For example, they noted that by increasing the yield from the affinity chromatography step to 90% led to a decrease in the cost by 25%. Also, an increase to 2000 m g / m L of expression level of active, refolded heparinase I leads to a 12% reduction in cost. These numbers may be compared to the base case. In conclusion, Ernst et al,^ emphasize that one should consider the economic aspects of a process early on in the development of a bioprocess. They indicate that during the development of a process based on recombinant expression in £. coli, there is often a choice whether to select a soluble or insoluble protein expression. In general, soluble protein expression results in lower product accumulation during fermentation. This leads to large volumes of broth for a given production rate. Finally, this lands up as a higher cost. Insoluble protein expression generally requires more processing steps, such as solubilization and refolding. The authors utilized their technique to help design a production scale process based on laboratory-scale data. They compared figures for heparinase I production using either a soluble or an insoluble expression form. Their results indicate that the insoluble expression method was more economical than the soluble expression method. They do point out that this choice between a soluble expression and an insoluble expression is critically dependent on the concentration at which protein refolding may be carried out for the insoluble expression process. The detailed analysis method presented by these authors is of particular value especially if a reliable breakdown of the cost for each process step is made available. The sensitivity analysis provides a good framework for the profitability function for a wide range of system parameters. This is of value, as indicated earlier, in a highly competitive and dynamic environment. Besides, the sensitivity analysis provides an overall perspective and potential of the economics of the bioprocess. The analysis presented is of a general enough nature and may be applied to the design of other bioprocesses to obtain economic data. The application and extension of this analysis to the design of other bioprocesses would be of considerable value, since it would provide a useful framework and considerably enhance the reliability of the analysis process. Finally, the authors do come up with a paradigm of some importance which is that "process steps which are downstream of the most expensive steps should be the most advantageous to optimize." They do emphasize, and caution, that changes should always be done within an economic framework.

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D. Example 7: A Process and Economic Evaluation of ^-Glucuronidase (rGUS) Production from Transgenic Corn^^ Plant biotechnology offers an alternative and attractive method for the production of many important recombinant proteins. Many plants have been investigated with this purpose in mind. The advantage of using these transgenic plants is that they are relatively inexpensive to grow and maintain. In many cases, the protein production can be simply scaled up by increasing the plant acreage. Also, by using appropriate promoters and growth factors the desired protein can be expressed and stored in the natural protein storage organs (such as tubers and seeds). This provides stable storage for extended periods of time.^"^ Moreover, postharvest handling and crop processing are also well established.^^ As is the case with other biotechnological products, the extent of protein purification depends on the final intended application of the product. There are applications where the plant tissue can be directly used, and hence purification is not needed. In other situations particularly pharmaceutical products administered parenterally, there are stringent purity requirements, necessitating complete removal of viral particles, endotoxins and other contaminants. There are very few published reports that make quantitative and characterize the extraction and purification of proteins from transgenic plants. Furthermore, there are practically none dealing with the economics of their downstream processing. Evangelista et alP proposed a downstream processing scheme based on the bench-scale extraction and purification of rGUS in their laboratory. The transgenic corn seed containing about 0.005% rGUS was first ground, and then extracted with a buffer. The slurry was centrifuged and filtered. Purification was done in three steps. The first step involved the adsorption on a DEAE-Toyopearl column (Supelco, Bellofonte, PA). This was followed by a hydrophobic interaction chromatographic step on an Octyl Sepharose column (Pharmacia, Piscataway, NJ). Finally, there is an ion-exchange step on a DEAE-Toyopearl column. Thus, the process model can be divided into three sections: (a) a front end consisting of a milling section; (b) a middle section for the aqueous extraction of proteins, removal of spent solids, concentration of the crude extract and drying of the spent solids; and (c) finally, a purification step comprising three-stage chromatography, diafiltration, and freeze-drying of purified rGUS. Evangelista et alP utilized a Superpro Designer 2.7 (Intelligen, Scotch Plains, NJ) for the process simulation and economic analysis. Note that the previous two examples have also utilized this software. As a base case, a production plant processing 4545 kg (10,000 lb) of transgenic corn per batch containing 0.015% rGUS was selected. The batch time was estimated to be 61 hr. Assuming a 90% efficiency, a plant operating for 7500 hr/year could process 383 batches producing 137.2 kg of rGUS annually. A final purification yield of about 58% and a purity of 83% is achieved. It was estimated that the total equipment cost was $1.69 million for this process. The major portion of this equipment cost was attributed to the chromatographic columns which was about 49%. The installation of this

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equipment along with the auxiHary faciHties cost around $5.71 milHon. The total plant costs including the direct fixed capital and other indirect costs comes to about $10.51 milUon. If we add the working capital and the startup costs we can envisage a total capital investment of about $11.14 million. The total annual operating costs (AOC) amounted to about $5.9 million. Cost items related to the direct fixed capital contributes to about 3 3 % of the AOC. Raw materials and other consumables contributed to about 30%. The major chunk (about two thirds) of the capital-related cost occurred in the purification section mainly because of the four chromatographic columns. The stringent FDA regulations ensure that one pays attention to waste treatment. Waste treatment accounts for 3.4% of the AOC. One can sell the spent soUds as animal feed. Thus, basically one has to worry about the Hquid wastes. 7 1 % of these wastes are generated from the ultrafiltration unit and the remaining from the purification section. After having estimated the economics and the scale of the operation it would be worthwhile to briefly review the current market status of rGUS, and the profitability in launching an operation on the above scale. rGUS obtained from E. coli (marketed by Sigma Chemical Co.) has a current market value of $2000-$60,000 per gram (based on purity). rGUS from transgenic corn could be sold for about $10,000 per kilogram. If one is able to sell rGUS at the above price and the spent solids at $0.10 per kilogram, the total annual revenue will be $13.9 million. At 137 kg per year the unit production cost (UPC) will be $43,000 per kilogram. The return of investment (ROI) will be 52% and this gives us a payback time of 1.9 years. The overall purification yield of recombinant enzyme is affected by the scale-up and may vary from plant to plant. The profitability at lower yields and at a constant selling price was also determined. A 10% reduction in yield resulted in a 12.9% decrease in the ROI. Conversely, the effect of increasing the plant capacity between 4545 (base case) and 45,450 kg of corn per batch on rGUS production, capital investment, UPC and ROI was estimated. A five-fold increase in capacity resulted in a reduction of 30% in the UPC, and an increase in the ROI from 52 to 9 1 % . Further increase in the capacity did not have a significant effect on the UPC and ROI. With a five-fold increase in the capacity the total capital investment increased by a factor of 3.3, labor, administrative, and overhead expenses doubled, and the rest of the operating costs increased proportionally with the capacity. Another important factor that has an influence on the process and consequently on the economics is the expression level of rGUS. Generally, it is in the range of 0.015 to 0.15% of corn dry solids. This estimate is pretty realistic, because an expression level of 0.015% of rGUS has been achieved by ProdiGene, Inc. UPC costs can be decreased dramatically if one is able to increase the expression levels from the current 0.015%. At an expression level of 0.08% (five-fold increase) the UPC decreased by 75%, from $43,000 to $12,000 per kilogram. The ROI increased by about 28% for every 0.01% increase in the expression level. By increasing the expression level by five-fold the same level of production can be achieved with lesser amounts of raw materials. TCI, UPC, labor, administrative, and overhead costs drop by 50, 48, 20 and 50-80%, respectively. The ROI almost doubles.

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Thus, the factors that are critical in lowering the manufacturing costs, in the production of rGUS from transgenic corn^^ have been briefly analyzed. Note that the figures and the estimates provided here can be used just as a guideline to predict the type of costs that are involved w^hile undertaking an operation on such a scale. Actual costs and figures may vary depending on the situation and the requirements. It can be seen from the preceding analysis that rGUS can be produced and profitably marketed even at a 0.015% expression level. Just like any other dov^nstream process, the bulk of the AOC was incurred on the protein extraction and purification sections. The cost of the transgenic corn contributed to about one third of the raw material cost. Ways by which this cost may be reduced should be sought. Large amounts of consumables were used up in the extraction and purification sections. Ways to reduce this by processing the corn to remove components that are of no value should be examined further. Optimizing the milling and protein extraction steps could result in an increase of the recombinant product in the extract. This will concomitantly lower the water requirement, the ultrafiltration load, and the liquid waste. Lowering the protein load will also be beneficial in the purification step using chromatographic columns. This will subsequently result in a reduction in the column size and in the resin cost. The production cost was also sensitive to the capacity below a certain threshold value and also on the expression level which has a dramatic effect on the UPC. All these factors should be given careful consideration and optimum conditions must be chosen, keeping in mind the situation and the requirements to design a process which is both technologically sound and economically feasible resulting in maximum profitability. REFERENCES 1. Burrill, G. S., and Roberts, W. J. (1992). Biotechnology and economic development: The winning formula. Bio/Technology 10, 647-653. 2. Business and Regulatory News (1997). Financial outlook rosy for biotech in 1997. Nat. BiotechnoL 15, 113. 3. Davidson, S. (1997). New clot busters threaten genentech's tPA. Nat. BiotechnoL 15, 405. 4. Ronsohoff, T. C , Murphy, M. K., and Levine, H. L. (1990). Automation of biopharmaceutical purification processes. Biopharm 3(3), 20-26. 5. Ernst, S., Garro, O. A., Winkler, S., Venkatraman, G., Langer, R., Cooney, C. L., and Sasisekharan, R. (1997). Process simulation for recombinant protein production: Cost estimation and sensitivity analysis for heparinase I expressed in Escherichia coli. BiotechnoL Bioeng. 53(6), 575-582. 6. van Brunt, J. (1985). Scale-up. The next hurdle. Bio/Technology 3(5), 419-424. 7. Raab, G. K. (1992). A natural selection. Cover story. Chief Exec, May, pp. 35-39. 8. Stone, R. (1995). Chem. Eng. News, June 5, p. 17. 9. Hassler, S. (1995). Managed innovation (editorial). Bio/Technology 13, 529. 10. Scarlett, J. A. (1996). Outsourcing process-development and manufacturing of rDNAderived products. Trends BiotechnoL 14, 239-244. 11. Furst, I. (1997). Amgen's NESP heats up competition in lucrative erythropoietin market. Nat. BiotechnoL 15, 940. 12. Sigmaplot (1993). "Scientific Graphing Software," User's Manual. Jandel Scientific, San Rafael, CA. 13. Datar, R. (1986). Economics of primary separation steps in relation to fermentation and genetic engineering. Process Biochem. 21(1), 19-26.

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14. Bridgewater, A. V. (1973). The build-up of costs. Chem. Eng. 279, 537-544. 15. Petrides, D., Sapidou, E., and Calandranis, J. (1995). Computer-aided process analysis and economic evaluation of biosynthetic human insulin production—a case study. Biotechnol. Bioeng. 48, 529-541. 16. Barfoed, H. C. (1987). Insulin production technology. Chem. Eng. Prog. 83, 49-54. 17. Petrides, D. M. (1994). BioPro Designer and advanced computing environment for modeling and design of integrated biochemical processes. Comput. Chem. Eng. 18, S621-S625. 18. Prouty, W. F. (1992). How^ to recover recombinant protein products. Chemtech, October, pp. 608-615. 19. Cooney, C. L., Petrides, D., Barrera, M., and Evans, L. (1988). Computer-aided design of a biochemical process. In "The Impact of Chemistry on Biotechnology: Multidisciplinary Discussions" (M. P. Phillips, S. P. Shoemaker, R. D. Middlekauff, and R.M. Ottenbrite, eds.), pp. 3 9 - 6 1 , Am. Chem. Soc, Washington, DC. 20. Evans, L. B., and Field, R. P. (1988). Bioprocess simulation: A nev^ tool for process development. Bio/Technology 6, 200-203. 21. Ernst, S., Langer, R., Cooney, C. L., and Sasisekharan, R. (1995). Enzymatic degradation of glycosaminoglycans. CRC Crit. Rev. Biochem. Mol. Biol. 30, 387-444. 22. Sasisekharan, R., Moses, M. A., Nugent, M., Cooney, C. L., and Langer, R. (1996). Method for inhibiting angiogenesis using heparinase. U. S. Pat. 5,567,417. 23. Evangehsta, R. L., Kusnadi, A. R., How^ard, J. A., and Nikolov, Z. L. (1998). Process and economic evaluation and purification of recombinant /3-glucuronidase from transgenic corn. Biotechnol. Prog. 14, 607-614. 24. Symons, P. C , Dekker, B. M. M., Schrammeijer, B., Verwoerd, T. C , van der Elzen, P. J. M., and Hoekema, A. (1990). Production of correctly processed human serum albumin in transgenic plants. Bio/Technology 8(3), 217-224. 25. Whitlam, G. C , Cockburn, B., Gandecha, A. R., and Ov^en, M. R. L. (1993). Heterologous protein production in transgenic plants. Biotechnol. Genet. Eng. Rev. 11, 1-29.

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FUTURE DEVELOPMENTS S. AHUJA Ahuja Consulting, Calabash, North Carolina 28467

I. INTRODUCTION II. THE PARTNERSHIP OF PROTEINS A N D NUCLEIC ACIDS III. BIOTECH DRUGS A. War against Cancer B. Targeting Messenger RNA C. HIV D. Rheumatoid Arthritis E. Amyloid Diseases IV. ASSURING PRODUCTION A N D PURITY A. Liquid-Liquid Partitioning B. HPLC C. lEF D. Membrane Adsorption E. Displacement Chromatography F. Antibodies G. Combination Methods V. GENOMICS VI. LAB O N CHIP VII. RECOVERY OF BIOLOGICAL PRODUCTS A. First Steps B. Chemical Reactions — Intended and Not C. Dealing with Real and Potential Infectious Agents D. Bioseparations at the Molecular Level E. Purification Methods: Alternatives to Chromatography F. Process Design, Simulation, and Economics G. Chromatography H. Coping with the Output from Genomics I. Industrial Case Studies J. Making and Managing Process Changes K. New Recombinant Feedstocks and Techniques REFERENCES

INTRODUCTION The field of bioseparations is very dynamic. As a result, new developments are being constantly made in the techniques that have been discussed in this book. At the same time, new techniques are also evolving that will have a Separation Science and Technology, Volume 2 Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

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future impact on this field. The biomolecules of interest discussed in this book are proteins, nucleic acids, and oligonucleotides. In addition, monoclonal and recombinant antibodies are covered because they are the largest class of proteins currently in clinical trials and they are of enormous therapeutic interest in this area. The large focus of this book is on proteins. It may be recalled that proteins were named by Berzelius in 1838, based on the Greek word proteios, meaning in the first rank. Proteins enjoy an interesting partnership with nucleic acids (see Section II). Nucleic acids come in a wide range of molecular mass. Single-stranded oligonucleotides are composed of short strands of both natural and chemically modified nucleic acids. It is generally understood that the length of synthetic oligonucleotides is in the range of 5-150 bases, corresponding to 1500-50,000 MW (molecular weight). Synthetic single-stranded oligonucleotides are used in a number of ways in molecular biology and biochemical research, e.g., as primers for DNA sequencing and polymerase chain reaction (PCR) experiments, probes for gene isolation and diagnostics, linkers and adapters for blotting and ligation experiments, cloning, site-directed mutagenesis, and gene alteration. DNA sequencing mixtures contain single-stranded oligonucleotides, most often 300 to more than 1000 bases long. Indubitably, the biotechnology industry has evolved enormously in the last two decades. Let's recall that human insulin was the first recombinant therapeutic agent that was Food and Drug Administration (FDA) approved in the United States and that was less than 20 years ago. Since then, over 75 other recombinant proteins have been introduced as therapeutic agents. The fist comprises cytokines, hormones, monoclonal antibodies, and vaccines. There are about 1300 companies in the United States competing for this market, and the current sale of these products comprise approximately 10% of the sales of all therapeutic products sold in the United States. One such product, erythropoietin (an erythropoiesis-stimulating factor, also known as epogen; it is a circulating glycoprotein that stimulates red blood cell formation in higher organisms) has worldwide sales in excess of $1 biUion U.S. (Table 1)} The financial potential of these products is indeed great. This is apparent from the fact that over 500 biotechnology-related drugs are currently in clinical trials. Bioseparations or separations of biological interest have played a significant role in the development and growth of this industry.^ These separations must be performed both on analytical and industrial scales and everything in between. In the 1980s, monoclonal antibodies were hailed as magic bullet therapeutics for the treatment of cancer, autoimmune disorders, and infectious diseases. Humanized monoclonal antibodies (MAbs) are now succeeding as drugs where mouse MAbs failed. Fully humanized MAbs are in the development pipeline, and therapeutic-biospecific MAbs are extending the versatility of nature's magic bullets. Several MAbs are now FDA-approved drugs (Table 2). Several MAbs are also in development (Table 3).^ It is generally recognized that the commercial success of biotechnology products is largely due to the successful development of high-powered analyt-

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TABLE I

Leading Biotech Drugs

Product

Biological basis

Activity/use

Epogen Neupogen

Eythopoietin Colony-stimulating factor Eythopoietin Insulin a-Interferon Hepatitis B Glucocerebrosidase Tissue plasminogen activator Somatropin GPIIb-IIIa antibody Interferon beta-la Somatrem-somatropin Dornase alpha Interleukin Colony-stimulating factor

Red blood cell growth White blood cell growth

Procrit Humulin Intron-A Engerix-B Cerezyme Activase Humatrope ReoPro Avonex Protropin-Nutropin Pulmozyme Proleukin Leukine

Sales ($ millions)

Red blood cell growth Diabetes Anticancer; infections Vaccine Genetic deficiency Heart attack/stroke Growth deficiencies Prevents blood clots Multiple sclerosis Growth deficiencies Cystic fibrosis Cancer White blood cell growth

$1161 1056 1000 936 598 584 333 261 260 254 240 224 92 71 53

Note that figures are unavailable for Roferon a-interferon, Recombinvax hepatitis B vaccine, and antihemophilia blood clotting such as Kogenate and Recombinate. 1997 estimated worldwide sales data from Thayer.^

TABLE 2

Humanized MAbs Approved by the FDA

Monoclonal antibody

Connpany

Target

Indication

Synagis

Medimmune

RSV F protein

Zenapax

PDL/Hoffman-La Roche

IL-2Ra

Herceptin

Genentech

Her-2/neu

Respiratory syncytial virus infection Acute rejection episodes after renal transplant Breast cancer

Data from Kling.^

TABLE 3 Some MAbs in Development Biospecific monoclonal antibody

Target

Indication

MDX-210 MDX-220

Her-2/neu TAG-72

MDX-447

EGF receptor

Prostate, renal, and colon cancer Lung, colon, prostate, endometrial, pancreatic, ovarian, and gastric cancer Breast, head and neck, brain, non-small cell lung, and bladder cancer

Data from Kling.^ All trigger arms bind CD64.

Stage of development Phase II Phase I/II

Phase II

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ical methods applied in this field. A number of difficult challenges have to be overcome to achieve this goal: • • • • • •

Protein characterization Carbohydrate mapping DNA removal Establishment of genetic stability Retrovirus clearance Identification of host-cell contaminants

These developments have mutually stimulated the fields of analytical chemistry and biotechnology. Some of the future developments in analytical methodologies and process-scale separations are discussed in Section IV.A. II. THE PARTNERSHIP OF PROTEINS AND NUCLEIC ACIDS Proteins and nucleic acids enjoy an interesting partnership in that proteins are constructed by nucleic acid, and nucleic acid reproduces w^ith the help of proteins. In today's biology, v^e recognize that RNA acts as a messenger and information transfer agent for DNA, the keeper of the master genetic code. Hov^ever, some types of RNA have a surprising ability to catalyze reactions as enzymes do. As a result, it has been thought that RNA could have been the original machinery of life. It is conjectured that the original duties v^ere eventually supplanted by better-suited DNA and enzymes. The studies into self-replication of peptides and oligonucleotides and the origin of life will help us better understand these biomolecules and their role in gene-based diseases. III. BIOTECH DRUGS A large number of drugs are in development to treat a variety of diseases (Table 4). The largest number of drugs are aimed at cancer. Discussed in this section are also drugs that are being developed by biotechnology for a variety of diseases. Gene therapy is discussed in Section V. A. War against Cancer Since selectivity and ubiquity characterize the tumor suppressor gene p53, this gene has emerged as one of the top targets in the war against cancer. It plays an integral role in activating the programmed cell death (apoptosis) and is mutated in 55% of the tumor types. Potential reUef strategies include replacing the defective gene, injecting viruses into tumors, and reactivating the gene's apoptotic function with small molecules. Future development in bioseparations will help us better understand gene-based diseases and their treatment (see genomics in Section V). Several years ago, researchers at Johns Hopkins University developed a method called serial analysis of gene expression (SAGE) that enables quanti-

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TABLE 4

69 I

Biotech Drugs

Target

In development

Approved

Cancer related Infectious diseases AIDS-HIV related Heart disease Neurologic disorders Respiratory diseases Autoimmune disorders Skin disorders Transplantation Diabetes Genetic disorders Digestive disorders Blood disorders Growth disorders Infertility Eye conditions Miscellaneous

151 36 29 28 26 20 19 14 14 13 10 9 8 4 4 3 22

14 10 7 5 4 2 2 0 6 4 2 0 9 9 3 1 1

Data from Thayer.^ "" Through June 1998.

tative analysis of thousands of transcripts simultaneously."^ The SAGE method is based on the isolation of unique sequence tags from individual transcripts and concatenation of tags serially into long DNA molecules. Rapid sequencing of concatement clones reveals individual tags and enables quantitation and identification of cellular transcripts. More recently, SAGE has been applied to the analysis of gene expression profiles in gastrointestinal cancers, to the identification of transcriptional targets of p53 that regulates apoptosis and mediates G2 arrest, and to connecting APC tumor suppressor and MYC oncogenic pathw^ays. The expectation is that the identification of such differentially expressed genes w^ill furnish insights into the pathophysiology of cancer and open up possibilities for useful diagnostic and therapeutic approaches. A prostate secretory protein PSP^"* has been reported to suppress prostate cancer growth in a mouse disease model (Procoyn Biopharma, London, Ont.). The results suggest that protein induces cell death specifically in cancer cells refractory to human hormones. B. Targeting Messenger RNA Antisense drugs are planned to have unique ability to bind to targeted messenger RNA (mRNA) while avoiding attachment onto other proteins. Antisense oligonucleotides are generally 20-30 bases in length The following milestones of antisense oligonucleotide therapeutics may be of interest^: 1961 Oligonucleotides complementary to RNA are reported to block protein translation.

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1978 Zamecnik and Stephenson first illustrate the idea of an antisense drug by using antisense oligonucleotides to inhibit the replication of the Rous sarcoma virus in vitro, 1986 Automated DNA oligonucleotide synthesis is commercialized. 1988 Phosphorothiolate oligonucleotides are first used as antiviral agents in research. 1995 First clinical trials of antisense oligonucleotide (ISIS 2105) are initiated for treatment of genital warts caused by human papilloma virus. 1998 Vitravene, a phosphorothiolate oligonucleotide, receives FDA approval for treatment of cytomegalovirus (CMV) retinitis in HlV-l-infected individuals. Vitravene binds the mRNA sequences containing the CMV major immediate early region 2 (IE2), w^hich encodes several protein-regulating expression of genes essential to CMV production. Vitravene binding inhibits IE2 protein synthesis, thus inhibiting viral replication. This class of drugs that originated from a simple idea based on inhibiting protein expression by binding complementary mRNA turned out to be much more complex. A successful antisense oligonucleotide must also possess numerous properties such as efficient cell uptake, specificity for target mRNA, high mRNA hybridization affinity, stability tov^ard nucleases, activation of RNase H for cleavage of duplex RNA and an acceptable side-effect profile. It took 20 years from initial inception to first approval of the antisense drug Vitravene (Isis Pharmaceuticals) to treat CMV retinitis in HIV-1-infected individuals (for further discussion on HIV see Section II.C). In spite of decades of research, arguments still remain about the mechanism of action of many drugs. Bioseparations can help us better understand these mechanisms and create more exciting drug therapies. One important reason for industrial and diagnostic applications is the use of oligonucleotides in research and development of automatic synthesizers.^ Electrophoresis is v^ell suited for size separation of nucleic acids, since the electric field acts on the w^hole molecule independent of its orientation and its local charge distribution. Media such as polyacrylamide and agarose w^ere first thought to suppress convective mixing; they have additional effect of reducing the electrophoretic mobility as a function of size. Because the apparent pore size distribution of polyacrylamide and agarose can be varied in a defined w^ay, mobility differences for a given size can be induced. Capillary gel electrophoresis (CGE) w^ith on-line detection and data evaluation is also suitable for the separation of both single- and double-stranded DNA fragments. C. HIV Aptamers are single- or double-stranded pieces of DNA or RNA, usually 15-40 bases long. They work by binding target molecules such as proteins with high affinity and specificity. Researchers recognized the medical and pharmaceutical potential of the interaction years ago, which led to the

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launching of large-scale screening of combinatorial libraries to find aptamers that might be valuable for applications such as inhibiting HIV infection. The molecular recognition of these compounds is most likely similar to antibodies, i.e., they rely on the interaction of the host's three-dimensional structure w^ith the target. Yokota and cov^orkers observed that protein-DNA complexes frequently appeared as V-shaped structures. Using a fluorescence method, the researchers demonstrated that the sharp bend in the DNA is coincident w^ith the proteinbinding site and that the protein-induced DNA bend can be used to localize the site of protein binding to DNA.^ A novel chemical cross-linking strategy has helped to clarify the longsought structure of HIV reverse transcriptase (RT) bound to its natural substrates—double-stranded DNA and a deoxynucleoside triphosphate.^ RT is a polymerase that transcribes HIV's single-stranded RNA genome into double-stranded DNA. The double-stranded DNA is incorporated into the genome of an infected host cell, beginning a cycle of infection. AIDS drugs such as zidovudine (AZT) inhibit RT, thus short-circuiting the life cycle of HIV. But the virus has a tendency to mutate very deftly away from the drugs' range of efficacy, rendering them ineffective. The new structure helps clarify how the virus develops resistance against RT inhibitors and thus could point the way to better drugs. It is important for us to understand the structure of enzyme with its substrates, when it is working, to help design inhibitors and to better understand mutations. Three enzymes with anti-HIV activity have been identified in the urine of women in the early months of pregnancy.^ Lee-Huang and her colleagues have now traced the anti-HIV activity to two ribonucleases and a lysozyme. The ribonucleases can digest RNA from a variety of sources, including cells infected with HIV-1. These proteins offer promise as anti-AIDS agents. The exact mechanism as to how these enzymes work needs to be worked out. D. Rheumatoid Arthritis Rheumatoid arthritis (RA) is a form of arthritis that results from the immune system's attacking joint tissues. This leads to pain, deformity, and damage that is often permanent. Enbrel (etanercept), a genetically engineered protein made by Immunex, is the first drug developed by biotechnology that was approved recently in 1998 by the FDA for the treatment of RA.^^ It consists of two soluble tumor necrosis factor (TNF) receptors fused to the Fc fragment of human immunoglobulin. TNF is a cytokine that plays an important role in the cascade of reactions that cause inflammation reaction in RA. Enbrel works by competitively inhibiting the site of TNF to its receptor sites. Some patients receiving Enbrel have a mild to moderate inflammation reaction at the injection site. Concern exists that Enbrel might interfere with the normal immune function. Further studies are needed in development of new drugs in this area where bioseparation will play a major role. Abgenix has begun clinical trials investigating a human monoclonal antibody (ABX-IL8) that binds interleukin-8 (IL-8), a protein that mediates inflammation process. Other studies have demonstrated that elevated levels of

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IL-8 in the synovial fluid of RA patients correlates with an increase in the number of infiltrating immune cells. Additional studies have shown that antibodies to the protein reduce immune infiltration and synovial membrane damage. E. Amyloid Diseases Many proteins can be made to clump into fibrous amyloid deposits like those seen in Alzheimer's disease, Creutzfeldt-Jakob disease (the human counterpart of mad cow disease), and other serious ailments. To help prove this point, a natural enzyme to convert to amyloid fibrils—insoluble protein aggregates with a /3-pleated sheet structure—simply by maintaining protein for some time in the unfolded state. Until now, scientists have generally believed that only specific proteins such as amyloid j8-protein and prions are capable of being converted into amyloid fibrils.^^ A variety of spectroscopic techniques have been used to confirm the gradual development of amyloid fibrils and to verify the fibrils' predominant j8-pleated sheet structure. In the partially unfolded intermediates that form under denaturing conditions, hydrophobic amino acid residues and polypeptide backbone normally buried inside fully folded structures become exposed. Further work is needed to confirm and advance these findings.

IV. ASSURING PRODUCTION AND PURITY The limiting factors in using recombinant proteins for bioprocessing include purification and overall yield of active protein.^^ Since many recombinant proteins are refolded from insoluble aggregates, a major factor limiting protein yield is in the development of individual protein folding schemes. The molecular chaperones are a group of proteins that are effective in vitro and in vivo folding aids and show a well-documented affinity for proteins lacking tertiary structure. Purified chaperones have been found to be efficient in refolding chemically and thermally denatured proteins. The affinity recapture of protein folding aids provides an economic process for the refolding of denatured proteins. The initial emphasis in analytical biotechnology was on broad safety concerns that translated into detection of host-cell components such as DNA, endotoxins, Escherichia coli proteins, and retroviral contamination.^ The detection of these impurities requires development of high-sensitivity assays that are based primarily on antibodies [e.g., enzyme-linked immunosorbent assay (ELISA) for £. coli proteins) or radioactivity (e.g., dot-blot assays for DNA detection). New developments are focused on low-sensitivity detection, characterization, and removal of undesirable target sequence variants. Bioseparations play an important role even after a product has been isolated and shown to contain a low level of contaminants for initiation of clinical studies. The focus shifts to achievement of a reproducible, large-scale manufacturing process. At this stage, analytical methods provide essential informa-

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tion such as host-cell contaminants or variants of the target sequence to assure better process development. Chromatographers play a key role in process development as is obvious from the degree of difficulty of preparing recombinant DNA-derived protein in large amounts with contaminants from bacterial or mammalian host-cell line at parts per million levels. Bioseparations are a key part of both recovery and analytical processes. Aside from mistranslation, the main route for protein variants is degradation resulting from proteolysis, deamidation, or aggregation. The future developments in bioseparations must be continually directed toward reducing the cost of operation while maintaining optimum quality.

A. Liquid-Liquid Partitioning Liquid-liquid partitioning is a convenient and often economical method for bioseparations. L. Gu (personal communication, 1999) has shown that an acetonitrile-water system can be used for separation of proteins. This system partitions into two phases under subzero temperatures with the top phase containing more acetonitrile and water. The low temperature and the presence of water in both phases help reduce protein denaturation. An added advantage is that sample solution can be directly applied to reversed-phase high-performance liquid chromatography (HPLC) for further purification. Aqueous liquid-liquid partitioning is likely to remain an attractive choice for the separation of proteins, and exploration of new systems will continue.

B. HPLC

The success of a particular analytical or preparative HPLC strategy for polypeptides or proteins is determined by the ease of resolving to a predefined level of a desired component from other substances, many of which may exhibit similar separation selectivities, but are usually present at different levels (M. Hearn, personal communication, 1999). To carry out high-resolution purification procedures, multistage high-recovery methods must be developed and utilized. To minimize losses and improve productivity, on-line, real-time evaluation of each of the recovery stages is an essential objective. Furthermore, overall optimization and automation of the individual unit operation must be achieved. In the next few decades, there must be greater enhancement of understanding of the molecular basis of interactions of the biomacromolecules with various classes of ligates. The fundamental nexus that links the thermodynamics of interaction with extrathermodynamic physicochemical properties of all peptides and proteins in interactive HPLC systems will take on greater significance, providing an avenue to more precisely interpret the molecular binding, the docking event, and atomic forces involved. The development and application of new generations of on-line detectors capable of monitoring the structure-function-retention characteristics of biopolymers represents a pressing challenge for spectroscopists and chromatographic scientists alike.

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R. Shiloach (personal communication, 1999) believes that ion-exchange adsorbents are a powerful tool in the purification of proteins. They can be used in the initial steps of the protein recovery process by capturing the proteins, enrichment, or polishing. It is likely that nev^ and improved adsorbents v^ill continue to be introduced.

C. lEF There is considerable interest in the development of a means for more effectively combining isoelectric focusing (lEF) and sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) gels of 2-D PAGE on a single support (D. Garfin, personal communication, 1999). Such a gel may become a reality in the not too distant future. This development w^ill allows for simple automation of the 2-D PAGE process and w^ill make 2-D PAGE as convenient as one-dimensional gel electrophoresis. Lov^-abundance proteins are of considerable interest in proteomics. This calls for improvement in detection capabilities. The separations of large proteins, on the other hand, v^ould require development of new^ support matrices. Efforts are likely to center on the development of new^ and improved polymers for gel formation.

D. Membrane Adsorption The applications of membrane chromatography have been limited by the size of membrane systems (R. Deshmukh, personal communication, 1999). The major scale-up barrier has nov^ been removed with development of large membrane modules (8 m^). Pilot plants of relatively modest size have demonstrated that a relatively small, inexpensive, and streamlined system are capable of producing metric tons of purified products within one year. Reducing dead volume is likely to further improve capability and lead to development of the larger systems. Further developments demand that the cost of these membrane modules approach that of standard sterile microporous filters. Future developments in membrane chromatography are likely to allow greater processing speed with compact inexpensive systems.

E. Displacement Chromatography Displacement chromatography offers an attractive alternative to the elution mode of operation for preparative purification (S. Cramer, personal communication, 1999). Further investigations must be carried out to identify more cost-effective, nontoxic, and readily detectable displacers that are commercially available to the biotechnology industries. Displacers with high affinities in a range of commercially available stationary phases must be identified to facilitate the development of displacement steps on these materials. This will require significant advances in our understanding of the nature of affinity of these systems.

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F. Antibodies Depending on the targeted disease, it is frequently necessary to produce and purify large amounts of a given antibody (E. Boschetti, personal communication, 1999). This requires significant progress in technological approaches that are used for purification. Emerging technologies such as fluidized sorbent beds w^ith solid phases of very low^ density appear to be promising. Further developments w^ould require that they accept unmodified feedstocks at their pH and ionic strength. Expression systems w^ill also play an important role in the future. Protein engineering v^ill continue to make changes in antibody molecules vv^ith new generation of fusion constructs better targeted for the disease in question. Novel and effective bioseparation techniques must be continuously researched and developed for profitable removal of proteins and other bioproducts of interest from very dilute solutions (A. Ramakrishnan and A. Sadana, personal communication, 1999). There appear to be two techniques that have tremendous potential for commercial applications: the reverse micelle technique and aqueous two-phase extraction. Before these techniques achieve their potential, it will be necessary to further delineate the effect of mass transfer, interactions at the interfaces, and other parameters that affect both the quality and quantity of proteins separated by these techniques. It is also necessary to have a large data bank of a wide variety of proteins and bioproducts with regard to their characteristics and stability to assist future improvements in bioseparations.

G. Combination Methods A therapeutically relevant potency assay is an essential requirement in drug development.^^ It must be able to quantify the dose, ensure product consistency, and quantify biological activity. To accomplish these goals and satisfy regulatory authorities, three complementary methods have been developed for analyzing Myloral (a complex mixture of proteins and lipids developed for oral tolerance therapy for multiple sclerosis): A reversed-phase HPLC method is used to quantify one representative protein myelin basic protein (MBP), which was chosen as a basis of dosing because it contains multiple human T-cell epitopes and is one of the labile protein components in the drug; SDS-PAGE is used to assess consistency in the overall protein profile; and ELISA is used to quantify immunological epitope availability. Eighty percent of human proteins are glycosylated. Hancock believes that developing well-characterized biological glycoproteins is most challenging because of extreme microheterogeneity due to carbohydrate moieties. Among the techniques employed to analyze such complex molecules are bioassays, immunogenecity, HPLC, capillary electrophoresis (CE), mass spectrometry (MS), nuclear magnetic resonance (NMR), circular dichroism, chemical and enzymatic probes, and compositional analysis. A combination of CE and off-line matrix-assisted laser desorption-ionization-time-of-flight (MALDITOF) MS is particularly invaluable.^"^

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Matrix-assisted laser desorption-ionization ionizes molecules with molecular masses of 100-1,000,000 Da for analysis by MS and provides high sensitivity, high throughput, and simplicity of operation. MALDI combined w^ith enzymatic reactions and protein chemistry can provide very useful information on molecular masses, peptide maps, and primary structure.^^ MALDI-TOF MS can quickly and accurately determine unfractionated mixtures at concentrations below^ 100 fmol per liter. The obtained data are then calibrated with internal standards, monoisotopic masses are assigned for all prominent peaks, and the peptide list thus generated is used to identify the protein by using a suitable database.^^ Matrix-assisted laser desorption-ionization mass spectrometry (MALDIMS) appears to work well for RNA analysis; it is fast and can be automated.^^ Intact ions of large molecules with molecular weights up to hundreds of megadaltons can also now be formed and analyzed. The combination of electrospray ionization (ESI) and Fourier transform MS (FTMS) is becoming a powerful method for structural analysis of large biomolecules.^^ ESI-FTMS and FTMS performance increases with the magnetic field strength and newer instruments with higher magnetic field strengths are being constantly developed.

Y. GENOMICS Genomics, functional genomics, pharmacogenomics, and proteomics are various areas of interest that operate at the interface of biology and chemistry. The structure elucidation of DNA by Watson and Crick in 1953, which suggested the genetic code (the information flow from DNA to RNA to proteins), led to a growing understanding of living systems on a molecular level. DNA is truly at the center of molecular biology and biotechnology. DNA sequencing has achieved paramount importance in molecular biology today. Gene therapy attempts to fix various problems in the DNA script by artificially providing a properly edited version to compensate in those cases where both copies in the patient are bad. Or, it can attempt to provide an added piece of script to counter the action of the bad disease-producing gene. The main objective is to make the bad copy unreadable so that the bad enzyme is never coded in the first place or to produce another gene product that interferes with its action. The state of gene therapy today is given in Table 5}' New strategies are being sought to disable Mycobacterium tuberculosis, Mycobacterium genitalium, and malaria-causing Plasmodium falciparum. These investigations will lead to new approaches to development of antibiotics. Genomics provides a way to find out what proteins are expressed by a microbe during infection and which of those are essential to the microbe's survival so that they can be targeted for developing new drugs. It is possible to select a few likely genes and, using bioinformatics tools, determine within a few minutes whether certain genes occur among a number

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TABLE 5 The State of Gene Therapy Disease

Status of therapy

ADA deficiency ("bubble boy syndrome") AIDS Cystic fibrosis Diabetes Gaucher's disease Hemophilia Hypercholesterolemia Ischemia/blood constriction Lung cancer Metastatic solid tumors Ovarian cancer Parkinson's disease

Human clinical trials; functional cure Human clinical trials Human clinical trials Rodent trials; apparent cure Human clinical trials Rodent trials; apparent cure Human clinical trials Human trials; 75% clinical success for legs Human trials; limited tumor suppression Human trials; limited tumor suppression Human trials; limited tumor suppression Rodent trials; apparent cure

Adapted from Lesney.^^ Note that human trials are begun after positive indications are obtained in test animals and/or in human cell cultures.

of related pathogens. Data mining and new developments in bioinformatics software are likely to further advance this field. Genomic analysis requires miniaturization and integration of sample preparation steps (see next section). An important current development in CE, namely integrated CE (ICE) on micromachined chips addresses these issues.^^ Note however, that chip array is not the only technology that is useful for unraveling the complexities of gene expression. CE is destined to contribute to both genome and proteome aspects of the human genome project (see Chapter 4). The potential of ICE for genomic analysis and other significant problems that can be solved with this technique and that require high throughput at low cost will continue to grow.

VI. LAB ON CHIP

In the early 1990s, microfabrication techniques were borrowed from the electronic industry to create fluid pathways in materials such as glass and silicon.^^ The objective was to scale down the size of the analytical platform, while automating handling and other procedures could improve throughput and eliminate losses and other sources of human errors. The focus of these methods is analysis of biologically sourced material for which existing analytical methods are cumbersome and expensive. The major advantages of these methods are that a sample size 1000-10,000 times smaller than with the conventional system can be used, high-throughput analysis is possible, and a complete standardization of analytical protocol can be achieved. A large number of sequence analyses must be performed in order to build a statistically relevant database of sequence variation versus phenotype for a given gene or set of genes.^^ For example, patterns of mutation in certain genes confer resistance to HIV for various antiretrovirals. Sequence analyses

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of many HIV samples in conjunction with phenotype data will enable researchers to explore and design better therapeuticals. Similarly, the relationship of mutation in cancer-associated genes, such as p53, to disease severity can be addressed by massive sequence analyses. DNA probe array systems are likely to be very useful for these analyses. Active DNA microchip arrays with 25-400 test sites are being developed for both research and diagnostic applications. For example, DNA chip arrays with 400 test sites and onboard semiconductor switching controls are useful for genomic research applications and evolving genetic disease and cancer diagnostics. These approaches can lead to high-throughput screening (HTS) in drug discovery and development. For HTS you must decide whether to use cell-based or non-cell-based assays, which specific test is best (ELISA, mRNA assays, or reporter gene assays), and which detection method and instrument to use. The researchers looking for HTS assay detection methods can choose from a number of technologies such as fluorescence, luminescence, and scintillation proximity assays. The Human Genome Project has been a major driving force in the development of suitable instruments and methods for genome analysis. The companies that can identify genes that will be useful for drug discovery will reap the harvest in terms of new therapeutic agents and therapeutic approaches and commercial success in the future. VII. RECOVERY OF BIOLOGICAL PRODUCTS Included here are short summaries of various oral presentations made at the Ninth Conference on Recovery of Biological Products, held on May 23-28, 1999, in Whistler, Canada. This conference has become the preeminent meeting in the field of bioseparations, and it provides exposure to the latest developments in the state-of-the-art downstream processing. Dr. S. Cramer of Rensselaer Polytechnic Institute was kind enough to cover this meeting, and his report follows. Various session titles are listed in the order of presentation. A. First Steps Michael Flickinger (University of Minnesota) showed results for capturing human serum albumin (HSA) from yeast cells in expanded-bed systems, using fluoride-modified zirkonia materials. These stationary phase materials can be used as ion-exchangers or as hydrophobic interaction chromatography (HIC) supports, depending on the operating pH. The stability in alkaline solutions makes these dense particles well suited for large-scale processes. This expanded-bed method appears to have potential utility as an alternative to filtration, followed by chromatography; however, further process development is required to deal with variable binding capacities observed with different cell concentrations. In addition, mathematical modeling was carried out using a general rate model of chromatography to identify the relative importance of various mass transfer steps and adsorption-desorption kinetics.

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Shishir Gadam (Merck) presented a rational approach for evaluating and comparing clarification techniques. Centrifugation and filtration techniques, such as hollows-fiber tangential flow^ filtration, flat sheet tangential flov^ filtration. Dean vortices, and co-current filtration, w^ere examined to study the impact of the most critical parameters on performance. A synthesis of the experimental data, mathematical treatment, and engineering constraints for these technologies w^as offered, v^ith emphasis on understanding the underlying design trade-offs. The results indicated that the optimal method depended on the most important criteria for the integrated dow^nstream design (e.g., clarification pov^er, productivity, product recovery and quality, system requirements, and sensitivity to fouling). Carsten Jacobsen (Novo Nordisk) presented results on protein crystallization in preclarified, concentrated fermentation broths. In particular, the impact of filtration rate on the formation of favorable large diamond versus rod shapes was examined. By adding seed crystals just above the solubility curve, w^here no nucleation occurred, the authors v^ere able to produce 30% larger crystals as compared to an unseeded crystallization. Although there v^as minimal recovery and characterization data, this technique may prove very beneficial for dealing w^ith difficult feed streams. While the w^ork presented in this talk w^as done at the laboratory scale, scale-up experiments will be required to confirm the suitability of this approach for industrial process applications. Ole Jentoft Olsen (DSS Danish Separation Systems) presented a large-scale industrial example on using ultrafiltration for the production of antibiotics. Within 15 hr, a volume of 100 metric tons of biomass was processed. The design of membrane module, operational conditions, and the overall plant were presented, along with detailed cost considerations for this process.

B. Chemical Reactions—Intended and Not This session focused on advances in the use of affinity tags for facilitating separations. Although affinity tag technology has many distinct advantages, several problems (e.g., nonspecific cleavage within the protein of interest, presence of the cleavage enzyme as a contaminant in the final product) have limited the usage of this approach to date. In this session, several advances in the state of the art of affinity tags were presented. Georges Belfort (Rensselaer Polytechnic Institute) presented results on using inteins—self-splicing proteins—to improve affinity tag technology. In this approach, they used a controllable autocatalytically cleaving protein linker for insertion between an affinity group and a product protein and demonstrated that the resulting fusion product could be effectively employed in an affinity separation scheme. This novel approach may obviate many of the problems associated with affinity tags. John Pedersen (Unizyme Laboratories) demonstrated how multienzyme systems can be used along with His affinity tags and subtractive IMAC to produce industrial enzymes in both packed or expanded bed adsorption systems. The efficiency of the approach was demonstrated with the produc-

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tion of native human tumor necrosin factor a (hTNFa) and human growth hormone (hGH). Finally, Charles E. Glatz (Iowa State University) demonstrated how oilseeds such as canola can act as effective hosts for the production of recombinant proteins. For canola, it was shown that there are "windows" in the properties of the contaminating native proteins that may allow for rapid chromatographic purification. Purification fusions were employed to move recombinant proteins into those windows, and their rapid purification was demonstrated. Clearly, with the explosion of new proteins from proteomics, affinity tag technology will become increasingly important.

C. Dealing with Real and Potential Infectious Agents The risk of viral contamination is a feature common to all biotechnology products derived from cell lines. In this session, we heard several talks related to viral contamination and viral clearance. Takao Hayakawa (National Institute of Health Sciences, Japan) presented a talk on the International Conference on Harmonization (ICH) viral safety guidelines. This guideline, which has been signed off by the three regulatory parties from the European Union, the United States, and Japan, is concerned with testing and evaluating the viral safety of biotechnology products derived from characterized cell lines of human or animal origin. It outlines data that should be submitted in the marketing application-registration package. Major areas described in the viral safety document include: (1) cell line qualification, (2) testing for viruses in unprocessed bulk, (3) rationale and action plan for viral clearance studies and virus tests on purified bulk, and (4) evaluation and characterization of viral clearance procedures. Arindam Bose (Pfizer Central Research) further discussed the ICH documents and presented a rationale for the recommended combination of test procedures and process clearance validations required to demonstrate that marketed biopharmaceuticals are free of adventitious agents. He showed that testing of Pre-Seed Stock (PSS), the Master Cell Bank (MCB), and the Working Cell Bank (WCB) is required to demonstrate that they are free from contamination by mycoplasma, bacteria, molds, and yeasts. In addition, viral clearance validation studies must be performed on scaled down versions of each chromatographic step and the viral inactivation/removal step employed in the product purification scheme. Finally, clearance studies must be conducted with a panel of relevant and model viruses (typically three to four) to establish that the purification scheme will indeed purge any viruses that may be inadvertently introduced during processing. Joachim Walter (Boehringer Ingelheim Pharma KG) presented results on the use of microwave technology for virus inactivation in biopharmaceutical products. By using microwaves, viral inactivation can be carried out with the exposure time of the product solution to high temperatures being limited to 500 ms or less. For antibodies, viral inactivation can be effectively carried out using peak temperatures up to 95°C with hold times as low as 5 ms.

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The talks in this session made it clear that the management of risks from adventitious agents has entered a period of concept stabilization. Some of the new^ developments to w^atch in this area are TSEs, MVM, and other potential threats as well as generic approaches and improved methods. D. Bioseparations at the Molecular Level This session w^as quite fascinating and included three talks on the development and use of novel affinity adsorbents, as well as a talk on how Raman spectroscopy can be used to provide insight into protein secondary structure in various bioseparation environments. The first talk, by Chris Lowe (Institute of Biotechnology, United Kingdom), focused on new affinity adsorbents for the purification of glycosylated biopharmaceuticals. This paper demonstrated how recent advances in computer-aided molecular design, the increased access to X-ray crystallographic data, and solid-phase combinatorial chemistry can be employed for the rational design of small synthetic affinity ligands targeted at specific sites on proteins. Previous work by the author on designing affinity ligands for a variety of proteins was extended in this paper to the purification of recombinant glycoforms with defined glycosylation. A detailed assessment of protein-carbohydrate interactions was used to identify key residues that determine monosaccharide specificity and which were subsequently exploited as the basis for the synthesis of a library of glycoprotein-binding ligands. Triazine-based ligands were identified as putative glycoprotein-binding ligands, since they displayed particular affinity for glycoproteins. This synergistic use of molecular design and combinatorial chemistry is a powerful approach that can be expected to produce a variety of effective affinity ligands in the future. The next paper, presented by Ruben Carbonell (North Carolina State University), focused on affinity ligands produced from combinatorial peptide libraries. The use of this technology for designing peptide affinity ligands for the purification of s-protein, von Willebrand's factor, and fibrinogen was presented. The effects of peptide density and orientation on the interaction of proteins with the chromatographic surface was also described. Brian Kelly (Genetics Institute) presented results on using peptide affinity ligands developed by DYAX (Cambridge, MA) for the purification of a recombinant therapeutic protein. Optimal polypeptide sequences were identified, using phage display technology where individual bacteriophages were selected from various libraries for their ability to bind the target protein under specified conditions of pH and ionic strength and to release the target molecule under mild elution conditions where the protein is stable. The performance of affinity chromatographic resins containing these polypeptides was presented and the general applicability of this method to the development of affinity purification techniques was discussed. This talk was important in that it demonstrated how combinatorial affinity ligand design can indeed be successfully employed for an industrial process. This will be discussed further in the talk by Inger Mollerup described below. The final talk in this session, by Todd Przybycien (Carnegie Mellon University), focused on the structural impacts of bioseparation operations on

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target proteins. This paper demonstrated that amide I band Raman spectroscopy can be successfully employed as a quantitative, in situ probe of protein secondary structure content in separations environments. This tool was used to assess protein behavior in reversed-phase liquid chromatography (RPLC) and in salt-induced precipitation (salting-out) environments. A series of model proteins v^ere examined v^ith linear alkyl-based RPLC media, and the results indicated that model protein retention behavior w^as strongly correlated v^ith adsorbed state structure. Furthermore, for precipitation systems, the results indicated that it is often the self-association of protein in the precipitate phase that leads to the structural perturbation rather than exposure of protein to high salt concentrations. These results indicate that Raman spectroscopy may be an extremely useful tool (particularly w^hen used in concert with other techniques such as NMR) for gaining insight into protein structure in bioprocessing environments. E. Purification Methods: Alternatives to Chromatography This session presented several examples of nonchromatographic purification methods. Mike Johns (University of Queensland) presented work on an empirical correlation that has been found to quantify the nucleation rate for a self-nucleating batch crystallizer. This correlation was obtained from selfnucleating batch crystallizations of glucose isomerase. The source of nuclei are believed to be twofold: first, from the aggregation of protein molecules in solution that reach some critical size necessary for a phase change, and second, from lattice fragments that break off when crystals collide. This correlation provides missing information in the population balance that is needed to eliminate the variability and lack of control in self-nucleating batch crystallization processes. Ralf Kuriyel (Millipore Corporation) addressed some of the issues related to the use of Dean vortices, formed during the flow of fluids in curved conduits, to enhance the performance of cross-flow filters by increasing the back transport of solutes. Results were presented on coiled hollow fibers with a varying radius of curvature, fiber diameter, and solution viscosity, to characterize the relationship between the back transport of solutes and hydrodynamic parameters. A performance parameter relating back transport to the Dean number and shear rate was derived, and a simple scaling methodology was developed in terms of the performance parameter. The use of Dean vortices may result in membrane systems with less fouling and improved performance. The last talk in this session, by Andrew Lyddiatt (University of Birmingham, United Kingdom), showed how liquid-liquid extraction can address some of the problems associated with the purification of nanoparticulates (e.g., viral and nonviral gene delivery vehicles). Nanoparticles (particle size range 20-150 nm) with low diffusivity and low molalities in culture feedstocks pose unique process engineering problems in the design and implementation of selective recovery and formulation operations. This paper demonstrated how aqueous two-phase partition systems (polymer-polymer and polymer-salt) can circumvent the process bottlenecks posed by the use of

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scale-limited, high-performance centrifugation in the primary processing of viral products. The behavior of nanoparticulate inclusion bodies (average diameter of 150 nm and available in gram quantities) in unit operations of centrifugation, microfiltration, adsorption, and aqueous two-phase partition w^as discussed in the context of sign-posting valuable technologies for processing and fractionating gene therapy vectors. F. Process Design, Simulation, and Economics Richard Blackmore (Baxter Hemoglobin Therapeutics) presented v^ork on a fully automated small-scale model of a pilot-scale purification process for recombinant hemoglobin. This paper employed the scaled-dov^n system to examine how the system will respond to feedstock changes. Interestingly, the operating temperature of the post cell lysis heat step was found to influence both the Q column yield and final product quality in an unexpected manner. The use of a fully automated, unattended, multistep purification system offers distinct advantages with respect to speed and efficiency in purification, as well as an analytical tool to understand interactions within the recovery and purification process. The last talk of the session, by Zhi-Guo Su (National Key Laboratory of Biochemical Engineering, People's Republic of China), discussed some of the unique challenges in producing modern biotechnology products in a developing country like China. To design an economical separation process for pharmaceutical protein production in China, energy cost and material cost were shown to be the major constraints, since labor costs can be minimal. Process integration and optimization thus become very important to create economic processes for biotech products in China. G. Chromatography Abraham M. Lenhoff (University of Delaware) demonstrated how confocal laser scanning microscopy can be used to visualize intraparticle phenomena during protein adsorption. By taking a detailed look into the stationary phase, experimental evidence can be obtained which encourages improvements to current approaches to modeling and simulation. An interesting phenomenon was observed where a focused peak appears at the concentration front advancing through the particle. The authors indicated that this may be explained by a coupling of electrophoretic and diffusive transport with nonlinear adsorption. This electrophoretic contribution to uptake transport, which may arise due to the high charge density on the stationary phase, may help in explaining unexpectedly high uptake rates and anomalous trends in band broadening that have been reported in the literature. Steve Cramer (Rensselaer Polytechnic Institute) presented the next talk on the use of modeling for displacer design and comparison of the performance of chromatographic materials. He attempted to show how modeling can be employed to provide insight into the relative efficacy of various stationary phase materials for different modes of nonUnear chromatography.

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A straightforward methodology was presented for determining various transport parameters, and dimensionless groups were employed to estimate the relative importance of the various transport mechanisms and to select appropriate models for a given material. Results were also presented on the use of modeling to guide the rational design of high-affinity low molecular weight displacers for a variety of chromatographic systems. Several examples were presented on the applications of low molecular weight displacer technology for bioprocessing of extremely difficult mixtures in both ion exchange and hydrophobic interaction systems. Alex Schwarz (Nextran, Inc.) demonstrated that synthetic ligands with an affinity to a whole group of proteins (e.g., thiophilic ligands-immunoglobulins) can be an interesting alternative when the development of a target molecule specific ligand is too complex or time-consuming. The use of a new resin that combines a thiophilic ligand with charge induction chromatography was shown to have significant potential, although new methods to further improve the purification factor in these systems need to be explored. Finally, Luuk A. M. Van der Wielen (Delft University of Technology) showed that as we move to very large processes, alternatives to conventional (batch) techniques will have to be considered. This talk focused on simulated moving-bed (SMB) technology, which is well established for small-molecule separation and can be readily transferred to protein recovery. In addition, a general method was presented to detect and efficiently eliminate azeotropes in SMB systems. H. Coping with the Output from Genomics This session highlighted the challenges in expressing cDNAs as the products of functional genomics programs. The paradigm of drug discovery has changed as a result of the efforts to advance the Human Genome Project. These efforts have made available in both public and private databases large numbers of nucleic acid sequences whose functions are unknown. The primary challenge is to "mine" these databases using the tools of bioinformatics to highlight cDNAs that represent potential targets. For a subset of these targets, a secondary challenge is to express and purify these as recombinant proteins for functional analysis. Proteins that are active in a biological model of disease represent potential drug targets. A key constraint in characterizing the products of these cDNAs is that often these sequences have no known function. Therefore, in expressing these cDNAs as recombinant proteins, one does not have an assay for activity to follow purification. Consequently, efforts to express these recombinant proteins rely on fusion constructs, multiple expression systems, and purification schemes that depend entirely on the presence of affinity tags. Expression and purification methods are designed to be independent of the characteristics of the protein of interest. Milton Hearn (Monash University) described his work to design novel affinity ligands that are capable of recognizing secondary structure motifs of proteins. Such ligands could speed the introduction of new tag sequences for use in fusion constructs.

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Karen de Jongh (Zymogenetics) discussed characteristics of different expression systems that she has used to express products of Zymogenetics' functional genomics program. In her experience, no single expression system works well for all proteins. She has tested expression in yeast (Pichia pastoris), bacteria ( £ . colt), insect (baculovirus), and mammalian cells (BHK, CHO). She recommends that multiple expression systems be used to insure expression of any given protein. She expresses proteins of interest as fusions with affinity tags. In addition, she underscored the importance of good analytical tools to characterize purified protein products. Expression in yeast (Saccharomyces cerevisiae) was the focus of the presentation by Alois Jungbauer (University of Agriculture, Vienna, Austria). He pointed out that expression of a cDNA as a fusion protein using both secreting and nonsecreting systems has been useful in his work. For secretion, he prefers a cleavable fusion construct in which a FLAG antibody epitope is used for both detection and purification. For nonsecretion, the construct contains also ubiquitin, the presence of which facilitates proper processing in yeast. Furthermore, he emphasized the importance of a proper reading frame in achieving a functional protein.

I. Industrial Case Studies This is always one of the most important session(s) of the meeting and is a unique feature of the Recovery of Biological Products Series. Joanne Beck (Amgen) presented a case study in process integration and direct cost analysis. In this paper, the link between the harvest step and downstream purification was examined. Both scale-down and pilot-scale experiments were employed to study the different harvest methods and their corresponding links to downstream processing. Process changes were shown to be driven by both cost and potency. A key issue in this presentation was the effect of culture titer on the downstream process. By changing the expression systems from E. colt to CHO a significant reduction in costs could be achieved. Gary Forrest (Wyeth-Ayerst Research) demonstrated some of the complex/multisite/outsourced manufacturing and containment issues for a monoclonal antibody conjugate consisting of an antitumor antibiotic linked to an antibody that targets the cancer cells. Because of the highly cytotoxic nature of the antibiotic, the derivatized antibiotic, and the antibody conjugate, special care must be taken to protect the safety of the manufacturing staff. Brent Pollock (Biomira Inc.) presented a talk on the rational development of a second-generation production process for recombinant human interleukin-2 that incorporates recent advances in cell expression systems, fermentation optimization, protein extraction, refolding, and purification. A key concept of this talk was to "know thy protein." The driving forces for process changes were regulatory concerns/robustness, economics, the ability to use standard equipment, and the elimination of bottlenecks in the process. Brian Turner i^ASF Bioresearch Corporation) presented a case study on the purification and characterization of human anti-TNF from the milk of transgenic goats. >4 collaboration with Genzyme Transgenics Corporation

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(Framingham, MA) resulted in the development of four transgenic founder animals, one of which produced kilogram quantities of human anti-TNF in 1998. In this paper, it was demonstrated that a 10,000 liter CHO cell culture equals approximately 50 goats. Interestingly, similar downstream processes were able to be employed for the different sources of bioproduct once the initial milk-processing steps were carried out. The economic advantages of transgenic animals for this particular application were not clear. Vikram Paradkar (Monsanto) presented a paper on the extraction and purification of proteins from transgenic plants. The production of several proteins was shown, including industrial enzymes, food-processing enzymes, and therapeutic proteins such as monoclonal antibodies, blood proteins, and cytokines. This paper showed that transgenic plants can produce 0.5-2 g protein per kilogram seed. Furthermore, the cost of producing 300 kg product per year was estimated to be $20M as compared to $50-100M for cell culture. Interestingly, 95% purity was achieved after the initial extraction step. Clearly, this technology has significant potential for large-scale production of bioproducts. The final talk in this session was given by Inger MoUerup (Novo Nordisk A/S) who presented work on the design and development of an affinity ligand for the purification of rFVIIa. Affinity ligands were developed in collaboration with Chris Lowe's group (Institute of Biotechnology, United Kingdom). The design process involved five stages: selection of an appropriate site on the target protein, design of a complementary ligand compatible with the supposed crystallographic structure of the site, construction of a limited solid-phase combinatorial library of near neighbor ligands, evaluation of the library, and solution synthesis of the most promising ligand prior to immobilization on an adsorbent. As part of the performance criteria, the ligand was designed to bind to rFVIIa only in the presence of calcium ions and to be released on disruption of the calcium complex. This protocol allowed for gentle elution and also mimicked the function of the antibody currently used in production. For this work to be successfully carried out, it was important to optimize the ligand density and spacer design. This paper served as an excellent example of the importance of academia-industry relationships. J. Making and Managing Process Changes William Adams (Merck & Co.) discussed process changes for biologies, such as live virus vaccines that cannot be well characterized because of their complex nature. Vaccine manufacturers are often inhibited from making process changes that will affect the product in subtle ways, which could ultimately result in changes in safety or efficacy. On the other hand, as issues such as TSEs and new viral agents continue to emerge, regulatory agencies are now requesting that manufacturers change their processes and remove these components to reduce the risk of introduction of infectious agents into their products. This paper also included a case study of how to implement process changes while reducing the risk of affecting final product safety and efficacy. An important point made in this talk was that change will happen.

FUTURE DEVELOPMENTS

709

and it is important to plan for it and, if possible, to think it through ahead of time. Jill Myers (Biogen) discussed preapproval and postapproval process changes. She showed how the process used to produce AVONEX® (Interferon beta-la) that was used in the pivotal phase 3 clinical trial differed from the commercial, approved process. The differences included the use of a different cell line and also the employment of different bioreactor and purification methods and production in different facilities located in different countries. The comparability of the two products was verified by extensive analysis and validation. Some of the key points of this talk were that: (1) efficient change is possible even late in process implementation, (2) early characterization can "light the pathway to change" (i.e., know the protein), (3) it is important to maintain process identity during transfers, and (4) it is also important to validate process changes. Morrey Atkinson (Targeted Genetics Corporation) discussed changes in production of gene therapy vectors. In viral vector production processes, assumptions that are valid for recombinant protein production do not always hold true. In recovery and purification efforts, novel contaminants, such as other viruses and human DNA become an issue. This presentation focused on the evolution of a production process for a viral gene therapy vector from the preclinical stages through phase 2 clinical trials, with an emphasis on decision making at critical points in the development cycle. This talk demonstrated that precise characterization of "living" systems is possible if the system is chosen carefully. In addition, the importance of defining and controlling changes in upstream as well as downstream processes was discussed. Finally, the validation of the genetic stability as well as the process was discussed. The last talk, by Bob Yetter (CBER), was on process development and regulatory decision making. The key points in this talk were that: (1) change happens—plan for it, the earlier the better; (2) communication is critical; and (3) regulatory decisions should not be made in a vacuum. K. New Recombinant Feedstocks and Techniques The first talk, given by Bob Bridenbaugh (Megabios), focused on the manufacturing of plasmid DNA at the hundred-gram scale. Unlike plasmid-based vaccines, where 10 g of DNA represents millions of doses, many cancer indications require much larger doses. This translates to the need to prepare hundreds of grams per run. Preparing and handling this amount of DNA presents many technical hurdles. The shear sensitivity and high viscosity of plasmid DNA in the 2000 to 25,000 base-pair range (1.2 miUion to 15 million Da) makes normally simple process activities, such as mixing, pumping, and filtration, problematic. Equally problematic is that the size of the plasmid DNA limits its access to the internal volume of most chromatography resins, making the dynamic binding capacity of commercial resins very low. This talk discussed how these and other issues with plasmid DNA manufacture can be addressed at the hundred-gram scale. Scott Fulton (Genzyme Transgenics) presented a paper on the production and recovery of protein products in transgenic milk. Recovery of proteins

710

S. AHUJA

from transgenic milk offers a number of advantages and significant challenges. Advantages include a high product concentration in the feedstream, negligible protease contamination, and constant product and feedstream characteristics across a broad range of scales. Challenges include dealing v^ith high particulate load and bioburden in milk (including potential viral contamination); meeting demanding scale, purity and cost requirements; and making the dov^nstream production system as scale-flexible as the expression system. This paper presented an upstream clarification and capture process for transgenic milk products, combining tangential flow filtration with chromatography in a single-unit operation. This process provides complete elimination of particulates and bioburden, high viral clearance, removal of milkspecific small molecules, and typically > 90% protein purity in a single step. The material produced by the process is suitable for final polishing in a standard good manufacture practice (GMP) protein purification operation. Data was presented on the use of this process for purification of antithrombin III used in current clinical trials and for monoclonal antibodies. This talk also discussed different approaches to "handshaking" transgenic milk production with conventional GMP downstream operations and explored the advantages and disadvantages of each alternative from a technical, operations management, and business perspective. The final presentation of the meeting was by Erno Pungor (Berlex Laboratories). This paper addressed the need for a detailed characterization of recombinant virus products through physical, chemical, biochemical, and in vitro biological analysis. The ability to develop an analytical definition of the product, as opposed to the product's being defined by the production process, enables one to change or optimize the production process without the need for clinical comparisons. The performance of a scalable purification process involving chromatographic and membrane separations was discussed, as well as the product characterization strategy.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Thayer, A. (1998). Chem. Eng. News, August 10, p. 30 Hancock, W., Wu, S., and Frenz, J. (1992). LC-GC 5, 30 Kling, J. (1999). Mod. Drug Discovery, March/April, p. 33. (1998). Scientist, January 18, p. 24. (1999). Mod. Drug Discovery, January/February, p. 68. (1998). Am. Lab., April, p. 59. (1999). Anal. Chem., May 1, p. 297A. (1998). Chem. Eng. News, November 30, p. 7. (1999). Chem. Eng. News, March 22, p. 25. (1998). Mod. Drug Discovery, November/December, p.12. (1999). Chem. Eng. News, April 5, p. 7. Walsh, M. K. (1998). 46th Am. Chem. Soc. Meet., Boston, 1998. Abst. 102. Zabrecky, J., Fowler, E., Bernardy, J., Brown, E., Compton, B., and Kretschmer, M. (1998). Biopharm. October, p. 30. 14. (1997). Am. Lab., p. 30. 15. (1997). Anal. Chem., November 1, p. 663. 16. (1998). Today's Chemist, October, p. 52.

FUTURE DEVELOPMENTS

17. 18. 19. 20. 21. 22.

(1999). Anal. Chem., February, p. 83A. (1998). AnaL Chem., March, p. 179A. Lesney, M. S. (1998). Today's Chemist, November, p. 61. Effenhauser, C. S., Bruin, G. J. M., and Paulus, A. (1997). Electrophoresis 18, 2203. (1998). Am. Lab., November, p. 22. Kreiner, T. (1996). Am. Lab., March, p. 39.

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INDEX

Acidic fibroblast growth factor, 58 Acrylamide, 274, 277 Acrylates, 252 Actin-myosin, 424 Acyl aryl amidase, 358 Adsorbed coatings, 250 Adsorption expanded-bed, 431-451 isotherms, 482, 483 kinetics, 187 Adsorptive membranes, 453 for bioseparations, 453 Affinity chromatography, 371, 421, 465, 572 adsorbents, 703 hgand, 703, 706, 708 partitioning, of biomolecules, 356, 357 plot, 396 risks, 617 tags, 701 Affinity-like techniques, 598 Agarose beads, 421 Agarose gels for isoelectric focusing, 278 Alanine, 331 Albumin, 383 bovine, 372

human serum, 257, 425, 492, 700 Albumin a^, 256 Albumin a2, 256 Albumin j8, 256 Albumin y, 256 Alcohol dehydrogenase, 358 Alcohols, solvent extraction of, 336 Alteplase, 672 Alzheimer's disease, 694 Amines, 249 Amino acids, 331, 382 isoelectric points of, 335 solvent extraction of, 335 a-Aminobutyric acid, 331 a-Aminocaproic acid, 331 p-Aminophenyl boronic acid, 589 Ampholytes carrier, 268, 269, 276, 278 compositions, 271 removal from proteins, 289 a-Amylase, 358 Amyloid /3-protein, 694 Amyloid diseases, 694 Analytical isoelectric focusing, 276-285

Analytical methodologies, 3 see also specific method Anion exchange (AX) chromatography, 457, 514, 516, 524, 607, 612 20 mer phosphorothioate, 516 stationary phase, 383 see also AX Antibiotics, 331, 701, 707 solvent extraction of, 337, 338 Antibodies, 17-18, 697, 707 from cell culture, 548 chimeric, 543 effector functions of, 540 light- and heavy-chain extremities, 538 molecular properties, 546 from plasma, 547 precipitation of, 554 separation by liquid chromatography, 535 separation methods, 546 structural properties, 538 from transgenic animals, 549 from transgenic plants, 550 variable regions of, 540 see also Antibody

713

714 Antibody capture, 440, 563, 564, 566, 604 conjugation, 54 fragments, 543 purification, 18, 537 see also Antibodies Antigenic vaccine protein, purification of, 400, 401 Antisense drug, 692 Antisense oligonucleotides, 16, 512 large-scale separation, 471 purification, 405 small-scale separation, 470 AOT (sodium bis(2-ethylhexyOsulfocuccinate), 341 AOT-iso-octane system, protein solubilization, 343 Apomyoglobin, 304 Aptamer, 692 Aqueous two-phase (ATP) system, 10, 349, 352, 370, 371 extraction of nucleic acid, 375 extraction of TDH, 376 partition coefficient, 371, 375 partitioning, 348-352 Aspartase, 358, 371 Aspartate /3-decarboxylase, 358 Association constant, 139 Association equilibrium constant, 573 ATP, see Aqueous two-phase (ATP) system Avidin, 321, 322 -biotin mixture, 322 AX purification of DMT-off oligonucleotides, 522, 524, 525 of ISIS 2302, 526 of oligonucleotides, 525 see also Anion exchange Axial dispersion, 187, 215

Bacterial nucleic acids, 368 Band shape in isoelectric focusing, 292 Band width, 246 Batch adsorption model, 190 Batch process control, 662 Bed expansion, of STREAMLINE DEAE, 421 Bed-packing quality. 111 Beta-glucuronidase, 682, 683 Beverage industry, 635

INDEX

Bio Pro Designer, 676, 677 Bioactivity property. 111 Biological product recovery, 700 steps, 365 Biologies license application (BLA), preparation, 2 Biomass, 433 Biomass separation, 636, 639 basket centrifuge, 642 cartridge filtration, 643 centrifugal filtration, 642 centrifugation, 645 continuous centrifuge filter, 642 cross-flow filtration, 643 decanter centrifuge, 646 disk stack centrifuge, 646 filtration processes, 640 gravity filter, 641 pressure filtration, 640, 641 pretreatment, 639 rotary drum filter, 642 sedimentation, 640 solid bowl centrifuge, 646 tubular centrifuge, 645 vacuum filtration, 641 Biomolecules, 243 affinity partitioning, 356, 357 dynamics of, 74 large-scale aqueous two-phase partitioning, 357 partition coefficient, 331 pK^ values, 334 MS, 299 topology, 74 see also Nucleic Acids, Oligonucleotides, Proteins, Peptides, specific proteins Biopharmaceutical industry, 2, 9, 634 Bioreactor control, 661 Bioremediation, 424 Bioseparation systems applications, 662 chromatography, 663 operating data, 663 product data, 663 status data, 663 electrophoresis, 663 extraction, 664 Bioseparation type, selection criteria, 19, 638 costs, 639 product location, 638 production scale, 638 program issues, 639 quality, 638 regulatory environment, 638

waste disposal, 638 waste production, 638 Bioseparations, 2, 19, 463, 634, 636 adsorptive membranes for, 453 economics, 667 future developments, 2 0 - 2 1 , 687-710 molecular level, 703 see also specific topic Biotechnology, 1, 688 advances, 365 drugs, 24, 688, 690, 691 Biotechnology of Industrial Antibiotics, 338 Biotin binding to avidin tetramer, 320, 321 derivatives, 321 Bisacrylamide, 274, 277 /3-Blockers, 498 Boronic acid ligands, 589 Bovine colostrum, 588 Bovine cytochrome c, 388 Bovine cytochrome, ESI-MS, 318 BTBAC, as displacer, 390 Buffer-pair pH gradients, 274-276 Buffers acrylamido buffer composition, 273-274 see also Capillary electrophoresis, pH gradients

Capacity factor, 79 Capillaries dynamic surface modification of, 247, 248 competing ions, 248 effect of pH, 248 static surface modification of, 247, 250 Capillary electrochromatography (CEC), 4, 119, 239 Capillary electrophoresis (CE), 5, 6,8,29,41-43,47,237,238, 240, 302 block diagram, 240 buffer additives, 245 buffer selection, 244-245 complexing agents, 246 cyclodextrins, 246 detectors, 240 divalent amines, 246 free solution, 43

715

INDEX

high-performance, 239 HPLC comparison, 241 ion-pairing reagents, 245 modes, 238 operation, 240 organic solvents, 246 protein separations, 252 surfactants ionic, 245 nonionic, 245 temperature effect in, 252 Capillary gel electrophoresis (CGE), 5, 45-46, 239, 253-255, 692 advantages, 254 disadvantages, 254 Capillary isoelectric focusing, 44-45, 291 Capillary zone electrophoresis (CZE), 5, 43-44, 239 Caprylic acid, 555 Carboxyhc acids partition coefficient of, 337 solvent extraction of, 336 Caseins, 260 Catalase, 331, 355 Cation exchange, 382, 499, 606, 607, 609,611 membranes, 468 CBER, see Center for Biologies Evaluation and Research CE, see Capillary electrophoresis CEC, see Capillary electrochromatography Celesticetin, 331 Cell disruption, 648 Cellulose acetates, 250 Center for Biologies Evaluation and Research (CBER), 2, 3 Cerebrospinal fluid (CSF), 256 CGE, mixture of proteins, 255 see also Capillary gel electrophoresis cGMP, see Current good manufacturing practices Cheese w^hey, 588 Chemical industry, 635 Chemical potential, 87 Chemiluminescence, 53 Chimeric antibodies, 543 Chiral epoxide, 498 Chlorophyl a / b protein, 358 Chromatography, 659 modes in MA application, 465 process, 475 separation, 649 techniques, 601, 602, 604 see also specific techniques

Chromatography membranes scale-up, 460 general considerations, 460 see also Scale-up Chromatophores, 358 Chymotrypsin, 372 CIP, 423 see also Cleaning in place Cleaning in place (CIP), 422, 437, 635 see also CIP CMV retinitis, 469 Coagulation, 635 Column configuration. 111 Columns disinfection of, 618 germ reduction of, 618 sanitization of, 618 by ethanol mixtures, 619 by hypohalogenites, 619 by peracetic acid, 619 by sodium hydroxide, 619 by sodium hypochlorite, 619 sterilization of, 618 Combination methods, 697 for antibody purification, 605 Combinatorial chemistry, 593 Combinatorial nanochemistry, 75 Commercial-scale operation, 633 Comparative separation procedures, 114 Complex molecules, 490, 492 Computer simulations of isoelectric focusing, 285 Computer-aided process simulation, 668 Computer-assisted separation strategies, 120 Conalbumin, 390 Concanavalin, 597 Concentration, effect of, 352, 354 Conformation changes in, 314, 315, 316 effects of, 160, 168 interconversions of, 187 requirements of, 159 species, 161 Constant region, 539 Control, food processing, 662 Coomassie brilliant blue G-250,32 Coomassie briUiant blue R-250, 32, 55, 284 Corticosteroids, 382 Countercurrent distribution apparatus, ?>59 of enolase, 359

Countercurrent system, 476, 479, 487 Craig extraction tubes, 434, 435 Crystallization, 650 Current good manufacturing practices (cGMP), 670 Cycloheximide, 331, 339 Cyclosporine, 493, 494 Cytochrome c, 315, 317, 342, 343, 319, 391, 392, 458 dynamic affinity plot, 396 Cytokines, 1 CZE, see Capillary zone electrophoresis

Data station, 301 Debye-Boltzmann equation, 123 Delayed extraction, 306 Desalting, 490, 491, 599, 500 Designer dyes, 424 Detection of protein bands, 284 Detector, 301 see also specific methods Determining pH gradients, 283 Determining pi, 283 Detritylation, 522, 523 of phosphorothioates, 523 Dextran-silica, 422 Diagnostic tools, 16 Differential extractors, 347 Diphtheria, 441, 444 Dispersion processes, 78 Displacement chromatography, 11, 379, 526, 696 alternative modes, 390 industrial case studies, 400 methods development, 393, 399 retained pH gradients, 393 sample, 390 schematic, 381 selective, 390 Displacement separation, developing, 399 Displacer, 11, 380 Dissociation equilibrium constant, 573 Distribution coefficient, 561 DMT (4,4'-dimethoxytrityl), 513 DMT-off oligonucleotides, AX purification of, 525 DMT-on antisense oligonucleotides hydrophobic interaction chromatography of, 524 RP purification, 519

716 DNA, 318, 325, 369, 465, 519, 571, 668, 690, 698 analogues, 16, 512 fragments purification, 254, 518, 529 microchip arrays, 700 reduction, 467 removal, effect of flow rate, 468 sequencing, 261, 313 Downstream processing, 20, 329, 384, 417, 514, 659, 668 Downstream product recovery, 633 Drug product, 25 Drug substance, 25 Drugs, see specific names Dye interaction chromatography, 590 Dynamic adsorption capacity, 181 Dynamic affinity plot, 395 of cytochrome c, 396 E. coli, 27, 369, 370, 402, 424, 425, 426, 427, 445, 673, 678, 680, 681, 683 Economics, 705 of bioseparations, 3, 667 of oligonucleotide purification, 530 of separations, 19 Effector functions, of antibodies, 540 Egg yolk, 551, 588, 615 antibodies, 551 Electrode reactions, 269 Electrofocusing, 282 Electroosmotic flow (EOF), 245 see also EOF Electrophoresis, 238, 659 Electrophoretic mobility, 292 Electrospray ionization, 7, 112, 299 see also ESI Electrospray ionization-mass spectrometry (ESI-MS), 301, 314-315 ELISA, 54, 540, 694 see also Enzyme-linked immunosorbent assay Enbrel, 693 Endotoxin, 465, 467, 468, 668 reduction, 467 Engineering process control, 19, 659, 660 Enzymatic cleavage, of IgGI, 545 Enzyme-linked immunosorbent assay (ELISA), 9, 48-49

INDEX

Enzymes, large-scale purification of, 370 EOF, 247, 249, 252 see also Electroosmotic flow EPO, 670, 671, 672 see also Erythropoietin Epogen, 2 Epoxy coatings, 252 EquiUbrium constant, 574 Equilibrium distribution coefficient, 79, 121 Equipment, plant and process, 3, 633 Erythromycin, 331, 340 Erythropoietin (EPO), 1, 670 see also EPO Escherichia coli, 1, 260, 465, 694 see also E. coli ESI, 7, 8, 309 comparison with MALDI, 307 disadvantages, 307 source, 302, 303 interfaced to HPLC, 302 see also Electrospray ionization ESI-MS, 301, 310. 314, 315, 316 see also Electrospray ionization-mass spectrometry (ESI-MS) ESI-QqQ MS, 308 Exotoxin a, 445-447, 449 Expanded-bed adsorption, 12, 431-451, 648 for protein purification, 417 Expanded-bed affinity chromatography, 419, 425 Expanded-bed chromatography, 4, 559 applications, analytical, 427 columns, 208 conditions, 77 operation of system, 418 temperature effect on, 212 Expansion behavior, 419 applications, 424 dense particles, 422 sanitation, 422 support material, 421 viscosity, 423 Expression systems, 707 Extraction, 659 Extrathermodynamic relationship, 219

FDA, 2, 469, 536, 638 document, 2,3 from CBER, 3

Web site, 3, 24 see also U. S. Food and Drug Administration Feedstock, 418, 419, 425, 426, 431,433,435,537,547,549, 565, 569, 709 Filtration, 635 Flat sheet disk membrane adsorbers, 455 Fluid velocity on MA performance, 459 Fluidized bed, 14, 433, 558 adsorption, 12 conventional, 420, 432, 433 Fomivirsen sodium, 469, 512 Food industry, 635 Formaldehyde dehydrogenase, 358 Formate dehydrogenase, 358 Fractoflow hollow-fiber membrane adsorber, 457 Free solution capillary electrophoresis, 43 Free-energy change, 122 Free-energy selectivity parameter, 152 Freeze-dryers, 652 Freundlich-Jovanovic isotherm, 184 Frontal chromatography, 501 Fuel industry, 635 Fumarase, 331, 358 Fused silica capillaries, modifications of, 247 Future developments, 2 0 - 2 1 , 687-710

j8-Galatosidase, 358, 383, 673 activity, 428 Gel electrophoresis, 8, 29-34 Gel filtration, 599, 600, 602, 606, 609 media selection, 601, 606 Gene machines, 16, 512 Gene p53, 690, 700 Gene therapy, 698, 699 Genomics, 261, 698 analysis, 699 output, 706 Glucose dehydrogenase, 358 Glucose isomerase, 331, 358 Glucose-6-phosphatedehydrogenase, 358 a-Glucosidase, 358 /3-Glucuronidase, 682, 683 Glutamic acid, 331

717

INDEX

Glycine, 331 Glycoproteins, 2, 258 microheterogeneity in, 258 GMP, 467, 470, 519, 522, 524, 655, 710 see also Good manufacturing practices Good manufacturing practices, 117 see also cGMP, GMP Gradient instability, 272-273 Gramicidin, 331 Guidelines, 27, 613, 614

HB Ai, 259 HBA2,259 HB F, 259 HB S, 259 HCIC, 589, 602, 605 see also Hydrophobic chargeinduction chromatography H - D exchange, 316, 317 Hemoglobin, 259 adsorption, dynamic capacity curve, 464 Heparinase I, 678, 679, 680, 688 Herceptin, 689 Hexakinase, 358 HIC, 524, 568 reversed phase, 465 see also Hydrophobic interaction chromatography High molecular w^eight displacers, 385 High-performance capillary electrophoresis, contributors to, 239 High-performance liquid chromatography (HPLC), 8, 29, 3 4 - 4 1 , 302, 475 see also HPLC High-throughput screening, 700 HIV, 692 reverse transcriptase (RT), 693 Hofmeister series, 127 Hormones, 1 Horse heart cytochrome c, 388 Host-cell protein impurity, 58 HPLC, 4, 34, 72, 74, 515, 695 biospecific and biomimetic affinity, 5, 75 efficiency, 156 gel permeation, 77, 119 hydrophilic interaction, 5, 75 hydrophobic interaction, 75

ion exchange, 4, 75 metal ion affinity, 75 molecular, 218 multiple-stage, 169 normal phase, 4, 75 optimization of separations, 158 overload effects, 111 peak capacity, 160 peak dispersion, 156 peak efficiencies, 133 phase ratio, 140 pore size. 111 porous particles, 192 pressure drop, 77 reversed phase, 4, 75 size-exclusion, 77, 119 van Deemter-Knox relationships, 134 void volume, 78 zero dead volume, 78 see also High-performance liquid chromatography HSA, 258 see also Human serum albumin Human insulin, 659, 675 see also Insulin Human serum albumin (HSA), 257, 425, 492, 700 see also HSA Human tumor necrosing factor, 702 Humanized MAbs in development, 689 FDA-approved, 6S9 see also Monoclonal antibodies Hydrodynamics, 484 Hydrodynamics-mass transfer limitation, 484 Hydrogen bond acceptor, 146 Hydrophilic proteins, 341 Hydrophobic charge-induction chromatography, (HCIC), 587, 606, 612 ligands, 587 Hydrophobic interaction chromatography (HIC), 36-37, 389, 517, 566, 602 of DMT-on antisense oligonucleotides, 524 see also HIC Hydroxyapatite, 517, 570, 602, 605, 606 Hydroxyapatite chromatography, 611 D-2-Hydroxyisocarproate dehydrogenase, 358

L-2-Hydroxyisocarproate dehydrogenase, 358 Hypervariable region, 538

ICH, see International Conference on Harmonization Identification of host-cell protein impurities, 5 4 - 5 7 IgA, 538, 583, 594 IgG, 538, 541, 544, 555, 575, 576, 582-585, 596, 601 structure, 538 IgGI, enzymatic cleavage of, 545 IgM, 538, 545, 556, 583, 594, 596, 601 structure, 539 IMAC, 465, 591 Immobilized pH gradients (IPCs), 273 Immunoaffinity chromatography, 595 Immunoassay, 9, 47-48 Immunoglobulin binding domain, 576 Immunoligand assay, 9, 52-53 Immunoprecipitation, 313 Impurity detection, 3 2 - 3 3 , 38-39 in capillary electrophoresis, 47 in gel electrophoresis, 32-33 in host-cell protein, 54-57 in HPLC, 38-39 in immunoassays, 53-54 Impurity, 25, 26, 27 see also Impurity detection Inactivation kinetics, 620 Industrial bioseparation equipment, 19 Industrial case studies, 707 displacement chromatography, 400 Industries using bioseparations, 634 Insulin, 1, 675 see also Human insulin Interactions of polypeptides and proteins dipole interactions, 84, 105 electrostatic, 84, 93 hydrogen bond, 84, 88 hydrophobic, 84, 85, 119, 125 Lifshitz-van der Waals, 84, 99 metal ion coordination interactions, 84, 100 Interferon, 358, 673

718 Interleukin-8 (IL-8), 448, 449, 693 International Conference on Harmonization (ICH), 3, 24 Web site, 3 International Union of Pure and Applied Chemistry (lUPAC), 330 Ion exchange, 383, 465 capture of antibodies, 562, 563 Ion pairs effect, 334 Ion source, 300 Ion trap-MS, 308 Ion-exchange chromatography, 37-38, 560, 606, 610 capacity factor, 560, 567 displacement chromatography, 385 Ionic molecules, 490, 495, 496 Ion-pairing RP chromatography (IP-RP), 517 Ion-pairing strategies, 115 IR studies, 316 Isodesmic interactions, 211 Isoelectric focusing (lEF), 4, 6-7, 31, 239, 263-298, 312, 400, 696 analytical, 276-285 definition, 263 mechanism, 265 practical considerations, 266-268 principles, 264 Isoelectric point (pi), 244, 289 of amino acids, 335 determining, 283 Isoelectric precipitation, 556 Isolation, 649 of host-cell protein impurities, 55-56 Isoleucyl-tRNA synthetase, 358 Isopropanol dehydrogenase, 358 lUPAC, see International Union of Pure and Applied Chemistry

Jacalin, 597

Lab on chip, 699 Lactate dehydrogenase (LDH), 388 D-Lactate dehydrogenase, 358 L-Lactate dehydrogenase profile elution and breakthrough, 425 Lactoferrin, 387 a-Lactoglobulin, 260 j3-Lactoglobulin, 260, 383, 384

INDEX

/3-Lactoglobulin A, 260, 384, 393, 398, 399 jS-Lactoglobulin B, 260, 384, 393, 398, 399 j8-Lactoglobulin C, 260 Langmuir equation, 181, 182 Large-scale aqueous two-phase partitioning, 357, 358 Large-scale chromatographic purification, oligonucleotides, 511 Large-scale production of antisense oligonucleotides, 512 Large-scale purification of enzymes, 370 of therapeutic oligonucleotides, 519 Laser scanning microscopy, 705 LC-ESI MS analysis, 312, 313 Lectin affinity separation, 597 Lectins, 598 Leucine dehydrogenase, 358 Lewis acid, 100 Ligand, 424, 464, 586 leakage, 580 selection for expanded bed adsorption, 423 Lincomycin, 331 Linear elution chromatography, 380 Lipoproteins, 259 high-density (HDL), 259 low-density (LDL), 259 very low density (VDLD), 259 Liquid chromatography (LC) purification of antibodies, 556 separation of antibodies, 535 Liquid-liquid distribution, 9-10, 329, 695 Liquid-liquid extraction, 704 reversed micelles, 341 Low molecular mass displacers, 384,387 Low molecular weight displacers, 386, 396 design of, 406-410 structural characteristics, 408 Lysine, 331 Lysozyme, 315, 342, 343 355, 390, 391, 392, 493, 693 MA, see Membrane adsorbers. Membrane absorption MAbs, see Humanized monoclonal MAbs, Monoclonal antibodies

MADC (metal affinity displacement chromatography), 388 MALDI, 7, 8, 306 comparison with ESI, 307 disadvantages, 307 see also Matrix-assisted laser desorption/ionization (MALDI) MALDI PSD spectrum, 313 MALDI TOE mass spectrum, 314 MALDI-TOE MS, 303 comparison with ESI-QqQ MS, 308 Manganese chloride, 369 Manganous sulfate, 369, 372, 373, 374, 375 MAs, see Membrane adsorbers (MAs) Mass analyzer, 301 Mass balance, 184 Mass spectrometry (MS), 4, 7-8, 300 applications to biological research, 309 of biomolecules, 299 see also specific name Mass transfer, 194 limitation, 484 resistances, 178 Mass transport processes, 132 eddy diffusion, 132 mobile phase, 132 stagnant mobile phase, 132 stationary phase, 132 Material balance, stages of recovery, 368 see also Stages of recovery Matrix-assisted laser desorption/ionization (MALDI), 112, 299 MECC, see Micellar electrokinetic chromatography MEKC, see Micellar electrokinetic chromatography Mellitin, 382, 383 Membrane adsorbers (MAs), 14, 453, 454, 456 applications to preparative bioseparations, 463 commercially available, 455 cylindrical, 456 flat sheet disk, 455 Eractoflow hollow-fiber, 457 Membrane adsorption, 696 applications, chromatographic modes , 465

719

INDEX

Membrane chromatography, 453 comparison to traditional chromatography, 458 capacity, 458 resolution, 459 cleanability, 459 sterilization, 459 velocity, 458 Membrane-like chromatographic media, commercial, 457 Messenger RNA (mRNA), 691, 692 Metal chelate affinity chromatography, 591 Methodology montage, 8 Micellar electrokinetic capillary chromatography, 46-47, 239, 255 Microwave technology, 702 Milk proteins, 260 Mimetic ligand, 593 Modeling, 705 Molecular chromatography, 74 Molecular dynamic procedures, 82 Monobromobimane, 61 Monoclonal antibodies (MAbs), 1, 17, 425, 536, 688, 689 purification, 15, 468 see also Antibodies Monomodal sorbent, 77 Moving-bed system, 476 Multicomponent separation, 480 Multimodal sorbent, 77 Multistep purification procedures, 165 Multizoning phenomenon, 169 Mycobacterium genitalium, 698 Mycobacterium tuberculosis, 698 Myo, 389 Myoglobin, 315, 387, 493

NAD kinase, 358 Natural product peptide, purification, 242 Negative chromatography, 467 Neutral polymers, 250 Noncovalent interactions, 318 Nonspecific interaction on displacer affinity, comparison, 410 Novel erythropoeisis stimulating protein (NESP), 670 Novobiocin, 331 N-terminal sequencing, 56, 59 N-terminal variants, 62-64

Nuclear magnetic resonance (NMR), 314, 315, 316, 320 Nuclease treatment, 370 Nucleic acids, 365, 390 bacterial, 368 partition coefficient, 372, 375 precipitants, 369 precipitation of, 368, 373 removal from cell homogenate, 375 Oligonucleotide composition and analysis, 515 process-related species, 515 production, schematic, 514 purification, 15, 16, 469 economics of, 530 strategies, 516 techniques, affinity, 518 AX, 525 gel permeation, 518 hydrophobic interaction, 518 hydroxyapatite, 518 ion exchange, 518 IP-RP, 518 large-scale chromatographic, 518 reversed phase, 518, 520 slalom chromatography, 518 see also Oligonucleotides Oligonucleotides, 322, 390 phosphorothioate, 513, 515 self-displacement of, 527 see also Oligonucleotide OligoProcess, 513 Operating regime plot, 396 milk protein system, 398 Optical isomer, 490, 497, 498 Optimum performance, 195 Organic molecules, 490, 496 Organic solvents, 555 Outsourcing companies, 669 Packed beds, 419, 557 reactors, 513 systems, 431, 647 Particle density, 436, 437 Particulate matter, 12 molecular w^eight effect, 353 Partition coefficient, 331, 333, 337, 352 of biomolecules, 331 of carboxylic acids, 337 nucleic acid effect, 372

PEG, 10, 357, 360, 371, 554, 556 PEG 3400-dextran 5000-v^^ater phase diagram, 349, 351 PEG molecular weight, effect of, 353 PEG-dextran-water system, 349, 352-355 PEG-potassium phosphate-water system, 349 Penicillin acylase, 358 Penicillin F, 331 Penicillin G, 338, 339 Penicillin K, 331 Penicillin V, 338 Pentaerythritol-based dendritic polyelectrolytes, 387 Peptide mapping, 39-41 molecular peak, 307 Peptides, 238, 311, 382, 389, 390 and proteins migration behavior, 243 pH effect, 333 on protein solubilization, 343 pH gradients, 268-276 acrylamido buffers, 274 buffer pairs, 276 carrier ampholytes, 269-272, 276 determination of, 283 Phage display Hbraries, 544 Pharmaceutical industry, 634 Pharmacogenomics, 698 Pharmacokinetics, 17 Phosphofructokinase, 358 Phospholipase, 358 Phosphoramidite chemistry, 513, 515 8 mer Phosphorothioate, purification of, 528, 529 20 mer Phosphorothioate anion exchange trace, 516 purification of, 528, 530 24 mer Phosphorothioate nucleotide, preparative displacement chromatography, 406 Phosphorothioate oligonucleotides, 513 Phosphorothioate, synthesis, 515 Phosphorylase, 358 Photoiode-array detection, 38 Phytol stereoisomers, 497 Pichia pastoris, 425, 444 Pilot-scale processing, 634 pKg values of biomolecules, 334 see also Biomolecules

720 Plasmids, purification of, 529 Plasmodium falciparum, 698 Plug flow, 434 "Points to Consider in the Production and Testing of New Drugs and Biologicals Produced by Recombinant DNA Technology," 2, 24 Polishing, 605 Polyacrylamide gels, 33, 267 carrier ampholyte isoelectric focusing in slab gels, 276, 279 casting gels, 280 experimental methods, 279 sample application, 281 sample preparation, 281 Polyamines, 251 Polyethylene oxide, 251 Polyethyleneimines, 251, 369 Polymers, 251 molecular weight, effect of, 352, 353 Polymyxin antibiotics, 382 Polypeptides and proteins, see Interactions of polypeptides and proteins Polyvinyl alcohol, 250 Power law equation, 672 Prazinquatel, 498 Precipitation, 659 by manganous sulfate, 372 by protamine sulfate, 372 by streptomycin sulfate, 372 see also Antibodies Predictive cost function, 671 Preparative bioseparations, applications with membrane adsorbers, 463 Preparative chromatography, 11-12,380,410,453 Preparative isoelectric focusing, 287-291 Preparative-scale protein purification, 389 Preservation of gels, 284 Prions, 694 Process bioseparation industry, 14 Process changes, making and managing, 708, 709 Process control, 660 calculation of adjustment, 660 manipulation of, 660 measurements of variables, 660 Process design, 705 Processing plants, 633 equipment, 18

INDEX

Process-related impurities, 25, 27 Process-scale bioseparation, 636-639 see also Bioseparation type Process-scale considerations, 653 automation, 654 construction materials, 653 GMP, 655 hygienic design, 656 location, 655 mechanical design, 653 safety, 654 validation, 656 Product purification, 649 Product-related impurities, 25, 28 Product-related substances, 26 Production, and purity, 694 Protamine sulfate, 369, 372 Proteases, 423 Protein adsorption of, 246 analysis, 309 crystallization, 701 fluorescence, 38-39 folding, 261, 314 dynamics, 317 impurity, 4, 28 mass, 244 mobility, 244 purification, 290, 383, 432 purity, 4 recovery, 425 separation, temperature effect on, 252 see also Interactions, Proteins, specific protein Protein A (SpA), 575 affinity chromatography, 575, 610 Fc-binding domain, 578 mimetic molecule, 594 see also SpA Protein bands, detection of, 284 Protein-DNA complexes, 321 Protein-free cell cuhure media, 549 Protein G, 582 Protein L, 583 Protein LG, 583 Protein P, 583 Protein profile, elution and breakthrough, 425 Protein-protein multilayer phenomenon, 184 Protein purification expanded-bed adsorption, 417 on MA, 464

Proteins, 2, 13, 258, 259, 260, 331,341,365,375,384,388, 390, 402, 490, 425, 492, 688 capture, 13 intracellulary produced, 367 ion-exchange displacement chromatography of, 385, 386 nucleic acids, separation, 10-11 poHshing, 13 purification, 13 see also Protein, specific protein Proteomics, 698 Pseudomonas aruginosa, 336 exotoxin A, 60, 425 Pseudomonas putida, 11, 372, 375 Pullulanase, 358 Purification antigenic vaccine protein, 400, 401 of 8 mer phosphorothioate, 529 of 20 mer phosphorothioate, 530 see also specific examples Purification methods, alternatives to chromatography, 704

Quality assurance (QA), 2 Quantitative structureretention-function relationships, 75

RA, see Rheumatoid arthritis Raman spectroscopy, 703 Recombinant antibodies, 536 see also Antibodies Recombinant DNA technology, 23, 659 Recombinant proteins, 259, 463 expressed in £. coli, 425 from variants, 402 see also Protein, Proteins, Recombinant proteins Recombinant vaccine protein, 463 purification of, 15, 465 Recombinant virus products, 710 Recovery, of biological products, 700 Reflectron-TOF MS, 305 Registration authorities, 613 Regulatory considerations, 2, 24, 612 Regulatory principles, 613 Residence time distribution (RTD), 420

721

INDEX

Resolution, 156 contour plots, 169 in isoelectric focusing, 294-295 Retention mechanism, 161 Retention time, 78 Reteplase, 672 Retroviral contamination, 694 Reversed micelles forw^ard and back extraction, 342 liquid-liquid extraction of, 341 Reversed-phase chromatography, 517 Reversed-phase purification, 514 Reversed-phase HPLC, 34-46, 60, 241, 243, 520, 522 Reversed-phase liquid chromatography (RPLC), 389 see also RPLC Rezulin, 673 rGUS, 683, 684 see also beta-glucuronidase Rheumatoid arthritis (RA), 693 rHu-BDNF purification, 404 Ribonuclease, 693 Ribonuclease B, 391, 392 Ribozymes, purification, 529, 530 Richardson-Zaki equation, 420 RNA, 369, 372, 519, 690 analogues, 16, 512 precipitation of, 369 purification, 529 RNase A, 387, 389 Rotofor cell, 287-290 RotoLytes, 289 RP purification of DMT-on oligonucleotides, 521 of oligonucleotides, schematic, 520 RPLC, 389, 403

Sah effect of, 355 selectivity, 152, 172 Sample application for isoelectric focusing, 281 Sample displacement chromatography, 390 Sample preparation for isoelectric focusing, 281 Scale-up, 380 heuristic approaches, 172 multimodule systems for pilotscale purification, 462 productivity considerations, 172

SDS-PAGE, 8, 2 9 - 3 1 , 50, 253, 254, 285, 400, 696 see also Sodium dodecyl sulfate polyacrylamide gel electrophoresis SEC, see Size-exclusion chromatography Secondary parameters. 111 Sedimentation, 635 Selective displacement chromatography, 390 Selectivity, 79 Self-associated (isodesmic) species, 80 Self-displacement chromatography, of oligonucleotides, 527 Separation and purification methods, 3, 9 Separation process, 604 classification of, 366 Sepharose, 421 Serial analysis of gene expression (SAGE), 690, 691 Shake-flask experiment, monitoring, 428 Sieve-tray extraction tower, 348 Silanols, 106 Silver staining, 33 Simulated moving-bed (SMB) chromatography, 15-16,475, 476, 480, 481, 706 advantages over batch chromatography, 507 applications, 490 classical implementation, 481 calculation, 488, 503-504 column number, 488 countercurrent, 475, 479 desalting, 491 developments, 490 diameter, 488 length, 488 multicomponent separation, 481 principle of, 478 Simulation, 705 Size-exclusion chromatography (SEC), 37 SMA parameters, estimation, 394 SMB, see Simulated moving-bed (SMB) chromatography Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 285

Solubility parameter, 90 for solvents, 332 Solvent extraction, 9-10, 329, 330 of alcohols, 336 of amino acids, 335 of carboxylic acids, 336 electrically enhanced, 344 equipment, 344 "Solvent Extraction in Biotechnology," 330 Solvent gradient CEC, diagram, 257 Solvent selection, 331 Solvophobic theory, 85 Sorption processes, 647 SpA, 576-578, 580-581 see also Protein A Spray dryers, 652 Stages of recovery, material balance, 368 see also Material balance Staphylococcal protein A-/3-galactosidase hybrid, 358 Step elution procedures, 131 Stereoisomers, 497 Steric mass action (SMA), 394 see also SMA Stoichiometric models, 93 Streamline, 420, 436, 438-440, 442-444, 447, 448 Strepavidin, 321 Streptomyces species, 339, 340 Streptomycin, 391 Streptomycin sulfate, 369, 372 precipitation of nucleic acids, 373 Structure-function relationships, 159 Sugars, 490, 491, 492, 499 separation of, 490, 491, 492, 499 Super Pro Designer, 679, 682 Surface tension, 87 molal, 125 Surfactants, 248, 249 Synagis, 689 Synthetic ligands, 706

Targeting messenger RNA, 691, 692 TDH, 368, 373, 375 extraction from cell homogenate, 376 partition coefficient, 375 Temkin isotherm, 184

722 Temperature-dependent heat capacity, 141 Temperature, effect of, 354 3,7,1,15-Tetramethyl-2hexadecen-l-ol,C2oH4oO, 497 Tetrapeptide, 311 see also Peptides Therapeutic ohgonucleotides, large-scale purification, 519 Thermal dryers, 651 Thermal separation, 651 Thiadiazin, 498 Thiophilic chromatography, 584, 606 Thiophilic ligand structures, 586 Threonine, separation of isomers, 498, 499 Thrombolytic drugs, 672 Time-of-flight(TOF), 301 see also TOF analyzer Time-resolved ESI MS, 317 Tiselius, 382 Tissue plasminogen activator (tPA), 672 TMB, 488, 502 see also True moving bed TNF, 693, see also Tumor necrosis factor TOF analyzer, 305, 306 TOMAC, 342 tPA, see Tissue plasminogen activator Traditional chromatography, comparison to membrane chromatography, 457 see also Membrane chromatography Transforming grov^th factor a, 60 Transgenic animals, 549

INDEX

Transgenic corn, 682, 684 Transgenic milk, 552, 615, 709-710 Transgenic plants, 550, 552, 708 Transmissible degenerative encephalopathies (TDE), 615 Transport equations in isoelectric focusing, 293 Transthyretin, 313 Transthyretin amyloidosis (ATTR), 311 Trioctylmethylamonium chloride (TOMAC), 335 True moving bed (TMB), 477 calculation, 485-488, 502-503 principle of, 477 see also TMB Trypsin, 372 Tryptic peptides chromatographic separation, 310 mapping, 40 Tumor necrosis factor (TNF), 693 2D-PAGE, 8, 696 see also Two-dimensional polyacrylamide gel electrophoresis Two-dimensional polyacrylamide gel electrophoresis (2DPAGE), 8, 282, 285, 300 Two-dimensional SDS-PAGE, 31 Two-stage aqueous two-phase partitioning, illustration, 360

U. S. Food and Drug Administration, 65S, 674 see also FDA Vaccines, 1 Valence, 244 Validation program, 2 van't Hoff plot behavior, 136 Variable light chain (VL), 539 Variable region, 540 Viral contamination, 702 Viral DNA, reduction of, 15, 467 Virginiamycin, 340 Viruses, 615, 616 clearance of, 616 detection of, 616 inactivation of, 616 removal of, 468, 617, 668 Vitravene, 692 Volt-hours, 283 Waste water treatment, 635 Western blot analysis, 4 9 - 5 2 , 61, 540 Westfalia countercurrent extraction decanter, 346 Whey, 358 Whey proteins, 384, 388 X-ray crystallography, 314, 315, 319, 320 Yeast RNA, 372

Ubiquitin, 315, 317 bovine, 316 Ultrafiltration, 553 Upstream fermentation, 633 Urea PAGE, 31

Zenapax, 689 Zeta potential suppressors, 248 Zidovudine (AZT), 693 Zwitterions, 248, 335

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