New Developments and New Applications in Animal Cell Technology

New Developments and New Applications in Animal Cell Technology

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New Developments and New Applications in Animal Cell Technology Proceedings of the 15th ESACT Meeting

Edited by

Otto-Wilhelm Merten Pierre Perrin and

Bryan Griffiths

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ORGANIZING COMMITTEE: Otto-Wilhelm Merten (Meeting Secretary) Wolfgang Noé (Sponsoring, Trading)

Georgia Barlovatz-Meimon

Andres Crespo, Génopoïétic Jean-Marc Engasser Fabrice Geoffroy Jean-Marc Guillaume Jan Lupker

Annie Marc Pierre Perrin

v

SPONSORS: The Organizing Committee acknowledges the financial support to :

Companies: Bayer B. Braun Biotech International Biolnvent Production AB Boehringer Ingelheim Bioproducts Boehringer Mannheim Covance Laboratories Ltd. Dr. Karl Thomae GmbH Genzyme Transgenics Corp. Hoffmann-La Roche Inc. Hyclone Europe Intervent International bv JRH Biosciences Laboratoires Serono SA Life Technologies MA BioServices Merck KG Merck Sharp & Dohme MicroSafe B.V. Nature-MacMillan Magazines Novo Nordisk Nunc A/S Pharmacia Biotech AB Promega Q-One Biotech Ltd. Sanofi Recherche Schering AG SmithKline Beecham Biologicals Institutions: City of Tours European Commission - DGXII - Biology

vi

ESACT Executive Committee, 1996-1997 C. MacDonald

Chairperson

University of Paisley, Paisley, U.K.

L. Fabry

Secretary

Smith Kline Beecham, Rixensart, B

B. Griffiths

Treasurer

CAMR, Porton, U.K.

O.-W. Merten

Meeting Secretary

Généthon II, Evry, F

M. Carrondo

IBET/CTQB, Oeiras, P

H. Hauser

GBF, Braunschweig, D

J. Lehmann

University of Bielefeld, Bielefeld, D

W. Noé

Dr. Karl Thomae GmbH, Biberach, D

vii

15th ESACT Meeting, Tours, France Session chairpersons :

Session

1st Chairperson

2nd Chairperson

1.

J. Lehmann

E.T. Papoutsakis

2.

G. Barlowatz-Meimon

W. Scheirer

3.

A. Crespo

J.-M. Guillaume

4.

B. Griffiths

J. Lupker

5.

L. Fabry

F. Geoffroy

6.

H. Hauser

C. MacDonald

7.

M. Carrondo

J.-M. Engasser

8.

M. Al-Rubeai

J. Bailey

Workshop

S. Barteling

viii

CONTENTS Introduction

xxiii

HYCLONE Lecture Genetic manipulation of the mammalian genome (Abstract) R. Jaenisch

1

ESACT Lecture Engineering glycosylation in animal cells J.E. Bailey, E. Prati, J. Jean-Mairet, A. Sburlati, P. Umaña

5

Sessions on : Biosynthesis and post-translational modifications of recombinant proteins, and Cell physiology and metabolic engineering of animal cells

25

Gene expression :

27

Enhanced recombinant protein expression in insect cells in the presence of dimethylsulfoxide (DMSO) L. Ramos, J.F. Kane, A.A. Murnane

29

Studies of the hepatitis C virus recombinant protein production and its effects on Sf-9 insect cells metabolism C.M. Charon

35

Characterization of phosphorylation mutants of the human multidrug transporter (MDR1) expressed in the baculovirus-Sf9 insect cell system K. Szabo, E. Bakos, E. Welker, A. Varadi, H.R. Goodfellow, C.F. Higgins, B. Sarkadi Glutamine synthetase transfected cells may avoid selection by releasing glutamine P. Bird, E. Bolam, L. Castell, O. Obeid, N. Darton, G. Hale Study of different cell culture conditions for the production of a reshaped Mab in NS0 cells A.J. Castillo, A. Fernandez, T. Boggiano, P. Pugeaud, I.W. Marison Increasing monoclonal antibody productivity by semicontinuous substitution of production medium for growth medium

39

43

51

55

x Expression of recombinant human insulin in Chinese hamster ovary cells is

complicated by intracellular insulin-degrading enzymes S.C.O. Pak, S.M.N. Hunt, M.J. Sleigh, P.P. Gray High yield expression of recombinant plasma factors : use of recombinant endoprotease derivatives in vivo and in vitro U. Schlokat, A. Preininger, M. Himmelspach, G. Mohr, B. Fischer, F. Dorner

The role of cell cycle in determining levels of gene expression in CHO cells D.R. Lloyd, V. Leelavatcharamas, D.C. Edwards, A.N. Emery, M. Al-Rubeai Heterogeneity within DHFR-mediated gene amplified population of CHO cells producing chimeric antibodies N.S. Kim, S.J. Kim, G.M. Lee

Immunofluorescence detection of recombinant monoclonal antibody as a tool in the characterization of chinese hamster ovary cell lines

59

69

77

81

85

C.A.S. Kinney, R.E. Trala, L.C. Hendricks, T.D. Hill Electrolyzed reduced water which can scavenge active oxygen species supresses cell growth and regulates gene expression of animal cells S. Shirahata, S. Kabayama, K. Kusumoto, M. Gotoh, K. Teruya, K. Otsubo, T. S. Morisawa, H. Hayashi, K. Katakura Differential gene expression of cytochrome P450 in immortalised hepatocyte cell lines

93

97

H.T. Kelly, K. Anderson, E. Hill, H. Grant, C. MacDonald Post-transcriptional control of recombinant gene expression : mRNA retargeting and regulation of expression

101

K.A. Partridge, A. Johannessen, A. Tauler, I.F. Pryme, J.E. Hesketh Transient transfection in mammalian cells. A basic study for an efficient and costeffective scale-up process E.-J. Schlaeger, L.Y. Legendre, A. Trzeciak, E.A. Kitas, K. Christensen, U. Deuschle, A. Supersaxo Large scale transient gene expression in mammalian cells

E.-J. Schlaeger, K.Christensen, G. Schmid, N. Schaub, B. Wipf, A. Weiss

105

113

xi

Synergistic enhancement of transient expression by dioleoyl-melittin (DOM) and polyethylenimine (PEI) in mammalian cells in suspension culture E.-J. Schlaeger, J.Y. Legendre, A. Trzeciak, E.A. Kitas, K. Christensen, U. Deuschle, A. Supersaxo

117

Transient expression of a soluble and secreted form of heterodimeric T-cell receptor in HEK-293 M. Jordan, C.L. Blanchard, I. Bernasconi, I. Luescher, F.M. Wurm

121

Measurable parameters of cells and precipitate predict transfectability with calcium phosphate M. Jordan, F. Wurm

125

Glycosylation:

129

Influence of Na-butyrate on the production and sialylation of human interferon-g by a2,6-sialyltransferase engineered CHO-cells

131

D. Lamotte, L. Monaco, N. Jenkins, A. Marc Elevated inhibits the polysialylation of the neural cell adhesion molecule in CHO MT2-1-8 cell cultures J.A. Zanghi, T.P. Mendoza, R.H. Knop, W.M. Miller

135

Influence of cultivation conditions on glycosylation pattern - a fed-batch and continuous culture study N.A. Schill, M.Z. Rosenberg, R.L. Dabora

141

Effects of different production systems on glycosylation pattern of murine monoclonal IgA F. Schweikart, E. Lüllau, R. Jones, G.J. Hughes

149

Identification of altered glycosylation as the major difference between intracellulary accumulated and secreted b-trace protein produced in baculovirusinfected insect cells G. Rodriguez, H.S. Conradt, V. Jäger

153

xii

How ammonium dominates the metabolism of in vitro cultivated mammalian cells A. Çayli, M. Wirth, R. Wagner

157

Study of human recombinant GM-CSF produced in different host systems using monoclonal antibodies M. Etcheverrigaray, M. Oggero, M. Bollati, R. Kratje

163

Metabolic engineering :

166

Modification of hybridoma cells metabolism J.J. Cairó, C. Paredes, F. Gòdia, E. Prats, F. Azorín, Ll. Cornudella

167

Cloning and expression of a cytosolic sialidase from CHO cells in a glutathione S-transferase (GST)-encoding expression vector M. Burg, J. Müthing Construction of a novel CHO cell line coexpressing human glycosyltransferases and fusion PSGL-1 - immunoglobulin G

175

181

B. Vonach, B. Hess, C. Leist

Production of defined glycosylation variants of secreted human of secreted human glycoprotein therapeutics by coexpression with human recombinant glycosyltransferases in BHK-21 cells P. Schlenke, E. Grabenhorst, J. Costa, M. Nimtz, H.S. Conradt

185

Antisense RNA for the elimination of NeuGc residues from recombinant glycoproteins A. Gregoire, A. Visvikis, A. Marc, J.-L. Goergen

191

Cell proliferation, proliferation control, apoptosis:

195

Intracellular fatty acid composition affects cell yield, energy metabolism and cell damage in agitated cultures M. Butler, N. Huzel, N. Barnabé, L. Bajno, T. Gray

197

Lipid requirements of a recombinant Chinese hamster ovary cell line (CHO) J.T. McGrew, C.L. Richards, P. Smidt, B. Dell, V. Price

205

xiii

Sustained expression in proliferation controled BHK-21 cells P.P. Müller, S. Kirchhoff, H. Hauser

209

Cell growth inhibition by the IRF-1 system A.V. Carvalhal, J.L. Moreira, P. Müller, H. Hauser, M.J.T. Carrondo

215

Correlation between BHK cell specific productivity and metabolism H.J. Cruz, J.L. Moreira, E.M. Dias, C.M. Peixoto, A.S. Ferreira, M.J.T. Carrondo

219

Effect of growth arrest in BHK metabolism: on line monitoring by 13C and 31P NMR spectroscopy P.M. Alves, A.V. Carvalhal, J.L. Moreira, H. Santos, M.J.T. Carrondo

223

Cell survival in CHO cell cultures grown in a defined protein free medium N. Chatzisavido, C. Fenge, L. Häggström

227

Enhanced apoptosis in insect cells cultivated in simulated microgravity N. Cowger, K. O’Connor

231

Modulation of apoptosis by bcl-2 expression following amino acid deprivation and in high cell density perfusion cultures R.P. Singh, D. Fassnacht, A. Perani, N.H. Simpson, C. Goldenzon, R. Pörtner,

235

M. Al-Rubeai

Effect of bcl-2 expression on hybridoma cell growth during stressful conditions D. Fassnacht, S. Rössing, , M. Al-Rubeai, R. Pörtner

Inhibition of c-jun expression in F-MEL cells causes cell cycle arrest and prevention of apoptosis Y.H. Kim, T. Iida, E.V. Prochownik, E. Suzuki Hippocampal cells in culture as a model to study neuronal apoptosis I. Figiel, J. Jaworski, L. Kaczmarek

243

247

255

Development of carboxy SNARF-1-AM and annexin V assays for the

determination of apoptosis in heterogeneous cultures A. Ishaque, M. Al-Rubeai

259

xiv

Session on : Integrated bioprocessing in animal cell technology

263

Physical environment, cells and products :

265

Diauxic cell behavior enables detoxification of CHO cell culture medium during fed batch cultivation H. Lübben, G. Kretzmer

267

Effects of polysaccharide derived from tea on growth of human cell lines in serum free culture H. Kawahara, M. Maeda-Yamamoto, K. Osada, K. Tsuji

273

Metabolic demands of BTI-TN-5B1-4 (High Five™) insect cells during growth and after infection with baculovirus E. Chico, V. Jäger

277

Effects of ammonium and lactate during continuous hybridoma fermentations in a fluidized-bed bioreactor H. Heine, M. Spies, M. Biselli, C. Wandrey

281

Serum concentration and pH affect the CD13 receptor content of HL60 cells cultured in stirred bioreactors C.L. McDowell, E.T. Papoutsakis

285

Four regulation-friendly serum-free media for mammalian and insect cells T.W. Irish, S.E. Lenk, L.A. Bugner, K.J. Etchberger

293

Proteolytic activities in the baculovirus-insect cell expression system G. Schmid, A. Bischoff

303

Monitoring and control:

307

Development of proteinase assays for improved CHO cell cultures C. Tans, C. vander Maelen, S. Wattiaux-de Coninck, M.-M. Gonze, L. Fabry

309

Digital image analysis: quantitative evaluation of colored microscopic images of animal cells K. Falkner, E.D. Gilles

317

xv

The viable cell monitor: a dielectric spectroscope for growth and metabolic studies of animal cells on macroporous beads Y. Guan, R.B. Kemp

321

Measurements of changes in cell size distribution to monitor baculovirus infection of insect cells E. Chico, V. Jäger

329

Dead cell estimation - a comparison of different methods A. Falkenhain, Th. Lorenz, U. Behrendt, J. Lehmann

333

Fed-batch culture development based on biomass monitoring E. de Buyl, A. Maxwell, L. Fabry

337

On-line monitoring of protein and substrate/product concentration in mammalian cell cultivation H. Lübben, J. Hagedorn, T. Scheper

343

Process control and on-line feeding strategies for fed-batch and dialysis cultures of hybridoma cells J.O. Schwabe, R. Pörtner

347

Metabolic network analysis of a hybridoma cell line using mass balances and labelled glucose and glutamine P.-A. Ruffieux, I.W. Marison, U. von Stockar

351

Dynamic medium optimization by on-line heat flux measurement and a stoichiometric model in mammalian cell culture Y. Guan, R.B. Kemp

355

Modeling of glycoprotein production by chinese hamster ovary cells for process monitoring and control J. Stelling, R.K. Biener, J. Haas, G. Oswald, D. Schuller, W. Noé, E.D. Gilles

359

The temperature effect in mammalian cell culture. An Arrhenius interpretation G. Kretzmer, T. Buch, K. Konstantinov, D. Naveh

363

Integrated processes and scale-up:

367

Dielectrophoretic forces can be exploited to increase the efficiency of animal cell perfusion cultures N. Kalogerakis, A. Docoslis, L.A. Behie

369

xvi Use of a new microcarrier with two-dimensional geometry for the culture of

anchorage-dependent cells in sonoperfused, continuously stirred tank reactors C. Gatot, F. Trampler, M.-P. Wanderpepen, J. Harfield, A. Oudshoorn, A. Johansson, V. Nielsen, A.O.A. Miller

377

Evaluation of porous microcarriers in fluidized bed reactor for protein production by HEK 293 cells M.A. Valle, J. Kaufman, W.E. Bentley, J. Shiloach

381

Fluidized bed technology: influence of fluidization velocity on nutrient consumption and product expression G. Blüml, M. Reiter, Th. Gaida, N. Zach, A. Assadian, C. Schmatz, H. Katinger

385

High density and scale-up cultivation of recombinant CHO cell line and hybridomas with porous microcarrier cytopore C. Xiao, Z. Huang, W. Li, X. Hu, W. Qu, L. Gao, G. Liu

389

Cell-settler perfusion system for the production and glycosylation of human interferon-γ by clumped cells D. Lamotte, J. Straczek, A. Marc

395

Disposable bioreactor for cell culture using wave-induced agitation V. Singh

399

Use of miniPERM system for an efficient production of mouse monoclonal antibodies

409

M.L. Nolli, R. Rossi, A. Soffientini, D. Zanette, C. Quarta

Production of anti-EGF receptor hMAb in hollow fiber bioreactors for in vivo diagnosis M.A. Arias, A. Valdés, D. Curbelo, O.M. Morejón, I. Caballero, J. Villán, J.A. Gómez, A. Fernandéz, J. Rodriguez, A. Morales, T. Boggiano, A. Castillo, L. Bouzó, C. Hermida, G. García, P. Vitón, N. Pérez, T. Rodríguez Optimisation of the production of VLP’s in 2 Lt stirred bioreactors P.E. Cruz, A. Cunha, C.C. Peixoto, J. Clemente, J.L. Moreira, M.J.T. Carrondo

Direct capture of monoclonal antibodies with a new high capacity streamline SP XL ion exchanger C. Priesner, N. Ameskamp, D. Lütkemeyer, J. Lehmann

417

421

429

xvii

The effect of different purification schemes on the activity of a monoclonal antibody D.J. Black, J.P. Barford, C. Harbour, A. Fletcher

Interfacing of protein product recovery with an insect cell baculovirus production system I.A. Carmichael, M. Al-Rubeai, A. Lyddiatt Preparation of plasmid DNA using displacement chromatography R. Freitag, S. Vogt Novel methods for large-scale preparation of nutrient media and buffered salt solutions D.W. Jayme Large scale application of the Semliki Forest Virus system: 5-HT3 receptor production H.D. Blasey, B. Brethon, R. Hovius, K. Lundström, L. Rey, H. Vogel, A.-P. Tairi, A.R. Bernard

From applied research to industrial application: The success story of monitoring intracellular ribonucleotide pools S.I. Grammatikos, K. Tobien, W. Noé, R.G. Werner New technology of production recombinant human erythropoietin for peroral administration T.D. Kolokoltsova, N.E. Kostina, E.A. Nechaeva, N.D. Yurchenko, O.V. Shumakova, A.B. Rizikov, N.A. Varaksin, T.G. Ryabicheva

433

437

441

445

449

457

463

General safety aspects:

467

Significance of multiple testing on murine leukemia virus of mouse hybridomas E.J.M. Al, T. Jorritsma, A. Blok, H.M.G. Sillekens, P.C. van Mourik

469

Murine retrovirus detection using Mus dunni cells and S+L- cells P. Seechurn, B. Mortimer, P. Newton, C. Martin

473

The removal of model viruses during the purification of human albumin using chromatographic procedures R. Cameron, C. Harbour, Y. Cossart, J.P. Barford

481

xviii Inactivation of hepatitis A virus during the production of Nordimmun D.W. Birch, P. Kaersgaard, C. Martin

Session on: Viral vector production for gene therapy Adeno-associated viral vectors: Principles and in vivo use O. Danos Novel retroviral packaging cell lines: Improved vector production for efficient hematopoietic progenitor cell transduction

485

491 493

503

S. Forestall, R. Rigg, J. Dando, J. Chen, B. Joyce, R. Tushinski, C. Reading, E. Böhnlein

Scale-up and otpimisation issues in gene therapy. Viral vector production J.E. Boyd, L. Borland, C.E.Fisher

509

Comparison of manufacturing techniques for adenovirus production J.M. Ostrove, P. Iyer, J. Marshall, D. Vacante

515

Safety considerations in the development of new retroviral and adenoviral vectors for gene therapy D. Morgan, I. Forgie, J. Ostrove, M.H. Wisher Gene therapy for neuromuscular disorders : Macrophages as shuttles for widespread targeting

523

531

E. Parrish, E. Peltékian, L. Garcia Gene transfer into dog myoblasts

541

S. Braun, C. Thioudellet, C. Escriou, M.-C. Claudepierre, F. Längle-Rouault, E. Jacobs, R. Bischoff, D. Elmlinger, H . Homann, Y. Poitevin, M. Lusky, M. Mehtali, F. Perraud, A. Pavirani

Biodistribution analysis of a gene therapy vector using the polymerase chain reaction (PCR) technique

545

J. Proffitt, M. Leibbrandt, K. Jarvis, C. Martin Session on: Developments for immunologicals and vaccines

The production of influenza virus by immobilised MDCK cells D. Looby, J. Tree, A. Talukder, K. Hayes, H. House, G. Stacey

549

551

xix

Serum-free grown MDCK cells: An alternative for influenza vaccine virus production N. Kessler, G. Thomas, L. Gerentes, M. Aymard

555

Evaluation of the new medium (MDSS2N), free of serum and animal proteins,

for the production of biologicals H. Kallel, P. Perrin, O.-W. Merten Production of high titre disabled infectious single cycle (DISC) HSV-2 from a microcarrier culture T.A. Zecchini, R.J. Smith New form of the live measles vaccine for oral administration E.A. Nechaeva, E.A. Kashentseva, A.P. Agafonov, N.A. Varaksin, T.G. Ryabicheva, A.P. Konstantinov, V.N. Bondarenko, T.D. Kolokoltsova, I.V. Kits, T.Yu. Sen’Kina, N.V. Zhilina

561

569

573

Study of Leningrad-16 vaccine strain of measles virus reproduction in cell cultures perspective for biotechnology E.A. Nechaeva, T.N. Getmanova, T.Y. Sen’Kina, N.D. Yurchenko

577

Recent advances with new vaccine adjuvants from preclinical to clinical development (Abstract) T. Voss

581

Vaccination with recombinant suicidal DNA/RNA P. Berglund, M. Fleeton, C. Smerdou, I. Tubulekas, B.J. Sheahan, G.J. Atkins, P. Liljeström

583

Growth of goat endothelial cells for the production of a veterinary vaccine P.M. Miranda, J.L. Moreira, M.J.T. Carrondo

593

Expression in insect cells of the major parasite antigen associated with human resistance to Schistosomiasis L.Argiro, C. Doerig, S. Liabeuf, A. Bourgois, J.L. Romette

597

Modulation of CD4 expression on helper T lymphocytes and U937 cells by ganglioside GM3 and its derivatives D. Heitmann, P. Budde, J. Frey, J. Lehmann, J. Müthing

601

Human serum in leukocyte cultures producing human interferon alpha P. Mattana, L. Scapol, S. Silvestri, G.C. Viscomi

607

xx The new type of immunomodulator

613

M.V. Mezentseva, V.A. Mozgovoi, L. Yu. Mozgovaya, R. Ya. Podchernyaeva

In vitro immunization of human peripheral blood lymphocytes with cholera toxin B subunit A. Ichikawa, Y. Katakura, T. Kawahara, S. Hashizume, S. Shirahata Session on: New technologies for health care products

617

625

Analysis of cell growth in a fixed bed bioreactor using magnetic resonance

spectroscopy and imaging P.E. Thelwall, M.L. Anthony, D. Fassnacht, R. Pörtner, K.M. Brindle Expansion of human hematopoietic progenitor cells in a fixed bed bioreactor (Abstract)

627

635

P. Meissner, P. Werner, B. Schröder, C. Herfurth, C. Wandrey, M. Biselli

A novel assay to determine and quantify the regulation of cell motility and migration demonstrated on hematopoietic cells D. Möbest, S. Ries, R. Mertelsmann, R. Henschler

637

Immortalization of differentiated hepatocytes G.S. Jennings, M. Strauss

645

Fixed-bed reactors for animal cell cultivation: An approach to artificial organs R. Pörtner, S. Rössing, J. Stange, D. Fassnacht

657

High density perfusion culture of primary rat hepatocytes for potential use as a bioartificial liver device K. Bratch, A.J. Strain, M. Al-Rubeai Metabolic competence and hormonal regulation of primary porcine hepatocytes in a 3-D sandwich configuration (Abstract) A. Bader, D. Rocker, A. Acigköz, S. Schwintek, J. von Schweder, M. Maringka, V. Armstrong, R. Wagner, G. Steinhoff, A. Haverich Novel mini-bioreactors for islet cell culture A. Handa-Corrigan, I.C. Green, J. Mabley, S. Hayavi, G.N. Kass, R.H. Hinton,

L.M. Morgan, J. Wright

661

665

669

xxi

Cultivation of skin cells suitable for recovery of burn wounds T.D. Kolokoltsova, N.D. Yurchenko, N.G. Kolosov, O.V. Shumakova, E.A. Nechaeva

673

Tissue therapy for treatment of primary myodystrophies T.B. Krokhina, G.B. Raevskaja, S.S. Shishkin

677

Possibility of application of thyroid organ culture for the treatment of persistent

hypothyroidism I.P. Pasteur, N.D. Tronko, E.N. Gorban, V.I. Kravchenko ESACT Lecture Human cells as therapeutic agents (Abstract) H . Green

Session on: Use of animal cells for in vitro testing

681

685

687

Embryonic stem cell differentiation models: Cardio-vascular, myogenic and neurogenic development in vitro A.M. Wobus, K. Guan, J. Rohwedel, C. Strübing, M. Drab

689

Responses of human lung epithelial cells (A549) to pathogenic infection by Mycoplasma pneumoniae J. Goodman, K. Morley, P. Packer, T. Battle

705

Polar lipid profiling of Mycoplasma pneumoniae-infected human lung epithelial cells J. Goodman, R. Wait, T. Battle

713

Comparison of three dimensional (3-D) rat hepatocyte cultures in simulated microgravity conditions T. Maguire, H.J. Moulsdale, G. Stacey, T. Battle

717

High density culture of the human hepatoma cell line HepG2: Long-term culture for in vitro toxicology A. Handa-Corrigan, R.M. Traynor, I. Adamopoulos, J. Salway

721

Application of primary cultures of rat fetal neurons to the study of neurotrophic

action of peptides O.V. Dolotov, I.A. Grivennikov

725

xxii Development and validation of an image analysis system for single cell

characterization in cell monolayers D. Kaiser, M.A. Freyberg, G. von Wichert, P. Marenbach, H. Tolle, P. Friedl

729

Development of an optically accessible perfusion chamber for in situ assays and for long-term cultivation of mammalian cells M.A. Freyberg, P. Friedl

733

Analysis of mitogenic activity of proteins after separation by gel electrophoresis

737

O. Hohenwarter, G. Marzban, E. Jisa, H. Katinger Workshop on:

The use of animal cells versus the use of transgenic animals for the production of recombinant proteins

741

Secretion of fusion proteins into milk by transgenic mouse mammary epithelium (Abstract)

743

D. Pollok, L. Chen, H. Liem, B. Wilburn, J. Williams, M. Harrington, Y. Echelard, H. Meade The production of proteins in the milk of transgenic livestock: A comparison of

microinjection and nuclear transfer

745

I. Garner

Creation of mice expressing human antibody by introduction of a human chromosome H. Yoshida, K. Tomizuka, H. Uejima, H. Kugoh, K. Satoh, A. Ohguma, M. Hayasaka, K. Hanaoka, M. Oshimura, I. Ishida

751

Transgenic technology - A challenge for mammalian cell culture production systems R.G. Werner

757

General Workshop-Discussion

765

List of trade-fair-sponsors

773

Index

775

INTRODUCTION

New Developments and New Applications in Animal Cell Technology The 15th Meeting of the European Society for Animal Cell Technology (ESACT), entitled « Animal Cell Technology. New Developments - New Applications », was organized in Tours/France at the beginning of September 1997. The objective was to give an overview of the actual and future developments and on new applications in the classical fields covered by ESACT as well as in adjacent areas. Products, like vaccines and recombinant proteins are standard products of aanimal cell technology, and have been covered by ESACT-Meetings for many years. The actual trend in the use of animal cell technology is going towards new domains which have not been covered previously or sufficiently by ESACT, and of which ESACT should be more aware. This is because it can provide a lot of technological expertise: for instance, the development, production, and use of viral vectors for use in gene therapy is comparatively new and has a bright future, or the use of cell culture technology for the development of artificial organs which is steadily becoming more important, as specially as established technologies for entrapment and immobilisation of animal cells are of utmost importance in this field. ESACT is a Society of applied scientists and engineers. It is therefore very important for members to get a deeper insight into the actual developments in fundamental research, because in the near or further future they have to deal with the results of this research by, for instance, developing production, cultivation, or purification methods. In order to fulfil these requirements (getting a view on these subjects and also getting overviews on matters of general interest for applied biotechnologists), several keynote lectures were organized during the 15th ESACT-Meeting. It was possible to get five keynote speakers and the speaker of the opening session (F. Horaud, Inst. Pasteur, Paris/F) all of whom participating actively at this meeting. These speakers gave excellent talks on diverse subjects which included DNA methylation and its role in the genetic manipulation of the mammalian genome (R. Jaenisch, Whitehead Inst., Cambridge, MA); the use of human cells as therapeutic agents (H. Green, Harvard Medical School, Boston, MA), the engineering of glycosylation patterns in mammalian cells (J. Bailey, ETH Zürich, CH), and how genomics, target identification and structure based design could provide a complementary rather than competitive approach to animal cells for human therapy (T. Blundell, University of Cambridge, U.K.). Finally a more global overview on the reshaping of medicine through molecular biology was given by T. Bartfai (Hoffmann La Roche, Basel/CH). During the 15th ESACT-Meeting eight scientific sessions and one workshop were organized : 1. New technologies for health care products, 2. Use of animal cells for in vitro testing. xxiii

xxiv

3. Viral vector production for gene therapy,

4. Biosynthesis and post-translational modifications of recombinant proteins, 5. Developments for immunologicals and vaccines, 6. New cell lines: from transformed to differentiated cells/cell functions, 7. Integrated bioprocessing in animal cell technology, 8. Cell physiology and metabolic engineering of animal cells, and a workshop on: The use of animal cells versus the use of transgenic animals for the production of recombinant proteins. The scientific presentation of these Proceedings is in a slightly different fromate to that of the 15th ESACT-Meeting, nevertheless they reflect very closely the scientific contents

of the meeting. There are two main reasons for the modified presentation: 1. Scientifically, it was preferable to merge sessions 4 and 8 in order to get a more global presentation of the biosynthesis of recombinant proteins, cell physiology and metabolism (including metabolic engineering). 2. There is no chapter with respect to session 6, because only two manuscripts have been collected which were finally attributed to sessions 1 (paper by Jennings & Strauss) or 2 (paper by Wobus et al.). In order to get a better structure of this volume, the chapters corresponding to each session are introduced by a short overview on the contents.

It is obvious that these Proceedings will be most valuable to those who are actively involved in the field of animal cell technology. The chapters on Biosynthesis and posttranslational modifications of recombinant proteins and Cell physiology and metabolic engineering of animal cells and on Integrated bioprocessing in animal cell technology », representing the key-domaines of ESACT, count for about 60% of all presentations. Classical fields, like reactor engineering, development and application of new devices for high cell density rector systems, monitoring, and optimisation are still of large interest. However, the emphasis in optimisation of the product has changed from reactor engineering to cell engineering, for instance, for modifying glycosylation patterns or for reducing apoptosis in reactor cultures (= prolongation of the life time of the active biomass) becomes of growing interest. There are many highlights of general interest scattered through the other sessions; among others to be mentioned, several papers in the session on Viral vector production for gene therapy on the biology and the potential use of adeno associated virus (AAV) in gene therapy, the development of new packaging cell lines for the production of viral

vectors, and the application of gene therapy for the treatment of muscular diseases; papers presenting new approaches in vaccine development (e.g. new adjuvants and new viral vectors) in the session on (« Developments for immunologicals and vaccines ».) A topic in an adjacent field of ESACT but of general interest, is the use of high cell

density (tissue-like) for the development of artificial organs. Originally, techniques developed for hollow-fiber and general immobilisation systems were the base for developments in the field of artificial organs. Today, general animal cell technology profits by developments in this field, like techniques for determining the distribution of biomass in the extracapillary space of high density hollow fiber systems.

xxv

As already mentioned, due to the keen interest a workshop on The use of animal cells versus the use of transgenic animals for the production of recombinant proteins was organized. The production of recombinant proteins in transgenic animals might become a very important concurrence for animal cell technology. The aim of this workshop was to give a comparison between both technologies, to present the advantages and disadvantages of both technologies, followed by a general discussion. As a conclusion, it

seems that production in animals is more economical, however, the system is less defined and this may hamper the registration of the product. Furthermore, the production of pharmaceuticals such as insulin could leak into the blood and consequently impair the health of the animal, which is, of course, never a problem in animal cell culture. In conclusion, as for previous ESACT-Meetings, the whole animal cell production process was considered - from the initial genetic studies of the cells through the stages of production to the regulatory issues surrounding product release. The holistic approach to animal cell technology which is the base of each ESACT-Meeting is the main reason for the continual increasing interest in these meetings.

O.-W. Merten, P. Perrin, B. Griffiths Editors

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Hyclone Lecture GENETIC MANIPULATION OF THE MAMMALIAN GENOME R. JAENISCH Whitehead Institute for Biomedical Research and Department of Biology M.I.T. Cambridge, MA 02142, USA Abstract: Over the last decade methods have been developed that allow us to precisely alter genetically the germ line of mice, permitting the generation of mouse models for human diseases. In this talk I will first review the technology of gene targeting. I will then illustrate the application of this technology for studying the role of DNA methylation in embryonic development and genomic imprinting. DNA methylation is the most important epigenetic change because DNA methylation is an epigenetic modification : that is, it is altered during normal development as well as during various diseases such as cancer. The implications of DNA methylation for biomedical research will be discussed. Discussion Danos:

The other drugs that have been used for re-activation of gamma globulins, like hydroxyurea (HU), reduce methylation. Can they be used in a cancer setting as well?

Jaenisch:

I believe HU gets cells into cycling and that is how you may get different types of cells; maybe foetal cells which express gamma globulin genes. I am not sure what the mechanism is, but there is no real evidence that HU leads to direct de-methylation of those genes. I should add that HU only works for sickle cell anaemias and not for telosaemias.

Faff:

Is there an enzymatic assay for methyl transferase?

Jaenisch:

Yes, there are very good in vitro assays. You take the purified enzyme, or an extract, label the methyl group and then measure the transfer of methyl to a substrate.

Singhvi:

Are you working with any pharmaceutical company on the development of drugs for de-methylation?

Jaenisch:

Yes, I am very keen on that.

Sasaki:

Would it be possible to summarise what you know about the enzyme responsible for de novo synthesis? 1

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 1-3. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

2 Jaenisch:

When I say this is a maintenance methylase, it not that strong a statement. The enzyme in vitro prefers a methylated substrate but it also accepts an un-methylated substrate although this is less efficient. So in vitro it has de novo methylase activity. In vivo we know that by using retroviruses to introduce virus into ES cells, they can become de novo methylated very efficiently. One reason that gene therapy trials of stem cells offers nothing is that the virus gets turned off and methylation plays a role here. This is one of the major complications of trying to infect stem cells with retroviruses. When you try to differentiate ES cells there is no de novo methylation activity, so I believe that the enzyme is turned off. It does not exclude the possibility that the maintenance methylase has a low efficiency of de novo methylase activity in vivo. However, we know that there is a separate enzyme which is very efficient in

de novo methylase activity;

we have the following genetic

information. When we take methyl transferase negative cells and infect them with retrovirus, the retrovirus gets de novo methylated with the same kinetics as a wild type cell. However, it never reaches the same level. This argues that the maintenance methylase

activity has been eliminated - there must be a totally independent encoded activity which does that. Biochemically we do not see that

activity, so it is either unstable or we do not have the right assay. Sasaki:

Has any attempt been successful to isolate the genes using homology?

Jaenisch:

Homology studies have been totally unsuccessful. This is surprising

because this enzyme has a highly conserved domain and is very similar in both prokaryotic and mammalian enzymes so antibodies recognise one or the other. There has been no success in finding another enzyme by sequence searches which is convincingly the de novo methyl transferase.

Al-Rubeai:

Is it possible that the drug 5 aza DC influences methylations by affecting the methyl transferase during the cell cycle by preventing cell division? The cells would be blocked in the Gl, thus allowing

another gene to commit the cell to death. So methyl transferase would not be an oncogene itself but a cell cycle gene and, therefore, demethylation will allow the cell to be blocked and committed to

death. Jaenisch:

So this would be a toxic effect. These mice are totally normal so the heterozygous mice age normally. They are protected against tumours by a factor of 3. So there is no difference in the cell cycle of heterozygous mice. We are not at the stage of de-methylation where apoptosis occurs. Apoptosis does occur when you differentiate mutant ES cells, or in the embryos which do not have this enzyme and die. The reason why they die is not known. We

3

believe that they lose the X chromosome gene expression and this would be sufficient to explain the death but is probably not the whole answer. We do not really know what the role of methylation is in development. We know it is important for gene expression and we have evidence for its importance in gene stability. It might be important for repair processes. In prokaryotes, methylation is crucial to mark the parental from daughter strain so you know whether replication errors occur. There is no evidence for this role in mammals but it is probable. Methyl transferase is not an oncogene in the conventional usage but I think it is an oncogenic determinate. Bernard:

Can you make a partially inactive mutant of methyl transferase in vitro?

Jaenisch:

We have partially inactivated mutants in mice, so we can adjust the enzyme levels genetically down to any level (2%, 5%, 10%). In vitro the truncated enzyme has activity. In answer to your question, I am sure you can but I am not sure why one would want to do it.

Aunins:

Can you speculate on whether methylation has a role in productivity of recombinant cell lines in vitro?

Jaenisch:

The old evidence is that a primary cell, which produces all sorts of proteins when put in tissue culture, very soon shows de novo methylation of CPG islands of genes which are tissue specific. So many genes turn off which are not needed in tissue culture. You turn off genes which inhibit cell growth, eg myo D is never methylated in vivo, only in culture. In tissue culture I think de novo methylation is a growth promoting process.

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ENGINEERING GLYCOSYLATION IN ANIMAL CELLS

J. E. Bailey, E. Prati, J. Jean-Mairet, A. Sburlati, P. Umaña

Institute of Biotechnology, ETH Zürich, CH-8093 Zürich, Switzerland

1. Abstract

Glycosylation can have significant effects on activity, pharmacokinetics, targeting, immunogenicity, and stability of a glycoprotein. Therefore, glycosylation engineering of animal cells which express cloned glycoprotein products is an enabling technology for generating molecular and functional diversity of these products. This review considers potential targets in modifying oligosaccharides on particular glycoprotein pharmaceuticals, strategies available to guide genetic design of a modified oligosaccharide biosynthesis pathway which will achieve the desired end-product, the

current challenges and limitations, technologically and scientifically, in achieving industrially significant results, and progress being made to address these challenges. 2. Introduction

Glycoproteins mediate many essential functions in human beings, other eucaryotic organisms, and some procaryotes, including catalysis, signalling, cell-cell communication, and molecular recognition and association. The function of glycoproteins can be profoundly influenced by their oligosaccharide component(s). Unfortunately, few simple, general principles are available which unify understanding of oligosaccharide structure - glycoprotein relationships (here subsequently referred to more briefly as “structure-function relationships”). Accordingly, the scope of scientific and technological investigation in this field is vast and cannot be comprehensively summarized here.

There are two main results from prior work which are critical in this discussion. 1. The oligosaccharide structures which are assembled starting from particular initiation sites on the polypeptide backbone are functions of the polypeptide amino acid sequence, the host cell in which the glycoprotein is synthesized, and that cell’s environment. 2. With polypeptide sequence, host cell, and environment fixed, the glycoprotein molecules which are synthesized differ with respect to their oligosaccharide structures. 5 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 5-23. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

6

These related yet distinct molecular species are called “glycoforms.” Thus, reference to a

particular glycoprotein is, usually, shorthand for a population of glycoforms. A definition of “glycosylation engineering” is needed here in order to focus this presentation (Further, a definition which reasonably delimits the field under discussion is essential to give that definition any functional utility). Here glycosylation engineering is considered as a subset of metabolic engineering, defined as “the improvement of cellular activitites by manipulation of enzymatic, transport, and regulatory functions of the cell with the use of recombinant DNA technology” [1]. That review included this perspective on glycosylation engineering, presenting several examples in which genetic engineering of the oligosaccharide synthesis pathway achieved altered glycosylation of glycoproteins expressed in mammalian cell culture. A later review took a broader view of glycosylation engineering, including effects of mutations and cell environment on glycosylation [2]. Although environmental effects belong to the field of bioprocess engineering in the current lexicon of our field, some highlights from such bioprocess research are also included here. Another specification is required here: what kinds of glycoproteins will be discussed, and

in what context? Here we focus on cloned, heterologous, secreted glycoproteins produced in animal cell culture, insect cell culture, and, to a lesser degree, in transgenic animals. These glycoproteins, some of which are listed in Table 1, are the backbone of the biotechnology industry which has emerged in the last two decades and which has redefined the scope and significance of animal cell technology in the same period. Of course glycosylation of viruses has major technological relevance, and glycosylation must be critically important, in ways still to be elucidated, in tissue engineering, but these topics must await a future presentation. Further information on the science of glycobiology, as well as introductions to basic concepts in the field, may be found in a number of excellent recent reviews [3,4,5,6,7],

The secreted proteins of interest here are used in many applications as injectable therapeutic agents. This then delimits the set of glycoprotein functional characteristics of greatest importance. Some of the available information on these is summarized next,

7

concentrating specifically on particular glycoproteins which are current or pending products of the biotechnology industry.

3. Oligosaccharide Structure-Glycoprotein Function Relationships

The advent of biotechnology enabled synthesis of human polypeptides in heterologous cells. This new combination of polypeptide and host gave rise to glycoproteins which had not previously existed, but which were under development as potential therapeutic agents. This motivated extensive development of more sophisticated methods for

characterization of glycosylation, and also research to explore the implications of glycosylation on the therapeutic properties of glycoproteins. As summarized in Table 2, this research has revealed that, in particular cases, glycosylation can influence the physical properties, immunological interactions, biological activity, and pharmacokinetics of particular glycoproteins. Unfortunately, it can be very misleading to generalize from an example for a specific glycoprotein to other glycoproteins. Oligosaccharides have different roles on different glycoproteins with different functions. In fact, one group of biochemists believes that oligosaccharides usually have only generalized, physicochemical effects, while another emphasizes evidence of specific biological functions of oligosaccharides. Evidence for both viewpoints exists, prompting the cogent title of one of the foremost reviews of the

subject; “Biological roles of oligosaccharides: all theories are correct” [4].

8

There are a few points which admit generalization: absence of sialic (neuraminic) acid on the termini of complex oligosaccharides results in more rapid clearance, as does the presence of high mannose oligosaccharides. These implications of glycosylation have motivated choice of host cell lines for production of biologicals (to avoid high mannose structures), and also great emphasis on sialylation in process and product development and in product characterization.

4. Why do Glycosylation Engineering?

Glycosylation engineering offers a way to generate glycoproteins which are novel in the sense that they carry different oligosaccharides compared to previously available material. In view of the sensitivity of function of many early products of the biotechnology industry to glycosylation, glycosylation engineering is an obvious strategy for generating “second generation” products. A prominent example is “novel erythropoiesis-stimulating protein” (NESP), moving very rapidly now through clinical trials. This product is a preparation of one or several high-activity glycoforms of EPO [I8]. While such a material can be produced by fractionation of a first-generation product, glycosylation engineering offers an alternative route to obtain novel glycoforms, including those not available from the original production cell line.

Glycosylation engineering provides a means to optimize a new product with respect to function, and also with respect to business position. Novel glycoforms are a possible basis for patent protection of a product. For example, U.S. Patent 5,547,933 [19] claims “A non-naturally occurring EPO glycoprotein product... (with) a higher molecular weight than human urinary EPO..”. Here no glycosylation engineering was involved - glycosylation different from that of human urinary EPO arises simply because the EPO referred to is made in recombinant CHO cells. Nonetheless, by choice of a different host, modification of the host by glycosylation engineering, or by innovative purification strategies, clear opportunities exist for creating new materials which can be patented.

Glycosylation engineering may also be employed to facilitate purification (e.g., by installing a residue or linkage recognized by a particular lectin), to simplify crystallization (in the limiting case, by modifying the amino acid sequence so that it is not glycosylated), or to reduce glycoform heterogeneity in a glycoprotein preparation [2,20].

With these practical benefits as motivations, we next review the factors which influence glycosylation of a polypeptide, which leads us to questions of how glycosylation can differ among glycoforms, or among different glycosylated polypeptides. Each of these types of characteristics, and these different ways of influencing them, provide an avenue for glycosylation engineering to play a role, and for differentiating the glycoprotein produced from presently available preparations.

9 5. Decision Points in Oligosaccharide Biosynthesis

Glycosylation occurs at particular amino acid residues on a polypeptide - known as glycosylation sites - as the polypeptide moves from the endoplasmic reticulum through the Golgi apparatus. However, the oligosaccharide structure attached to a glycosylation site on a given polypeptide can vary over a large set of chemical and structural diversity - beginning with the possibility that the site may have an oligosaccharide attached in some glycoforms but have no oligosaccharide there in other glycoforms (site occupancy). The polypeptide itself, the host cell, and that cell’s environment all can influence substantially the distribution of glycoforms produced. The situation is extremely complicated, making it scientifically and commercially rich in unresolved questions and opportunities. Unfortunately, there is no way at present to predict what types of glycoforms will be produced when an arbitrary polypeptide is expressed in a given host under given conditions (except in the trivial case in which, due to the polypeptide sequence or the host, no glycosylation occurs). Experience is accumulating to allow reasonable hypotheses in some cases about how a change from a given situation will change the glycoform distribution, but, even in this more limited perspective, many uncertainties remain. A summary of many of the ways in which glycoforms can differ, with corresponding examples taken frequently from the set of products listed in Table 1, is provided in Table

3. In some cases, the basic mechanism which determines a particular “decision” in oligosaccharide biosynthesis can be identified - for example cells will not produce bisected glycoforms or glycoforms containing sialic acid in to nonreducing end-galactose residues (found in humans), if they lack, respectively, or Different polypeptides expressed in similar hosts under similar conditions can be glycosylated much differently, clearly indicating that the polypeptide itself exerts a controlling role for its own glycosylation [3,10,23,24]. A particularly interesting and technologically relevant example, which also illustrates this connection at the level of individual amino acid substitutions, is the development of TNK-tPA [25]. A single mutation, T103N, introduced a new glycosylation site carrying complex-type

oligosaccharides, and at the same time changed glycosylation at native site N l 1 7 from a high mannose to a complex pattern. This mutant had a 10-fold lower plasma clearance than wild-type tPA, but only one third of the fibrin binding activity of the wild-type glycoprotein. This activity could be restored to wild-type level by removing the native glycosylation site at position N117. The net effect in the resulting double mutant T103N, N117Q tPA is to shift one site from position 117 to 103 and change

glycosylation pattern from high-mannose to complex type.

the

10

6. Tools and Strategies for Manipulating Glycosylation

6.1. CHOICE OF HOST It is the capability of animal cells to accomplish glycosylation in a manner compatible with human application which has created a special niche for animal cell technology in the biotechnology industry. Commonly used bacterial hosts do not glycosylate their proteins, and yeast glycosylate with high mannose oligosaccharide structures which are not suitable for injection into humans. Similarly, baculovirus expression systems, as well as plants, also synthesize oligosaccharides on glycoproteins which are undesirable for pharmaceutical applications. Even within the sphere of cultured animal cells and whole animals, different cell lines and different animals produce different glycoforms following translation of an identical

polypeptide. A few examples of such phenomena are listed in Table 4. extensive list of examples can be found in Reference 11 .

A more

11

6.2. MUTANTS AND METABOLIC ENGINEERING There are two different classes of genetic manipulation which can influence oligosaccharide biosynthesis. The first is modification of expression level of enzymes and related proteins involved in oligosaccharide biosynthesis which are encoded in the genome of the host cell. There are in turn two alternative technologies for obtaining host cells with such altered expression levels of its homologous proteins. One, which has played a critical role in the development of glycobiology, is screening for mutant hosts which exhibit altered glycosylation. These are typically identified by detection of

the corresponding modification in oligosaccharides displayed on the outer cell surface. Further information on such glycosylation mutants and their application in glycobiology can be found in a recent review [2]. It is reasonable to expect that, at least for some cloned, secreted heterologous glycoproteins, use of glycosylation mutants will result in an alteration of product protein glycosylation. However, to date there are no publications confirming this possibility. A second approach for modifying expression of host cell proteins involved in oligosaccharide biosynthesis involves several different methods based on recombinant DNA technology. By gene activation or by installing additional copies of chosen genes with appropriate transcriptional controllers, particular enzymes can be amplified. Conversely, antisense and other methods can be employed in order to down-regulate expression of targeted host cell genes.

In many potential applications of glycosylation engineering, the oligosaccharide biosynthesis capabilities of the host are extended by introduction into that host of one or more genes encoding heterologous oligosaccharide biosynthesis enzymes, or possibly other proteins including precursor transporters, into a heterologous host. This can be achieved by recombinant DNA methods but not by mutation. A limited number of experiments employing this approach have so far been published. These involve a relatively smallset of heterologous enzymes, in part because the working pool of cloned enzymes for this type of glycosylation engineering, at least in the public domain, is still small. However, these experiments have so far produced positive and encouraging results, in the sense that installation of a heterologous enzyme resulted in the

12

corresponding modification in glycosylation for at least a detectable quantity of a particular glycoprotein which has been analyzed. Table 5 summarizes all such publications known to the authors as of the submission date of this manuscript.

It is by no means guaranteed that genetic manipulation of the activity of a glycosylation enzyme will result in a corresponding change or any change at all in the glycosylation

of particular glycoproteins. Failures in this respect are rarely documented, but there is

one published example. Overexpression of cloned in F9 carcinoma cells (which already express this enzyme) gave no detectable change in the glycosylation of endogenous lysosomal membrane protein 1 (LAMP-1), although the intracellular level of the enzyme was significantly increased [47]. Glycosylation engineering has also been achieved in a transgenic animal [48J. In this case, human was trangenically expressed in the mammary gland of mice. Milk samples from transgenic animals contained soluble, active forms of the

large quantities of a free oligosaccharide product of this

enzyme (2'-fucosyllactose), as well as modified glycoproteins. In contrast, milk from control animals lacked these glycoconjugates. Glycosylation engineering to modify glycoforms of a cloned glycoprotein product can be expected also to influence glycosylation of some set of host cell glycoproteins [49,50], potentially influencing their function. This could disturb growth, productivity, development [51], or viability of the host, complicating achievement of the desired product glycosylation. Engineering global changes in glycosylation capabilities of a cell, such as modifying a yeast to glycosylate like a human ce11, is extremely difficult for this and another reason: oligosaccharide synthesis depends not only on the enzymes directly involved but on synthesis and transport capabilities for nucleoside sugar cosubstrates and their soluble reaction products, requiring that any missing functions in this entire complex system must also be installed to enable the new biosynthetic activity.

As presently understood, oligosaccharide biosynthesis achieves the observed end result by sophisticated organization and segregation of various enzymes, and the reactions they

13

catalyze, into different organelles. Therefore, achieving synthesis of human oligosaccharides in a procaryotic cell seems unattainable, whatever new genes and functions are introduced by metabolic engineering. However, the potential sensitivity of glycoform distribution to synthesis enzyme localization suggests a new avenue for manipulating product glycosylation: The enzyme genes can be engineered to alter targeting of enzymes within the endoplasmic reticulum and Golgi apparatus. Calculated results indicating the conceptual feasibility of this have recently been presented [52], and amino acid sequences implicated in targeting proteins among these compartments are known [53,54], providing essential molecular reagents to undertake such “localization engineering” (!).

6.3. TECHNOLOGIES FOR EFFECTIVE GLYCOSYLATION ENGINEERING Success in metabolic engineering depends critically upon availability of a powerful set of design and implementation tools in order to define potentially effective strategies and in order to execute them. One area of genetic technology development which is potentially very important but still relatively undeveloped is antisense methodology in order to reduce expression of chosen enzymes. Experience with antisense technology and

other applications has shown that it has a greater chance of success if full length antisense messages are used, although even in this case the extent to which expression of the targeted protein will be reduced is unpredictable. Antisense technology is only

now beginning to enter the arena of animal cell technology, as presented elsewhere in this volume. 6.3.1. Antisense Reduction in Cell-Cell-Adhesion for CHO Cells No prior reports have described successful modification of the oligosaccharides synthesized by Chinese hamster ovary (CHO) cells by antisense methods. Here such a

study is summarized. A CHO mammalian cell line that stably expresses FucTVI activity and the cell-surface was previously engineered. This cell line was transfected with an antisense-Fuc-TVI full length gene. The FucTVI activity was measured in three antisense stable cell clones (anti-Fuc-TVI clones) and compared to the and

wild type CHODG44 cell lines (Table 6).

14

The three antisense clones showed different levels of reduced Fuc-TVI activity. The expression level of in the different clones was therefore investigated (Table 7). An monoclonal antibody (CSLEX-1) coupled to IgM-FITC was used for the expression assay.

The

expression in the three antisense clones was lower when compared with the cell line, and null for the wild type CHODG44. As reduced expression should result in by a lower binding capacity to, for example E-selectin, a simple adhesion assay of (transfected and untransfected) cell lines to HUVEC cells (both in the presence and absence of human recombinant which is needed to induce the expression of E-selectin) was performed. The results are shown in Table 8.

15

Anti-Fuc-TVI clones showed the expected reduced E-selectin binding capacity. These results indicate that is definitely possible to regulate the expression of the epitope by antisense technology which specifically targets the fucosyltransferase activity responsible for its synthesis. This antisense technique can be applied to other glycosyltranferases to regulate different points of the glycosylation pathway. Antisense technology of CHO cells, alone or associated with other glycosylation engineering methods, may bring new possibilities for the production of novel glycoproteins.

6.3.2. Mathematical Models of Complex Glycosylation Pathways The network of enzyme-catalyzed reactions which conduct oligosaccharide biosynthesis in animal cells is complicated by two types of general phenomena. First, it is common for a single species or intermediate in this pathway to serve as a substrate for several different enzymes, giving raise to correspondingly different further intermediates or oligosaccharide end-products. Second, certain oligosaccharide biosynthesis enzymes act on large numbers of different biosynthetic intermediates. These two features render intuitive or mental simulation of oligosaccharide biosynthesis very difficult. Therefore, mathematical models which provide a systematic representation of all of the simultaneous and competitive phenomena involved and which can suggest expected consequences from the operation of this network, or, more importantly, from perturbations in it, are indispensable for glycosylation design in the future. A first model for N-linked oligosaccharide biosynthesis has been presented recently, and its results show the types of non-obvious qualitative insight which can be obtained by this

approach [52]. The first step of the pathway has also been recently modelled [26]. This mathematical model incorporates different factors that determine the extent of the oligosaccharyl-transfer reaction and provides useful insights for the engineering of this step.

6.3.3. Vectors for Regulated Multicistronic Expression More sophisticated glycosylation engineering in the future will require coordinated expression of several sense and/or antisense messages, perhaps at a particular time in a production process. Recently, convenient multicistronic cloning and expression vectors for animal cells have been described which can be used in these applications [55].

7. Bioprocess effects on glycosylation

Although it is not the main topic of this review, a few highlights from previous research exploring interactions between the process environment of a recombinant animal cell expression system and glycosylation of product proteins may be of interest. Also, genetic engineering may play a future role in modifying these host cell responses to environmental changes in a way that makes product glycosylation less sensitive to these changes or which enhances the consistency and quality of the final recovered, purified product.

16

Bioprocess effects on glycosylation can be divided into two categories. First, certain conditions in the bioreactor influence activities within the cell, changing glycosylation of proteins secreted by those cells. Second, conditions in the bioreactor, and also in downstream processing, may influence the nature of the end product, including its glycosylation. It should be noted that the possibility of oligosaccharide modification after glycoprotein secretion from the cells has not always been considered explicitly in previous bioreactor studies, so that a rigorous assignment of the phenomena involved as intracellular or extracellular cannot be made in several cases. 7.1. EFFECTS ON GLYCOPROTEIN BIOSYNTHESIS Changes in culture conditions such as and ammonia concentration can cause shifts in the glycoform distribution produced. Several examples which illustrate such phenomena are given in the Table 9. Mechanisms responsible for coupling between external conditions and the internal, compartmentalized, enzyme-catalyzed oligosaccharide biosynthesis reactions which determine glycosylation are not defined, but some reasonable possibilities have been suggested in some situations. For example, because it is a weak base and its unionized form is freely diffusible across cellular membranes, ammonium in the external medium causes changes in intracellular of hybridoma cells [56,57,58]. This is consistent with, for example, associated changes in sialylation of O-linked glycosylation of GCSF, via the relationship of the enzyme Olinked

17

Another bioprocess-glycosylation connection has been elucidated in the elegant study of Jenkins, James, and coworkers on glycoform distributions of cloned human produced by recombinant CHO cells [65]. Glycosylation of shifted significantly as a function time of sampling during a batch cultivation. In terms of

product quality control and process consistency, this work raises profound questions and challenges for the bioprocess engineer. Such questions could be even more acute for long-term cultivations with extended times of product harvest, such as fed-batch and perfusion culture. 7.2. POST-SYNTHESIS EFFECTS

Glycoproteins exit the cell and enter the culture medium where they are exposed to other proteins secreted by the cells, proteins released from lysed cells, and probably serum proteins as well (although medium is changed between cell growth and production phases in several manufacturing processes, this does not completely eliminate serum proteins, as anyone who has tried to wash away serum proteins by extensive treatment of the cells in the laboratory before doing SDS-PAGE analysis knows). Neuraminidase, an enzyme which cleaves sialic acid residues on oligosaccharides, has been detected at potentially significant levels in recombinant CHO cell cultures, and has

been shown to reduce sialylation of secreted glycoprotein products [66]. Activities of several oligosaccharide hydrolases have been detected in insect cell (Sf9) cultures [67]. These findings invite genetic manipulation to reduce expression of these product-degrading enzymes [68].

8. In Vitro Biosynthesis

Oligosaccharide

Fractionation,

Remodeling

and

Obviously the processing pathway to the packaged product does not end with the bioreactor. Different types of technology can be applied downstream to modify glycoforms in the final product. Simplest among these are separation methods which fractionate the product into different glycoforms, allowing selective environment of

preferred molecules. Glycoforms can be fractionated to some extent on the basis of charge or hydrophobicity, while highly specific separations can be achieved using various lectins (e.g., immobilized on a chromatography matrix) which offer exquisite selectivities for a wide variety of particular glycosidic linkages [69]. Many enzymes are known, and some commercially available, which synthesize or which hydrolyze particular glycosidic bonds. These are important tools in current technologies for oligosaccharide analysis, and they are also potentially useful to accomplish in vitro modifications of glycoproteins. In the extreme case many or all of the oligosaccharides on the glycoprotein could be synthesized in vitro. One major barrier to this concept is

18

cost of the enzymes, and especially of the nucleoside sugar donor substrates required for glycosidic bond synthesis. For decades organic chemists have dreamed of replacing microorganisms for synthesis of natural products such as cephalosporin. This remains a dream, but chemistry - including

enzyme catalysis - is a vital contributor in modifying natural products to obtain an optimized pharmaceutical. It seems likely that a similar scenario will unfold concerning glycosylation. 9. Concluding Remarks

By modifying oligosaccharide synthesis processes in the production host, glycosylation engineering interacts with process and polypeptide influences to determine product glycosylation which in turn can influence many practically important functional characteristics of that glycoprotein (Figure 1). The future potential of this technology to contribute to new and improved pharmaceuticals is great. Progress in the field now is limited by lack of a greater number of cloned genes for enzymes and other proteins

involved

in

oligosaccharide

biosynthesis,

by

insufficient

knowledge

about

oligosaccharide structure-glycoprotein function relationships, and by underdeveloped tools for genetic design and manipulation of glycosylation. However, motivated by the scientific and commercial importance of this field, rapid progress in all of these areas is evident, expanding future horizons.

19 10. Acknowledgements The authors’ research in glycosylation is supported by the Swiss Priority Program in Biotechnology (SPP BioTech).

11. References 1. Bailey, J. E. 1991. Toward a science of metabolic engineering. Science 252: 1668-1675. 2. Stanley, P. 1992. Glycosylation engineering. Glycobiology 2: 99-107. 3. Gumming, D. A. 1991. Glycosylation of recombinant protein therapeutics: control and functional

implications. Glycobiology 1: 115-130. 4. Varki, A. 1993. Biological roles of oligosaccharides: all theories are correct. Glycobiology 3: 97-130. 5. Rademacher, T. W., Parekh, R. B., Dwek, R. A. 1988. Glycobiology. Annu. Rev. Biochem. 57: 785-838. 6. Lis, H., Sharon, N. 1993. Protein glycosylation: Structural and functional aspects. Eur. J. Biochem. 218: 1–27.

7. Natsuka, S., Lowe, J. B. 1994. Enzymes involved in mammalian oligosaccharide biosynthesis. Curr. Opin. Struct. Biol. 4: 683-691. 8. Goochee, C. F., Gramer, M. J., Andersen, D. C, Bahr, J. B. 1992. The oligosaccharides of glycoproteins: Factors affecting their synthesis and their influence on glycoprotein properties, p. In: P. Todd, S. K. Sikdar and M. Bier (ed.), Frontiers in Bioprocessing II. American Chemical Society,

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Enzyme Microb. Technol. 16: 354-364. 10. Wyss, D. F., Wagner, G. 1996. The structural role of sugars in glycoproteins. Current Opinion in Biotechnology 7: 409–416. 11. Jenkins, N., Parekh, R. B., James, D. C. 1996. Getting the glycosylation right: implications for the biotechnology industry. Nature Biotechnology 14: 975-981. 12. Dwek, R. A. 1995. Glycobiology: more functions for oligosaccharides. Science 269: 1234-1235.

13. Koenig, A., Rakesh, J., Rakesh, V., Norgard-Sumnicht, K. E., Matta, K. L., Varki, A. 1997. Selectin inhibition: synthesis and evaluation of novel sialylated, sulphated and fucosylated oligosaccharides,

including the major capping of GlyCAM-1. Glycobiology 7: 79-93. 14. Graham, R. A., Burchell, J. M., Taylor-Papadimitriou, J. 1996. The polymorphic epithelial mucin: Potential as an immunogen for a cancer vaccine. Cancer Immun. Immunother. 42: 71-80.

15. Lloyd, K. O., Burchell, J., Kudryashov, V., Yin, B. W. T., Talor-Papadimitriou, J. 1996. Comparison of O-linked carbohydrate chains in MUC-1 mucin from normal breast epithelial cell lines and breast carcinoma cell lines. Demonstration of simpler and fewer glycan chains in tumor cells. J. Biol. Chem. 271: 33325-33334.

16. Malhotra, R., Wormald, M. R., Rudd, P. M., Fischer, P. B., Dwek, R. A., Sim, R. B. 1995. Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the mannose-binding protein. Nature Med. 1: 237-240. 17. Misiaizu, T., Matsuki, S., Strickland, T. W., Takeuchi, M., Kobata, A., Takasaki, S. 1995. Role of antennary structure of N- linked sugar chains in renal handling of recombinant human erythropoietin. Blood 86: 4097-4104. 18. Fürst, I. 1997. Amgen’s NESP heats up competiton in lucrative erythropoietin market. Nature Biotech. 15: 940.

20 19. Fu-Kuen, L. 1996. Production of erythropoietin. U.S. patent 5,547,933.

20. Davis, S.J., Puklavec, M.J., Ashford, D.A., Harlos, K., Jones, E.Y., Stuart, D.I., Williams, A.F. 1993. Expression of soluble recombinant glycoproteins with predefined glycosylation: applications to the

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22. Lee, E. U., Roth, J., Paulson, J. C. 1989. Alteration of terminal glycosylation sequences on N-linked oligosaccharides of Chinese hamster ovary cells by expression

sialyltransferase. J. Biol. Chem. 264: 13848-13855. 23. Shao, M. C., Wold, F. 1995. The effect of the protein matrix proximity on glycan reactivity in a glycoprotein model. Eur. J. Biochem. 228: 79-85. 24. Do, K. Y., Fregien, N., Pierce, M., Cummings, R. D. 1994. Modification of glycoproteins by Nacetylglucosaminyltransferase V is greatly influenced by accessibility of the enzyme to oligosaccharide acceptors. J. Biol. Chem. 269: 23456-23464.

25. Keyt, B. A., Paoni, N. F.. Refino, C. J., Berleau, L., Nguyen, H., Chow, A., Lai, J., Peña, L., Pater, C., Ogez, J., Etcheverry, T., Botstein, D., Bennett, W. F. 1994. A faster-acting and more potent form of issue plasminogen activator. Biochemistry 91: 3670-3674. 26. Shelikoff, M , Sinskey, A. J., Stephanopoulos, G. 1996. A modeling framework tor the study of protein

glycosylation. Biotechnology and Bioengineering 50: 73-90. 27. Kumar, V., Korza, G., Heinemann, F. S., Ozols, J. 1995. Human oligosaccharyltransferase: isolation,

characterization, and the complete amino acid sequence of 50-kDa subunit. Arch-Biochem-Biophys. 320: 217-223. 28. Schachter, H. 1986. Biosynthetic controls that determine the branching and microheterogeneity of protein- bound oligosaccharides. Biochem. Cell Biol. 64: 163-181. 29. Moremen, K. W., Trimble, R. B., Herscovics, A. 1994. Glycosidases of the asparagine-linked oligosaccharide processing pathway. Glycobiology 4: 113-125. 30. Kumar, R., Yang, J., Larsen, R. D., Stanley, P. 1990. Cloning and expression of Nacetylglucosaminyltransferase I, the medial Golgi transferase that initiates complex N-linked carbohydrate formation. Proc. Natl. Acad. Sci. USA 87: 9948-9952. 31. Tan, J., D'Agostaro, A. F., Bendiak, B., Reck, F., Sarkar, M., Squire, J. A., Leong, P., Schachter, H. 1995.The human UDP-N-acetylglucosamine:alpha-6-D-mannoside-beta-l,2-N-acetylglucosaminyltransferase II gene (MGAT2). Cloning of genomic DNA, localization to chromosome 14q2l, expression in insect cells and purification of the recombinant protein. Eur. J. Biochem. 231: 317-328.

32. Yoshiada, A., Minowa, M. T., Hara, T., Takamatsu, S., Oguri, S., Iguamatsu, A., Ikenaga, H., Takeuchi, M. 1997. Two novel isoforms of N-acetylglucosaminyltransferase IV. Glycoconjugate J. 14: S46. 33. Shoeribah, M., Perng, G. S., Adler, B., Weinstein, J., Basu, R., Cupples, R., Wen, D., Browne, J. K.,

P. Buckhaults, Fregien, N., Pierce, M. 1993. Isolation, characterization, and expression of a cDNA encoding N-acetylglucosaminyltransferase V. J. Biol. Chem. 268: 15381-15385.

34. Tsuji, S. 1996. Molecular cloning and functional analysis of sialyltransferases. J. Biochem. Tokyo 120: 1-13. 35. Takeuchi, M., Kobata. A. 1991. Structures and functional roles of the sugar chains of human erythropoietins. Glycobiology 1: 337-346. 36. Yan, S. B., Chao, Y. B., van Halbeek, H. 1993. Novel Asn-linked oligosaccharides terminating in are present in recombinant human Protein C expressed in human kidney 293 cells. Glycobiology 3: 597-608.

21 37. Demnan, J., Hayes, M., O'Day, C., Edumnds, T., Bartlett, C., Hirani, S., Ebert, K. M., Gordon, K., McPherson, J. M. 1991. Transgenic expression of a variant of human tissue-type plasminogen activator in goat milk: purification and charaterization of the recombinant enzyme. Biotechnology N.Y. 9: 839843. 38. James, D. C., Freedman, R. B., Hoare, M., Ogonah, O. W., Rooney, B. C., Larionov, O. A.,

Dobrovolsky, V. N., Lagutin, O. V., Jenkins, N. 1995. N-glycosylation of recombinant human produced in different animal expression systems. Bio/Technology 13: 592-596. 39. Sawada R, Lowe J B, Fukuda M. 1993. E-selectin-dependent adhesion efficiency of colonic carcinoma cells is increased by genetic manipulation of their cell surface lysosomal membrane glycoprotein-1 expression levels. J.

Biol. Chem. 268: 12675-12681.

40. Sako, D., Chang, X. J., Barone, K. M. et al. 1993. Expression cloning of functional glycoprotein ligand for Pselectin. Cell 75:1179-1186.

41. Bierhuizen, M. F. A., Maemura, K., Fukuda, M. 1994. Expression of a differentiation antigen and poly-Nacetyllactosaminyl O-glycans directed by a cloned core 2 Chem. 269:4473-4479.

J. Biol.

42. Grabenhorst, E., Hoffmann, A., Nimtz, M. et al. 1995. Construction of a stable BHK-21 cells coexpressing human secretory glycoproteins and human Gal(-l-4)GlcNAc 2,6-sialyltransferase. -2,6 Linked NeuAc is preferentially attached to the Gal(-l-4)GlcNAc(-l-2)Man(-l-3)-branch of diantennary oligosaccharides from

secreted recombinant-trace glycoprotein. Eur. J. Biochem. 232:718-725. 43. Minch, S. L., Kallio, P. T., Bailey, J. E. 1995. Tissue plasminogen activator coexpressed in Chinese hamster ovary cells with contains Biotechnol. Prog. 11: 348-351.

44. Li, F., Wilkins, P. P., Crawley, S., Weinstein, J. et al. 1996. Post-translational modifications of recombinant Pselectin glycoprotein ligand-l required for binding to P-selectin and E-selectin. J . Biol. Chem. 271:3255-3264.

45. Wagner, R., Liedtke, S., Kretzschmar, E. et al. 1996. Elongation of the N-glycans of fowl plague virus hemagglutinin expressed in Spodoptera frugiperda (Sf9) cells by coexpression of human

acetylglucosaminyltransferase I. Glycobiology 6:165-175. 46. Grabenhorst, E., Costa, J., Conradt, H. S. 1997. Construction of novel BHK-21 cell lines coexpressing Golgi resident or soluble forms of human and together with secretory glycoproteins. In: Carrondo M.J.T., Griffits B Moreira J.L.P., eds. Animal Cell

Technology: Kluwer Academic Publishers., The Netherlands: 481-487. 47. Youakim, A., Shur, B. D. 1993. Effects of overexpression of beta-1,4-galactosyltransferase on glycoprotein biosynthesis in F9 embryonal carcinoma cells. Glycobiology 3: 155-163. 48. Prieto, P. A., Mukerji. P., Kelder, B., Erney, R., Gonzalez, D., Yun, J. S., Smith, D. F., Moremen, K. W.,

Nardelli, C., Pierce, M., Li, Y., Chen, X., Wagner, T. E., Cummings, R. D., Kopchick, J. J. 1995. Remodeling of mouse milk glycoconjugates by transgenic expression of a human glycosyltransferase. J.

Biol. Chem. 270: 29515-29519.

49. Bailey, J. E., Umaña, P., Minch, S., Harrington, M., Page, M., Sburlati, A. 1997. Metabolic engineering of N-linked glycoform symthesis systems in Chinese hamster ovary (CHO) cells, in M.J.T. Carrondo, B. Griffiths and J.L.P. Moreira (eds.), Animal Cell Tecnology, Kluwer academic publishers, Dordrecht, pp. 489-494. 50. Minch, S. L. 1996. Engineering of Protein Glycosylation in Chinese Hamster Ovary Cells: Genetic Manipulations, Global Glycoprotein Analysis, and Studies of Environmental Influences. Ph. D. Thesis, California Institute of Technology, Pasadena, California. 51. Livingston, B. D., De Robertis, E. M., Paulson, J. C. 1990. Expression of sialyltransferase blocks synthesis of polysialic acid in Xenopus embryos. Glycobiology 1: 39-44 52. Umaña, P., Bailey, J. E. 1997. A mathematical model of N-linked glycoform biosynthesis. Biotechnol. Bioeng. 55: 890-908. 53. Nilsson, T., Rabouille, C., Hui, N., Watson, R., Warren, G. 1996. The role of the membrane-spanning domain and stalk region of N-acetylglucosaminyltransferase I in retention, kin recognition and structural

22 maintenance of the Golgi apparatus in HeLa cells. J. Cell Biol. 109: 1975-1989. 54. Russo, R. N., Shaper, N. L., Taatjes, D. J., Shaper, J. H. 1992. Beta-l, 4-Galactosyltransferase: A short

NH-2-terminal fragment that includes the cytoplasmic and transmembrane domain is sufficient for Golgi retention. Journal Of Biological Chemistry 267: 9241-9247.

55. Fusseneger, M., Mazur, X., Bailey, J. E. 1997. A novel cytostatic process enhances the productivity of Chinese hamster ovary cells. Biotechnol and Bioeng. 55: 927-939. 56. McQueen, A., Bailey, J. E. 1990. Effect of ammonium ion and extracellular metabolism and antibody production. Biotechnol. Bioeng. 35: 1067-1077.

on hybridoma cell \

57. McQueen, A., Bailey, J. E. 1990. Mathematical modelling of the effects of ammonium ion on the intracellular pH of hybridoma cells. Biotechnol. Bioeng. 35: 897-906.

58. McQueen, A., Bailey, J. E. 1991. Growth inhibition of hybridoma cells by ammonium ion: correlation with effects on intracellular pH. Bioprocess Eng. 6: 49-61. 59. Andersen, D. C, Goochee, C. F. 1995. The effect of ammonia on the O-linked glycosylation of granulocyte colony-stimulating factor produced by Chinese hamster ovary cells. Biotechnol. Bioeng. 13: 98-105.

60. Hayter, P. M., Curling, E. M., Baines, A. J., Jenkins, N., Salmon, I., Strange, P. G., Tong, J. M., Bull, A.

T. 1992. Glucose-limited chemostat culture of Chinese hamster ovary cells producing recombinant interferon-g. Biotechnol. Bioeng. 39: 327.

61. Tachinaba, H., Taniguchi, K., Ushio, Y. et al. 1994 Changes of monosaccharide availability of human hybridoma lead to alteration of biological properties of human monoclonal antibodies. Cytotechnology

16:151-157.

62. Maiorella, B. L., Winkelhake, J., Young, J., Moyer, B., Bauer, R., Hora, M., Andya, J., Thomson, J., Patel, T., Parekh, R. 1993. Effect of culture conditons on IgM antibody structure, pharmacokinetics and

activity. Bio/Technology 11: 387-392. 63. Pels Rijcken, W. R., Overdijk, B., Van den Eijnden, D. H., et al. 1995. The effect of increasing nucleotide-sugar concentrations on the incorporation of sugars into glycoconjugates in rat hepatocytes. Biochem. J. 305: 865-870.

64. Borys M C, Linzer D H, Papoutsakis E T. 1993. Culture pH affects expression rates and glycosylation of

recombinant mouse placental lactogen proteins by Chinese hamster ovary (CHO) cells. Bio/Technology 11:720-724. 65. Hooker, A. D., Goldman, M. H., Markham, N. H., James, D. C., Ison, A. P., Bull, A. T., Strange, P. G., Salmon, I., Baines, A. J., Jenkins, N. 1995. N-glycans of recombinant human change during batch culture of Chinese hamster ovary cells. Biotechnol. Bioeng. 48: 639-648. 66. Gramer, M. J., Goochee, C. F., Chock, V. Y., Brousseau, D. T., Sliwkowski, M. B. 1995. Removal of

sialic acid from a glycoprotein in CHO cell culture supernatant by action of an extracellular CHO cell sialidase. Bio/Technology 13: 692-698. 67. Licari, P. J., Jarvis, D. L., Bailey, J. E. 1993. Insect cell hosts for baculovirus expression vectors contain endogenous exoglycosidase activity. Biotechnol. Prog. 9: 147-152. 68. Ferrari, J., Gunson, J., Lofgren, J., Nayak, N., Krumment, L., Sliwkowski, M., Warner, T. G. 1997. Constitutively expressed sialidase antisense RNA results in increased sialic acid on recombinant

glycoprotein expressed in Chinese hamster ovary cells. Glycoconjugate J. 14: S119. 69. Merkle, R. K., Cummings, R. D. 1987. Lectin affinity chromatography of glycopeptides. Methods Enzymol. 138: 232-259.

23

Discussion

Lupker:

An important thing about glycosylation is the in vivo function and, in this respect, are you aware of groups trying to make knock-out mice for some of the glycosylation enzymes?

Bailey:

Yes, but there was no possibility to cover this approach in this talk. This will show how host changes can be tolerated and we will probably find large sensitivities.

Lupker:

How many genetic diseases exist in humans that concern glycosylation, or are they all lethal?

Bailey:

I do not know the answer to this good question.

Bernard:

You mentioned that we have to be cautious when we produce targets in baculovirus insect cells. Do you have specific examples of proteins that we could not study in vitro function because of the specific glycosylation occurring in insect cells?

Baily:

No, this is another area where the structure/function data is absent. There are a few examples of the polypeptide structure depending upon which glycoform you have. We could question whether deglycosylated polypeptides used for X-ray structure analysis, and subsequent drug design, are the right configurations.

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SESSIONS ON : BIOSYNTHESIS AND POST-TRANSLATIONAL MODIFICATIONS OF RECOMBINANT PROTEINS, and CELL PHYSIOLOGY AND METABOLIC ENGINEERING OF ANIMAL CELLS

Over the last decade the expression of recombinant proteins in animal cells has evolved from a fine art reserved to specialists to almost a routine business. The focus has changed from the quantitative aspects of expression to the quality of the protein produced with a special emphasis on post-translational modifications such as glycosylation. In addition, considerable interest has now been invested in understanding, modifying and improving metabolic and synthetic pathways in animal cells. Despite the relative ease with which many recombinant proteins can be manufactured by

animal cells today, we should not forget that some proteins are extremely difficult to produce and that, for others production is far from optimal. These problems present the justification for the continuation of fundamental research on gene expression and transfection systems. Of equal importance, as discussed in the papers of the glycosylation chapter, is the effect of manipulating the environment on post-translational modifications in general, and on glycosylation in particular. Among other parameters, the carbohydrate sources and ammonium/glutamine concentrations have been shown to have significant effects. Considerable interest has now been invested in understanding and modifying (or redirecting) metabolic pathways in animal cell lines, when this cannot be achieved by cultural means. The section on metabolic engineering comprises papers dealing with the possibility for modifying the rapid glycolysis and glutaminolysis that generate high levels of waste metabolites by overexpressing the glutamine synthetase gene and thus eliminate the need for glutamine supplementation. Other matters of interest are the modification of glycosylation (novel glycoforms) of proteins by genetic engineering means. This can be achieved either by expressing new sugar transferases in cells, or by eliminating certain transferase activities by an antisense RNA-approach. Cell proliferation and apoptosis are two interlinked fundamental processes which are important for the industrial production of drugs. Therefore, these processes must be fully understood in order to be able to regulate them. On one side, cell growth should be reduced in order to increase the specific productivity. However, this can lead either to increased cell death or, in the case of induced growth reduction (IRF-1 fusion protein -

presence of estradiol leads to a growth reduction) to reduced productivity. By using genetic means, this negative effect can be avoided. The productive cell mass may be reduced by premature cell death (usually apoptotic) caused by culture stresses. Different means to reduce or avoid premature apoptosis were pre-sented, based for instance on the overexpression of bcl-2 or c-jun antisense genes, leading to a prolonged growth phase and often to an increased productivity. These papers indicate 25 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 25-26. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

26

that key cell-cycle control, and apoptosis genes and the proteins they encode provide targets for new approaches to improve large-scale production. Considering that the main reason for choosing an animal cell production system over cheaper and easier alternatives is for their ability to process proteins optimally for bioactivity as therapeutic or diagnostic agents, this area of research is very important. It should enable culture processes to become more efficient resulting in more economical and effective bioproducts. Thus the topics of both sessions on Biosynthesis and posttranslational modifications of recombinant proteins, and on Cell physiology and metabolic engineering of animal cells are very much interrelated, and represent key areas for the development of efficient and reproducible animal cell technology.

B. Griffiths, J. Lupker, M. Al-Rubeai, and J.E. Bailey Chairpersons

GENE EXPRESSION

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ENHANCED RECOMBINANT PROTEIN EXPRESSION IN INSECT CELLS IN THE PRESENCE OF DIMETHYLSULFOXIDE (DMSO)

LUCIANO RAMOS, JAMES F. KANE, & AMY A. MURNANE

SmithKline Beecham Pharmaceutical, Philadelphia PA, U.S.A.

1. Abstract The use of insect cells to express many recombinant homologous and heterologous proteins is reported throughout the scientific literature. A popular insect promoter is the metallothionein (Mtn) promoter which can be induced by the addition of heavy metals such as In this report we present data from Drosophila melanogaster Schneider 2 (S2) cells that have been stably transfected and express several different proteins under the control of the Mtn promoter. Selection was achieved using the hygromycin B-resistant gene derived from Escherichia coli. Our data show that such stably transfected insect cells expressing several different types of proteins, such as fusion

proteins, soluble receptors and antigens, have enhanced product expression when the cells are treated with 1% dmethylsulfoxide (DMSO) prior to induction with When DMSO is added at least 12 hours before the addition of 750 uM protein expression increased 5 to 8-fold. The addition of DMSO at other times, was not as effective in stimulating expression. Furthermore, DMSO did not affect expression from the D. melanogaster constitutive actin promoter.

2. Materials and Methods Cell lines: Three independent stable cell lines of D. melanogaster cells were selected following transfection with plasmid DNA encoding a fusion protein (S2-F), a soluble receptor (S2-R) or an antigen (S2-A) using procedures previously described (4,5). All of these genes were under the transcriptional control of the D. melanogaster Mtn promoter. Growth Medium: Growth medium is a proprietary SmithKline Beecham serum-free

insect media designated as SB insect media . Experimental conditions: Cells were propagated in 250 ml Bellco spinner flasks or Corning Erlenmeyer disposable 250 ml flasks. Cells were seeded in these flasks by means of dilution into SB insect media to attain a seeding density of about

in a volume of 100 ml per flask. Cultures were incubated at 24°C on a shaker base set at 120 RPM. Cell counts were performed using the ZM Coulter counter and

viability assessed using trypan blue dye exclusion. For production studies, cells were incubated for 3-5 days and induced at a cell density of and incubated for an additional 3-5 days. For experiments where DMSO was used, cultures were treated similarly except that DMSO was added to achieve the desired concentration used in our studies. 29 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 29-33. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

30

Product assay: Quantitation of product expressed by the various cell lines was performed using ELISA based assays

31

32

33

4. Conclusions We have examined three proteins stably expressed in D. melanogaster S2 cells under the transcriptional control of the Mtn promoter. In each case we observed that the addition of 1% DMSO 24 hours prior to induction with (see figs. 1-4) resulted in significant increases in productivity. In addition, there was no negative effect of 1% DMSO on cell viability or growth. While the mode of action of DMSO in D. melanogaster S2 cells is not understood, we propose that the effect is linked to stimulating the specific Mtn promoter used in these studies. This is based on the following observations. First, we have seen DMSO dependent increases in Mtn controlled mRNA. Second, DMSO did not affect expression from the constitutive D. melanogaster actin promoter. Third, DMSO did not effect recombinant protein expression in recombinant CHO containing constitutive promoters. Finally, Waalkes and Wilson observed that DMSO increased metallothionein expression in cultured liver cells (2). 5. References Reid S., and Greenfield P.F. 1992. Improved production of recombinant protein by the baculovirus expression system. In Animal Cell Technology:

Basic & Applied aspects, pp. 419-424. H. Murakami et al. (Eds), Kluwer Academic Publishers. and Wilson M. J. 1987. Increased synthetic capacity for metallothionein in cultured liver cells following dimethyl sulfoxide pretreatment. Experimental Cell Research 169:25-30.

and Swetly P. 1982. Interferon production in human hematopoietic cell lines: Response to chemicals and characterization of Interferon's. Journal of Interferon Research 2:261-270. A., Johansen H., Sweet, R., and Rosenberg, M. 1988. Efficient

expression of foreign genes in cultured Drosophila melanogaster cells using hygromycin B selection. Invertebrate and Fish Tissue Culture, pp. 131-134. Kuroda, Y., Kurstak, E., and Maramorosch K., eds., Springer-Verlag, Berlin. van der Straten, A., Oto, E., Maroni, G., and Rosenberg, M. 1989. Regulated expression at high copy number allows production of lethal oncogene product in Drosophila Schneider cells. Genes & Development 3:882-889. Shea, C., Sant R. G. 1997. Intravesical dimethyl sulfoxide (DMSO) for interstitial cystitis-a practical approach. Urology 49:105-107.

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STUDIES OF THE HEPATITIS C VIRUS RECOMBINANT PROTEIN PRODUCTION AND ITS EFFECTS ON SF-9 INSECT CELLS METABOLISM CÉLINE. M. CHARON University of Westminster, School of Biological & Health Sciences, 115 New Cavendish Street, London W1M 8JS, UK.

ABSTRACT. The recombinant hepatitis C protein BHC 11 was produced in serum-free medium using the baculovirus expression vector (BEV). Protein synthesis in suspension culture of Spodoptera frugiperda Sf-9 insect cells was achieved in controlled and uncontrolled oxygen environment. The same multiplicity of infections (MOI = 0.1 and MOI = 1) and time of infection were used in each experiment and compared. N-glycosylation of the protein was investigated with different lectins.

1. Introduction

Product productivity depends on different parameters such as, the type of medium used [1], the MOI and the time of infection. The effect of dissolved oxygen tension (D.O.T) on recombinant protein production is controversial. Maintaining a constant level of

D.O.T can improve [4] or decrease product accumulation [4, 6]. In some cases the level of D.O.T was shown to have no effect on transient protein production [3, 5]. The presence of nutrients is also important and the limiting factor for protein synthesis is still unknown. The following experiments compare the protein productivity in small scale culture (100 ml) and larger scale culture (1 L). In the latter the D.O.T was kept at 30%, the pH was monitored and nutrient consumption rates were determined. 2. Materials and Methods

2.1. SUSPENSION CULTURES In small scale culture insect cells were grown in 100 ml SF-900 Gibco medium using 250 ml shaken flasks (128 rpm). Larger scale culture experiments were performed

in a 2L stirred (70 rpm) tank bioreactor (LSL Biolafitte) and maintained under 30% D.O.T. The medium (1 L working volume) was aerated by sparging air. pH remained ~6. 35 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 35-37. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

36

Viable cell density was assessed in a Neubauer counting chamber (BDH) using the trypan blue exclusion test [7]. 2.2. PROTEIN ANALYSIS

The recombinant protein BHC11 (97 kD) was extracted at 24, 48, 72, 96 and 120 hours post infection (hpi). Transient protein expression was studied on western blots and quantified by ELISAs (Murex Diagnostics). N-glycosylation studies were carried out using two biotinylated lectins Galanthus nivalis and Concanavalin A (Vector laboratories) for ECL western blot analysis (Amersham). 2.3. BIOCHEMICAL ANALYSIS Substrates (glucose & glutamine) and product (lactate) were analysed under 30% D.O.T as previously described [2].

3. Results 3.1. PROTEIN PRODUCTION IN UNCONTROLLED OXYGEN ENVIRONMENT

Concentrations of protein BHC11 per unit volume Erleruneyer flask with MOIs of 1 (338 were found at 72 hpi (Figures 1 & 2). Western blot analysis of BHC11 with an MOI = 1 showed accumulation of the product at 48, 72 and 96 hpi.

3.2. PROTEIN PRODUCTION IN STIRRED TANK BIOREACTOR Protein production was studied for each MOI under 30% D.O.T. The volumetric productivity with an MOI of 1, produced at 48 hpi and 72 hpi was 38

37

The specific glucose consumption rate and the specific glutamine consumption rate in the first 48 hours of infection with an MOI of were higher than those recorded with uninfected cells or Sf-9 cells infected with an MOI of 0.1. The lactate concentration remained low (2 mM) in infected and uninfected batch cultures. Glycosylation studies showed that the recombinant protein BHC11 was not Nglycosylated. Western blot analyses also revealed that the recombinant protein BHC11 was more stable when using an MOI of 1 rather than 0.1 (results not shown). Recombinant protein production in batch fermentation under 30% D.O.T was limited compared with non-oxygen limited shaken flask cultures. 4. References 1. Akhnoukh R., Kretzmer G and Schügerl K. (1996): On line monitoring and control of the cultivation of Spodoptera frugiperda Sf-9 insect cells and production by Autographa californica virus vector. Enz. Microb. Technol. 18, 220-228. 2. Charon C. (1995): Infection of the insect cells Spodoptera frugtperda SF-9 with the recombinant baculovirus BHC11 in serum-free medium. In: Animal cell technology: Developments towards the 21st century. Beuvery E.C.,

Griffiths J.B and Zeijlemaker W.P. (Eds). Kluwer academic publishers. Dordrecht, pp 131-135. 3. Hensler W.T and Agathos S.N. (1994): Evaluation of monitoring approaches and effects of culture conditions on recombinant protein production in baculovirus-infected insect cells. Cytotechnology. 15, 177-186. 4. Jain D.. Ramasubramanyan K., Gould S., Lenny A., Candelore M., Tota M, Strader C., Alves K., Cuca G., Tung J.S., Hunt G., Junker B., Buckland B.C and Silberklang M (1991): Large scale recombinant protein production using the insect cell baculovirus expression vector system: antistatin and receptor. In: Spier R.E.,

Griffiths J.B., Meignier B (Eds). Production of biologicals from animal cells in culture. Butterworth - Heinemann Press. Oxford, pp 345-351. 5. Kamen A.A., Bédard C., Tom R., Perret S and Jardin B. (1996): On-line monitoring of respiration in recombinant-baculovirus infected and uninfected insect cell bioreactor cultures. Biotech. Bioeng. 50, 36-48.

6. Reuveny S., Kim Y.J., Kemp C.W and Shiloach J. (1993): Effect of temperature and oxygen on cell growth and recombinant protein production in insect cell cultures. Appl Microb Biotechnol. 38, 619-623 7. Summers M.D and Smith G.E. (1987): A manual of methods for baculovirus vectors and insect cell culture procedures. Texas Agricultural Experiments Station, and Texas A&M University, College Station. Bulletin No

1555. pp 1 0 - 1 8 .

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THE HUMAN MULTIDRUG TRANSPORTER (MDR1) EXPRESSED IN THE BACULOVIRUS-SF9 INSECT CELL SYSTEM 1, 2 3

SZABÓK , 1BAKOS,É., 1,2WELKER,E., 1VÁRADI,A., GOODFELLOW,H.R., 3HIGGINS,C.F., AND 2SARKADI,B.

1

Inst. of Enzymology, Hung. Acad. Sci., and 2Natl. Inst. of Haematology and Immunology, Research Group of the Hung. Acad. Sci., Budapest, Hungary; 3Inst. of Molecular Medicine, University of Oxford, UK. NIHI, Budapest, Daródczi u. 24., H-1113, Hungary

Introduction Overexpression of the human multidrug transporter (MDR1 or P-glycoprotein) is

responsible for the phenomenon of multiple drug resistance in various cancer cell types. MDR1 is an integral plasma membrane protein which acts as an ATP-

dependent efflux pump to reduce the intracellular concentration of diverse hydrophobic compounds [1]. MDR1 was described as a phosphorylated glycoprotein, but no clear role for phosphorylation in the transporter activity of MDR1 has yet been established [2,3,4,5]. The “linker” region represents the only

documented target for phosphorylation both in vitro and in vivo. We have constructed MDR1 mutants in which the serines and/or threonines in the linker region of the protein were replaced by alanincs (to prevent phosphorylation) or glutamic acid residues (to mimic the charge of permanent phosphorylation). In

one set of mutants the three serine residues phosphorylated in vivo (Ser 661, 667 and 671) were replaced to generate 3A and 3E mutants. In the other set all the eight serines and threonines providing possible additional phosphorylation sites

in the linker region were replaced ( 8A and 8E mutants), in order to investigate the possible role of secondary phosphorylations in this region. The wild-type and mutant transporters, expressed using the baculovirus-Sf9 insect cell system, were tested for their expression level and correct membrane insertion, and characterized by analyzing their ATP and drug affinity in terms of drugstimulated ATPase activity. 39 O. - W. Merten et al. (eds.). New Developments and New Applications in Animal Cell Technology, 39-41. © 1998 Khuwer Academic Publishers. Printed in the Netherlands.

40

Summary of the results The results presented here indicate that phosphorylation of serine residues in the linker region of the MDR1 modulates its interaction with the certain transported hydrophobic substrate molecules. There is no measurable effect of phosphorylation on the ATP-concentration dependence of the multidrug transporter [6]. 1. A similar high expression level of all the phosphorylation site variants could be achieved as that of the wild-type MDR1 protein, and the normal

membrane insertion pattern was also preserved for all these mutants expressed in the baculovirus-Sf9 insect cells system. 2. The maximum level of drug-stimulated MDRl-ATPase activity was similar for the wild-type and the mutant proteins using several MDRl-interacting compounds. 3. The half-maximum activation of the MDRl-ATPase activity of the mutants 3E and 8E, which mimic the phosphorylated MDR1, and the wild-type MDR1, known to be phosphorylated in Sf9 membranes, was achieved at significantly lower concentrations of verapamil, rhodamine 123 and vinblastine

than that for the nonphosphorylatable mutants 3A and 8A. Fig. 1A shows the data obtained by varying the concentration of verapamil for the 3A and 3E mutants. 4. The ATPase activation in the wild-type MDR1 and its phosphorylation mutants was indistinguishable by calcein-AM or valinomycin.

5. The kinetic analysis presented here may be interpreted to mean that MDR1 has multiple drug-interacting sites, which are characteristically modulated

by the phosphorylation of the serine residues in the linker region of this protein. In the non-phosphorylated state these sites show an apparent negative cooperativity or significantly different drug affinities, while in a fully phosphorylated form of the enzyme this phenomenon can not be observed. Fig. 1B shows data from Fig. 1A analyzed using a simple Michaelis-Menten kinetic approach and presented in a linearised (Lineweaver-Burk) plot. Fig. 1C presents a Hill plot analysis of the same data points. 6. The dependence of the MDRl-ATPase activity on ATP concentration was identical in the wild-type and the mutant proteins, and 7. Hill-plots indicated that more than one ATP-binding/interacting sites are present in the enzyme and these sites interact with a positive cooperativity. 8. The 8A and 8E mutants showed a similar behaviour in all the above experiments as their 3A or 3E counterparts, indicating that the phosphorylation of one or more of the targeted 3 serine residues is fully responsible for the druginteraction effects observed, and there is no additional role of serine or threonine phosphorylation in this region in terms of drug-stimulated ATPase activity, even if three serines are already replaced by alanines. Acknowledgements Participation of this work at the meeting was supported by ESACT.

41

References 1. Gottcsman, M.M., and Pastan, I. (1993) Anna. Rev. Biochem. 62, 385-427

2. Germann, U.A., Chambers, T.C., Ambudkar, S.V., Pastan, I., and Gottesman, M.M. (1995) J. Bioenerg. Biomembr. 27, 53-61 3. Gottesman, MM., Hrycyna, C.A.. Schocnlein, P.V., Gemann, U.A., and Pastan, I. (1995) Anna. Rev. Genet. 29, 607-649 4. Germann, U A., Chambers, T.C., Ambudkar, S.V., Licht, T., Cardarelli, C.O., Pastan, I., and

Gottcsman, M.M. (1996) J. Biol. Chem. 271,1708-1716

5 Goodfcllow, H.R., Sardini, A., Ruetz, S., Callaghan, R.. Gros, P., McNaughton, P.A., and

Higgins, C.F. (1996) J. Biol. Chem. 271, 13668-13674 6. Szabó, K., Bakos,É., Welkcr.E., Müller.M., Goodfellow.H.R., Higgins, C.F., Váradi, A., and

Sarkadi, B. (1997) J. Biol. Chem. 272, 23165-23171

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GLUTAMINE SYNTHETASE TRANSFECTED CELLS MAY AVOID SELECTION BY RELEASING GLUTAMINE

P.BIRD, E. BOLAM, L.CASTELL*, O.OBEID#, N. DARTON and G.HALE. Therapeutic Antibody Centre, Sir William Dunn School of Pathology, University of Oxford Old Road, Headington, Oxford, 0X3 7JT, UK. Tel +44-1865-744845 Fax +44-1865-741291. *Cellular Nutrition Research Group, Dept of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU. #Dept. of Human Nutrition, The Royal London Hospital, Whitechapel, London El 1BB.

1. Abstract Glutamine synthetase (GS) is a popular marker for selection of plasmid expression in mammalian cell lines. Positive selection is maintained simply by omitting glutamine (gln) from culture medium. We have studied the stability of GS transfectants which produce humanised monoclonal antibodies. Spent culture medium of GS transfectants contained up to gln which we conclude is released from the cells. Using a new assay to detect intracellular Ig, cells were detected with low levels of Ig after a short time of culture in medium with as little as

added gln and also in hollow fibre cultures fed with gln free

medium. These variants comprised up to 50% of the cell population and secreted very little antibody. It may be hard to maintain selection pressure in practical large scale cultures particularly in hollow fibre systems where, with a high cell density, the local gln concentrations may be substantial.

2. Introduction The Therapeutic Antibody Centre (TAC) was set up 7 years ago in Cambridge as a centre for the small scale production of therapeutic monoclonal antibodies available for clinical trials under DDX certification. Two years ago the centre moved to a new purpose-built facility in Oxford. In total more than 600g of therapeutic antibody has been produced to treat more than 3000 patients. Seven humanised monoclonal antibodies have been grown in the TAC using hollow fibre based production in seven Acusyst machines (Cellex) and an Acusyst P3X where six hollow fibre devices are incorporated in a single flowpath. Humanised antibodies have been expressed in CHO or NS0 cells using either dihydrofolate reductase or glutamine synthetase for plasmid selection. Glutamine synthetase (GS) is a popular marker for selection of plasmid expression in mammalian cell lines. Positive selection is maintained simply by omitting glutamine (gln) from culture medium (Bebbington et al 1992). The plasmid for transfection is licensed by Lonza. Using this plasmid to transfect NS0, we have produced four different humanised IgG1 antibodies. When these cell lines are grown in

hollow fibre we expect to see an increase in antibody concentration from 0.1-0.4 mg/ml in saturated cell supernatants in small scale culture to 1-4 mg/ml in the Acusyst harvest. Each cell line has a characteristic

product concentration. One of our GS transfectants (cell line 1) showed considerable variation in mean product concentration from day 50-100 post inoculation in three different runs (1.0, 1.7, and 3.1 mg/ml) whilst another (cell line 2) failed to show any significant increase in concentration of product when grown

in hollow fibre (maximum 0.3 mg/ml) compared with small scale tissue culture (0.1 mg/ml). This variability in hollow fibre production by these two cell lines has been examined. We looked for the presence of low or negative antibody producing cells in each cell line by analytical

43 O.- W Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 43-49. © 1998 Kllnver Academic Publishers. Printed in the Netherlands.

44 cloning in medium containing 0 or 1 mM gln. Additionally we developed an assay to detect intracellular immunoglobulin (ICIg) in individual cells by permeabilising the plasma cell membrane with saponin prior

to incubation in FITC conjugated anti- human Ig and assaying individual cell fluorescence on a FACScan. A considerable amount of cell death occurs in hollow fibre cell culture. Our hypothesis was that the death of high Ig producing cells may release intracellular synthesised gln that can be used by lower Ig producing cells that have downregulated both GS and Ig synthesis. Therefore we have measured gln concentrations in small scale tissue cultures and in samples taken from the extracapillary side of hollow fibre cultures of these lines.

3. Methods 3.1. MEDIA. Two types of gln free media were compared. Media 1 was IMDM (Sigma I-2762) with the standard Lonza recommended nucleoside, glutamic acid (glu) and asparagine (asn) additions. Media 2 was a special TAC formulation of IMDM lacking gln but with nucleosides and a higher level of glu (2600 and asn Dialysed fetal calf serum (Gibco ultra low Ig) was added to both media at 5%. The gln content of the FCS was below post dialysis final).

3.2. INTRACELLULAR IMMUNOGLOBULIN (ICIg) ASSAY Actively growing cells were washed into PBS and fixed in 4% formaldehyde/PBS for 15 min and then permeabilised with 0.5% saponin ( Sigma S-2149)/0.5% BSA/PBS for 30 min. Cells from hollow fibre cultures were separated first over Histopaque to enrich for live cells. Fixed cells were incubated in optimal dilution of FITC conjugated sheep anti- human IgG (Fc specific) in permabilising buffer for 30 min. After

washing the cells were resuspended in PBS/BSA/azide to close the membrane pores and fluorescence assayed on a Becton Dickinson FACScan. A sample of line1 cells grown in gln free medium was used as a positive control and non transfected NS0 cells as a negative control in every assay. During analysis of gated 'live' cells, the % positive cells (using a marker to define NS0 as <2% positive), the % strong positive cells (using a marker to define line 1 cells as >95% positive), and the geometric mean of FL1 were recorded. An additional control of staining positive cells with FITC conjugated anti-rat Ig gave similar intensity of FL1 staining as NS0. 3.3. GLUTAMINE ASSAY BY HPLC. Cell culture supernatants samples were stored frozen. Deproteinisation was performed with an equal volume of 10% sulphosalicylic acid. Amino acid derivatives of o-phthalaldehyde were separated on a Hichrom spherisorb reverse phase ODS C18 column as in Mann et al (1988). Peak areas were integrated

and amino acid concentrations calculated from peak areas by reference to the area of an internal standard homocysteine peak.

4. Results and Discussion. 4.1. INTRACELLULAR IG ASSAY. 4.1.1 Assay validation This assay distinguished high Ig producing cells (1.1A: line 1 cells d 236 in culture, from untransfected negative controls (1.1B: NSO cells d 42, by their intensity of green (FL1) fluorescence. Premixing positive and negative cells (1.1C) gave similar % of positive cells and FL1 geometric means to that seen when cells were mixed after staining (LID). Additionally, in a number of cell stability tests a drop in the FL1 geometric mean and % strong positive cells by ICIg analysis has been seen to precede a drop in secreted Ig production by about 10 days, (for example see figure 2) suggesting that

this assay is an early indicator of cell instability.

45

4.2. THE DEVELOPMENT OF LOW IG PRODUCING LINE 1 CELLS IN MEDIA WITH VARYING GLN CONCENTRATIONS. Line1 cells were passaged twice weekly in 1 ml of TAC media with gln added back from Saturated supernatants of passed cells were collected and analysed for Ig concentration by ELISA and percent of strongly positive intracellular Ig cells was also assayed with time (figure 2). At all gln

concentrations tested, low Ig producing cells developed as detected by reduced Ig production and intracellular Ig levels. Only in gln free medium were no low producers detected over 120 days of culture. As little as gln was sufficient to allow low Ig producers to survive in line 1 cultures. (Line 2 cells

developed low Ig producing cells in the complete absence of added gln (data not shown).

46

4.3 GLUTAMINE CONCENTRATIONS IN CELL CULTURES.

4.3.1 In hollow fibre culture Three different hollow fibre cultures of line1 sampled on days 153 and 214 respectively contained 50 - 90 gln in the extracapillary (cell growing) side. Considering that there is continuous dialysis across the hollow fibre it could be that the cells are bathed in higher local concentrations of gln. We have shown that line 1 cells can lose productivity in as little as gln (figure 2) and therefore local production of gln in the hollow fibres may affect productivity.

4.3.2 In flask cultures Supernatant from line 1 cell cultures in TAC medium were collected over a growth period of 15 days. Cell numbers and viability were correlated with gln levels in the supernatant (figure 3). Gln levels rose from 0 to over on day 10 of culture and this increase coincided with the loss of cell viability suggesting that release of cell synthesised gln on cell death is a likely cause of the gln detected in these cultures and also in the hollow fibre cultures. When these cell cultures were repeated in standard media with lower asn and glu concentrations, lower levels of gln(maximum were detected in the media. We are currently trying to find optimal levels of media constituents that allow high Ig production but low gln

accumulation.

47

5. Conclusions A number of factors will affect GS transfectant stability, such as integration site, copy number, and product. GS transfectants exist which retain stability in the presence of gln. Our GS transfectant cell line1

is stable for over 200 days in gln free medium when passaged twice a week in flask culture but not in the presence of or more gln nor in long term hollow fibre culture. We have shown here that GS transfectants can release up to gln into culture medium in association with cell death and that hollow fibre cultures of GS transfectants can also develop high levels of gln on the cell side. We propose

that the loss in productivity of this transfectant in hollow fibre culture results from the level of gln produced locally. This gln related instability may limit the growth of similar GS transfectants in hollow fibre devices. 6. Acknowledgements This work was funded by MRC and Leucosite inc.. We wish to thank Hilary Metcalfe and Sue Bright at Lonza for their helpful discussions and Andy Beevers for his help.

7. References Bebbington, C.R., Renner.G., Thomson, S., King, D., Abrams,D., and Yarranton G.T.. (1992) High level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker, Biotechnology 10, 169-175. Mann G.E., Smith S.A., Norman P.S.R., and Emery P.W. (1988). Fasting refeeding and diabetes modulate free amino acid concentrations in the rat exocrine pancreas: role of transstimulation in amino acid efflux,

Pancreas 3, 67-76.

48

Discussion

Ozturk:

Your data are very consistent to the original CellTech data which showed that if glutamine was present, the stability decreased. They suggested using methionine sulphoxamine. Have you tried it?

Bird:

Yes, we tried it for cell line 2, but unsuccessfully. Possibly the product is a bit more unstable for cell line 2. Also, as glutamine is

by-passing the need for glutamine synthetase and MSO blocks this enzyme as it is in such low concentrations, the fact that glutamine is around may by-pass that inhibition. Ozturk:

In your hollow fibre system you are getting 3 g/l antibody. Does the membrane cut-off affect productivity as the yield looked very high?

Bird:

We inoculate a 200 ml culture at cells and they grow in the presence of foetal calf serum, so you get a very high cell concentration within the EC side. This is what leads to the very high antibody concentration. The MW cut-off is 10,000 of the kidney dialysis membranes which we use.

Griffiths:

I am wondering whether there is a straight-forward nutritional explanation for the glutamine effects which you observe. Whether an amino acid is essential or non-essential depends upon the cell density. The higher the density, the fewer the amino acids that are

essential. Above 50 million/ml, glutamine ceases to be essential. So at the cell densities you are using, there is no advantage for the cell to have glutamine synthetase. In fact, it maybe a disadvantage, especially with all the asparagine and amides that are present. So it may well be just a question of the advantage that glutamine synthetase has for the cell is completely lost at high densities. Petrie:

Have you observed loss of glutamine synthetase activity also at low density where there is less cell death? Also, have you observed this with other cells, such as CHO and non-NS0 cells?

Bird:

Cell line 1 is a very stable producer in small-scale culture in glutamine-free medium. We have cultured them for nearly 1 year, passaging them twice weekly, and the ICIG levels remain high. Each transfectant will very slightly in its characteristics, but at low cell density there is not a problem. With CHO cells, we have not seen this problem.

49

Berthold:

I always struggle with the naming of cell lines when they are not apparently genetically stable. Assuming that the de-selection of what you see is due to genomic diversity, would it be possible to have alternative strategies to somehow constrain these cell lines to more mono-clonality? Do you have steps to ensure mono-clonality because it is well known, for these cell lines in particular, that the chromosomes are not a set but a histogram, so you have all types of de-selection possibilities?

Bird:

The easy answer is, no, we have not. We can routinely check for low IG producers. We have varied the media to see if we could get lower glutamine levels.

Berthold:

Do you use single-cell cloning from time to time?

Bird:

Yes, we set up each run from a master cell bank containing a minimum number of cell passages before freezing, although of course the hollow fibre runs are long.

Aunins:

I would have thought that you will eventually come to a steadystate even with low producing clones present. Have you run your experiments out to see if you get a steady-state population?

Bird:

This is seen clearly in cell line 2, with or without glutamine present. The percentage of high versus low producers changes very slowly, but you do get to a steady-state where the amount of glutamine produced by the high-producers is just enough and they are growing with a constant division time.

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STUDY OF DIFFERENT CELL CULTURE CONDITIONS FOR THE PRODUCTION OF A “RESHAPED” MAB IN NSO CELLS.

A.J. CASTILLO1, A. FERNANDEZ1, T. BOGGIANO1, P. PUGEAUD2, I. W. MARISON2. 1) Center of Molecular-Immunology (CM), P.O. Box 16040, Havana City 11600, Cuba, 2) Institute of Chemical Engineering and Bioengineering, Swiss Federal Institute of Technology, CH-1015, Lausanne, Switzerland.

Abstract: The recombinant cell line R3/T16 expresses a “reshaped” Mab against epidermal growth factor receptor (EGF-R) in NS0 myeloma. In this study the ability of two culture media to mantain good levels of growth and production of this cell line under serum reduced conditions have been compared and the effect of supplementation of aminoacids and vitamins at 1% of fetal calf serum was investigated.

Furthermore, production and growth yields of R3/T16 cell line in cultures with different macroporous microcarriers have been compared. Results showed better performances for the immobilized cell cultures in fluidized bed reactor, suggesting that such systems are suitable for a large scale production of this recombinant Mab. Introduccion

themain aims in developing commercial processes is to lower production costs by increasing unit production. To achieve this goal, high density systems capable of volumetric scale-up and long term operation are required. These aims could be obtained using culture systems with cell immobilization in macroporous microcarriers. Immobilization is also reported to improve cell specific production rate [2]. By these reasons overall aims of this study were to optimize a basal medium that could support good levels of growth and production of R3/T16 recombinant cell line at low levels of serum supplement and to test different macroporous microcarriers in order to

There are a number of problems associated with the presence of serum in culture medium used for production, including high cost, regulatory considerations and the difficulty in removing serum proteins when purifying the product of interest. By these reasons the reduction of serum supplement or the use of serum-free medium for the culture of genetically engineered mammalian cells offers many advantages. Previously it has been shown that NSO cells are cholesterol auxothrophs, that renders very difficult their adaptation to serumfree medium. However some authors have reported the use of serum-free media to culture this cell line [1]. One of 51

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 51-53. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

52

grow this cell line in medium with low concentration of serum supplement.

and other metabolites were measured by RP-HPLC methods (4).

Materials and methods

Results

The R3/T16 cell line is a NSO myeloma that produces an humanized monoclonal antibody against EGF receptor [3]. Protein free media Turbodoma FMX-B1 (FMX Std) and HP 1 (THP1) were obtained from Dr. F. Messi, Cell Culture Technologies and all other basal media and fetal calf serum (FCS) were purchased from Gibco BRL. All other reagents were acquired from Sigma. Previously to culture experiments for

Growth curves were carried out in 6 well plates with a seed cell density of cells/ml using FMX-1 and PFHM-II serum free media supplemented with 8, 3 and 1 percent of FCS. From this study (Fig. 1) we selected Turbodoma medium, due to its best support for cell growth and production at low serum levels, Kinetic studies in T-flasks were carried out in DMEM/F-12 medium supplemented with 8 % FCS (control)

testing different medium supplements,

and in THP1 medium supplemented with

cells were passaged at least 7 days in order to adapt them. For each culture

9 and 1 % of FCS. It was observed an increase in specific glucose consumption

experiment in spinners or Tflasks cells were grown in duplicates. Cultures in well plates were carried out in triplicates. Cytopore I (Pharmacia Biotech) and Cultispher G (Hyclone) microcarriers were tested in spinner flasks (Integra Biosciences). One culture was done in the Cytopilot Mini fluidized bed reactor (FBR) with 200 ml of Cytoline I microcarrier (Pharmacia Biotech). In all cases seed density was cells/mL and culture medium was Turbodoma HP1 supplemented with 1 % of FCS. Cell count was peformed by trypan blue exclusion method. IgG concentration was measured by an anti-human heavy chain ELISA developed at CIM. Aminoacids

and lactate production rates (Table 1), when reducing serum supplement. Glutamine specific consumption rate was higher in THPl medium with respect to control. However growth and production parameters were very similar in all cases, Critical aminoacids were determined and supplemented following the criteria previously described by Stoll et al. [5]. Kinetic studies in T-flasks using THP 1 medium with 1 % of FCS and different supplements were done. Not significative differences were observed for growth and production behaviour (Fig. 2) , as well as for specific consumption and production rates of different metabolites (data not shown) with different medium supplements.

53

very similar in THP1 supplemented both with 9 or 1 % of FCS compared with control medium (DMEM-F12 + 8% FCS). Non positive effect was observed respect to biomass, cumulative product and key metabolic parameters when the adding aminoacids and vitamins, indicating that these metabolites are not limiting steps when this cell line is cultured under serum reduced conditions. R3/T16 cell culture in FBR with Cytoline I microcarriers showed the best performances respect to determined values of Qab. Further experiments with longer culture time are required in order

to investigate full potentialities of this type of system. References 1.- Keen M. J. and Steward T.W. (1995). Adaptation of cholesterol-requiring NSO mouse myeloma cells to high density growth in a fully defined protein-free

Values for overall production rates (Qab) and average IgG concentrations for cultures with different microcarriers are showed in Table 2. Best value for Qab was obtained for cell culture in FBR using Cytoline I microcarriers. Conclusions

Turbodoma culture medium showed better support for cell growth and antibody production at low serum supplement. Growth parameters were

and cholesterol-free culture medium. Cytotechnology 17; 203-211. 2.- Lee G. M. and Palsson B. O. (1990).

Immobiliization

can

improve the

stability

of

hybridoma antibody production in serum-tree medium. Biotechnology & Bioengineering 36; 1049-

1055. 3.- Mateo C., Moreno E., Amour K., Lombardero J., Harris W. and Perez R. (1997). Humanization of a

mouse monoclonal antibody that blocks the epidermal growth factor receptor: recovery of antagonistic activity. Immunotechnology 3; 71 -81. 4.- Stoll T., Pugeaud P., Von Stockar U. and Marison I. W. (1994). A simple HPLC technique for accurate

monitoring

of

mammalian

cell

metabolism.

Cytotechnology 14; 123-128. 5.- Stoll T., Muhlethaler K., Von Stockar U. and

Marison I. W. (1996). Systematic improvement of a chemically-defined protein medium for hybridoma growth and monoclonal antibody production. Journal of Biotechnology 45 (2); 111-123.

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INCREASING MONOCLONAL ANTIBODY PRODUCTIVITY BY SEMICONTINUOUS SUBSTITUTION OF PRODUCTION MEDIUM FOR GROWTH MEDIUM and K. ŠRÁMKOVÁ Institute of Molecular Genetics, Fundamental Cytotechnology Laboratory, CZ-14220 Praha 4, Czech Republic

Introduction Hybridoma culture in perfusion mode, both in homogeneous suspension and in heterogeneous systems, offers a daily harvest of monoclonal antibody (Mab) synthesized in a relatively small working volume by a high-density cell population over a long period of time [1]. Because high perfusion rates are needed to provide sufficient quantity of substrates and to remove toxic waste products, the simple variant of perfusion culture has some disadvantages. The Mab concentration in the harvest fluid is generally lower than that achievable in an optimized fed-batch culture. The volume of medium consumed per mass unit of the product is relatively high, and the utilization of the substrates is incomplete.

Improvements to the perfusion process have been developed based on addition of nutrient concentrates [2,3], bleeding of homogeneous perfusion cultures [2,4], or separate use of concentrated medium for feeding and buffer solution for dialysis [3]. In this work we report on the development of a semicontinuous process in which an efficient Mab batch production employs cells transferred from a cell growth modul run in perfusion mode. The philosophy of this strategy arises from the notion that optimal medium for Mab production is different from optimal medium for growth. In this process we apply the knowledge of suppression of apoptotic death by addition of some amino acids [5-10] to the cell growth medium. Daily cell bleeds from the growth modul served to set up short-term production cultures in batch mode, in the course of which the Mab concentration rose to multiples of the starting value. Material and Methods Mouse hybridoma ME-750, an IgG2a producer, was cultured in DMEM/F 12/RPMI 1640 (2:1:1) medium supplemented with BME amino acids, 2.0 mM glutamine, 0.4 mM each of alanine, serine, asparagine and proline, 15 mM HEPES, 2.0 g/1 sodium bicarbonate and with the iron-rich protein-free growth-promoting mixture containing ferric citrate [11].The values of the culture parameters were determined as described before [5-11]. The cell growth modul consisted of a strirred reactor with a working volume of 1 liter equipped with a sedimentation cone serving as the cell-retention device. The perfusion-mode hybridoma cultures were carried out at the dissolved oxygen tension of 10% air saturation. The pH was kept at the value of 7.0. 55 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 55-57. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

56

Results and Discussion Upon reaching viable cell density cells/ml the cell growth modul was run in perfusion mode Cell bleed volumes (0.3 -0.4 1) were withdrawn daily when the viable cell

density increased to cells/ml. The cell growth modul was runnig for several weeks under steady-state conditions (viability 90-95%, viable cell density cells/ml, residual glucose 8-10 mM, residual glutamine 1-2 mM, ammonia 4-5 mM, lactate 15-20 mM and Mab concentration 80-110mg/l). Batch cultures were initiated by supplementing the cell bleed withdrawn from the growth modul with a volume of fresh medium and with essential amino acids (Figs. 1 and 2). Our novel strategy changes substantially the functions of perfusion and batch modes in the process (Table 1).

Conclusions

1. A hybridoma perfusion culture can be maintained at steady state (viability >90%, cell density up to cells/ml) for arbitrary time period, thanks to the enrichment of the medium with apoptosis-suppressing amino acids, and to the application of a high cell bleed rate. 2. The perfusion cell growth modul with a working volume of 1.0 1 can serve as a source of cells available daily for initiation of short-term batch cultures. 3. Due to high initial cell density, and substantial exhaustion of nutrients in the growth medium, the batch cultures behave as if initiated at their stationary phase.

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4. If the medium is sufficiently enriched with the building stones ofthe Mab molecule, i.e. the essential amino acids, and with the main energy source, glutamine, the synthesis of Mabs proceeds at high specific rate. The exhaustion of glutamine, but not of glucose, acts as the stopsignal for Mab synthesis. 5. The Mab concentration in the medium harvested from the batch cultures is several-fold higher than in the medium harvested directly from the perfusion cell growth modul. Acknowledgement. The work was supported by the Contract No. BIO4 CT95-0207 of the Commision of the EC , DGXII/ Contract No. OK120 of the Czech State Budget, and the Grant No. I/68957 of the Volkswagen Foundation. References 1. Griffiths, IB. (1992) Animal cell culture processes - batch or continuous. J. Biotechnol. 22, 21-30. 2. Büntemayer, H., Wallerius, C., and Lehmann, J. (1992) Optimal medium use for continuous high density perfusion processes. Cytotechnology 9, 59-67. 3. Pörtner, R., Lüdemann, I. and Märkl, H. (1997) Dialysis cultures with immobilized hybridoma cells for effective production of monoclonal antibodies. Cytotechnology 23, 39-45. 4. Hiller, G.W., Clark, D.S. and Blanch, H.W. (1993) Cell-retention-chemostat studies of hybridoma cells: Analysis of hybridoma growth and metabolism in continuous suspension culture on serum-free medium. Biotechnol. Bioeng. 42, 185-195. 5. F. (1995) Starvation-induced programmed death of hybridoma cells: Prevention by amino acid mixtures. Biotechnol. Bioeng. 45, 86-90. 6. F., and K. (1997) Unbalanced media for hybridoma cell culture: an alternative reality, in M.J.T. Carrondo et al. (eds.), Animal Cell Technology. From Vaccines to Genetic Medicine, Kluwer Academic Publishers, Dordrecht, pp. 675-680. 7. F., and ChládkováK. (1995) Apoptosis and nutrition: Involvement of amino acid transport system in repression of hybridoma cell death. Cytotechnology 18, 113-117. 8. F. and K. (1996) Cell suicide in starving hybridoma culture: survival-signal effect of some amino acids. Cytotechnology 21, 81-89. 9. F., and K. (1996) Protection of B lymphocyte hybridoma against starvation-induced apoptosis: survival-signal role of some amino acids, Immunol. Lett. 52, 139-144. 10. F., and K. (1997) Amino acid signaling for growth or death, in K. Funatsu et al. (eds.), Animal Cell Technology: Basic & Applied Aspects, Vol.8, Kluwer Academic Publishers, Dordrecht, pp. 131-135. 11. F., Vomastek, T. and J. (1992) Fragmented DNA and apoptotic bodies document the programmed way of cell death in hybridoma cultures. Cytotechnology 9, 117-123.

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EXPRESSION OF RECOMBINANT HUMAN INSULIN IN CHINESE HAMSTER OVARY CELLS IS COMPLICATED BY INTRACELLULAR INSULIN-DEGRADING ENZYMES

S.C.O. PAK, S.M.N. , M.J. SLEIGH* & P.P. GRAY Department of Biotechnology and the CRC for Biophurmaceutical Research, University of New South Wales, Sydney 2052 Australia; of Molecular Oncology, Westmead hospital; *Peptide Technology Limited, Sydney 2009, Australia

Abstract

To investigate the role of insulin degrading enzymes in heterologous insulin gene expression, Chinese hamster ovary (CHO) cells were transfected with a mammalian expression vector carrying the cDNA for the human pre-proinsulin under the control of the RSV promoter. Stable transfectants were isolated and characterised in regard to insulin transcription, translation, processing and secretion. Levels of insulin expressed by these cells were extremely low, peaking at around cells/24 hours. Analysis of the secreted product indicated inefficient processing with less than 10% of the total product being fully processed. degradation assays revealed insulin degrading activity in the cytosol and in the conditioned medium of CHO cells. In both cases the insulin degrading activity was significantly inhibited by the addition of bacitracin, a peptide antibiotic and an inhibitor of insulin degrading enzyme (IDE). No noticeable effects were seen with the addition of a general protease inhibitor, PMSF. When transfected CHO cultures were supplemented with small quantities of bacitracin insulin expression improved considerably. These results combined support the hypothesis that IDEs are directly involved in inhibiting overexpression of recombinant human insulin in transfected CHO cells. Introduction

Insulin is a potent mitogenic protein naturally synthesised by endocrine islet cells of the pancreas. Although insulin expression has been extensively studies in many different mammalian cell lines, attempts to overexpress insulin in non-endocrine cell lines have largely been unsuccessful (Groskreutz et al., 1994; Moore et al., 1983; Quin et al., 1991; Vollenweider et al., 1992). The reason for this is currently unclear.

Insulin degrading enzymes (IDE) are neutral thiol metalloproteinases that are involved in specifically degrading insulin molecules (reviewed by Duckworth, 1988). When insulin binds to it's receptors on the cell surface a wide range of metabolic/anabolic pathways are 59 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 59-67. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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stimulated. This is followed by endocytosis of the receptor-ligand complex to form an intracytoplasmic vesicle called an endosome. The pH in the endosoine rapidly drops to 5.6 resulting in dissociation of the insulin-receptor complex. Although some receptors are degraded, the majority are recycled to the cell membrane. The internalised insulin, on the other hand, undergoes rapid degradation by insulin-specific enzymes that are present in the cytosol. A large number of cells (including the liver, kidney, muscle, pituitary and fibroblast) are known to be rich in IDEs. To investigate the role of insulin degrading enzymes in recombinant insulin expression in non-endocrine cells, CHO cells were transfected with a mammalian expression vector carrying the human preproinsulin cDNA under the control of the RSV promoter. Consistent with previous reports, insulin expression and processing by recombinant CHO cells was extremely poor. Further characterisation revealed considerable insulin degrading activity in the cytoplasm and culture supernatant of CHO cells. In this report we provide evidence to support the hypothesis that intracellular IDEs complicate overexpression of recombinant human insulin in transfected CHO cells. Materials and Methods

Cell Culture. Recombinant CHO cells expressing human insulin were generated by transfecting CHO-K1 cells (ATCC CCL-61) with a plasmid (pRSVIns) carrying the human pre-proinsulin cDNA under the control of the RSV promoter. The basal medium used in this paper is a custom-made preparation from Gibco (USA) which consisted of a 1:1 mix of Dulbecco's Modification of Eagle's Medium (DMEM) and Coon's F12 medium supplemented sodium selenite and is refer to in this paper as serumfree (SF) medium. Recombinant CHO-Ins cells were cultured in serum-free medium supplemented with human apo-transferrin (Sigma, USA) and is referred as SF+Tf. Sample Collection. To assess the recombinant cell lines for the production of insulin, conditioned medium was collected from confluent T-75 cultures. Cells were grown to confluence in 10% FCS medium, rinsed twice with PBS and incubated in 10 ml SF+Tf for 24 hours. Cell lysates were prepared by directly applying 10 ml of lysis buffer [140 mM NaCl, 4 mM KC1, 1 mM NaPO4, 20 mM Tris (pH 7.4), 1% NP-40, PMSF] to the cells. Culture supernatant and cell lysates were clarified by centrifugation and stored at -20 °C until required. Analysis of insulin mRNA in CHO cells. Cytoplasmic RNA from CHO cells were analysed for insulin mRNA by Ribonuclease Protection Assay (Ambion, Inc., Texas) or by Northern blot as previously described (Hunt et al., 1996). Analysis of insulin. Insulin in the cell lysate and in the culture supernatant was measured by an automated Microparticle Enzyme Immunoassay (MEIA) courtesy of Mr Kris Tan (Royal Prince Alfred Hospital, Sydney) and by an immunoenzymetric assay using a

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MEDGENIX INS-EASIA kit ( M E D G E N I X Diagnostics). Total immunoreactive insulin (IRI) was measured by a radioimmunoassay (RIA).

degrading assay. A confluent monolayer of cells from a T-flask was detached and washed with ice-cold PBS before centrifuging at 2000 rpm for 2 minutes to pellet the cells. The pellet was then resuspended in 3 ml ice-cold Buffer A (10 mM Tris-Cl, 10 mM KC1, 0.06% pH 8.1) and homogenised for 20 minutes on ice. The homogenate was spun at 14,000 rpm for 60 minutes at 4 °C to pellet cellular debris. A small aliquot of the supernatant was removed for determination of protein concentration using a BioRad protein assay kit. A of the cytosolic fraction was preincubated with of Buffer B (125 mM potassium phosphate, 0.25% BSA, pH 7.4) and of a protease inhibitor (PMSF: 100 mM; Bacitracin: 350 units/ml) for 20 minutes at 37 °C. A no protease inhibitor control was also included for comparison. After the preincubation, of insulin [ICN] was added to each tube and incubated at 37 °C for 0, 15, 30, 50 and 80 minutes. Reactions were stopped at the specified times by addition of ice-cold 40% TCA. Following incubation on ice for 30 minutes the tubes were centrifuged for 20 minutes at 14,000 rpm. Supernatant was discarded and the radioactivity in the pellet was measured using a Packard Auto-Gamma 5650 counter. The decrease in the radioactive counts in the pellet indicated the extent of insulin degradation. Conditioned medium for insulin degradation assay was prepared by culturing CHO-K1 cells in roller bottle containing 10% FCS. Upon reaching confluence, cells were rinsed twice with PBS and incubated in 50 ml of SF medium for 24 hours. After centrifugation, culture supernatant was preincubated with or without of 350

units/ml solution of bacitracin for 20 minutes at 37 °C. Assays were then initiated by addition of of insulin [ICN] to each tube. After incubation at 37 °C for 0, 15, 30, 60, 90 and 120 minutes, reactions were stopped and the radioactivity

in the pellet measured as described above. Results and Discussion

Expression of Insulin by CHO-Ins cells To generate stable insulin-expressing cells, of pRSVIns and DNA were co-transfected into CHO cells and exposed to of active G418 for 6 weeks. Individual clones (78) were isolated by limiting dilution and tested for insulin expression by RIA. Consistent with previous studies (Yanagita et al., 1992; Vollenweider et al., 1992), levels of insulin expressed were extremely low with less than 7 ng of cells/24 hours being produced by the best clone (Figure 1, A). In an effort to delineate the factors involved in the low level expression of insulin, cytoplasmic RNA from CHO-Ins cells were analysed by Northern blot. Insulin-specific mRNA was detected in ail the transfected cells but not in the control indicating active transcription of the stably integrated genes (Figure 1, B).

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Characterisation of secreted insulin

To determine whether CHO cells processed insulin correctly, CHO-Ins conditioned medium was analysed using methods which distinguish mature (fully processed) insulin from partially processed intermediates. Results indicate that over 90% of the total immunoreactive insulin secreted by CHO cells is partially processed, mainly in the form of proinsulin (Figure 2). In an attempt to improve furin processing, site-directed mutagenesis was explored to change the DNA sequence at the C-peptide/A-chain junction of the insulin gene from Leu-X-Lys-Arg to the optimal furin cleavage sequence Arg-X-Lys-Arg. Although cells transfected with the mutated insulin gene processed insulin well, the overall levels of insulin expressed remained below degradation by CHO cell extract To investigate the possibility of intracellular insulin degradation, known amounts of

radiolabelled were incubated with of cytosolic CHO cell extracts over a period of 80 minutes in the presence and absence of protease inhibitors (Figure 3). In the absence of any protease inhibitors, greater than 95% of the added insulin was rapidly

degraded as measured by a decrease in radioactivity of the TCA precipitated intact insulin

(a). In the presence of PMSF, a general protease inhibitor, degradation was only slightly inhibited (b). However, in the presence of bacitracin, a peptide antibiotic and an inhibitor of insulin degrading enzyme (IDE), insulin degradation was almost completely inhibited (c). These results indicate that there is substantial insulin degrading activity in the cytosol of CHO cells and that the majority of that activity is due to IDEs. degradation by CHO culture supernatant

To test for insulin degrading activity in the conditioned medium of CHO cells, insulin degradation by cell-free, conditioned medium was measured at 37 °C in the presence and absence of bacitracin (Figure 4). In the absence of any protease inhibitors, the CHO conditioned medium degraded 80% of the added within 2 hours of incubation (a). On the other hand, when labelled insulin was co-incubated with bacitracin, degradation was significantly inhibited (b). Effect of Bacitracin on recombinant insulin secretion

degradation experiments have indicated that there is significant insulin degrading activity in the CHO cell extract and culture supernatant. To determine whether these enzymes are involved in inhibiting expression of recombinant human insulin in CHO cells, transfected CHO cells were cultured in SF+Tf medium with various concentrations of bacitracin (Figure 5). In the absence of any bacitracin, CHO-Ins cells expressed 0.13 pmoles of cells/24 hours. In comparison, the same cells cultured in the presence of bacitracin showed improved insulin expression proportional

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to bacitracin concentration. When bacitracin was present at a concentration of 1 mg/ml, insulin expression was shown to improve by around 250%. Conclusion

Recombinant insulin expression has been extensively studied in a number of different mammalian host cell lines. Whilst endocrine cells express insulin well, attempts to overexpress insulin in non-endocrine cells have largely been unsuccessful. Although insulin remains as the most well studied protein, the factors influencing its expression in non-endocrine cells have not been fully established. In an effort to investigate the role of IDEs in heterologous insulin expression, we transfected CHO cells with a plasmid expressing the cDNA for human preproinsulin. Active transcription of the stably integrated insulin gene was confirmed by Northern blot analysis (Figure 1. B). Consistent with previous studies, the levels of insulin expressed were below cells/ 24 hours (Figure 1. A). Analysis of the conditioned medium revealed inefficient insulin processing as indicated by the presence of insulin and proinsulin at a ratio of 1:9 (Figure 2). The inefficient processing was thought to be due to the presence of a suboptimal furin recognition sequence at the C-peptide/A-chain junction of the insulin molecule. Thus to improve insulin processing site-directed mutagenesis was explored to alter the furin recognition sequence. By changing one amino acid, from leucine to arginine, it was possible to improve insulin processing considerably (up to 100% efficiency). However, despite this improvement, the overall levels of insulin secreted did not improve (data not shown).

Insulin-degrading enzymes (IDEs) are enzymes involved in switching off the insulin signal by rapidly degrading internalised receptor-bound insulin. Considerable insulin degrading activity was detected in the cytosol of CHO cells as measured by the degradation of insulin by the CHO cell extract (Figure 3). Analysis of the

cell extract showed presence of IDEs as determined by bacitracin inhibition of insulin degradation. It may be possible that these enzymes are rapidly degrading newly synthesised recombinant insulin and thus prevent the release of intact insulin. Although pulse-chase studies would provide concrete evidence for intracellular insulin degradation, attempts were frustrated by the low levels of insulin expressed by these cells. Instead, evidence for the involvement of IDEs was provided by monitoring insulin expression by cells cultured in the presence and absence of bacitracin. In the presence of bacitracin, intracellular IDEs were inhibited resulting in improved insulin expression in a manner dependent on bacitracin concentration (Figure 5). Investigations are currently under way to identify the sites of IDE binding and cleavage. It is believed that introducing favourable mutations at these sites will reduce intracellular degradation and improved insulin expression. Some evidence for this has already been supplied by Groskreutz and colleagues (1994). These investigators obtained better insulin expression by altering the amino acid at position B-10 from histidine to aspartic acid. By introducing further mutations to the insulin gene it may be possible to engineer an insulin molecule that is completely resistant to IDE degradation.

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References Duckworth, WC (1988) Insulin Degradation: mechanisms, products and significance. Endocrine Reviews 9: 319-345 Groskreuiz DJ, Sliwkowski MX and Gorman CM (1994) Genetically engineered proinsulin constitutively processed and secreted as mature, active insulin. J Biol Chem 269:6241-6245 Hunt SMN, Tail AS, Gray PP and Sleigh MJ (1996) Processing of mutated human proinsulin to

mature insulin in the non-endocrine cell line, CHO. Cytotechnology 21:279-288 Moore H-PH, Walker MD, Lee F and Kelly RB (1983) Expressing a human proinsulin cDNA in a mouse ACTH-secreting cell. Intraccllular storage, proteolytic processing and secretion on stimulation. Cell 35:531-538 Quin D, Orci L, Ravazzola M and Moore H-PH (1991) Intracellular transport and sorting of mutant human proinsulin that fails to form hexamers. J Cell Biol 113:987-996 Vollenweider F, Imiinger JC, Gross DJ, Villa KL and Halban PA (1992) Processing of proinsulin by transfected hepatoma (FAO) cells. J Biol Chem 267:14629-14636 Yanagita M. Hoshino H, Nakayama K and Takeuchi T (1993) Processing of mutated insulin with

tctrabasic cleavage sites to mature insulin reflects the expression of furin in nonendocrine cell lines. Endocrinology 133:639-644

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Discussion

Barteling:

You are working with human insulin. Did you try to incorporate

hamster-type insulin as the system might work differently? Pak:

We have not tried that but other groups have used rats and insulin from various other species. They have all found similar results.

Ozturk:

What happens if insulin is not added to the cells? Can you select a clone that is insulin independent?

Pak:

The isolation of insulin independent mutants has been tried successfully. However, the growth rates were considerably slower and since the cell line was intended for recombinant biopharmacological production, it was essential to get a fast growing cell.

Sasaki:

You add insulin to stimulate CHO cell growth or to maintain viability. Does bacitracin stimulate cell growth?

Pak:

No, bacitracin at high concentrations will inhibit cell growth. I included it in this talk to demonstrate that insulin-degrading enzymes were involved in preventing insulin expression in CHO cells. We have expressed ILGFI, and that escapes degradation and stimulates cell growth.

Aunins:

I want to clarify a previous answer on insulin mutants. As I understand it, you did your selection in just G418. Did you have insulin present when selecting your clones?

Pak:

It was present.

Ryll:

With insulin being a secreted protein targeted to the Golgi, how do you envisage the intracellular cytosolic activity as being responsible for the degradation?

Pak:

Although the majority of the insulin-degrading activity is found in the cytosol, there are insulin-degrading enzymes in the cell membrane and also in the culture supernatant. Those enzymes were also preventing the accumulation of insulin extracellularly.

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Bader:

You describe how the insulin is taken up by receptors and goes into an endosome, and is then suddenly degraded by a cytosolic enzyme. How is that possible? Why would insulin not permeate through the membrane? What is your definition of cytosolic?

Pak:

Although insulin is internalised through vesicles, these enzymes have been shown to be permeable and do enter and cleave these molecules.

Meade:

I am interested to know why someone is interested in making insulin in mammalian cells since E. coli produces it so well. We tried to do it in milk and got 20 mg/ml. The animals get in trouble but we had no problems in getting it secreted out. So does it just happen in a tissue culture system rather than in a natural system?

Pak:

Endocrine cells are quite capable of producing insulin but, unfortunately, CHO cells are not endocrine, therefore they lack the necessary functions to easily secrete insulin.

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HIGH YIELD EXPRESSION OF RECOMBINANT PLASMA FACTORS: USE OF RECOMBINANT ENDOPROTEASE DERIVATIVES IN VIVO AND IN VITRO U. SCHLOKAT1,3, A. PREININGER 1 , M. HIMMELSPACH 1 , G. MOHR 1 , B. FISCHER 2 , AND F. DORNER Departments of Molecular Cell Biology1 and Protein Chemistry2, Biomedical Research Center, IMMUNO AG, Uferstr. 15, 2304 Orth/ Donau, AUSTRIA; for correspondence3: e-mail [email protected]; fax +43-2212-2716; phone +43-1-20100-4144

1. Abstract Posttranslational proteolytic processing steps are often required to render proteins mature and functionally active. However, upon high yield expression of recombinant forms of these proteins, proteolytic processing becomes severely l i m i t i n g . In this report, the mammalian endoprotease Furin was used in order to achieve complete processing of recombinant von Willebrand Factor and Factor X precursors. Significant processing of rvWF was found to be mediated primarily by naturally secreted 'shed' rFurin accumulating in the cell culture supernatant, rather than intracellularly, upon coexpression. Coamplification of rvWF/rFurin resulted in increases in rvWF yield but, at best, maintenance of rFurin expression, implying that rFurin becomes lethal to its host cell upon overexpression. In order to establish a system allowing for the processing of large quantities of recombinant protein precursor molecules with comparably small amounts of rFurin molecules in vitro, C-terminally truncated, epitope-tagged rFurin derivatives were constructed. The fate of these molecules upon expression was investigated and suitable candidates were purified to homogeneity by a one step procedure. These molecules were used for in vitro processing in solution, in reactions consisting solely of defined components, and allowing for recycling and re-use of the rFurin. Initial attempts towards the development of a downstream processing system employing matrix bound rFurin indicate that complete proteolytic processing of large amounts of precursor molecules by small quantities of rFurin is feasible. 2. Introduction High yield expression of desired biopharmaceuticals is routinely achieved by amplification of the foreign gene/cDNA copies in their host cell. Unfortunately, posttranslational modifications, such as or proteolytic processing, which frequently are required for maturation and/or exertion of functional activity, often become limiting at overexpression. Human von Willebrand Factor (vWF) and Factor X (FX) are glycoproteins circulating in the blood which exhibit pivotal functions in haemostasis: vWF mediates adhe69 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 69-76. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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sion of platelets to the subendothelium at the site of injury, is involved in platelet-platelet interactions, and stabilizes circulating Factor VIII. The conversion of FX to FXa is a mandatory reaction in the coagulation cascade ultimately leading to the formation of the fibrin clot. vWF and FX require proteolytic removal of a propeptide for maturation; in addition, processing of the single chain precursor to the light and heavy chains is necessary in order to render FX functionally active. Upon overexpression of recombinant vWF 1 (rvWF) and these proteolytic cleavage reactions become limiting. The mammalian endoprotease Furin 3 has recently been shown to mediate processing of a variety of plasma proteins, e.g. rFIX and rProtein C precursors, C-terminal to the consensus cleavage site arg - x - lys/arg - arg. Here, we describe the construction, expression, and fate of full length and truncated rFurin molecules and their use in order to achieve complete processing of desired precursor molecules in vivo upon coexpression as well as in vitro. 3. Results

3.1. PROTEOLYTIC PROCESSING IN VIVO: CO-EXPRESSION OF RvWF AND WILDTYPE RFURIN CHO cell clones expressing rvWF were established; these cells secreted x day. As shown in Fig. 1, only propeptide free, completely processed rvWF molecules were detectable in the conditioned medium derived from individual cell clones. Upon dihydrofolate reductase/methotrexate mediated amplification, rvWF expression increased to x day at At this expression level, however, propeptide removal by the endogenous proteolytic machinery had become insufficient: approximately 50% of the secreted rvWF molecules still possessed the propeptide (Fig. 1 A).

In an attempt to improve propeptide removal in intermediate yield CHO-rvWF cells, a CHO-rvWF cell clone, secreting x day, was further transfected with a vector mediating human wild-type, full length rFurin expression. Cell clones isolated from this transfection exhibited different degrees of rvWF precursor processing due to their varying degrees of individual rFurin expression (Fig. 1B). A CHO-rvWF/rFurin cell clone exhibiting complete rvWF precursor processing in 24 hour supernatants was further characterized4. Unexpectedly, significant amounts of rvWF precursor molecules were detectable upon frequent media changes, e.g. every 8 hours, but the precursor was absent from subsequent 24 hour supernatants (Fig. 2, top left panel) 5 . In contrast, 8 hour and 24 hour conditioned media derived from CHO-rvWF cells did not reveal any significant changes in the rvWF precursormature rvWF ratio when compared to each other (Fig. 2, top right panel).

In conditioned media derived from CHO-rvWF/rFurin cells, significant amounts of rFurin were identified (Fig. 2, lower left panel). Even though Furin resides, via its trans-membrane domain, mainly in the trans-Golgi network, a naturally secreted form termed 'shed' Furin has recently been identified6-8. This form results from proteolytic cleavage N-terminal to its transmembrane domain. The degree of rvWF propeptide removal correlated well with the amount of 'shed' rFurin in the conditioned media. Subsequent mixing experiments employing conditioned media containing rvWF precursor molecules and shed rFurin confirmed that the 'shed' rFurin mediated

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rvWF propeptide removal in vitro 5 , contrasting previous reports by others using an experimentally truncated and slightly larger rFurin molecule 9,10. By titration western blotting, rFurin levels were found to exceed endogenous Furin levels by at least a factor of 100 (not shown). Since intracellular rFurin did not noticeably contribute to processing despite this enormous overexpression, the enzyme might either be miscompartmentalized or non functional/silenced, as long as it is located intracellularly, and activated or accessible only once it is extracellular 11.

A variety of attempts to establish CHO-rvWF/rFurin high yield coexpressing clones upon rvWF/rFurin/dhfr triple transfection and subsequent amplification failed. While the rvWF yield could be improved, rFurin expression always uncoupled, resulting in expression of propeptide containing rvWF molecules, although at higher yields (not shown). Presumably, the failure to increase the rFurin yield any further resulted from rFurin becoming lethal to its host cell upon overexpression 12 . The ability to process large amounts of rvWF precursor therefore requires the establishment of an efficient in vitro processing procedure. 3.2. PROTEOLYTIC PROCESSING IN VITRO: USE OF RFURIN DERIVATIVES In order to produce sufficient quantities of rFurin for purification, a variety of C-terminally truncated rFurin molecules were constructed. To test the function of these molecules, they were transiently expressed in 293 HEK cells, as summarized in Fig. 3. Removal of the transmembrane domain that anchors Furin in the membrane of the trans-Golgi network resulted in enhanced secretion of rFurin molecules into the cell

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culture supernatant. Interestingly, rFurin molecules with deletions affecting amino acids N-terminal to amino acid residue 577, i.e. the 'middle' domain , were strongly expressed intracellularly; however, secreted forms of these derivatives were not detected in the conditioned medium, indicating either blockage of secretion or, alternately, rapid degradation during the secretory process.

Expression of resulted in the secretion of two molecular species, visualized as a double band in western blots. The lower/faster band migrated at a position identical to that exhibited by shed rFurin. Thus, it can be concluded that the cleavage site in rFurin leading to shed rFurin is located in the vicinity of and N-terminal to amino acid 708 (not shown). When permanently expressed in CHO cells, trans membrane domain deleted rFurin molecules generally led to 10 to 20 fold increased amounts of secreted rFurin compared to wt/shed rFurin expression.

In order to facilitate purification, poly-histidine epitopes were added to the C-termini of the deletion mutants and separated from the residual rFurin sequence by spacers of varying length. Interestingly, one construct in which eight histidines were substituted with a threonine/isoleucine rich stretch of amino acids was not secreted any longer. Thus, very few amino acids at the C-terminus may decide upon the molecules' fate and/or intracellular destination. cell clones were established, and from serum free conditioned media was purified to homogeneity on a matrix by a one step procedure as seen in Fig. 4A. The peak functional activity, determined by a fluorogenic Furin specific substrate test5, and the Furin immunoreactive material in a western blot coincided with the 200mM and 500mM

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imidazole eluates; Furin is the only protein band in these fractions, as visualized by

silver staining.

After dialysis to remove the imidazole, purified and rvWF precursor were mixed in a buffer containing ions. Under these defined minimal conditions, rvWF precursor was successfully processed (Fig. 4B). A mockpurified preparation from unmanipulated CHO cells did not mediate rvWF precursor processing.The polyhistidine tail allows for easy re-purification of the rFurin derivative upon completion of the processing reaction. Thus, this enzyme can be 'recycled'.

In this manner, small quantities of rFurin can be used for the maturation of large amounts of recombinant target molecules. Similarly, both rFX propeptide removal and the processing of the single chain precursor to the heavy and light chains were successfully performed by rFurin derivative in vitro. Strikingly, the rFX propeptide, which does not contain a typical Furin site, is cleaved by a different endoprotease, as determined by expression in Furin deficient CHO cells and subsequent purification2. With the aim of establishing an industrial scale in vitro processing procedure, the histidine tagged rFurin molecules were tested for their functional activity when bound to the resin. Initial experiments employing matrix bound and a Furin specific fluorogenic substrate5 demonstrated successful substrate conversion and, hence, the feasibility of this approach. Optimization of the length and sequence of the spacer between the epitope and the residual rFurin sequence, and choice of resin, e.g. tentacle-gels, will allow proteoly-

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tic maturation of large amounts of incompletely processed recombinant protein precursors by small quantities of rFurin.

4. Summary

In high yield CHO-rvWF cell clones, removal of the propeptide from rvWF precursors was incomplete. Additional wild-type, full length rFurin expression in these cells improved processing. Unexpectedly, complete rvWF precursor processing was mediated by a naturally secreted form of rFurin, termed 'shed' rFurin, in the conditioned medium. The contribution of intracellular wild-type rFurin was limited despite tremendous overexpression of this enzyme compared to endogenous Furin. Attempts to further improve rFurin expression by amplification did not meet with success, presumably due to toxicity of rFurin to its host cell upon overexpression. In contrast, rvWF amplification could successfully be demonstrated. In order to achieve complete processing of large amounts of rvWF precursor with comparably small amounts of rFurin, the use of rFurin for in vitro processing was

investigated. A variety of C-terminally truncated and, hence, secreted, epitope-tagged rFurin derivatives were constructed and expressed. The extent of the truncation, the type and length of the spacer between the residual rFurin sequence and the epitope, and the amino acids constituting the new C-terminus determined the fate of the

rFurin derivatives upon expression and their individual properties. Using a one step

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procedure, rFurin derivatives were purified to homogeneity and used in in vitro recombinant target protein precursor processing reactions consisting solely of defined components. Preliminary experiments have demonstrated the feasibility of resinbound rFurin mediated in vitro processing. 5. Acknowledgements We are grateful to Wim van de Yen, John Creemers, and Stephen Leppla for material, Gary Thomas, Nabil Seidah, Anton Roebroek and Wolfgang Garten for discussions, and Carol P. Gibbs for critical reading of the manuscript. 6. References 1. Fischer B, Mitterer A, Schlokat U, DenBouwmeester R, Dorner F (1994). Structural analysis of rceombinant von Willebrand factor: identification of hetero- and homonmultimers. FEBS Lett. 351: 345 - 348. 2. Himmelspach M, Pfleiderer M, Fischer B, Antoine G, Gritschenberger W, Falkncr FG, Schlokat U, Dorner F. Recombinant Human Factor X: High Yield Expression And Maturation By Furin Mediated In Vitro Processing. Submitted. 3. Van de Ven WJM, Voorberg J, Fontijn R, Pannekoek H, van den Ouweland AMW, van Duijnhoven HLP, Roebroek AJM, Siezen RJ (1990). Furin is a subtilisin-likc pro-protein processing enzyme in higher eucaryotes. Mol. Biol. Rep. 14: 265 - 275. 4. Fischer BE, Schlokat U, Mitterer A, Reiter M, Mundt W, Turecek PL, Schwarz. HP, Dorner F (1995). Structural analysis of recombinant von Willebrand factor produced at industrial scale fermentation of transformed CHO cells co-expressing rccombinant furin. FEBS Lett. 375: 259 - 262. 5. Schlokat U, Fischer BE, Herlitschka S, Antoine G, Preiningcr A, Mohr G, Himmelspach M, Kistncr O, Falkner FG, Dorner F (1996). Production of highly homogeneous and structurally intact recombinant von Willebrand Factor multimers by furin mediated pro-peptide removal in vitro. Biotechnol. Appl. Biochem. 24: 257 - 267. 6. Vey M, Schäfer W, Berghöfer S. Klenk HD, Garten W (1994). Maturation of the trans-Golgi Network Protease Furin: Compartmentalization of Propeptide Removal, Substrate Cleavage, and COOH-terminal Truncation. J. Cell Biol. 127: 1829 - 1842. 7. Molloy SS, Thomas L. VanSlyke JK, Stcnherg PE, Thomas G (1994). Intracellular trafficking and activation of the furin proprotein convertase: localization to the TGN and recycling from thc cell surface. EMBO J. 13: 18 - 33. 8. Vidricaire G, Denault JB, Leduc R (1993). Characterization of a secreted form of human furin endoprotease. Biochem Biophys. Res. Comm. 195: 1011 - 1018. 9. Rehemtulla A, Kaufman RJ (1992). Preferred Sequence Requirements for Cleavage of Pro-von Willebrand Factor by Propeptide-Processing Enzymes. Blood 79: 2349 - 2355. 10. Rehemtulla A, Dorner AJ, Kaufman RJ (1992). Regulation of PACE propeptide-processing activity: Requirement for a post-cndoplasmic reticulum compartment and autoproteolytic activation. Proc. Natl. Acad. Sci. USA 89: 8235 - 8239. 11. Anderson ED, VanSlyke JK, Thulin CD, Jean F, Thomas G (1997). Activation of the furin endoprotease is a multiple-step process: requirements for acidification and internal propeptide cleavage. EMBO J. 16: 1508 - 1518. 12. Creemers JWM (1994). Structural and Functional Characterization of the Mammalian Proprotein Processing Enzyme Furin. Ph.D. Thesis at the University of Leuven, Belgium.

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Discussion

Massie:

The over-expression of furin seems to result in some sort of secretion of the protease in the medium. Do you have any idea why; for instance, is the Golgi saturated or is some other type of event occurring?

Schlokat:

There is a cleavage site. It is not an auto-proteolytic cleavage by furin. If you express wild type furin at some point it is a natural process for furin to be secreted from the cell. In CHO cells even without the expression of any additional furin, just endogenous furin, you have secretion of furin-like activity which mediates the processing of furin-like substrate in the conditioned medium. So it is just part of a natural process.

Massie:

Have you considered using an inducible system to over-express furin at sufficient levels to overcome the toxicity effect?

Schlokat:

It is an alternative but we think it is better to do it just in vitro.

Pak:

My experience is that furin is expressed intracellularly. Could the detection of furin in the supernatant be a result of cell lysis rather than secretion?

Schlokat:

I do not think so as there are such massive amounts of furin in the supernatants, even when you grow cells at low densities where there is no detectable lysis. It is not the wild type furin but a shorter molecule. The trans-membrane domain of the wild type furin molecules is missing. If what you suggest is happening, you would see the whole molecule including the trans-membrane domain. So I do not think it is lysis.

THE ROLE OF CELL CYCLE IN DETERMINING LEVELS OF GENE EXPRESSION IN CHO CELLS

D.R. LLOYD, V. LEELAVATCHARAMAS, D.C. EDWARDS, A.N. EMERY & M. AL-RUBEAI Centre for Bioprocess Engineering, School of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.

1. Abstract

Understanding relationships between cell cycle and protein expression is critical as a means to optimising media and environmental conditions for successful commercial operation. Using flow cytometry and centrifugal elutriation together with synchronisation techniques, the possible role of CHO cell cycle dependent features in product expression was evaluated. The production of . was found to be time dependent. Maximum specific productivity was achieved at the mid-exponential phase of batch culture and was always associated with maximum cellular concentration of protein and a high proportion of S phase cells. However, in experiments using sorted S and phase cells the apparent correlation between S phase and productivity was shown not to be causal since there was no significant difference in specific productivity between the different cell cycle phases. 2. Introduction

Growth of mammalian cell cultures is determined by cell cycle duration, cell death rate and the number of dividing (mitotic) cells. The cell cycle state of a culture can be deconvoluted by flow cytometry. Although the duration of the cell cycle or its individual phases may vary between different cell lines, cell cycle events always occur in sequence and checkpoints ensure that if preceding events are not completed correctly, the cell is arrested and will eventually die. Defining the relationship between cell cycle phase and protein expression is crucial to understanding the dynamics of product elaboration which, in turn, must be incorporated in culture protocol design for successful commercial operation. The relationship between cell cycle and protein expression varies with cell line and gene expressed. The aim of this work is to determine the relative effects of cell cycle phase and culture environment on the (specific) productivity of recombinant CHO cells. 77 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 77-79. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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3. Materials and Methods

Recombinant CHO cells were grown in stirred batch suspension cultures at 37°C; CHO 320, producing human was grown in RPMI 1640 & 5% FCS & 1 µM methotrexate (MTX); CHO tF70R, producing human tissue plasminogen activator (t-PA), was grown in a proprietary serum free medium (Biopro 1, BioWhittaker, Belgium). Both constructs use the SV40 promoter/enhancer. Cultures examined were either synchronised by late exponential nutrient deprivation or phase enriched fractions separated using a Beckman JE-6B centrifugal elutriation system with a standard chamber.

Product was measured by ELISA; using Genzyme DuoSeT kit product number 803932-00 for and Biopool Imulyse tPA kit product number 101005 for tPA. The DNA content of propidium iodide (PI) stained, RNAse treated, ethanol fixed cells was measured by flow cytometry (Coulter EPICS Elite). Cell cycle analysis was performed using Multicycle software (Phoenix Flow Systems). Cell size was determined either by Coulter principle using a Coulter Multisizer or as relative to flow

cytometric forward angle light scatter (FS). After staining formaldehyde fixed cells with was determined as

FITC conjugated anti-human intracellular the ratio measurement of FITC: FS by flow cytometry.

4. Results 4.1. CHO 320 SYNCHRONIZED BY NUTRIENT DEPRIVATION

Intracellular interferon concentration & specific productivity profiles follow that of percentage of S phase cells but also follow the growth curve. Thus, intracellular interferon concentration and specific productivity are related both to the cell cycle and culture growth phases. 4.2. APPLICATION OF CENTRIFUGAL ELUTRIATION TO THE STUDY OF CELL CYCLE RELATED PRODUCTIVITY

Cultures separated into fractions on the basis of size, allowing preparation of phase enriched samples which all have the same culture history and whose physiology has not been disturbed by nutrient starvation or chemical synchronisation techniques. 4.3. PRODUCTIVITY OF ELUTRIATED FRACTIONS Elutriated CHO tF70R fractions were cultured for 4 hours and the specific productivity/cell/ hour was determined (Figure 1). Preliminary observations that

79

productivity was related to cell cycle phase proved groundless on further investigation.

No fraction was shown to have a specific productivity which was significantly different (p=0.03) from that of any other fraction. Overall, the mean specific productivity was 43 fg/cell/hr. These results also indicate that the SV40 promoter in the construct has not influenced the regulation of any cell cycle dependant protein production mechanism.

5. Conclusions

Gene expression (intracellular product concentration) may be partly cell cycle related because it varies with cell cycle phase and with batch phase; the same was thought to be true of specific productivity. However this work, using centrifugal elutriation, has shown that the cell cycle phase of a population has no significant effect on the specific productivity of that population. The apparent cell cycle association seen previously in batch culture is thus shown to be a coincident effect of batch phase transition from lag to exponential, rather than a causal relationship. Thus it must be the batch phase, possibly due to the environmental conditions (medium condition), which is the major determinant of recombinant CHO productivity. Further work will confirm these observations and clarify the effect of different promoters on cell cycle/size related specific productivity. 6. Acknowledgements This work is supported by the BBSRC UK and EC Framework IV programme.

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HETEROGENEITY WITHIN DHFR-MEDIATED GENE AMPLIFIED POPULATION OF CHO CELLS PRODUCING CHIMERIC ANTIBODIES No Soo Kim, Sang Jick Kim, and Gyun Min Lee Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Kusong-Dong, Yusong-Gu, Taejon 305-701, Korea ABSTRACT Recombinant Chinese hamster ovary (CHO) cells expressing a high level of chimeric antibody were obtained by cotransfection of heavy and light chain cDNA expression vectors into dihydrofolate reductase (dhfr) deficient CHO cells and subsequent gene amplification in medium containing stepwise increments in methotrexate (MTX) level such as 0.02, 0.08, 0.32, and 1.0 µM. In order to determine the clonal variability within amplified cell population in regard to antibody production stability, 20

subclones were randomly isolated from amplified cell population at 1 µM MTX and their antibody production stability was characterized. During 8 weeks of cultivation in the absence of selective pressure, the specific antibody productivity decreased significantly. However, the relative extent

of decrease in was varied among subclones, ranging from 30 % to 80 %, showing that clonal analysis allows one to screen the clone with better stability. Southern and Northern blot analyes showed that this decreased resulted mainly from the loss of amplified immunoglobulin (Ig) gene copies and their respective cytoplasmic mRNAs. Furthermore, since the stability of subclones regarding is not related with either their or Ig gene copies at the beginning of long-term culture,

their stability during long-term culture cannot be predicted based on their initial

or Ig gene copies.

INTRODUCTION The degree of gene amplification, in most cases, is proportional to the level of gene expression (Pensde

et al., 1992). When subjected to successive rounds of selection in medium containing stepwise increments in methotrexate (MTX) concentration, recombinant CHO (rCHO) cells with specific

antibody productivity as high as were obtained by Page and Sydenham (1991). Not only the expression level but the stability of cell lines used is critical for large-scale production of recombinant antibodies. When drug-resistant cells are propagated in the absence of the selective agent, the amplified genes may be maintained or lost. Changes in rCHO populations after extended culture in the presence and absence of selective pressure have been reported (Kaufman et al., 1985; Sinacore et

al., 1995; Weidle et al., 1988; Zettlmeissl et al., 1987). Most of these studies were based on the average cell population. However, CHO cells become quite heterogeneous in regard to

in the course of

dhfr-mediated gene amplification, though they are derived from a single parental clone (Kim et al., 1997). Likewise, their stability during long-term culture may also be heterogeneous. If so, the clonal analysis of the entire cell population allows one to screen the clone with better stability. However, it requires extensive efforts. If the initial properties of clones such as specific growth rate (µ) and gene copy numbers help predict their stability, lots of efforts in screening a stable clone will be saved. MATERIALS AND METHODS Cell line and Cell Culture

Parental CHO cells expressing a chimeric antibody directed against the S surface antigen of hepatitis B virus (HBV) were made by cotransfecting the light and heavy chain expression plasmids into dhfr CHO cells (DG44). Amplified 20 subclones investigated in this study were randomly isolated from

high producer CS 13-1.0 resistant at 1.0 µM MTX using limiting dilution method. For long-term culture in the absence of MTX, 20 subclones were cultivated as monolayer in 6 well tissue culture plates containing 3 mL of with 5 % dialyzed FBS in mixture, humidified at 37°C. The viable cell concentration were determined by the trypan blue dye exclusion method and samples for antibody, nutrient, and metabolic assays were taken every other passage and kept frozen at -80 °C. Chemical and Antibody Assays Glucose and lactate concentration were measured using a glucose/lactate analyzer (Yellow Spring Instruments, Model 2300 STAT) and secreted chimeric antibody was quantified using an ELISA

81 O.-W. Merten el al. (eds.), New Developments and New Applications in Animal Cell Technology, 81-83. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

82 Flow Cytometry Intracellular antibody content was determined using a FACScan flow cytometer (Becton Dickinson). FITC-labeled goat anti-human IgGs specific for heavy chain was used to stain the cells. Southern Blot Analysis Chromosomal DNAs of subclones at the beginning and end of long-term culture were prepared using protocols described in kit manual (NEB). Heavy and light chain cDNAs (1,600 and 900 bp, respectively) were used as templates for heavy and light chain probes, respectively. The probes were labeled by randomly primed incorporation of DIG-labeled deoxyuridine-triphosphate. Prehybridization, hybridization, and subsequent colorimetric detection were conducted as described in the DIG user's guide (BM). The gene copy number was determined by comparison of band intensities on each Southern blot using a UltraScan laser densitomer. Northern and Slot Blot Analysis

For Northern and slot blottings, cytoplasmic mRNA was isolated from each subclone at the beginning and end of culture using NP-40 method. For analyses, 5 µg mRNAs of each subclone were loaded and other procedures were performed as described in Southern blotting. Evaluation of Specific Growth Rate, Specific Antibody Productivity, and Specific Glucose Consumption Rate and Lactate Production Rates Specific growth rate (µ), specific antibody productivity and specific glucose consumption and lactate production rates were evaluated as described earlier. RESULTS AND DISCUSSION

1. Changes in µ and of Subclones during Long-term Culture Clonal analysis shows that CS13-1.0 cells became quite heterogeneous in regard to µ and in the course of dhfr-mediated gene amplification, though they were derived from a single clone. No significant change in µ was observed during long-term culture. However, the of all 20 subclones significantly decreased during the culture. The average of 20 subclones at the end of long-term culture was which is approximately 47 % of the initial average The

antibody production characteristics were varied among the subclones, though the of all 20 subclones decreased during the culture. The of A6, which was most stable, gradually decreased for first 20 days and thereafter was stabilized at The of D1, which was most unstable, gradually decreased throughout the culture and was at the end of long-term culture. 2. Characterization of Subclones by Southern Blot Analyses In order to investigate whether clonal variability with respect to production stability is related to the variation of changes in immunoglobulin (Ig) gene copy numbers of subclones during culture, genomic DNA isolated from 20 subclones at the beginning and end of long-term culture was characterized by

Southern blotting. With heavy and light chain probes, 2.3 kb and 7.1 kb major band were detected, respectively. The relative gene copy numbers of all 20 subclones were summarized in following table. In general, the decrease in of most subclones except for a few subclones was mainly due to the loss of heavy and light chain gene copies during long-term culture. However, the extent of decrease in gene copy numbers was varied significantly among the subclones, suggesting that the initial gene copy numbers of subclones do not help predict their long-term production stability. 3. Characterization of Subclones by Northern and Slot Blot Analyses Northern and slot blotting were carried out in order to quantify the relative heavy and light chain mRNA content of subclones at the beginning and end of long-term culture. In Northern analysis, the major bands of heavy and light chain mRNA were positioned at the similar size of 18s rRNA and below 18s rRNA, respectively. On the whole, a trend obtained in the quantification of the heavy and light chain mRNA content of subclones was similar to that in 4. Characterization of Antibody Production Stability of Subclones by Flow Cytometry

In order to assess the degree of population heterogeneity after long-term culture, the intracellular antibody content of each subclone at the beginning and end of long-term culture was measured by flow cytometry. The changes in intracellular antibody contents of stable A6 and B3 subclones after long-

term culture were insignificant. On the other hand, the intracellular antibody content of unstable D4 and C5 subclones decreased significantly. Accordingly, the changes in intracellular antibody contents of subclones correlate with those in suggesting the potential of flow cytometric measurement of intracellular antibody as a useful tool determining production stability during long-term culture.

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CONCLUSIONS 1. CHO cells become quite heterogeneous in regard to antibody production stability in the course of dhfr-mediated gene amplification, though they are derived from a single parental clone 2. Clonal analysis enables one to screen the stable clone. 3. The antibody production stability of each clone cannot be predicted based on itsinitial and Ig gene copy numbers. REFERENCES 1. Kaufman. R.J., Wasley, L.C., Spiliotes, A.J., Gossels, S.D. Latt, S.A., Larsen, G.R., Kay, R.M. 1985. Coamplification and coexpression of human tissue-type plasminogen activator and murine dihydrofolate reductase sequences in Chinese hamster ovary cells. J. Mol. Biol. 5:1750-1759. 2. Pendse, G.J., Karkare, S., Bailey, J.E. 1992. Effect of cloned gene dosage on cell growth and hepatitis B surface antigen synthesis and secretion in recombinant CHO Cells. Biotechnol. Bioeng. 40: 119-129. 3. Page, M.J., Sydenham, M.A. 1991. High level expression of the humanized monoclonal antibody Campath-lH Chinese hamster ovary cells. Bio/Technology 9: 64-68. 4. Sinacore, M.S., Brodeur, S., Brennan, S., Cohen, D., Fallon, M., Adamson, S.R. 1995. Population analysis of a recombinant Chinese hamster ovary cell line expressing recombinant human protein cultured in the presence and absence of methotrexate selective pressure, pp 63-67. In: E.C. Beuvery, J.B. Griffiths, and W.P. Zeijlemaker (eds.), Animal cell technology, Kluwer Academic Publishers, Dordrech. 5. Weidle, U.A., Bucke, P., Wienberg, J. 1988. Amplified expression constructs for human tissue-type plasminogen activator in Chinese hamster ovary cells: instability in the absence of selective pressure. Gene 66:193-203. 6. Zettlmeissl, G., Ragg, H., Karges, H. 1987. Expression of biologically active human antithrombin I I I in Chinese hamster ovary cells. Bio/Technology 5: 720-725.

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IMMUNOFLUORESCENCE DETECTION OF RECOMBINANT MONOCLONAL ANTIBODY AS A TOOL IN THE CHARACTERIZATION OF CHINESE HAMSTER OVARY CELL LINES

CHERYL ANN S. KINNEY, ROBIN E. TRALA, LINDA C. HENDRICKS, and TIMOTHY D. HILL SmithKline Beecham Pharmaceuticals, Biological Process Sciences, PO Box 1539, King of Prussia, PA 19406, USA

1. Abstract A highly sensitive and specific method was developed for direct immunofluorescence analysis of recombinant Chinese Hamster Ovary (rCHO) cells expressing IgG monoclonal antibody (mAb). Fluorescence signal-to-noise ratio using labeled goat anti-human IgG was improved through optimization of fixation, staining and mounting protocols. These improvements enabled measurement of relative cell to cell differences and the detection of non-producing cells when they represented only 2.5% of a mixed cell population. Heterogeneity of fluorescence signal with both intense and low-staining cells was noted for some rCHO cell lines. Low mAb-specific signal was unrelated to cell viability since low-staining cells excluded the viability indicator ethidium monoazide bromide. Furthermore, positive staining of the soluble ER resident protein BiP in dual labeling studies with anti-mAb reagents indicated that low level staining was not an assay artifact related to poor access of fluorescent antibody into secretory compartments. Correlations of signal heterogeneity to long-term culture stability and process performance is under investigation. 2. Introduction CHO cells are a convenient expression system for the generation of protein biopharmaceutical therapeutic agents. Productivity levels for monoclonal antibodies (mAb) achieved in rCHO effectively compete with traditional hybridoma/myeloma cell systems, thus enabling rCHO to serve as an economical antibody production system. In addition, although rCHO contain non-infectious retroviral-like particles, they have been extensively characterized and found free of adventitious viruses, making them a safe substrate for production of biopharmaceuticals. Cell lines developed for industrial processes often vary in productivity and clonal stability. Current methods for evaluating rCHO cells rely on whole population 85 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 85-92. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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measurements, such as supernatant titer by HPLC or ELISA, and typically require optimization of growth conditions and qualified assays, while providing no information about homogeneity of cell populations. Immunofluorescence microscopy methods were developed to rapidly profile individual cells within a population, regardless of culture conditions. Through use of immunofluorescence, information critical to selection of the lead process cell line may be available earlier in the development process, before costly development resources are committed. 3. Methods 3.1 CELL LINE

Suspension-adapted host CHO cells (DG-44 clone as described by Urlaub et. al., 19831) and genetically modified recombinant CHO (rCHO) were maintained in SB proprietary medium at a minimum culture viability of 85%. The gene-minus control cell line was generated by transfection with plasmids lacking antibody genes. 3.2. CELL PREPARATION, FIXATION AND PERMEABILIZATION

3.2.1. Slide Preparation

HTC™ coated slides (Cel-Line Assoc., New Field, NJ, 4mm wells) were pre-treated for 2 min at room temperature with 2% (v/v) 3-amino-propyl silane (Sigma Chemical , St. Louis, MO) diluted in acetone, washed 3X in water for injection (WFI), then dried 2hr at 37°C. 3.2.2. Cell Attachment Cells were washed 1X with Dulbecco's phosphate buffered saline (PBS), then cells were added to slide wells and incubated in a humidified environment for 30 minutes at 37°C. PBS was carefully aspirated prior to fixation. 3.2.3. Fixation/Permeabilization

Cells were fixed for 15 minutes at room temperature in 1% buffered paraformaldehyde (ultrapure, Polysciences,Inc.,Warrington, PA), washed twice in PBS, then permeabilized by 2 minute treatment with 0.2% Triton X-100 in PBS, followed by a 5 minute wash in PBS. To reduce background signal associated with free aldehydes, cells were treated for 5 minutes in 0.1M glycine

(J.T. Baker, Phillipsburg, NJ) in PBS, followed by two washes in PBS. To enhance reagent uptake, cells were dehydrated stepwise in ethanol (50%, 70%, 95%, and 100%) at room temperature. Slides were air dried prior to addition of probe.

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3.3. IMMUNOFLUORESCENCE STAINING

3.3.1. Antibodies and Reagents Cy3-conjugated goat anti-human IgG (Fc specific) was obtained from Sigma

ImmunoChemical, St. Louis, MO. Cy2-conjugated goat anti-human IgG (Fc specific) and Cy3-conjugated goat anti-mouse IgG (H&L) were obtained from Jackson ImmunoResearch, West Chester, PA. Mouse anti-rat Grp78(BiP) was obtained from StressGen Biotechnologies, Victoria, BC. Ethidium monoazide bromide (EMA), a fluorescent photoaffinity label used as a viability indicator, was purchased from Molecular Probes, Eugene, OR. Cy3 and Cy2 are registered trademarks of Amersham International, Inc.

3.3.2. Direct Detection of Intracellular Antibody Cy2- or Cy3-conjugated anti-IgG probes were diluted in PBS containing 1% heatinactivated (65°C, 1hr) fetal bovine serum (FBS). Probe was applied to permeabilized samples on slides and incubated 30 minutes at 4°C in the dark, followed by wash in 1% FBS at 4°C, wash in cold PBS, and final wash at room temperature in WFI. 3.3.3. DNA staining with Ethidium Monoazide Bromide(EMA) EMA is a fluorescent photoaffinity label that after photolysis, binds covalently to nucleic acids in cells with compromised membranes.2 Viability assessment with EMA was performed prior to cell attachment and fixation. Cells were transferred to polypropylene

plates (Costar) on ice and incubated 15 min with 4ug/ml EMA dissolved in PBS, then exposed at 15 cm under a 13 watt fluorescent light source to activate EMA. Following exposure, cells were washed 2X in PBS, and processed for attachment, fixation,

permeabilization and immunofluorescent staining. 3.3.4. Indirect Detection and Dual Labeling Permeabilized cell samples were incubated 30 minutes at 4°C with anti-BiP antibody diluted in PBS containing 1% heat-inactivated FBS, then washed 2X in 1% FBS/PBS. Samples were incubated an additional 30 minutes at 4°C with secondary Cy3-anti-mouse IgG antibody, washed 2X in cold PBS and 1X in room temperature WFI. 3.3.5. Mounting of coverslips Residual WFI was removed from processed slides, then samples were mounted under coverslips using AntiFade reagent (Molecular Probes, Eugene, OR) according to manufacturers instructions. Slides were stored in the dark under low-humidity.

3.4. IMAGE ANALYSIS Images were acquired using a Nikon Optiphot epifluorescence microscope fitted with fluorophore-specific filter sets from Omega Optical (Brattleboro, VT), and captured at

1000X magnification using an IS-2000 CCD camera system and (Alpha Innotech, CA).

software

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4. Results

Immunofluorescence methods were developed to generate high resolution images of rCHO cell intracellular targets. Fluorescent-labeled antibodies specific for the Fc portion of human IgG were used to detect intracellular mAb in the secretory pathway of CHO cells. Figure 1A and C illustrate the high signal specificity of anti-mAb probes within secretory pathway compartments exemplified by fluorescent staining pattern typical of endoplasmic reticulum and Golgi elements. Non-transfected parental CHO or geneminus rCHO generated by transfection with plasmids lacking mAb genes were tested as negative controls and appeared as faint opaque spheres, lacking any definition of intracellular membranes and requiring extended exposure times for visualization (Fig 1B and D).

89

The limit of detection was determined by mixing up to 50% gene-minus negative control cells with CHO cells expressing mAb (Fig.2). Data demonstrated that the method is highly sensitive, capable of detecting a low level of negative cells (2-3 per 100) within a population, and that fluorescence intensity differences are readily distinguished across a population.

Clonally selected rCHO cells derived from lines genetically engineered to secrete mAb were characterized at the single cell level using anti-mAb immunofluorescence. Subpopulations of cells weak in fluorescence intensity, ranging from 5-50% were observed in various rCHO cell lines (Fig. 3). Low staining cells show structural patterns consistent with higher intensity cells(Fig. 3B). Detection of subpopulations varying in

mAb expression suggests that lines are unstable and /or no longer clonal. Thus, this technique may be useful, particularly early in development, to support cell line selection.

90

To test whether weakly stained or negative rCHO cells represent a non-vital subpopulation of the original culture, a dual labeling method was developed to simultaneously detect intracellular mAb with Cy2-antibody probe, and prefixation viability using EMA, a fluorescent photoaffinity label that, after photolysis, binds covalently to nucleic acids in cells with compromised membranes.2 EMA is excluded from viable cells. Cells weakly stained for mAb (Fig. 4A) were impermeable to EMA (Fig. 4B), indicating lack of mAb-specific fluorescence was not related to viability. In some cases cells were found containing dual label, demonstrating nonspecific adsorption of anti-mAb probe to dead cells (data not shown).

Since recombinant protein is synthesized at very high levels in transfected CHO it was necessary to determine whether low levels of staining were due to poor access of

fluorescently labeled antibody probes to 'saturated' secretory compartments. In addition, since detection of cells which stained weakly for mAb required long exposure times, a reagent was needed to allow visualization of all cells in a field. BiP (IgG heavy chain

binding protein, Grp78) is an ER lumenal protein which functions in post-translational

protein folding 3,4 . BiP was selected as a convenient marker to delineate cells and to test reagent accessibility to ER lumenal targets to determine whether low-/ non-staining cells were an assay artifact. rCHO cells were dual labeled to simultaneously probe mAb and BiP. Cells with low anti-mAb (Fig. 5C) signal had correspondingly high anti-BiP signal (Fig. 5D), demonstating reagent access to secretory compartments.

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5. Discussion

A highly specific and sensitive immunofluorescence method for intracellular characterization of individual rCHO cells has been developed utilizing Cy2- and Cy3-

labeled antibody probes and the viability indicator EMA. Targets detected include expressed mAb, constitutive BiP and viability status. Preliminary application of this method proved valuable in detecting unexpected heterogeneity of intracellular mAb

across a population of rCHO cells generated by clonal selection. Immunofluorescence offers the ability to rapidly profile cell lines at the single cell level during early stages of process development. Early detection of unstable and/or non clonal lines may allow channeling of costly development resources into cell lines of highest potential. In addition, work is underway to combine this technique with quantitative imaging analysis to allow prediction of cell line productivity very early in the development process.

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6. References 1

Urlaub, G., Kaas, E., Carothers, A.M., Chasin, L., (l983),Cell, 33:405-412.

2

Haughland, R.P., ed. Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Eugene, OR., 6:150.

3

Warren, G., (1987), Nature, 327:17-18. Bole, D.G. et al (1986) Posttranslational Association of Immunoglobulin Heavy Chain Binding Protein with Nascent Heavy Chains in Nonsecreting and Secreting Hybridomas, Journal of Cell Biology. 102:15581566.

4

ELECTROLYZED REDUCED WATER WHICH CAN SCAVENGE ACTIVE OXYGEN SPECIES SUPRESSES CELL GROWTH AND REGULATES GENE EXPRESSION OF ANIMAL CELLS.

S. Shirahata, S. Kabayama, K. Kusumoto, M. Gotoh, K. Teruya, K. Otsubo*, J.S. Morisawa*, H. Hayashi** and K. Katakura

Graduate School of Genetic Resources Technology, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-81, Japan; *Nihon Trim Co. Ltd., Meiji Seimei Jusou Building 6F, 1-2-13 Shinkitano, Yodogawa-ku, Osaka 352, Japan; **Water Institute, Nisshin Building 9F, 2-5-10 Shinjuku, Tokyo 160, Japan. Abstract:

Active oxygen species are considered to cause extensive oxidative damage to biological macromolecules, which bring about a variety of diseases as well as aging. Reduced water produced near cathode during electrolysis of water exhibits high pH, low dissolved oxygen, extremely high dissolved molecular hydrogen, and extremely negative redox potential values. Recently we found that strongly electrolyzed reduced water scavenges active oxygen species and protects DNA from oxidative damage (Shirahata, S. et al., Biochem. Biophys. Res. Commun., 234, 269-274 (1997)). Electrolyzed reduced water suppressed the growth of human normal fibroblast TIG-1, human lung adenocarcinoma A549, and human uterine cervix cancer HeLa, indicating that reduced water affects the signaling pathway of cell cycle. The expression of the interleukin-6 gene was enhanced by reduced water as well as ascorbic acid, (+)-catechin and tannic acid when added to the culture of human osteosarcoma MG-63 cells, suggesting that reduced water acts as a reductant to cells. Introduction

Active oxygen species such as singlet oxygen superoxide anion radical hydrogen peroxide and hydroxyl radical (OH) are considered to cause extensive

oxidative damage to biological macromolecules (DNA, membranes, enzymes and so on), which bring about a variety of diseases as well as aging (1, 2). Antioxidative enzymes such as superoxide dismutase, catalase, and glutathione peroxidase can scavenge active

oxygen species. Daily intake of antioxidants such as vitamin C, vitamin E, (+)-catechin is also important to protect cells from oxidative damage. In spite of these

protective mechanism, a chronic state of oxidative stress exists in cells because of an imbalance between prooxidants and antioxidants. Recent heavy environmental pollution

seems to strengthen oxidative stress to our bodies. Water is a most abundant compound on the earth and indispensable for existence of life. More than a half century ago, domestic devices to reform water have been developed in Japan. The principal is to separate reduced water near cathode from oxidized water near anode by semipeamiable membrane during electrolysis of water. Electrolyzed reduced water exhibits alkaline pH and negative redox potential. The devices to reform water are admitted as therapeutic devices by the Japanese ministry of public welfare because reduced water is effective to suppress abnormal fermentation in intestine and hyperacidity, although the action mechanism is unclear. Based upon the interesting clinical improvement of a variety of diseases by intake of reduced water, Hayashi proposed the hypothesis “water regulating theory’ since 1985 (3). Recently we found that electrolyzed reduced water contains high concentration of dissolved hydrogen (DH), scavenges active 93 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 93-96. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

94 oxygen species and protects DNA from oxidative damage (4). We proposed a new hypothesis that active hydrogen in reduce water may scavenge active oxygen. Here we report that electrolyzed reduced water suppresses the cell growth and regulates the gene expression in animal cells. Materials and methods

Electrolysis of water. Ultrapure water produced by an ultrapure system (ULTRAPUR LV-10T, TORAY, Tokyo) was added 0.1 g/l NaCl to elevate electrical conductance (EC) to about 20 ms/m. The water was then electrolyzed with various voltages (0 - 40 V) by an electrolyzing device (Type TI-7000S and TI-7000SL, Nihon Trim Co., Osaka) equipped with platina-coated titanium electrode to produce reduced water which exhibited various RP. Ultrapure water containing NaCl was sent to the electrolyzing device using a water current pump at a rate of 0.5 1/min to 1.0 l/min. Electrolyzing voltage was changed to several to several tens V and currents several to 10 A. RP, EC, dissolved oxygen (DO) and DH were measured using a RP meter (type, HM-14P), a EC meter (CM-14P), a DO meter (DO-14P) and a DH meter (DHDI-1) of Toa Electronics Ltd. (Tokyo) at 25°C. pH was measured using a pH meter (Beckman, Type pHI32) at 25°C. Assay of scavenging effect of reduced water against superoxide onion radical. The scavenging effect of reduced water against was examined by hypoxanthine-xanthine oxidase system using luciferine analog and a chemiluminometer as described previously (4). All reactions were performed in neutralized condition using 40 mM sodium phosphate buffer (pH 7.0). Measurement of growth curve of human normal and cancer cell lines. Normal human fibroblast cell line TIG-1, human lung adenocarcinoma cell line A549 and human uterine cancer cell line HeLa were inoculated into 24-well microplates and cultivated in 10% fetal calf serum-MEM medium containing electrolyzed reduced water at 37°C under an atmosphere of 5% Since strongly electrolyzed reduced water often contains HOCl which was produced near anode, the effects of reduced water on the cell growth were compared with those of HOCl of the same concentration. Tap water must contains more than 0.1 ppm of residual chlorine. The concentration of HOCl was determined by otolidine method. Analysis of the interleukin-6 gene expression in MG-63 cells. Human osteosarcoma MG-63 cells were cutured in 5% FBS-MEM medium at 37°C under an atmosphere of 5% The cells were treated in PBS (+) containing 2.7% reduced water, 0.1 µM ascorbic acid, 0.33 µg/ml (+)-catechin or 40 µM tannnic acid in the presence of for 2 hour. RT-PCR was performed after extraction of mRNA. Results and discussion

Characteristic of electrolyzed reduced water Strongly electrolyzed reduced water exhibited high pH, low DO, high DH and extremely negativeRP values. Marked changes in these values occur in water after electrolysis. For example, the value of RP +350 mV in water before electrolysis changes to -659 mV after electrolysis; 4.08 of pH changes to 10.47; 4.46 mg/1 of DO changes to 3.38 mg/l and 0.0046 mg/l of DH changes to 0.467 mg/l, respectively. It should be noticed that DH value is higher in reduced water than in the original water by two orders of magnitude. Strongly reduced water completely scavenged produced by hypoxanthine-xanthine oxidase system (4). Reduced water was shown to scavenge It prevented singlestrand breakage of plasmid DNA caused by the Cu(II)-catalyzed oxidation of ascorbic acid, suggesting that reduced water can also scavenge OH and The scavenging activity of reduced water against was stable in closed bottle at 4 °C for about a month

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but lost by opened autoclave or closed autoclave in the presence of tungsten trioxide which is knwon to be a specific adsorbent of active hydrogen.

Suppression of the growth of several human cell lines by electrolyzed reduced water. Electrolyzed water exhibits strong scavenging activity against active oxygen species. In order to examine the effect of reduced water on animal cells, the growth of human normal fibroblast TIG-1, lung adenocarcinoma A549, and human uterine cervix cancer HeLa were examined in the medium containing reduced water. Since reduced water contained

HOCl which was produced near anode, effect of the same concentration of HOCl was examined as control. As shown in Fig. 1, the growth of TIG-1 was suppressed by reduced water and the cells reached confluence at lower cell density in medium containing reduced water than in medium containing no reduced water. The morphology of cells in medium containing reduced water seemed to be normal. One and 0.5 ppm of HOCl slightly stimulated the growth of TIG-1 cells, suggesting that oxidative stress affects the growth of the cells. The growth of lung cancer A549 cells was remarkably suppressed by reduced water. HOCl did not affect the cell growth of A549 cells. The growth of uterine cancer HeLa cells were significantly suppressed by reduced water, but HOCl did not affect the growth of HeLa cells. These results suggested that reductive stress by reduced water may affect the signaling pathway of cell cycle to slower the rate of cell division.

Sensitivity of animal cells to reductive stress may be different depending upon cell types.

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Induction of the interleukin-6 gene expression in human osteosarcoma MG-63 cells by electrolyzed reduced water and reducing substances.

In order to examine if reduced water regulates some gene expression in animal cells, we examined the expression of the interleukin-6 gene in human osteosarcoma MG-63 cells. Super-induced MG-63 cells produce interferon The expression of interleukin6 gene was examined by RT-PCR method. Treatment of MG-63 cells with reduced water induced the expression of the interleukin-6 gene (Fig. 2). Since reducing substances such as ascorbic acid, (+)-catechin, and tannic acid also induced the expression of the interleukin-6 gene, the effect of reduced water may be due to its reducibiltiy. Here we first demonstarted that electrolyzed reduced water can regulate the growth of animal cells and gene expression. Water can permeate everywhere in the body and penetrates every membrane in eluding the blood-brain barrier. To neutralize the toxic action of active oxygen species, electrolyzed-reduced water may be an ideal and very powerful antioxidant and may be applied for prevention and therapy of various diseases. Further intensive investigation on the effect of reduced water on cell biology, immunology and oncology should be promoted. REFERENCES

1. Sohal, R. S. and Weindruch, R. (1996) Oxidative stress, caloric restriction, and aging.Science 273, 59-63. 2. Feig, D. I., Reid, T. M. and Loeb, L. A. (1994) Reactive oxygen species in tumorigenesis. Cancer Res. 54, 1980s-1984s. 3. Hayashi, H. (1995) Water, the chemistry of life, part IV. Explore 6, 28-31. 4. Shirahata, S., Kabayama, S., Nakano, M., Miura,T., Kusumoto, K., Gotoh, M.,

Hayashi, H., Otsubo, K., Morisawa, S., and Katakura, Y. (1997) Electrolyzedreduced water scavenges oxygen species and protects DNA from oxidative damage. Biochem. Biophys. Res. Commun. 234, 269-274 (1997). 5. Kabayama, S., Osada, K., Tachibana, H., Katakura, Y. and Shirahata, S. (1997) Enhancing effects of food components on the production of interferon from animal cells supressed by stress hormones.Cytotechnology 23, 119-125.

DIFFERENTIAL GENE EXPRESSION OF CYTOCHROME IMMORTALISED HEPATOCYTE CELL LINES

P450

IN

H.T.KELLY1, K.ANDERSON2, E.HILL 1 , H.GRANT2, C.MacDONALD1 Biological Sciences1, University of Paisley and Bioengineering Unit2, University of Strathclyde, Glasgow, UK.

INTRODUCTION Cytochrome P450 isoenzymes and the associated mixed function oxidase system are

important factors in the metabolism and detoxification of xenobiotics in the liver. However these functions are amongst the most labile activities in primary cultures of hepatocytes (Steward et al., 1985; Wortelboer et al., 1990). As an alternative approach to the use of primary hepatocyte cultures for the study of xenobiotic metabolism and toxicity several immortalised rat hepatocyte cell lines have been generated by either calcium phosphate precipitation (MacDonald et al, 1994) or electroporation of SV40 DNA (Yin et al., 1996,). In order to examine the retention of drug metabolising enzyme activity in our immortalised lines we employed the reverse transcriptase polymerase chain reaction (RTPCR) to determine the expression of P450 isoform mRNA, RNA arbitrarily primed polymerase chain reaction (RAP-PCR) to examine differential gene expression of cytochrome P450 and immunoblotting to determine the presence of the P450 protein. METHODS 1.Cell Culture: Hepatocytes were isolated from male Sprague Dawley rats by collagenase perfusion and transfected with SV40 early region DNA by either calcium phosphate precipitation ('C' lines) or electroporation ('LQC' and 'KC' lines) and cultured as described previously (MacDonald et al., 1994; MacDonald and Willett, 1997; Yin et al., 1996). 2.RNA Preparation: Total RNA from freshly perfused hepatocytes and immortalised cell lines was prepared using the total RNA isolation reagent TRIzol (Life Technologies). The RNA obtained was subsequently used for RTPCR and RAP-PCR. 3. First Strand Synthesis: Total RNA (5µg/sample) was converted to cDNA using the Superscript First Strand Synthesis kit and method (Life Technologies). Oligo(dT)12-18 was used to prime the first strand synthesis of all cDNAs prepared in this study. 4. PCR: A 10mM primer mix specific for either 2B1, 2B2 or 3A1 isoforms of CYP450 was used here (Omiecinski et al., 1990). To a thermowell tube (Costar) 5µl of each primer mix was added. In addition 5µl of 10xReaction buffer [500mM KCl, 15mM

100mM Tris HCl (pH9 at room temp.)] (Pharmacia Biotech), 4µl 10mM dNTP mix (10mM dATP, dTTP, dCTP, dGTP), 30.75µl and 0.25µl Taq DNA polymerase (Pharmacia Biotech) were added to each reaction and pulse spun. cDNA solution (5µl) was subsequently added to each tube and the total reaction overlayed 97 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 97-99. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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with mineral oil. The reaction was pulse spun and placed in the thermocycler (Techne) to undergo a series of denaturation, annealing and elongation cycles as follows: 1 cycle @ 93°C for 4min, 30 cycles @ 93°C for 1min, 54°C for l.5min and 72°C for 1min and 1 cycle @ 72°C for 5min. The resulting PCR products were resolved on a 1.5% agarose DNA gel and visualised by ethidium bromide staining. A 100bp DNA ladder was used as a sizing marker. 5.RAP-PCR: For the purpose of RNA fingerprinting a RAP-PCR kit (Stratagene ) was used. cDNA synthesis was performed as described above using an 18mer arbitrary primer Al (Stratagene) to replace oligo(dT). To a thin wall tube of 10x reaction buffer, _ mix (25mM each dATP, dTTP, dCTP, dGTP), Taq DNA polymerase RAP-PCR primer Al, of 1:10 cDNA dilution and were added. The reaction was gently mixed, pulse spun and overlayed with mineral oil. RAP-PCR was performed using the following cycling conditions: 1 cycle of 1min @ 94°C, 5 min @ 36°C and 5min @ 72°C; 40 cycles of 1min @ 94°C, 2min @ 50°C and 2min @ 72°C and finally 1 cycle @ 72°C for 10min. To analyse the product of the above reaction was added to of a stop solution containing 95% formamide, 20mM EDTA, 0.05% Bromophenol Blue and 0.05% xylene cyanol FF and the mixture heated to 80°C for

2min. of the reaction was run in a 6% acrylamide sequencing gel in 1xTBE buffer. 6. Western Blotting: All microsomes used in the immunoblotting were prepared from cell homogenates by ultracentrifugation. The samples were separated by electrophoresis, transferred onto nitrocellulose and stabilised in a 3% (w/v) gelatin solution. To immunostain 20ml of relevant primary antibody (a gift from Glaxo/Wellcome) were added for 2h. The protein samples on the nitrocellulose were then washed twice in TTBS (Tween 20 Tris Buffered Saline) and the secondary antibody, goat conjugated with alkaline phosphatase (Biorad), added for 2h. The samples were subsequently washed once in TTBS and once in TBS (Tris Buffered Saline). An alkaline phosphatase detection kit (Biorad) was used for colour development after which the nitrocellulose paper was washed with 3 rinses of water and dried between sheets of blotting paper before being photographed. The immunoblots were analysed using densitometry on a Biorad GS 690 scanning densitometer using Biorad Molecular Analyst software. RESULTS The presence of PCR products specific for the 2B1, 2B2 and 3A1 isoforms of cytochrome P450 was detected in primary hepatocytes using RTPCR. In contrast however larger PCR products, specific for the 2B1, 2B2 and 3A1 primers used here, were identified in both the calcium phosphate transfected and electroporated immortalised hepatocyte cells. Immunoblotting with antibodies specific for 1A1, 2B(1/2), 3A(l-9), 2C6 and 2C11 isoforms of P450 demonstrated that freshly isolated hepatocytes had a higher level of staining for all isoenzyme forms compared with that observed for 24h cultured primary hepatocytes. Immortalised cells, in addition to staining positively for the CYP450

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isoenzyme proteins examined, also demonstrated multiple stained products smaller than that expected for the proteins studied. Using densitometry analysis the majority of immortalised lines were found to stain positively for CYP1A1, 2B(l/2), 2C6, 2C11 and 3A(1-9) albeit at levels much lower than that observed in the primary cultures. In order to compare the RNA present in both the primary hepatocytes and immortalised cells, arbitrarily primed PCR (McClelland et al., 1995) was used to generate RNA fingerprints of these two hepatocyte populations. Using this technique RAP-PCR products were identified in the immortalised hepatocytes that were absent in the primary hepatocytes. CONCLUSIONS RTPCR analysis demonstrated that mRNAs encoding the cytochromes 2B1, 2B2 and 3A1 are present in our immortalised lines although the product amplified appeared larger than that observed with primary hepatocytes. Cytochrome P450 isoenzyme proteins were detected in both freshly isolated and 24h cultured primary hepatocytes with the levels of isoenzyme lower in the latter cells. In addition to giving the expected protein band, multiple bands were identified for the immortalised cells suggesting cross-reactions had occurred. This was unexpected as monoclonal antibodies were used throughout and no cross reaction was observed with other proteins in freshly isolated or primary rat hepatocytes. These proteins were also smaller in the immortalised cells and some degradation of the cytochrome P450 may have occurred. RNA fingerprinting, using RAP-PCR, identified differences in the RNA transcripts

from immortalised cell lines compared with those obtained from primary hepatocytes. This suggests the occurrence of differential gene expression in our immortalised hepatocytes and highlights RAP-PCR as a useful screening technique for identifying those immortalised lines which retain the most 'normal' phenotype in relation to primary hepatocytes. REFERENCES 1. MacDonald C., Vass M., Willett B., Scott A., Grant M.H. (1994). Expression of liver functions in immortalised rat hepatocyte cell lines. Human Experimental Toxicology 13, 439-444. 2. MacDonald C and Willett B. (1997). The immortalisation of rat hepatocytes by transfection with SV40

sequences. Cytotechnology 23, 161-170. 3. McClelland M., Mathieu-Daudi F., Welsh J., (1995). RNA fingerprinting and differential display using arbitrarily primed PCR. TIG 11, 242-246.

4.Omiecinski C.L., Hassett C., Costa P., (1990). Developmental expression and in situ localisation of the phenobarbital-inducible rat hepatic mRNAs for cytochromes CYP2B1, CYP2B2, CYP2C6 and CYP3A1. Molecular Pharmacology 38, 462-470.

5. Steward A.R., Dannan G.A., Guzelian P.S., Guengerich F.P., (1985). Changes in the concentration of seven forms of cytochrome P450 in primary cultures of adult rat hepatocytes. Molecular Pharmacology 27, 125-132. 6. Wortelboer H.M., de Kruif C.A., van Lersel A.A.J., Falke H.E., Noordhoek and Blaauboer B.J. (1990). The isoenzyme pattern of cytochrome P450 in rat hepatocytes in primary culture, comparing different enzyme activities in microsomal incubations and in intact monolayers. Biochemical Pharmacology 40, 25252534. 7. Yin L., MacDonald C., Grant H. (1996). Electroporation conditions for retention of rat hepatocyte viability. The Genetic Engineer and Biotechnologist 16. 27-34.

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POST-TRANSCRIPTIONAL CONTROL OF RECOMBINANT GENE EXPRESSION: mRNA RETARGETING AND REGULATION OF EXPRESSION

K.A. PARTRIDGE1, A. JOHANNESSEN2, A. TAULER3, I.F. PRYME2 and J.E. HESKETH1,* 1 Intracellular Targeting Group, Rowett Research Instititue, Bucksburn, Aberdeen, AB21 9SB, UK; 2Department of Biochemistry and Molecular Biology, University of Bergen, Arstadveien 19, 5009 Bergen, Norway; 3 Unit of Biochemistry, Faculty of Pharmacy, University of Barcelona, Diagonal 643, 08028 Barcelona, Spain; *author for correspondence.

1. Introduction Mammalian cell lines are used to produce commercially significant amounts of recombinant proteins in cell factories. There are numerous examples where normally

secreted proteins are being produced in this way (e.g. Lin et al, 1986), as well as a number of commercial vectors for the secretion of heterologous proteins. However such systems have been used only for the production of secreted proteins rather than proteins which are normally intracellular. The ability to secrete intracellular proteins would greatly increase the range of potential products which could be made by cell factories and therefore the secretion of intracellular proteins in mammalian expression systems is an important aim in biotechnological research (James and Simpson, 1996). Secreted proteins are synthesised on the endoplasmic reticulum (ER) and the secretion of an intracellular protein from a cell factory would require as a first step the redirection of the mRNA for synthesis of the protein to the ER.

2. Experimental Approach

Targeting of mRNAs coding for secreted and membrane proteins is achieved by a signal peptide in the nascent polypeptide chain whilst there is growing evidence for targeting of mRNAs coding for intracellular proteins to different cytoplasmic domains by signals in the 3'untranslated region (3'UTR) (Hesketh, 1996). The feasibility of retargeting a mRNA coding for a recombinant intracellular protein to the ER was investigated using stable transfected cell lines expressing gene constructs containing the reporter sequences and luciferase linked to different combinations of 5' and 3' signals. In particular we have studied the effects of adding the 5'albumin signal sequence in combination with either the native globin 3'untranslated region (3'UTR) or the 3'UTR from the c-myc mRNA since the latter is known to contain a signal for perinuclear localisation and association with the cytoskeleton (Veyrune et al, 1996). The subcellular localisation and stability of the transcripts were determined. 101 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 101-103. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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3. Results and Discussion

Expression of the exogenous gene in the stable transfected fibroblast cell lines was assessed by Northern hybridisation to detect globin transcripts. The highest levels of globin expression from all four genes was greatest in cells at 70% confluence and all subsequent experiments were carried out at that stage of growth. Detergent/salt fractionation (Vedeler et al, 1991; Veyrune et al, 1996) was carried out to investigate the compartmentation of the transcripts between free polysomes, cytoskeletal-bound polysomes and membrane-bound polysomes. Each cell line was fractionated, polysomes pelleted, and then RNA extracted from the pellets and analysed by Northern hybridisation for abundance of globin transcripts. Globin transcripts with either the native 3'UTR or with the 3'UTR of c-myc were found to be present predominantly in free/cytoskeletal-bound polysomes. In contrast, globin transcripts with the native globin 3'UTR and the albumin signal sequence were found at highest abundance in the membrane-bound polysomes derived from the ER. Globin transcripts with both albumin signal sequence and c-myc 3'UTR showed a markedly lower enrichment of the mRNA in the ER fraction. Reprobing of filters with the 18S rRNA probe allowed correction for RNA loading and quantification of the hybridisation data per unit RNA. This data confirm that the addition of the albumin signal sequence redirects the globin mRNA to the ER (p=0.01). The signal sequence redirected globin with the c-myc attached (p<0.05) but presence of both c-myc 3'UTR and the signal sequence reduced the extent to which redirection occurred (p<0.05).

The data show that the addition of the signal sequence from rat albumin leads to efficient redirection of the globin mRNA with the native 3'UTR to membrane-bound polysomes. The presence of the c-myc 3'UTR reduced the extent of redirection, suggesting that there is competition between 5' and 3' localising signals in the chimaeric mRNA. Measurement of mRNA stability showed that the addition of the signal sequence does not destabilize the mRNA. Translational efficiency was found to be unaffected by addition of the signal sequence or replacement of the native 3'UTR by that of c-myc. Overall the results show that it is possible to retarget a mRNA from free/cytoskeletalbound polysomes to the ER while maintaining stability and translational efficiency but that removal of 3'UTR sequences may be necessary in order to achieve efficient retargeting. This is a prerequisite for secretion of an intracellular protein and is the first step in developing appropriate technology to achieve secretion of intracellular proteins from a cell factory; as such it has important implications for the development of vectors to promote secretion of intracellular proteins from cell factories. Acknowledgements: This work was supported by a grant from the European Union Biotechnology Programme (Bio2-CT94-3069) and the Scottish Office Agriculture, Environment and Fisheries Department. We thank Eli Johannesen and Gill Campbell for excellent technical assistance.

103 References: Hesketh, J.E. (1996) Sorting of mRNAs in the cytoplasm: mRNA localisation and the cytoskeleton. Expt. Cell

Res. 225, 219-236. James, J. and Simpson, B.K. (1996) Application of enzymes in food processing. Crit. Rev. Food Sci. Nutr. 36, 437-463. Lin, F-K., Lin, C-H., Lai, P-H., Browne, J.K., Egrie, J.C., Smalling, R., Fox, J.M., Chen, K.K., Castro, M. and Suggs, S. (1986) Monkey erythropoietin gene: cloning, expression and comparison with the human erythropoietin gene. Gene 44, 201–209. Vedeler, A., Pryme, I.F. and Hesketh, J.E. (1991) The characterization of free, cytoskeletal and membrane-bound polysomes in KrebII ascited and 3T3 cells. Mol. Cell Bioch. 100, 183-193. Veyrune, J-L., Campbell, G.P., Wiseman, J., Blanchard, J-M. and Hesketh, J.E. (1996) A localisation signal in the 3'untranslated region of c-myc mRNA targets c-myc mRNA and beta-globin reporter sequences to the perinuclear cytoplams and cytoskeletal-bound polysomes. J. Cell Science 109, 1185-1194.

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TRANSIENT TRANSFECTION IN MAMMALIAN CELLS A basic study for an efficient and cost-effective scale up process E.-J. Schlaeger , J.Y. Legendre*, A..Trzeciak, E.A. Kitas, K. Christensen, U. Deuschle

and A. Supersaxo F. Hoffmann La Roche Ltd. PRPN-G, PRPV CH-4070 Basel, Switzerland *UPSA Laboratoires, 128 rue Danton, 92506 Rueil-Malmaison Abstract

The aim of the presented work was to develop a cost-effective and easily scaleable transient transfection system with mammalian cells grown in serum-free suspension culture. For this purpose the cationic polyethylenimine (PEI) and the novel hybrid molecule dioleoyl-melittin (DOM) were used. Both substances are highly efficient transfection reagents for mammalian cells and have been described recently. The transfection complexes were made directly within the cell culture by consecutively adding plasmid and PEI (direct method). Alternatively, the DNA-PEI transfection complexes were performed in fresh medium (1/10 culture volume) and then added to the cells (indirect method). Here we have optimized the PEI-mediated transfection conditions for HEK293 and 293EBNA cells grown in serum-free suspension culture for large scale experiments. In a second part of the investigation the efficiency of gene expression was studied in labscale as well as in fermentor scale by combining DOM with PEI to form transfection complexes.

Our data show the PEI-mediated transfection into EBNA cells can be used as a costeffective transfection system in spinner culture scale up to 2-5L, to produce milligram amounts of recombinant protein. Fermentor scale experiments, however, are less efficient because the PEI- mediated transient gene expression is inhibited by the conditioned medium. Transfection experiments performed in the present of both DOM and PEI exhibited synergistic enhancement of gene expression in EBNA cells. The synergistic enhancement between DOM and PEI at low dose DNA (0, l-0,2µg/ml) was seen in spinner scale as well as in 10L and 23L fermentor Keywords : Transient transfection, HEK293 cells, serum-free, gene expression

Introduction

Efficient gene delivery in gene therapy using non-viral vectors where the DNA is generally complexed with cationic lipids or polycationic polymers has recently become of great interest (1, 2). More recently some of these transfection agents were conjugated with proteins or peptide in order to target then to specific cell types (3, 4). In addition to being important for gene therapy protocols improved non-viral gene delivery techniques are also of significant interest in gene expression in cell culture. 105 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 105-112. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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Obtaining stable cell clones is generally a time consuming processs lasting from 4-6 weeks untill 6 months. In order to speed up the whole process of expressing milligram amounts of recombinant proteins we were interested in scaling up the transient gene expression system in suspension culture. Here we have optimized the PEI-mediated transfection conditions of HEK293(EBNA) cells grown in serum-free suspension culture for large scale experiments. In a second part of the investigation the efficiency of gene expression was studied by combining dioleoyl-melittin (DOM), which represents a new class of peptide-based reagent for efficient transfection of mammalian cells, with PEI to form transfection complexes (5,6). Materials and Methods Cell culture Cell line: HEK 293EBNA (Invitrogen) adaptated to growth in suspension Medium: HL serum-free, Ca-free with transferrin, insulin, Primatone RL, Pluronic F68, fructose, maltose) (7 ). Feed solution.: Mixture of gln, glucose, essential and non-essential. amino acids, vitamins, Primatone RL Culture vessel: Spinner flasks (Bellco), 12 L bioreactor, stirred (MBR) Transfection Transfection reagents: Polyethelenimine (Fluka), dioleoyl-melittin (Roche) Reporter gene: Extracellular domain of human TNF receptor p55 (TNFRp55). Plasmid: pREP 7-TNFRp55 (EBV-based expression vectors containing the EBV ori P and the EBNA-1 gene which replicate extrachromosomally in dividing cells, Invitrogen). Transfection assay HEK293(EBNA) cells grown to a cell titer of 0,6-1.0x10e6cells/ml were centrifuged, washed once and resuspended in fresh medium. The cell titer was adjusted to 5-6xlOe5 cells/ml. Preparation of the transfection complexes Direct method: The DNA-PEI transfection complexes were preformed directly in the cell culture by adding DNA and PEI consecutively Indirect method: The DNA-PEI transfection complexes were preformed in fresh medium (1/10 volume) and then added to the cells. The DNA-DOM-PEI transfection complexes were prepared using the indirect method

Incubation The transfected culture was grown for 2-5 days.After the second day the culture was fed once daily with a nutrient mixture of of sugars, amino acids and vitamins.

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Product analysis The recombinant TNFRp55 were measured by ELISA. Results and Discussions First we adaptated the adherently growing HEK293 cells and the subclone 293EBNA to serum-free growth in suspension culture using HL medium. In order to quickly generate a large set of data on the transient gene expression, experiments were performed in 12 well dishes by transferring 1 ml suspended cells into each well. The transfection complexes were then formed by adding first the plasmids followed by the cationic polymer PEI directly into the culture (direct method). First the PEI nitrogen : DNA phosphate ratio was examined, which is very important for high transfection efficiency (6). The relatively high ratio of 26:1 which was found to be optimal could partially explained by the DNA-PEI complex formation in the culture in the presence of cells. This ratio was subsequently used for gene delivery experiments in spinner culture. Fig. 1 shows the expression levels from an experiment in which 1µg/ml DNA was complexed either by using the direct method or by the indirect method in 1/10 volume of fresh medium. Two clear results were obtained. First the expression levels in EBNA cells were significantly higher compared to 293 cells if the direct method was used. This could be due to the higher growth rate observed during the post-transfection time, (see the final cell density). In contrast, the 293 cell titer is lower after 3 days incubation. Secondly, the transient expression of TNFRp55 displays a significantly lower titer if the indirect method was used, indicating that the conditions of the separately prepared DNA-PEI complexes is suboptimal.

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In an additional experiment the scaleability of the direct method was investigated using 1 µg/ml plasmid. In Table 1 we demonstrate scaleability of the spinner culture system. No difference in expression levels were observed if increased culture volume up to 2L was used in corresponding spinner flasks. From this finding it is evident that milligram amounts of recombinant proteins can be produced in a cost-effective way using this simple indirect method of DNA-PEI complex formation.

A lot of effort was invested to reduce the DNA concentration in transient transfection experiments further. From previous experiments we know that the transient gene expression was always slightly if PEI (Mw 25kDa) was used instead of PEI (800kDa) higher compared to data obtained with PEI Mw 800 kDa. Therefore, studies were performed using both 25 and 800 kDa PEI to determine the optimal ratios of PEI: DNA with respect to plasmid concentration and the method used for complex formation. The results clearly shows that in the presence of 1µg/ml DNA the ratios of PEI:DNA is different depending on the method and DNA concentration, see Table 2. While the direct method again shows the ratio of 26:1 the indirect complex formation shows a N : P ratio of 13: 1. The latter finding confirms results described in the literature (6). Highly remarkable is the fact that at low DNA concentration

(0,2µg/ml) high expression levels were measured if the amount of PEI was increased . The optimal ratio of PEI: DNA was found to be 33 : 1. Table 2 shows the TNFRp55 titer of transient transfection experiments with different DNA concentrations and different molecular weights of PEI ( 25 and 800 kDa), using the direct and indirect method.

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Before each transfection experiment 293EBNA cells were transferred into fresh medium. This step is very important for the DNA uptake process as is shown in the next experiment. EBNA cells were washed and diluted into fresh medium, followed for 48 hrs. incubation. During culturing the medium become conditioned. After 24 and 48 hrs. incubation cells were adjusted to a cell titer of 5×10e5 c/m1 with

the corresponding spent medium and transfected using the indirect method with DNAPEI complex formation in 1/10 culture volume with fresh medium. In the control sample, cells from the 48 hrs. time point were carefully transferred into fresh medium and then transfected as described. The results in Fig.2 clearly demonstrate that transient gene expression is strongly inhibited by condioned medium depending on the age of the culture. However, the cells are still competent for DNA uptake as illustrated by the control result. The inhibition ot transient transfection by conditioned medium presents a serious problem in large scale experiments. To overcome this difficulty we have used the combination of two transfection agents dioleyl-melittin (DOM) and PEI. DOM is highly toxic in serum-free suspension culture if the described DNA-DOM ratio of 1 : 10 was used. By lowering the DNA concentration from 1µg/ml to 0.1-0.2 µg/ml the toxic effect is drastically reduced but no TNFRp55 could be measured. However, if PEI was also added to the DNA-DOM complexes (here we used the 25 and the 800 kDa polymer) a significantly increase of the expression levels was observed as shown in Fig.3. This clearly demonstrates a synergistic enhancement of transient transfection efficiency in the presence of DOM and PEI. In a set of experiments we optimized the ratios of DNA-DOM-PEI. Conditions could be found which show high transfection efficiency at low DNA concentration. Furthermore, the inhibitory effect of conditioned medium could be significantly reduced by using DOM and PEI together. From this it is evident that if no medium exchange is performed in a large scale transient transfection experiment DOM and PEI must both be used and the culture must be diluted with fresh medium to give the correct cell titer.

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In a last experiment we show a transient gene expression experiment using a 12L bioreactor. EBNA cells were transferred into fresh medium and used to inoculate the fermentor. If the cell titer of 5-6x10e5 c/ml was achieved then 1L of fermenter volume was replaced by 1L freshly prepared transfection complexes (DNA-DOM-PEI). The released 1L portion was transfected under identical conditions and incubated in a spinner flasks. The results of this fermentor scale experiments is illustrated in Fig. 3. About 16 milligram TNFRp55 was produced within 5 days post-transfection.

Conclusions PEI-mediated gene transfer into HEK293EBNA cells can be used as a simple transfection system to produce milligram amounts of protein in a cost effective way during 3-5 days in serum-free medium. Transient transfection is inhibited in conditioned culture. The combination of DOM and PEI significantly reduces the inhibitory effect and yields higher expression values >Furthermore, the action of both agents together are extremely useful in fermentor scale transfection at low DNA concentration.

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References

1. X Gao and L Huang (1995) Cationic liposome-mediated gene transfer, Gene Therapy 2, 710-722. 2. Barthel F., Remy J.-S., Loeffler J.-P. and Behr J.P. (1993) Gene Transfer Optimization with Lipospermine-Coated DNA, DNA and Cell Biology 12, 553-560. 3. Gottschalk S., Sparrow JT., Hauer J, Mims MP., Leland FE, Woo SLC and Smith LC. (1996) A novel DNA-peptide complex for efficient gene transfer and expression in mammalian cells, Gene Therapy 3, 448-457. 4. Kircheis R., Kichler A., Wallner G., Kursa M., Ogris M., Felzmann T.

Buchberger M. and E. Wagner (1997) Coupling of cell-binding ligands to polyethelenimine for targeted gene delivery, Gene Therapy 5. Legendre JY., Trzeciak A., Bohrmann B., Deuschle U., Kitas E.A. and Supersaxo A. (1996) Dioleoylmelittin as a Novel Serum-Intensitive Reagent for Efficient Transfection of Mammalian Cells, Bioconjugate Chem. 8, 57-63 6. Boussif O., Lezoualc'H F., Zanta M., Mergny M., Scherman D., Demeneix B. and Behr J.-P. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethelenimine, Proc. Natl. Acad. Sci.. USA 92,72977301. 7. Schlaeger E.-J. (1996) The protein hydrolysate, Primatone RL, is a cost-effective multiple growth promotor of mammalian cell culture in serum-free media and displays anti-apoptosis properties, J. Immunol. Methods 194, 191-199.

112

Discussion

Wurm:

What is the transfection efficiency in the reactor?

Schlaeger:

I do not know as I only looked for the accumulation of TNF receptor. From earlier experiments 30-60% of the cells were transfected.

Mueller:

Some time ago NC2 precipitation of DNA was used to transfect cells and those reactions were very strongly pH dependent. I would expect the same in your system. Have you looked for pH dependence on your precipitation efficiency?

Schlaeger:

No. The whole system works very well on the plate, and we have used different types of medium, but the pH was constant at about 7.3. I do not believe that there is any difference in the complex formation with pH. The system is good in that it is simple, but we have a problem with the conditioning medium in the 60 1 fermenter because of the number of passages.

Merten:

I know that DOM is still produced in your laboratory. Will it become available on the market as it would be useful in large-scale applications?

Schlaeger.

You can buy the 2 molecules separately but you have to do the conjugation yourself. It has been given to various companies for testing, but I do not know what the decision was.

LARGE SCALE TRANSIENT GENE EXPRESSION IN MAMMALIAN CELLS E.-J. Schlaeger, K. Christensen, G. Schmid, N. Schaub, B. Wipf, and A. Weiss F. Hoffmann-La Roche Ltd., Pharmaceutical Division - PRP Department PRPN-G, CH-4070 Basel, Switzerland

Keywords: transient transfection, plasmid production, plasmid purification

• The aim of this work was to develop a fast, robust and costeffective integrated process for the production of mg

amounts of protein for research purposes. The process consists of 3 distinct parts: Plasmid production, plasmid purification and large-scale transient transfection of mammalian cells grown in serum-free suspension culture.

• We investigated plasmid amplification using various E. coli strains cultivated in different complex media followed by the establishment of plasmid production conditions in 10 L fermenters

• The efforts in the area of plasmid purification were geared towards the development of a fast and cost-effective method that generates material suitable for our PEImediated transfection protocol

• Different plasmid preparations were tested in PEI-mediated transient transfection experiments with HEK293 (EBNA) cells grown in serum-free suspension culture at lab and bioreactor scale 113 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 113-116. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

114

Process flow diagram for the purification of plasmid DNA for transient transfection

Alkaline Lysis and 2-Propanol Precipitation

115

Cell culture Cell line: Medium: Culture vessel: Plasmid: Reagent:

HEK293(S), 293EBNA (Invitrogen) HL-0%FCS w heparin Spinner flaks (Bellco), 12L bioractor (MBR) pREP7, pC1,(soluble human TNFRp55) pGL3-CMV(luciferase) Polyethylenimine (PEI), Fluka, Aldrich Dioleolyl-melittin (DOM), Roche

Transfection assay: Log cells(5xl0e5c/ml) in HL w/o heparin Formation of DNA-PEI transfection complexes: indirect method: The complexes were performed in fresh medium (1/10 volume) an then added to the cells. Incubation: 3-5 days, spinner flask, 100rpm, (feed) Test: sol. human TNFRp55 ELISA

116

Summary • We established an E. coli fermentation process for plasmid production in 10 L bioreactors yielding between 50-150 mg of unpurified plasmid depending on the plasmid and the host strain. The E. coli strain

•

was selected to give a high yield of monomeric supercoiled pREP7-TNFRp55 DNA.

•

A standard alkaline extraction method was scaled up and a 1-step chromatographic procedure yielded plasmid DNA suitable for our transfection protocol.

•

Transient gene expression with different plasmid preparations exhibited high transfection efficiencies.

•

We developed a cost-effective large scale transient transfection process to produce mg amounts of recombinant protein using HEK293 (EBNA) cells grown in serum-free suspension culture.

Synergistic enhancement of transient expression by dioleoylmelittin (DOM) and polyethylenimine (PEI) in mammalian cells in suspension culture E.-J. Schlaeger , J.Y. Legendre*, A. Trzeciak, E.A Kitas, K. Christensen, U. Deuschle and A. Supersaxo F. Hoffmann La Roche Ltd. PRPN-G, PRPF, CH-4070 Basel, Switzerland *UPSA Laboratoires, 128 rue Danton, 92506 Rueil-Malmaison, France Abstract

Dioleoyl-melittin, which is a conjugate of dioleoyl-phosphatidyl-ethanolamine-N-{3-(2pyridyldithio)propionate} with the amphipatic peptide melittin { Gly-Cys} 1 represents a new class of peptide-based reagent for efficient transfection of mammalian cells. In this work we investigated the transfection efficiency of dioleoyl-melittin (DOM) combined with polyethylenimine (PEI) using HEK293(EBNA) cells grown in serumfree suspension cultures. Gene expression was monitored using the luciferase reporter gene and the human soluble TNF receptor p55 gene (TNFRp55) inserted into different vectors. Our data clearly show that DOM together with PEI exhibited synergistic enhancement for gene expression in EBNA cells. At the optimal DNA-DOM-PEI

weight ratio the efficiency of transfection increases significantly compared to corresponding PEI and DOM transfection at low DNA concentration. In summary, our data show that dioleoyl-melittin and polyethylenimine act synergistically in transfecting 293(EBNA) cells grown in serum-free suspension culture. Keywords : HEK293, serum-free culture, transient transfection, bioreactor

Introduction

Efficient gene delivery in gene therapy by using non-viral vectors has recently become of great interest (1 ). Substantial work has been done to improve DNA uptake where the DNA is generally complexed with cationic lipids or polycationic polymers. More recently some of these transfection agents were conjugated with proteins or peptide in order to target it to specific cell types (2,3). In this context we were interested in dioleylmelittin (DOM) which is a novel peptide-based gene transfer agent as described recently (4 ). The authors described that the highest expression levels for gene delivery experiments were found by using the ratio DNA:DOM of 1:10. If we transferred the weight ratio of DNA: DOM to our transfection system it became clear that if lµ g/ml DNA was used in a transfection experiment the corresponding amount of DOM is highly toxic especially in serum-free suspension culture. Therefore, it was very important to minimize the cell toxicity by reducing the plasmid concentration. In this work we investigated the transient transfection efficiency of dioleoyl-melittin (DOM) combined with polyethylenimine (PEI) at low DNA concentration using HEK293(EBNA) cells grown in serum-free suspension cultures. 117 O. - W. Merten et al. (eds.), Mew Developments and New Applications in Animal Cell Technology, 117–120. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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Materials and Methods Cell culture Cell line: HEK 293EBNA (Invitrogen) serum-free HL medium (5). Culture vessel: Spinner flaks (Bellco), 12 L bioreactor, stirred (MBR) Transfection Transfection agents: Polyethelenimine (PEI), dioleoyl-melittin (DOM) Reporter gene: Extracellular domain of human TNF receptor p55 . Plasmid: pREP7-TNFRp55 (EBV-based expression vectors (Invitrogen) Transfection assay HEK293(EBNA) cells were centrifuged, washed once and resuspended into fresh medium. The cell titer was adjusted to 5-6xl0e5 cells/ml. Preparation of the transfection complexes The DNA-PEI transfection complexes were prepared in fresh medium (1/10 volume) and than added to the cells (6). The DNA-DOM-PEI transfection complexes were prepared in fresh medium (1/10 volume) and then added to the cells.

Incubation: The transfected culture was grown for 2-5 days.After the second day

the

culture was fed once daily . Product analysis: The secreted TNFRp55 were measured by ELISA.

Results

and

Discussions

Before establishing data for a scaleable transient gene expression system we first adaptated the adherently growing HEK293 cells and the subclone 293EBNA to serumfree growth in suspension culture using HL medium . In a set of experiments we tested the expression levels of DOM-mediated transient transfection by using different DNA and DOM concentrations. The results clearly indicate significantly toxic effects on cells if more than DNA and DOM is used. However, lowering the DNA concentration further caused a significant drop in efficiency ( data not shown). From our experience with PEI-mediated transient gene expression we tried to combine both transfection agents in order to improve the gene delivery system in suspension cell culture. Fig. 1 shows the time course of a transfection experiment in which 0.25 DNA was incubated with DOM in the absence and in the presence of PEI. Using the DNA-DOM complexes high cell viability was observed (> 85%). However, 3 days post-infection only background expression levels were measured. Similar results were obtained if DNA-PEI transfection complexes were used. Quite different results were obtained in the sample where PEI was added with the DNA-DOM complex. A high yield of TNFRp55 was accumulated in the culture medium. The conclusion of this simple experiment clearly indicates that DOM and PEI act synergistically in transfecting 293EBNA at low plasmid concentration. This result was subsequently confirmed on several occasions. The concentrations of DNA, DOM and PEI were optimized in order to improve transfection efficiency.

119

After establishing lab-scale data on transient gene expression we scaled up this technique. First we applied the transfection conditions in spinner culture up to 2L volume without lost of expression levels, demonstrating the possibility of production of several mg recombnant protein. Finally transient gene expression was performed in a 12 L bioreactor. Figure 2 shows the results. About 2,5L cells were grown up to a cell titer of 1,2×l0e6 cells/ml, transferred into fresh medium by a centrifugation step and used to inoculate the fermentor giving a final volume of 11L.

120

Conclusion

The results of this study clearly show that DOM together with PEI exhibited synergistic enhancement for gene expression in EBNA cells at low DNA concentration. At the optimal DNA-DOM-PEI weight ratio (0.2-0.15-0.5) the efficiency of transfection increases significantly (up to 10-fold) compared to corresponding DNA-PEI and DNADOM transfection. No toxic effect was observed. Therefore, the combination of DOM and PEI exhibits a cost effective transient gene expression system with high transfection values and is extremely useful for large scale production of milligram amounts of recombinant proteins at low plasmid concentration.

References 1. X Gao and L Huang (1995) Cationic liposome-mediated gene transfer, Gene Therapy 2, 710-722. 2. Gottschalk S., Sparrow JT., Hauer J, Mims MP., Leland FE, Woo SLC and Smith LC. (1996) A novel DNA-peptide complex for efficient gene transfer and

expression in mammalian cells, Gene Therapy 3, 448-457. 3. Kircheis R., Kichler A., Wallner G., Kursa M., Ogris M., Felzmann T. Buchberger M. and E. Wagner (1997) Coupling of cell-binding ligands to polyethelenimine for targeted gene delivery, Gene Therapy 4. Boussif O., Lezoualc’h F., Zanta M., Mergny M., Scherman D., Demeneix B. and Behr J.-P. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethelenimine, Proc. Natl. Acad. Sci.. USA 92,72977301. 5. Schlaeger E.-J. (1996) The protein hydrolysate, Primatone RL, is a cost-effective multiple growth promotor of mammalian cell culture in serum-free media and displays anti-apoptosis properties, J. Immunol. Methods 194, 191-199. 6. Legendre JY., Trzeciak A., Bohrmann B., Deuschle U., Kitas E.A. and Supersaxo A. (1996) Dioleoylmelittin as a Novel Serum-Intensitive Reagent for Efficient Transfection of Mammalian Cells, Bioconjugate Chem. 8, 57–63

TRANSIENT EXPRESSION OF A SOLUBLE AND SECRETED FORM OF HETERODIMERIC T-CELL RECEPTOR IN HEK-293

M. JORDAN* , C. L. BLANCHARD°,1. BERNASCONI°, I. LUESCHER° and F. M. WURM*

* Center of Biotechnology UNIL-EPFL, Swiss Federal Institute of Technology, 1015 Lausanne, Switzerland °Ludwig Institute for Cancer Research, University of Lausanne, 1066 Epalinges, Switzerland

1. Abstract Biologically active human T-cell receptor (TCR) was expressed as a secreted recombinant protein in 293 cells.

Correct

chain pairing was facilitated by

complementary charged peptides (leucine zipper) at the C terminus. Two plasmids, coding for the chains were constructed and prepared using commercially available purification kits. After optimizing transfection conditions for secretion of heterodimeric TCR in 12 well plates, transfections were performed in spinner flasks. The transfection efficiency was determined with a co-transfected expression vector and correlated with TCR levels as determined by ELISA. Product was isolated by affinity chromatography. Biological activity, i.e. ligand binding was tested. Transiently produced recombinant soluble TCR showed similar affinity to the ligand as receptors purified from cell membranes. 2. Introduction For determination of the structure of TCR, mg amounts of soluble protein are desirable. Before a quantitative ELISA with a purified standard was available, relative amounts of heterodimers were detected by an ELISA using antibodies against both chains. Biological activity was measured using ligand. Stable expression in CHO cell lines gave poor signals in both assays, indicating that the formation of heterodimers is not very efficient and that TCR might be difficult to express at high levels. This article describes the transient expression of TCR in the human kidney cell line 293 T. 3. Materials and Methods Plasmids and vectors: The chain were cloned seperately into the pCI-neo expression vector (Promega). In order to have a soluble and secreted version of TCR the transmembrane domain and cytoplasmic tail were deleted. In addition, a leucine zipper 121 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 121-123. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

122

sequence was introduced into each chain, which should facilitate formation of heterodimers. The Qiagen kit was used to purify mg amounts of plasmids. Transfections in plates: Exponentially growing 293 T cells were seeded at 10-20 hours before transfection in DMEM/F12 medium supplemented with 2% FCS1. Per 10 ml of medium, . of 250mM containing a total of DNA was mixed with HEPES buffer (1.5mM 140mM NaCl, 50 mM HEPES, pH = 7.05). The transfection cocktail was added to cells after 1 minute and the plates were incubated for 4 hours at . Precipitate was removed at this point by exchanging the medium. Transfections in spinner flasks: For 1 L spinner flask, cells were seeded in 500 ml of fresh medium at Immediately before the precipitate was formed 15 ml of 250mM was added into the spinner flasks to increase the calcium concentration. Subsequently, 20 ml of transfection cocktail was added2. After 4 hours, the culture was diluted by adding 500 ml of fresh medium. Protein was harvested after 2 - 3 days. 4. Results and Discussion IS OPTIMAL FOR HETERODIMER EXPRESSION Expression of TCR was first optimized in 12 well plates to identify an optimal ratio of the two chains, encoded by two different expression vectors. Ratios of 1:9 through 9:1 were tested (Fig. 1). ELISA data as well as photo-affinity labeling tests indicated that the is limiting and that at an excess of expression vector significantly improves transient expression.

4.2. T-FLASKS VERSUS SPINNER FLASKS TCR was transiently expressed in T-flasks or in 1 L spinner flasks. The ratio of chain expression vectors was 1:2 . For the spinner experiment, cells were adapted for one month to grow in suspension containing single cells and small aggregates (less than 10 cells per aggregate). Transfections of this adapted population in plates showed that the cells still grew adherent and there was no change in transfection efficiency. The expression of TCR in the spinner flasks was comparable to the T flasks (Table 1). In other experiments no difference could be found between 12 well plates and T-flasks.

123

4.3. DOES AN INTERNAL CONTROL CORRELATE WITH PRODUCT TITERS? Although the transfection procedure is well defined, the problem of variation in transfection efficiency between different experiments remains. For the transient expression of proteins such variation can cause a problem, in particular when there is no quantitative assay available for the protein to be expressed. To minimize variation

and to guarantee at least a reasonable transfection efficiency before efforts for product isolation are started, we included in one series of transfections in 100 ml spinner flasks as an internal standard 10 % of pCMV The cells were fixed 24 hours after transfection and stained for 4 hours with X-gal. Positive cells were counted in the microscope and compared with the relative ELISA-signal (Fig.2). Although none of the assays were optimized for a high accuracy, the internal marker seems to correlate with the levels of produced TCR. Such an internal standard, co-transfected with any protein has a clear advantage, because it allows detection with one assay all the variations in transfection efficiency, which would directly affect the product yield.

5.

Conclusions

Different chains seem to be expressed at different efficiency, even when both chains are cloned into the same expression vector. For two chain proteins the levels of transient expression can be improved by optimizing the ratio of the two vectors used for co-transfection. Transfections in spinner flasks resulted in the same level of expression as transfections in T flasks or in 12 well plates. Co-transfection of a marker (10% or less of total DNA) correlates with the product level for a particular protein and generally can be used as a control for reproducible transfection efficiencies. 200 µg of recombinant TCR was purified from 2.5 L of supernatant harvested 2 days after transfection.

References 1

Jordan, M., Schallhorn, A. and Wurm, F.M. (1996) Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res. 24, 596-601

2

Jordan, M., Koehne, C. and Wurm, F.M. (1997) Calcium phosphate mediated DNA transfer into HEK293 cells in suspension: control of physicochemical parameters allows transfection in stirred media. Cytotechnology, in press

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MEASURABLE PARAMETERS OF CELLS AND PRECIPITATE PREDICT TRANSFECTABILITY WITH CALCIUM PHOSPHATE

M. Jordan and F. Wurm Center of Biotechnology UNIL-EPFL, Swiss

Federal Institute of

Technology, 1015 Lausanne, Switzerland

1. Abstract Transient expression of proteins in mammalian cells at 1 - 100 L scale has the potential to become a powerful technique for the rapid production of research proteins. We have shown that the calcium phosphate technique is compatible with routine handling in bioreactors and that the yield of proteins is comparable to that seen in standard transient transfections 1 . However, although the reproducibility of the method is

excellent within triplicates, there can be dramatic variations in transfection efficiencies with different batches of cells, solutions, plasmid preparations etc. In addition, expression levels of novel proteins may differ from those seen with proteins like tPA or hGH. There is a need therefore, to monitor the transfection procedure itself and to predict protein expression through defined and quantitative assays. We suggest two

tests: the first assesses, through OD-measurements of the transfection cocktail, the quality of the DNA-carrier complexes. The second employs a commercially available vector encoding a strongly fluorescent green fluorescent protein (GFP). Transfection with this vector provides, within a day, a quantitative signal. Together, these assays

allow a fast assessment of transfections with calcium phosphate for optimization purposes. Moreover, co-transfecting GFP with the vectors of interest allows, to a certain degree, prediction of transfectability and expression of the desired protein. 2. Introduction

Transfection efficiency depends on physical and biological parameters, some of them not known or difficult to control. For the calcium phosphate technique in particular, physical parameters of the precipitation buffers are most critical 2 . The effect of such parameters on the precipitation of DNA can be studied by a turbidity assay described earlier3. Here we suggest a modified protocol without the addition of DNA, to test and to compare new batches of transfection solutions rapidly. Transfectability of cells is more difficult to control. Even with the same cell line, transfection results can vary several fold depending on the actual status of the cell population. It is desirable therefore, to include an internal standard in order to monitor 125 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 125-128. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

126

the actual transfection efficiency. Such an internal standard should be applicable for various cell lines and different methods applied. In addition, it should allow comparison of transfection efficiencies at various scales. 3. Materials and Methods

Turbidity assay: One volume of 250 mM (without any DNA) is quickly mixed with one volume of HEPES buffer HEPES, pH = 7.05). After 1 minute, the absorption at 320 nm is measured. GFP measurements:The plasmid pEGFP-Nl (Clontech) is transfected in multiwell plates using the calcium phosphate method. Fluorescent signals are directly measured from 12 well plates containing cell culture medium (CytoFluor II, extinction at 485/20, emission at 530/25). Since plastic and cell culture medium significantly contribute to the signal, a control measurement immediately taken after the transfection when fresh medium had been added, is to be subtracted from the derived values. Alternatively, cells were counted and resuspended in PBS. cells per well (96

well quartz plate) typically resulted in good signals much above the background from negative cells.

4. Results and Discussion 4.1. CONTROL OF NEW BATCHES OF SOLUTIONS

Although many parameters have been described to optimize the solutions needed to form the precipitate, only a transfection experiment with a known reference solution proves equivalency. It is obvious that this can be tricky. A more direct method is the quantification of the turbidity of the forming precipitate itself. There is no absorption at

320 nm in the absence of "precipitate" particles. When the phosphate concentration in the precipitation buffer is increased, the physical nature of the precipitate changes and the turbidity increases in a typical mode (Fig. 1.). For transfections, an absorption of 0.13 - 0.145 was found to be optimal for precipitates formed in the presence of 25µg of DNA per ml of precipitate. Once this

value is known for a transfection protocol, the turbidity can be used as a quick assay assessing the capacity of the used solutions to generate an efficient DNA-carrier complex with calcium

phosphate.

127 4.2. GFP SIGNAL IS PROPORTIONAL TO DNA AMOUNT ADDED PER WELL

Preliminary results with showed that an internal standard correlates with product titers, but too many variations were seen with this assay. We investigated the use of GFP protein, eliminating lysis of cells, enzymatic reaction etc. A major drawback of GFP is that signals are relatively weak, which makes detection not very sensitive. Second generation GFP's overcame this limit. We tested the sensitivity by transfecting different amounts of GFP plasmid into 293 cells and measured the signal directly in the 12 well plates (Fig. 2). Even at the lowest amount of DNA, 200 ng per well, the signal was clearly above the background level. Up to a dose of 1 µg per well, signal intensity increased linearly with the amount of DNA, yet became saturated above 2.5 µg per well.

4.3. GFP SIGNAL INCREASES FOR 70 HOURS AFTER TRANSFECTION

3 – 5 hours after the addition of precipitate to the cells, first positive cells can be

identified in the microscope. The quantification of fluorescence however is difficult at time point earlier than 12 hours. Intensities of signals increase for the first 3 days and stay almost constant for a few more days (Fig. 3). The plateau after 3 days was seen at all the DNA concentrations tested. It appears therefore that such a plateau does not reflect a saturation of intracellular GFP accumulation, but a loss of expression after a certain period of time. A similar behaviour can also be observed for transient expression of

secreted proteins.

128

5. Conclusions The turbidity at 320 nm quantitatively describes the precipitate. Solutions can be quickly tested without the addition of DNA. The EGFP-N1 vector in combination with an efficient transaction method results

within a day after transfection in fluorescent signals detectable directly from growing cells. Since the signal derives directly from the expressed protein, any pretreatment of samples or incubation steps can be avoided. Expression of GFP appears not to interfere with the expression of other proteins, when representing 10% or less of plasmid quantity of the transfection cocktail. Even at this low molar ratio, the product concentration is high enough to produce easy detectable

fluorescent signals. Signals increase for about 70 hours and reflect GFP expression with a high accuracy. Intracellular accumulation of GFP shows a trend typical for the expression of secreted

proteins, such as tPA or rDNase (data not shown). The fluorescent signal might not only correlate with transfection efficiency, but with protein expression in general as well.

References 1

2

3

Jordan, M., Koehne, C. and Wurm, F.M. (1997) Calcium phosphate mediated DNA transfer into HEK293 cells in suspension: control of physicochemical parameters allows transfection in stirred media. Cytotcchnology in press Jordan, M. and Wurm, F.M. (1995) High level transient expression in mammalian cells: identification and optimization of physico-chemical parameters of the calcium phosphate transfection method, in E.C. Beuvery et al. (eds.) Animal Cell Technology: Developments towards the 21st Century, Kluver Academic Publisher, Dordrecht. 49-55 Jordan, M., Schallhorn, A. and Wurm, F.M. (1996) Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res. 24. 596-601

GLYCOSYLATION

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INFLUENCE OF Na-BUTYRATE ON THE PRODUCTION AND SIALYLATION OF HUMAN INTERFERON- BY 2,6-SIALYLTRANSFERASE ENGINEERED CHO-CELLS D. LAMOTTE1, L. MONACO2, N. JENKINS3 & A. MARC1 1 LSGC-CNRS, BP 451, 54001 Nancy Cedex - France 2 DIBIT, via Olgettina 60. 20132 Milano - Italy 3 De Montfort University, L91 9BH Leicester - UK 1. Introduction Mammalian cells are widely used for the production of recombinant drugs because of their ability to perform extensive post-translational modifications. However, they exhibit low production yields in comparison with procaryotic cells. Nevertheless, the use of stimulating agents such as sodium butyrate in medium formulation can enhance the synthesis of recombinant glycoproteins 1,2 . Some conflicting results demonstrated that Na-butyrate addition affects the cell's glycosylation machinery. For some, sodium butyrate may reduce the sialyltransferase synthesis 3,4 . Conversely, an increase in the specific activity of sialyltransferases in CHO cells was pointed out as a result of sodium butyrate addition 5 . Although the CHO-derived glycan structures are similar to those present on natural human proteins, they are not identical6. In CHO cells, sialic acids are exclusively added to terminal galactose via Since it is known that the absence of linkages can dramatically enhance clearance of a drug from bloodstream 6 , we have recently expressed the cDNA coding for the rat into the CHO 320 producing (IFN) line and allowed the presence of on IFN7. Here, we report the effect of 1 mM sodium butyrate on cell growth, IFN production and sialylation. Experiments were performed in controlled bioreactor cultures with parental and engineered CHO cells, in order to determine the influence of Na-butyrate on the IFN sialylation. 2. Materials & Methods The producing IFN CHO cell line (CHO 320) was supplied by the Wellcome Foundation Laboratories. The engineered producing IFN CHO cell line (CHO C5) was constructed as described in ref. 7. Cultures were performed with a serum-free medium based on RPMI-1640 supplemented with BSA, insulin and salts. The culture conditions were similar to those described in ref. 8. Sodium butyrate was added from a stock solution (500 mM in PBS - Sigma B2503) 24 or 36 hours further the seeding of the bioreactor. IFN purification and HPLC resolution and detection of sialic acids to discriminate between and are described in ref. 7.

131 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 131-133. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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3. Results 3.1. INFLUENCE OF Na-BUTYRATE ON GROWTH AND IFN PRODUCTION

Since preliminary shake-flask cultures have shown that the 1 mM Na-butyrate concentration is optimal for enhancing the IFN production without compromising the cell viability, bioreactor cultures were performed with 1 mM Na-Butyrate. 1 mM Na-butyrate concentration restricted the maximal viable cell density and increased the final IFN concentration by 90-130 % (fig. 1). Butyrate exposure resulted in a major increase in the maximal specific IFN production rate reaching 45 and 150 for CHO 320 and CHO C5 respectively (data not shown). For both cell lines, the production of the IFN occurred exclusively during the growth phase. 3.2. INFLUENCE OF Na-BUTYRATE ON IFN SIALYLATION IFN was purified from culture supernatants at the end of the growth phase of the batch cultures (80-100 hours of culture) performed with and without butyrate addition. The percentages of sialic acids and the molar ratio of total sialic acids to IFN molecules were determined (table 1). In control conditions, transfection of the cDNA into CHO 320 cells to produce C5 resulted in 68% of total sialic acids linked in the and nearly double the overall IFN sialylation, in comparison with the IFN from the parental cell line.

133

The effect of adding butyrate was determined for the two cell lines. As expected, 100 % of sialic acids on IFN from CHO 320 were still in the in the culture performed with Na-butyrate. Conversely, 82 % of sialic acids were in the conformation on IFN secreted by CHO C5 line with Na-butyrate compared to 68% without butyrate. Furthermore, the overall degree of IFN sialylation was twofold higher for CHO C5 cells subjected to butyrate, whereas it was not enhanced by butyrate exposure in the CHO 320 cell line. Therefore, the increase in the overall degree of sialylation on IFN produced by CHO C5 cells with butyrate results

predominantly from an increase in the molar ratio of total (+130%) rather than sialic acids (+15%).

sialic acids

4. References 1. Chevalot I., Dardenne M., Cherlet M., Engasser J.-M. and Marc A. (1995) Effect of sodium butyrate on protein production in different culture systems, in Beuvery E.C., Spier R. and Griffiths B. (eds.), Animal Cell Technology: Developments towards the 21st century, Kluwer Academic Publishers, Dordrecht, pp. 143-147 2. Oster T., Thioudellet C., Chevalot I., Masson C., Wellman M., Marc A. and Siest G (1993) Induction of recombinant gamma-glutamyl transferase by sodium butyrate in transfected V79 and CHO Chinese hamster cells, Biochem Biophys Res Comm 193, 406-412.

3. Shah S., Lance P., Smith T.J., Berenson C.S., Cohen S.A., Horvath P.J., Lau J.T.Y. and Baumann H. (1995) n-butyrate reduces the expression of in Hep G2 cell. J Biol Chem 267, 10652-10658. 4. Li M., Andersen M.L. and Lance P. (1995) Expression and regulation of glycosyltransferases for N-

glycosyl oligosaccharides in fresh human surgical and cultured cell lines, Clin Sci 89, 397-404. 5. Chotigeat W., Watanapokasin Y., Malher S. and Gray P.P. (1994) Role of environmental conditions on the expression levels, glycoform pattern and levels of sialyltransferase for hFSH produced by recombinant CHO cells, Cytotechnology 15, 217-221. 6. Jenkins N., Parekh R.B. and James D.C. (1996) Getting the glycosylation right: implications lor the biotechnology industry, Nature Biotechnology 14, 975-981. 7. Monaco L., Marc A., Eon-Duval A., Acerbis G., Distefano G., Lamotte D., Engasser J.-M., Soria M and Jenkins N. (1996) Genetic engineering of in recombinant CHO cells and its effect on the sialylation of recombinant interferon-gamma, Cytotechnology 22, 197-203. 8. Lamotte D., Eon-Duval A., Acerbis G., Distefano G., Monaco L., Soria M., Jenkins N., Engasser J.-M and Marc A. (1997) Controlling the glycosylation of recombinant proteins expressed in animal cells by genetic and physiological engineering, in Carrondo M.J.T., Griffiths B. and Moreira J.L.P. (eds.), Animal Cell Technology: From Vaccines to Genetic Medicine. Kluwer Academic Publishers, Dordrecht, pp. 761-765.

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ELEVATED INHIBITS THE POLYSIALYLATION OF THE NEURAL CELL ADHESION MOLECULE IN CHO MT2-1-8 CELL CULTURES

JAMES A. ZANGHI1,3, THOMAS P. MENDOZA1, RICHARD H. KNOP2, WILLIAM M. MILLER1,# 1

Department of Chemical Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208-312 2 Division of Medical Oncology, Evanston Hospital, 2650 N. Ridge Avenue, Evanston, IL 60201-1794 3 Present location: Institute of Biotechnology, Swiss Federal Institute of Technology, ETH Hönggerberg, HPT, CH-8093 Zürich, Switzerland. Email: [email protected] # Corresponding author. Phone: (847)491-4828. Email: wmmiller@nwu. edu

Animal cells are important for the production of complex glycoproteins for use in therapeutics and diagnostics. Conditions during culture, such as the depletion of nutrients and accumulation of metabolites, may adversely affect the glycosylation process or increase the deterioration of glycoproteins after secretion (1, 2). Glycosylation has a critical role in protein quality by regulating its physiochemical and biological properties such as specific activity, thermal stability, solubility, clearance rate from blood stream, and immunogenicity (1). We and others have shown that ammonia, a metabolic waste product of animal cells, can specifically inhibit protein glycosylation (3, 4, 5, 6). Using the neural cell adhesion molecule (NCAM) as our model cell surface glycoprotein (Fig. 1), we showed that ammonia inhibits the NCAM polysialylation in Chinese hamster ovary (CHO) and small cell lung cancer (SCLC) cells in a dosedependent and reversible manner (6). The response was very strong, with a 90% decrease in CHO cell PolySia surface content following an exposure to 10 mM for 4 days.

A possible explanation for ammonia inhibition of sialylation or polysialylation is decreased (poly)sialyltransferase activity due to perturbations in the trans golgi pH

where the enzymes reside. The weak base readily diffuses across biological membranes, while its conjugate acid is virtually impenetrable so that the base accumulates in acidic compartments in the cell, including the trans golgi, resulting in collapse of the pH gradient (7, 8, 9, 10).

Carbon dioxide is another important waste product of animal cells, though problematic only under specific conditions such as high density culture systems where significant differences between the mass transfer coefficients of oxygen and carbon dioxide leads to a buildup of dissolved 135 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 135-140. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

136

By neglecting follows (13):

in water may be summarized as

Analogous to ammonia, freely diffuses across cellular membranes while the conjugate base is essentially membrane impermeable. For this reason, cells exposed to elevated at constant pH experience an initial decrease in pH. While the trapping effect observed for ammonia is less likely to occur with because of specific transporters such as the antiporter and symporter (14), the decrease in intracellular pH may or may not return to the original value (15, 16). We therefore hypothesized that elevated may also disrupt intracellular pH and other ion gradients, and thus inhibit polysialylation in a manner analogous to ammonia.

Elevated inhibits CHO cell growth and protein production (12, 17, 18). Kimura et al. (18) showed that CHO cell growth inhibition was more severe when osmolality was allowed to increase with while an increase in osmolality alone had no effect. In addition, it was shown that elevated had no effect on recombinant tissue plasminogen activator (tPA) glycosylation with the exception of an increase in Nglycolylneuraminic acid (Neu5Gc; balance N-acetylneuraminic acid, Neu5Ac) (19). A similar observation was reported by Grampp (20). Here we examined the effects of elevated as well as pH and osmolality, on CHO MT2-1-8 cell NCAM polysialylation. The experiments were conducted with the same CHO MT2-1-8 cell line, culture medium and set-up as that previously published for the studies with tPA (18, 19, 21, 22) (Fig. 2). This allows a direct comparison of the effects of on the glycosylation of the more robust tPA molecule and the hyper-sensitive NCAM

137

polysialic acid. Cells were cultured for 1-5 days under an atmosphere of 38 (control, 5% , 90, 140, or 195 torr and subsequently analyzed by flow cytometry using anti-NCAM and -PolySia mAbs. We separately measured the effects of osmolality and pH on NCAM polysialylation by adjusting the NaCl or NaOH concentration in the

medium, respectively.

The results of the experiments are summarized in Table 1 (23). At normal levels, both increasing pH and osmolality inhibited polysialylation. The effect was most pronounced at a pH above 7.4 and when the osmolality exceeded 400 mOsmol/kg. As increased the effect was much greater, with PolySia content decreasing until no immunoreactivity remained (i.e. anti-PolySia mAb fluorescence was identical to background fluorescence) after four days exposure to 140 at pH 7.5. At moderately elevated levels (90 torr), PolySia was unaffected or only slightly inhibited at pH 7.2 but was significantly reduced at pH 7.5. In all cases, NCAM was not affected, suggesting that the increase in pH, osmolality and were specific to polysialic acid.

138

Kimura et al. showed that increasing osmolality exacerbates the growth (18). In addition, increases with increasing pH which may disrupt exchangers in the cell membrane or other cell functions. Cell counts prior to flow cytometry confirm that there was a synergism between increased pH and elevated At pH 7.5, CHO cell growth was completely inhibited inhibition caused by elevated

at 195 torr, and there was considerable cell death at 250 torr This contrasts the results obtained at pH 7.2, where 195 torr and 250 torr decreased cell growth by 24% and

44%, respectively (18). In addition, at values of 195 and 250 torr, there were gross changes in morphology, including larger cell size and decreased adherence to the culture flask only at the higher pH. An increase in increases the buffering capacity of the medium, thus requiring more acid or base to change the pH.

As

illustrated in Fig. 3, small changes in pH will greatly affect the amount of NaOH added to the cell culture at elevated which will also affect osmolality. For ammonia, an increase in extracellular pH results in greater accumulation of ammonia in the cell and greater inhibition of both cell growth and sialylation (4, 6, 24, 25). It is clear the carbon dioxide has a similar effect. The increased buffering capacity at elevated

will likely reduce proton gradients within the cell, especially in acid

organelles where pH can be a low as 5.0, and increase the energy required to maintain the ATPase activity needed to pump protons from the organelles. If exchangers are

present in membranes of these organelles, then elevated may directly disrupt intracellular pH gradients by way of Either way, changes in pH may result in a decrease in the activity of pH-sensitive enzymes within these organelles and may explain the observed decrease in NCAM polysialylation.

In addition to the relevance to biotechnology, the regulation of PolySia by factors in the extracellular environment can have broad implications.

NCAM

polysialylation is developmentally regulated and mediates a variety of cell-cell adhesive interactions including cell migration during morphogenesis (26) and malignant growth

(27). Conditions present in large scale, high density culture systems such as elevated ammonia and carbon dioxide levels are also present within fast-growing solid tumors

139

such as SCLC.

The data provide evidence of a link between the tumor microenvironment and metastasis through the regulation of NCAM polysialylation. Acknowledgments. We thank Merck, Sharp & Dohme for sponsoring the oral presentation of this work at the 15th ESACT meeting. We also acknowledge the organizing committee for making this possible through a bursary. We thank Albert Schmelzer for his laboratory assistance.

This research was supported by NSF grants BCS-9058416 and BES-9402030 (W.M.M.) and the Jean Ruggles Romoser Research Endowment (R.H.K).

References 1. 2. 3. 4.

C. F. Goochee, T. Monica, Bio/technol. 8, 421-426 (1990). D. C. Anderson, C. F. Goochee, Curr. Opin. in Biotechnol. 5, 546-549 (1994). D. C. Anderson, C. F. Goochee, Biotechnol. Bioeng. 47, 96-105 (1995). M. C. Borys, D. I. H. Linzer, E. T. Papoutsakis, Biotechnol. Bioeng. 43, 505-514 (1994).

5.

M. Gawlitzek, U. Valley, M. Nimtz, R. Wagner, H. S. Conradt, in Animal Cell Technology:

Developments towards the 21st Century. E. C. Beuvery, Ed. (Kluwer Academic, Netherlands, 1995). 6. J. A. Zanghi, T. P. Mendoza, R. H. Knop, W. M. Miller, J. Cell Physiol. submitted (1997). 7. W. F. Boron, P. de Weer, J. Gen. Physiol. 67, 91-112 (1976). 8. R. T. Dean, W. Jessup, C. R. Roberts, Biochem J. 217, 27-40 (1984). 9. C. de Duve, et al., Biochem. Pharmacol. 23, 2495-2531 (1974). 10. R. G. Anderson, R. K. Pathak, Cell 40, 635-643 (1985). 11. S. S. Ozturk, Cytotechnol. 22, 3-16 (1996). 12. D . R. Gray, S. Chen, W. Howarth, D. Inlow, B. L. Maiorella, Cytotechnol. 22, 65-78 (1996). 13. W. W. Umbreit, in Manometric and biochemical techniques W. W. Umbreit, R. H. Burris, J. F. Stauffer, Eds. (Burgess Publishing, Minneapolis, 1972) pp. 20-29. 14. I. H. Madshus, Biochem. J. 250, 1-8 (1988).

15. 16.

R. Krapf, C. A. Berry, R. J. Alpern, F. C. J. Rector, J. Clin. Invest. 81, 381-389 (1988). L. Simchowitz, A. Roos, J. Gen. Physiol. 85, 443-470 (1985).

17.

D. Drapeau, Y.-T. Luan, J. C. Whiteford, D. P. Lavin, S. R. Adamson, Paper presented at the Annual

Meeting of the Society of Industrial Microbiology, Orlando, FL (1990). R. Kimura, W. M. Miller, Biotechnol. Bioeng. 52, 152-160 (1996). R. Kimura, W. M. Miller, Biotechnol. Prog. 13, 311-317 (1997). G. E. Grampp, et al., Paper presented at the Cell Culture Engineering IV meeting, San Diego, CA (1994). 21. R. Kimura, PhD Thesis, Northwestern University (1996). 22. J. A. Zanghi, PhD Thesis, Northwestern University (1997). 23. J. A. Zanghi, T. P. Mendoza, A. Schmelzer, R. H. Knop, W. M. Miller, Manuscript in preparation. (1997). 24. C. Doyle, M. Butler, J. Biotechnol. 15, 91-100 (1990). 25. I. Lüdemann, R. Pörtner, H. Märkl, Cytotechnology 14, 11-20 (1994).

18. 19. 20.

26.

G. M. Edelman, Ann. Rev. Biochem. 54, 135-169 (1985).

27.

E. P. Scheidegger, P. M. Lackie, J. Papay, J. Roth, Lab.Invest. 70, 95-106 (1994).

140

Discussion

Ozturk:

It is very difficult to separate the effects of pH and bicarbonate and because they are all coupled together. So I was wondering if it is possible to decouple the bicarbonate and

Zanghi:

Constant pH and osmolality was maintained and we varied the but the bicarbonate level went up. It may be possible to alter the pH and together and maintain the bicarbonate concentration.

Ozturk:

Bicarbonate maybe more important than the

Zeng:

Have you measured, or calculated, the

Zanghi:

The

itself. data?

was measured using a blood-gas analyser in all cases

INFLUENCE OF CULTIVATION CONDITIONS ON GLYCOSYLATION PATTERNA FED-BATCH AND CONTINUOUS CULTURE STUDY

NATASCHA A. SCHILL, MORRIS Z. ROSENBERG, REBECCA L. DABORA Biogen Inc., 14 Cambridge Center, Cambridge MA 02142, USA

1. Introduction

Glycosylation is a post-translational event taking place in the Golgi apparatus of eukaryotic cells. The glycosylation pattern of a protein may have a significant effect on

its physical, chemical and biological properties. Therefore, process development for a potential therapeutic glycoprotein is only successful if it can be demonstrated that the product is consistently glycosylated from batch to batch. One of the most critical process steps with respect to glycosylation pattern of a protein is the cell culture

process (e.g. Jenkins et al. 1996). From the literature it appears as though factors

affecting glycosylation are different from protein to protein; both environmental and kinetic variables were reported to influence the glycosylation pattern of proteins. This paper discusses the influence of different process variables on the glycosylation pattern of a recombinant fusion protein which contains eight N-linked glycosylation sites. The carbohydrate groups contribute approximately 30% to the total molecular mass of the protein. The protein is produced by a recombinant CHO cell line in fed-

batch mode. It was observed that minor changes in cultivation methods, timing of feed addition or time of product harvest, could lead to a significantly different glycosylation pattern of the protein. To identify the process variables influencing glycosylation of this fusion protein, the study included fed-batch and continuous cultivations. Results suggest that only specific productivity affects the glycosylation pattern of this recombinant fusion protein. 2. Materials and methods A CHO derived cell line was used in the study. It grew in serum-free low protein medium. Methotrexate (MTX) was always present in the medium to maintain selective

pressure. All fed-batch and continuous cultures were performed in bench-scale reactors controlled at 36 C, pH 7.2 and 50% air saturation. Appropriate amounts of fusion protein were purified in a one column step, and glycosylation pattern was analyzed by IEF (isoelectric focusing), asialo-FACE (fluorophore assisted carbohydrate 141 O.-W. Merten et al. (eds.). New Developments and New Applications in Animal Cell Technology, 141-147. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

142

electrophoresis), OGS assay (Oxford Glyco-systems), and SEC (size exclusion chromatography). 3. Results and Discussion 3.1. FED-BATCH CULTIVATION Changes in protein glycosylation pattern during a typical fed-batch cultivation were

followed by analyzing samples taken during exponential, stationary and death phase. Results from IEF, SEC and OGS analysis show that the glycosylation pattern of the fusion protein changed over the course of the fed-batch culture. Between days 6 and 8 the IEF banding pattern shifted towards more acidic pH (see Figure 1), suggesting an increase in the sialic acid attached to the carbohydrate groups of the fusion protein. Indeed, analysis of sialic acid content confirmed that the sialic acid content of the fusion protein increased by 20% during the cultivation. In addition to changes in sialic acid content it was noted that the antennary structure was also altered. Figure 2 shows the antennary structure of the fusion protein at different cultivation times, as measured

by OGS analysis: biantennary structures decreased, whereas tetraantennary structures increased with cultivation time. The observation that the cells only started to produce a more completely glycosylated protein after the exponential growth phase suggested that one or more process variables

could be responsible for this. Throughout the cultivation specific growth rate specific productivity (qP), growth phase, nutrient composition, ammonium concentration, and culture osmolality changed. Because the culture environment is

altered steadily with time during fed-batch or batch cultivation, these methods are not amenable to identifying critical process parameters affecting glycosylation microheterogeneity. However, continuous culture experiments allow the control of the culture environment independent of time. This technique is thus suitable for a thorough investigation of the relationship of each of these growth variables with the glycosylation pattern of the fusion protein. 3.2. CONTINUOUS CULTURE EXPERIMENTS

During long-term continuous cultures selective pressure was maintained with methotrexate (MTX) to avoid a loss in fusion protein gene copy number. During the experiment the following groups of variables were tested for their influence on product quality: 1) environmental variables, i.e. asparagine concentration, glutamine

concentration, and ammonium concentration; and 2) kinetic variables, i.e. specific growth rate and specific productivity. The glycosylation pattern was determined by

IEF, SEC, asialo-FACE, and OGS analysis. None of the environmental variables tested in pulse and/or shift-experiments affected

the protein glycosylation pattern, although concentrations were increased one to three fold for the amino acids, and seven fold for the ammonium concentration.

143

Steady states at dilution rates of 0.35/d and 0.20/d were established to investigate the influence of specific growth rate on glycosylation pattern. Analysis by IEF, OGS and

SEC showed that glycosylation pattern was similar for both steady states although the difference in growth rate was nearly 2-fold. Thus, the glycosylation pattern of the fusion protein was independent of specific growth rate. Interestingly, glycosylation pattern resembled the pattern obtained at the end of fed-batch cultivations

The glycosylation pattern did change significantly during a transient increase in ferric citrate concentration (Figure 3), which resulted in an increase in both specific growth and specific productivity. Coincident with these changes were observed an increase in the amount of aggregation from 5.6 to 13.9 percent, and a decrease in terminal sialylation as evidenced by the disappearance of the more acidic bands on an IEF gel (not shown). Figure 4 shows that the change in ferric citrate concentration also resulted in a decrease in tetraantennary glycoforms, with a corresponding increase in the fraction of biantennary structures. The observation that the complexity in antennary structure and sialic acid content decreases when the specific productivity increases, agrees with the results obtained during the fed-batch cultivation discussed above. For example, Figure 5 illustrates the relationship between sialic acid content and specific productivity as observed during fed-batch and transient continuous cultures. This strongly suggests that the specific productivity is affecting the glycosylation of the fusion protein. 3.3. CHANGES IN INTRACELLULAR PROTEIN GLYCOSYLATION DURING FED-BATCH CULTIVATION

There are two possible explanations for the observed relationship between glycosylation pattern and specific productivity: 1) at high specific productivities the glycosylation enzymes are saturated and can not glycosylate all proteins flowing through the Golgi apparatus; or 2) the residence time of the protein in the Golgi apparatus is too short to ensure complete glycosylation. The following experiment was performed to investigate which of the two explanation is most likely for the fusion protein of interest. A typical fed-batch experiment was performed in duplicate reactors, where samples were taken throughout the cultivation to analyze intracellular fusion protein quantity and quality. Figure 6 shows a SDS PAGE Western blot of the intracellular fusion protein at

different time points of cultivation. The intensity of the protein band on the SDS PAGE Western blot increased with cultivation time indicating an increase in intracellular fusion protein concentration. This suggests that the fusion protein is accumulating in the Golgi apparatus over the course of the fed-batch cultivation. As a consequence, an increasing fraction of the product would be exposed to the glycosylation enzymes for a longer period of time, and therefore, become more completely glycosylated. That intracellular fusion protein becomes more fully glycosylated with cultivation time is supported by the following three analysis: 1) the molecular mass of the intracellular fusion protein appears to increase with cultivation time as evidenced by a shift to a

144

higher molecular mass of the protein band on the SDS PAGE Western blot (Fig. 6); 2) the mass of glycan per gram of protein increased with cultivation time as shown by the carbohydrate structure analysis (not shown); and 3) a clear shift towards more acidic pH with cultivation time was observed on the IEF Western blot (not shown), suggesting an increase in sialic acid content.

In summary, these results show that for studied cellular system, glycosylation is inversely related to the specific productivity of the cell. Results further indicate that the residence time of the protein in the Golgi apparatus is crucial for the extent of glycosylation. However, this hypothesis would need confirmation by a pulse/chase type experiment, which allows flow rates through the Golgi apparatus to be measured. 4. Conclusions

This project focused on the relationship between cultivation conditions and glycosylation pattern of a recombinant fusion protein produced by a CHO derived cell line. During the investigation it was found that the glycosylation pattern changed

during fed-batch cultivation: sialic acid content and antennary complexity increased

with cultivation time although ammonium concentration and most likely glycosidase activities - two factors which reportedly can decrease sialic acid concentration increased simultaneously. Continuous cultures were performed to identify which environmental and kinetic variables impact the glycosylation process. A change in glycosylation pattern was only found when the specific productivity during a continuous culture was increased transiently by an increase in ferric citrate concentration. This indicated that it is a kinetic variable which influences the glycosylation of the studied fusion protein. Further experiments focused on the glycosylation of intracellular fusion protein and support the hypothesis that specific productivity, or rather the residence time of the protein in the Golgi apparatus, is crucial to the extent of glycosylation. Notably, concentration, molecular weight, and sialic acid content of the fusion protein increased with cultivation time, suggesting that a prolonged residence time in the Golgi apparatus leads to more complete glycosylation.

5. References Jenkins N., Parekh R.B. and James D.C. (1996) Getting the glycosylation right: implications for the biotechnology industry. Nature Biotechnol. 14: 975-982.

145

146

147

Discussion

Franek:

What are your ideas on the mechanism of how ferric citrate influences the sialylase?

Schill:

I do not think it is ferric citrate which influences glycosylation. In continuous culture, we chose to decrease the ferric citrate concentration as we did not want to limit the cultures by glucose or another energy source. It is not the effect of the ferric citrate, but rather of increasing the specific productivity.

Kempken:

In principle, you could use a very low perfusion rate and could verify a glyco-pattern that is similar to the end of a batch culture. You could then use a very high perfusion rate and verify a pattern that you have in an early stage of a batch fermenter.

Schill:

We established two steady-states. The higher one was close to the exponential growth rate. To establish a steady-state you need to allow 7 residence times to stabilise the system, and with mammalian cells this can take months. So the restriction to doing this is time.

Kempken:

I agree, but you might then find a compromise between your required product structure and the productivity of the system.

Schill:

Specific productivity is not linked to the specific growth rate in this system.

Butler:

In establishing your steady-states, do you know what your limiting nutrient was and, if so, have you considered that the limiting nutrient might affect the interpretation of the data?

Schill:

I think ferric citrate was the limiting factor in the cultures, but it was still at measurable levels, ie not that low. We do not know the affinity of the cell for ferric citrate, which could be higher as normally transferrin is used as a transport factor.

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EFFECTS OF DIFFERENT PRODUCTION SYSTEMS ON GLYCOSYLATION PATTERN OF MURINE MONOCLONAL IgA SCHWEIKART, F.1; LÜLLAU, E. 2 ; JONES, R.1 ; HUGHES, G.J.1 1 Dept. of Medical Biochemistry at the Medical Center, Faculty of Medicine, University of Geneva, 1 rue Michel-Servet, CH-1211 Geneva 4, Switzerland; 2 Biomedical Research Institute, Glaxo Wellcome Research and Development SA, 14 chemin des Aulx, CH-1228 Plan-les-Ouates, Switzerland

Introduction

Immunoglobulin A is one of the key components in mucosal immune defence in mammals. Monoclonal IgA was produced under

protein free conditions by a murine hybridoma cell line (ZAC3) in a 0.5 1 hollow fibre (HFR), a 2.15 1 continuous stirred tank (CSTR) and a 2 1

fluidized bed reactor (FBR). The IgA was directed against the LPS-antigen of vibrio cholerea. IgA from the three different production systems was purified by DEAE anion exchange, hydroxyapatite and size exclusion chromatography [1]. The glycosylation pattern of the alpha chain was examined by two different approaches outlined in Fig. 1. Approach 1 gives information on glycosylation at specific sites by analysing selected peptides from a peptide map. The heterogeneity of the glycostructures at a given site can be examined. In a second approach HPLC profiling of the total oligosaccharide content was performed after PNGase F cleavage of the whole IgA and labelling of the released oligosaccharides. This was followed by analysis of the separated, derivatized oligosaccharides by matrix-assisted laser desorption time of flight mass (MALDI-TOF) spectrometry. This gives quantitative information about relative amounts of certain structures.

Murine IgA glycosylation Mouse has 4 potential N-glycosylation sites. Only two of them were originally found to be glycosylated [3]. One is located in domain and the other in the 149 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 149-152. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

150 tailpiece of domain (329NVS). The other potential sites for N-glycosylation are 99NCS (domain ) and 314NFT (domain ). Glycosylation at a third site has been

described for an chain expressed in an another myeloma cell line[4]. Myeloma IgA's were shown to contain no N-acetylgalactosamine and therefore no O-linked oligosaccharides [2, 5]. The light chains contain no N-linked glycosylation sites in the constant region. Results

All IgA’s examined carry a large variety of different Nlinked oligosaccharide structures. The glycosylation of

the chain varies according to the IgA production system. IgA chain from all three bioreactors is shown to carry N-linked oligosaccharides only at position 38NVS (domain ) (Fig.2). The expected site in the tail piece (329NVS, domain ) is surprisingly not glycosylated : the corresponding tryptic peptide was found in a nonglycosylated form in the peptide map

and it could be identified by Edman sequencing and precise mass assignment. A MALDI-TOF mass spectrometric analysis of the glycosylated tryptic peptide (peak 1) is shown in Fig. 3. The spectrum

reveals a great variety of structures all of which could be independently confirmed by following the second approach. Here, by achieving a monoisotopic resolution, a more detailed and accurate analysis of the oligosaccharide structures could be made. N-linked oligosaccharides Some m/z values

and the corresponding proposed structure are indicated. After

treatment of the glycopeptide with PNGase F an average mass of 4291 could be determined (not shown); the nominal mass of the peptide moiety

appeared with a mass difference of 16, presumably due to the oxidised

A small portion of the peptide

in the position

37. This mass difference makes a differentiation between e.g. NeuAc~NeuGc or Hex~Fuc in the glycosylated peptide impossible. More detailed information are only available by following the approach 2.

151

were released and labelled with PMP (Fig.4). The UV-profiles (Fig. 5) represent the compositions of

chain oligosaccharides derived from IgA's produced in the three

bioreactor systems. The proportion of di- and oligosialylated structures decreased in the following order: CSTR>FBR>HFR; the HFR derived IgA contained almost no sialylated oligosaccharides. The proportion of

monosialylated structures decreased in the same order but to a lesser extent. Selected HPLC-fractions were analysed by MALDI-TOF mass spectrometry. The monosaccharide compositions were deduced from the very accurate monoisotopic mass determination of the whole PMP-labelled

oligosaccharide.

The

error

in

mass

determination varied between 0.005 and 0.02%. By measuring mass differences after digestion with exoglycosidases a further confirmation of several structures could be obtained. High mannose structures were deduced after treatment; sialylated structures either by direct

measurement of Cl-methylated compounds or after sialidase treatment. At least 21 different oligosaccharide structures could be deduced. A further 20 additional, less abundant oligosaccharide structures could be detected. HFR IgA contained more of a GlcNac bisected hybrid structure (peak 1, 2) than FBR IgA. In CSTR IgA no such structure could be detected. IgA's from all reactor systems contained a quite high proportion of high mannose structures (HFR peak 3,4; CSTR peak 5,6). Otherwise mainly complex oligosaccharides of the biantennary type and only low amounts of triantennary complex structures were found. N-Glycolylneuraminic acid was present on a large number of oligosaccharides from CSTR derived IgA. Many of the oligosaccharide

structures were fucosylated. The large number of different structures suggests that many of them are incompletely

processed or truncated. Although ammonium in the cell culture medium is known to be

152 an important factor influencing the sialylation level of expressed glycoproteins, in these studies no significantly higher or longer-lasting ammonium concentration was observed during the fermentation in the three production systems. Our work indicates that different production systems profoundly influence the glycosylation pattern of a glycoprotein. Although these systems were operated under conditions as similar as possible (protein free, pH, they represent quite different environmental conditions for the cells. Given that specific oligosaccharide structures can inhibit bacterial adhesion

at mucosal surfaces [6], the different glycosylation patterns seen with different production systems may influence the efficacy of antigen-specific IgA when used for therapeutic intervention [7]. References [1]

Lüllau, E., Schweikart, F., Huser, M., von Stockar, U. and Hughes, G. J., Biotech. Bioeng., submitted

[2]

The numbering of amino acid residues was taken over from that used in the SWISS-Prot entry P01878 Robinson, E. A. and Appella, E., Proc. Natl. Acad. Sci. USA.77, 4909-4913 (1980)

[3] [4]

Taylor, A. K. and Wall, R., Mol. Cell. Biol., 8, 4197-4203 (1988)

[5]

Lipniunas, P. et al., Arch. Biochem. Biophys., 300, 335-345 (1993)

[6]

Boren, T., Falk, P., Roth, K. A., Larson, G. and Normark, S., Science, 262, 1892-1895 (1993)

[7]

Weltzin, R., Hsu, S. A., Mittler, E. S., Georgakopoulos, K. M. and Monath, T. P., Antimicrob. Agents Chemother., 38, 2785-2791 (1994)

Acknowledgement

This work was supported by research funds from the Swiss National Science Foundation, SPP Biotechnology Priority Program

IDENTIFICATION OF ALTERED GLYCOSYLATION AS THE MAJOR DIFFERENCE BETWEEN INTRACELLULARY ACCUMULATED AND SECRETED PROTEIN PRODUCED IN BACULOVIRUS-INFECTED INSECT CELLS. GRYSSELL RODRIGUEZ1*, HARALD. S. CONRADT2 and VOLKER JÄGER 1 . 1

Cell Culture Technology Dept., 2Protein Glycosylation Dept., Gesellschaft für Biotechnologische Forschung mbH, Mascheroder Weg 1, D-38124 Braunschweig, Germany. * Permanent address: Centro de Inmunología Molecular, POBox 11600 Habana, Cuba. Introduction

The baculovirus expression vector system (BEVS) has become widely used for the production of recombinant proteins due to its ability to express large amounts of foreign gene products inserted under the transcriptional control of the polyhedrin or p10

promoters. Another advantage is that insect cells perform most of the post-translational modifications of proteins observed in higher eukaryotes. However, the intracellular

accumulation of substantial amounts of protein can be observed frequently for numerous recombinant proteins. Recombinant human a glycoprotein bearing

two N-glycosylation sites, was selected as a model protein. Isolated from human cerebrospinal fluid, serum, plasma and urine its biological significance as a prostaglandin-D synthetase remains disputed (3). This work is focussing on the comparison of intracellularly accumulated and secreted produced by IPLB-SF21AE and High Five™ insect cells and the identification of the intracellular accumulation of recombinant protein, apparently associated to the glycosylation process. Results and Discussion Comparison and characterization of intra- and extracellular in insect cell lines

SDS-PAGE analysis (Figure 1) revealed that

protein expressed

protein from insect cells runs

slightly faster than the protein secreted by recombinant BHK21 cells or isolated from hemofiltrate of patients. This difference in the electrophoretic mobility implies smaller N-glycan structures than those found in protein from recombinant BHK21 cells or hemofiltrate (1,3). Two insect cell lines, High Five and Sf21, were infected with a recombinant baculovirus for expression of For both cell lines the pattern of intracellular protein was characterized by three bands at the same positions than those of the secreted protein (Figure 2). In addition, intracellularly accumulated protein revealed a band with a higher molecular

weight of 28 kDa, which was of increasing intensity with time post infection. 153 O.-W. Merten el at. (eds.), New Developments and New Applications in Animal Cell Technology, 153-155. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

154

Another band with a molecular weight of about 24 kDa was detected in protein from Sf21 cells. In our opinion this band corresponds to an intermediate stage in the processing of the secreted protein inside the cell, but not to an intracellularly accumulated form., because the intensity did not alter after infection. The extracellular protein produced by both cell lines was represented by three bands with molecular weights of about 25.6 kDa, 22.7 kDa and 21 kDa. From 4 days post infection onward, after the majority of cells had died due to viral infection, a smeary band appeared near to 28 kDa. This smear was proved to become finally a clear band (data not shown). The presence of this band in the supernatant, at the same position than the intracellular band with 28 kDa, suggests an accumulation of the corresponding protein inside of the cell, which is released into the supernatant only after cell lysis.

Oligosaccharide processing, limiting step in the protein secretory pathway

The intracellular accumulation of substantial amounts of recombinant protein has been reported frequently for baculovirus-infected insect cells (2, 4, 5). A detailed carbohydrate analysis of both intracellular and secreted IgG was carried out recently (5). In contrast to the secreted glycoprotein, the N-glycans of intracellular IgG from insect cells included more than 50% of high mannose type structures, indicating a significant level of incomplete oligosaccharide processing for a fraction of the intracellular immunoglobulins. It

was hypothesized that these intracellular immunoglobulins may not reach the subsequent cellular compartments in which carbohydrate trimming takes place, probably due to retention or slow secretory pathway processing of some immunoglobulins in the

endoplasmatic reticulum or Golgi apparatus of baculovirus-infected cells. Using protein as a model we wanted to check if glycosylation was playing a crucial role in the protein accumulation. For that purpose it was checked if glycosylation was responsible for the differences in the SDS-PAGE patterns between intra- and extracellular protein.

155

Both protein fractions, intra- and extracellular were digested with N-Glycosidase F enzyme. After enzymatic digestion, the electrophoretic pattern of recombinant intra- and extracellular protein changed dramatically. All bands except the lowest one representing the non-glycosylated form of protein disappeared and the remaining band became more intensive (Figure 3). These results clearly demonstrate that the different bands from intracellular protein correspond to different glycosylation stages of the same peptide, and provide an evidence about a possible bottle neck in the secretion of protein, apparently due to a problem

during the glycosylation process.

Conclusions SDS-PAGE/western blotting of protein from supernatant as well as from lysed virusinfected insect cells (IPLB SF21 AE, BTI Tn5Bl-4) revealed the presence of an intracellularly acumulated species, which was demonstrated to have a molecular weight different to those observed in the supernatant. Enzymatic digestion with N-Glycosidase F has proved that the difference between the intra-and extracellular protein pattern was due to different glycosylation stages of the recombinant protein. The results suggest the presence of a bottle neck in the glycosylation pathway of baculovirus-infected insect cells secreting protein. Acknowledgements We want to acknowledge the financial support from ESACT for G. Rodriguez to participate in this conference. References 1.

Grabenhorst, E., Hoffmann, A., Nimtz, M., Zettlmeissl, G. and Conradt, H.S. (1995) Construction of stable BHK-21 cells coexpressing human secretory glycoprotein and human Gal

GlucNAc-R

3.

sialyltransferase NeuAc is preferentially attached to the Gal Gluc NAc ) Man of diantennary oligosaccharides from secreted recombinant protein. Eur. J. Biochem. 232, 232-718. Hasema, C.A and Capra, J.D. (1990) High-levels production of a funtional immunoglobulin heterodimer in a Baculovirus Expression System. Proc. Natl. Acad. Sci. USA. 87, 3942-3946. Hoffmann, A.,Nimtz, M., and Conradt, H.S. (1997) Molecular characterization of protein in

4.

human serum and urine: a potencial diagnostic marker for renal disease. Glycobiology 7, 499-506. Hsu, T.A, Eiden, J.J., Bourgarel, P., Meo, T. and Betenbaugh, M.J. (1994) Effect of co-expressing

2.

chaperone BIP on funtional antibody production in the baculovirus system. Protein Expr. Purif. 5, 95-603. 5. Hsu, T.A., Takahashi, N., Tsukamoto, Y., Kato, K., Shimada, I., Masuda, K., Whiteley, E.M., Fan, J.-Q., Lee, Y.C and Betenbaugh, M.J. (1997) Differential N-Glycans patterns of secreted and intracellular IgG produced in Trichoplusia ni cells. J. Biol. Chem. 272, 9062-9070.

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HOW AMMONIUM DOMINATES THE METABOLISM IN VITRO CULTIVATED MAMMALIAN CELLS

OF

Aziz Çayli1, Manfred Wirth2 and Roland Wagner1 1 Cell Culture Technology Department, 2Department of Gene Regulation and Differentiation, Gesellschaft für Biotechnologische Forschung mbH, Mascheroder Weg 1, D-38124 Braunschweig, Germany

Introduction

The accumulation of ammonium ions during cultivation of mammalian cells used for the production of recombinant biopharmaceuticals affects growth rate and productivity (Backer et al. 1988; Butler and Spier, 1984; Glacken, 1988; Ito and McLimans, 1981; Jensen and Liu, 1961) and the quality of the synthesized product, particularly with regard

to glycosylation pattern (Gawlitzek et al., 1995, 1997, Maiorella, 1992; Thorens and Vasselli, 1986). Recently we have reported that the intracellular content of UDP-activated N-acetyl hexosamines (UDPGNAc) is substantially elevated under higher ammonium

concentrations in the medium (Ryll et al, 1994). Ammonium ions are channeled into the pathway of UDPGNAc formation at the amination step of fructose-6-phosphate (Frc6P) to form glucosamine-6-phosphate (GlcN6P). UDPGNAc is then formed by a bifurcated pathway whereby UTP and N-acetyl glucosamine-1-phosphate (GlcNAclP) condense to UDPGlcNAc which subsequently is transformed to UDPGalNAc. We could show that 15N of UDPGNAc, a precursor of carbohydrates in glycoproteins, is found in the N-glycans (Valley, 1996) resulting in the formulation of the hypothesis, that the effects of ammonium ions on protein glycosylation are mediated via the ammonium-induced elevation of the UDPGNAc pool (Grammatikos et al., 1998, Ryll et al., 1994). Here, we present the glucosamine-6-phosphate isomerase (GPI) as the key enzyme responsible for ammonium incorporation. Moreover, we propose two strategies to inhibit its activity. Firstly, a strategy based on metabolic engineering by antisense RNA expression and secondly a strategy based on process control by the addition of an inhibitor to the culture. Results The enzyme. The glucosamine-6-phosphate isomerase has been identified as the key enzyme accepting ammonium ions and elevating the intracellular UDPGNAc content. GPI

was extracted from BHK21 cells and purified to homogeneity by affinity chromatography (Calcagno et al, 1984). The enzyme is a hexamer with a molecular weight of approximately 180 kd. Sequence analysis of the amino terminus revealed a high similarity to the human form of the enzyme. The reaction is reversible but the equilibrium is far on the side of Frc6P. We could show that the enzyme exclusively accepts but no glutamine. Glutamine is used by the glucosamine-6-phosphate synthase (GPS) which catalyzes the 157 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 157-161. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

158

same reaction and which is controlled by an end product inhibition of UDPGNAc (Fig. 1). We assume that GPI is cut out for the role to control the stationary intracellular concentrations of intermediate metabolites of the UDPGNAc pathway by regulating GlcN6P in vivo where never high ammonium concentrations are reached. However, under in vitro conditions in the presence of high ammonium concentrations, this regulation system causes serious problems on the synthesis of UDPGNAc followed by an effect on the expression of carbohydrate structures. The inhibitor. GPI was characterized with respect to effectors to be used as modulators in cultivation processes of mammalian cells. Mannose-6-phosphate (Man6P) was shown to be a strong inhibitor whereas glucose-6-phosphate, glutamine and N-acetyl-glucosamine6-phosphate (GlcNAc6P) act as activators. By using mannose in appropriate concentrations in combination with glucose as carbon source we have shown that the formation of higher UDPGNAc concentrations could be prevented in the presence of ammonium in the culture medium indicating that mannose which is intracellularly transformed to Man6P is an efficient inhibitor in bioprocesses to control N-glycan expression of glycoproteins and glycolipids.

Antisense strategy. Due to the fact that synthesis and regulation of UDPGNAc can be exclusively performed by the GPS alone we planned to inhibit GPI by antisense RNA expression. Two different plasmids were constructed using human cDNA and directed to

159

the 5´- and 3´-end of the enzyme under the control of the SV40 early promotor (Table 1). In addition a further plasmid harboring the 5´-end in sense direction with the same promotor was used as control.

As a result the transfected cell lines showed a significant decrease in the GPI enzyme activity (Fig. 2). Plasmid 1 and 2 revealed an enzyme expression of only 30 and 50% of the control, respectively. Cotransfection of both plasmids, however, resulted in 90% of repression. Only 10% of the expression found in the control cells could be detected.

160

Conclusion Glucosamine-6-phosphate isomerase has been identified as the ammonium accepting enzyme resulting in an elevated UDPGNAc content under high ammonium concentrations. GPI was efficiently inhibited using two different strategies: The addition of mannose to culture media and the genetic modification of the cells using antisense RNA expression. References Backer, M.P., Metzger, L.S., Slaber, P.L., Nevitt, K.L., Boder, G.B. 1988. Large Scale production of monoclonal antibodies in suspension culture. Biotechnol. Bioeng. 32: 993-1000. Butler, M., Spier, R.E. 1984. The effects of glutamine utilisation and ammonia production on the growth of BHK cells in microcarrier cultures. J. Biotechnol. 1: 187-196

Calcagno, M., Campos, P.J., Mulliert, G., Suastegui, J. 1984. Purification, molecular and kinetic properties of glucosamine-6-P isomerase (deaminase) from Escherichia coli. Biochim. Biophys. Acta. 787: 165173 Gawlitzek, M., Valley, U., Wagner, R. 1995. Effects of Ammonia and Glucosamine on the Glycosylation Pattern of Recombinant Proteins Expressed from BHK-21 Cells. In: Animal Cell Technology

'Developments towards the 21st Century'; Beuvery, E.C., Griffiths, J.B., Zeijlemaker, W.P., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, pp. 379-383

Gawlitzek, M., Valley, U., Wagner, R. 1997. Ammonium ion/glucosamine dependent increase of oligosac charide complexity in recombinant glycoproteins secreted from cultivated BHK-21 cells. Biotechnol. Bioeng, in press Glacken, M.W. 1988. Catabolic Control of Mammalian Cell Culture. Bio/Technology 6: 1941-1050.

Ito, M., McLimans, W.F. 1981. Ammonium Inhibition of Interferon Synthesis. Cell Biol. Int. Rep. 5: 661-666.. Jensen, E.M., Liu, O.C. 1961. Studies of Inhibitory Effect of Ammonium Ions in Several Virus-Tissue Culture Systems. P.S.E.B.M. 107: 834-838. Maiorella, B.L. 1992. In Vitro Management of Ammonia's Effect on Glycosylation of Cell Products Through pH Control. US Patent, Number 5,096,816. Ryll, T., Valley, U., Wagner, R. 1994. Biochemistry of Growth Inhibition by Ammonium Ions in Mammalian Cells. Biotechnol. Bioeng. 44: 184-193 Thorens, B., Vassalli, P. 1986. Chloroquine and ammonium chloride prevent terminal glycosylation of immunoglobulins in plasma cells without affecting secretion. Nature 321: 618-620.

Valley, U. 1996. UDP-N-acetyl-hexosamines as central metabolites in growth and potein glycosylation of recombinant BHK cells. Thesis, Technical University Braunschweig.

161

Discussion

Singhvi:

Do you have any idea of the rates of UDP molecules that go by the two different pathways of synthetase and isomerase? Would this be reduced by inhibiting the isomerase and by how much?

Cayli:

Normally the disproportionately high level of UDPGNAC is due to the activity of isomerase because in animal cell culture we do have 4-5 mM ammonium. These high ammonium levels drive the reaction to UDPGNAC. I do not have any data on the synthetase activity. I guess if the isomerase was inhibited, the concentration of UDPGNAC should be very low.

Konstantinov:

You mentioned that you had tried mannose in your culture medium. Could you comment on whether this affected glycosylation?

Cayli:

There are 2 steps: first, adding mannose to the culture medium and measuring UDPGNAC; second, the analysis of the glycostructures. We have just measured the UDPGNAC pool in the cell because it is much quicker. We just optimised the mannose level in the medium and just checked the decrease of UDPGNAC in the cells. We have no data yet on the structures of the proteins.

Konstantinov:

You need mannose for glycosylation. So I was wondering, if you add it to the medium whether it helps glycosylation by a different mechanism?

Cayli:

We have no data on the effect of pure mannose on protein glycosylation. So what you say is possible.

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STUDY OF HUMAN RECOMBINANT GM-CSF PRODUCED IN DIFFERENT

HOST SYSTEMS USING MONOCLONAL ANTIBODIES

M. ETCHEVERRIGARAY, M. OGGERO, M. BOLLATI, R. KRATJE Instituto de Tecnología Biológica (INTEBIO). Facultad de Bioquímica y Ciencias Biológicas. Universidad Nacional del Litoral Ciudad Universitaria - C.C. 530 - (3000) Santa Fe. ARGENTINA

Abstract GM-CSF is a glycoprotein that activates growth and differentiation of hemopoietic progenitor cells, usefull to reverse or prevent chemotherapy secondary leukopenias. In spite of the differences in the chemical structure, due to the variable glycosydic content of the forms produced in bacteria, yeast and mammalian cells, all of them have biological activity. Independently, antigenicity of non glycosylated recombinant human proteins may have relevance in the choice of the host system for the production of factors for clinical use. To study the undesirable immune response in the results of the therapy, we used the following strategy: i) To obtain the glycosylated hormone, CHO dhfr - cells were manipulated by

genetic methods to produce human GM-CSF under the control of the adenovirus major late promoter. ii) A panel of GM-CSF MAbs was produced from hybridoma cells obtained in our lab. We analyzed the specificity of the MAbs taking into account their neutralizing

capacity. Some of them neutralized the in vitro biological activity of the hormone, with different affinities for GM-CSF from several sources. Patients under treatment with the commercial non-glycosylated GM-CSF develop antibodies and undesirable responses. Competition experiments between the neutralizing MAbs and these human antibodies would demonstrate the importance of the immune response in the choice of the host system for its production. Introduction

Granulocyte macrophage colony stimulating factor (GM-CSF) was expressed in different systems: bacteria, yeast and mammalian cells. All of them have biological activity. Despite

the fact that the low levels that can be achieved by recombinant mammalian cells are disappointing for the production of GM-CSF, there are important reasons to support and improve this host system. It was observed that the therapeutical administration of nonglycosylated or partially glycosylated GM-CSF produces activation of the immune system [1,2] and development of undesirable responses that undoubtly cause the inefficacy of the treatment together with many disagreeable side effects. The recognition of antibodies specificity in patients sera under treatment with GM-CSF may be important in the choice

of the appropriate host system for the production of this hormone. Materials and Methods

Expression vectors for CHO cells were obtained from Genargen S.R.L., Buenos Aires, Argentina. CHO dhfr - cells were transfected by lipofection to produce human GM-CSF 163 O.-W. Merten et al. (eds.). New Developments and New Applications in Animal Cell Technology, 163-165. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

164

under the control of the adenovirus major late promoter. The vector contained the gen for dihydrofolate reductase (dhfr) to allow the selection of stable transfectants. Among the several cell lines achieved, the clone was selected for further experiments, taking into account its higher productivity [3]. Non-glycosylated GM-CSF was the E. coli derived GM-CSF International Standard (NIBSC, 88/646, UK), and the E. coli derived GM-CSF purchased from Schering-Plough Co., Ireland, that was used as internal standard previously valorized against the International Standard. Glycosylated GM-CSF was the CHO derived hormone prepared in our lab. The GM-CSF MAbs obtained in our lab are shown in Table I.

To test the biological activity of E. coli and CHO derived GM-CSF, we used the factordependent cell line TF-1 (ATCC CRL-2003). The proliferation assay was optimized in our lab for GM-CSF valoration. TF-1 cell proliferation was determined measuring dehydrogenase-enzyme´s activity as marker for the biological activity, using a commercial colorimetric kit (Cell Titer 96™, Promega, USA). MAbs were evaluated for their ability to inhibit the proliferation of GM-CSF stimulated TF-1 cells, i.e. to neutralize in vitro GM-CSF activity. For this purpose we used either six different MAbs: ascites 2E11, 8B10, 15G5 and 32H6, and purified 1B8 and 7E10 MAbs by protein A affinity chromatography . Serial dilutions of the antibodies were tested both with glycosylated and non-glycosylated GM-CSF. The results were expressed as neutralization percent. Results and Discussion

The isotype MAbs IB8 and 7E10 were purified with 82% and 70% recovery, respectively. The MAb 32H6 was heated at 56°C for 30 minutes. The IgM isotype ascites were used without previous treatment. Fig. 1 shows an example of the different behaviour of neutralizing and non-neutralizing antibodies. The neutralizing effect of the purified MAbs 7E10 and IB8 is shown in Fig. 2. The behaviour of MAb 7E10 may be related with heteroclytic antibodies [4], i.e. antibodies of higher affinity with a related molecule than with the immunogen (Table II). In contrast, the other antibodies that reacted with the glycosylated hormone correspond to cross-reactive antibodies, being E. coli derived GM-CSF the immunogen. It must be taken into account

165

that antibodies present in patients´ sera may be of both types and play an important role in the development of undesirable responses: loss of response in future treatments and crossreaction with the own hormone. The choice of host systems for the production of clinicals has to consider not only the biological activity of the product but also the immune system, wich plays, in this case, an important role and once it is stimulated there is no possibility

to abolish its effect. References: 1- Wadhwa, M., Bird, C., Fargerberg, J., Gaines-Das, R., Ragnhammar, P., Mellstedt, H. and Thorpe R. (1996). Clin. Exp. Immunol. 104:351

2- Gribben, J. G., Devereux, S., Thomas, N. S. B., Keim, M., Jones, H. M., Goldstone, A. H. and Linch, D. C. (1990). The Lancet 335:434 3- Bollati, M., Kratje, R. and Etcheverrigaray, M. (1997). Encuentro de Investigadores Jóvenes. UBA. Argentina.

4- Mäkelä, O. (1965). J. Immunol. 95:378

METABOLIC ENGINEERING

MODIFICATION OF HYBRIDOMA CELLS METABOLISM

J.J. Cairó, C. Paredes and F. Gòdia* Departament d'Enginyeria Química. Facultat de Ciències. Edifici C. Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona. Spain. E. Prats, F. Azorín, LI. Cornudella Departament de Biologia Molecular i Cel.lular. Centre d'Investigació i Desenvolupament. CSIC. Jordi Girona Salgado, 18-26. 08034 Barcelona. Spain.

* To whom correspondence should be addressed.

SUMMARY Two different genetic modifications of hybridoma cells are studied. First, cells are

transfected with the glutamine synthetase gene, and important modifications in their physiology can be observed, that are reflected on the corresponding analysis of the intracellular metabolic fluxes. For the modified cell line the need for glutamine feeding in the culture has been eliminated and the ammonium production suppressed. Moreover, the glucose uptake rate has been reduced to half with respect to the parental strain, while maintaining a similar growth pattern. Second, antisense RNA techniques were employed to inhibit partially the expression of two glycolytic enzymes in order to create some rate limiting steps in the glycolytic pathway. The preliminary results obtained with the glucose transporter and enolase genes show the potential of such approach.

INTRODUCTION. Typical batch cultures of hybridoma cells have a number of drawbacks : low cell concentration, low level of product expression, limited viability of the cells and complex nutrient requirements. The design of more efficient processes for monoclonal antibody production and the development of optimized operation strategies1 has to be based in a much better knowledge of the cell metabolism. Many authors have 167 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 167-174. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

168 demonstrated that the metabolism of several cells can be redirected to the obtention of desired products by using the metabolic engineering approach 2 . Hybridoma cell cultures typically produce lactic acid, ammonia and some amino acids when metabolizing the carbon and energy sources, glucose and glutamine. The byproduct generation from its metabolism represents a poor exploitation of these substrates and can inhibit cell growth due to lactate and ammonium accumulation 3 . In order to overcome the high glutamine uptake rate, the generation of high levels of ammonium as well as the high glycolytic flux and the lactate generation two approaches have been performed. First approach consists in the expression of the glutamine synthetase in a hybridoma cell line. Second approach is based on the use of antisense RNA techniques to partially inhibit the glucose transporter and the enolase gen. Antisense control of gene expression has become widely used method for specifically interfering with gene function 4 . MATERIALS A N D METHODS Cells : KB-26.5 murine hybridoma, producing an

against antigen

of red cells.

Medium : DMEM supplemented with 2% foetal calf serum. Viable cell number : haemocytometer, viability as trypan blue exclusion. Batch and continuous cultures : Spinner flask, 250 ml working volume, 40 rpm, 37°C, Continuous cultures at a dilution rate of

, average cell population

cell/ml. Glutamine and Glucose feeding decreasing stepwise one at a time 5 .

Glucose and Lactate : YSI 2700 Ammonium concentration : flow injection analysis system 6 . Glutamine and other amino acids concentrations : HPLC. Glutamine Synthetase : pCMGS.gpt 7 kindly provided by Celltech. Selection in glutamine free medium. Clone selected by serial dilution. Antisense constructions : 450 bp fragment downstream the ATG codon of both glucose transporter gene GLUT1 and enolase gene. Fragments were cloned in antisense orientation into the BamHI site of plasmid pcDNA3. Selection by resistance against Neomycin. Transfection : lipofection method. Stoichiometric model : As described in [8]. RESULTS A N D DISCUSSION Hybridoma cell line KB-26.5, like all tumoral cells, can be considered as an energetically efficient system, since is able to take energy from different sources (glucose and glutamine), but this efficiency exists as long as cells are in their ideal surroundings, as in body tissue. The body provides the cells with an extremely controlled environment, in which the inefficiency observed when cells are in culture does not exist. Glucose and glutamine concentrations in batch and fed-batch cultures are usually much

169 higher than the physiological ones found in tissues, and cells will consume tar greater amounts of both substrates, due to the high transport and glycolysis and glutaminolysis rates, and produce excessive amounts of lactic acid and ammonium. This point was corroborated in continuous culture experiments performed in two different ways: maintaining a constant glucose concentration in the feed medium while decreasing the glutamine concentration stepwise, and maintaining glutamine concentration constant while decreasing glucose stepwise. The results of this series of experiments are summarized in Figure 1a and 1b. It can be observed that as the glutamine concentration found in the medium increases (so, more excess of glutamine is provided to the cell metabolism), higher amounts of glutamine are consumed and ammonium produced. The same behaviour can be observed for glucose and lactate. Thus, when excessive amounts of glutamine are added to the hybridoma culture, glutamine is not efficiently used for cell growth, but rather to produce glutamine-related by-products such as ammonium, alanine and proline. On the other hand, as glucose level in the exhaust medium increases, more lactate is produced. Thus, as this cellular level was the same, it seems that there is not a real need to consume those great amounts of glucose and glutamine in order to keep the cell growing.

In order to overcome the high glutamine uptake rate and the generation of high levels of ammonium, the glutamine synthetase gene has been expressed in the KB-26.5 cell line. The physiological consequences of this expression can be seen in Figure 2. Transfected cell line show a lower growth rate than the original one but

170 specific production rate of ammonium is zero. Similarly specific glucose uptake rate is reduced to a half of that of the untransformed cell line, and the Lactate/Glucose molar yield has slightly decreased.

To elucidate which are the metabolic reasons tor this physiological change a stoichiometric model was constructed to estimate the intracellular fluxes. In brief it

consists in the derivation of a mass balance for each considered metabolite including the transport rates across membranes and their production or consumption rates by the

intracellular reactions considered. All these data makes possible the estimation of the intracellular fluxes by means of a least squares procedure.

The simplified results obtained in the estimation of the corresponding intracellular fluxes are presented in Figure 3. For the non modified hybridoma cells, the glucose and glutamine high consumption rates generate high amounts of lactate and ammonium as it has been stated before. A possible explanation of these results could be a low efficiency in the shuttle system for mitochondrial reoxidation of the large amounts of NADH produced in the glycolytic pathway. As a consequence NADH should be regenerated via lactate dehydrogenase using pyruvate as a substrate rather than through the electronic transport chain. Thus, the pyruvate amount that can be incorporated into TCA cycle is very small and the rapid glutaminolysis rate collapses and unbalances the

TCA cycle. Simultaneously the great amount of ammonium generated from glutamine and other amino acids deamination overflows the cell capacity and it is excreted as tree ammonium, alanine and proline. Moreover as alanine is produced in large amounts the availability of pyruvate for the cell metabolism decreases (alanine is formed from

pyruvate and glutamic acid).

171

The results obtained with the transfected cell line show similar trends with

respect to N A D H regeneration via lactate production. The main difference is found in the glycolytic flux, that in the transfected cell has been reduced to half of that for the parental strain (Figure 2). The effect of the glutamine synthetase expression on the ammonium excretion could he explained as a result of a change in the way that the cells use in order to eliminate the excess of ammonium ions generated from glutamate metabolism, not requiring neither ammonium excretion nor animation of pyruvate to alanine. Last factor could be the reason for the reduction of the glycolytic flux as a consequence of a lower need for pyruvate in the transfected cell line. The carbon circulation w i t h i n the TCA cycle is not collapsed by glutamic acid. On the other hand, it can be seen from Figure 3 that the exchange between mitochondrial and cytoplasmic malate has been inverted for the transfected cells when compared to the parental ones. In order to overcome the high glucose uptake rate and the generation of high levels of lactate, an antisense R N A approach has been used. It is generally assumed that the mechanism of inhibition by nuclear-derived antisense R N A entails sequence-specific hybridization of antisense RNA transcript to the target m R N A in the nucleus that blocks protein synthesis 4 . Under optimal conditions an antisense RNA

172

approach could inhibit absolutely protein translation. Thus under suboptimal conditions it can be used to decrease the amount of the target protein. If that protein is an enzyme its is possible to lower the flux through the enzymatic reaction that it catalyses lowering the amount of enzyme. In the present work this possibility has been used in order to create a rate-limiting step in the glycolytic pathway. Two points in the glycolytic pathway were chosen as a target; first the transporter protein for glucose (GLUT 1) and second the enzyme enolase. This enzyme catalyses the conversion from 2phosphoglyceric acid to phosphoenolpyruvic acid. The physiological consequences of this approach can he seen in Figure 4. Both cell lines show higher growth rates than the control. This fact can he explained by the fact that integration in the genome occurs randomly and there will he some random effect or by the fact that cells carrying antisense construct have some advantage over non-carrying ones. Specific glucose uptake rate has decreased about 20% for enolase construction and 45% for glucose transporter construction. From these values it is likely to suppose that both constructions are able to generate a rate-limiting step in the glycolytic pathway. The molar yield for lactate/glucose remains almost unchanged showing that these approach does not affect to the cytosolic NADH regeneration.

CONCLUSIONS

When glucose and glutamine are added in excess to the culture medium they are consumed at high rates, with low efficiency for the cells, and generation of by-products. The use of the glutamine synthetase gene allows the reduction of ammonia production and unexpectedly decreases glycolysis rate to one half. Some potential decrease of the glycolysis rate could he introduced generating a rate-limiting step by means of antisense RNA techniques. Further work is required to corroborate these results, assess the stability of the transformed cells and the consequences in product formation.

173 Acknowledgements CellTech Therapeutics kindly provided the glutamine synthetase gene. This work was supported by CICYT (project BIO94-0288) and was carried out in the framework of the Centre de Referencia de Biotecnologia. References 1 .Bibila, T.A., Robinson, D.K. (1995) In Pursuit of the Optimal Fed-Batch Process for Monoclonal Antibody Production, Biotechnology Progress 11, 1-13. 2.Bailey, J.E. (1991) Toward a Science of Metabolic Engineering, Science 252,1668-1675.

3.Ozturk, S.S., Riley, M.R., Palsson, B.O. (1992) Effects of ammonia and laclate on hybridoma growth, metabolism, and antibody production. Biotechnology and Bioengineering 39,418-431. 4.Pestka S. (1992) Antisense R N A . History and Perspective in R. Baserga and D.T. Denhardt (eds.), Annals of the New York Academy of Sciencies, Vol 660. New York Academy of Sciencies. New York pp. 251-262. 5.Sanfeliu A., Paredes C., Cairó J . J . , Gòdia F. (1997) Analysis of Glucose and Glutamine Metabolism of

Hybridoma C e l l s by Continuous Culture Experiments, in M.J.T. Carrondo, B. Griffiths and J.L.P. Moreira (eds.), Animal Cell Technology. From Vaccines to Genetic Medicine, Kluwer Academic Publishers, Dordrecht, pp. 785-789.

6.Campmajó, C., Cairó, J . J . , Sanfeliu, A., Martínez, E., Alegret, S., Gòdia. F. (1994) Determination of ammonium and L-Glutamine in hybridoma cell cultures by sequential flow injection analysis. Cytotechnology

14,177-182. 7.Bebbington.C.R., Rentier, G., Thompson, S., King, D., Abrams, D., Yarranton, G.T. (1990) High level expression of a recombinant antibody from myeloma cells using a glulaminc synthetase gene as an amplifiable selectable marker. Bio/Technology 10,169-175.

8.Sanfeliu, A. (1995) Producció d’ Anticossos Monoclonals Mitjancant el Cultiu in vitro d’ Hibridomes en Bioreactors : Anàlisi de la Fisiologia i Metabolisme C e l . l u l a r , Ph. D. Thesis, Universitat Autònoma de Barcelona.

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Discussion

Franck:

How do the changes in metabolism influence the specific productivity of the antibody?

Godia:

We do not have quantitative data to answer your question. We focus on genetic modification and metabolic pathway changes, rather than productivity.

Singhvi:

Why did you choose the two enzymes for your anti-sense targets? Was it random or based on preliminary work?

Godia:

The rationale was that the glucose transporter was a clear target as it is at the beginning of all processes. We also knew, by means of DNA probes, which was the glucose transporter active in our cell line and we could make it more easily than other ones. For the inulase we tried to select one enzyme that was well down the glycolytic pathway and so it would not affect the initial steps.

Cayli:

What kind of gene did you use for your anti-sense construct? Is it from the hybridoma or another cell line? Also, are your anti-sense clones stable?

Godia:

I cannot tell how stable the clone is because we have only had it for one month but we have to check for this. We obtained the gene from a cDNA library using the oligonucleotide primers and by PCR.

CLONING AND EXPRESSION OF A CYTOSOLIC SIALIDASE FROM CHO CELLS IN A GLUTATHIONE S-TRANSFERASE (GST)-ENCODING EXPRESSION VECTOR

M. BURG and J. MÜTHING University of Bielefeld, Faculty of Technical Sciences, Institute of Cell Culture Technology, P.O.Box 100131, 33501 Bielefeld, Germany

1. Introduction Many proteins, produced by recombinant DNA technology for clinical and therapeutical purpose, are glycosylated in their native state. Sialic acids, which are terminally linked to the oligosaccharide chains, play an important role in determining the in vivo fate of a glycoprotein in the circulatory system concerning its activity [1] or hepatic clearance rate [2]. Recent publications revealed cytosol-derived sialidase activity in cell-free supernatant of Chinese hamster ovary (CHO) and hybridoma cells [3] in bioreactor cultures with potential for glycoprotein desialylation [4]. Detailed studies published by our laboratory showed the effect of CHO cell derived sialidase towards sialylated oligosaccharides of recombinant human antithrombin III [5,6] (Fig. 1). In continuation of this work, we attempted to clone and express the cytosolic sialidase of CHO cells in E. coli strains. In order to distinguish sialidases of the CHO donor cells from the E. coli host, we used an expression vector encoding for glutathione S-transferase (GST). A fragment with almost the entire open reading frame was generated for subsequent cloning. 175 O. - W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 175-179. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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2. Materials and Methods

POLYMERASE CHAIN REACTION (PCR) EXPERIMENTS Reverse transcriptase PCR (RT PCR) Single strand cDNA was synthesized by reverse transcription of CHO-K1 total RNA with oligo (dT) primers using the GeneAmp® RNA PCR kit (Perkin Elmer). Double strand cDNA fragments with almost the entire open reading frame of the cytosolic sialidase and in frame restriction sites were amplified using the primers shown in Fig. 2 and a mixture of Klen

Therm DNA polymerase

and Accu Therm DNA polymerase (Gene Craft). The samples were subjected to a temperature cycle step of 94°C (40 sec) and 68°C (2 min) for a total of 35 cycles, followed by a 7 min extension

step at 72°C after the final cycle. SUBCLONING The Original TA Cloning® kit (Invitrogen) was applied to subclone the PCR product in the pCR® 2.1 vector by direct insertion and transformation of E. coli F’. The generated plasmids, isolated with the Quantum Prep Plasmid Miniprep kit (BioRad) from the cultivated subclones, were tested by restriction analysis. The inserted fragment was cut off the ligated vector, isolated by gel extraction and used for further cloning experiments.

CLONING AND EXPRESSION The pGEX-2T plasmid vector of the GST Gene Fusion System (Pharmacia) was employed for cloning experiments. This vector encodes for GST at the amino terminus of the generated protein. The plasmid encoding the cytosolic CHO cell sialidase was constructed by inserting a BamHI-EcoRI cDNA fragment into the multiple cloning site of the vector. E. coli M15 harbouring pREP4 was transformed with the products of ligation. The clones were screened by restriction analysis and two were sequenced by the dye termination method with an EBI 377 (Perkin Elmer) sequencer from both insert directions and with two additional internal nested primers (worked out by IID Biotech Bioservice GmbH) for double strand sequencing of the whole insert. The clones were

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induced by IPTG and the expression was assayed by SDS-PAGE running the total protein extracts on 8-25 % gradient gels (Pharmacia). 3. Results AMPLIFICATION OF A cDNA FRAGMENT BY RT PCR We amplified a cDNA fragment of the cytosolic sialidase derived from CHO cells encoding different restriction sites for cloning by using the designed primer sequences.

Fig. 3 A (lane 2) shows the generated RT PCR product on a 2% agarose gel. The fragment was used for the subsequent cloning experiments.

INSERTION OF THE RT PCR PRODUCTS IN THE pCR® 2.1 VECTOR To eliminate enzymatical modifications the product was subcloned in the pCR® 2.1 vector. Several M15 pREP4 transformands were produced and plasmid isolations of the

subclones (scl) scl1, scl2 and scl3 were tested by restriction analysis. The insert is flanked by a BamHI and a EcoRI restriction site and encodes for one ScaI site (Fig. 3 B). The sc12 contains the successfully ligated plasmid pMBU4-2 (Fig. 3 B, lane 2).

CLONING AND EXPRESSION OF THE CYTOSOLIC SIALIDASE FROM CHO

CELLS IN pGEX-2T The BamHI-EcoRI fragment (Fig. 3 C, lane 1) was cut off the pMBU4-2 plasmid and cloned into the pGEX-2T plasmid vector, which encodes for GST. The screening of the transformands revealed four clones (cl1, cl2, cl6 and cl8) with the constructed plasmid

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encoding the CHO cell sialidase sequence, identified by its PstI restriction sites (Fig. 4). Sequencing of the clones cll and cl2 ruled out base replacements or amplification errors. SDS PAGE of the total protein extracts after induction revealed a new protein at about 70 kDa (Fig. 5). This correlates with the molecular weight of a fusion protein

composed of the GST (29 kDa) and the cloned cytosolic CHO cell sialidase (43 kDa).

4. Discussion Regarding the published cDNA sequence of the CHO cell derived cytosolic sialidase [7]

and with reference to the cloning and expression data of the cytosolic sialidase of rat cells [8], we successfully cloned a CHO cell sialidase cDNA fragment with the almost entire open reading frame in a pGEX-2T expression vector. The GST-sialidase fusion protein was found to be expressed in four M15 pREP4 clones, identified by SDS-PAGE. Next we will purify the GST-sialidase fusion protein by affinity chromatography on Gluthatione Sepharose 4B (Pharmacia), cleave the cytosolic sialidase from the GST by thrombin digestion (using the thrombin specific recognition site encoding by the pGEX

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plasmid), and utilize the isolated sialidase to produce specific antibodies against cytosolic CHO cell sialidase.

5. References [1] Chavin, S. I., Weidner S. M., (1983) Blood clotting factor IX, J. Biol. Chem. 259: 3387-3390 [2] Briggs, D. W., Fisher, J. W., George, W. J., (1974) Hepatic clearance of intact and desialylated erythropoietin, Am. J. Physiol. 227: 1385-1388 [3] Gramer, M. J., Goochee, C. F., (1993) Glycosidases activities of the 293 and NS0 cell lines, and of an antibody-producing hybridoma cell line, Biotechnol. Bioeng. 43: 423-428 [4] Gramer, J. M., Goochee, C. F., Chock, V. Y., Brousseau, D. T., Sliwkowski, M. B., (1995) Removal of sialic acid from a glycoprotein in CHO cell culture supernatant by action of an extracellular CHO cell sialidase, Biotechnology 13: 692-698 [5] Munzert, E., Müthing, J., Büntemeyer, H., Lehmann J., (1996) Sialidase activity in culture fluid of chinese hamster ovary cells during batch culture and its effect on recombinant human antithrombin III integrity, Biotechnol. Prog. 12: 559-563 [6] Munzert, E., Heidmann, R., Büntemeyer, H., Lehmann, J., Müthing, J., (1997) Production of recombinant human antithrombin III on 20-L-bioreactor scale: corellation of supernatant, neuraminidase activity, desialylation, and decrease of biological activity of recombinant

glycoprotein, Biotechnol. Bioeng., in press [7] Ferrari, J., Harris R., Warner, T.G., (1994) Cloning and expression of a soluble sialidase from chinese hamster ovary cells: sequence alignment similarities to bacterial sialidases, Glycobiology 4: 367-373 [8] Miyagi, T., Konno, K. , Emori, Y., Kawasaki, H., Suzuki, K., Yasui, A., Tsuik, S., (1993)

Molecular cloning and expression of cDNA encoding rat skeletal muscle cytosolic sialidase, J. Biol. Chem. 268: 26435-26440

6. Acknowledgements

This work was financed by the Deutsche Forschungsgemeinschaft (DFG), Graduiertenkolleg ,,Zelluläre Grundlagen biotechnischer Prozesse“. We would like to thank the ESACT for a bursary, which enabled MB to attend the 15th ESACT Meeting.

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Construction of a novel CHO cell line coexpressing human glycosyItransferases and fusion PSGL-1 - immunoglobulin G B. Vonach, B. Hess and C. Leist. Novartis Pharma AG, Biotechnology Development and Production, CH-4002 Basel, Switzerland.

Introduction The sialyl Lewisx (sLex) determinants serve as ligands in the selectin-mediated adhesion of leukocytes to activated endothelium or platelets. The most frequently used mammalian host cell lines (CHO, BHK) for the production of recombinant glycoproteins are incapable to produce sialyl Lewisx oligosaccharides. Therefore, we have constructed CHO cell lines expressing human N-acetyl-Dglucosaminyltransferase and 1,3-fucosyl-transferase III. Stable clones were obtained which were subsequently transfected with a plasmid encoding human PSGL1-lgG. The recombinant PSGL-1 molecule was recognized by specific antisialyl Lewisx antibodies that only bind to the correctly glycosylated protein. Furthermore, the recombinant PSGL-1 bound to P-selectin, its natural ligand, showing that it was properly glycosylated.

Expression of recombinant human glycosyltransferases Plasmids used:

ƒ pGNT-his, coding for the N-acetyl-D-glucosaminyltransferase and the enzym histidinol-dehydrogenase ƒ pFucTIII-zeo, coding for the 1,3-fucosyltransferase III and zeocine resistance ƒ pPSGL-1-lgG1-hyg, coding for the P-selectin glycoprotein ligand 1immunoglobulin G1 (Fc region) fusion protein and hygromycin resistance CHO SSF3 cells were first cotransfected with pGNT-his and pFucTIII-zeo. pGNThis expressed the dominantly acting hisD gene of Salmonella typhimurium encoding the enzym histidinol-dehydrogenase. The presence of this gene allowed the cells to survive in media without histidine supplemented with 1mM Lhistidinol. Positive transfected clones were picked and grown in 96-well plates until analysis. 181 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 181-183. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

182 Immunostaining and FAGS analysis The presence of the sLe x determinant on the surface of cotransfected cells (CHO SSF3 /GNT/FucTIII) was verified by immunostaining using a specific mouse anti-sialyl-Lex IgM (Becton Dickinson) and an anti-mouse-lgM-peroxidase conjugated antibody (Jackson Dianova). The specificity of the reaction was shown by the absence of signal in untransfected cells, indicating that these CHOSSF3 cells normally lack the enzyms N-acetyl-D-glucosaminyltransferase and fucosyl-transferase III. When analysing cotransfected cells a brown DAB (Diamino-benzidin tetrahydrochloride)/ cobalt and nickel chloride precipitate was clearly detectable by microscopy (enlargement:20x). The presence of membrane-bound expressed sialyl Lex was checked on the surface of cotransfected CHO cells (CHO SSF3/GNT /FucTIII) by FACS analysis. The cells were treated with the specific mouse anti-sLex IgM followed by the fluorescence dye DTAF. Untransfected cells were used as negative control. The samples were analyzed by flow cytometry on a FACStar Flow Cytometer (Becton Dickinson). The fluorescence of the positive clone was above 97% indicating that the newly constructed cell line was almost pure (Fig.1).

Expression of PSGL-1-lgG1 in the newly constructed cell line

CHO SSF3/GNT/FucTIII cells were transfected with the plasmid pPSGL-1-lgG1hyg, coding for the P-selectin glycoprotein ligand 1 - immunoglobulin G fusion protein. The recombinant PSGL-1 molecule was recognized by specific anti-sialyl Lewisx antibodies that only bind to the correctly glycosylated protein.

183

We also evaluated binding of recombinant PSGL-1-lgG to P-selectin, the natural ligand. The immobilized P-selectin only bound minor amounts of recombinant PSGL-1-lgG purified from the supernatant of B5 cells (only transfected with the plasmid pPSGL-1-lgG1-hyg) (Fig.2). However, the binding was significantly increased when adding recombinant PSGL-1-lgG purified from the supernatant of H4 cells (transfected with the plasmids pGNT-his, pFucTIII-zeo and pPSGL-1lgG1-hyg). There was no detectable difference between the proteins obtained from cells grown in the absence or presence of fetal calf serum.

Conclusion The CHO SSF3 cell line used as host for our DNA transfections has been previously adapted for growth in suspension cultures in serum-free media. The recombinant CHO SSF3 cells efficiently returned to growth in serum-free suspension culture after extended culture in monolayers using serumsupplemented media. This indicated that the preadapted cell phenotype was stable after transfection and selection of heterologous genes. Furthermore, the recombinant PSGL-1-lgG protein expressed by CHO SSF3 previously cotransfected with plasmids encoding both the N-acetyl-D-glucosaminyltransferase and 1,3-fucosyltransferase presented a "human-like" glycosylation pattern, which was specifically recognized by anti-Lewisx antibodies. The recombinant PSGL-1 bound to P-selectin, its natural ligand, showing that it was properly glycosylated. The transfection of mammalian cell lines with plamids encoding human glycosyltransferases of defined specificity provides a tool to generate novel stable host cell lines for the production of recombinant proteins with tailored glycosylation pattern.

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PRODUCTION OF DEFINED GLYCOSYLATION VARIANTS OF SECRETED HUMAN GLYCOPROTEIN THERAPEUTICS BY COEXPRESSION WITH HUMAN RECOMBINANT GLYCOSYLTRANSFERASES IN BHK-21 CELLS

Schlenke, P., Grabenhorst, E., * Costa, J., Nimtz, M., and Conradt, H.S. Dept. of Protein Glycosylation, GBF, Mascheroder Weg 1, D-38124 Braunschweig, Germany, *IBET, ITQB, Apartado 12, 2780 Oeiras, Portugal

1. Abstract To investigate the stability of a BHK-21 A cell line transfected with human EPO (huEPO) and the human 2,6-sialyltransferase ST6N (huST6N), the cell line was cultivated over a time period of 18 days in a 2.5 L perfused bioreactor in protein- and serum-free medium and under physiological stress after addition of ammonium. The stability of gene expression and the activity of the newly introduced huST6N and the endogenous 2,3-sialyltransferase ( 2,3-ST) were analyzed during the different cultivation conditions by RT-PCR and in vitro assays. Furthermore, the purified secreted huEPO was analyzed by western blot and the liberated N-glycans were characterized by HPAEC-PAD analysis. The results confirmed the stability of the cell line with respect to the expression of the newly introduced human proteins even under physiological stress. The elevated ammonium concentration led to reversible changes on the EPO N-glycans. 2. Introduction When expressed from mammalian host cell lines, the glycan moieties found on recombinant human glycoprotein therapeutics depend on the cell line used for production, the polypeptide and the culture conditions. Several common motifs found on proteins from natural sources like the terminal 2,6-sialylation of N-glycans which is characteristic for human serum proteins or the Lex or sLex structures are not synthesized from the most frequently used host cell lines BHK or CHO because these cell lines do not express the specific glycosyltransferases. The differences in the glycan structures between recombinant proteins and the natural human counterparts are of relevance for glycoproteins produced for clinical applications because these motifs are involved in numerous biological phenomena, e.g.: in-vivo half-live, inflammatory processes, antigenicity. We have shown that it is possible to alter the glycosylation potential of host cell lines by rransfection with plasmids encoding glycosyltransferases not expressed from the parent cell line (1,2). By coexpression of human glycoproteins with glycosyltransferases it is possible to obtain stable cell lines producing reproducible human-type glycosylated products. In addition, coupled with analysis of the resulting products by mass spectrometry, the system offers a reliable approach to investigate the in vivo substrate 185 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 185-190. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

186

specificity of glycosyltransferases. By using this system together with in vitro assays the substrate specificity of the human fucosyltransferase III was elucidated (3). To investigate the stability of a BHK-21A cell line transfected with huEPO and the human 2,6-sialyltransferase, the cell line was cultivated in a 2.5 L perfused bioreactor in protein- and serum-free medium with or without addition of ammonium and ammonium/mannose. It is known that ammonium which accumulates during cell culture due to amino acid metabolism and degradation of glutamine affects the glycosylation of proteins (8,9). The BHK-21A cells used for the reactor experiment differ from BHK-21B cells by the high amount of terminal GalNAc structures found on the N-glycans (1). Due to the fact that the endogenous 2,3-sialyltransferases is unable to act on these structures, Nglycans produced from this cell line are undersialylated. This would result in a rapid hepatic clearance of the corresponding proteins in humans. We could show that the transfection of the BHK-21A cell line with the huST6N increases the sialylation state of the coexpressed recombinant huEPO because the human enzyme was shown to be able to act in vivo on terminal Gal as well as on terminal GalNAc structures (2). 3. Material and Methods Cell line and culture conditions: A BHK-21A cell line transfected with huEPO and huST6N was cultivated in a 2.5 L perfused bioreactor for 3 days in protein- and serumfree standard medium (SMIF 6, Gibco-BRL), followed by a cultivation period of 5 days in SMIF 6 supplemented with 15 mM After return to standard conditions for 3 days the perfusion was started with standard medium supplemented with 15 mM and 4 mM mannose. The process was finished after a cultivation period of 3 days in standard medium (see Fig. 1). Total cell number and viability was determined as described (4). During the different culture conditions, the supernatant and cells were harvested for further investigations. EPO purification, characterization and analysis of N-glycans: Western analysis of the secreted protein was performed before and after removal of the N-glycans by PNGase Ftreatment. The secreted EPO was purified from the concentrated cell culture supernatants by immunoaffiniry chromatography (2). After the purity of the preparation was verified by SDS/PAGE and Coomassie staining, the protein was digested with trypsin and the resulting peptides were separated by RP-HPLC. The N-glycans were liberated from the peptides by PNGase F and separated by RP-HPLC. The native and desialylated oligosaccharides were desalted and analyzed by HPAEC-PAD as described previously (5). PCR analysis: Total cellular RNA was prepared by Tri-Reagent followed by cDNA synthesis using standard protocols (6). The cDNA samples were normalized with respect to the signals obtained after actin PCR, electrophoretic separation of the PCR products in agarose gels and densiometric analysis of the resulting bands. The normalized cDNA samples were used for PCR analysis with primers corresponding to the endogenous 2,3-ST (E. Grabenhorst, unpublished results) and the newly introduced huST6N,

187 resulting in PCR fragments of 774 bp and 699 bp, respectively. Analysis of the PCR products was performed as described. Preparation of cell extracts and sialyltransferase assay: After harvesting, cells were washed with PBS, spun at 1000 x g and resuspended at a concentration of cells/ml in ice-cold extraction buffer (10 mM MES/NaOH, pH 6.5, 2% Triton CF-54, 1 mM PMSF, 10 µg /ml aprotinin, 10 µg /ml leupeptin) and stored at -70 °C. Prior to analysis the samples were thawed and incubated for 1h on ice. Sialyltransferase activity was tested with an 8-methoxycarbonyloctyl glycoside type II acceptor in reaction mixtures containing in a volume of 100 ': 50 mM MES/NaOH buffer, pH 6.5, 27 cell extract, 90 nmol acceptor, 5 nmol of CMPNeu5Ac (60.000 cpm/nmol), 20 mM , 100 mM NaCl and 0.2 nmol ATP. The mixture was incubated at 37 °C for 5h, diluted to 1 ml with ice-cold and applied to Sep-Pak cartridges. After washing with 15 ml of water the products were eluted with 1.5 ml of methanol. The eluat was dried under vacuum and resuspended in 700 sialidase buffer (50 mM Na-acetate pH 5.0, 5 mM , 0.02% ). Aliquots

of 200 were incubated for 4h at 37 °C with a) 10 mU Vibrio Cholerae sialidase (removes 2,6 and 2,3 linked sialic acid), b) 5 mU Newcastle disease virus sialidase (removes only 2,3 linked sialic acid) and c) without sialidase. Subsequently, the reactions were stopped by the addition of water and the mixtures were applied to the Sep-Pak cartridges as described. After elution with 1.5 ml of methanol the incorporation

of

Neu5Ac onto the acceptor was determined by liquid scintillation counting.

4. Results and Discussion By cotranfection of mammalian host cell lines with plasmids encoding glycoprotein therapeutics and glycosyltransferases not expressed from the parent cell lines, it is possible to obtain therapeutics with human-type glycosylation characteristics (1,2). In order to investigate the stability of a genetically engineered BHK-21A cell line transfected with huEPO and the huST6N, the cell line was cultivated over a time period of 18 days in a perfused 2.5 L bioreactor. During the cultivation in protein- and serumfree medium and under physiological stress induced by the addition of 15 mM ammonium with or without 4 mM mannose to the standard medium, the cellular growth rate and viability was determined. Furthermore, the stability of gene expression and the activity of the newly introduced huST6N and the endogenous α2,3-ST was investigated using RT-PCR analysis and in vitro sialyltransferase assays, respectively. The secreted huEPO was characterized by immunoblotting after purification by affinity chromatographie. The liberated N-glycans were analyzed by HPAEC-PAD. Cell growth: Ammonium is known as a harmful substance accumulating during cell culture. Several authors described a reduction of the cell density due to elevated ammonium concentrations (see ref. 7 for review). The degree of growth reduction by

ammonium varies between a wide range and probably reflects the different sensitivity of the cell lines used for the studies as well as differences in the cell culture conditions. In the present study, no significant effect of the elevated ammonium concentration on cell

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growth rates or the viability of the BHK-21 A cell line (82-95%) was observed (see Fig. 1). RT-PCR analysis: RT-PCR analysis of the transcripts encoding the newly introduced huST6N and the endogenous 2,3-ST revealed a stable expression of the corresponding genes during the cultivation period even under high ammonium concentration. The analysis indicated a constant transcription rate of both, the huST6N and the 2,3-ST, which was not affected by the culture conditions. Northern analysis revealed a l0fold stronger signal for the huST6N compared to the endogenous enzyme (not shown).

Sialyltransferase assay: Similar to the transcription rate of the sialyltransferases, the enzyme activity detected in cellular extracts remained stable during the production process. A total sialyltransferase activity between 0.18 and 0.34 cells was determined. With the acceptor substrate used for the assay, the human sialyltransferase showed a significantly higher activity as the endogenous enzyme (about 83% of the total activity).

Immunoblot: Western blotting analysis of the supernatants collected during the different culture conditions confirmed a stable secretion rate of the recombinant EPO during the cultivation process. The analysis revealed a reversible increased mobility of the protein in SDS-PAGE when produced under elevated ammonium concentration. Removal of the N-glycans from the protein by PNGase F-treatment eliminated the

differences indicating a variation in N-glycosylation being responsible for the observed differences in mobility of EPO after addition of ammonium. The protein part as well as

the ratio of O-glycosylated and non O-glycosylated protein remained unaffected during the different culture conditions. Glycan analysis: Differences in the oligosaccharide structures produced under elevated ammonium concentrations were also observed by HPAEC-PAD analysis of the separated native and desialylated N-glycans. Predominantly tetraantennary structures that were highly sialylated were detected when synthesized under standard conditions. On the contrary, stress conditions (application of ammonium with or without mannose) led to a decrease in sialylation state and to the formation of partially agalacto tetraantennary structures as shown by MALDT-TOF MS analysis of the glycans (data not shown). The oligosaccharide structures observed during standard conditions were regained after switching from the stress conditions back to the standard conditions again. These results clearly indicate the stability of the genetically engineered BHK-21 A cell line during the production process with respect to the expression of the recombinant proteins even under high ammonium concentrations. As expected, the addition of ammonium to the culture medium led to changes in the N-glycan structures found on the secreted EPO. Thorens and Vassalli (8) reported the complete inhibition of sialic acid

transfer to terminal galactose residues of immunoglobulins secreted by plasma cells in the presence of 10 mM Similar to the results obtained in the present study, no influence of ammonium on the secretion rate was observed. The detection of truncated glycan structures on the recombinant huEPO produced under elevated ammonium concentrations is consistent with results published by Borys et al. (9). The authors described the inhibition of glycosylation of mouse placental lactogen-I expressed from CHO cells by increasing levels of ammonium in a pH-dependent manner. Their results

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indicated that ammonium has the potential not only to affect the terminal sialylation but the entire glycosylation process, resulting in truncated structures. The reversibility of the effects observed after the addition of ammonium confirmed the stability of the BHK21A cell line investigated. The results revealed that it is possible to obtain stable cell lines by transfection of BHK21 cells with glycosyltransferases not expressed from the parent cell lines. These cell lines can be used for the production of human-type glycosylated therapeutics.

5. References 1. Grabenhorst, E., Hoffmann, A., Nimtz, M., Zettlmeissl, G., Conradt, H.S.: Construction of stable BHK-21

cells coexpressing human secretory glycoproteins and human Eur. J. Biochem. 232 (1995), 718-725

--sialyltransferase,

2. Schlenke, P., Grabenhorst, E., Wagner, R., Nimtz, M., Conradt, H.S.: Expression of human α2,6sialyltransferase in BHK-21A cells increases the sialylation of coexpressed human erythropoietin, In: Animal Cell Technology, Kluwer Academic Publisher, Dordrecht, (1997), 475-480 3. Costa, J., Grabenhorst, E., Nimtz, M., Conradt, H.S.: Stable expression of the golgi form and secretory

variants of human fucosyltransferase III from BHK-21 cells, J. Biol. Chem. 272 (1997), 11613-11621 4. Gawlitzek, M., Valley, U., Nimtz, M., Wagner, R., Conradt, H.S.: Characterization of changes in the glycosylation pattern of recombinant proteins from BHK-21 cells due to different culture conditions, J. Biotechnol. 42 (1995), 117-131

190 5. Nimtz, M., Martin, W., Wray, V., Klöppel, K.-D., Augustin, J., Conradt, H.S.: Structures of sialylated oligosaccharides of human erythropoietin expressed in recombinant BHK-21 cells, Eur. J. Biochem. 213 (1993), 39-56 6. Sambrook, J., Fritsch, E.F., Maniatis, T.: Molecular cloning: a laboratory manual, 2 nd edn, Cold Spring Harbor Laboratory, Cold Spring Harbor NY (1989) 7. Schneider, M., Marison, I.W., Stockar, U.v.: The importance of ammonia in mammalian cell culture, Minireview, J. Biotechnol. 46(1996), 161-185 8. Thorens, B. & Vassalli, P.: Chloroquine and ammonium chloride prevent terminal glycosylation of immunoglobulins in plasma cells without affecting secretion, Nature 321 (1986) 618-621 9. Borys, M.C., Linzer, D.I.H., Papoutsakis, E.T.: Ammonia affects the glycosylation pattern of recombinant mouse placental lactogen-I by Chinese Hamster Ovary cells in a pH-dependent manner, Biotechnol. Bioeng. 43 (1993), 505-514

Discussion Handa-Corrigan:

How stable is your protein in terms of processing and freezing and thawing?

Schlenke:

You can freeze it several times without affecting the activity.

ANTISENSE RNA FOR THE ELIMINATION OF NEUGC RESIDUES FROM RECOMBINANT GLYCOPROTEINS

A. GREGOIRE, A. VISVIKIS*, A. MARC, J-L. GOERGEN Laboratoire des Sciences du Génie Chimique, CNRS-INPL, BP 172, F 54505 Vandoeuvre-lès-Nancy * Centre du Médicament - Fac. Pharmacie, 30 rue Lionnois - 54000 Nancy

1. Abstract

In order to inhibit the CHO CNAH translation, responsible for the conversion of NeuAc to NeuGc residues, fragments of different sizes from the mouse cnah cDNA have been cloned as antisense fragments. It appeared that the different antisense species tested were able to inhibit the CNAH translation in vitro. However, when decreasing the amount of the antisense fragments by half, different efficiencies were found according to the

fragment's length or its sequence. 2. Introduction

CHO cells have become one of the most favoured way for the production of complex recombinant proteins destined for human therapy (1,2,3). While, in rodent cells, the CMP-N-acetylneuraminic-acid-hydroxylase (CNAH) converts CMP-N-acetylneuraminic acids (CMP-NeuAc) to CMP-N-glycolylneuraminic acids (CMP-NeuGc) (4), in adult human cells this enzyme is absent and thus NeuGc-bearing proteins can produce a strong immune response when injected (5). The recent cloning of the mouse cnah gene (6) can be used, in an antisense strategy, to stop the production of NeuGc residues bearing proteins in CHO cells. In this study, different pieces of the mouse cnah cDNA have been used to generate antisense RNAs, which have then been tested for their ability to inhibit the CNAH expression in vitro. 3. Materials and Methods Plasmid constructions : Classical molecular biology techniques were performed according to Sambrook et al., 1989 (7). The 400nt antisense fragment is a XbaI/SacI fragment isolated from the pBSCNAH (plasmid containing the mouse cnah cDNA, kindly given by T. Kawano) and subcloned in the pGEM3Z vector. The 800nt antisense fragment was amplified by PCR, using the Eurogentec TAQ DNA polymerase, and cloned into the pCR2.1 vector issued from the "TA cloning kit" (Invitrogen). In vitro transcription & translation : In vitro transcription reactions were performed using the "Riboprobe in vitro Transcription System". In vitro translation reactions were performed using the "Rabbit Reticulocyte Lysate System" and chemiluminescent detection was possible due to the "Transcend™ non-radioactive Translation Detection System" (Promega). 191 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 191-193. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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4. Results & Discussion

4.1 ANTISENSE RNA PRINCIPLE AND STRATEGY

Since initial experiments (8, 9) showed that artificial antisense oligonucleotides may be used to interfere with gene expression, there has been a growing body of literature on antisense nucleic acids. As a first attempt, the lack of generally applicable rules for the

design of efficient antisense led us to test, in vitro, the ability of different antisense RNAs to inhibit the translation of the mouse cnah mRNA. We thus tested the efficiency of antisense RNAs of different lengths by cloning fragments from the mouse cnah cDNA. Three different antisense RNAs could then be produced : a full length (FlAs of ca. 1700nt), a 800nt and a 400nt, schematically represented on fig.l.

4.2 TRANSCRIPTION AND TRANSLATION INHIBITION TESTS The mouse cnah cDNA and the antisense fragments were transcribed by either T7 or T3 RNA polymerase and the RNAs were loaded on an agarose gel (fig. 2). The RNA lengths could be verified by comparison with 4 control RNAs of 250, 1525, 1065 and

2346nt. The band intensity reflected the amount of RNA obtained, allowing an estimation of the sense and antisense RNAs quantities necessary for the in vitro translation tests.

As shown in fig. 3, lane CNAH, a 66Kda protein was synthetised during the translation reaction in the tube containing the cnah mRNA. The next step was then to inhibit

CNAH expression adding antisense RNAs in the reaction. The first in vitro translation antisense experiments were performed with a same amount of sense and antisense RNAs. In presence of any of the antisense RNAs, no band corresponding to the CNAH protein

appeared (fig. 3A, Lanes FlAs, 800As and 400As), indicating that the 3 antisense RNAs

193 are able to stop the cnah mRNA translation. With one half of antisense RNA, the full length and the 400nt antisense fragments were still very efficient (fig. 3B, Lanes FlAs and 400As), whereas a decreased inhibition was observed when the 800nt antisense RNA was present (fig. 3B, Lane 800As). The 800nt antisense RNA is less efficient than the shorter 400nt piece, probably because : (i) of a particularly stable secondary structure adopted by the 800nt RNA; (ii) the 800nt RNA begins to hybridize to the cnah mRNA just at the ATG codon, which is not the case for the 2 other antisense RNAs beginning their hybridization earlier on 5' (fig. 1).

5. Conclusion

We were able to inhibit the in vitro mouse cnah mRNA translation by RNA antisense technology. Three different mouse antisense RNAs were tested, and all of them efficiently inhibited the CNAH translation. However, probably due to the region it hybridizes to the mRNA, the 800nt antisense was less efficient. The shorter and most efficient antisense (400As) will then be tested for its translation inhibition ability in vivo in CHO cells. 6. References 1. Archer, R., Wood, L. (1992) in R.E. Spier, J.B. Griffiths, C. Macdonald (eds.),: Animal Cell Technology: Developments, Processes and Products , Butterworth-Heinemann, pp. 403-408.

2. Lubiniecki, A., Arathoon, R., Polastri, G., Thomas, J., Wiebe, M., Garnick, R., Jones, A., van Reis, R.,

Builder, S. (1989) in R.E. Spier, J.B. Griffiths, J. Stephens, P.J. Crooy (eds.), Advances in Animal Cell Biology and Technology for Bioprocesses., Butterworth-Heinemann, pp.442-449.

3. Cole, E.S., Lee, K., Lauziere, K., Kelton, C., Chappel, S., Weintraub, B., Ferrera, D., Peterson, P., Bernasconi, R., Edmunds, T., Richards, S., Dickrell, L., Kleeman, J.M., McPherson, J.M., Pratt, B.M. (1993) Biotechnology 11, 1014-1024. 4. Kawashima, I., Ozawa, H., Kotani, M., Suzuki, M., Kawano, T., Gomibuchi, M., Tai, T. (1993) J. Biochem. 114, 186-193. 5. Muchmore, E.A., Milewski M., Varki, A., Diaz, S. (1989) J. Biol. Chem. 264, 20216-20223. 6. Kawano, T., Koyama, S., Takematsu, H., Kozutsumi, Y., Kawasaki, H., Kawashima, S., Kawasaki, T., Suzuki, A. (1995) J. Biol. Chem. 270, 16458-16463. 7. Sambrook, J., Fritsch, E.F., Maniatis, T. (1989) Cold Spring Harbor Laboratory Press. 8. Paterson, B.M., Roberts, B.E., Kuff, E.L. (1977) Proc. Acad. Sci. USA 74, 4370-4374. 9. Zamecnik, P.C., Stephenson, M.L. (1978) Proc. Natl. Acad. Sci. USA 75, 280-284.

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CELL PROLIFERATION

PROLIFERATION CONTROL APOPTOSIS

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INTRACELLULAR FATTY ACID COMPOSITION AFFECTS CELL YIELD, ENERGY METABOLISM AND CELL DAMAGE IN AGITATED CULTURES M.BUTLER, N.HUZEL, N.BARNABÉ, L.BAJNO AND T.GRAY Department of Microbiology, University of Manitoba, Winnipeg, Canada R3T 2N2

Abstract Continuous passage of cells in serum-free media requires the presence of micronutrients and growth factors to compensate for the lack of serum. Fatty acid supplementation is essential to ensure an adequate composition of the structural lipid components of the cell. We have shown that the unsaturated fatty acids, oleic and linoleic independently enhance cell yield and Mab productivity. The cellular content of the fatty acids gradually increased during continuous culture passage with no evidence of regulatory control. Most of the fatty acid accumulated in the polar lipid fraction and the unsaturated/ saturated fatty acid ratio of all cellular lipid fractions increased significantly. This caused a substantial decrease in the rate of glutamine metabolism and an increase in the rate of glucose metabolism. The changes in energy metabolism were reversed when the cells were removed from fatty acidsupplemented medium. The most plausible explanation for this effect is an altered rate of transport of glutamine via the cell membrane. An observed change in the phospholipid composition of the membrane also caused a significant protective effect on the cells in agitated cultures. The life-span of fatty acid-loaded cells showed a improvement compared to controls in cultures stirred at high rates of agitation. Introduction The unsaturated fatty acids, linoleic acid and oleic acid have been shown to be essential for the repeated passage of hybridomas in serum-free cultures. In previous reports (1,2) we showed that linoleic or oleic acid enhances significantly the cell yield and monoclonal antibody productivity of a B-lymphocyte hybridoma (CC9C10). However, continued culture passage with the unsaturated fatty acids leads to a lipid-loaded state in which cells maintain a high capacity for growth but a decreased capacity for antibody production. A similar differential effect of fatty acids on product secretion and cell growth has been reported previously for the secretion of cytokines from human peripheral lymphocytes (3) and for recombinant protein productivity from BHK cells (4). The mechanism of growth-promotion of these fatty acids may be related to their importance in the synthesis of cellular membranes (5,6) which may have a significant effect on membrane fluidity (7). We now present data to show that the fatty acids cause significant changes in the phospholipid composition of the cell membrane. This may lead to altered rates of transport 197 O.-W. Merten et al. (eds.). New Developments and New Applications in Animal Cell Technology, 197-203. ©1998 Kluwer Academic Publishers. Printed in the Netherlands.

198

of key energy substrates into the cell and reduced fragility under conditions of culture agitation. Materials and methods

Cell line: The murine B-lymphocyte hybridoma (CC9C10), which secretes a monoclonal antibody against insulin, was obtained from the ATCC (No HB123). The cells were adapted to a serum-free medium over several passages and grown for at least 10 passages in this medium prior to the described experiments. Insulin was one component of the serum-free formulation. However, substitution with a recombinant analogue of insulin-like growth factor was equally effective with respect to growth promotion. Growth yields in the

presence of either insulin or recombinant IGF were enhanced by 20%. Culture: The basal medium consisted of DMEM: Ham‘s F12 (1:1 v/v) supplemented with a

serum-free mixture of hormones and micronutrients (1). The cultures were also supplemented, where indicated, with fatty acid-free bovine serum albumin complexed with a specific concentration of oleic or linoleic acid. Total cell concentrations were determined by a Coulter counter. Viable cell concentrations were determined by counting a cell suspension diluted 1:1 v/v with 0.2% trypan blue using a Neubauer

haemocytometer.

Results 1. The effect of fatty acids on growth and antibody production Cells were adapted from a serum-supplemented to a serum-free medium and grown for at least 10 passages before there was a noticeable decrease in cell yield. The original cell yields were restored by the addition of oleic or linoleic acid to the serum-free cultures at 25-50 µM. Cell yields were enhanced by as much as 300%. The doubling time in the presence of the fatty acids was around 17 h. Ten other fatty acids were tested for growth promotion in the serumfree medium but none produced similar effects to these unsaturated fatty acids. Cultures of hybridomas grown over several passages in media supplemented with linoleic acid (25 µM), oleic acid (25 µM) or an equimolar mix of oleic/ linoleic (25 µM) showed a consistent enhancement of cell yield compared to fatty acid-free control cultures. Growth yields were greatest in cultures containing an oleic/ linoleic acid mix >linoleic acid >oleic acid >control. The effect of growth enhancement was reversible. When cells that had been passaged continuously in the presence of fatty acids were re-introduced into unsupplemented medium, the growth advantage over control cultures was lost within 2 to 3 passages. The Mab yield increased significantly to 90 µg/ml at the first passage of growth in the presence of fatty acids. However, the yield of Mab gradually declined over the subsequent 3 passages of growth with fatty acid until there was no difference in Mab yield (60 µg/ml) from the control culture. The effect of removing the fatty acids was to temporarily restore the higher Mab yield of the cells previously grown in fatty acid. However, this advantage was eroded after 4 passages in the absence of fatty acids.

199

2. Metabolic state of cells grown with fatty acids

The results of the growth experiments suggest a model set out in Table 1. Initially (state 1) the cells were starved of critical fatty acids following continuous growth in serum-free medium without a fatty acid supplement. State 2 arises following a brief exposure to linoleic (or oleic) acid (25-50 µM). State 3 arises from prolonged exposure to fatty acids and can be partially reversed to state 2 following the removal of fatty acids from the growth medium.

The fatty acid composition of each of these states is significantly different. In state 1,82% of cellular fatty acids could be accounted for by palmitic, oleic and stearic acids. The linoleic acid concentration was low (<5%). In state 2 the linoleic content increased to 70% and in state 3 to 90%, whilst the content of the other fatty acids decreased progressively over this period to 5%. Further evidence of metabolic differences between cells in these 3 states comes from the analysis of energy metabolism. We have shown by radioactive incorporation experiments that the oxidative metabolism of glucose increases (x2) and the oxidative

metabolism of glutamine decreases (x8) as cells progress from state 1 to state 3. Cells in state 3 grown in the absence of fatty acid revert to state 2 and demonstrate a partial reversal of this oxidative pattern. In all states the rate of fatty acid oxidation was low in relation to the total

utilisation of fatty acid and in relation to glucose and glutamine oxidation (8).

200

3. Regulation of fatty acid uptake It would appear that the cells have a requirement for linoleic and/or oleic acid but are unable to regulate the rate of uptake to maintain optimal intracellular metabolism (state 2 in Table 1). In order to verify this , we determined the rate of incorporation of radioactively labelled fatty acids from the media into 3 intracellular lipid fractions (Fig. 1). The pattern of fatty acid utilisation was determined for cells grown in the presence or absence of linoleic acid for at least 5 passages. Most of the fatty acid was incorporated into the polar lipid fraction (>74%). This fraction included the phospholipids derived from cell membranes. There was no significant difference in the total incorporation of linoleic acid between control and fatty acid-grown cells. This suggests that there is no regulatory mechanism to prevent an over-accumulation of fatty acid into the cell. A similar result was obtained with respect to the incorporation of oleic acid into these lipid fractions.

4. Uptake of energy substrates in the presence of fatty acids The metabolic profile of cells grown in linoleic or oleic acid was found to be significantly

different from control cells. The possibility that the changes were associated with an altered membrane permeability to substrate uptake was investigated by short-term radioactive uptake

experiments. The rates of cellular uptake of glucose and glutamine were determined from radioactive incorporation into cells over a 3 min incubation period. In separate incubations glucose was added at concentrations up to 100 mM and glutamine in concentrations up to 20 mM (Fig. 2).

Hyperbolic curve fitting indicated Michaelis-Menten-type kinetics of substrate uptake with respect to concentration. This data shows a significant difference in glutamine uptake between the linoleic acid-grown and control cultures but no significant difference for glucose uptake.

201

The kinetic parameters of substrate uptake were determined by statistical analysis using the soft-ware program, HYPER (Table 2). The was no apparent difference in the Km values for glucose transport in the linoleic acid-grown cells. However, the Km for glutamine uptake in linoleic acid-grown cells at 23±5 mM was almost an order of magnitude higher than the value of 2.7±0.5mM for control cells. At the glutamine concentration (6 mM) used in the standard growth medium the glutamine uptake rate was 2.5-fold lower in linoleic acid-grown cells compared to control cells. This indicates that the incorporation of the unsaturated fatty

acids into the membrane lipids caused a significant decrease in the transport of glutamine.

5. The effect of culture agitation on fatty acid grown cells Suspension cells agitated in spinner flasks are susceptible to shear damage caused by gas entrainment. We determined that a measurable rate of loss of cell viability occured in spinner flasks at an agitation rates between 470 - 630 rpm (tip speed 120-165 cm/sec). At the higher value (630 rpm) the viable cell concentration decreased to zero after 1.5 h, whereas at the lower value (470 rpm) the viable cell concentration decreased to half the original value within 1-3 h. Fig 3a shows the time-dependent decrease of viable concentration of linoleic acidgrown and control cells over a period of 5 h. This is a representative of a series of curves generated within the agitation range from which we determined the half-life of the viable cell population. Fig. 3b shows that the half life values determined for the linoleic acid-grown cells were significantly higher than those of control cells up to 550 rpm. At the higher agitation rates the half-lives were extremely short and no significant difference was found for the values for the two cell types. At 470 rpm the determined half-life for linoleic acid-grown cells was x3 the equivalent value of control cells. This result indicates that the effect of the fatty acid is to improve the robustness of the cells significantly in agitated culture.

202

Cells were inoculated at 5xl05 cells/ml into 200 ml medium contained in a spinner flask which was agitated at a selected speed for 5 h. The viable cell concentration was determined at intervals during this period. (A) The change in viable cell concentration for cultures agitated at 470 rpm. (B) The half-life of the viable cell population at various agitation speeds. Values are means ±SEM for n=3 Conclusions Supplementation of a serum-free culture of a hybridoma with the unsaturated fatty acids, oleic and linoleic causes :- a substantial growth enhancement (300%) - an initial enhancement of Mab yield (60%) - a significant reduction in glutamine uptake (60%) - an increased tolerance to culture agitation For maximal productivity of the hybridomas, the fatty acid composition of the cell requires to be finely balanced between a lipid-starved and lipid-loaded state. As there appears to be no regulatory mechanism for fatty acid uptake, the lipid-balanced state of the cells in culture must be maintained by a controlled feeding regime References 1. Butler, M. and Huzel, N. (1995) J. Biotechnol. 39, 165-173 2. Butler,M., Huzel,N. and Barnabé,N. (1997) Biochem J. 322, 615-623. 3. Karsten, S., Schafer, G. and Schauder, P. (1994) J. Cell Physiol. 161, 15-22 4. Schmid, G., Zilg, H., Eberhard,U. and Johaiinsen, R. (1991) J. Biotechnol. 17, 155-167 5. Rockwell, G.A., Sato, G.H. and McClure, D.B. (1980) J. Cell Physiol. 103, 323-331 6. Rintoul, D.A, Sklar, L.A. and Simoni, R.D. (1978) J. Biol. Chem. 253, 7447-7452 7. Calder, P.C., Yaqoob, P., Harvey, D.J, Watts, A. andNewsholme, E.A. (1994) Biochem. J. 300, 509-518 8. Butler, M. and Huzel, N. (1997) ESACT 14, 657-662.

203

Discussion

Grammatikos:

How do you add the fatty acids to the medium?

Butler:

We attached the fatty acids to de-lipidated BSA.

Grammatikos:

When you incorporated linoleic acid into cells you showed a much

lower arachidonic acid level. It is known, theoretically, that linoleic acid can lead to increases in arachidonic acid. Can you speculate on your result - is it just not processed to arachidonic acid?

Butler:

The figures may have been misleading. I quoted moles percent to show that the proportion of linoleic acid goes up dramatically. The amount of arachidonic remains about the same.

Jordan:

Do you have air bubbles in the bioreactor at 470 rpm?

Butler:

We were not sparging; it was a spinner flask, so we had a vortex and head space.

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LIPID REQUIREMENTS OF A RECOMBINANT CHINESE HAMSTER OVARY CELL LINE (CHO) JEFFREY T. MCGREW, CHERYL L. RICHARDS, PAULINE SMIDT, BRADLEY DELL, AND VIRGINIA PRICE Immunex Corporation, Department of Cell Sciences, 51 University Street, Seattle, WA 98101 USA

1. Abstract

We have tested the effect of a wide variety of lipid supplements and precursors on the growth of a recombinant Chinese Hamster Ovary (CHO) cell line. This cell line is adapted to grow in protein-free media and grows slowly in the absence of any exogenous lipid

source. Several exogenous lipid sources stimulated robust growth including Intralipids. Intralipid (Kabi Pharmacia) is a filterable lipid emulsion consisting of 10% soybean oil, 1.2% egg yolk phospholipids, and 2.25% glycerin in water. Soybean oil consists of a mixture of neutral triglycerides. In order to determine if the growth promoting activity could be due to one of these components, purified triglycerides and phospholipids were tested alone and in combination. We found that the growth promoting activity required

both triglycerides and phospholipids. We also found that the phospholipid growth factor lysophosphatidic acid stimulated CHO cell growth. 2. Introduction

Growth of mammalian cells in culture typically requires the addition of exogenous lipids. The requirement for lipids can be met with serum, partially purified serum fractions, lipid rich albumin, or protein-free lipid emulsions. Exogenous lipids become incorporated in the cell membranes and play a nutritional role for cell growth [1]. Lipids are also involved in signaling intracellularly and extracellularly [2]. Previous studies demonstrated that addition of exogenous lipids to Chinese Hamster Ovary (CHO) cells improved growth and

production of recombinant proteins [3,4]. For manufacturing purposes lipid supplements should be chemically defined, filterable, and easy to prepare [5]. In addition, a lipid supplement would preferably not be derived from serum thus avoiding adventitious contaminants. The objective of this study is to identify commercially available lipid supplements that support robust growth of CHO cells and are suitable for manufacturing.

3. Results and Discussion 3.1 GROWTH OF CHO CELLS IN RESPONSE LIPIDS Table 1 shows the results of a growth stimulation assay of several lipids and lipid precursors. Consistent with previous results, both Ex-cyte and cholesterol rich lipids stimulated growth of CHO cells [4]. Intralipid, a protein-free lipid emulsion also supported robust growth of CHO cells. Figure 1 shows the stimulation of cell growth in response to increasing concentrations of Intralipid. Intralipid is an emulsion of 10% soybean oil,

1.2% egg yolk phospholipids. 205 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 205-207.

© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

206

Since Intralipid supported cell growth, we sought to determine if either soybean oil or phosphatidyl choline, the primary component of egg yolk phospholipids, would stimulate

CHO cell growth independently. Soybean oil or phosphatidyl choline on their own supported little, if any, cell growth (data not shown). An emulsion was prepared consisting of soybean oil, phosphatidyl choline, and F68. This emulsion did stimulate some cell growth, but not as much as Intralipid (data not shown).

207

Intralipid is a potentially useful lipid supplement for manufacturing. It is chemically well defined and relatively inexpensive. The mean particle size of Intralipid is 0.37 microns which is larger that the pore size of a 0.2µ filter [6]. Since emulsion particles should be somewhat flexible, we pressure filtered Intralipid prior to adding it to media and found that Intralipid easily penetrated a 0.2µ filter. If, however, Intralipid was added to media prior to filtration, we found that the filter would clog suggesting that the particle size of the emulsion increases in the presence of media. Filtering Intralipid prior to adding it to media did not influence performance of CHO cells in shake flask or bioreactor cultures (data not shown). 3.2 LYSOPHOSPHATIDIC ACID STIMULATES GROWTH OF CHO CELLS Lysophosphatidic acid (LPA) is a simple phospholipid that stimulates growth of many cell types when added to cells extracellulary [2]. LPA bound with albumin is a component of

serum, and many of the growth factor effects of serum can be accounted for by LPA [2]. Since some serum-free media does not contain any polypeptide growth factors, LPA in or produced from lipid supplements could potentially account for some of the growth stimulatory effects of exogenous lipids. LPA was added in increasing concentration to CHO cells in the presence of fatty acid free-BSA as a lipid carrier (Figure 1B). LPA stimulated modest growth of CHO cells with a saturating concentration of about 20 µg/ml. In Figure 1B, a saturating concentration of Intralipid supported indicating the lysophosphatidic acid was not as potent as Intralipid. This suggests that some of the

effects of exogenously added lipids may be due to growth factor like properties rather than a nutritional properties. LPA also stimulated growth of cells in the absence of BSA, but less than that in the presence (data not shown). 4. References 1. Schimd, G. (1991) Lipid metabolism of animal cells in culture-A review, in Production of Biological from Animal Cells in Culture: Research, Development and Achievements. R. E. Spier et al., (Eds.) Butterworth Oxford, pp61-65

2. Moolenaar, W.H.: Lysophosphatidic Acid, a Multifunctional Phospholipid Messenger, J. Biol. Chem. 270 (1995), 12949-12952 3. Darfler, F. J. : Preparation and use of lipid microemulsions as nutrition supplements for culturing mammalian cells, In Vitro Cell Dev. Biol. 26 (1990), 779-783 4. Jenkins, N., Castro, P., Menon, S., Ison, A., and Bull, A.: Effect of lipid supplements on the production and glycosylation of recombinant interferon-g expressed in CHO cells, Cytotechnology 15 (1994), 209-215 5. Seamans, T.C., Gould, S.L., Distefano, D.J., Silberklang, M., and Robinson, D.K.: Use of Lipid Emulsions as Nutritional Supplements in Mammalian Cell Culture, Ann. N.Y. Acad. Sci. 245 (1994) 240-243 6. Mehta, R.C., Head L.F., Hazrati, A.M., Parr, M., Rapp,R.P., DeLuca,P.P.:Fat emulsion particle -size distribution in total nutrient admixtures, Am. J. Hosp. Pharm. 49 (1992) 27492755

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SUSTAINED EXPRESSION IN PROLIFERATION CONTROLED BHK-21 CELLS Keywords: BHK-21, IRF-1, proliferation control, growth regulation, expressions

PETER P. MÜLLER, SABINE KIRCHHOFF AND HANSJÖRG HAUSER GBF - National Center for Biotechnological Research, Mascheroder Weg 1, 38124 Braunschweig, FRG Tel.: ++49 (0)531 6181 250

Fax.: ++49 (0)531 6181 262 E.mail: [email protected]

Abstract

We have established proliferation controled BHK cell lines employing IRF-1 (Interferon

Regulatory Factor 1), a transcriptional activator that inhibits cell growth. The activity of IRF-1 has been made hormone dependent by fusing IRF-1 to the hormone-binding

domain of the estrogen receptor. The growth rate of cells expressing the IRF-l-estrogen receptor fusion protein can be reduced by adding estradiol to the medium. Productivity of

constitutively expressed proteins decreases when growth is downregulated. Expression during both, unrestricted growth and the growth controled phase is obtained by using two separate transcription units. Long-term stability of the regulation has been achieved by coupling the expression of IRF-1 with a selection marker on a dicistronic mRNA construct. The results demonstrate that stable proliferation control and sustained expression can be achieved in a biotechnologically relevant cell line.

Introduction Cell culture processes are initiated from a small number of cells that are expanded

manyfold to achieve high densitiy. In an ideal continuous culture process initial cell growth is rapid, followed by a production phase in which proliferation is downregulated. After reaching an optimal high cell density proliferation is no longer required, only the production is of interest. Proliferation of most cell lines is lower at a high densities, however, not sufficiently low for continuous culture. In a proliferation inhibited state, protein synthesis and metabolic activity of the cell could be devoted largely to the

production of the desired recombinant protein. Advantages of a proliferation inhibited production phase would be

- lower release of intracellular hydrolizing enzymes, leading to a higher uniformity and stability of the product, - higher consistency of the product by keeping the cells in an identical state of proliferation during the whole production period, - genetic drift is reduced due to the reduced proliferation, resulting in a higher stability of the production - reduced media consumption as a consequence of lower metabolic turnover.

Understanding molecular processes that control cell proliferation and progress in the technology of genetic manipulation of mammalian cells (Hauser, 1997) has allowed us to

209 O.-W. Merten et al. (eds), New Developements and New Applications in Animal Cell Technology, 209-214. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

210 influence the proliferation of cell lines which are of biotechnological interest. IRF-1 overexpression inhibits the proliferation of a number of cell lines (Kirchhoff et al.,

1993, 1995, 1996). To demonstrate the feasability of growth control in a biotechnologically relevant cell line, IRF-l-estrogen receptor fusion protein encoding

gene has been transfected into a producer BHK cell line. Upon estrogen addition to the growth medium, cell proliferation is reduced to various degrees depending on the estrogen concentration. After three days in the presence of low estrogen concentrations (30nM) inhibition is about five- to tenfold. After five days of IRF-1 activation the cells are still viable as determined by trypan blue staining (Kirchhoff, unpublished). Cell morphology as examined by microscopic observation shows membrane blebbing as well as the accumulation of granula within the cytoplasm in most of the cells. Whereas native

BHK-21 cell nuclei are not uniformely sized and often show a typical kidney form, IRF-1 activity leads to a rounded nuclear morphology. Indications for apoptosis were not

detected (Kirchhoff, unpublished). IRF-1 does not lead to a cell cycle specific arrest. Instead, there is a slow-down of all cell cycle phases (Kirchhoff et al., in preparation). The slight accumulation of the cells in Gl phase cannot account for the drastic inhibition of cell proliferation. The most likely cause is interferon secretion induced by IRF-1 imposes the weak Gl arrest. A proportional

extension of other cell cycle phases could explain the reduced proliferation. IRF-1 activation leads to reduced gene expression, depending on the promoter used.

Productivity of a recombinant immunoglobulin decreases after three days of growth arrest (Kirchhoff et al 1996). Since IRF-1 is a DNA sequence specific transcriptional activator,

transcription from promoters containing IRF-1 binding sites is enhanced in IRF-1 proliferation controled cells. Both constitutive expression during unrestricted growth and during the controled growth phase are obtained by employing two separate expression

constructs, one with a constitutive promoter and a second one with an IRF-1 inducible promoter. Since growth control imposes a strong selection pressure, stability of the

regulatory control is essential. Long-term stability of this regulatory system has been obtained by tightly coupling expression of the IRF-1 fusion gene to a selection marker on a dicistronic construct.

Material and Methods DNA constructs Constructions were carried out by standard procedures (Sambrook et al., 1989). The IRF-1 fusion with the human estrogen receptor fragment has been described (Kirchhoff et al., 1993). An EcoRI-NheI restriction fragment from the plasmid pMC-2P carrying

an IRES element from Polio Virus and the pac gene as a selection marker has been inserted downstream of the IRF-1-hER gene to create a dicistronic expression cassette (Dirks et al., 1994). The dicistronic transcript is driven by the constitutive MPSV

promoter. Luciferase is constitutively expressed from the SV40 promoter or IRF-1 inducible expressed from a from a synthetic promoter that contains IRF-1 binding sites. Cell culture and gene transfer BHK-21 (ATCC CCL10) cells maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum were stably transfected by calcium phosphate coprecipitation method. 5 µg plasmid, 5 µg of high molecular weight DNA and 0,5 µg of selection plasmid pSV2pac (Vara et al., 1986) or pAG60 (Colbère-Garapin et al. 1980) per cells was used. Cells from a stable cell clone were transfected with the expression plasmid encoding the fusion protein IRF-1-hER and single cell clones were

isolated and expanded. Clones were cultured in the presence of 5 µ g/ml puromycin and 800 µ g/ml G418 (Gibco). Luciferase activity was measured using the Luclite kit

(Packard) and normalized against LDH activity. Other procedures were performed as described earlier (Kirchhoff et al., 1996).

211 Results 1. Establishment of stable proliferation control in BHK cells

No obvious phenotype on cell growth was found in cells constitutively expressing 1RF1 fusion protein in the absence of estrogen. Nevertheless, long-term cultivation of such cell clones leads to a loss of estrogen dependent growth regulation. Possibly subtle growth advantages of cells that have lost the 1RF-1 fusion gene expression lead to overgrowth by these non-expressing cells. To stabilize the growth regulatory system we coupled the expression of the IRF-1 fusion gene to the expression of a puromycin resistance marker gene on a dicistronic construct (Fig. 1). The regulation has been stable in all experiments and no reversion of the growth controled phenotype has been

observed. This selection pressure has been required during cloning and manipulation of cells, but could be relieved during actual production periods.

2. Recombinant gene expression in proliferation controled cells

Recombinant genes are commonly expressed from strong constitutive promoters. Cell growth and expression from the highly active MPSV promoter determined 3, 4 and 5 days after inducing growth restriction showed that productivity is not significantly

altered during this period but decreases from 40 % to 60 % during the fourth day and to about 20 % at the fifth day (Kirchhoff et al. 1996). The concentrations of estrogen used

do not significantly influence the productivity from these constructs whereas cell growth is further reduced at higher estrogen concentrations. To improve expression in growth controled cells we have made use of the fact that IRF-1 activates transcription of promoters with IRF-1 inducible elements (Kirchhoff et al., 1993, Tamura et al., 1995). Although basal expression is low, induced expression levels are in the same range as from strong viral promoters. That these promoters are inducible by estrogen in BHK-21 cells that express the IRF-1 fusion protein shows that

it functions at the same time to reduce the growth rate and activate transcription of IRF-1 dependent promoters. Both, constitutive expression and sustained expression during growth restriction is achieved when cells contain two different expression constructs, one driven by the constitutively active SV40 promoter and a second construct with an IRF-1 inducible promoter (Fig. 2).

212

Discussion

We demonstrate the feasability of IRF-1 mediated proliferation control by i) controling cell growth in the biotechnologically relevant BHK-21 cell line using an estrogen activatable IRF-1 fusion protein ii) stabilizing the growth regulatory system by coupling the expression of the IRF-1 fusion protein to the expression of a selectable marker gene on a dicistronic mRNA, and iii) expression of a recombinant protein could be sustained

during both unrestricted growth and the growth control phase by expression from two separate constructs that are driven by a constitutively active promoter and and IRF-1

inducible promoter, respectively. Carvalhal et al. (this volume) show that high viability in 1RF proliferation controled cultures can be maintained. The combination of genetic

manipulation with cultivation techniques moves this regulatory system from an academic interest oriented research project towards an applicable biotechnology project.

Acknowledgements We thank H. Conradt, L. Kongerslev, K. Scharfenberg, R. Wagner, D. Wirth and M. Wirth for suppport and discussions. The work was supported financially by an EC-grant BIO4-CT95-0291.

213 References Colbère-Garapin, F., Horodniceau, F., Khourilsky, P. and Garapin, A.C. (1981). A new dominant selective marker for higher eukaryotic cells J. Mol. Biol. 150, 1-13. Dirks, W., Schaper, F. Kirchhoff, S., Morelle, C, and Hauser, H.. (1994). A multifunctional vector family for gene expression in mammalian cells. Gene 149, 387-388 Hauser, H., (1997) in Mammalian cell biotechnology in protein production (Hauser, H. and Wagner, R., eds.). Walter de Gruiter, Berlin, 3-27. Kirchhoff. S., Schaper, F., and Hauser, H. (1993). Interferon regulatory factor 1 (IRF-1) mediates cell growth inhibition by transactivation of downstream target genes. Nucleic Acids Res. 21: 2881-2889. Kirchhoff. S., Koromilas, A., Schaper. F., Grashoff, M., Sonenberg, N. and Hauser, H. (1995). IRF-1 induced cell growth inhibition and interferon induction requires the activity of the protein kinase PKR., Oncogene 1 1 , 439-445 Kirchhoff. S., Kröger, A., Cruz. H., Tümmler, M., Schaper, F. and Hauser, H. (1996). Regulation of cell growth by IRF-1 in BHK-21 cells. Cytotechnology 22, 147-156. Sambrook, J., Frisch, E. F., and Maniatis, T. (1989). Molecular Cloning: A laboratory manual. Cold Spring Harbor Press (New York) Tamura, T., Ishihara, M., Lamphier, M S., Tanaka, N., Oishi, I , Aizawa, S., Matsuyama, T., Mak, T W., Taki, S. and Taniguchi, T. (1995). An IRF-1 dependent pathway of DNA-damage-induced apoptosis in mitogen-cativated T lymphocytes. Nature 376, 596-599. Vara J, Portela A, Ortin J and Jimenez A (1986). Expression in mammalian cells of a gene from Streptomyces alboniger conferring puromycin resistance. Nucleic Acids. Res. 14: 4617-4624.

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Discussion

Massie:

When your cell cycle growth slows down and you look at viability, do you see any induction of apoptosis?

Müller:

We have tried to find signs but there is no indication that the cells died of apoptosis. The cells accumulate a lot of intracellular vesicles and grow larger. After prolonged exposure to estradiol they burst.

Massie:

Do you think that this is a side-effect of estradiol, or regulation of other genes by your transcription factors?

Müller:

I think that it is an effect of over-activation of IRF-1 because cells which lose expression of IRF-1 fusion protein do not show any effect to the action of estradiol.

Al-Rubeai:

Have you looked at these vesicles?

Müller:

We do not know what is in them.

Konstantinov:

I think that your system is flexible but complex. What is its advantage compared with a simple decrease of temperature in the fermenter?

Müiler:

Can tell me how decreasing the temperature can adjust the growth of stromophilia to the growth of haemopoetic stem cells? You need to control the growth specifically of one particular cell line. You cannot just change the overall conditions by taking away one

limiting medium component, or by changing the temperature.

Kostantinov:

Some groups have successfully shown modulation of BHK cell metabolism by adjusting the temperature.

Müller:

You can do that within a certain range.

CELL GROWTH INHIBITION BY THE IRF-1 SYSTEM A.V. CARVALHAL1 ; J.L. MOREIRA1; P. MÜLLER2, H. HAUSER2; M.J.T. CARRONDO1,3 1 - IBET/ITQB, Apartado 12, 2780 Oeiras, Portugal 2 - GBF, Mascheroder Weg 1, 38124 Braunschweig, Germany 3 - Lab. Eng. Bioq., FCT/UNL, 2825 Monte da Caparica, Portugal

1. Abstract A genetic approach, based on the growth regulatory protein interferon-regulated-factor-

1 (IRF-1), activated by the addition of estradiol, has been applied to regulate BHK cell growth. With the addition of 100 nM of estradiol 48 hours after inoculation, growth inhibition occurs within 24 hours but is followed by a significant decrease in cell viability, whereas rec-protein specific productivity is not significantly altered. Viability decrease is not due to estradiol effect and is independent upon the culture system. In order to define strategies to extend the stationary growth phase, several parameters have been studied: estradiol concentration, time post inoculation for estradiol addition and time span of estradiol exposure. When the time of contact between the cells and estradiol is reduced the cell viability increases, achieving similar values of the control, without the estradiol, and leading to a stationary growth phase extension.

2. Materials and Methods Cell line and medium: BHK clones were obtained from Dr H. Hauser (GBF, Braunschwig, Germany) and grown in DMEM supplemented with 10% (v/v) FBS, 4.5g/l of glucose, 5 mg/1 of puromycin and 5 mg/1 of vitamin Kl. Culture system: The cells were grown in T-flasks and inoculated with a seeding density of . Cell concentration and viability were measured by the trypan blue dye exclusion method. Estradiol was directly added to the culture medium using a 100 fold stock solution and, unless indicated, the final concentration was 100 nM. Estradiol removal from the culture medium (whenever indicated) was performed by replacing the total supernatant by fresh medium. 215 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 215-217. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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3. Results and Discussion 3.1. IRF-1 OVEREXPRESSION INDUCTION EFFECT ON: 3.1.1. Cell growth Estradiol addition to the culture medium 48

hours after inoculation leads to an effective growth inhibition (Figure 1A) but this is followed by a significant decrease in cell viability (Figure 1B) (up to 50% five days after estradiol addition). This decrease is faster than the normal pattern of a static batch culture, as can be seen by the control test, where no

estradiol was added. The results presented in Figure 1 are independent on the clone under test and the culture method (static or stirred cultures).

3.1.2. Rec-protein productivity Rec-protein productivity is not significantly affected by the estradiol addition (specific productivity of 130 ± 20 I.U.).

3.2. PARAMETERS AFFECTING CELL GROWTH INHIBITION In order to eliminate the cell viability decrease 48 hours after estradiol addition, the effect of several parameters on cell growth inhibition were evaluated.

3.2.1 Estradiol concentration There is a clear difference in cell growth pattern between 10 nM and 100 nM: cell growth inhibition is more efficient when 100 nM is used. However, in both cases a similar sharp decrease in cell viability is observed, similar to Figure 1B and independent upon estradiol concentration.

3.2.2. Time post inoculation for estradiol addition 100 nM estradiol was added at two different times, 48 or 96 hours after inoculation; at the end of the exponential phase (96 hours), a similar pattern to the control was observed (the cell viability pattern did not decrease differently from the control).

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3.2.3. Time of contact between the cells and the estradiol In the previous reported experiments there is no estradiol removal after its addition. However, as presented in Figure 2B, cell viability increases when the time of contact between the cells and estradiol is reduced to 72 hours. The recovery of viability is due to the removal of estradiol and not to the exchange to fresh medium (data not shown). Similar results were obtained with the producer clone (data not shown).

If the media is changed again 3 days after estradiol removal by fresh estradiol-free medium, the cells

start to grow again (Figure 2A), clearly indicating that the cell growth inhibition process can be reversed.

4. Conclusions

• The induction of IRF-1 overexpression leads to a clear growth inhibition by the addition of estradiol (100 nM) at the beginning of the exponential growth phase, but is followed by: . decrease in cell viability (faster than in the control); . no significant change in rec-protein specific productivity.

• The significant decrease in cell viability can be avoided when the time of contact between the cells and estradiol is reduced.

• IRF-1 control proliferation system can be reversed by the removal of the estradiol from the medium. References Kirchhoff S , Schaper F; Hauser H (1993), Nuclei Acids Res 21:2881-2889. Kirchhoff S; Kroger A; Cruz H; Tümmler M; Schaper F; Köster M; Hauser H (1996), Cytotechnology 22:147156.

Acknowledgements The authors acknowledge and appreciate the financial support received from the European Commission (B1O4-CT95-0291) and from Junta Nacional de Investigação Cientifica-Portugal (BIC 1788). The authors are grateful to Ms. Maria do Rosário Clemente for technical support.

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CORRELATION BETWEEN BHK CELL SPECIFIC PRODUCTIVITY AND METABOLISM H. J. CRUZ1, J. L. MOREIRA1, E. M. DIAS1, C. M, PEIXOTO1, A. S. FERREIRA1 & M. J. T. CARRONDO1,2

1 - IBET/ITQB, Ap. 12, 2780 Oeiras, Portugal 2 - Lab. Eng. Bioq., FCT/UNL, 2825 Monte da Caparica, Portugal

1. Abstract

A rBHK21 clone producing an antibody/cytokine fusion protein was used to study the dependence of cell metabolism on the glucose and glutamine levels in the culture

medium. Results indicate that glucose and glutamine consumptions and lactate and ammonia productions show hyperbolic behaviors. The estimated values for were 1.39 ± 0.11 mM for lactate production as a function of glucose and 0.59 ± 0.17 and 0.15 ± 0.04 mM for ammonia production as a function of glutamine, at glucose concentrations of 0.05 and 1.0 respectively. At very low glucose concentrations the glucose to lactate yield decreased markedly, showing a metabolic shift towards lower glucose fermentation. At very low glutamine concentrations, the glutamine to ammonia yield increases, showing a more efficient glutamine metabolism. The cell specific productivity can be increased significantly by manipulating nutrient concentrations. 2. Introduction

The metabolism of transformed cells shows new properties like high glycolysis and glutaminolysis rates, glucose and glutamine being the main nutrients utilized by mammalian cells [1]. Mammalian cells grown in culture excrete lactic acid and

ammonia in quantities that may inhibit cell growth and thus decrease final product

liters. High glucose and glutamine concentrations lead to overflow metabolism while limiting concentrations provoke the use of more efficient energy pathways and reduce formation of inhibitory by products [2]. 3. Materials and Methods Cell lines and cell growth systems rBHK 21A cells were obtained from Dr. Hansjörg Hauser (GBF, Braunschweig, Germany). Cells were grown in DMEM with 5% PCS. Studies were performed in static flasks containing 10 ml of medium and as inoculum. Metabolite analysis Glucose- Sigma # G20, glutamine- Sigma # GLN-2, Lactate- Boehringer Mannheim # 139084, Ammonia- Boehringer Mannheim # 1112732. 219 O. - W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 219-221. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

220

4. Results and Discussion 4.1. GLUCOSE METABOLISM The results obtained from the glucose uptake as a function of glucose concentration show a hyperbolic behavior, with value of 1.64 ± 0.17 mM (similar to the reported 2 mM in chick embryo fibroblast cultivation [3]) and (the range of values obtained includes those found in the literature [4]: 12 nmol at 20 mM of glucose). For the lactate production a similar behavior to that found for glucose was obtained, with lactate production being very low under low glucose concentrations The values obtained for are higher than those found in the literature [4] but are consistent with the ones obtained in the glucose uptake assay. The glucose to lactate yield decreases dramatically at very low glucose levels. This clearly indicates a metabolic shift from a high lactate to a low lactate producing metabolism- more efficient pathways are involved. This shift is observed for glucose below 1.64 mM or which is comparable with results obtained for hybridomas [5]. 4.2. GLUTAM1NE METABOLISM The glutamine uptake has a hyperbolic behavior with and mM for glucose 0.05 and respectively. These values are lower than those found in the literature that range from 2 mM in normal rat kidney cells to 4.5 mM in extracts of Ehrlich ascite cells [6]. The values obtained for were for glucose and respectively. These values are comparable to those in the literature: 3.0 to 4.9 nmol cells at 3 mM glutamine [4] and 5.7 nmol cells for MDCK cells in medium with fructose instead of glucose [7]. The glutamine consumption being higher at the lower glucose concentration shows the interactive and complementary nature of both these main nutrients for animal cells. The ammonia production showed a similar behavior to that found for glutamine uptake, with and at glucose 0.05 and respectively. Reported results showed that ammonia production of MDCK cells can be reduced by maintaining the glutamine concentration at less than 1.0 mM [7]. The values obtained for were and at glucose 0.05 and 1.0 respectively. The glutamine to ammonia yield increases at very low glutamine, meaning that a more efficient glutamine metabolism is occurring [2]; although the yields are higher, the overall ammonia production will be lower at low glutamine concentrations.

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4.3. PRODUCTIVITY

A significant variation (approximately 2 fold) in the cell specific productivity is observed with the glucose concentration. With glutamine the same behavior is observed at glucose but at glucose the trend is to increase productivity with decreasing glutamine. 5. Conclusions

Significant reductions in the glucose consumption were observed for low glucose concentrations; the same occurred with lactate production. Lactate showed a significant decrease in yield from glucose at very low glucose concentrations, showing that a shift towards a more efficient glucose metabolism occurred. Significant reductions in the glutamine consumption were observed for low glutamine

concentrations; the same occurred with ammonia production. Ammonia showed an increase in yield from glutamine at very low glutamine concentrations which shows higher efficiency in glutamine metabolism. Glutamine consumption and ammonia production increased significantly with the decrease in glucose concentration. The cell specific productivity can be significantly increased by manipulating nutrient concentrations. Acknowledgements The authors acknowledge the financial support received from the European Commission (B1OTECH PL933069) and Junta Nacional de Investigação Cientifica e Tecnológica- Portugal (PBICT/BIO/20333/95 and PRAXIS XXI/BD/2764/94) and thank Dr. Sadettin Ozturk for the revision and discussion of this work.

References 1. McKeehan WL (1982) Mini-review: glycolysis, glutaminolysis and cell proliferation. Cell Biol Int Rep 6: 635-649. 2. Ljunggren J & Häggström L (1994) Catabolic control of hybridoma cells by glucose and glutamine fed batch cultures, Biotechnol Bioeng 44: 808-818, 3. Fagon JB & Racker E (1978) Determinants of glycolytic rate in normal transformed chick embryo fibroblasts. Cancer Res 38: 749-758 4. Neermann J, Wagner R (1996) Comparative analysis of glucose and glutamine metabolism in transformed mammalian cell lines, insect and primary liver cells. J Cell Physiol 166: 152-169, 5. Zhou W, Rehm J & Hu WS (1995) High viable cell concentration fed-batch cultures of hybridoma cells through on-line nutrient feeding. Biotechnol Bioeng 46: 579-587. 6. Xie L & Wang DIC (1994) Fed-batch cultivation of animal cells using different medium design concepts and feeding strategies. Biotechnol Bioeng 43: 1175-1189. 7. Glacken MW, Fleischaker RJ & Sinskey AJ (1986) Reduction of waste product via nutrient control: Possible strategies for maximizing product and cell yields on serum in cultures of mammalian cells. Biotechnol Bioeng 28: 1376-1389.

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EFFECT OF GROWTH ARREST IN BHK METABOLISM: ON LINE MONITORING BY AND NMR SPECTROSCOPY

P.M. ALVES 1, A.V. CARVALHAL1, J.L. MOREIRA1, H. SANTOS1, M.J.T. CARRONDO1,2 1

Instituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia Quimica e Biológica, Apt. 12,2780 Oeiras, Portugal. 2 Faculdade de Ciências e Tecnologia/Universidade Nova de Lisboa, 2825 Monte da Caparica, Portugal.

1. Introduction

Controlling proliferation of cell growth has been successfully applied recently [1]. Nevertheless, the physiological and metabolic behaviour of the cells after growth arrest is not well understood. Nuclear magnetic resonance (NMR) techniques are potentially very attractive to

explore cell metabolism due to their unique non-invasive characteristics. With a suitable perfusing system it is possible to monitor biochemical processes in real time under physiological conditions and various metabolic manipulations can be simulated. The low-sensitivity of the NMR method requires high cell densities in the detection zone; basement membrane gel (BMG) threads are a good strategy for cell immobilization because their matrix can support a high cell number and allow protection from shear damage [2]. The aims of this work were: on line monitoring of cells energetic status by NMR spectroscopy and on line monitoring of glucose uptake and lactate production by NMR spectroscopy, after estradiol addition to a BHK cell line growth regulated by the IRF-1 (interferon-regulated-factor-1) system. 2. Materials and Methods 2.1 Cell line and growth systems

BHK-21 cells genetically modified to depend on estradiol addition to regulate growth were obtained from Dr Hansjörg Hauser (GBF, Braunschweig, Germany). Cells were immobilized in BMG threads as described before [2]. An inocullum of cells was used for 1.5 ml of BMG. After immobilization, the threads were placed in 14 cm culture dishes containing 50 ml of Dulbecco’s modified Eagle’s medium (DMEM) with lg/1 glucose and supplemented with 10% fetal bovine serum (FBS). Cells were mantained for 2 days in a humidified atmosphere of 10% CO2/90% air at 37 °C. 2.2 On line NMR For NMR measurements the immobilized cells were transferred to a 10 mm tube fitted with a perfusion apparatus [2]. Cells were perfused at 1.5 ml/min with DMEM 223 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 223-225. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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containing

glucose (1 g/1) and estradiol (100 nM). The media was oxygenated with and mantained at 37 °C.During the experiment, the medium was changed or supplemented with glucose in order to avoid nutrient limitations and inhibition of cell metabolism by waste products. NMR spectra and NMR spectra were obtained using a 10 mm broad band probehead on a Brucker DMX500 NMR spectrometer. 3. Results and Discussion

3.1. Effect of Growth Inhibition on Cell Energetic Status Growth inhibition was successfully achieved after estradiol addition as no increase on NTP levels was observed during the following 100 hours (figure la).

During the first 24 hours after estradiol addition, NTP drops steadily. After that period a slight decrease was observed and NTP levels almost reach a plateau (figure 1b). This behaviour is consistent with the induction of growth arrest as an adaptation of cells energetic state to the new metabolic conditions is expected. As can be seen in the spectra (figure la), GPC/NTP, PC/NTP and PE/NTP ratios were affected with the time-course of the experiment, which indicates that phospholipid content of the cells is changing. The effect of estradiol on membrane fluidity is well reported [3] and further studies are necessary to confirm the integrity of cell membrane after long periods in contact with estradiol. 3.2. Effect of Growth Inhibition on Glucose and Lactate Metabolism

Using labelled substrates, as glucose, it is possible to monitor the distribution of the label within various metabolites during the time-course of an in vivo NMR experiment. As can be seen in the spectra (Figure 2), labelling from glucose was

225

incorporated mainly in lactate. Following time trajectories for labelling from glucose into lactate it is possible to compare glucose uptake and lactate production rates along time after estradiol addition.

For the first 12 hours, a higher rate for glucose consumption and lactate production was observed as compared to the following 80 hours and to the rates obtained before growth inhibition (data not shown). This can be due to the fact that cells are adapting their metabolism immediately after growth arrest. This is in agreement with the decrease in the NTP levels observed in the

spectra. An interesting result is that the glucose uptake rate slightly increases again after the re-feed done at time 60h, with no significant increase in lactate production rate. Further experiments have to been performed in order to evaluate the importance of this behaviour.

4. Conclusions and Perspectives

• BHK cells can be immobilized in gel threads (BMG) and perfused inside a NMR tube for long periods of time (up to 100 hours) without significant losses in cell viability. • Estradiol addition is efficient to induce growth arrest (NTP does not increase with time) and no significant changes in glucose metabolism were observed. Differences in phospholipid content of the cells were detected after growth inhibition. • Work using different labelling -glucose and other labelled precursors is in progress in order to evaluate metabolic fluxes in BHK before and after growth arrest. Further studies are necessary to elucidate the effect of estradiol on phospholipid metabolism. References:

1 Kirchhoff, S., Kröger, A., Cruz, H., Tümmler, M., Schaper, F., Köster, M. and Hauser, H. (1996) Regulation ofcell crowth by IRF-1 in BHK-21 cells, Cytotechnology 22,147-156.

2 Alves, P.M., Flögel, U., Brand, A., Leibfritz, D., Carrondo, M.J.T., Santos, H. and U. Sonnewald (1996) Immobilization of primary astrocytes and neurons for on-Line monitoring of biochemical processes by NMR, Dev. Neurosci. 18, 478-483. 3 Schwarz, S.M. (1988) Estrogen modulates ileal basolateral membrane lipid dynamics and Na+-K+ATPase activity. Am. J. Physiol. 254(5), G687-G694 Ackowledgments: The authors acknowledge the financial support received from European Comission BIO4-

CT95-0291, Fundação para a Ciência e Tecnologia PBICT/BIO/20333/95 and PRAXIS XXI/BD/2721/94.

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CELL SURVIVAL IN CHO CELL CULTURES GROWN IN A DEFINED PROTEIN FREE MEDIUM

ABSTRACT

Animal cells, primarily hybridomas, important for diagnostic and pharmaceutical production have been shown to die almost exclusively via apoptosis when entering the stationary phase. Recently similar reports have been presented for CHO (Chinese Hamster Ovary) cells grown in batch cultures under optimal conditions for high product yield 1. Experiments performed in small scale using a defined protein free medium, with the sole

addition of recombinant insulin, enabled us to investigate the importance of physiological parameters and their effect on cell survival. We show here that a CHO cell line engineered to produce a recombinant therapeutic protein is capable of inducing the apoptotic programme under batch culture conditions in a 2 L reactor, as

measured by the TUNEL assay (DNA end-labeling). Cells are arrested in the Gl phase of the cell cycle though insulin is present in excess and no apparent depletion of nutrients is at hand. Proliferation ceases and viability

decreases although the concentration of inhibiting metabolites is low. INTRODUCTION

Animal cells in culture are constantly exposed to environmental changes. There are several factors determining

the health status of the cells in a bioreactor: the availability of growth and survival factors, the availability of nutrients, the accumulation of both toxic and beneficial metabolites, variations in pH and DO levels and the physical shear forces encountered by the cells 2. If exposed to sudden toxic insult animal cells die by necrosis, morphologically characterised by swelling and the flocculation of nuclear chromatin, with subsequent cell lysis. Cells which experience mild environmental perturbations in culture conditions respond by undergoing an active, energy dependent programmed cell death called apoptosis. Apoptosis is characterised morphologically by cell shrinkage, condensation and fragmentation of the nucleus and biochemically by cleavage of DNA into nucleosomal fragments as a result of induction of transglutaminases, proteases and nucleases 3. A number of agents are reported to induce apoptosis in vivo and in vitro. Hormone and growth factor deprivation and loss of matrix attachment triggers apoptosis in different cell systems. Damage related inducers such as heat shock, viral infections, activation of oncogenes, or the tumour suppressor

as well as exposure of cells to nutrient

deprivation, free radicals and high concentrations of antimetabolites results in cell death4 .

METHODS

Batch growth studies were performed both in spinners and in a controlled 2 L stirred tank bioreactor (Belach bioteknink AB, Sweden). pH and dissolved oxygen (DO) were monitored and controlled at optimal levels. pH was controlled on line through additions of

on demand and oxygenation by bubble free

aeration using air and pure oxygen. The reactor culture was characterised in respect to consumption of nutrients (glucose and amino acids), the growth factor (insulin RIA) and formation of metabolites (lactate and ammonia).

Glucose and lactate was determined with a YSI 2700 analyser; amino acids with Waters PicoTag method and ammonia using an Orion electrode. Temperature was maintained at 37° C. Cells were grown in a proprietary defined serum free medium supplemented with 10 mg/L recombinant insulin, (nucellin-Zn, Eli Lilly Inc.).

227 O.-W. Merten et al. (eds.). New Developments and New Applications in Animal Cell Technology, 227-229. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

228 Cell count and viability were assesed using a particle counter (CASY, Schärfe system, Germany) and by Erythrosin B dye exclusion with a Bürker chamber. Cell cycle distribution analysis were performed using the method of Vindeløv. Apoptosis was measured by several independent methods: cells were harvested twice daily and fixed according to the protocol for the TUNEL reaction, (Boehringer Mannheim), and analysed with an Epics Elite flow cytometer. Different modified protocols for DNA fragmentation were employed according to ”Techniques in Apoptosis”, T. G Cotter and S. J. Martin, 1996. Additional experiments were performed in 100 ml stirred spinner cultures where the effect of decreased concentrations and consecutive supplementations of the growth factor were investigated, as well as different inoculum densities during batch growth.

RESULTS

The proliferation of CHO cells in batch culture stops after 100 h of culture at a cell density of approximately 2,5X106 cells/ml (Fig. 1). Practically all cells are viable throughout the growth phase. The cessation of growth is immediately followed by rapid cell death which obviously occurs almost exclusively by apoptosis as measured by TUNEL whereas no DNA fragmentation could be detected in the viability ranges examined. At this time (100 h) neither the level of glucose were limiting nor were the accumulated levels of lactate and ammonium inhibiting

(Fig. 2). The consumption and accumulation patterns of the amino acids show that only a few of them were used to any great extent (Fig. 3). The consumption of aspartate and asparagine was most significant and it seems likely that the cells are using these amino acids for their energy metabolism. Almost no uptake of the dipeptide

glycyl-glutamine (gly-gln) occured and the concentration in the medium did not change until the viability dropped. At this point both glutamine and glycine were accumulated in the culture medium which indicates an

extracellular cleavage of the dipeptide probably due to the release of proteases from the dying cells. The uptake of serine may also have contributed to the accumulation of glycine. Alanine was the sole additional amino acid which accumulated extracellularly. The concentrations of the remaining amino acids, (ser, glu, met, cys, val, leu, ile, pro, lys, arg, thr, his, tyr, phe and trp) were not affected significantly during the growth phase. Analysis of insulin concentrations in the medium showed that nucellin-Zn levels were high during almost the entire culture period and dropped to lower levels only during the last day, i.e. long time after cell death was initiated (Fig 1). Cell cycle distribution analysis from cells grown in spinner cultures (Fig. 4) revealed that the cells arrest in G1 phase of the cell cycle long before the viability is negatively affected and while the insulin still is plentiful. Insulin concentrations below 2,5 mg/L resulted in a reduced growth rate and decreased viability whereas total omission of the growth factor caused a rapid cell death. To investigate whether the insulin present was bioactive, additional nucellin-Zn (10 mg/L) was added intermittently to the spinner cultures at 4 time points after the commencement of the cultures. No improvement of the survival, nor any augmentation of the proliferation could be observed in this test indicating that a sufficient amount of nucellin-Zn is present (Fig. 5). Using different inoculum densities with the medium containing 10 mg/L nucellin-Zn, we could obtain an increasing amount of maximal total cell concentration, which would indicate an autocrine growth factor effect.

References 1

”Apoptosis in CHO cell cultures, examination by flow cytometry”, A Moore et al, Cytotechnology 17: 1-17, 1995.

2

”Cell death (apoptosis) in cell culture sytems”. T G. Colter and M Al-Rubcai. TIBTECH vol 3 pp 150-155, April 1995

3

”The genetic regulation of apoptosis”, A. H. Wyllie, Current Opinion in Genetics and Development, 5: 97-104, 1905.

4

”Apoptosis in the pathogcnesis and treatment of disease”, C. B. Thompson, Science vol 267, pp 1456-1462, March 1995.

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ENHANCED APOPTOSIS IN INSECT CELLS CULTIVATED IN SIMULATED MICROGRAVITY N. COWGER and K. O’CONNOR Tulane University, Chemical Engineering Department, and Molecular & Cellular Biology Program, New Orleans, LA 70118

Abstract

There is a shift from necrosis to apoptosis when batch cultures of Spodoptera frugiperda are grown in the low turbulence environment of NASA’s High-Aspect Rotating-Wall Vessel (HARV) versus a shaker flask. The NASA vessel was developed for the cultivation of animal cells in an environment which simulates microgravity on earth. Fluorescence microscopy with the DNA stains acridine orange and ethidium bromide showed 33% of S. frugiperda cells were apoptotic in the HARV when the viability was

50%, while cells grown in shaker flask had no more than 11% apoptotic cells at the same viability. We believe that the shift to apoptosis in the HARV is primarily the result of reduced hydrodynamic forces. Background

The HARV bioreactor used in our research was developed by NASA in 1990 for the cultivation of animal cells in an environment which simulates the effects of microgravity here on earth. As described in our earlier publications [1,2], the combination of endover-end mixing, bubble-free aeration and solid-body rotation greatly reduces the magnitude of hydrodynamic forces within the HARV relative to conventional bioreactors. Since its introduction, the HARV has mostly been applied as a tool for tissue engineering studies [1]. In addition, our laboratory chose to study the usefulness of the HARV for insect-cell suspension culture. Our work with insect cells was the first application of simulated microgravity to a non-mammalian and a freely suspended cell line. Insect cells were chosen primarily because their reported sensitivity to hydrodynamic forces made them amenable to the quiescent environment of the HARV. Our previous studies [2] have revealed profound differences in the growth profile and metabolism between S. frugiperda cells grown in the HARV compared to those in shaker flask. The shaker was selected as a control vessel since it is one of the most gentle forms of conventional cultivation. Our studies showed less nutrients consumed and less wastes produced in HARV insect-cell cultures. The metabolic differences are evidence for the use of alternate metabolic pathways in the HARV culture. In the quiescent HARV, the 231 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 231-233.

© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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energy required for cell repairs may be redirected to other metabolic processes. Earlier studies [3] also showed changes in morphology and DNA fragmentation indicative of increased apoptosis in the HARV culture. It is only recently that apoptosis has been documented [4] during routine subculturing of S. frugiperda cells with no viral infection present. Methods The cultivation conditions for IE1FB2 cells, a subclone of Sf9 S. frugiperda, were as presented previously [3]. In the fluorescence microscopic protocol [5], cell samples were stained with acridine orange and ethidium bromide and viewed on an Olympus BX60 microscope with UPlanFl objective at 40x magnification.

Results and Discussion Figure 1A shows representative growth curves for S. frugiperda cells. Though maximum cell densities were virtually identical in the two vessels, stationary phase in the HARV was much extended and the death rate constant in the HARV was a factor of 20 to 90 times less than for the control culture [3]. The increased longevity of insect cells in the HARV is not an artifact from experimental conditions, such as feeding interval and cell density, and most likely results from the HARV’s combination of reduced shear and lower accumulation of waste.

Fluorescence microscopy was performed to quantify the subpopulations appearing in the two cultures during death phase. The protocol used relies on the differential uptake of the DNA-binding dyes acridine orange and ethidium bromide. Cells are classified as viable, early apoptotic, late apoptotic, or necrotic based on their staining characteristics and morphology. From Figures 1B and 1C, it can be seen that apoptosis is dominant for the HARV culture, while necrosis is the controlling mechanism for shaker cells. When the viability is 50%, up to 33% of total cells are apoptotic in the HARV. In comparison, cells grown in shaker flask had no more than 11% apoptotic cells at the same viability. Thus, necrotic cells at this stage represented approximately 17% of the population in the HARV and 40% in the shaker.

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In a previous publication [6], we discussed some of the factors which may influence cell death. One pertinent example is that the intensity of a mechanical stress may affect the relative occurrence of apoptosis and necrosis, as shown in a study [7] where animal cells were subjected to different agitation rates in a stirred-tank bioreactor. In our studies, since there was less turbulence in the HARV than in the shaker flask, a higher percentage of apoptosis relative to necrosis may have been favored in the microgravity vessel. Other cultivation conditions have been shown to enhance apoptosis, for example, glutamine, serum, or glucose deprivation, and ammonia toxicity. These do not appear relevant to the HARV culture, particularly since it is characterized by reduced glucose utilization and reduced ammonia accumulation relative to the control culture.

Conclusion

Knowledge of death mechanisms in insect-cell cultures could potentially impact commercial processes by offering a means of control over the culture to improve productivity. During long-term cultivation of Spodoptera frugiperda insect cells in simulated microgravity, there is an apparent shift to apoptosis relative to a shaker control. Our work with HARV cultivation has further shown significant effects on the metabolism, morphology, and growth profiles of insect cells. We believe, first, that these phenomena were primarily the result of reduced hydrodynamic forces in the HARV, and second, that these results would not have been readily achieved with conventional means. Acknowledgments This work was funded by a grant from NASA (NAG 9-826). We are also grateful to Dr. Carol Burdsal and Dr. Kyriakos Popadopoulos for the use of their microscopes. References

1.

2. 3. 4

Clejan. S., O’Connor, K.C., Cowger, N.L., Cheles. M.K.. Haque, S., and Primavera, A. (1996) Effects of

simulated microgravity on DU 145 human prostate carcinoma cells. Biotechnol. Bioeng 50. 587-597 Francis, K.M., O’Connor. K.C., and Spaulding, G.F. (1997) Cultivation of fall armyworm ovary cells in simulated microgravity. In Vitro Cell Dev. Biol. 33, 332-336. Cowger, N.I., O’Connor. K.C., and Bivins. J.E. (1997) Influence of simulated microgravity on the longevity of insect-cell culture. Enzyme Microb. Technol. 20, 326-332. Palli, S.R., Sohi. S.S., Cook, B.J., Primavera. M., and Retnakaran. A. (1997) Screening of 12 continuous cell lines for apoptosis, in K. Maramorosch and J. Mitsuhashi (eds.), Invertebrate Cell Culture: Novel Directions and Biotechnology Applications, Science Publishers, Inc., Enfield, New Hampshire, pp. 5360.

5.

Duke. R.C. and Cohen. J.J. (1992) Quantitation of apoptotic index and cell viability using fluorescent dyes, in J.E. Coligan, A.M. Kruisbeek, D.H. Margulies. E.M. Shevach, and W. Strober (eds.), Current

Protocols in Immunology, Greene Publishing Associates and Wiley-lnterscience, New York. pp. 3.17.13.17.3. 6.

7.

Cowger, N.L. and O’Connor. K.C. (1997) Application of simulated microgravity to insect-cell culture, in K. Maramorosch and J. Mitsuhashi (eds.), Invertebrate Cell Culture: Novel Directions and Biotechnology Applications, Science Publishers, Inc., Enfield, New Hampshire, pp. 131-138. Al-Rubeai, M., Singh. R.P., Goldman. M.H., and Emery, A.N. (1995) Death mechanisms of animal cells in conditions of intensive agitation. Biotechnol Bioeng. 45, 463-472.

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MODULATION OF APOPTOSIS BY BCL-2 EXPRESSION FOLLOWING AMINO ACID DEPRIVATION AND IN HIGH CELL DENSITY PERFUSION CULTURES

R. P. Singh*, D. Fassnacht**, A. Perani*, N. H. Simpson*, C. Goldenzon*, R. Pörtner** and M. Al-Rubeai* * School of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham, B15 2TT, U.K. and **Technical University of HamburgHarburg, Bioprozess und Bioverfahrenstechnik, Dnickestr. 15, D-21071, Hamburg, Germany. Key words: Apoptosis, Hybridoma, Bcl-2, Amino Acids, Perfusion Culture 1. Introduction

A number of studies have now demonstrated that cell death during commercial cell cultures proceeds by the active, genetically determined mechanism called apoptosis, rather than the passive accidental form of cell death known as necrosis (Al-Rubeai et al., 1990; Franek and Dolnekova, 1991; Mercille and Massie, 1994; Singh et al., 1994; Perreault et al., 1994). Suppression of apoptosis significantly improves the robustness of the cells, thus raising culture productivity. However, there are still major gaps in our knowledge in terms of the role of specific components of the culture environment in terms of the induction and suppression of apoptosis. Studies conducted thus far have demonstrated that the deprivation of glutamine, threonine and cysteine results in the induction of high levels of apoptosis (Mercille and Massie, 1994; Perreault and Lemieux, 1994; Singh et al., 1994). However, it is not clear whether this is a feature specific to these particular nutrients, or an effect which is seen following the deprivation of any of the amino acids found in culture medium. In order to answer this question, we have identified the mechanism of cell death following deprivation, individually, of all amino acids. We then went on to investigate the effect of over-expression of the anti-apoptosis gene bcl-2 under these conditions. Finally, the impact of bcl-2 overexpression on antibody productivity in the extremely stressful environment of high cell density perfusion culture systems was studied.

2. Materials and Methods 2.1 CELL LINES AND CELL MAINTENANCE

The cell lines used in this study were TB/C3.bcl-2 and TB/C3.pEF derived from the murine hybridoma TB/C3 which was transfected with the expression vector pEF bcl-2235 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 235-241. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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MC1neopA and the control vector pEF-MC1neopA respectively (see Simpson et al., 1997). Cells were maintained at 37°C in RPMI 1640 medium (Gibco, UK.) supplemented with 5% (v/v) Foetal Calf Serum (FCS) (Sigma, UK.). Bcl-2 expression levels were monitored by immunostaining of the Bcl-2 protein followed by flow cytometric analysis as previously described ( Simpson et al., 1997). The medium used for the fixed bed culture was a 1:1 mixture of Iscove's MDM and Ham's F12 supplemented with 2 mmol 1 -1 L-glutamine, 2 g 1-1 NaHCO3, 0.01% PEG and 50 mmol 1 -1 ethanolamine supplemented with 1% (v/w) of a protein-free iron-rich supplement (IR) containing 20 mmol 1-1 ferric citrate. 2.2 AMINO ACID DEPRIVATION: EXPERIMENTAL PROCEDURE

48 Hours Deprivation. Cells taken from the mid exponential phase of batch cultures were centrifuged for 5 minutes at 1000 rpm. They were washed once in phosphate buffered saline (PBS) and then resuspended in the Select-Amine basal medium with 5% dialysed FCS containing all amino acids except the one under investigation at a target viable cell number of 2xl0 5 /ml in a 15 ml volume. The cells were then dispensed in 5 ml aliquots into 3x5 ml plastic wells on 12 well plates, giving triplicate cultures for each experiment. The Plates were incubated for 48 hours at 37°C in a humidified 5% CO2/air incubator. Prior to analysis, the cells were trypsinized using a trypsin EDTA solution (Sigma UK), centrifuged at 1000 rpm and resuspended in the original 5 ml of culture medium (to ensure that any floating dead or viable cells in the original culture medium were included in the analysis). Levels of apoptosis, necrosis and viability were assessed by fluorescence microscopy as previously described in Simpson et al., 1997. Time Course Study Following Deprivation of Individual Essential Amino Acids. Cells

were prepared as described above at a target cell number of 2xl0 5 /ml in 90 ml of selectamine medium (containing all amino acids except for the one under study). The cells were dispensed in 5 ml aliquots into 18x5 ml wells. At regular intervals, 2 wells were “sacrificed” and cells removed by trypsinization and assessed for viable cell number, viability and levels of apoptosis and necrosis as described above. This allowed for the analysis of the cultures at 9 time points during the course of the culture. 2.3 PERFUSION CULTURES

Fixed-bed Reactor. The fixed bed reactor (Meredos, Germany) used in this study consists of a 100 ml fixed-bed in which the cells are immobilised on porous carriers and a 1 litre conditioning vessel. During operation, the medium was constantly pumped from the conditioning vessel through the fixed-bed and back. Bubble aeration took place in the conditioning vessel to supply the cells with oxygen. The maximum superficial flow velocity in the fixed-bed was kept below 1.1 mm s-1 in order to prevent cells from being washed out of the fixed-bed. The pH was measured and controlled

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automatically by adding CO2 to the air. The medium in the conditioning vessel was agitated using magnetic stirrer and the temperature maintained at 37°C.

Porous glass bead carrier with a diameter of 3 to 5 mm (Siran, Schott, Germany) were used. The fixed bed was inoculated with 1xl06 cells (ml fixed-bed)-1 which corresponds to a cell density of 1xl05 cells (ml suspension)-1. Monoclonal antibody titre was measured by an IgG ELISA as described in Simpson et al., 1997. Hollow Fibre System (Tecnomouse). Hollow fibre studies were carried out using a Tecnomouse bioreactor (Integra Biosciences, UK) with two OxyCell hollow fibre

cassettes, one for each cell line. Temperature was controlled at 37°C and the cultures were aerated with 5%CO2/air. Medium was recirculated through the cassette at a flow

rate of l00ml/hr from a 2 L reservoir bottle. Samples were harvested from the extracapillary space at weekly intervals and assayed for levels of mouse IgG using a sandwich enzyme-linked immunosorbent test as described previously (Simpson et al., 1997). Glucose levels were determined using a Reflolux II® system (Boeringher, Germany) The medium bottle was replaced at regular intervals such that the glucose concentration remained above 3 mM. 3. Results and Discussion 3.1 INDUCTION OF APOPTOSIS BY AMINO ACID DEPRIVATION: INFLUENCE

OF BCL-2 EXPRESSION

The percentage of viable, necrotic and apoptotic cells in the control cell line cultures was determined following 48 hours of deprivation of each amino acid. The majority of cell deaths in each case occurred by apoptosis. However, there were clearly two distinct groups of amino acids. Deprivation of essential amino acids exhibited the greatest loss of viability, with over 70% of all cells being apoptotic. Deprivation of the non-essential amino acids had the least effect on viability , although apoptosis still accounted for most of the dead cells.

The effect of bcl-2 expression on the viability of the culture was also assessed. For most amino acids, the viability of the bcl-2 cultures was greater than 70 % after 48 hours, representing a substantial improvement in viability over control cultures. Time course studies were then conducted to examine the death profile following deprivation of each essential amino acid. As illustrated by the 3 examples given in Figure 1, the cultures could be divided into two phases: an initial high viability phase, followed by a rapid death phase. Both of these phases were significantly extended during the bcl-2 cultures. However, the specific level of protection offered varied from one amino acid to the next, which is clearly illustrated by comparing threonine to

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isoleucine. In the former, the viability fell constantly between 40 and 80 hours, whereas in the latter, there was a significantly lower fall in viability. Development of fed batch culture processes is an iterative process which relies on feeding of exhausted nutrients and is therefore dependent on intensive monitoring of nutrient levels. However, in many cases the exhaustion of a particular amino acid willresult in the induction of apoptosis before feeding has commenced. The use of bcl-2 transfected cell lines should prevent this from happening, thus considerably speeding up the process of feeding strategy development by removing the need to constantly having to re-initiate the culture from the beginning.

3.2 INFLUENCE OF BCL-2 ON ANTIBODY PRODUCTIVITY IN HIGH CELL DENSITY PERFUSION CULTURES

The high cell numbers achieved in perfusion culture systems normally lead to an exacerbation of nutrient and oxygen limitations. Therefore, it is expected that bcl-2 transfected cells would have a significant survival advantage in the relatively stressful

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environment encountered in such systems. In order to investigate this possibility, growth of bcl-2 transfected and control cell lines was studied in fixed bed and hollow fibre perfusion systems. Direct estimation of viability is impossible, although a flourometric technique was applied to the estimation of the free DNA concentration in the culture medium from the fixed bed reactor. This revealed a higher concentration of free DNA in the control cell line culture, indicating a significant level of cell death when compared to the control cell line. The difference in the antibody productivity of the two cell lines in each culture system is shown in Figures 2 and 3. In both systems, the antibody titre in cultures using the bcl-2 transfected cell line was approximately 100% higher than that of the control cell line cultures. This increase must reflect the reduction,, in the bcl-2 transfected cultures, of the amount of perfused medium required to generate replacement cellular biomass. 4. References Al-Rubeai, M., Mills, D., Emery, A. N. (1990) Electron Microscopy of hybridoma cells with special regard to monoclonal antibody production. Cytotechnology. 4:13-28.

Franek, F. and Dolnikova, J. (1991) Nucleosomes occurring in protein-free hybridoma cell cultures. Evidence for programmed cell death. FEBS Letters 248:285-287. Mercille, S. and Massie, B. (1994a). Induction of apoptosis in nutrient-limited cultures of hybridoma cells. Biotechnology and Bioengineering: 44, 1140-1154. Perreault, J. and Lemieux, R. (1994). Essential role of optimal protein-synthesis in preventing the apoptotic death of cultured b-cell hybridomas. Cytotechnology. 13, 99-105. Simpson, N., Milner, A. N. and Al-Rubeai, M. (1997) Prevention of hybridoma cell death by bcl-2 during suboptimal culture conditions. Biotechnology and Bioengineering 54, 1-16. Singh, R. P., Al-Rubeai, M., Gregory, C. D., and Emery, A. N. (1994). Cell death in bioreactors: a role for apoptosis. Biotechnology and Bioengineering 44, 720-726.

5. Acknowledgements

This work is supported by the EC Framework IV Programme

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Discussion

Massie:

What is the stability of the clones expressing bcl-2, not only in terms of the expression of the genes but also the phenotype following passaging?

Singh:

We have passaged the cells for 6 months and found very high levels of stability in bcl-2 expression, even in the absence of geneticin. The same is true for antibody productivity.

Massie:

I assume that there is no selection pressure in the perfusion system?

Singh:

No.

Massie:

A question concerning the relative amounts of apoptotic cells in batch culture in the bcl-2 transfectant: do you still see cells dying by apoptosis in a typical batch culture?

Singh:

Yes, we do see the classical apoptosis morphology but at a lower level. This may reflect the rate of apoptotic death and progression of cells into a secondary necrotic state, so it becomes more difficult to clearly identify them morphologically as apoptotic.

Massie:

Do you think that there is a relationship between the level of bcl-2 and protection? Can you saturate the dominant effect of protection which bcl-2 confers on hybridoma cells?

Singh:

I think that you probably can but we have not reached that point with these cells. Clearly, in the amino acid deprivation experiments, the cells are still dying of apoptosis.

Ryll:

You found that cells over-expressing bcl-2 have reduced amino acid uptake rates. Is that something one would expect? Did you do this with just on clone that you selected, or is it a population? If it was a clone, you could have selected for reduced amino acid uptake by chance.

Singh:

There are mixed populations, not clones, for that precise reason. The cells are under starvation conditions, so one would expect the

cells to adapt by down-regulating metabolic activities. This is evidenced by the utilisation rates.

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EFFECT OF BCL-2 EXPRESSION ON HYBRIDOMA CELL GROWTH DURING STRESSFUL CONDITIONS D. FASSNACHT1, S. ROSSING1, F. M. AL-RUBEAI3, R. PORTNER1 1 Technische Universitat Hamburg-Harburg, und Bioverfahrenstechnik, D-21071 Hamburg, Germany 2 Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, CZ-14220 Praha 4, Czech Republic 3 Centre for Biochemical Engineering, School of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK

1. Abstract Two transfected hybridoma cell lines TB/C3-bcl2, (overexpressing the Bcl-2 protein) and TB/C3-pEF (control cell line), were compared to assess the potential of Bcl-2 in preventing apoptosis. The conditions examined were batch cultures, growth in diluted media, and long-term fermentations in a high cell density fixed-bed reactor. The membrane intact index (percentage of cells with intact membranes determined by trypan blue staining) of the TB/C3-bcl2 cell line decreased much slower than that of the control cell line during the dying phase of the batch cultures. No significant difference in the consumption (glucose, glutamine) and production rates (lactate, ammonia, antibodies) was notable in the exponential phase of the experiments. When comparing the cell lines in diluted media the advantage of the Bcl-2 overexpressing cell line was again a higher membrane intact index at increasing dilution steps. Comparing both cell lines in a fixed-bed reactor at various dilution rates revealed a significant difference in antibody production. TB/C3-bcl2 produced twice as many antibodies as the control cell line for all steady-states. But no difference in the consumption (glucose, glutamine) and production (lactate, ammonia) rates was observable. 2. Cell Line and Culture Conditions Both mouse hybridoma cell lines produce an IgG antibody against an antigenic determinant in the hapten domain in the Fc region of human IgG. These cell lines were transfected with the Bcl-2 (pEF bcl2-MClneopA) and control (pEF-MClneopA) plasmids by Simson et al. (1997). 243 O.-W. Merten et al. (eds.). New Developments and New Applications in Animal Cell Technology, 243-245. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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For growth, a 1:1 mixture of Iscove’s MDM and Ham’s F12 supplemented with L-glutamine, 0.01% PEG and ethanolamine was used as basal medium. This basal medium was either supplemented with 1% (v/w) of a protein-free iron-rich supplement (IR) containing ferric citrate and Dolníková, 1991) or 3% (v/w) horse serum (HS). The total amount of glutamine in both media was and for glucose. For the batch experiments, cultivation took place in stationary flasks. The flasks were inoculated with exponentially growing cells at an initial cell density of Growth curves were obtained by removing a doublet of flasks each day for counting and analyses. A fixed-bed reactor (meredos, Germany) was used for continuous fermentation. The reactor consists of a 100 ml fixed-bed in which the cells are immobilised on porous carriers and a 1 litre conditioning vessel. The carrier used was Siran (Schott, Germany) which proved especially suitable for hybridoma cells (Pörtner et al., 1997). pH was measured and controlled automatically by adding CO2 to the air. The medium of the conditioning vessel was heated to 37 °C by an electrical heater. No separate heating was required for the fixed-bed because it was integrated in the conditioning vessel. This also assured a convenient handling of the whole system.

3. Results 3.1. BATCH EXPERIMENTS The main aspect of comparing the two cell lines in batch cultures was to verify the effect of Bcl-2 during ideal growth conditions (exponential phase) as well as in sub-optimal conditions (death phase). These experiments were performed with two different medium supplements: 3% HS and 1% IR to clarify if the medium supplement has an effect on the behaviour of the cell lines. The two cell lines grew in both media to the same maximal cell density of approx. (Fig. 1). In the HS medium, Bcl-2 seemed to prolong the exponential phase and to delay the dying of the cells for 48 hours compared to the TB/C3-pEF cell line. The growth of both cell lines in the IR medium did not only show the delay of the death phase but also a significant reduction in the specific death rate for the TB/C3-bcl2 cell line. Fifty percent of the TB/C3-bcl2 cells in

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the IR medium still had intact membranes after 9 days of cultivation whereas no viability was detected in all other experiments after this time. There was no significant difference in glucose and glutamine consumption as well as lactate and ammonia production for the exponential phases of the experiments. This was also true for the antibody titre, which reached a concentration of approx. 50 mg l-1 in all experiments. 3.2. GROWTH IN DILUTED MEDIA These experiments were carried out in diluted media in order to compare the two cell lines under nutrient limitation. Both hybridoma cell lines reached a viable cell density of 6.105 to 7.105 cells ml-1 in the undiluted IR medium after 72 hours. The membrane intact index was close to 90%. The reduction of the content of nutrients, achieved by medium dilution, resulted both in a lower relative viable cell density and in a lower membrane intact index. The TB/C3-bcl2 cell line displayed higher resistance to the starvation stress. At the verge of starvation-induced death, i.e. in 40% to 50% medium, the membrane intact index clearly documented the superiority of the TB/C3-bcl2 cell line. Here the membrane intact index was approx. 15% higher than that of the control line. 3.3 FIXED-BED REACTOR Both cell lines were also compared in the fixed-bed bioreactor at various dilution rates. Comparing the results of the cell lines revealed a significant difference in antibody

production. TB/C3-bcl2 produced twice as many antibodies as the control cell line for all steady-states. But no difference in the consumption (glucose, glutamine) and production (lactate, ammonia) rates was observable. For details of the results, please refer to R. P. Singh et. al ‘Manipulation of apoptosis by Bcl-2: Enhancement in survivability after amino acid deprivation and in intensive culture systems’. 4. Conclusions

The overexpression of Bcl-2 did not have an influence on the metabolism of cells growing under ideal conditions (exponential phase of batch cultures). But the Bcl-2 overexpressing cell line survived much better when induced to stressful conditions such as the late exponential phase in batch experiments, growth in saline diluted media or during continuous fermentations in fixed-bed reactors at low dilution rates. References F., and Dolníková, J, 1991. Hybridoma growth and monoclonal antibody production in iron-rich protein-free medium: Effect of nutrient concentration. Cytotechnology 7: 33-38 Pörtner, R., Rössing, S., Koop, M., Lüdemann, I. 1997. Kinetic studies on hybridoma cells immobilized in fixed bed reactors. Biotechnology and Bioengineering 55: 535-541 Simpson, N. H., Milner, A. E., Al-Rubeai, M. 1997. Prevention of hybridoma cell death by Bcl-2 during sub-optimal culture conditions. Biotechnology and Bioengineering 54: 1-16

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INHIBITION OF c-jun EXPRESSION IN F-MEL CELLS CAUSES CELL CYCLE ARREST AND PREVENTION OF APOPTOSIS

YON HUI KIM 1 , TAKEHIKO IIDA 1 , EDWARD V. PROCHOWNIK 2 , EIJI SUZUKI 1

1. Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

2. Division of Pediatric Hematology/Oncology, Children's Hospital of Pittsburgh, Pittsburg, PA 15213-2583, USA ABSTRACT

We are able to provide an evidence that will support the suppression of cell proliferation to withdrawal from the cell cycle and suppression of onset apoptosis with conditional expression of c-jun antisense transcript in F-MEL cells. F-MEL cells were transfected with c-jun antisense gene located downstream of a glucocorticoid-inducible MMTV promoter, and named as c-jun AS cells. By treating the c-jun AS cells with dexamethasone (DEX) in DMEM supplemented with 10% serum, the growth could be suppressed for duration of 16 days with high cell viability of 92%. When DEX-treated c-jun AS cells were serum deprived, the cell viability remained at high of 86% for upto 10 days, the onset of apoptosis was suppressed, and the internucleosomal cleavage of DNA was not detected upto 8 days. In contrast, when wild type F-MEL cells were serum deprived, the cell viability is low (50%.) and the onset of apoptosis is induced within 2 days, and internucleosomal cleavage of DNA was detectable.

1. INTRODUCTION Biologically active proteins such as cytokines, vaccines, and antibodies are produced by mammalian cell culture. The protein production of such cultures usually increases with increase in cell integration, the viable cell number integrated with respect to culture time. Viable cells in the late logarithmic growth phase and the following non-growth viable phase are major contributors to cell integration. Cells often produce protein at a rate higher in the non-growth viable phase than in the logarithmic growth phase (Suzuki and Ollis, 1990). However, cells tend to die quickly after reaching the maximum cell density due to adverse conditions caused by over-growth. Hence the viable non-growth culture period may be as short as only two to three days (Duval et al., 1990; Vomastek and Franek, 1993). Therefore, preventing cell death which starts in the late logarithmic growth phase (Vomastek and Franek, 1993) and maintaining them viable in batch culture for longer time

period should increase protein production of the culture. The cells die due to depletion of nutrients such as amino acids (Franek and Chladkova-Sramkova, 1995) and glucose (Marcille and Massie, 1994; Singh et al., 1994), limitation of growth factors such as serum components(Singh et al., 1994), or accumulation of toxic metabolites (Glacken et al., 1988; Tohyama et al., 1990). The over-growth inevitably leads to some of these cell death inducing conditions. Cell death may follow two distinct patterns: necrosis and apoptosis. Apoptotic cell death follows a well-defined sequence of events characterized by cell shrinkage, nuclear 247 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 247-254. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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condensation, and fragmentation of the cell into discrete membrane enclosed bodies. In the early apoptotic death process, an endogenous endonuclease is activated and cleaves DNA within the internucleosomal spacer regions. Hence, a characteristic ladder pattern consisting of multiplies of 180 bp fragments is seen when the DNA from apoptotic cells is electrophoresed on an agarose gel. Reportedly, the cell death in the late logarithmic growth and stationary phases of batch culture is mostly apoptosis (Mercille and Massie, 1994). Proto-oncogene c-jun known as the immediate early response' gene is rapidly induced in response to mitogenic stimuli, and it plays a critical regulatory role in the commitment of the cell to proliferate (Ryseck et al., 1988, Smith and Prochownik, 1992). A nuclear phosphoprotein c-Jun is a major component of the AP-1 trans-acting transcriptional activator complex that promotes transcription of various genes required for progression of the cell cycle (Turner and Tjlan, 1989). The selective inhibition of c-jun expression caused exit from the cell cycle, in other words, it blocked cell growth (Smith and Prochownik, 1992; Soprano et al., 1992). The possibility of c-Jun being the inducer for natural cell death, apoptosis, was reported (Colotta et al., 1992; Estus et al., 1994; Rampalli and Zelenka, 1995; Anderson et al., 1995). Interleukin (IL)-6- and lL-2-dependent mouse cell lines underwent programmed cell death alter the growth factor deprivation; in this death process, c-jun gene was rapidly induced; and the antisense oligonucleotides directed against c-jun reduced this c-jun induction, while improving survival of the cells after the growth factor deprivation (Colotta et al., 1992). The antisense oligonucleotides for c-jun prevented HL-60 cells from apoptosis induced by ceramide (Sawai et al., 1995). Using this understanding that the c-jun gene plays an important role in the progression of the cell cycle and induction of apoptosis or programmed cell death. The objective of the present work is to establish a cell line in which growth can be switched on and off without induction of apoptosis. And we were successful in creating a cell line c-jun AS that can be induced by glucocorticoid hormone to block cell proliferation and at the same time suppress apoptosis even under growth factor deprivation. This growth and apoptosis controllable cell line can be utilized to keep high viability in batch, fed-batch, or perfusion culture. The cell line will be also suitable to culture for which use of any serum components is prohibited. 2. MATERIALS AND METHODS Cell Line and Cell Culture Conditions Friend murine erythrolcukemia (F-MEL) cells were supplied by Riken Cell Bank. FMEL cells and all the cells derived from F-MEL cells in the present work were cultured in Dulbecco's modified Eagle's medium (DMEM) (Nissui, Tokyo), supplemented with 10% fetal bovine serum (v/v) (JRH, Biosciences), 4 mM glutamine, 100 mg/ml kanamycin, 0.2% NaHCO3 (complete medium), unless otherwise specified. The cells were maintained in the logarithmic phase of growth in 25-cm2 plastic T-culture flasks (Sumitomo Bakelite. Tokyo) at 37°C in a 5% CO2-95% air humidified incubator. For the induction of the transfected sense or antisense c-jun transcripts, cells were cultured in the complete or serum deprived medium supplemented with dexamethasone (Sigma) at a concentration of 1x10 -6 M. Viable cell count was performed by the trypan blue dye exclusion method employing a hemocytometer.

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Transfection, S e l e c t i o n , and Gene A m p l i f i c a t i o n pMS-G c-jun AS dhfr vector contains an antisense fragment of c-jun gene downstream of a glucocorticoid-inducible murine mammary tumor virus (MMTV) promoter and a dhfr (dihydrofolate rcductase) gene. The antisense c-jun gene is the inversely inserted sequence that consists of 287 bp of 48 bp of coding sequence derived from the extreme end of the coding region, and 929 bp of c-jun cDNA. pMS-G c-jun dhfr vector contains a complete sense c-jun cDNA instead of the antisense cjun gene (Smith and Prochownik, 1992). For transfections, 2 x 1 0 6 F-MEL cells were pelleted at 500 g for 10 min, washed twice in phosphate-buffered saline (PBS), and resuspended in 200 ml of PBS. 20 mg of BamHI-linearized pMS-G c-jun AS dhfr DNA or pMS-G c-jun dhfr DNA plus 2 mg of NdeI-linearized pSV2neo plasmid DNA in a total volume of 50 ml of sterile water were added to the cell suspension. Transfection was accomplished by electroporation with a homemade apparatus at settings of 650 voltage with 10 pulses at resultant time of 250 msec. The cells were grown for 24 h in the complete medium, and then G-418 was added to a final concentration of 0.4 mg/ml. G-418 resistant clones were detected within 4 to 8 days. Pooled G-418 resistant clones were then cultured in the complete medium containing methotrexate (MTX) at a final concentration of 50 nM for amplifying the sense or antisense c-jun gene together with the dhfr gene for several months. For cloning the cell , the dilution plating techniques were applied using 96-weIl plates. Detection of c-Jun Protein by Western B l o t t i n g

To assess the effect of c-jun antisense transcript induction on c-jun protein levels, the c-jun AS cells were cultured in the complete medium containing 10 -6 M dexamethasone for 48 h. The 5x10 5 cells were harvested, pelleted at 2500 g for 10 min, washed twice in phosphate-buffered saline (PBS) and resuspended in lysis buffer (1% Triton x-100, 0.15 mM NaCl, 10 mM Tris, pH 7.8) at 4°C for 30 min. The cell lysates were boiled in sodium dodecyl sulfate (SDS) sample buffer for 5 min, and electrophoresed on a 10% SDSpolyacrylamide gel, and then electroblotted onto nitrocellulose filter overnight. The blotted nitrocellulose filter was blocked with 0.5% skim milk in TBST for 3 h at room temperature. At size of 39-kDa, c-jun protein was detected by rabbit polyclonal antibodies to c-jun /AP-1 protein (Medac, Diagnostika) to which peroxidase-conjugated goat antirabbit Ig polyclonal antibody (Bio Source International, Inc. Tago Products) bound. The cJun-specific band was then visualized by use of the ECL Western blotting system (Amersham, Life Science). F-MEL cells and the c-jun AS cells were cultured free from DEX, and treated in the same manner for Western blotting. DNA Fragmentation Assay by Electrophoresis 1x106 cells were pelleted and lysed by vortexing vigorously in 0.5 ml of TE buffer (10

mM Tris-HCl, pH 7.4 and 1 mM EDTA) containing 0.2% Triton X-100, and centrifuged for 10 min at 13,000g at 4°C. DNA was extracted after addition of 0.1 ml of 5 M NaCl and 0.7 ml of isopropanol. The supernatant was vortexed, placed at -20°C overnight, and then centrifuged for 10 min at 13,000 g at 4 °C. The DNA pellet was dissolved in TE buffer and treated with RNase prior to electrophoresis on a 2% agarose gel. To visualize the fragmented DNA bands, the gel was stained with 1 mg of ethidium bromide per ml.

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3. RESULTS and DISCUSSION Blocking Cell-Growth by Antisense c-Jun Expression F-MEL cells were transfected with the linearized pMS-G c-jun AS dhfr vector carrying the antisense c-jun gene as described in Materials and Methods. The vector contains a glucocorticoid-inducible MMTV promoter and dhfr gene. The dhfr gene was applied for amplifying the vector by culturing cells with methotrexate (MTX), an inhibitor of dihydrofolate reductase. MMTV promoter was used for turning on or off the transcription of the antisense gene depending on the presence or absence of a synthetic glucocorticoid, dexamethasone (DEX). The transfected cells were selected with G418, and then cultured in MTX for amplifying the antisense gene (generally 50 to 200 copies/cell) (Prochownik and Kubowska,1986). The obtained cell line was named c-jun AS. First we assessed the effect of c-jun antisense transcript induction on c-jun protein levels. We exposed the c-jun AS cells to 10-6 M DEX for 48 h. The cells were harvested and subjected to SDS-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose. Control lysates were prepared from the wild type F-MEL cells. Rabbit polyclonal antibodies to c-Jun and AP-1 proteins was used to detect c-Jun. Next we examined whether we can block the growth of the c-jun AS cells and maintain the cells in

viable state. The c-jun AS cells in logarithmic growth phase in the complete medium were collected and resuspended in 10 ml of the fresh complete medium at day 0 in each of 25 cm 2 T-flasks. For inducing the antisense c-jun to block the cell growth, DEX was added to the medium at 10-6 M on day 0. The viable cell density and viability are shown in Fig. 1-a and -b, respectively. For comparison, we cultured in the same manner the wild type F-MEL cells and those transfected with the complete sense c-jun gene. The growth of the c-jun AS cells was almost blocked two days after DEX addition. After that the viable cell density was kept almost unchanged for 14 days. A viability greater than 86% was maintained for 16 days. In the absence of DEX, the c-jun AS cells grew to the over-growth condition and then started dying. The wild type F-MEL cells also grew to over-growth , and then started dying either in the presence or absence of DEX. Jointly these results indicated that the c-jun AS cell line was a desired cell line, that is, a viably growtharrestable cell line. Suppressing Apoptosis by Antisense c-Jun Expression

At least two groups reported that antisense oligonucleotides directed for c-jun gene suppressed apoptosis (Colotta et al., 1992; Sawai et al., 1995). This suggested that the cjun AS cell line is possibly free from apoptosis when cultured in the presence of DEX. Use of oligonucleotides is limited to research purpose, because they are expensive and not stable in culture. Contrarily, the antisense c-jun transcripts in the c-jun AS cells can be induced with relatively inexpensive DEX when necessary. Therefore, we examined the

apoptosis resistance of the c-jun AS cells by applying serum-deprivation, one of typical apoptosis inducing conditions. The logarithmically growing c-jun AS cells in the complete medium were collected, and washed three time in PBS. Then the cells were placed in the serum-deprived medium, and cultured in the presence or absence of DEX. The wild type FMEL cells were also subjected to the same procedure. The viable cell density and viability are shown in Fig.2-a and -b, respectively. Under serum-deprivation, the viability of the

251

wild type F-MEL cells decreased to 50% in 3 days, while that of the c - j u n AS cells was maintained above 80% for 8 days. In the absence of DEX, the c-jun AS cells, of which c-jun expression was partially

suppressed by the antisense c-jun expression due to the leakiness of MMTV promoter, survived and grew well under serum-deprivation (Fig.2-a and -b). This meant that we could unexpectedly establish a F-MEL cell line that can grow without any serum component. We interpreted this result as c-jun expression in the c-jun AS cells cultured in the absence of DEX was high enough to drive cell cycling and too low to induce apoptosis. We examined emergence of the ladder pattern, a land mark of apoptotic cells, on electrophoreses of DNA extracted from the cells of the above experiments. The c-jun AS cells cultured with DEX under serum-deprivation were collected on day 4, 6, 8, 10, and 12.

DNA samples extracted from the cells were treated as described in Materials and Methods for detecting DNA fragmentation. The wild type F-MEL cells cultured under the serumdeprivation were similarly examined. The clear DNA ladder was detected for the wild type F-MEL cells on day 4, but not for the c-jun AS cells treated with DEX (Fig.3). Jointly with the result of Western blotting for detection of c-jun protein, this result indicated that suppression of c-jun expression inhibits apoptosis. A cell line c-jun AS that can be reversibly and viably growth-arrested was established by transfecting F-MEL cells with inducible antisense c-jun gene. The cell line is resistant to apoptosis induced by scrum-deprivation , or by cell cycle arrest. The c-jun AS cells can grow even under serum-deprivation when cultured free from dexamethasone, while the wild type F-MEL cells die quick. Jointly with several other reports on the relation between apoptosis and c-jun expression, our result strongly suggested that c-jun expression is required for onset of at least some types of apoptosis. References Anderson, A. J., Pike, C. J. and Cotman, C. W. 1995. Differential induction of immediate early

gene proteins in cultured neurons by b-amyloid (Ab): association of c-Jun with Ab - induced apoptosis. J. Neurochem. 65: 1487-1498. Colotta, F., Polentarutti, N., Sironi, M. and Mantovani, A. 1992. Expression and involvement of c-fos and c-jun protooncogenes in programmed cell death induced by growth factor deprivation in lymphoid cell lines. J. Biol. Chem. 267: I8278-18283. Duval, D., Demangel, C., Geahel, I., Blondeau, K. and Marcadcd, A. 1990. Comparison of various methods for monitoring hybridoma cell proliferation. J. Immunol. Meth.

134: 177-185. Estus, S., Zaks, W. J., Freeman, R. S., Gruda, M., Bravo, R., and Johnson, E. M. Jr. 1994.

Altered gene expression in neurons during programmed cell death: identification of c-jun as necessary for neuronal apoptosis. J. Cell Biol. 127: 1717-1727. Franek, F, and Chladkova-Sramkova, K. (1995) Apoptosis and nutrition: Involvement of amino acid transport system in repression of hybridoma cell death. Cytotechnology 18: 113-117.

Glacken, M. W., Adema, E, and Sinskey, A. J. 1988. Mathematical descriptions of hybridoma culture kinetics: I. initial metabolic rates. Biotechnol. Bioeng. 32:491-506. Mercille, S. and Massie, B. 1994. Induction of apoptosis in nutrient-deprived cultures of

hybridoma and myeloma ells. Biotechnol. Bioeng. 44: 1140-1154. Rampalli, A. M. and Zelenka, P. S. 1995. Insulin regulates expression of c-fos and c-jun and

suppresses apoptosis of lens epithelial cells. Cell Growth Differ. 6: 945-953. Ryseck. R. P., Hirai, S. I., Yaniv, M. and Bravo, R. 1988. Transcriptional activation of c-jun

during the G0/G1 transition in mouse fibroblasts. Nature 334: 535-537. Sawai, H., Okazaki, T'., Yamamoto, H., Okano, H., Takeda, Y., Tashima, M., Sawada, H.,

Okuma, M., Ishikura, H., Umehara, H., and Domae, N. 1995. Requirement of AP-l for ceramide-induced apoptosis in human leukemia HL-60 cells. J. Biol. Chem. 270: 27326-

27331.

252 Singh, R. P., Al-Rubeai, M., Gregory, C. D., and Emery, A. N. 1994. Cell death in bioreactors: a role for apoptosis. Biotechnol. Bioeng. 44 : 720-726. S m i t h , M. J. and Prochownik, E. V. 1992. I n h i b i t i o n of c-jun causes reversible p r o l i f e r a t i v e

arrest and withdrawal from the cell cycle. Blood 79: 2107-2115. Soprano, K. J., Cosenza, S. C., Yumet, G., and Soprano, D. R. 1992. Use of antisense oligomers to study the role of c-jun in G1 progression. Ann. N. Y. Acad. Sci. 6 6 0 : 231 -239. Suzuki, E. and Ollis, D. F. 1990. Enhanced antibody production at slowed growth rates: experimental demonstration and a simple structured model. Biotechnol. Prog. 6 : 231 -236. Tohyama, N., Karasuyama, H. and Tada, T. 1990. Growth autonomy and t u m o r i g e n i c i t y of i n t e r l e u k i n - 6 - d e p e n d e n t B cells transfected w i t h i n t e r l e u k i n - 6 cDNA. J. Exp. Med. 1 7 1 : 389400.

Turner. R. and Tjlan, R. 1989. Lcucine repeats and an adjacent DNA b i n d i n g domain mediate the formation of f u n c t i o n a l cFos-cJun heterodimers. Science 2 4 3 : 1689-1694

Vomastek, T. and Franek, F. 1993. Kinetics of development of spontaneous apoptosis in B cell hybridoma cultures. Immunol. Lett. 35 : 19-24.

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254

Discussion Handa-Corrigan:

Dexamethasone is a gluco-corticoid and most clinicians look at this molecule as giving cells the ‘kiss of life’. The change in the biochemistry of the cell is quite acute as the cell will start producing glucose, rather than utilising it, and revert to using amino acids and fatty acids instead of glucose. So when we add dexamethasone to cells we find that there is a slowing of the growth rate and they live slightly longer. In your study, what happened to the parent cell line in the presence of dexamethasone?

Kim:

The F-MEL cells were not affected at all by dexamethasone in any respect.

Hauser:

Can you use the system with cells that have endogenous dexamethasone receptors? This would be a problem for changing to other cells. You would probably have to change to another inducible system. Also, have you tried to get a stable expression of your growth anti-sense regulator, and can your cells sustain stability with this type of regulator?

Kim:

It is a problem in that sometimes dexamethasone does induce apoptosis in some cell lines. So there is a limit to this model. A year ago I did have a stability problem but, as I selected more and more, I now have very strong expression even without dexamethasone. So I believe that it is very strong, once selected.

Hatzfeld:

Did you try adding for short cultures oligonucleotide anti-sense?

Kim:

No, I have not tried oligonucleotides.

HIPPOCAMPAL CELLS

IN

CULTURE AS A MODEL TO

STUDY

NEURONAL APOPTOSIS

I. FIGIEL, J. JAWORSKI and L. KACZMAREK Nencki Institute of Experimental Biology Pasteura 3, Warsaw 02-093, Poland

1. Abstract

Glutamate toxicity (excitotoxicity) has been well documented and proposed to play a role in a broad spectrum of neurological diseases. There is a growing interest in defining glutamate-mediated neurotoxic mechanisms, but the high degree of complexity of the intact nervous system hampers a detailed analysis. Thus, cell cultures may provide a useful alternative. We have applied a culture of hippocampal granule cells of postnatal rats to study glutamate-evoked toxicity. These cells are known to be particularly resistant to excitotoxic insult in the brain in situ. However, we have found that granule neurons grown in culture die when they are exposed to high concentrations of glutamate. Moreover, the dying cells display morphological and biochemical features characteristic of apoptosis, a mode of cell death typical of physiological neuronal elimination during development. To further investigate molecular correlates of this phenomenon we have developed an improved calcium phoshate coprecipitation procedure to transiently introduce DNA into cultured neurons. Using this technique we have been able to investigate direct involvement of particular transcription factors in the regulation of glutamate excitotoxicity. The results of our study may be helpful to understand the molecular mechanisms of neuronal apoptosis.

2. Introduction

Glutamate is the major excitatory neurotransmitter, that, in addition to interneuronal communication, can also be neurotoxic. In the past several years there have been many studies on isolated neuronal populations treated with glutamate, which have lead to the hypothesis that toxicity is caused by an influx of calcium through receptor-controlled ion channels, which in turn triggers a cascade of events leading to neuronal cell death (Choi and Rothman, 1990; Randall and Thayer, 1992). However, details of the complex process are still uknown. Recently it has been suggested that excitotoxicity may have an apoptotic character, i.e., may belong to programmed cell death phenomena (for review see: Dragunow and Preston, 1995). 255

O.-W. Merten et al. (eds.). New Developments and New Applications in Animal Cell Technology, 255-258. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

256

Since apoptosis is supposed to be an active process, it is believed that it should involve gene expression. Regulation of gene expression is brought about by transcription factors. Activation of glutamate receptors causes an increase in expression of AP-1 (activator protein-1) transcription factor (Morgan and Curran, 1991; Kaczmarek 1994; Kaminska et al., 1994). Major components of AP-1 are Fos (cFos, Fos B, Fra-1, Fra-2) and Jun (c-Jun, Jun B, Jun D) proteins. Possible role of AP-1, and more specifically c-Fos and c-Jun proteins, in active cell death has been initially supported by results of experiments carried out on non-neuronal cells (Colotta et aI., 1992). In the studies on the central nervous system, Smeyne et al. (1993) using c-foslacZ transgenic mice expressing β-galactosidase under control of c-fos regulatory element, observed that c-Fos expression is associated with apoptotic cell death following kainate injection. Dragunow et al. (1993), on the other hand, reported a prolonged increased in c-Jun immunoreactivity in a model of status epilepticus coupled to neurodegeneration. Recently, Estus et al. (1994) and Ham et al. (1995) have demonstrated that c-Jun is induced in sympathetic neurons undergoing programmed cell death after NGF withdrawal, and moreover, blocking of AP-1 activity with either microinjection of specific antibodies or introduction into the cells of dominant negative mutant of c-Jun prevented neuronal death, whereas overexpression of c-Jun promoted the neuronal death. Hence, it is expected that identification of genes as well as their regulatory mechanisms involved in control of apoptosis should be of great value for both understanding the process and development of therapeutic strategies. 3. Experimental approach and results

In our studies we have employed cultures of hippocampal dentate gyrus, a brain area known to be rather resistant to excitotoxic insult. Dentate granule neurons were

obtained from 5-day-old rat pups and cultured as previously described (Figiel and Kaczmarek, 1997). Cells grown for 6 days in vitro were exposed to glutamate. Unexpectedly, in our culture conditions, we have observed an extensive neuronal loss after treating the cultures with 0.5 mM glutamate. Generally, we noted somal rounding, thining and fragmentation of dendrites. In some cases cell body became enlarged, the nucleus vacuolated, and eventually the cell body detached from the substrate. We have used the specific DNA stain, Hoechst 33258, in order to assess changes of nuclear structure following exposure to glutamate. Several features indicative of apoptosis, such as the condensation of the nuclear chromatin and fragmentation of the nucleus, were observed (Fig. 1) These apoptotic-like morphological changes were acompanied by DNA fragmentation, as revealed by the method of TdT-mediated dUTP-digoxigenin 3’ end labeling (TUNEL) (Gavrieli et al., 1992).

We have also employed immunocytochemistry to investigate expression of c-Fos and c-Jun, two AP-1 transcription factor components, in the control and glutamatetreated cells. We found that following glutamate treatment the level of c-Fos protein was significantly increased in comparison to control (untreated) cultures. However the pattern of this immunoreactivity was almost identical, regadless of the concentration of

glutamate. Moreover, there was much higher number of neurons expressing c-Fos, than the number of dying cells identified with an aid of the aforementioned approaches. On the contrary, c-Jun expression was found only in dying cells.

257

To test the hypothesis that c-Jun contributes to the induction of neuronal apoptosis, we employed calcium phosphate transfection developed by M.E. Greenberg (Xia et al., 1995) with modifications. PS65T-C1 plasmid (Clontech, GenBank accession #U36202) encoding red shifed variant of GFP under control of CMV promoter was used for optimalization of transfection. Preliminary results are shown in Fig.2.

4. Conclusions

In conclusion, we have demonstrated that excitotoxicity produced by glutamate in cultured hippocampal dentate neurons induces cell death by an apoptotic mechanism. Results of our studies based on transient expression of c-jun dominant negative mutant may show the correlation between cell death and c-Jun induction.

5. References Choi, D. and Rothman, S.M. (1990) The role of glutamate neurotoxicity in hypoxic-ischaemic neuronal death Annu. Rev. Neurosci. 13, 171-182. Colotta, F., Polentarutti, N., Sironi, M., and Mantovani, A. (1992) Expression and involvement of c-fos and c-jun protooncogenes in programmed cell death induced by growth factor deprivation in lymphoid cell lines, J. Biol. Chem. 267, 18278-18283. Dragunow, M. and Preston, K. (1995) The role of inducible transcription factors in apoptotic nerve cell death, Brain Res. Rev. 21, 1-28. Dragunow, M., Young, D., Hughes, D., Mac Gibbon, G., Lawlor, P., Singleton, K., Sirimanne, E., Beilharz, E., and Gluckan, P. (1993) Is c-Jun involved in nerve cell death following status epilepticus and

258 hypoxic-ischaemia brain injury? Mol. Brain Res. 18, 347-352. Figiel, I. and Kaczmarek, L. (1997) Cellular and molecular correlates of glutamate-evoked neuronal programmed cell death in the in vitro cultures of rat hippocampal dentate gyrus, Neurochem. Int. 31, 229-240. Gavrieli, Y., Sherman, Y., and Ben-Sasson, S.A. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation, J. CellBiol. 119, 493-501. Ham, J., Babij, C., Whitfield, J., Pfarr, C.M., Lallemand, D., Yaniv, M., and Rubin, L.L. (1995) A c-Jun dominant negative mutant protects sympathetic neurons against programmed cell death, Neuron 14, 927-939. Kaczmarek, L. (1994) Glutamate-evoked gene expression in brain cells - Focus on transcription factors,

Amino Acids 7, 245-254.

Kaminska, B., Filipkowski, R.K., Zurkowska, G., Lason, W., Przewlocki, R., and Kaczmarek, L. (1994) Dynamic changes in composition of the AP-1 transcription factor DNA dinding activity in rat brain following kainate induced seizures and cell death, Eur. J. Neurosci. 6, 1558-1566. Morgan, J.I. and Curran, T. (1991) Stimulus-transcription coupling in the nervous system: involvement of the inducible protooncogenes fos and jun, Annu. Rev., Neurosci. 14, 421-451. Randall, R.D. and Thayer, S.A. (1992) Glutamate-induced calcium transient triggers delayed calcium overload and neurotoxicity in rat hippocampal neurons, J. Neurosci. 12, 1882-1895. Smeyne, R.S., Vendrell, M., Hayward, M., Baker, S.J., Miao, G.G., Schilling, K., Robertson, L.M., Curran, T., and Morgan, J.I. (1993) Continuous c-fos expression precedes programmed cell death in vivo, Nature 363, 13-23. Xia, Z., Dickens, M., Raingeaud, J., Davis, R.J., and Greenberg, M.E. (1995) Opposing effects of ERK and JNKp38 MAP kinases on apoptosis, Science 270, 1326-1331.

DEVELOPMENT OF CARBOXY SNARF-1-AM AND ANNEXIN V ASSAYS FOR THE DETERMINATION OF APOPTOSIS IN HETEROGENEOUS CULTURES

A. Ishaque & M. Al-Rubeai Centre for Bioprocess Engineering, School of Chemical Engineering, University ofBirmingham, Edgbaston, Birmingham, U.K.

1. Abstract Accurate identification and quantitation of apoptosis is essential for developing efficient strategies for optimisation of culture survivability and productivity. Flow cytometry in conjunction with several fluoroprobes is increasingly used to identify apoptotic cells. We have examined the possibility of using carboxy SNARF-1-AM, a pH sensitive fluoroprobe and FITClabelled annexin V, a probe specific to phosphatidylserine exposed on the outer surface of apoptotic cells. Intracellular acidification was shown to precede the occurrence of apoptosis thereby proving to be an early indicator of cellular deterioration and cell death. Annexin V in combination with propidium iodide enabled identification of viable, transient apoptotic and necrotic cells in heterogeneous cultures. Metabolic activity (pHi), and cell death population dynamics (viable/ apoptotic/ necrotic fraction) were therefore effectively and reliably determined using flow cytometry. key words: flow cytometry, apoptosis, pHi and annexin-V.

2. Introduction

The presence of dead cells significantly limits the productivity of hybridoma cells in culture. Cell death via apoptosis represents the predominant mode of cell death in culture prosesses, especially in the batch culture of hybridoma cells (Singh et al., 1996). Therefore, monitoring the development of early apoptotic cell state will significantly facilitate the control and optimisation of bioreaction processes. Flow cytometry is the best method for detecting and analyzing apoptosis in heterogeneous culture, revealing information on the state of cells in a near on-line mode and time scale (Al-Rubeai et al., 1991). In the present study Annexin-V (AV) (a naturally occurring phospholipid which has a high affinity for phosphatidylserine exposed on the surface of early membrane intact apoptotic cells) conjugated to FITC was used in a simple flow cytometric assay for the identification of apoptosis.

FITC-AV in combination with the DNA binding dye

propidium iodide (PI) entered plasma membrane damaged cells to identify necrotic cells. Cell death population dynamics (viable/apoptotic/necrotic cells) were therefore assessed in batch culture of hybridoma cells using the AV-assay. Intracellular acidification may represent a signal for the integration of a number primary signals

to enhance the development of apoptotic cell death. This hypothesis was tested by determining the intracellular pH of hybridoma cells, using a pH sensitive fluoroprobe carboxy-SNARF-1-AM, undergoing apoptotsis either spontaneously in batch culture or by induction after exposure to campothecin. 259 O. - W. Merten et al (eds.), New Developments and New Applications in Animal Cell Technology, 259-261. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

260 3. Materials and methods

Transfected control hybridoma cell line (Pef), and hybridoma cells transfected with the antiapoptotic protein bcl-2 were cultured at 37 °C in RPMI 1640 basal medium supplemented with 5% (v/v) foetal calf serum. Cultures were maintained in 50 ml or 250 ml Duran-bottles stirred by magnetic followers.

Pef and bcl-2 cells undergoing spontaneous apoptosis in batch culture were double stained with FITC-AV and PI. Cells were washed in PBS (1%) and then resuspended in binding buffer (10 mM Hepes/NaOH, pH 7.4, 150 mM KCl, ImM MgCl 2 and 1.8 mM CaCl2). FITC-AV was added to give a final concentration of 2.5 The mixture was incubated in the dark at room temperature for 10 mins. PI (1mg/ml) was added 5 mins before flow cytometric analysis.

Pef and bcl-2 hybridoma cells induced to undergo apoptosis by campotheicn treatment or from the death phase of batch culture were loaded with carboxy-SNARF-1-AM (1 mM in DMSO), to give a final concentration of 10 in a HEPES-buffered medium. Cells were incubated for 30 min at 37 °C and then analysed by a Coulter Epic Elite flow cytometer, with excitation at 488 nm to produce an acidic emission signal at 575 nm and a basic emission signal at 635 nm. The ratio of basic and acidic forms gave an indication of the pHi. All cells were correspondingly assessed for cell viability by fluorescent microscopy using a mixture of acridine orange and PI. 4. Results

4.1. FITC-AV staining of hybridoma cells in batch culture AV/PI staining of control (Pef) hybridoma cells allowed the transition from viable to necrosis via apoptosis to be followed in batch culture (fig.l). At 72 hrs the viable population [AV(-ve)/PI(-

ve)] moved into an early apoptotic compartment [AV(+)/PI(-)]. The emergence of early apoptotic cells coincided with fluorescent microscopy results. At 84 hrs of culture the entire apoptotic population migrated to a necrotic cell gate [AV(+)/PI(+)]. Bcl-2 transfected cells migrated

directly from [AV(-ve)/PI(-ve)] to [AV(+)/PI(+)] compartments during batch culture, therefore by-passing apoptotic cell death demonstrated by phosphatidylserine externalization on cells with intact plasma membranes. 4.2. Carboxy-SNARF-1-AM staining of hybridoma cells exposed to campothecin Exposure of hybridoma cells to the DNA topisomerase I inhibitor campothecin (CAM) led to the

appearance a subpopulation of cells with a lower fluorescent ratio than the untreated cells. The apoptotic population had a reduced forward scatter which was due to cellular shrinkage. In the CAM treated cells a population with lower ratio (acidic and apoptotic ) appeared 3 hrs before the maximum proportion of apoptotic cells could be detected by fluorescent microscopy.

261

4.3. Carboxy-SNARF-1-AM staining of hybridoma cells in batch culture Carboxy-SNARF-1-AM staining of Pef hybridoma cells growing, in batch culture showed an increase in pHi during exponential growth (48 hrs), probably due to an increase in the percentage of S-phase cells. Decrease in pHi at the decline phase was, however, a manifestation of both an increase in G1-cells and apoptosis. The reduction in pHi occurred prior to the identification of

apoptosis by fluorescent microscopy. In hybridoma cells over expressing bcl-2 in batch culture there was an initial slight increase followed by a continuous but slow decline in pHi with time which coincided with an accumulation of cells in G1-phase of the cell cycle 5. Conclusion

Acidification and phosphatidylserine externalization demonstrate early events in apoptotic cell death. Intracellular acidification is linked to cell growth inhibition and to apoptosis. Monitoring acidification and phosphatidylserine externalization by flow cytometry provide earlier identifications of apoptosis compared to other techniques and therefore can be used to control bioreaction processes.

6. Acknowledgments This work is funded by the E.C. Framework IV Programme 7. References 1. Al-Rubeai M., Emery A.N. & Chalder S. Flow cytometric study of cultured mammalian cells. J.Biotechnol. 1991, 19:67. 2. Singh R.P., Al-Rubeai M , Emery A.N. Apoptosis: Exploiting novel pathways to the improvement of cell culture processes Genetic Engineer and Biotechnologist 1996, 16:227-251.

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SESSION ON INTEGRATED BIOPROCESSING IN ANIMAL CELL TECHNOLOGY

This area still represents the main line of activity of many of the ESACT members and thus it provides the organisers with a large choice, so that, as in the earlier meeting in Vilamoura, it was considered that no invited speaker was required. Indeed, as in the past, the meeting organising committee had a very difficult task in choosing only ten oral presentations out of the sixty two submitted abstracts. In order to decrease the unfairness of the process and to balance out the oral and poster sessions, there was an attempt to balance presentations along three topics of research and development: 1- Physical environment, cells and products - at the cross roads between the physical environment which is becoming more controllable and the cell physiology, yielding more reliable production: 2- Monitoring and control - even though sensors (on line and off line) have not

yet delivered as much as is expected of them, the relevance of this area for quality assurance and economic production is becoming more firmly established; 3- Integrated processes and scale-up - an area which plenty of congress organisers want to present but which is only slowly yielding results for public discussion, as companies and teams become more assured of what they are doing and risk presenting.

From the overall presented papers, a few can be briefly described to give the flavour of the session.

Konstantin Konstantinov (Bayer, Berkeley, CA, USA) presented a production process model for high-density perfusion cultures showing that dynamic, profit based optimisation of feeding is essential for the efficient manufacturing of therapeutic proteins; it is only to be expected that other protein manufacturers will add depth to this trend setting process of economically pushing animal cell technology. The use of the single round infective Semliki Forest Virus which targets a broad range of host cells for the transient expression of proteins at high level was described by Horst Blasey (GlaxoWellcome, Geneva, Switzerland) for the production of 15mg of the 5HT3 receptor protein in an 11.5 litre batch reactor. Stephanos Grammatikos (Karl Thomae, Biberach, FRG) provided industrial credibility for the use of intracellular ribonucleotide pools to indicate the metabolic status of the cell. Monitoring of nucleotide ratios such as UTP to UDP-N-acetylhexosamine in very large scale (up to 10000 1) can be used to predict the behaviour of a culture up to two days before any hint of physiological changes is given by traditional cell number/cell viability estimations - optimisation and economic modelling are envisaged. At the other extreme, Nicolas Kalogerakis (NYSU, Buffalo, NY, USA) described a dielectrophoresis-based cell separation/filter being worked out on a very small scale using microfabrication tools which is under test for separating viable 263 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 263-264. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

264

from non viable cells in perfusion systems, and speculated on the inherent capability of this filter for the preferential removal of apoptotic bodies from bioreactor effluent streams. Pedro Cruz (IBET, Oeiras, Portugal) described the importance of optimising the conditions in the bioreactor in order to obtain high quality virus like particles from baculovirus infected insect cells. The use of sonoperfusion to keep single mammalian cells inside continuously stirred tank reactors was described (A. Miller, Mons, Belgium). The effect of bioreactor parameters on less commonly described receptor production (CD 13 in HL60 cells) was presented by T. Papoutsakis (U.S.A.). Given the increasing relevance of fed batch production, feeding and monitoring strategies have been presented for the more common industrial cell types (CHOs, insect cells). Control of proteolytic activities during fermentation were described for both mammalian and insect cells. Indeed, the winner of the poster prize (courtesy of Promega) was Ernesto Chico for work performed at GBF with insect cells; for these cell types, L.

Häggström (Royal Inst. Technol., Stockholm, Sweden) used osmotic shock to increase growth rate and final cell density. Amongst the various control strategies described, a couple of imaginative imaging processes were presented by groups in Stuttgart and Berkeley, whereas the group of R. Kemp (U. Wales, UK) presented a viable cell monitor based on a dielectric spectroscope and proposed on line heat flux measurement for animal cell culture modelling in what might be an upscaleable fed batch system.

The recurrent utilisation of internal settlers in continuous culture was elegantly presented by the group of A. Marc (Nancy, France) for the production of properly glycosylated gamma interferon. Different groups evaluated fluidized bed systems for production of therapeutic proteins (M. Biselli, Jülich, Germany; J. Shiloach, NIH, MD, USA; H. Katinger, Vienna, Austria) while the advantages of temperature control namely to temperature lower than 37° C, were presented by G. Kretzmer (Hannover, Germany) and the Californian teams at Bayer and Genentech.

Different options for the purification of proteins were presented, ranging from the expanded bed adsorption (again from Genentech, J.T. Beck) as well as the direct protein sequestration concepts put forward by A. Lydiatt (Birmingham, UK); even though regulatory issues might hinder the use of such concepts they deserve more work to prove

their feasibility. Integrated approaches and the influence of the chromatographic steps used upon structure and activity of proteins were also covered (S. Fletcher, Sidney, Australia; H. Vogel, GBF, Germany) whereas displacement chromatography was tested for DNA preparation (R. Freitag, ETH Lausanne, Switzerland).

Appropriately for systems meant for production of proteins, different processes of virus clearance and validation and other safety issues (General safety issues) were also covered in what was a very holistic and expert dominated session. M. Carrondo Chairperson

PHYSICAL ENVIRONMENT

CELLS AND PRODUCTS

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DIAUXIC CELL BEHAVIOR ENABLES DETOXIFICATION OF CHO CELL CULTURE MEDIUM DURING FED BATCH CULTIVATION

Holger Lübben and Gerlinde Kretzmer, Institut für Technische Chemie, 30167 Hannover,Germany

Introduction

In fed batch cultures usually the feeding of the main substrates as glucose and glutamine leads to an increase in cell count and product concentration, but very often a dramatic decrease in cell viability due to the enormous toxification of the cell culture broth with lactate and ammonia at high cell density is observed. Especially the excretion of large amounts of lactate is known to be responsible for the growth inhibiting effect, finally ending in abrupt cell death. Using a special feeding technique the recombinant CHO celline SSOA2 producing human antithrombin III is

able to grow on its waste product lactate as a carbon source and completely detoxifies the cell culture broth from this major metabolite demonstrating a functional gluconeogenesis of mammalian cells in vitro. Inducing the diauxic cell behaviour a new way for extended cultivation spand, increased biomass and product titre as well as optimal viability is generated. Material and Methods

Different feeding techniques have been applied : In the first experiments a pulsewise feeding of different substrates has been applied (glucose and glutamine, asparagine and glutamine). The pulses were given at small portions to the cultivation (1g/L for glucose and 100 mg/L glutamine) in order to prevent excessive metabolisation as have been described earlier. Later on a continuous feeding technique of glutamine and asparagine was developed. The intension was to keep the glutamine concentration constant at 200 mg/L during the feeding phase. Although asparagine is a key substrate and essential for the occurrence of the lactate consuming ability it was not possible to measure the asparagine concentration on-line. For that reason the feeding mix consists of a molar ratio of 4:1 glutamine to asparagine, respectively 0.1 m gln and 0.025 m asn. This ratio is equal to the ratio of the specific consumption rates. The nutrient flow rate was controlled either manually (210 mg/L.d) or by means of on-line FIA analysis (PID-controller). Gluconeogenesis is not very common in mammalian cell culture. In this metabolic state previously excreted metabolites as pyruvate, citrate and lactate are remobilized 267 O.-W. Merten et al. (eds.). New Developments and New Applications in Animal Cell Technology, 267-271. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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(Figure 1). To enable several steps in the gluconeogenetic pathway, energy in form of ATP is required. Without the amino acid precursors for energy production no gluconeogenesis is possible. The functionality of TCA cycle is maintained by anaplerotic input of glutamine via 2-oxoglutarate and aspartate via oxaloacetat. The following figure illustrates a hypothesised mechanism of the pathway during the gluconeogentic phase. The proposed reactions are in accordance with the biochemical literature and the experimental measured data.

Huge amount of excreted succinate (1g/L) was observed and could not be explained by a simple mass balance of the reactions that fill the TCA cycle. For explanation reasons some of the reactions in the TCA must be reversed to some extend. At oxaloacetate two directions are hypothesised. First: A part of oxaloacetate enables the gluconeogenetic pathway by reversed reduction to malate, hence leaving the mitochondrium. Another part is needed for maintaining the flux within the TCA for further oxidation of acetyl-CoA. The excretion of succinate is thought to be due to the reversed function of the mitochondrial malate dehydrogenase. Excreted succinate is supposed to originate from

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glutaminolysis, but also, in order to close the carbon balance, from lactate which maintains the TCA cycle running via acetyl-CoA.

Results and discussion

The application of various feeding techniques induces different metabolic behaviour. Applying a pulsewise feeding of glucose and glutamine an excessive metabolisation of both nutrient sources was observed, although just small portions were given to the culture. The velocity of glucose and glutamine consumption is enormous. Glycolysis and TCA are completely unbalanced. As a consequence high excretion rates of TCA metabolites (citrate and succinate) as well as lactate were observed. Already in the beginning of the feeding phase cell growth is inhibited due to the large lactate production and consequently no further recombinant product was obtained. Obviously the relation between productivity and growing is due to the genetic construction. A combination of glucose and glutamine feeding is not a successful way for process optimization. In order to prevent an ongoing lactate production in the feeding phase a pulsewise feeding of glutamine and asparagine was established. In this case the cells change their metabolic behaviour. As far as glucose has been depleted they switched from glycolysis to gluconeogenesis. This diauxic behaviour is well known in procaryontic systems and is here indicated by the consumption of the excreted waste

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product lactate and a reduced growth rate (0.715.d-1 during glucose metabolization vs. 0.308.d-1 in the lactate consuming phase). The ability to use lactate as a carbon source resulted in a prolonged exponential growth, mainly by reducing the inhibition of cellular growth by lactate. The cell count as well as the product titre is increased (40% more biomass; 30% more ATIII). See Figure 3. Although the glutamine demand in the gluconeogenetic phase is reduced (qGln= 0.061 (mg/(106ce.d)] compared to 0.176[mg/(106ce.d)] in the glucose consuming phase), a manual feeding of 100 mg/(L.d) was not sufficient because of the increasing cell number. For this reason a continuous feeding of asparagine and glutamine was applied to prevent the limitation of these amino acids. Due to the availability of these anaplerotic amino acids the specific growth rate in gluconeogenetic phase was increased up to 0.44.d-1 and a further optimization was achieved (cell count: 4.11.106ce/ml, product concentration: 36.45mg/L). The re-metabolisation of lactate is almost complete and results in a high viability up to 160 hours. The results of different feeding techniques on cell growth and productivity are summarised in Table 1.

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Conclusions For aims of process optimisation an increased cell and product concentration could not

be achieved by simple glucose and glutamine feeding due to the growth inhibiting tension of their waste products lactate and several organic acids. The experiments pointed out that any significant accumulation of lactate has to be avoided. By applying an intelligent feeding technique we could induce a so far not often seen remetabolisation of the toxic waste product lactate for biosynthesis purposes and therefore denote this phase as gluconeogenesis. The induction of the gluconeogenitic phase was achieved by a continuous feeding of TCA-cycle anaplerotic amino acids glutamine and asparagine. The utilisation of waste products diminishes the growth inhibiting effect. As a consequence of the removement of lactate a prolongation of cell growth was achieved, because of the occurrence of a second exponential phase when the cells grow on lactate as a carbon source. Although in this phase the growth rate was decreased the overall number of cells was improved. Increased biomass correlates with an increase of product titre.

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EFFECTS OF POLYSACCHARIDE DERIVED FROM TEA ON GROWTH OF

HUMAN CELL LINES IN SERUM FREE CULTURE

H.KAWAHARA, M.MAEDA-YAMAMOTO, K.OSADA, K.TSUJI National Research Institute of Vegetables, Ornamental Plants and Tea, MAFF, 2769 Kanaya, Shizuoka 428, JAPAN

1. Introduction

Serum free culture of mammarian cells is more advantageous method that culture environment can be designed freely for various bio-assay than usual culture method

containing serum. We try to investigate various effects of food components on cell function

of human immune cell lines. In such a case, if culture medium containing serum is used, it is difficulte to prove the function of those components because of unknown elements in serum [1]. Serum free culture is supposed that it is effective for our system. Growth factors such as insulin, trasferrin, ethanolamin, sodium selenite for serum free culture were known well [2]. However, every cell llines don't always grow in serum free culture containing such growth factors. In this report, we studied that tea polysaccharides added in serum free media effect on cell growth of human immunological cell lines.

2. Materials and Methods 2.1. CELL LINES AND MEDIA

Human immunological cell lines (Burkitt lymphoma RAJI, human T lymphoma PEER, human eosinophil cell line EoL-1, human basophil cell line KU812, human chronic granulocytic leukemia K-562) were cultured in ERDF basal medium containing 10%FBS.

Each cell lines was sure of growing in ERDF medium supplemented with

insulin,

273 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 273-275.

© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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10µg/ml transferrin, 20µM ethanolamine, 25nM sodium selenite(ITES). Cells were cultured in ITES-ERDF serum free medium for 2 days before testing. Each cell lines was sure of growing in ITES-ERDF medium. Cell numbers were measured by trypan blue dye exclusion method. 2.2. PURIFICATION OF HEMICELLULOSE FROM JAPANESE GREEN TEA Hemicellulose from the Japanese green tea (Camellia Sinensis var. 'yabukita') was obtained as follows. The extract from powdered green tea with acetone was mixed with 0.5N NaOH and 0.1%NaBH 4 . After shaking the mixture for 2 hours at room temperature, it was centrifuged at 5000xg for 30 min. The supernatant was obtained and adjusted to pH4.5 with acetic acid solution. Then, adding 7% trichloroacetic acid to the solution, it was centrifuged. The supernatant was dialyzed against water for 3 days, and the solution was mixed with ethanol to separate the polysaccharides fraction. After centrifugation, the

precipitate was dissolved in water and lyophilized. The lyophirized powder was dissolved in distilled water again, the solution was added to culture medium. 2.3. PREPARATION OF pH OF CULTURE MEDIUM ERDF basal medium adjusted to six point of pH6.0, 6.5, 7.0, 7.3, 7.5, 8.0 were applied to this experiment. Acidified or neutralized medium of pH range from 6.0 to 7.3 was performed with gas. Basic medium at pH7.5 or 8.0 was prepared by adding 1N NaOH to the medium.

3. Result and Discussion

We acquired several kinds of polysaccharides from tea by using various purification methods. In these extracts, the polysaccharide having the highest activity of cell growth was named TH, and used following experiments. We tried to add the TH at various concentration to ITES-ERDF medium. Figure 1 shows the maximum dose of TH for 5 kinds of cell lines. When the TH was added

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to ITES-ERDF medium at 10µ g/ml of final concentration, all kinds of cell lines except KU812 could grow in this serum free media. If TH over 10µg/ml was added to ITESERDF medium, the viability of cell was significantly decreased on microscopic observation. As shown in Figure 2, though lag of the cell growth was slightly prolonged by adding TH, saturated cell density of ITES-ERDF-TH was about 2 times higher than that of ERDF containing only ITES. We investigated that the effect of pH of serum free culture medium on viability of the cells. As shown in Figure 3, the viability of the cells cultured in ITES-ERDF-TH was kept on the higher level than that of the cells cultured in ITES-ERDF. Specially, on the higher pH than pH7.4, addding the TH to ITES-ERDF medium is more effective than the culture medium without TH. These results suggested that the TH had a effect to stabilize something change of medium with cultivation rather than to stimulate the cell growth like a growth factor.

4. References 1.Shinmoto,H., Kobori,M., Tsushida,T., and Shinohara,K. (1995) Protein-free culture of human macrophage like UM cell line, in E.C.Beuvery, J.B.Griffiths and W.P.Zeijlemaker (eds.), Animal Cell Technology, Kluwer Academic Publishers, Dordrecht, pp. 1147-1151. 2.Murakami,H. (1989) Serum-free media used for cultivation of hybridomas, Monoclonal Antibodies: Production and Application, Alan R. Liss, Inc. pp. 107-141.

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METABOLIC DEMANDS OF BTI-TN-5B1-4 (HIGH FIVE™) INSECT CELLS DURING GROWTH AND AFTER INFECTION WITH BACULOVIRUS ERNESTO CHICO1, 2 and VOLKER JÄGER1 1 Gesellschaft für Biotechnologische Forschung mbH Mascheroder Weg 1 D-38124 Braunschweig, Germany. 2 Center of Molecular Immunology POBox 16040 Habana 11600, Cuba. Introduction • Because of its high productivity BTI-Tn-5Bl-4 (High Five™) cell line has become an attractive alternative to Spodoptera fugiperda cells for recombinant protein production using the Baculovirus expression system. • In this work we present the main metabolic demands of this cell line and discuss the major challenges for growing these cells at high cell density for infection and subsequent recombinant protein production. Experimental conditions High Five cells were cultivated in Ex-Cell 401 serum-free medium fortified with glucose, asparagine and glutamine. Suspension cultures were carried out in Techne spinner flasks as well as in membrane aerated bioreactors for studying the oxygen demand. For infection, a recombinant baculovirus was used expressing human protein. Synchronous infection of the cell population was achieved using a MOI of 5. Results and Discussion General Metabolic Pattern of High Five cells Fig. 1 shows the general metabolic behaviour of High Five cells during a batch culture which is characterized by following observations: The exponential growth phase ends with the almost simultaneous depletion of Gln and Asn. From all of the amino acids measured only the concentrations of Ala, Asn and Gln change substantially throughout the culture time. Alanine accumulates during the exponential phase of growth and its concentration remains constant after the depletion of asparagine and glutamine. Final values of 10 - 20 mM of can be found at the end of a batch cultivation of High Five cells. These values are extremely high in comparison with most insect as well as mammalian cell lines. Baculovirus infection does not affect the general metabolic behaviour of High Five cells (data not shown). 277

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 277-279. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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Specific uptake rates of glutamine and asparagine (qGln and qAsn) during growth and infection are presented in Fig. 2. Whereas qGln shows a clear trend to reach a saturation in the experimental concentration range, qAsn still increases at concentrations higher than 10 mM of Asn. This fact is important for the design of feeding strategies for High Five cells. Although the general metabolic behaviour of

279 cells is not affected during baculovirus infection the cell specific consumption of Asn and Gln is significantly higher than during exponential growth.

Oxygen demand The specific oxygen uptake rates of High Five and IPLB Sf21 AE cells were measured

in membrane-aerated bioreactors. Results in Table I indicate that High Five cells have the highest cell specific oxygen consumption rate reported in the literature for insect as well as mammalian cell lines.

High Five cells showed a 270% higher cell specific oxygen consumption rate when compared to Sf21 cells. This difference is reduced to approximately 30% if the oxygen consumption rate is expressed per volume of the cell population due to the significantly bigger cell size of High Five cells (See Table I).

The overall oxygen uptake rate (OUR) of the bioreactor was measured online from the

DO controller response during growth and after infection (data not shwon). During exponential growth the OUR correlated well with the cell density. This correlation is only altered during the first 30 hours post infection (hpi), suggesting that there is a peak of the cell specific oxygen uptake rate which is associated to cellular events during the early phase of baculovirus infection. Conclusions High Five cells are characterized by a very active metabolism, by which Asn and Gln are consumed at very high rates. The use of feeding strategies, aimed to keep the concentrations of these amino acids in a region of lower qAsn and qGln, is strongly recommended to avoid nutrient limitations without extensive medium exchange. Due

to the high specific oxygen demand of High Five cells, the growth of this cell line up to high cell densities could be limited by the transfer capabilities of current animal cell bioreactor systems. An additional increase in the oxygen demand must be expected during the first 30 hours post infection. References 1. 2.

Eyer K, Oeggerli A, and Heinzle E (1995). Biotechnol. Bioeng. 45, 54-62. Taticek RA, Hammer DA, and Shuler ML (1995) in: Shuler ML, Wood HA, Granados RR, and Hammer DA (eds.), Baculovirus expression system and biopesticides, Wiley-Liss, New York, pp. 131-174

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EFFECTS OF AMMONIUM AND LACTATE DURING CONTINUOUS HYBRIDOMA FERMENTATIONS IN A FLUIDIZED-BED BIOREACTOR H.Heine, M. Spies, M. Biselli, C. Wandrey Institut für Biotechnologie 2, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany

Introduction Cell immobilization in porous microcarriers in a fluidized-bed bioreactor is an efficient method to reach very high cell densities in continuous fermentations [1]. High medium fluxes have to be used to prevent cells from being limited by nutrient exhaustion. Accumulation of inhibitory compounds like ammonium or lactate [2] limits the degree of supplementation of the culture media. The aim of this work is to compare kinetic effects of ammonium and lactate in different fermentation systems. Hybridoma cells cultured in a fluidized-bed bioreactor using porous glass carriers have been compared with suspension batch culture in spinner vessels. It turned out that significant effects of three dimensional environment in porous carriers occur.

Material and Methods The cell line used is a mouse-mouse hybridoma secreting a monoclonal antibody of the subclass Suspension cultures were carried out in batch spinner flasks. The immobilized cultivations were performed in a fluidizedbed bioreactor (Fig. 1) with 60 ml carrier and 250 ml total volume. The carriers have a size of 400-710 µm and are coated with gelatine [3]. The perfusion rate was adjusted to after startup phase. Suspended cells were counted using a hemacytometer with Erythrosin B-staining, immobilized cells were lysed using a 100 mM citric acid buffer, with subsequently staining of nuclei with crystal voilet. The culture medium consists of a 3:1 mixture of DMEM and Ham´s F-12, supplemented with insulin, amino acids, transferrin, BSA, fatty acids, vitamins and trace elements (all Boehringer Ingelheim, Germany). For the ammonium experiments was added to final concentrations of 2, 4, 6, 8, 10, 12, 14 mmol/1. For the lactate experiments Na-lactate was added to a final concentration of 2, 4, 8, 12, 16, 20, 25 mmol/1. 281 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 281-283. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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Results and Discussion ADDITION OF AMMONIUM: In continuous immobilized culture addition of ammonium results in a reduction of immobilized cell density whereas suspended cell density and antibody titer remain constant (data not shown). This indicates an increasing growth rate due to more space on the carrier (Fig. 2a). Though the growth rate increases in immobilized culture the cell cycle distribution remains nearly constant at every ammonium concentration. But there is a significant difference between suspended cells and immobilized cells in the fluidized bed bioreactor. Indicating a faster proliferation, the greater part of the immobilized cells are in the S-phase compared to the suspended cells. The specific antibody production rate is coupled to growth (Fig. 2a). The metabolic rates increase by factor six (data not shown). Suspended cells in batch culture show a different

response to ammonium addition (Fig. 2b), their growth rate remains nearly constant whereas the specific productivity slightly decreases. Nevertheless the growth rate in immobilized culture is lower than in suspension culture and only at the highest ammonium concentrations growth rate is higher than in batch culture. The specific productivity of immobilized cells in continuous culture is much higher than in batch suspension culture. ADDITION OF LACTATE The addition of lactate has no influence on immobilized cell density, suspended cell density and antibody titer. The growth rate and specific productivity remain more or less constant (Fig. 3a). Due to the lower immobilized cell density in the continuous experiment compared to the ammonium experiment the initial rates are higher compared to Fig. 2a. In suspended spinner culture there is also no effect on the growth rate and the specific productivity (Fig. 3b).

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Summary Our experiments show an influence of the three dimensional environment of the cells

during the addition of ammonium. Ammonium leads to decreasing immobilized cell densities and increasing metabolic rates in porous carriers. In suspended batch culture

growth rate remains constant whereas cell specific productivity decreases slightly. The addition of lactate shows no effect of the three dimensional environment. In both cases the cell cycle distribution shows differences between immobilized cells and suspended

cells in the fluidized bed bioreactor. References

[1] Thömmes, J. et al:The influence of dissolved oxygen tension on the metabolic activity of an immobilized hybridoma population, Cytotechnology 13 (1993), 29-39. [2] Miller, W.M., Wilke, C.R., Blanch, H.W.: Transient responses of hybridoma cells to lactate and ammonia pulse and step changes in continuous culture, Bioprocess Engeneering, 3 (1994), 113-122 [3] Lüllau, E. et al: Immobilization of animal cells on chemically modified carrier, in: Spier, Griffith (eds.): Animal cell technology, Butterworth-Heinemann,(1992), 469475 Acknowledgement

The authors thank the European Commission Directorat General XII for the sponsoring of this work.

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SERUM CONCENTRATION AND pH AFFECT THE CD13 RECEPTOR CONTENT OF HL60 CELLS CULTURED IN STIRRED BIOREACTORS C. L. MCDOWELL AND E. T. PAPOUTSAKIS Dept. of Chemical Engineering, Northwestern University 2145 Sheridan Road, Evanston, IL 60208-3120, USA

Keywords: antigen expression, agitation, pH, serum concentration

Abstract The effects of serum medium concentration and culture pH on CD 13 receptor content and mRNA levels of HL60 (human promyelocytic leukemia) cells were examined using flow cytometry and Northern blotting. Increasing the serum concentration from 5% to 10% increased the CD 13 receptor content and mRNA levels of HL60 cells cultured at 80 rpm in a 2 L bioreactor. When the agitation rate was increased to 300 rpm, 10% FBS increased the HL60 apparent growth rate, but decreased the CD 13 receptor content and

mRNA levels. Decreasing the culture pH from 7.4 to 7.2 (at 80 rpm) did not affect the

HL60 apparent growth rate, but increased the CD 13 receptor content. Unlike the results of the serum experiment, changes in CD 13 content in response to culture pH were not correlated with changes in CD13 mRNA levels. Introduction Large-scale culture of freely suspended cells is necessary for the production of whole cells for use in somatic therapies. Industry is now making use of agitated bioreactors, as they offer uniform culture conditions and the ability for growth to high cell densities. However, the high agitation rates and/or sparging often needed to satisfy the oxygen demand of high cell concentration cultures generate fluid-mechanical forces that affect cell physiology and/or injure cells. It is important to understand not only how hydrodynamic forces due to agitation affect cells, but also how other bioreactor parameters, such as serum medium concentration and pH, affect cell physiology. Of

particular interest is how bioreactor parameters affect receptor concentration on the cell surface, which has implications in the production of whole cells for somatic therapies. Somatic therapies involve interaction, through their surface receptors, of the transferred cells with other cells in the human body. Therefore, cells used in somatic therapies must be cultured in a manner that maintains the expression of key surface receptors. In addition to the requirement of intact and functional surface receptors, cells used in somatic therapies must be cultivated in numbers sufficient for treatment (7,9). Cultivation in agitated bioreactors could produce the cell numbers sufficient for treatment, but parameters such as agitation rate, serum medium concentration, and pH

could affect the expression of key surface receptors. Therefore, it is necessary to understand how agitation, serum medium concentration and pH affect cellular receptors. 285 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 285-291. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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This work examines the effects of serum medium concentration, agitation rate, and pH on the surface content of the CD 13 receptor (aminopeptidase N) of HL60 (human promyelocytic leukemia) cells. Materials and Methods CELLS AND CULTURE MEDIUM

HL60 cells (cell line ATCC CCL240) were cultured in IMDM supplemented with 5% or 10% fetal bovine serum (FBS), sodium bicarbonate, and sodium pyruvate. BIOREACTOR CULTURES Experiments were carried out in two Setric Genie 2 L bioreactors (Toulouse, France),

with an initial volume of 1.35 L. Temperature was maintained at 37°C. Dissolved oxygen concentration was maintained above 70% of air saturation. The pH was maintained at 7.4 or 7.2 by injection into the headspace. In the pH experiment, the cultures were grown at 80 rpm. In the FBS experiment, once the cultures reached midexponential growth, the agitation rate of both was increased to 300 rpm. Every 8-12

hours, samples were taken to determine cell concentrations, viability, CD13 receptor content, and CD 13 mRNA levels. FLOW CYTOMETRIC ANALYSIS OF SURFACE ANTIGENS

Samples from each experiment were analyzed in the same session using a Becton Dickinson FACSscan flow cytometer. Ten thousand cells were analyzed per sample. NORTHERN BLOT ANALYSIS Total RNA was purified using RNA STAT-60 (Tel-Test Inc., Friendswood, TX, USA). Northern blots and autoradiography were carried out as described previously (8). The amount of RNA was controlled by rehybridization of the blots with the glyceraldehyde-

3-phosphate dehydrogenase (GAPDH) probe. Probes were labeled with using random hexamer primers. CD13 cDNA was provided by Dr. A. Thomas Look (St. Jude's Children's Research Hospital, Memphis, TN, USA). GAPDH cDNA was received from ATCC. Results EFFECT OF 5% VERSUS 10% FBS AT 80 AND 300 RPM

As shown in Figure 1a, the cell concentrations of the 5% and 10% FBS cultures were similar before the agitation rate increase to 300 rpm. After the agitation rate increase to 300 rpm, however, the 10% FBS culture had a cell concentration higher than the 5% FBS culture. The viability of both cultures remained above 90% for the duration of the experiment (data not shown). Prior to the agitation rate increase, the 10% FBS culture

had a higher CD 13 receptor content as compared to the 5% FBS culture (Figure 1b). After the agitation rate increase to 300 rpm, however, the CD 13 content of the 10% FBS culture continued to decline, while the decline in the 5% FBS culture was arrested. The

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differences between the 5% and 10% FBS cultures in terms of CD 13 content correlated with differences in CD 13 mRNA levels (Figure 1c). EFFECT OF CULTURE pH 7.0 VERSUS 7.4 AT 80 RPM

Decreasing the culture pH from 7.4 to 7.2 had no affect on the HL60 apparent growth rate (Figure 2a). It also had no effect on cell viability, as the viability of both cultures remained at or above 90% (data not shown). However, decreasing the pH from 7.4 to

7.2 increased the CD13 receptor content, on average, by approximately 25% (Figure 2b). Unlike the FBS experiment, despite the differences in CD13 receptor surface content there were no differences in CD13 mRNA levels (Figure 2c). Discussion THE PROTECTIVE EFFECT OF FBS AT 300 RPM FBS protected HL60 cells from damage under conditions of agitation at 300 rpm. As

shown in Figure la, the 10% FBS culture had a higher cell concentration as compared to the 5% FBS culture. These results are consistent with experiments in the literature, which showed that serum protected cells cultured in agitated bioreactors (2, 3, 6). FBS DECREASES CD 13 RECEPTOR CONTENT AT 300 RPM

At 300 rpm, 10% FBS leads to a decrease in CD 13 receptor content. As shown in Figure 1b, the 10% FBS culture exhibited a continued decrease in CD 13 content at 300 rpm, while the 5% FBS culture exhibited an arrest in the decline of CD 13 content. The behavior of the 10% FBS culture is consistent with experiments that showed that HL60 cells cultured in 10% FBS exhibited a decrease in CD 13 content in response to agitation at 270 rpm, as compared to control cultures at 80 rpm (4). The behavior of the 5% FBS culture is consistent with previous experiments demonstrating that HL60 cells cultured in 5% FBS exhibited an arrest in the decline of CD13 content in response to agitation at 300 rpm, as compared to control cultures at 80 rpm (5). These results show that at 300 rpm, 10% FBS reduced the transduction of fluid-mechanical forces that affect CD 13 receptor content. In contrast, the 5% FBS culture sensed the fluid-mechanical forces due to agitation and responded by arresting the decline in CD 13 receptor content.

FBS INCREASES CD 13 RECEPTOR CONTENT AND mRNA LEVELS AT 80 RPM

As shown in Figure 1b, the 10% FBS culture had a higher CD13 receptor content at 80 rpm as compared to the 5% FBS culture. This observation is consistent with studies that showed that cytotoxic T lymphocytes cultured in serum had a higher CD38 and CD57 receptor content as compared to cells cultured in the absence of serum (10). As shown in Figure 1c, the higher level of CD 13 receptor content in the 10% FBS culture was correlated with higher levels of CD 13 mRNA. This higher level of CD 13 mRNA could be due to either an increase in the rate of CD 13 mRNA transcription, or a reduction in the rate of CD13 mRNA degradation. These results have implications for the culture of human hematopoietic cells with animal sera, or autologous serum or plasma in stirred tank reactors.

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pH AFFECTS CD 13 RECEPTOR CONTENT BUT NOT CD 13 mRNA LEVELS

Decreasing the culture pH from 7.4 to 7.2 increased the CD13 receptor content by approximately 25% (Figure 2b). This behavior is consistent with experiments that showed that a decrease in pH from 7.4 to 7.0 increased the atrial natriuretic peptide receptor content on bovine aortic endothelial cells by approximately 100% (1). In contrast to the FBS experiment, higher CD13 mRNA levels in the pH 7.2 culture were not correlated with higher CD 13 mRNA levels (Figure 2c). Since culture pH affected the CD 13 receptor content, but not mRNA levels, another receptor processing step must be affected. Culture pH could affect CD 13 receptor protein synthesis, trafficking to the cell membrane, internalization, degradation, or recycling.

Acknowledgments This work was supported by a National Science Foundation Graduate Fellowship awarded to C.L.M. and a Predoctoral Biotechnology Training Grant (NIH GM08449). References

1. Katafuchi, T., Hagiwara, H., Ito, T. and Hirose, S. (1993) A Dramatic pH Dependent Alteration in ANP Receptor Density: a Note for Using Cultured Cells, American Journal of Physiology 264, C1345-C1349. 2. Kunas, K. T., and Papoutsakis, E. T. (1989) Increasing Serum Concentrations Decrease Cell Death and Allow Growth of Hybridoma Cells at Higher Agitation Rates, Biotechnology Letters 11,525-530. 3. Kunas, K. T., and Papoutsakis, E. T. (1990) The Protective Effect of Serum Against Hydrodynamic Damage of Hybridoma Cells in Agitated and Surface-Aerated Bioreactors, Journal of Biotechnology 15, 57-70. 4. Lakhotia, S., Bauer, K. D., and Papoutsakis, E. T. (1993) Fluid-Mechanical Forces in

Agitated Bioreactors Reduce the CD13 and CD33 Surface Protein Content of HL60 Cells, Biotechnology and Bioengineering 41, 868-877. 5. McDowell, C. L., and Papoutsakis, E. T. (1997) Increased Agitation Intensity Increases CD 13 Receptor Surface Content and mRNA Levels, and Alters the Metabolism of HL60 Cells Cultured in Stirred Tank Bioreactors (submitted 1997). 6. Michaels, J. D., Petersen, J. F., Mclntire, L. V., and Papoutsakis, E. T. (1991) Protection Mechanisms of Freely Suspended Animal Cells (CRL 8018) from FluidMechanical Injury. Viscometric and Bioreactor Studies Using Serum, Pluronic F68 and Polyethylene Glycol, Biotechnology and Bioengineering 38, 169-180. 7. Rosenberg, S. A., Anderson, W. F., Blaese, M., Hwu, P., Yanelli, J. R., Yang, J. C., Topalian, S. L., Schwartzentruber, D. J., Weber, J.S., Ettinghausen, S. E., Parkinson, D. N., and White, D. E. (1993) The Development of Gene Therapy for the Treatment of Cancer, Annals of Surgery 218, 455-464. 8. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Plainview, NY. 9. Scheding, S., Franke, H., Brugger, W., Kanz, L. and Schmitz, S. (1995) How Many

Myeloid Post-Progenitor Cells Have to be Transplanted to Completely Abrogate Neutropenia After High-Dose Chemotherapy and Peripheral Blood Progenitor Cell Transplantation? Blood 86, 224a. 10.Trimble, L., Perales, M., Knazek, R., and Lieberman, J. (1996) Serum Enhances the Ex Vivo Generation of HIV-Specific Cytotoxic T Cells, Biotechnology and Bioengineering 50,521-528.

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Discussion Wurm:

Have you looked at the protease activity in the supernatant under those various conditions?

Papoutsakis:

No.

Wurm:

It might give an explanation for your various levels. If you reduce serum, you might increase hydrodynamic damage and release more proteases, which would affect protein on the cell.

Papoutsakis:

We have not studied this but did show that increased damage increased the receptor level, which goes against the idea of

proteolytic attack. Barteling:

Could the serum effect be due to a reduction in shear which you could mimic by the addition of pluronic?

Papoutsakis:

The effect of serum is nothing to do with shear, but is complicated by the presence of fluid mechanical forces.

Bernard:

The signal which you receive is by facs, I presume. Did you calibrate this for actual C13 by another method?

Papoutsakis:

Yes, you can do an enzyme assay. There is a complication in that the culture environment might be changing in glycosylation and, therefore, the recognition of a particular receptor. We have been

looking for a suitable receptor to look at the effect of glycosylation in the culture medium but it is practically impossible. The only one which comes close is CD34, but this is very complicated. Al- Rubeai:

When you look at changes in cell size, do you think that there is a possibility that the concentration of CD 13 has not changed but the amount has?

Papoutsakis:

No, if you normalise it by cell size, you see the same thing.

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FOUR REGULATION-FRIENDLY SERUM-FREE MEDIA FOR MAMMALIAN

AND INSECT CELLS

Thomas W. Irish, Susan E. Lenk, Lisa A. Bugner, and Karen J. Etchberger JRH Biosciences, Inc., 13804 West 107th Street, Lenexa, KS 66215 USA

Abstract Mammalian and insect cell lines are used commercially to produce products for both the human and animal health markets. Due to regulatory issues surrounding the use of animal-derived raw materials in culture media, JRH Biosciences has developed four regulation-friendly media formulated using raw materials that are

recombinant, synthetic or non-animal-derived. Cells can be transferred directly from serum-containing cultures into these media without extensive adaptation or

extended lag phases. EX-CELL™Vero SF supports the growth of Vero cells in stationary flasks as well as on microcarriers. EX-CELL™293-S supports the growth of 293 cells as single cells in suspension cultures. EX-CELL™302 was designed for the culture of CHO cells in suspension systems. In each of these media, cells achieve doubling times and final cell densities comparable to 10% FBS controls. EX-CELL™420 was optimized for growth of Sf9 and Sf21 insect cell lines, supporting cell densities of cells/ml (viability >95%) for more than 10 days.

Materials and Methods EX-CELL™302: CHO-K1 cells (ATCC CCL 61) previously adapted to

EX-CELL™301 were transferred to 250 mL spinner flasks with a seeding density of cells/mL in 100 mL of EX-CELL™302 (~60 rpm). Daily cell counts and viabilities were determined by trypan blue exclusion. EX-CELL™420: Sf9 cells (ATCC CRL1711) were transferred directly into EX-CELL™420 as attachment cultures from an ampule of ATCC supplied cells.

Cells grown in the serum free control medium were adapted from attachment cultures grown in Hink's TNM-FH + 10% FBS. Cells in both serum-free media were

adapted to suspension culture in 125 mL shaker flasks at 130 rpm. For growth curves, cells were seeded at either cells/mL or cells/mL in 50 mL volume of media. Daily cell counts and viabilities were determined by trypan blue exclusion. Virus production and activity assays were performed 293

O.-W. Merten et at. (eds.). New Developments and New Applications in Animal Cell Technology, 293-302. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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by Invitrogen (Carlsbad, CA). Sf9 cells grown in EX-CELL™420 and Grace's + 10% FBS were infected with a pVL941 CAT clone HTS at an MOI of 5. Viral liters were determined by plaque assays were performed on the culture supernatants. production by Sf9 cells grown in EX-CELL™420 and Grace's + 10% FBS was determined by spectrophotometric activity assays. EX-CELL™293-S: 293 cells (ATCC 1573) were weaned to EX-CELL™293-S from cultures grown in MEM + 10% DBCS. For adaptation to serum-free medium, the cells were passaged as attachment cultures into EX-CELL™293-S + 1% FBS for one passage followed by two passages in EX-CELL™293-S. Cell dissociation was accomplished with trypsin followed by protease neutralization with ultrapure soybean trypsin inhibitor (0.1 %). Cells were transferred to roller bottles and spinner flasks at a density of Cell counts and viabilities were determined by trypan blue exclusion. EX-CELL™VERO-SF: Vero (ATCC CCL-81) and MDCK (CCL 34) cells were adapted directly to EX-CELL™VERO-SF from cells grown in MEM + 10% FBS.

Serum-free attachment cultures were seeded at Cell dissociation was accomplished with trypsin followed by protease neutralization with ultrapure soybean trypsin inhibitor (0.1%). For growth curves, cells were counted at culture days three though seven. Cell counts and viabilities were determined by trypan blue exclusion.

EX- CELL™ 302

Data presented here demonstrates extended cellular growth in JRH Biosciences EX-CELL™302 for nine (9) days in spinner batch cultures. Cultures of CHO-K1 cells achieved an approximate average maximum cell density of cells/mL with exponential doubling times in the range of 20 hrs., maintaining viabilities greater then 70%. EX-CELL™302 has been formulated without using animal derived proteins. EX-CELL™302 does contain very low levels (> 50 ug/L total) of recombinant proteins having a molecular weight less the 10 KD. CHO-K1 cells, cryopreserved and adapted to serum free growth in JRH Bioscience’s EXCELL™301, were transferred directly into EX-CELL™302 without an adaptation period. JRH Bioscience's "regulatory friendly" EX-CELL™302 can be safely used for production of human therapeutical products because it does not contain animal derived proteins, amino acids, or other components considered to be questionable.

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Conclusions EX-CELL™302 can achieve cellular densities in suspension batch cultures greater then cell/mL. •

EX-CELL™302 can support batch cultures maintaining high viabilities for greater then 9 days without a refeed.

•

EX-CELL™302 does not contain any animal derived raw materials considered to be regulatory questionable.

•

Cells grown in serum free media can be directly transferred into EXCELL™302 without adaptation .

EX-CELL™420

Insect cells are commonly used for the expression of recombinant proteins and virus production in a variety of applications including vaccines, diagnostics, and biopesticides. Cells derived from the fall armyworm, Spodoptera frugiperda, have been the cells of choice for baculovirus infection and protein production. EXCELL™420 is a complete medium developed and optimized for the serum-free growth of Sf9 and Sf21 insect cell lines as either adherent or suspension cultures. This medium contains no proteins detectable by standard methods. Cells can be transferred directly from adherent cultures grown in serum-free or serum-containing media into EX-CELL™420 suspension culture without an adaptation period. Sf9 cells cultured in EX-CELL™420 routinely achieve cell densities of cells/mL with viabilities greater than 95% and can be maintained for more than 10 days at these densities with no loss of viability. Cultures seeded at low density cells/mL) over multiple passages do not exhibit the typical 24-48 hour lag phase seen with many serum-free media. Sf9 and Sf21 cultures have been carried in EXCELL™420 for more than 20 passages. Sf9 cells grown in EX-CELL™420 were infected with a pVL941 CAT clone HTS at an MOI of 5 and the resulting viral titer at 72 h was pfu/mL, approximately half a log less than the 1.3 x 10 pfu/mL titer obtained from cells grown in Grace's + 10% FBS. Expression of IL-6, CAT, and by Sf9 cells grown in EX-CELL™420 is comparable to that obtained from cells grown in Grace's + 10% FBS.

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298

299

Conclusions •

Cells can be transferred directly to EX-CELL™420 from a serumcontaining medium without an adaptation period.

•

Cells grown in EX-CELL™420 routinely achieve cell densities of cells/mL with viabilities of >95%.

•

Cultures can be maintained for more than 10 days with no loss of viability.

•

Cultures seeded at low density do not exhibit the typical 24-48 hour lag phase seen with many serum-free media.

•

Virus production and protein expression in EX-CELL™420 is comparable to that obtained in serum-containing media.

EX-CELL™Vero-SF

JRH Bioscience's EX-CELL™Vero-SF media has been formulated to support the long term growth of the Vero cell line. This medium has demonstrated the ability to grow both Vero cells and MDCK cells both of which are commonly used by the human pharmaceutical industry to produce human and animal vaccines and protein produces. Because this media is free of animal derived raw materials it is considered to be regulatory friendly. Vero cells grown in EX-CELL™Vero-SF media achieve culture densities comparable to densities achieved in a serum containing media. MDCK cells grown in EX-CELL™Vero-SF media achieve densities and growth rates greater then those achieved when grown in a media containing serum. Both cells lines can be directly transferred into EX-CELL™VeroSF media from serum conditions without adaptation or lose of growth rates or densities.

300

301

Conclusions •

Vero and MDCK cells can be grown in EX-CELL™Vero to densities comparable to or higher then those achieved in a serum containing media.

•

EX-CELL™Vero media does not contain any animal derived raw materials making this media regulatory friendly.

•

Both Vero and MDCK can be directly transferred into EX-CELL™Vero media without adaptation.

•

Population doubling are sustained over extended periods of time.

EX-CELL™293-S

The 293 cell line used for production of proteins, and adeno virus can be grown in JRH Biosciences EX-CELL™293-S as a suspension culture. This media does contain BSA as a protectant for cellular growth in spinner cultures and very low levels of recombinant growth factors. Cells can be quickly adapted to serum free growth in stationary culture and then transferred into suspension culture conditions. Adaption of 293 cells acquired from ATCC requires three separate steps, adaptation to serum free growth in static cultures, adaptation to suspension in roller bottles and adaptation to spinner growth. 293 cells grown in JRH Biosciences EXCELL™293-S grow as single cells or small, loose, aggregates that can be easily broken up by gentle pipetting. Doubling times and cellular densities achieve in serum free growth are comparable to those achieved in a serum containing media.

302

Conclusions •

Cells can be transferred from serum containing media into low serum conditions within two subpassages.

•

293 cells in low serum conditions (1% DBCS) will form loose cell aggregates and grow as suspension cultures.

•

Adaptation to serum free conditions require an additional one to two subpassages for sustainable growth.

•

293 cells grown in EX-CELL™293-S will achieve culture densities of 1 x in roller bottles 5 -7 days from a seed of

PROTEOLYTIC ACTIVITIES IN THE BACULOVIRUS-INSECT CELL EXPRESSION SYSTEM Georg Schmid and Andrea Bischoff F. Hoffmann-La Roche Ltd., Pharmaceutical Research Building 66/112A, CH-4070 Basel, Switzerland Introduction Proteolytic degradation of recombinant proteins produced in the baculovirus-insect cell expression system is an often encountered phenomenon. The system encompasses proteases of viral and cellular origin At least one viral proteinase (v-cath), a cathepsin Llike cysteine proteinase, has been identified to date. The activities of cellular proteases are often regulated by the cells' metabolic state and oxidative stress, heat shock or amino acid (substrate) starvation induce formation of various stress proteins, including proteases, that may have a significant impact on product quality in any given bioprocess. We investigated the occurence of aspartic, metallo-, serine and cysteine proteinases in non-infected and infected Spodoptera frugiperda (Sf9) insect cells using chromogenic substrates, zymogram gels and SDS-PAGE analysis of degradation products of model proteins. The secreted and intracellular activities were evaluated for spinner flask, laband pilot-scale bioreactor cultures operated under different physiological conditions.

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O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 303-306. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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MONITORING AND CONTROL

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DEVELOPMENT OF PROTEINASE ASSAYS FOR IMPROVED CHO CELL CULTURES C. TANS, C. VANDER MAELEN, S. WATTIAUX-DE CONINCK*, M.-M. GONZE AND L. FABRY SmithKline Beecham Biologicals S.A., Rixensart - *F.U.N.D.P., Namur, Belgium

1. Abstract Proteolytic activity in CHO cell culture supernatants is a key parameter to be considered towards an improved quality of secreted recombinant glycoproteins. In the present study, synthetic substrates have been used to evaluate the proteolytic activity in parental and recombinant CHO cell culture supernatants. Zymographic and isoelectric focusing analysis showed that modifications in the peptidic sequence of a model secreted glycoprotein are related to a protease activity detected in supernatants using the Z-GlyGly-Arg-AMC substrate. Protease measured by the substrate was suggested to be a form of plasminogen activator as its activity was inhibited by plasminogen activator inhibitor type 1 and its optimal pH was in the range of pH 8.5. Cell cultures performed at lower temperature, eg 29°C instead of 37°C resulted in a decrease of the protease activity in supernatants as assayed using the Z-Gly-Gly-ArgAMC substrate (3 fold decrease). Consequently, lower cell culture temperature is expected to be a key factor to improve the quality of recombinant secreted glycoproteins in CHO cell cultures. 2. Introduction The objective of the study was to evaluate the influence of CHO cell derived proteases on the integrity of secreted glycoproteins. The first step consisted of the characterization of proteases activity in CHO cell supernatants able to modify a model recombinant glycoprotein (rec. glycoprotein): potential synthetic substrate, inhibition profile, molecular weight. During the second step, changes in the culture conditions and their effects on the protease activity and rec. glycoprotein quality were evaluated. 309 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 309-316. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

310

3. Material and methods 3.1.

CHO CELL CULTURE

All cultures were carried out in serum-free medium. Parental (untransfected) CHO cellfree supernatants were obtained from suspension batch culture after 72 hours culture at 37°C. Suspension batch culture of rec. CHO cells were performed at different temperatures in spinner flask 1L. 3.2.

INCUBATION OF THE PURIFIED GLYCOPROTEIN IN PARENTAL CHO CELL CULTURE SUPERNATANTS

The purified rec. glycoprotein sample (4 % of N-terminal modifications) was incubated in the presence of parental CHO cell culture supernatants (5 fold concentrated by ultrafiltration with a cut-off limit of 10 kD) and 0.01 % merthiolate. Prior to incubation, pH was adjusted to pH 7.2 with 5N HC1 or 5N NaOH. Incubation time was 44 hours at 37°C. 3.3.

ISOELECTRIC FOCUSING/WESTERN BLOTTING (IEF/WB), IMMUNODETECTION TECHNIQUES

The glycoprotein samples were concentrated 15 fold by ultrafiltration (10kD cut off) prior to diafiltration and IEF/WB. Gel electrophoresis was performed under denaturing conditions as reported by O’Farrel (1975) and Lognonné (1994) in a vertical slab gel unit; a pH gradient between 3 and 6 was used; migration was stopped after 30 000 Vxhours. After several washes in a fixing solution, gels were blotted to PVDF membranes in the presence of SDS using a semidry transblot system. The blot was incubated with a polyclonal rabbit anti recombinant glycoprotein antibody and then with an anti-rabbit horseradish peroxidase conjugated antibody; the blot was developed with 3.3' diaminobenzamidine. The IEF/WB were analysed with a densitometer/image analyser (Bio-Rad). 3.4.

ZYMOGRAPHIC ANALYSIS OF PROTEOLYIC ACTIVITY IN CULTURE SUPERNATANT

Supernatants samples migration was realised in a 7.5 % acrylamid gel polymerized in presence of 0.2 % purified rec. glycoprotein at 4°C. After washes, gel was incubated at 37°C during 20 hours in 0.1 Tris-HCl buffer pH 8.5 and then stained with Coomassie blue (Heussen and Dowdle, 1980). 3.5.

ZYMOGRAPHIC DETECTION OF PROTEASE ACTIVITY AGAINST SUBSTRATE Z-GLY-GLY-ARG-AMC

Supernatants samples migration was realised in a 7.5 % acrylamid gel at 4°C. After gel washes, a membrane impregnated with Z-Gly-Gly-Arg-AMC was overlayed on the gel,

311

gel and membrane were incubated 3 hours at 37°C. Fluorescence picture was obtained using Bio-Rad Glyco Doc Analytical Software. 3.6.

PROTEASE ACTIVITY MEASUREMENTS

200 µl of CHO cell culture supernatants were incubated at 37°C in presence of 0.25 mM synthetic substrate, 0.1 M buffer (Glycine - Tris - Acetate); the reaction is stopped after 2 hours. 4. Results and discussion 4.1.

PROTEOLYTIC ACTIVITY IN PARENTAL CHO CELL CULTURE

SUPERNATANTS Four synthetic substrates containing a basic residue at the cleavage site have been used to detect the protease responsible for the N-terminal (N-term.) modifications of the model rec. glycoprotein. Maximal activity was detected at basic pH for all synthetic substrates. The results show that parental CHO cell culture supernatant contain proteolytic activity which can be easily measured using fluorescent synthetic substrates.

312 4.2.

DETERMINATION OF INHIBITION CHARACTERISTICS OF THE PROTEASE RESPONSIBLE FOR N-TERM. MODIFICATIONS OF THE REC. GLYCOPROTEIN

IEF/WB technique was used to analyse rec. glycoprotein isoforms and to evaluate the extent of rec. glycoprotein profile changes. Incubation of the rec. glycoprotein sample with CHO cell-free culture supernatant resulted in the appearance of five bands 1, 2, 2’, 3, 5 (figure 2 : positive control). When

incubation occured in fresh culture medium, the five bands 1, 2, 2’, 3, 5 did not appear (data not shown). N-terminal sequencing of bands 1, 2, 2’, 3, 5 in the IEF/WB profile revealed that they contained more than 70 % of N-modified forms of the rec. glycoprotein. The proposal was to use the relative amount of the bands 1, 2, 2’, 3, 5 in the IEF/WB profile as obtained from the densitometric analysis to assess the N-terminal integrity of the rec. glycoprotein. Incubation of parental CHO cell supernatant in presence of AEBSF,p-aminobenzamidine (data not shown) or human plasminogen activator inhibitor type 1 (hPAI-1) (figure 2) showed that the relative amount of the bands 1, 2, 2’, 3, 5 in the IEF/WB profile decreased significantly.

These data suggest that parental CHO cell culture supernatant contains protease(s) which can modify the glycoprotein of interest at the N-term. domain. The protease(s) responsible for the N-terminal modifications of the recombinant glycoprotein is a serine

protease inhibited by p-aminobenzamidme and could be a form of plasminogen activator.

313

4.3.

ZYMOGRAPHIC ANALYSIS OF PROTEOLYIC ACTIVITY IN PARENTAL CHO CELL CULTURE SUPERNATANT

Zymography was used to estimate the molecular weight of the protease(s) responsible of

rec. glycoprotein modifications in parental CHO cell culture supernatant (figure 3). Proteolytic activity against the rec. glycoprotein was observed mainly at 76 kD.

4.4.

ZYMOGRAPHIC DETECTION OF PROTEASE ACTIVITY AGAINST SUBSTRATE Z-GLY-GLY-ARG-AMC IN PARENTAL CHO CELL CULTURE SUPERNATANT

Z-Gly-Gly-Arg-AMC is described to be a good substrate to measure plasminogen

activator activity (Zimmerman & al, 1978). Z-Gly-Gly-Arg-AFC was used in zymography to determine if Z-Gly-Gly-Arg-AMC could be a good substrate to evaluate the activity of the protease responsible for the rec.

glycoprotein modifications (figure 4). Z-Gly-Gly-Arg-AMC is cleaved at the same molecular weight than the protease responsible for modifications of the rec. glycoprotein. Other experiments (data not shown) revealed that - Z-Gly-Gly-Arg-AMC cleavage in parental CHO cell supernatant is inhibited in the same way as the rec. glycoprotein modifications - rec. glycoprotein modifications are maximal at basic pH

- same zymographic results are obtained when analysing rec. CHO cell supernatants Z-Gly-Gly-Arg-AMC could be a good substrate to evaluate the activity of the protease responsible for rec. glycoprotein modifications.

314

4.5.

CHANGES IN CHO CELL CULTURE CONDITIONS TO REDUCE PROTEOLYTIC ACTIVITY

Culture temperature was modified in order to decrease proteolytic activity and increase rec. glycoprotein quality (figure 5-6).

Batch cultures conducted at different temperatures showed that culture temperature 29°C after 48 hours reduced significantly protease activity (about 3 times for 37-29°C

compared to 37°C). IEF profile confirmed the protease activity results : lower temperature could improve protein integrity, best conditions are a shift of temperature from 37°C to 33°C after 48 hours. Batch production at lower culture temperature could improve the glycoprotein quality in CHO cell culture supernatant.

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5. Conclusions

The study demonstrates that N-term. heterogeneity of a rec. secreted glycoprotein can result from CHO cell derived proteases in the culture supernatant. The protease responsible for the N-term. modifications was identified as a serine protease of 76 kD with an optimal basic pH. Inhibition of the protease can be obtained by PAI-1 suggesting that the protease could be a form of plasminogen activator. Specific assay (Z-Gly-Gly-Arg-AMC substrate) can be used to detect the protease in CHO cell culture supernatant. Batch production at lower temperature could improve the glycoprotein quality in CHO cell culture supernatant. 6. Acknowledgements

This work was supported by a F.I.R.S.T grant nr 2369 from the Walloon Region of Belgium 7. References Heussen, C. and Dowdle, E. B. (1980), Anal. Biochem. 102, 196-202. Lognonné, J.-L. (1994) 2-D analysis : a practical guide to principle critical parameters, Cell. Mol. Biol. 40 (1), 41-55. O’Farrell, P.M. (1975) High resolution two-dimensional electrophoresis of proteins, J. Biol. Chem. 250 (10), 4007-4021. Zimmerman, M., Quigley, J. P., Ashe, B., Dorn, C., Goldfarb, R. and Troll, W. (1978) Direct fluorescent assay of urokinase and plasminogen activators of normal and malignant cells : kinetics and inhibitor profile, Proc. Natl. Acad. Sci. USA 75 (2), 750-753.

DIGITAL IMAGE ANALYSIS: QUANTITATIVE EVALUATION OF COLORED MICROSCOPIC IMAGES OF ANIMAL CELLS

K. Falkner and E.D. Gilles Institut für Systemdynamik und Regelungstechnik Universität Stuttgart, 70550 Stuttgart, FRG E-Mail: [email protected]

Abstract

A fully automated PC-based image analysis system was developed to count and characterize animal cells in fermentation processes in a routine and reproducible way.

Chinese Hamster Ovary cells were used for the establishment of the system. Digital image analysis provided data for statistical evaluation and graphical representation of the cell population. This information improved the monitoring of the investigated processes and therefore facilitates the development of adequate process control

strategies. 1 . Introduction

Fast and accurate online determination of the number of viable cells plays an important role in fermentation processes with eucaryotic cells. Usually, the cells are stained, and then viable and dead cells are counted. There are different possibilities for counting

cells [1], e.g. Coulter Counter, flow cytometry and the conventional method by using the hemocytometer. In comparison to these methods, a digital image analysis (DIA) system has several advantages. It is easy to handle, the system is comparatively inexpensive and the results are operator independent. Fast characterization of the cells improves the monitoring of the investigated processes and therefore DIA is an important tool for the culture optimization. However, DIA can do much more than evaluation of staining methods: quantitative and reproducible determination of shape, size and morphology provides additional information about the state of the cells. DIA is therefor superior to the conventional manual method.

2. Material and Methods

CELL LINE, MEDIUM AND DYES. Chinese Hamster Ovary (CHO) cells were

cultivated in RPMI 1640 Medium (LIFE TECHNOLOGIES) supplemented with 5% fetal calf serum (LIFE TECHNOLOGIES) in culture flasks with 30 ml medium 317 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 317-319. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

318

at 37 °C in the presence of 5% The cell concentration was approximately cells/ml and after 3 days a cell passage was done. For the determination of the number of viable cells, the three different dyes Trypan Blue, Erythrosin B and Neutral Red were used. Before staining, 1 ml cell suspension was centrifugated for 5 min at 1500 rpm and subsequently resuspended in 0.5 ml PBS buffer (LIFE TECHNOLOGIES). Afterwards 0.4 % (w/v) dye solution prepared in 0.81 % NaCl and 0.6 % (w/v) was mixed with the cell suspension. Then, viable as well as non-viable cells were counted using a hemocytometer and a chamber (300µl) constructed for use with the DIA. IMAGE ANALYSIS. For image acquisition, a CCD color video camera (DXC-950P Power HAD, Sony) was coupled with an inverse microscope (Axiovert 10, Zeiss) and a PC equipped with an IMASCAN framegrabber. For image analysis the MS-Windows software package OPTIMAS (Stemmer) was used.

3. Results and Discussion For the fast determination of cell density and viability in fermentation processes, a fully automated DIA system was designed. The staining of dead CHO cells worked better with Erythrosin B than with Trypan Blue. Erythrosin B was taken up faster than Trypan Blue and the colouring was more intensive. Neutral Red was used to confirm the results from staining with Erythrosin B. Fig. 1 shows an original Erythrosin B stained image and the binary images resulting from image analysis. Table 1 shows the mean values of extracted data of a 3 days old culture of CHO cells.

For a comparison of the DIA system to the manual counting method, the data from all experiments were pooled for further statistical analysis. Table 2 shows that the DIA method provides better results, but still the confidence interval is only a little smaller than the confidence interval of the manual method. The variation of DIA data is mostly explained by the presence of cell heaps. If there are only single cells in the cell suspension, DIA is evidently superior to the manual counting method.

319 TABLE 2. A comparison of statistical results for the mean number of dead cells contained in 1 ml cell suspension

With the CHO cell line used in our experiments, it was not possible to omit the staining step and use only morphological characteristics of the cells for the determination of cell viability. In contrast to the results reported [2], we did not find any significant difference in size, shape or density of viable and non-viable cells. The results show that DIA is superior to the manual hemocytometer method, and an important additional advantage is the possibility of automation.

For automation of viability determination in fermentation processes, an online image processing system, developed for the characterization of morphology of mycelial microorganisms [3], can be used. Sampling, dilution, image acquisition and image analysis are done automatically under the control of a PC. Figure 2 shows the experimental set-up of the online DIA system.

4. Acknowledgements

The work was supported by the German Federal Ministry of Science and Technology (BMFT) and the Dr. Karl Thomae GmbH. The authors would like to thank Prof. Pfizenmaier for kindly providing the CHO cells and the possibility to perform the experiments at the Institut für Zellbiologie und Immunologie, Universität Stuttgart.

5. References [1] [2] [3]

Konstantinov, K. et al. (1994) Real-time biomass-concentration monitoring in animal-cell cultures, TIBTECH 12, 324-333 Frame, K. K. and Hu, W.-S. (1989) Cell Volume Measurement as an Estimation of Mammalian Cell Biomass, Biotechnol. Bioeng. 36, 191-197 Treskatis, S.-K. (1995) Wachstum, Morphologic und Antibiotikaproduktion von Streptomycten

in Submerskultur , PhD thesis, Universität Tübingen, Germany

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THE VIABLE CELL MONITOR: A DIELECTRIC SPECTROSCOPE FOR

GROWTH AND METABOLIC MACROPOROUS BEADS

STUDIES

OF ANIMAL

CELLS

ON

Y. GUAN and R.B. KEMP Institute of Biological Sciences University of Wales, Penglais, Aberyshvyth, SY23 3DA, UK

Abstract. One of the problems in using macroporous carriers to grow animal cells in culture has been to assess biomass on-line. The possibility that dielectric spectroscopy could be the solution was explored using a Viable Cell Monitor (VCM) optimised for animal cells. The instrument was validated using suspension-adapted CHO cells and was shown to measure the volume fraction of viable cells. It was then applied to

Cytopore 1 carrier cultures over 7-day periods, the results indicating that the technique was a reliable complement to metabolic studies. 1. Introduction

The biotechnological exploitation of animal cells to produce monoclonal antibodies

and recombinant proteins in culture has focused attention on the need to provide a precisely controlled environment for their growth. This was recognised more than a decade ago [1] but there is still a paucity of on-line biosensors to monitor culture variables [2], let alone use them to control the process. This is true even of the fundamental measurement of biomass where the only two methods available are optical density using laser light [3] and dielectric spectroscopy [4]. Whereas the former gives the total number (living and dead) of cells, the latter identifies only the living cells. Both sets of information are valuable in assessing percentage viability but viable cell number is of primary importance in metabolic studies and those relating to the efficiency of target protein production. Commercial dielectric spectroscopes designed to monitor the biomass of microorganisms have been available for several years [4, 5] and have been employed with limited success to measure the number of viable animal cells in culture [6]. It is only recently, however, that one optimised for that purpose has come on the market with the name of Viable Cell Monitor (VCM). The first aim of the present study was to evaluate the instrument using Chinese hamster ovary (CHO) cells growing in suspension. 321 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 321-328. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

322

The productivity per unit volume of animal cells in simple batch culture is poor because the cell number concentration is comparatively low at the end Various devices have been used to increase the numbers, including fed-batch and perfusion cultures, but one of the most promising ways is to allow the cells to stick to macroporous carriers [7]. A difficult problem with this method, however, is to ascertain the number and viability of the cells because many of them are buried deep within the bead. There was preliminary evidence that the dielectric spectroscopy could fulfil this role [8] and. thus, the second aim of the study was to see if the VCM was appropriate for this task. Earlier it had been shown that CHO cells could be grown in macroporous carriers for 3 weeks |9| and so these were used for this work.

2. Theoretical

A detailed theoretical treatment of the theory for measuring biomass by dielectric spectroscopy is available elsewhere [5] but. as a simplified account, the electric field set up by the 4 electrode pins of the VCM probe in the biorcactor creates a force field which pushes ions in opposite directions until stopped by intact cell plasma membranes [10]. Thus, a charge separation (polarisation) is set up at the poles which is measured as capacitance in Farads (F) by a phase shift between the outer electrodes (current) and the inner ones (voltage). The more the cells, the greater the amount of plasma membrane and the more the capacitance Dead cells and debris do not have intact and/or functioning plasma membranes and so do not contribute to capacitance. It is obviously necessary to reverse the field periodically and the rate of change in direction is measured by its frequency as Hertz (Hz). This has a profound effect on capacitance since the ions need a finite time to move up to and polarise the plasma membranes. The higher the frequency, the less time there is for polarisation and the smaller the value for capacitance. The curve of capacitance against frequency is called the The most appropriate frequency for animal cells has been found to be 0.5 Hz and the VCM is fixed at this point. The culture medium has a residual capacitance which must be deducted from the value for the cell suspension to give the capacitance increment of the As the biomass (cell number) concentration increases, so does the size of Capacitance of the cell suspension has a complex relationship to the conductivity of it. If the latter is constant during growth or only changes slightly, then no allowance for it needs to be made but there can be quite significant changes during long-term culture at high cell densities. For this reason, the VCM can be calibrated for a range of conductivity values simply by adding salt to a test sample of culture medium. Without this cross-talk calibration, an increase of from an initial value of can produce a shift in capacitance from 0 pF to 0.1 pF.

323

3. Experimental

The cells were CHO 320 which produce recombinant interferon when grown in a defined medium based on RPMI 1640 [11] with BSA free of fatty acids [12] For some experiments the cells were grown on Cytopore 1 macroporous carrier beads (Pharmacia) suspended at a mass concentration of The Applicon bioreator system (Applicon Ltd., Tewksbury. Glous.. UK) controlled the bicarbonatebuffered pH at temperature at and the dissolved oxygen concentration at 55% saturation [12]. It also received the analogue capacitance and conductivity signals from the VCM (Aber Instruments Ltd.. Aberystwyth. SY23 3AH, UK) operating at 0.5 MHz. All the digitised signals were logged by Applicon BioXpert and its moving average facility was used over 1 h to smooth the capacitance signal.

Off-line measurements [12] were made of cell density by Coulter counter Model D. (Coulter Ltd.. Luton, UK) and of cell size by Skatron Argus 100 flow cytometer (Skatron Ltd., POB 34, Newmarket, UK). The latter was calibrated with a range of highly monodisperse latex beads (Dyno Particles AS, POB 160, Lillestrom, Norway). Since these always underestimate cell size. 400 cells were photographed and measured for size distribution [12], The concentrations of glucose and lactate were measured, after deproteinisation, using respectively. Sigma kits 635 and 826. Protein was assayed by the BioRad DC protein assay kit. 4. Results and Discussion

When CHO 320 cells were cultured in suspension for 140 h. the capacitance curve gave the characteristic bell-shape (Figure 1A). depicting three phases; an increase for the first 80 h, followed by a plateau and then by a decline for the final 40 h. Statistical analysis gave a standard error for the curve of 0.085 pF. Calibrating capacitance in terms the viable cell number concentration gave the following semi-empirical correlation:

(1) where is the increment of viable cell number concentration and that of capacitance. There was considerable data scatter, but this was reduced by using the moving average smoothing technique over 1 h (see Figure 1A). As a result, the lowest measurable cell density was It was also calculated that 1 pF was equivalent to approximately It was necessary to establish what exactly capacitance measures in terms of biomass. Since animal cells take up a gross spherical shape in suspension, the dielectric principle of measurement is [15,12]. (2)

324

where K is a constant, N v is cell number concentration, V cell is the average individual cell volume and is volume fraction of the cell entity. Eq. (2) indicates that the capacitance measurements could relate to individual cell size and/or viable cell density and/or the volume fraction of viable cells. Estimates were made of all three variables and the results are shown in Figure 1B. In the first 100 h. the average cell volume varied by ca. 21%. In relation to Eq. (2). this finding implies that using the VCM signal to represent viable cell density would give a 20% relative error when compared to less than 10% for the volume fraction This difference is illustrated in Figure 1B which clearly shows that capacitance was better reflected in the volume fraction than in the viable cell density for the first 100 h. Cell counts were also unreliable at low number densities. This is common for Coulter counting and is due to sampling errors. After 100 h. there was a decline that correlated poorly with the viable cell volume fraction but may have been due to apoptotic cells, which are known to be smaller than living cells [13].

When the cells were grown on Cytopore 1 beads for long periods in batch culture, the medium had to be renewed by partial replacement after allowing the beads to settle for a short while by stopping the agitator. Thus, the growth as represented by the smoothed capacitance trace, was punctuated by sharp peaks (Figure 2A). Nevertheless, there was considerable growth over the 168-h period representing It is not possible to estimate the accuracy of this number because a direct measurement cannot be performed, but a similar increase with time was shown for the off-line protein measurement. Protein concentration in itself cannot be an accurate measurement of viable cells because (i) dead ones would accumulate in the carrier pores and (ii) BSA and other proteins can be trapped in the beads. However, the increase could be indicative of cell growth. The changing capacitance signal could not have been due to altered conductivity because there was very little change in it over the period (see Figure 2A).

325

Some indication of the metabolic rate of the cells could be gauged from the consumption of glucose (Figure 2A). With growth, the total glucose used by the cell population increased so that, with replacement taking place at regular intervals, the maximum amount of substrate available to the cells gradually decreased with time. One of the characteristics of cells in culture is that they produce considerable amounts of lactate. This can be seen in Figure 2B as a virtual mirror image of glucose consumption. Of course, some of it is removed as medium is replaced to give the sawtooth appearance. Lactate was excreted as a result of glycolysis and the oxidation of glutamine [11,12] but this was not due to anaerobic conditions, the oxygen saturation being maintained at 55%. Rather, it seems to be caused by the poorly designed medium which does not contain a sufficient amount of the required biosynthetic precursors [12,14], The cell has to resort to making some of the necessary components by oxidoreductive catabolic processes, leading to the formation of lactate as a by-product under fully aerobic conditions. Its excretion causes the pH of the culture medium to fall, necessitating the introduction of NaOH to neutralise it. The on-line record of this titration is probably a fairly accurate reflection of cell growth (Figure 2B), better than the protein estimation.

Although a great deal more needs to be done to authenticate the use of dielectric spectroscopy to measure biomass in beads, it is probable that it will become the standard method of assessment in due course. More confidence in its application could be derived from studies on whether it measures in proportion the number of cells in free-standing aggregates. Once validated for animal cell systems, the VCM will become a valued tool in metabolic studies because it makes these other measurements specific to size, in this case volume. This is already being done for the estimation of overall metabolic rate by combining the VCM with flow microcalorimetry to give heat flux [12]. a variable which can be used on-line to control the growth of animal cells in suspension culture.

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5. Acknowledgements The authors are thankful to the Nuffield Foundation (NUF-URB97) and the Wellcome Trust (VS/3/97) for vacation scholarships to Dr R.B. Kemp which enabled Miss Rachel Ryan and Mr Stuart Martin, respectively, to

assist in part of these studies. He gratefully acknowledges that the research was supported by BBSRC grants, 2/3680 and 2/TO7389.

6. References 1. Cartwright, T. (1994) Animal Cells as Bioreactors. Cambridge University Press, Cambridge, UK. 2. Zhou, W. and Mulchandani. A. (1995) Recent advances in bioprocess monitoring and control. American Chemical Society Symposium Series 613, 88–98. 3. Zhou. W.C. and Hu. W.S. (1994) On-line characterisation of a hybridoma cell-culture process. Biotechnology and Bioengineering 44, 170–177. 4. Harris. C.M., Todd, R.W., Bungard. S.J., Lovitt. R.W., Morris, J.G. and Kell. D.B. (1987) The dielectric permittivity of microbial suspensions at radio-frequencies: a novel method for the real time estimation of microbial bilmass. Enzyme and Microbial Technology 9, 181–186. 5. Davey, C.L. and Kell. D.B. (1995) The low-frequency dielectric properties of biological cells, in D. Walz. H. Berg and G. Milazzo (eds.), Bioelectrochemistry of Cells and Tissues, Birkhauser Verlag, Basel, Swizerland, pp. 159–207. 6. Cerckel. I., Garcia. A.. Degouys. V., Dubois. D., Fabry. L. and Miller, A.O.A. (1993) Dielectric spectroscopy of mammalian cells 1. Evaluation of the biomass o f HeLa- and CHO cells in suspension by lowfrequency dielectric spectroscopy. Cytotechnology 13, 185–193. 7. Blüml, G.. Reiter. M., Gaida. T.. Schmatz. C., Assadian, A.. Strutzenberger, K. and Katinger, H. (1994) Development of a new type of macroporous carrier, in R.E. Spier, J.B. Griffiths and W. Berthold (eds.), Animal Cell Technology, Butterworth-Heinemann, Newton, MA, USA, pp.267–269. 8. Degouys, V., Cerckel. I., Garcia. A.. Harfield. J., Dubois, D., Fabry. L. and Miller, A.O.A. (1993) Dielectric spectroscopy of mammalian cells 2. Simultaneous in-situ evaluation by aperture impedance pulse spectroscopy and low-frequency dielectric spectroscopy of the biomass of HTC cells on Cytodex 3. Cytotechno/ogy 13, 195–202. 9. Reiter. M., Borth. N., Blüml. G., Wimmer. K., Harant. H., Zach. N., Gaida. T., Schmatz, C. and Katinger, N. (1992) Flow cytometry and two-dimensional eletrophoresis (2-DE) for system evaluation of long term continuous perfused animal cell cultures in macroporous beads. Cytotechnology 9, 247–253.

10. Davey, C.L., Guan. Y., Kemp. R.B. and Kell. D.B. (1997) Real-time monitoring of the biomass content of animal cell cultures using dielectric spectroscopy, in K. Funatsu, Y. Shirai and T. Matsushita (eds.). Animal Cell Technology. Vol. 8, Kluwer Academic Publishers. Dordrecht, The Netherlands, pp.61–65. 11. Hayter. P.M., Curling. E.M.A., Baines. A.J., Jenkins, N., Salmon. I., Strange, P.G. and Bull, A.T. (1991) Chinese hamster ovary cell growth and interferon production kinetics in stirred batch culture. Applied Microbiology and Biotechnology 34, 559–564. 12. Guan. Y . Evans. P.M. and Kemp. R.B. (1998) Specific heat flow rate: .An on-line monitor and potential control variable of specific metabolic rate in animal cell culture that combines microcalorimetry with dielectric spectroscopy. Biotechnology and Bioengineenng (in press). 13. Wyllie, A.H. and Durall, E. (1992) Cell death, in J.O’D. McGee. P.G.Isaacson and N.A. Wright Oxford Book of Pathology, Vol. 1. Oxford University Press. Oxford. UK. Pp. 141–157. 14. Kemp, R.B. and Y. Guan (1998) Probing the metabolism of genetically-engineered mammalian cells by heat flux. Thermochimica Acta (in press).

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Discussion

Godia:

The capacitance measurement you make, is it not at a constant frequency? You also mention the importance of changes in conductivity in the medium and that you correct them by on-line conductivity measurements in parallel to your electrical measurements.

Kemp:

This is done at a set frequency of 0.5 MHz. This is not necessarily ideal for every mammalian cell, and the system can be changed to other frequencies. For conductivity measurements the instrument is calibrated with a range of conductivities by adding salt. The instrument will automatically adjust for changes in conductivity because conductivity will affect the capacitance measurement.

Konstantinov:

You stated that there are two basic methods for measuring cell density: the optical and the capacitance. I believe they are not conflicting, but complementary, because the optical method measures total cell density and the capacitance measures viable cell density, so you can combine both signals to measure viability. Can you comment on this possibility, and would you need 2 sensors to do this?

Kemp:

I think it is a good point. We have not tried it because we cannot afford to buy the additional sensors. I think that it is an excellent way of going forward.

Al-Rubeai:

Putting additional sensors into a culture gets complicated. Why is it necessary to have on-line measurement of cell number or viability? A one off sample gives you opportunities for flow cytometry, which measures viability very simply.

Kemp:

We want to measure the specific rate of heat production, and the combination of heat flow and biomass measurements will allow you to control the culture. The advantage of heat flow is that it is an instant rate, you do not have to measure concentrations of things. So when the cells start to decline you can immediately adjust the conditions.

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Ozturk:

Wurm:

A comment on Al-Rubeai’s question: if you are doing a batch culture, you do not need to measure cell density because you have to live with what you have. However, perfusion of fed-batch systems, would benefit from this especially in high density culture where conditions change fast. Do the size and number of gas particles in your reactor affect the electronics?

Kemp:

No, it is a sparged system and there is no problem because bubbles do not charge up.

MEASUREMENTS OF CHANGES IN CELL SIZE DISTRIBUTION TO MONITOR BACULOVIRUS INFECTION OF INSECT CELLS. ERNESTO CHICO1,2 and VOLKER JÄGER1. 1 Gesellschaft für Biotechnologische Forschung mbH Mascheroder Weg 1, D-38124 Braunschweig, Germany. 2 Center of Molecular Immunology POBox 16040, Habana 11600, Cuba.

Abstract We have found that the cell size distributions measured by a CASYTM cell counter can be used to follow the infection process of various insect cell lines. After infection, cell size deviates from the typical distribution of exponentially growing cells, shifting to an increased amount of cells with bigger cell diameters. This deviation has proved to be dependent on the MOI as well as time post-infection. A method is proposed to estimate the degree of infection of a population of insect cells based on the cell size distribution (CSD). The potential of using this method for measuring the ratio of infected to non-infected cells is discussed.

Experimental conditions Cell size distributions were measured using a CasyTM cell counter with a measuring capillary of 150 µ m. No more than 5000 cells per single measurement were counted to avoid particle clumping inside the capillary. Most of the experiments were carried out with the BTI-Tn-5Bl-4 (High Five™) cell line grown in suspension using fortified ExCell 401 serum-free medium. A recombinant AcNPV was used for infection. Main experimental observations During some stages of the baculovirus infection, there is a mixed population of

infected and non-infected cells in the culture. However, there is a lack of a simple experimental method to distinguish between infected and non-infected cells, which can be reliably used for a numeric estimation of the degree of infection (DOI) of the culture, refered as the fraction of infected cells in the population. Cell size distribution (CSD) of insect cells, measured by a CASY™ cell counter, do not change significantly during the batch growth curve (data not shown). On the contrary, during the infection process the CSDs of insect cells deviate from the typical distribution of exponentially growing cells, shifting to an increased amount of cells with bigger cell diameters. This deviation has proved to be dependent on the

multiplicity of infection (MOI) as well as on time post-infection as shown in Figs. 1 and 2. Our experiments prove that cell size clearly increases with the time post infection and with increasing MOI, becoming a potential early qualitative indicator of a successful infection process. This qualitative indicator of infection could help in decisions 329

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 329-331. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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concerning virus transfection and propagation as well as during recombinant protein production.

From qualitative monitoring to numeric estimation of the degree of infection

Based on our experimental data as well as on the statistical nature of the CSD measurements a calculation method is proposed for numeric estimating of the degree of infection in insect cell populations using its CSD.

331 Principles of the method: • A population of insect cells

after infection can be regarded as a mixture of infected and uninfected (growing) cells. • The fraction of cells in the population, which can not be statistically described as uninfected

cells,

will

be

considered as infected cells.

This method has been calibrated using samples of known degree of infection, resulting in good linearity and reproducibility. However, interference could be expected from other events affecting cell size (formation of cell aggregates, oxygen limitation,etc.) Conclusions From the changes in the cell size distribution of insect cells after baculovirus infection, it was possible to develop a method of monitoring the degree of infection of insect cell populations. Since the measurement of cell size distribution is a reproducible and rapid technique, the detection of changes in cell size after infection represents a more simple

approach to evaluate the success of baculovirus infection in comparison with other techniques such as PCR, plaque assay or detection of protein production. The approach for numeric estimation of the number of infected cells presented in this work

is simple, reliable, and has a lot of potential applications in process design and control, mathematical modelling and provides a tool for better understanding of the kinetics of baculovirus infection. Acknowledgement We want to thank Schärfe System GmbH for providing financial support allowing E. Chico to attend ihe 15th ESACT Meeting.

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DEAD CELL ESTIMATION - A COMPARISON OF DIFFERENT METHODS

A. FALKENHAIN , TH. LORENZ*,U. BEHRENDT*, J. LEHMANN Cell Culture Technology, P. O. Box 100131, University of Bielefeld, Germany *Boehringer Mannheim GmbH, Nonnenwald 2, 82377 Penzberg, Germany

1. Introduction

The success of a mammalian cell cultivation depends on the ratio of viable and dead cells - the viability. The viability is routinely measured by the trypan blue exclusion method [1]. In short cultivation processes the density of viable cells normally is high. Therefore, the trypan blue exclusion method gives an exact value for the viable as well as for the dead cells. For longer cultivation processes e.g. fed batch or dialysis

fermentations the trypan blue exclusion only shows a snapshot of the status of the culture: cells which died previously might have lysed and cannot be determined

microscopically. Therefore, further methods were evaluated to determine the total amount of cells which had been present during the cultivation process at a given time: conductive electronical cell count (CASY 1®; Schärfe System GmbH, Reutlingen, Germany) [2], biochemical determination of lactate dehydrogenase (LDH) [3] and biochemical determination of DNA [4]. 2. Material and methods

Murine hybridoma have been cultivated in RPMI based media in 1000 1-bioreactors [5, 6]. The cell densities were determined using the Trypan blue (Cat. No. 15250-061, Life

Technologies GmbH, Eggenstein, Germany) exclusion method and the CASY l ® - Cell Counter and Analyzer System (Schärfe System GmbH, Reutlingen, Germany).The principle of this method is the change of the conductivity along an aperture during the flow of a cell containing liquid. A pulse area analysis is performed. The result of a measurement is a size distribution curve. A 150 µm capillary was used to detect particles between 3.4 and 30 µm in diameter. The LDH activity was measured using a test combination (Cat. No. 139 084, Boehringer Mannheim GmbH, Germany) which has been adapted for the determination of LDH on a Hitachi 705 analyser. The DNA concentration was measured by staining with bisbenzimide (Cat. No. B 1782, SigmaAldrich Chemie GmbH, Germany). The DNA-bisbenzimide complex is detected with a fluorescence photometer. 333 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 333-336.

© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

334

3. Results and discussion

For the quantification of dead cells by analysis of LDH activity and DNA concentration their intracellular content had to be determined. The intracellular LDH activity is instable during a cultivation and has to be determined for each sample of a fermentation process. The intracellular DNA concentration is regarded to be constant and has to be

measured only once for a specific cell line. The number of dead cells was calculated as follows: Number of dead cells = c (LDH in supernatant) / c (LDH per cell) Number of dead cells = c (DNA in supernatant) / c (DNA per cell) where c means concentration. The size distribution curve of the CASY® method was

classified for three types of particles: 3.4 to 5 µm cell debris 5 to 10 µm dead cells and 10 to 30 µm viable cells. Distribution curves for samples of a cultivation taken at different fermentation times are shown in Figure 1A and 1B. During a course of fermentation the amount of cell debris ( 3 . 4 - 5 µm) as well as the amount of dead cells (5 - 10 µ m) increases significantly. For both values a linear correlation versus the number of dead cells estimated by the LDH method was observed. Since dead cells disintegrate in an unknown number of particles it is difficult to assign the counts (3.4 - 10 µm) to a number of dead cells. Therefore the counts in the range of 5 - 10 µm were used during the following considerations.

335

Figure 2 shows the time course of dead cells during a fermentation process determined by the four different methods.

The dead cells measured by the trypan blue exclusion method show an inconsistent course. The number is very low due to the fast disintegration of dead cells and depends on the operator. The DNA concentration gives a higher number of dead cells during the fermentation. During the last third of the process the concentration is constant which

might be due to an equilibrium between the release of DNA from the cells and the degradation of DNA in the culture supernatant. The increase of the DNA concentration is observed later than the increase of the LDH activity. This can be explained by the higher stability of the cells' nucleus in comparison to the cells themselves. An increase of the LDH activity during fermentation can already be measured after about 40 hours. This shows the high sensitivity of this parameter. Since the enzyme is highly stable (about 10 days in RPMI based medium) [3] cells which just have lysed as well as cells which lysed previously are included. The density of the dead cells determined by CASY 1® shows a similar course in comparison to the LDH method. Slightly higher values were obtained.

336

4. Acknowledgement Many thanks to ESACT which offered a bursary.

5. References 1) 2)

3) 4)

5)

Cook, J.A., Mitchell, J . B . (1989) Viability measurements in mammalian systems, ANALYTICAL BIOCHEMISTRY, 179, 1-7 Winkelmeyer, P., Glauner, B., Lindl, T. (1993) Quantification of cytotoxicity by cell volume and cell proliferation, ATIA, 21, 269-280

Goergen, J.L., Marc, A., Engasser, J. M. (1993), Determination of cell lysis and death kinetics in continuous hybridoma cultures from measurement of lactate dehydrogenase release, CYTOTECHNOLOGY, 11, 189-195 Loontiens, F . G . , McLaughlin, L. W., Diekmann, S., Clegg, R. M. (1991) Binding of Hoechst 33528 and 4', 6-Diamidino-phenylindole to self-complementary decadeoxynucleotides with modified exocyclic base substituents, BIOCHEM1STRY, 30, 182-189 Comer, M. J., Kearns, M. J., Wahl, J., Munster, M., Lorenz, Th., Szperalski, B., Koch, S., Behrendt, U., Brunner, H. (1990) Industrial production of monoclonal antibodies and therapeutic proteins by dialysis fermentation, CYTOTECHNOLOGY , 3, 295-299

6)

Behrendt, U, Koch, S., Gooch, D. D., Steegmans; U., Comer, M. J. (1994) Mass spectrometry: A tool for on-line monitoring of animal cell cultures, CYTOTECHNOLOGY, 14, 157-165

FED-BATCH CULTURE DEVELOPMENT BASED ON BIOMASS MONITORING

Eric DE BUYL1, Alan M AXWELL 2 and Luc FABRY1 1. SmilhKline Beecham Biologicals s. a., 89 rue de l’Institut, B - 1330 Rixensart 2. University of Paisley, High Street, Paisley PA12BE, Scotland

INTRODUCTION It is known that glucose level in the culture medium is an important parameter influencing mammalian cells physiology (for inst., see ref.l); growth, lactate accumulation, recombinant protein production and glycosylation are among the parameters likely to be affected. The aim of this work was to investigate the effect of glucose level on the growth parameters of a recombinant CHO cell line expressing a viral glycoprotein (glucose consumption, recombinant protein production); the development of a feeding policy was also tested; the principle of this policy was based on the knowledge of the specific consumption rate in various growth conditions and on the on-line measurement of the biomass with an optical density sensor.

M ATERIAL AND METHODS Batch, fed-batch and continous culture were realised at the 1,5L scale (B.Braun Biostat B bioreactors); the growth conditions were: proprietary protein-free medium, temperature 33°C (see ref.3 for a discussion on growth at sub-physiological temperatures), initial pH of 7,4, maintained above 7,2 through head-space aeration, dissolved oxygen 35% of air saturation through O2 sparging. During fed-batches, glucose level was maintained by glucose addition through manual adjustment of a peristaltic pump setting, based on off-line glucose concentration measurement (YSI glucose/lactate analyser). Seed culture were produced in disposable shake flasks (500 ml total volume, 150 ml of culture, 100 RPM), the PDL at the seeding being identical for each bioreactor scale.

Fed-batches were also realised at the 10L scale, in a bioréactor (MBR) equipped with a Wedgewood optical density sensor. Glucose level was maintained in the same way, except during automated feeding trial, where the setting of the pump was based on the optical probe reading through a calibration curve of glucose consumption vs. OD 337 O.-W. Merlen et al. (eds.), New Developments and New Applications in Animal Cell Technology, 337-342. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

338

The evaluation of the glucose effect was based on the determination of kinetic parameters : qs, specific glucose consumption rate; qp, specific rec.protein production rate; qlac, specific lactate production rate (expressed in mM/106 cells.hour for qs and qlac and in µg/106cells.hour for qp) and of the release of protease in the growth medium; cathepsin L was chosen as a marker because it was demonstrated previously for other recCHO lines that its release in the growth medium was strongly affected by growth conditions, even for equally high viabilities during the culture (always higher than 90% during all the cultures). The specific growth rate was estimated by fitting of the equation dX/dt = µX (SigmaPlot software) to the data of cell count in function of time.

The recombinant protein was analysed by Elisa and Western Blotting (Pharmacia Phast system), with detection either with a Mab or with various lectins, to obtain a preliminary assessment of the quality of its glycosylation (kit Boehringer n° 1210 238). RESULTS AND DISCUSSION

1. Glucose level effect on cell metabolism The biomass, glucose, lactate and recombinant protein concentration profile of three relevant 10L bioreactor trials are given in fig. 1.

339

The specific glucose consumption rate is decreasing during fed-batches (fig.3 a to d); the mean value of is higher for higher glucose concentrations (fig. 2); however, the value of is not very reproducible (trial E16 vs. trial E17 for instance); the effect of glucose concentration is less clear on (contrary to what has been observed by others (ref. 1); these tendencies are also observed during chemostat trials (fig. 2): is reduced two fold and five fold during glucose-limited chemostat (residual glucose 0,19 mM), but is hardly affected by glucose limitation.

340

2. Development of an automated feeding policy The linearity of the link between cell count and the reading of the Wedgewood probe is well established (fig.4). The specific glucose consumption rate is steadily decreasing during the cultures (fig.3a to 3d). A calibration curve, with plotted vs. optical density, was established on the basis of the results obtained during trial E12/E13 (fig 5). However, it can be concluded that the evolution of is not reproducible enough to be used in a robust automated glucose feeding system: the glucose level during the automated feeding trial (trials El6 and El7) increases indeed very quickly above the set point of 1.1 mM. The arrows indicates the point where manual adjustment of glucose feed is resumed (fig. 1)

341

A slight tendency toward a decrease in protease release with decrease in glucose concentration could be concluded from fed-batches results; this tendency is not confirmed by chemostat results (fig. 6).

Although it gives only a limited insight into the glycosylation pattern of the protein,

the blots (fig.7) revealed with MAA (Maackia amurensis) lectin suggests that only in the conditions of severe glucose limitation is the glycosylation affected (main band detected being splitted in two sub-bands); the blots for all fed-batches are similar to the one for chemostat at 8.5 mM residual glucose (results not shown). Since the protein of interest here is heavily glycosylated, the determination of its heterogeneity as described in ref.2 (densitometry of WB) is probably not possible (detection with other lectins gives a smear, presumably because of an heterogeneous glycosylation).

342

CONCLUSION The glucose concentration in the growth medium influences specific glucose consumption rate; however, a glucose limitation well below 1.1 mM is needed to modify the specific growth rate or the lactate production rate; the limitation of lactate production could be interesting to facilitate scaling up of the production, as far as pH control is concerned.

The specific recombinant protein production rate is not affected by glucose limitation; however, a detailed analysis of the glycosylation should be carried out on sample produced under glucose limitation; a modification of the glycosylation in these

conditions is indeed detected by a simple lectin blot. The extracellular protease release is hardly affected by the glucose level for this cell line. The growth is adequately described by fitting the cell counts with the equation dX/dt = µX (exponential growth); again, between 1.1 mM and 16 mM, specific growth rate is not affected by glucose concentration. The cell count is well predicted by the reading of the Wedgewood probe; the specific glucose consumption rate, however, is not reproducible enough to be used as part of a glucose regulation loop; this lack of reproducibility is probably partly due to slight, unnoticed, modifications of seed culture generation; a clear tendency of diminution of with glucose concentration can be detected in the trials carried out at various glucose concentration from 1.1 to 16 mM; however, this variation is not very reproducible in our conditions, leading to the failure of the feeding policy tested. KEYWORDS

rec CHO cell line, fed-batch, chemostat on-line measurement, optical density, specific glucose consumption, specific recombinant protein production.

REFERENCES 1. Fieder, J., Schorn, P., Bux, R. and Noé, W., Cytotechnology 14 (suppl. l, 2.15), 1994 Increase of productivity in recombinant CHO-cells by enhanced glucose level 2. Hayter, P. et al., Biotech.Bioeng. 39, pp.327-335, 1992 Glucose-limited chemostat culture of CHO cells producing recombinant hu-INF 3. Kretzmer, G. et al., Proceedings of the 14th Esact meeting, p. 319, 1997 Temperature - A factor influencing cell behaviour

ON-LINE MONITORING OF PROTEIN AND SUBSTRATE/PRODUCT CONCENTRATION IN MAMMALIAN CELL CULTIVATION PROCESS

H.Lübben, J. Hagedorn and T. Scheper Institut für Technische Chemie, 30167 Hannover, Germany

Introduction

The characterisation and optimisation of mammalian cell culture process requires an on-line monitoring of product as well as low molecular weight concentration. For this purpose an automated analyzer based on the principle of flow injection analysis (FIA) was developed. It is an modular and flexible three channel set-up containing pumps, different valves, oxygen electrodes with amplifiers, a fluorescence detector and space for buffer reservoirs. The system can be used to monitor protein concentration via immunoassay as well as substrat and product concentration by enzymatic reaction. Heterogenous immunoassay

For the heterogenous immunoassay product depending antibodies or protein G are immobilized on a polymer loaded in a flow-through cartridge.

After sample injection all target proteins bind to the antibodies and will be eluted after a washing step. The protein fluorescence is monitored at 280 nm (exc.) and 340 nm (em.), so that labeling of the analyte is not necessary. The advantages of this set up are: short analysis time in the range of 6-8 minutes for each cycle, no sample dilution is necessary. Furthermore the immuno components can be used several times, which is very economically due to the high costs of specific antibodies. 343 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 343-346.

© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

344

Enzymatic Analysis

For the analysis of low molecular weight metabolites, enzymes are immobilized on a epoxy activated support material. The detection is carried out by amperometric measurement of oxygen consumption. This method allows simultanous determination of different analytes such as glucose, maltose, L-amino acids, saccharose and L-glutamine.

345

Sampling

To obtain a representative, cell free sampling from the reactor for FIA analysis, a tubular in-situ filtration probe was applied during cultivation process (ESIP, EppendorfNetheler-Hinz, Germany). The sampling module is compatible with 19 mm or 25 mm standard port of a bioreactor. For the module a polypropylene micro filtration membrane with a pore size between 0,2 and 0,6 µm is used, providing a sterile barriere that cannot be penetrated by any microorganism. The module contains a small dead volume of less than 2 ml, a large filter surface of nearly 40 qcm and new membrane sealings made of PTFE ferrules well, known in HPLC technique. Continuous and discontinous performance with flow rates up to 2 ml/min are possible. The sampling device can be sterilised together with the culture medium inside the reactor. Experimental data

The major advantages of both techniques are their high selectivity, the short analysis time (6-8 or 3 minutes) and that the immobilized components can be used several times.

Product concentration:

The comparison of HIA showed a good correlation to ELISA data with a deviation below 5 %. A higher accuracy was achieved (CV < 5%) using a triplicate sample determination. The HIA detection in this case was performed off-line. A higher frequency seemed to be not appropriate regarding the low specific production rates of animal cells. Using the ESIP sampling module the detection frequency can be increased up to every 6 minutes. In order to preserve the activity of the immobilised antibodies a frequency every 30 minutes is recommended.

346

Substrate concentration: As a main substrate in all mammalian cell culture processes glucose was chosen as a target for on-line monitoring and as a modell system. The comparison of on-line and offline data (YSI 2700 analyser) showed sufficient correlation of the concentration values. See Figure 5.

The interruptions and interferences pointed out as (1) (2) and (3) were caused by air bubbles on the amperometric electrode or by blocking of the tubing after microbial contamination.

Aknowledgements The authors would like to thank the European Commission Directorat General XII for supporting the participation in this meeting and the presentation of our research. Reference Hilmer, J.-M., Scheper, Th.: A new Version of an In situ Sampling System for Bioprocess Analysis. Acta Biotechnol.16 (1996) 2-3

PROCESS CONTROL AND ON-LINE FEEDING STRATEGIES FOR FED-BATCH AND DIALYSIS CULTURES OF HYBRIDOMA CELLS J. O. SCHWABE, R. PÖRTNER Technische Universität Hamburg-Harburg, und Bioverfahrenstechnik, Denickestr. 15, D-21071 Hamburg, Germany

1. Abstract The concept of the fed-batch strategy was to minimise the formation of inhibiting metabolites and to increase the yield of monoclonal antibodies by carefully supplying substrates. A process control system based on fieldbus technology was used for monitoring and control. External program routines were implemented to control dissolved oxygen (DO) and to calculate the oxygen uptake rate (OUR) and cumulative

oxygen consumption (COC) simultaneously. Concentrated feed solution was supplied according to the off-line estimated stoichiometric relation between oxygen and glucose consumption (GC). The developed feeding strategy initiated feeding automatically when the OUR decreased because of substrate limitation. Hybridoma cells were cultivated in batch and fed-batch cultures in laboratory scale using media containing an iron-rich, protein-free supplement. The antibody concentration increased three- to four-fold compared to the conventional batch culture when applying this strategy. It was not possible to avoid inhibition by metabolic products such as lactate and ammonium during fed-batch phase. This was accomplished by using dialysis and ‘nutrient-split’ feeding in a membrane dialysis reactor [Pörtner et al., 1997] and resulted in a ten-fold increase of the antibody concentration compared to the batch.

2. Cell Line and Culture Conditions The hybridoma cell line IV Fl9.23 is producing monoclonal IgG1 antibodies (MAb) against penicillin-G-amidase for the application in an affinity chromatography. Cultivation was carried out in a 1:1 mixture of Iscove’s MDM and Ham’s F12. The media was completed with and 1 % (v/w) of a protein-free, iron-rich supplement and Dolníková, 1991]. During the fed-batch phase and for ‘nutrient-split’ feeding a 10fold concentrated, salt-free nutrient solution containing amino acids and vitamins with 50 mmol l-1 glucose and concentrated glutamine solution (200 mmol l-1, Life Technologies were supplied separately. Dialysis cultivation was carried out in a membrane dialysis reactor (Bioengineering AG, CH) described by Pörtner et al. (1994). 347 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 347-349. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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3. Process Control System The process control (Fig. 1) was based on a field bus technology under a Unixcompatible real-time environment. Control platform (X-Control, Direct Digital Control) and data management (X-Manager) were working independently for process safety reasons.

4. On-line Characterisation using OUR On-line characterisation is a basic approach to describe metabolism and physiological state of the cells on-line. A process control system stores and controls measurable data. An user-interface enables the operator to change process parameters and to provide data from off-line analysis. Non-measurable variables, e.g. the oxygen uptake rate OUR and cumulative oxygen consumption COC, were calculated to describe the current metabolism. Based on these variables the feed flow was calculated on-line. Metabolic rate ratios and stoichiometric coefficients were calculated from off-line analysis. The stoichiometric coefficient described the ratio of oxygen and glucose consumption. This parameter was not constant during cultivation and was therefore regularly updated from off-line glucose analysis.

349

5. Results 5.1. FED-BATCH STRATEGY The feeding strategy was based on the coupling of on-line and off-line measurements and enabled the fed-batch operation without expensive on-line substrate analysis. The cell growth was characterised on-line using the oxygen uptake rate OUR and the feed was supplied based on the stoichiometric relation of glucose and oxygen uptake. The viable cell concentration and oxygen uptake rate OUR of batch, fed-batch and dialysis fed-batch are shown in Figure 2. The cell concentration in fed-batch increased 1.5-fold and the antibody concentration about 3-fold compared to the conventional

batch. The fed-batch strategy was not able to avoid inhibition by accumulated metabolites after 80 h cultivation time. 5.2. DIALYSIS FED-BATCH

The removal of inhibiting metabolites such as ammonia and lactate from the cultivation chamber using a dialysis membrane resulted in a 10-fold increase in the viable cell and antibody concentration compared to batch cultivation. Substrate utilisation was very efficient due to nutrient split feeding strategy. The concentrated feed was supplied to the culture chamber and buffer solution was used as dialysate in the dialysis chamber.

6. References and Dolníková, J. 1991. Hybridoma growth and monoclonal antibody production in iron-rich

protein-free medium: Effect of nutrient concentration. Cytotechnology 7: 33-38 Lüdemann, I., Pötner, R. and Märkl, H. (1995) ‘Nutrient-Split’ feeding strategy for dialysis cultures of hybridoma cells. Proceedings 8th JAACT meeting, Iizuka, Japan Pörtner, R., Bohmann, A., Lüdemann, I. and Märkl, H. (1994) Estimation of specific glucose uptake rates in cultures of hybridoma cells. Journal of Biotchnology 34: 237-246

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METABOLIC NETWORK ANALYSIS OF A HYBRIDOMA CELL LINE USING MASS BALANCES AND C 14 -LABELLED GLUCOSE AND GLUTAMINE

P.-A. RUFFIEUX, I.W. MARISON, U. VON STOCKAR Swiss Federal Institut of Technology LGCB - 1015 Lausanne, Switzerland

1. Introduction Continuous cultures of a hybridoma cell line (Zac3) were performed with two C14 labelled substrates: glucose and glutamine. A simplified network was used to describe the metabolism of the cells [1], see figure 1. The measurement of the main species consumed or produced by the cells allows one to solve the network and determine the rate of the 20 reactions. The use of labelled glucose and glutamine allows determination of very small fluxes with high accuracy. Thus it was possible to determine the flux of carbon to biomass and also the ratio of substrate which is degraded into carbon dioxide from glucose and glutamine. These results could be confirmed from those obtained by the network, for example the CO2 production rate.

2. Materials and Methods A hybridoma cell line (ZacJ) was grown into a CSTR of 1.5 liter working volume. When the stationary phase is reached, the standard medium is changed to labelled medium, with exactly the same composition, the only difference is that a part of the glucose or glutamine is radioactive. The labelled atoms of carbon are followed in the different phases: the biomass is collected and the activity measured, the exiting the reactor is trapped into NaOH, and the activity of the medium is determined after liquid chromatography, which separate the different metabolites (glutamine, lactate, alanine.etc). This method is describe elsewhere[2]. The specific rate of consumption or production of all the major components is determined according to the following equations: (1) IN-OUT + PRODUCTION = ACCUMULATION; Where : F=Flow rate of species i X=biomass concentration consumption rate of Production rate of 351 O. - W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 351-353. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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The specific oxygen uptake rate is determined with a balance around the liquid phase. With a constant D.O. and a determined experimentally, it is possible to calculate the oxygen transfer rate from a knowledge of the molar fraction of oxygen in the gas phase[3].

3. Results

The concentration of the main metabolites was determined by off-line analysis and the specific rates of consumption/production are summarised in table 1:

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The use of glucose and glutamine enables determination of the fate of the C labelled atoms [2]. This supplementary information allows confirmation of the model used for the network and to determine some fluxes which are difficult to measure : production rate, due to the use of buffer to control pH. • Carbon assimilation into biomass, not possible to determine without tracers.

With the stoichiometric coefficients of the reactions in the network, a matrix A is defined and Ar=q. With r being a vector of the flux through the biochemical reaction and q the measured rates of change of extracellular metabolites. By solving this system, the rates r1 to r20 are obtained measured = calculated = Thus 92% of the 14C in glucose goes to lactate. In the network, this value could be approximate by the calculation of r 5 / r 4 = 89%

4. Conclusions This simple metabolic network gives a good idea of the main fluxes into the cell, especially for the catabolic reactions. This calculation requires only 7 rates of consumption. The use of labelled glucose and glutamine enables the metabolic routes to be followed with great accuracy, especially production rate. Normally this rate is difficult to measure due to buffer system used to control pH. Therefore it was possible to validate the results of the network. To use completely the information provide by the C-labelled experiments, it is necessary to write equations, which take into account the position of each C-atom in all reactions (Mapping equations). However, such networks are very complicated and it is difficult to perform the required measurements, since this would involve the use of substrate with the C-labelled at different positions of the structure, together with analysis of the position of the label in the product. 5. Reference 1. 2.

Zupke C. et. al. Intracellular flux analysis in hybridomas using mass balances and in vitro 13C NMR Biotechnol. Bioeng.(1994) Vol. 45 Pp. 292-303 Ruffieux P-A. et. al. Development of carbon balances for continuous animal cell culture using 14-C labelled glucose, Animal cell technology, (1997), Pp. 725-729

3. Oeggerli. A. et al. Online Gas-Analysis in Animal-Cell Cultivation .1. Control of Dissolved-Oxygen and pH. Biotechnol. Bioeng., (1995) Vol 45. Pp. 42-53.

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DYNAMIC

MEDIUM

OPTIMIZATION

BY

ON-LINE

HEAT

FLUX

MEASUREMENT AND A STOICHIOMETRIC MODEL IN MAMMALIAN CELL CULTURE

Y. GUAN and R. B. KEMP Institute of Biological Sciences, The University of Wales

Aberystwyth, SY23 3DA, UK

Abstract. Metabolic flow rate was detected on-line and in real time by a Heat Flux Probe for animal cell culture. For a model system, CHO320 cells producing recombinant human Interferon-γ (IFN-γ ), this probe identified different metabolic states during cell growth. These were then related to dynamic changes in glucose and glutamine consumption, which would allow optimization of the feeding medium. Based on heat flux measurement and the collected data from a stoichiometric model, dynamic optimization of the two substrates in a feeding medium would then be possible by automatically triggering two peristaltic pumps.

1. Introduction

To increase the production duration and the culturing density of animal cells, an important factor is to feed the cells with the correctly formulated medium. Except for continuous culture, cellular metabolic activity varies during the culturing time and therefore the composition of the medium should be changed to meet the dynamically altered optimal medium requirement [ 1.2]. To achieve this aim. a direct measurement of cellular metabolic activity is

necessary. Cells produce heat as an integral part of their metabolism and its flow rate thus is a measure of total metabolic rate. Heat flow rate is measured on-line by calorimetry and its quantity per amount of cell mass is known as heat flux. Among other methods dielectric spectroscopy was chosen to measure cell mass in real time. 2. Experimental

CHO320 cells were grown in an Applikon 3-L bioreactor system with BioXpert

software for data acquisition and controlled medium feeding. Measurement of heat flow rate was realized by an ex situ flow microcalorimeter and that of viable cell biomass by an in situ dielectric Viable Cell Monitor [3,4]. 3. Results and Discussion

The data depicted in the figure are used to justify, by a thermochemical approach, the following growth equation: 355 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 355-357. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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The growth reaction is easily be characterized by its set of stoichiometric coefficients,

An important concept in the present work on the stoichiometric growth equation is that the set (matrix) of stoichiometric coefficients has a one-to-one corresponding

relationship to the metabolic status of the entire cell population [5]. If the metabolic status is expressed by the metabolic rate, this means that: (3)

where is advancement of eq. (1) and is the metabolic rate in terms of the growth equation. Eq. (3) clearly delivers the experimental strategy for optimizing cell growth and the production of target proteins, i.e. the on-line

diagnostics of cellular metabolic requirement can be achieved by on-line measurement of heat flux. For empirically divided time intervals in growth, the data are incorporated into the equation to obtain stoichiometric coefficients, see table 1 below.

The results show that the heat flux probe is an early and sensitive indicator of the changing metabolic state of cells. Since the production of the target protein deteriorates

with these changes, action must be taken to retard the metabolic decline. Analysis of stoichiometric coefficients at different time intervals showed the changing cellular requirement for the relative amounts of major catabolites.

357

For the present purpose, metabolic states are considered to be directly associated with the cellular metabolic rate whereas external physiological conditions are the extracellular environmental conditions in which the cells grow. Although different, they decrease in the same direction for a deleterious physiological condition, which thus will negatively affect the metabolic state, reducing the metabolic flux either for the cell growth or for the target protein production, or both. This implies it is possible to detect worsening physiological conditions using heat flux and then decide when to add fresh medium. The classical approach to this problem is fed-batch culture. The

difficulties here are to know when to add more nutrient and in what quantities. The use of on-line heat flux measurement can solve these by at least the following: (a) timing of feeding — as the heat flux curve decreases to a certain extent, medium is automatically fed by a control loop; (b) quantity of feeding — the basic approach would be to feed the cells with the correct relative amounts of the major substrates. For instance, to

maintain the maximal growth of CHO320 cells, glucose and glutamine should be fed by a molar ratio of 2.7 to 1 in the fast growing period (Table 1).

A more refined approach would be to feed the cells continuously at a rate determined by heat flux and dependent on the consumption rate of major substrates in their correct molar ratio. The same approach would be possible for the same cells grown on macroporous beads. Furthermore, since the increase in the proportion of unwanted heterogeneous proteins is also a reflection of worsening physiological conditions, it is likely that heat flux can also be used to monitor the quality of the products, typically glycoproteins

The heat probe in this work is a robust, reliable and novel biosensor for on-line use in cell culture. It detects total metabolic rate and changes in it. which are related to alterations in substrate consumption rates leading to their depletion. The stoichiometric approach has shown the actual substrate requirement of CHO320 cells for growth. In the fed-batch method advocated here, the cells are given substrates in the molar ratio determined by the stoichiometric coefficients. In recombinant cells, this would sustain production of foreign proteins and should lead to much needed improvements in quality. Acknowledgments: Y.G. was the recipient of a generous bursary from ESACT. The project is funded by the Biotechnology and Biological Sciences Research Committee (UK) under grants 2/3680 and 2/TO3789.

4. References 1.Kemp, R.B., Evans, P.M. and Guan, Y. (1997) An enthalpy balance approach to the study of metabolic activity in mammalian cells. .J. Thermal Anal., 49, 755-770. 2.Kemp. R.B. and Guan, Y., (1997) Heat flux and the calorimetric-respirometric ratio as measures of catabolic flux in mammalian cells, Thermochim. Acta 300, 199-211. 3.Guan, Y., Lloyd, P.C., Evans, P.M. and Kemp, R.B. (1997) A modified continuous flow microcalorimeter for

measuring heat dissipation by mammalian cells in batch culture, J. Thermal Anal., 49, 785-794. 4.Guan, Y., Evans, P.M., Kemp, R.B. (1998) Specific heat flow rate: An on-line monitor and potential control variable of specific metabolic rate in animal cell culture that combines microcalorimetry with dielectric

spectroscopy, Biotechnol. Bioeng. (in press). 5.Guan, Y., Kemp, R.B., (1996) Medium design with the aid of heat flux measurement in mammalian cell culture, in BioThermoKinetics of the living cell, eds. H.V. Westerhoff, J.L.Snoep, F.E.Sluse, J.E.Wijker and B.N. Knolodenko, pp. 387-397, BTK, Amsterdam.

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MODELING OF GLYCOPROTEIN PRODUCTION BY CHINESE HAMSTER OVARY CELLS FOR PROCESS MONITORING AND CONTROL J. STELLING*, R. K. BIENER*, J. HAAS†, G. OSWALD†, D. SCHULLER† W. NOE† and E. D. GILLES* * Institut für Systemdynamik und Regelungstechnik, 70550 Stuttgart, FRG †Dr. Karl Thomae GmbH, 88397 Biberach, FRG Abstract

Substantial improvements of animal cell culture process performance can be achieved by model-based methods of process optimization and control. Here, a structured mathematical model describing the dynamics of Chinese hamster ovary (CHO) cell growth and recombinant glycoprotein production has been developed. It covers the main aspects of cell metabolism as well as aspects linked to cell cycle regulation. Model predictions fit well to experimental data obtained from fed-batch cultures and allow for insight into the complex behaviour of CHO cells in culture. Model-based improvement of feeding trajectories is shown. Further applications for process monitoring and control are discussed.

1 Introduction In animal cell culture technology, substantial improvements of process performance can

be achieved by model-based methods of rational medium design, feeding strategy optimization and on-line monitoring and control. When applied to fed-batch processes, these methods can enhance growth and production efficiency by reducing growth limitations due to depletion of essential nutrients (e.g. glucose and amino acids) and / or accumulation of toxic by-products (e.g. ammonia and lactate). [1,2] Whereas most research in this area is focused on monoclonal antibody production by hybridoma cell lines [1,2], here a structured mathematical model describing the dynamics of CHO cell growth and recombinant glycoprotein production has been developed. As an exemplary application, the model-based improvement of feeding trajectories in laboratory scale fed-batch fermentations is shown.

2 Model structure

The CHO model is composed of two main modules: A cell cycle part including the subpopulations of phase cells (producers), phase cells (non-producers) and dead cells is used to describe population dynamics. Phase transitions are assumed to be 359 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 359-361. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

360

governed by the accumulation of cell-cycle phase specific cyclins and by cell size. The mathematical formulation results in the two-dimensional population balance

(1) with f: number density function, cell volume, intracellular cyclin concentration, velocities, source/sink, subpopulation index and time. The complementary metabolic part has been designed as a cybernetic model. For a close representation of cell physiology, the model includes the main metabolic pathways (glycolysis and PPW, citrate cycle, amino acid metabolism), metabolite production, formation of cellular components (proteins, lipids, nucleotides) and product, energy metabolism (ATP, NADH) and growth control by essential amino acids / toxic by-products [1].

3 Simulation results A series of CHO fed-batch fermentations has been used for parameter estimation. As shown in Fig. 1, simulation results agree well with experimentally observed time series.

Analysis of model simulations furthermore allows for the identification of growth-limiting steps, e.g. amino acid uptake rates and ATP generation as well as the plausible prediction of unmeasured variables, e.g. share of subpopulations and average cell size (Fig. 2).

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4 Application: Feed design

The model-based optimization of the feed composition lead to a feed medium including lower glutamine concentration and higher levels of specific essential amino acids.

As a result - compared to a standard fed-batch fermentation - significantly higher cell yields and an increase in final product concentration have been achieved (Fig. 3).

5 Concluding remarks

The mathematical model presented is able to give a good description of CHO growth and production dynamics. Compared to purely metabolic models [1,2] or other combined cell-cycle / metabolic approaches [3], it provides a closer, modular representation of many aspects of cell biology. Model-based feed design serves only as one component of an integrated approach to process improvement. Further steps include complete off-line process optimization, on-line monitoring and (model-predictive) control [1].

6 Acknowledgements

This work was supported by the State Government of Baden-Württemberg (FRG) and Dr. Karl Thomae GmbH (Biberach, FRG).

7

References [1]

Biener, R.K. et al. (1996) Model-based control of animal cell cultures, in M.J.T. Carrondo, B. Griffiths and J.L.P . Moreira (eds.), Animal cell technology: from vaccines to genetic medicine, Kluwer Academic Publishers, pp. 639-645

[2]

Xie, L. and Wang, D.I.C. (1996) High cell density and high monoclonal antibody production

[3]

Martens, D.E. et al. (1995) A combined cell-cycle and metabolic model for the growth of hybridoma cells in steady-state continuous culture, Biotechnol. Bioeng. 48, 49-65

through medium design and rational control in a bioreactor,Biotechnol. Bioeng. 51, 725-729

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THE TEMPERATURE EFFECT IN MAMMALIAN CELL CULTURE AN ARRHENIUS INTERPRETTION

Gerlinde Kretzmer1, Torsten Buch1, Konstantin Konstantinov2, David Naveh2 1Institut für Technische Chemie, University Hannover, Germany 2Bayer Corporation, Pharmaceutical Division, Berkeley, USA

Introduction

Recently cultivation temperature has become of interest for process characterisation and optimisation for research and manufacturing. All chemical and biochemical reaction are known to be temperature dependent. Therefore, one expects reactions rates going down with reducing the cultivation temperature. Results show that the reduction in rates is different for different reactions. The primary metabolism is influenced by reducing the temperature, while the secondary metabolism especially the protein production is not effected or even improved. Cell growth rate as a bulk of reactions taking place is slowed down with temperature reduction in most cases. The extend to which the reduction will occur depends on the specific reaction studied. All reactions follow the thermodynamical rules and Arrhenius was the first to set up a mathematical equation to explain the relationship between chemical reaction rates and temperature. Material and Method

Data from Batch cultures of different cell lines were used for the calculations: adherent BHK 21, suspension rBHK, rCHO I, rCHO II, hybridoma. The cultivations of the adherent BHK were carried out in serum containing medium, the other in serum free medium. Data from continuous cultivation of rBHK (FVIII) were used.

For many reactions the rate expression can be written as a product of a temperaturedependent term and a composition term, or ri = f1 (temperature) * f2(composition) = k*f2(temperature) 363 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 363-366. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

364

For such reactions the temperature-dependent term, the reaction rate constant, has been found in practical all cases to be well represented by Arrhenius´law:

This was found true also for enzyme-activity-temperature relationship. For a small temperature range Arrhenius´law is also applicable for microbial cultivations.

For example the cell growth can be described as follows:

This can be plotted as an Arrhenius plot:

This equals Similar equations can be set up for metabolic reactions and for product formation. With quasi-linear regression of the following form

the activation energy and the frequency factor can be calculated. Results and discussion

Growth rate (Fig. 1) All cell lines show a decrease in growth rate with decreasing temperature. The non recombinant BHK and the hybridoma show a steady decrease whereas the recombinant cell lines show stable or better growth rate between 37 and 33°C.

365

Arrhenius plot growth rate (Fig. 2) All cell lines show two different slopes in their Arrhenius plots. The plot of the recombinant cell lines and the hybridoma have an area with a steep slope and one with

a flat slope. The non recombinant BHK cell line has two areas with nearly the same slope, separated by a plateau. Arrhenius plot growth rate and oxygen uptake rate of rBHK (Fig. 3) Again a two stage relationship occurs for the growth rate as seen with other

recombinant cell lines. The relationship between ln (OUR) and 1/T is linear.

366

Arrhenius plot glucose consumption rate and productivity of rBHK (Fig. 4) A linear relationship of ln (Glucose) and 1/T occurs. Specific glucose consumption rate is decreasing with decreasing temperature. The relationship between ln(Productivity) and 1/T is also linear but the slope is reverse. The specific productivity is increasing

with decreasing temperature Conclusions

The Arrhenius plot of the growth reaction is only in very small temperature range linear. For all the recombinant cell lines the activation energy at higher temperature differs from that a lower temperature. In between there is a temperature range where the activation energy does not change with temperature. There the activation energy.

is independent from

The kinetic reactions of glucose consumption, lactate production and oxygen uptake follow Arrhenius´ law linearly. The activation energy of those reaction lies in the range of 80 to 140 Ws/mol. The Arrhenius plot of the production reaction shows a negative slope at lower temperature. This would lead to a negative activation energy which is thermodynamical senseless.

The product formation is a very complex system build up of many single reaction steps. From the Arrhenius plot one can assume that at higher temperature one of the reactions must be inhibited . This occurs also by the cell growth: If the temperature is high enough the growth decreases because of the denaturation of proteins. Summary Cell growth and the primary metabolic reactions can be represented by Arrhenius´ law. That means Arrhenius´ law can be used for a process model in a small temperature range. For the product formation this simple approach is not sophisticated. The kinetic

approach for the production has to be studied furthermore.

INTEGRATED PROCESSES

AND SCALE-UP

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DIELECTROPHORETIC FORCES CAN BE EXPLOITED TO INCREASE THE EFFICIENCY OF ANIMAL CELL PERFUSION CULTURES N. KALOGERAKIS1, A. DOCOSLIS2 AND L. A. BEHIE3 1 Laboratory of Biochemical Engineering & Environmental Biotechnology

Technical University of Crete, Chania 73100, Greece Bioengineering Laboratory, Dept. of Chemical Engineering State University of New York, Buffalo, NY 14260 USA 3 Pharmaceutical Production Research Facility (PPRF), Faculty of 2

Engineering The University of Calgary, Calgary, Alberta, Canada T2N 1N4

1.

ABSTRACT

Dielectrophoresis is a well established and effective means for the manipulation of viable cells. Various applications have been found, ranging from electrofusion, to individual cell manipulation, and to differential separation from cell mixtures. Its effectiveness, however, greatly depends upon the utilization of very low electrical conductivity media. High conductivity media, as in the case with cell culture media result only in negative dielectrophoresis (i.e., induction of weaker repulsive forces) and excessive medium heating. Recently, a dielectrophoresis-based cell separation device (DEP-filter) has been developed for perfusion cultures that successfully overcomes these problems and provides a very high degree of viable cell separation while most of the nonviable cells are removed from the bioreactor by the effluent stream. The latter results in high viabilities throughout the culture period and minimization of lysed cell proteases in the bioreactor. However, an important question that remains to be answered is whether we have any adverse effects by exposing the cultured cells to high frequency dielectrophoretic fields for extended periods of time. A special chamber was constructed to quantitate the effect under several operational conditions. Cell growth, glucose, lactate and monoclonal antibody production data suggest that there is no appreciable effect and hence, operation over long periods of time of our DEP-filter should not have any adverse effect on the cultured cells. 2.

INTRODUCTION

Perfusion cultures represent the most cost effective mode of operation for the large scale production of therapeutic proteins, such as HBAg, tPA, erythropoietin, monoclonal antibodies, etc. The main characteristic of a perfusion culture is the retention of the cells in the bioreactor, i.e. no cells are removed by the effluent stream which results in high cell densities, increased volumetric productivity and reduced downstream purification requirements. A suitable cell retention device, located in the effluent stream of the bioreactor, is an important part of a cell perfusion bioreactor system. Existing cell retention systems are based on sedimentation, centrifugation or conventional filtration techniques and are subject to significant limitations. A recent device3, 17 that employs ultrasonic resonance fields to transiently aggregate animal cells and are intermittently

settled back in the bioreactor has also several limitations. The main disadvantage being its inability to selectively separate viable from nonviable cells and second the high power 369 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 369-375.

© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

370

requirements that can cause excessive medium heating with possible detrimental effects to cellular viability. As an alternative to the existing devices, we have developed a cell retention device based on the exploitation of dielectrophoretic (DEP) forces4. Dielectrophoresis is the motion of neutral particles (e.g., spheres, bubbles, biopolymers, biological cells, etc.) under the influence of a divergent electric field. Numerous techniques have been developed using dielectrophoresis that include electroporation,37 electrofusion, 1,14,18 electroinjection, 13 measurements of the dielectric properties of the cell,6,7,10 as well as cell separation in low conductivity media.2,8,1112,15 We have recently shown that there is a frequency range where selective separation of the viable from non-viable cells is achievable in high conductivity media typically found in cell culture systems4. As Fuhr et al.5 has also shown using anchorage dependent mouse fibroblasts, cell separation in highly conductive culture media can only be achieved under negative DEP, using high frequency AC fields. Their results were also encouraging in that no adverse effects due to continuous exposure of the cells to high frequency electric fields were detected. They found that high field frequencies (above 10 MHz) had no significant effect on cell growth. In this present work, we examine whether there are any adverse effects by exposing the cultured cells to high frequency dielectrophoretic fields for extended periods of time. A special chamber was constructed to quantitate the effect under several operational conditions. Cell growth, glucose, lactate and monoclonal antibody production data are presented here that have been obtained from cultured cells exposed to high frequency dielectrophoretic fields over long periods of time. Finally, the inherent capability of the DEP-filter for preferential removal of apoptotic bodies is also discussed. 3.

MATERIALS AND METHODS

The murine lymphocyte hybridoma HFN 7.1 producing an IgG antibody reactive with human fibronectin (ATTC: CRL-1606) was used in these experiments. The cell culture medium was DMEM (Dulbecco's Modified Eagle's Medium, Sigma) supplemented with 10% Fetal Bovine Serum (Gibco/BRL). In addition CHO/dhfr- cells (ATCC: CRL-9096) were grown in EMDM (Iscove's modified Dulbecco's medium) supplemented with 4 mM L-glutamine, 0.1 mM hypoxanthine and 0.01 mM thymidine and adjusted to contain 1.5 g/L sodium bicarbonate and 10% FBS after adaptation to grow in suspension.. The cells were grown in suspension in a suitably modified (open bottom) polycarbonate vial at 37°C in a 5% and humidity controlled incubator. The bottomless vial was glued onto an oxidized silicon wafer where a grid of micro-electrodes were deposited. In this case the silicon was not etched through in the areas between adjacent electrodes as the case is for the construction of the DEP filter. We simply wanted to grow the cells in the vicinity of the electrodes and quantify any adverse effects from the exposure to the high frequency electrical fields. The analysis of glucose and lactate was done by an YSI-2700 analyzer, the aminoacids by HPLC (HP1090), the MAb by ELISA and cell viability by trypan blue. 4.

THEORY

The net time-averaged induced dielectrophoretic force for AC electric fields is directly proportional to the cell volume, the real part of the medium permittivity, and the gradient of the electric field intensity squared, E 2 (rms value), i.e.,

371

where r is the cell radius and is the intensity of the electric field. The frequency dependence of the induced DEP force is given by the dimensionless Clausius-Mossotti function,

where the underlined parameters denote complex quantities. The complex permittivity of the surrounding medium is given by where and is the angular frequency of the applied field. According to the single-shell model,9,10 the cell can be represented as an ohmic, spherical particle, enclosed by a thin insulating shell (cellular membrane). The transmembrane conductance and surface conductivity are assumed to be negligible since mammalian cells do not have a cell wall and their membrane thickness is at least three orders of magnitude smaller than their cell radius15,16. If, furthermore, the cell cytoplasm is modeled as a linear ohmic dielectric fluid (i.e., there are no dielectric losses) of permittivity and conductivity the resulting effective cell permittivity is given by 9

where is the area-specific membrane capacitance and constants. As shown in Equation (1), the real part of The sensitivity of

are time is directly related to

to cell size and various other electric parameters over a wide

frequency range have been discussed elsewhere4. Having estimated the unknown parameters in through a series of levitation experiments, one can plot versus and determine the frequency range where we can have preferential cell retention of viable cells in the bioreactor through the action of negative dielectrophoretic forces and withdrawl of nonviable cells by the effluent stream4. 5.

DESCRIPTION OF THE DEP-FILTRATION SYSTEM

The DEP-filter, shown in Figure la, can be briefly described as a grid of micro-electrodes deposited on a silicon substrate, with the silicon etched through in the areas between adjacent electrodes. The filter was made using photolithography and silicon microfabrication techniques. The electrodes are made of gold deposited on a chromium layer. Structural details can be found elsewhere4. The DEP-filter is mounted at the bottom of a housing device and the whole unit is submerged in the cell suspension. A schematic diagram with details of the whole system is given in Figure 1. A schematic of the forces acting on viable and nonviable cells as well as their expected trajectories in the vicinity of the DEP-filter is shown in Figure 2. Our first results have shown that, at low flow rates, the retention of the viable cells can be very high (up to 98%) whereas it is extremely low, often below 15%, for nonviable cells regardless of the operating conditions. The latter is an indication that cell debris does not experience any dielectrophoresis. It was also shown that the retention of viable cells is subject to many factors, such as frequency of the applied electric field, voltage across the filter electrodes and flow rate of the effluent stream4.

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6.

EXPERIMENTAL RESULTS & DISCUSSION

A series of experiments were designed to examine whether cells staying in the vicinity of the DEP-filter electrodes suffer from adverse effects. In a typical perfusion culture due to bulk agitation, the cells are expected to arrive and stay near the DEP electrodes only for a short period of time. To emulate this, we applied a high frequency (10 MHz) electric field (25 V pk to pk) intermittently. However, we were unable to see any adverse effect. Hence, it was decided to expose the cells continuously and examine if any changes in their growth characteristics and metabolism can be observed. In figure 3, the results from three subcultures of HFN 7.1 cells are shown. Actually the last subculture was monitored throughout the stationary phase. Comparison of viable cell density, viability, glucose uptake, lactate production as well as monoclonal antibody production data between the DEP-culture and the control suggests that there are no adverse effects. Similar results were obtain for 15 aminoacids measured by HPLC analysis (data not shown). These experiments are quite conservative as in an actual perfusion culture, the cumulative exposure of the cells is expected to be much less. Similar results shown in Figure 4 are obtained from another series of experiments using CHO cells adapted to grow in suspension.

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374

7.

REFERENCES

1. Abidor, I.G. and Sowers, A.E. 1992. J. 61: 1557-1569. 2. Archer, G.P., Render, M.C., Betts, W.B. and Sancho, M. 1993. Microbios 76: 237-244. 3. Doblhoff-Dier, O., Gaida, T., Katinger, H., Burger, W., Gröschl, M. and Benes, E. 1994.

Biotechnol. Prog. 10: 428-432. 4. Docoslis, A., Kalogerakis, N., Behie, L.A., Kaler, K.V.I.S. 1997. Biotechnol. Bioeng. 54: 239-250.

5. Fuhr, G. Glasser, H., Müller, T. and Schnelle, T. 1994. Biochim. Biophys. Acta 1201: 353-360. 6. Gascoyne, P.R.C., Becker, F.F. and Wang, X.-B. 1995. Bioelectrochem. Bioenerget. 36: 115-125. 7. Gimsa, J., Marszalek, P., Loewe, U. and Tsong, T.Y. 1991. Biophys. J. 60: 749-760. 8. Huang, Y., Wang, X.B., Tame, J.A. and Pethig, R. 1993. J. Phys. D: Appl. Phys. 26: 1528-1535. 9. Kaler, K.V.I.S. and Jones, T.B. 1990. Biophys. J. 57: 173-182.

10. Kaler, K.V.I.S., Xie, J.P., Jones, T.B. and Paul, R. 1992. Biophys. J. 63, 58-69. 11. Krishna, G.G., Anwar, A.K.W., Mohan, D.R. and Ahmad, A. 1989. J. Biomed. Eng. 11: 375-380.

12. Markx, G.H., Talary, M.S. and Pethig, R. 1994. J. Biotechnol. 32: 29-37. 13. Neil, G.A. and Zimmermann, U. 1993. Methods in Enzymology 221: 339-361. 14. Neumann, E., Sowers, A.E. and Jordan, C.A. 1989. Electroporation and Electrofusion in Cell Biology. Plenum Press, New York-London.

15. Pohl, H.A. 1977. In: Methods of cell separation (N. Catsimpoolas, ed.), Plenum Press, New York, vol.1, 67-169. 16. Sukhorukov, V.L., Arnold, W.M. and Zimmermann, U. 1993. J. Membrane Biol. 132: 27-40 17. Trampler, F., Sonderhoff, S.A., Pui, P.W.S., Kilburn, D.G. and Piret, J.M. 1994. Bio/Technol. 12: 281-284.

18. Zimmermann, U. 1986. Rev. Physiol. Biochem. Pharmacol. 105:176-256.

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Discussion Al-Rubeai:

Have you done any work on the effect of cell density on cell retention?

Kalogerakis:

I only showed low cell densities (1 million cells/ml) as our prototype broke - it is very sensitive which is why we are moving away from it. There are problems, eg gas bubbles, so we incline the device by 40°, and pearl chains form, but we can overcome this problem with agitation. Your perfusion rate is going to be a constrained variable, so I was wondering if you have explored differences in field strength? Also, what are the practical limits for nano fabrication for the area?

Aunins:

Kalogerakis:

It is superficial velocity, and not perfusion rate, which limits the operation. The open area was 1.5 cm by 2 cm for a 1.5 1 fermenter working at 2 volumes per day. So it is not bulky and can be used in a modular battery.

Reuveny:

Do you look for the small ions in your medium?

Kalogerakis:

If you mean current going through the medium, yes, this is a serious problem. This is why we coat the electrodes with silicon dioxide, which is an insulator. An electrical field does exist and this poses problems in terms of manufacturing from the microfabrication point of view.

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USE OF A NEW MICROCARRIER WITH TWO-DIMENSIONAL GEOMETRY FOR THE CULTURE OF ANCHORAGE-DEPENDENT CELLS IN SONOPERFUSED, CONTINUOUSLY STIRRED TANK REACTORS

C. GATOT(1),

F. TRAMPLER(2), (3)

M.-P. WANDERPEPEN(1), (4)

J. HARFIELD , A. OUDSHOORN , A. JOHANSSON(5), (5) (6) V. NIELSEN , A.O.A. MILLER (1) Computer Cell Culture Center s.a. (Mons, Belgium) ; (2) Sonosep Biotech Inc (Vancouver, Canada) ; (3) Coulter Electronics Ltd (Luton, UK) ; (4) Applikon Dependable Instruments (Schiedam, Holland) ; (5) Nunc A/S (Roskilde, Denmark) ; (6) Biochimie/Technologie Cellulaire - Faculté de Médecine - Université de Mons-Hainaut (Mons, Belgique)

Dextran-based microbeads characterized by a 3-dimensional geometry (3D microsupport), on the surface of which anchorage-dependent cells (ADC's) attach and multiply, constitute a reference for the large scale cultivation of this type of cells. Using commercially available 3D microsupport at 5 g/L, allows one to reach cell concentrations around cells/ml, some 7 % of the culture volume being occupied by the microcarrier. Growing ADCs to higher cell densities can be obtained by increasing the concentration of the microsupport. However there rapidly conies a moment when the volume of the swollen microbeads becomes so high as to represent an important proportion of the culture volume, decreasing accordingly the volume of the growth medium available to the cells. Macroporous microbeads accomodate more cells per unit volume but their highly convoluted inner structure makes quantitative release of cells very difficult without extensive trypsinisation. Recently, a new generation of microcarriers with 2D geometry has been made available for evaluation. Photo 1 gives the rationale underlying their manufacture. It shows that the outside surface of the microbead available for cell growth equals the sum of the surfaces of two superimposed very thin discs situated at the equator. However the combined volumes of two such discs which becomes smaller the thinner the film used to manufacture them (Blodgett's monomolecular films would be ideal in this respect), represent only a very small fraction of the volume of the corresponding sphere (2D microhexagons cut from 25 microns thick polystyrene films have been preferred to discs since their manufacture generates less wastes (Photo 2). The characteristics of the 2D microhexagons used in this study are given in Table I. 377 O.-W. Merten et al. (eds.). New Developments and New Applications in Animal Cell Technology, 377-380. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

378

Photographs 3 and 4 give the growth kinetics of mouse L929- and Muntjac cells respectively.

379

Additional results also obtained from batch cultures conducted this time at at confluency) with 2D microhexagons suggest that still higher cell concentrating could be obtained provided the surface made available to cells is further increased and the growth medium renewed by perfusion. Acoustic perfusion is a re cently developed technology whereby isolated cells in suspension can be grown at concentrations of and higher. In this system, isolated cells in suspension accumulate at the nodes of a resonating acoustic wave. At regular intervals, in order to empty the resonating chamber, the accumulated cells are aspirated back into the culture by means of the suspension culture continuously recirculating at the apex of the resonating chamber at twice the perfusion rate. To adapt this technology to the cultivation of ADC's, recirculation is strictly prohibited since it would provoke extensive detachment of the cells from the microsupport. Sonoperfusion at 4L/day (4 culture volumes/day), of a 1L culture containing naked 2D microhexagons (8,75 g/L) shows few if any microsupport particles reaching the acoustic chamber. This effect is interpreted as being the result of the long (± 14 cm) large bore tube bringing the microcarrier suspension to the transducer playing the role of a settling tube. To compensate for the observed decreased density of cell-laden 2D microhexagons, a culture of 750 ml (inoculated with 7,5 g of microcarrier and

380

) is perfused at l,5L/day only (2 culture volumes/day). Three days after inoculation the cell concentration reaches allowing the collection of close to the maximum theoretical value of This 91,5 % recovery is obtained without recourse to trypsinisation by simply forcing several times the cell-laden 2D microhexagons suspension in 0,2 % EDTA-PBS through the orifice of a 25 ml capacity pipette. Provided means are found to prevent cell-laden 2D microhexagons from escaping through the resonating acoustic wave when very high perfusion rates are used, our results suggest that cell concentrations of can be routinely obtained with the microsupport occupying only some 30 % of the total culture volume, making it envisageable to reach even higher cell concentrations in the future. Discussion Anon:

Do you see aggregation of the carriers in the acoustic field or later in the reactor?

Miller:

The microcarriers-carriers align perfectly at the nodes of the wave and when they are returned back to the suspension they are totally isolated.

Ozturk:

What limits the cell density: just cell retention, particle number, oxygenation, or microcarrier concentration?

Miller:

We have enough microcarrier area to support 25 million cells/ml, theoretically.

Ozturk:

Do you sparge?

Miller:

No, we have tried horizontal semi-permeable tubing but the microcarriers sick horizontally. So now we are using a Braun device which is placed vertically.

Aunins;

Comparing the spherical and flat carriers, you need twice as many particles for the same surface area and they need inoculating on both sides. Do the inoculum requirements practically limit the multiplication ratios and scale-up?

Miller:

Our work is so preliminary that I cannot really answer your question. Starting in batch culture, we are reasonably confident that we can reach the theoretical results which we anticipate.

EVALUATION OF POROUS MICROCARRIERS IN FLUIDIZED BED REACTOR FOR PROTEIN PRODUCTION BY HEK 293 CELLS

M.A. VALLE1,2, J. KAUFMAN1, W.E. BENTLEY2, J. SHILOACH1 1. Biotechnology Unit, NIDDK, NIH Bethesda, MD 20892 USA 2. Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute, University of Maryland College Park, MD 20742 USA

1. Purpose of Study In an effort to obtain large quantities of the human parathyroid binding domain, a recombinant protein secreted extracellularly in very low quantities from the anchorage dependent HEK 293 cells, polyethylenesilica (Cytoline ) macroporous microcarriers in a fluidized bed reactor (Cytopilot) were examined. 2. Introduction Microcarriers allow the cultivation of anchorage-dependent cell lines with efficient use of bioreactor volume. For sensitive cell lines, macroporous microcarriers are more advantageous. The pores protect the cells from shearing and because of the larger available surface area, the carriers can hold high cell densities (Nilsson et al., 1986)(Shiragami et al., 1993). Fluidized bed reactors (FBR) are a good complement to macroporous microcarriers (Kratje and Wagner, 1992). In addition to providing a homogeneous environment under gentle mixing, they offer good mass transfer characteristics, are easy to monitor allow for high cell densities and are readily scaled up. Of special interest is the modular FBR, which eliminates the necessity of a recirculating loop with its associated handling problems (Reiter et al., 1991). Human embryonic kidney cell line 293 (HEK 293) is a good candidate for culture with macroporous microcarriers in FBRs. As the name indicates, 381

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 381-384. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

382

these cells are derived from secondary embryonic kidney cells transformed with sheared fragments of adenovirus (Graham et al., 1977). They are anchorage dependent and easily transformed to produce heterologous proteins (Berg et al., 1993)(Hamilton et al., 1993). Few attempts have been made to characterize this cell line and define the optimal conditions for its growth and protein production. This knowledge is essential if future scale-up is to be attempted. 3. Materials and Methods 3.1. CELL LINES AND MEDIA Human embryonic kidney cells (HEK 293) were obtained from ATCC (CRL 1573) (USA), and were routinely passaged in tissue culture flasks (Costar Corporation, USA) using DMEM (Biofluids Inc., USA) supplemented with 10% fetal bovine serum (Biofluids). HEK 293 transformed to produce the extracellular domain of the human parathyroid receptor (HEK 293add) were obtained from NPS Pharmaceuticals (USA) and passaged in T-flasks using DMEM containing 10% FBS and 200 u/mL Hygromicin B (Calbiochem Corporation, USA). Production medium for HEK 293add consisted of DMEM containing 293 Serum-Free Supplement (Shiloach et al., 1996), 2 mM glutamine (Gibco, USA), 200 U/mL Hyg B and 10,000 U/mL Penn-Strep (Gibco) . 3.2. REACTOR SYSTEM AND CULTIVATION CONDITIONS Stationary experiments were conducted in tissue culture flasks (Costar). Cells in mid-exponential to late exponential growth phase were inoculated at a density of and incubated at 37°C in 5% atmosphere. Bioreactor experiments were conducted in a 2L modular fluidized bed reactor (Cytopilot) (Pharmacia, Sweden) containing 300-400 mL (15-20% v/v) macroporous microcarriers (Cytoline , Pharmacia). pH and dissolved oxygen concentration were kept at values of 7.00 and 30% saturated air respectively sparging with and . 3.3. ANALYSIS OF SAMPLES Cell density was determined using a Coulter Counter ZM (Coulter Electronics, FRG).Glucose, lactate and glutamine concentrations were determined using a YSI analyzer (Yellow Spring Instruments, USA). Ammonia concentration was determined using a Biolyzer (Eastman Kodak Corporation,

USA).

383

4. Results and Discussion For evaluating the system, fermentations using HEK 293 and HEK 293add were conducted in the Cytopilotand T-flasks. Results from these fermentations are shown in Table 1. In the first 100 hours after attachment, HEK 293 cells in the Cytopilotshowed exponentially increasing consumption of glucose and production of lactate. After 100 hours, the rates of glucose and glutamine consumption and lactate and ammonia production became constant indicating that stationary or maintenance phase was reached.

The fermentation of HEK 293add in the Cytopilotdid not perform as smoothly as the HEK 293 fermentation due to interruptions caused by temperature and dissolved oxygen control problems. Nevertheless, during the first 70 hours after attachment, the cells exhibited exponential consumption of glucose and lactate production, with faster rates than the ones observed with the HEK 293 cells and a smaller yield of lactate on glucose. Rates of consumption of glutamine and production of ammonia during this phase were faster with the HEK 293add than with the HEK 293 and yield of ammonia on glutamine was also larger. After 330 hours of operation, the growth medium was replaced with production medium. Constant rates

384 of glucose consumption and lactate production indicated a maintenance phase. These rates were slower than the ones observed in the HEK 293 fermentation during maintenance phase even though yield of lactate on glucose was similar. The yield constants in the T-flasks were similar to the yield constants observed in the fermentor. However, the culture was not allowed to reach stationary phase. The fermentations in the Cytopilotwere conducted in repeated batch mode as opposed to perfused mode as suggested by the manufacturer. Some problems resulted from this decision. The small head space in the reactor did not allow efficient gas exchange and the filters often became clogged. Sampling was made difficult because the volume of medium that can be removed without affecting recirculation is small. Also, the process of medium replacement puts the beads under violent bubbling and mixing which might have a detrimental effect on the cells. References Berg, D.T., Mcclure, D.B. and Grinnel, B.W. (1993) High-level expression of secreted proteins from cells adapted to serum-free suspension culture, Biotechniques 14, 972978. Graham, F.L., Smiley, J., Russel, W.C. and Nairn, R. (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5, J. gen. Virol. 36, 59-72. Hamilton, B.J., Lennon, D.J., Im, H.K., Seeburg, P.H. and Carter, D.B. (1993) Stable expression of cloned rat GABA-A receptor subunits in a human kidney cell line, Neuroscience Lett. 153, 206-209. Kratje, R.B. and Wagner, R. (1992) Evaluation of production of recombinant human interleukin-2 in fluidized bed bioreactor, Biotechnol. Bioeng. 39, 233-242. Nilsson, K., Buzsaky, F. and Mosbach, K. (1986) Growth of anchorage-dependent cells on macroporous microcarriers, Bio/Technology 4, 989-990. Reiter, M., Blml, G., Gaida, T., Zach, N., Unterluggauer, F., Doblhoff-Dier, O., Noe, M., Plail, R., Huss, S., and Katinger, H. (1991) Modular integrated fluidized bed bioreactor technology, Bio/Technology 9, 1100-1102. Shiloach, J., Kaufman, J., Trinh, L. and Kemp, K. (1996) Continuous production of the extracellular domain of recombinant receptor from HEK 293 cells using novel serum free medium, in Carrondo, J.T., Griffiths, B. and Moreira, J.L.P. (eds.), Animal Cell Technology, Kluwer Academic Publishers, Dordrecht, pp. 535-540. Shiragami, N., Honda, H. and Unno, H. (1993) Anchorage-dependent animal cell culture using a porous microcarrier, Bioproc. Engin. 8, 295-299.

FLUIDIZED BED TECHNOLOGY: INFLUENCE OF FLUIDIZATION VELOCITY ON NUTRIENT CONSUMPTION AND PRODUCT EXPRESSION

G. BLÜML 1 , M. REITER 2 , TH.GAIDA2, N. ZACH2, A. ASSADIAN 2 , C. SCHMATZ2 and HERMANN KATINGER 2 1 )Pharmacia Biotech Europe, Vienna, Austria 2 )Institute for Applied Microbiology, Vienna, Austria

Introduction

Fluidized beds in combination with macroporous microcarriers are based on the perfusion technology. Perfusion technology was developed as it was recognised that cells in vivo are continuously supplied with blood, lymph, or other body fluids to keep

them in a constant physiological environment. To exploit the high productivity potential per reactor volume of a fluidized bed system optimisation of fluidization

velocities for anchorage dependent as well suspension cells immobilized in

macroporous matrices are needed to guarantee a sufficient nutrient supply similar to in vivo conditions.

Fluidized bed with internal circulation Fig 1 shows the principle of the internal circulation with microsparging for oxygenation. In the Cytopilot (Vogelbusch / Pharmacia Biotech) oxygen microbubbles are sparged homogeneously into the downcomer flow of the draft tube and uniformly

distributed by the impeller (Fig. 1). This minimizes on one hand pO2 gradients of the system and on the other hand increases theoretical height of the fluidized bed enormously. 385 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 385-387. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

386

Culture Run Comparison During the Fermentation run with Cytoline 2 a bed expansion of 120 % (fluidization velocity 12 cm/min) lead to a concentration gradient of nutrients throughout the carrier bed. The Volume stream was 2.1 L/min that equals one medium exchange in 8.3 min, which is insufficient for high density cultures. Fig 2a shows Mol produced lactate per Mol consumed Glucose. The Quotient was 50% higher than the CSTR and FBCytolinel which were well supported with Oxygen. After increasing the fluidization velocity to 25 cm/ min the culture of FBCytoline2 came nearly to the same level of lactate production per Glucose consumption. Fig 2 b (showing the relation between secreted product per consumed Glucose) demonstrates the same effect of circulation rate on sufficient nutrient supply. After increasing the circulation rate to 25 cm/min (day 57) the culture behaved similar to CSTR and fluidized bed culture with heavier beads. 2 Amino acids (Arginin and Serin) were analysed at different Glucose levels on all 3 culture runs. The CSTR and FBCytolinel (45 cm/min) are much more similar in spite of the different fermentation system than the slow circulating culture FBCytoline2 (Fig 2c,d)

Summary These fermentation runs showed that the minimum bed expansion for light beads like

Cytoline 2 is 200% at 25cm/min to give a reasonable nutrient supply to the cells in the pores. For Heavier beads like Cytoline 1 a bed expansion of 150% at 45 cm/min is enough. That implements a higher carrier load in the fermenter than with lighter

387

beads. The disadvantage of the higher speed is the higher shear stress especially for

sensitive cells like hu hybridoma cells in the attachment phase of the culture. The relation between volume specific productivity of hu-hybridoma and fluidization velocity shows an optimum of about 25 cm/min using Cytoline2 (sufficient medium supply and low shear stress). Using Cytolinel, CHO cells (expressing the same monoclonal antibody) and a fluidization rate of 45 cm/min (necessary for oxygen supply) the productivity could be increased by a factor of 3 compared to the fluidized bed with hu-hybridoma and by a factor of 30 compared to the CSTR (Tab 2).

The required fluidization velocity in Tab 3 are 3-4 times lower than the sedimentation

velocities of Cytoline 1 or 2. There is a small risk of flushing out the carriers from the upper chamber of the fluidized bed.

Product formation could be growth dependent for some cell lines. In that case a higher fluidization velocity could be useful to create cell bleeding out of the pores to keep the

growth rate as high as possible. The fluidization velocity could be reduced about 15 20 % by using macromolecules such as Dextran, Pluronic, Xanthan etc., maintaining

the same bed expansion.

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HIGH DENSITY AND SCALE-UP CULTIVATION OF RECOMBINANT CHO CELL LINE AND HYBRIDOMAS WITH POROUS MICROCARRIER CYTOPORE CHENGZU XIAO, ZICAI HUANG, WENGQING LI, XIANWEN HU, WENLU QU, LIHUA GAO AND GAOYAN LIU. Department of Cell Engineering, Institute of Biotechnology, 20 Dongdajie, Fengtai, Beijing 10007l, P.R. China Abstract Using porous microcarrier Cytopore and a low-serum medium supplement BIGBEF-3, we have successfully cultivated recombinant CHO cell line CL-11G producing prourokinase and hybridomas producing prourokinase monoclonal antibody in Celligen 1.5L or 5L bioreactor. The cell density could reached the yields of prourokinase and monoclonal antibody increased with increasing cell density. As all of these cells could release spotaneously and reattach to porous microcarriers, it was very easy to scale-up the cultivation. Thus the bead to bead cells transfer method has been used to scale up the culture of CL-11G cells in 20L bioreactor in the pilot production of prourokinase, and to scale-up the culture of hybridomas in the production of McAB for purification of prourokinase. Key Words antibody

Porous microcarrier;

recombinant CHO cells;

prourokinase;

monoclonal

1. Introduction The development of microcarrier culture by van Wezel in 1967 (1) made the large- scale industrial culture of anchorage dependent cells for the production of vaccine and interferon possible. In 1980s, a great advance of this procedure was achieved by the development of a series of porous microcarriers (2-4). In previous paper we reported that using porous microcarrier Cytopore, we had successfully cultivated a genetically-engineered CHO cell line and some hybridomas to produce pro-UK and McAB (5). This paper reports our successful results in using the bead to bead cells transfer method to sule up the cultivation of both the CHO cells and hybridomas in the pilot production of pro-UK and McAB. 2. Materials and Methods 2.1 .CELL LINES AND MEDIA The cell lines used were genetically-engineered CHO cell line CL-11G producing prourokinase ( 6 ) and hybridomas X15 and 38-1-7 cell lines producing prourokinase monoclonal antibody (McAB) ( 7 ). The medium for CL-1G cells was DMEM:F12 (1:1), with the addition of some amino acids and low-serum medium supplement BIGBEF-3 ( 5 ), and supplemented with 1% NCS, aprotinin and kanamycin. The medium for hybridomas was RPMI 1640, supplemented with 1% NCS and 0.1% pepton. 389

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 389-393. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

390 2.2. POROUS MICROCARRIER The dry Cytopore microcarriers ( Pharmacia Co. Sweden ) were hydrated and swelled in PBS before cultivation. After autoclaving at for 20 min, removed the supernatant and washed the microcarriers with culture medium once or twice. 2.3. CULTURE VESSELS Firstly spinner flask (Wheaton Co., USA) was used. The stirring rate was 30-40rpm. Then 1.5L or 5L Celligen bioreactor (NBS Co., USA) supplemented with a modified perfusion controller (8) and Biostat UC 20L bioreactor with spin filter was used. 2.4. CELLS COUNTING The citric acid-crystal violet method was used to count cells in porous microcarriers. In order to avoid a part of cells remaining in pores, the carriers were incubated for 4-5 hours and shaked several times during incubation. At the same time, the MTT ( 9 ) method was also used to confirm the satisfactory growth of cells inside the microcarriers. 2.5. GLUCOSE, PRO-UK AND McAB ASSAY The methods used in measuring glucose and pro-UK were the same as that mentioned in reference ( 10 ). The method for measuring McAB was ELISA assay ( 7 ).

3. Results 3.1. THE CULTURE OF CL-11G CELLS AND HYBRIDOMAS IN WHEATON SPINNER FLASK When the concentration of Cytopore was

, and the medium was exchanged once or

391 twice each day, the cell density of both the CL-11G and hybridomas could reach over ( Fig. 1 ). The pro-UK and McAB yields increased with increasing cell density .

3.2. CELLS TRANSFER FROM BEADS TO BEADS

Just like the culture with Biosilon (10) and MC-1 (11) microcarriers, CL-11G cells could detach from Cytopore porous microcarriers spotaneously and reattach on fresh ones. So it was also very easy to scale up the cultivation as reported by Kamiya ( 12 ). When the scale up ratio was 3, as shown in Fig 2,we withdrew 2/3 of culture from a spinner flask (200ml) and added the same volume of fresh midium and microcarriers at 18th day, and then transfered the whole 200ml culture into a larger flask with 500ml of fresh midium and microcarriers at 28th day, the final cell density could still reach over each time (Fig.2, 3).

392 3.3. SCALE-UP CULTIVATION OF CL-11G CELLS FROM 5L BIOREACTOR TO 20L BIOREACTOR When we used the bead to bead cells transfer method to scale up the cultivation of CL-11G cells from Wheaton spinner flask (700 ml ) to Celligen bioreactor ( 5L ), then to Biostat UC 20L bioreactor, the cell density could also reach , and the highest yield of proUK was ( Fig. 4 ).

3.4. SCALE-UP CULTIVATION OF HYBRIDOMAS FROM WHEATON TO 1.5L BIOREACTOR Using the same method we also successfully scaled up the cultivation of hybridomas from Wheaton spinner flask ( 700 ml ) to Celligen 1.5L bioreactor. The cell density reached , and the titer of McAB could reach ( Fig. 5 ). Using this McAB affinity chromatograph, we purified the prourokinase to a purity of over 95% by HPLC assay with the specified activity of (reported elsewhere).

4. Discussion In a privious paper (5) we demonstrated that porous microcarriers had a lot of advantages compared to solid microcarriers, such as : l.The concentration of carriers in the culture was much less than that of solid microcarrier.2. Cells grown inside pores were protected for damage by shearing stress, so that higher stirring speed could be used. 3. Cytopore porous microcarrier was not only suitable to culture anchorage-dependent cells such as CHO cells, but also suitable to culture anchorage-independent cells such as hybridomas. 4. As most of cells were neted inside pores, cells suspended in the supernatant were much less. In addition, the cells cultured inside the porous microcarriers were less depandent on attachment factor, so the serum used in the culture might be furtherly reduced. This time we find an another advantage,

393

that is it can also be used to scale up the cultivation with bead to bead cells transfer method. So we definitely consider that using porous microcarriers to large-scale culture geneticallyengineered cells for the production of recombinant products including prourokinase is a very prospective approach. 5.References

1. 2. 3. 4. 5.

van Wezel AL. Growth of cell-strains and primary cells on microcarriers in homogeneous culture. Nature, 1967; 216:64 Looby D, et al. Immobilization of animal cells in porous carrier culture. TIBTECH, 1990; 8:204 Gotoh T, et al. A new type porous carrier and its application to culture of suspension cells. Cytotechnology, 1993; 11:35 Shirokaze J, et al. High density culture using macroporous microcarrier. in: Spier RE. Griffith B eds. Products for today, prospects for tomorrow. Oxford: ButterwerthHeinemann, 1994; 261 Xiao CZ, et al. High density cultivation of recombinant CHO cell line and two strains of hybridomas with porous microcarrier Cytopore. Bull Acad Mil Med Sci, 1996; 20:191

6.

Li FZ, et al. High-level expression of pro-urokinase cDNA in Chinese hamster overy cell

7.

line (1). Bull Acad Mil Med Sci, 1993; 17:89 Ai X, et al. Preparation and characterization of monoclonal antibodies against uPA and their application for purification of uPA. Bull Acad Mil Med Sci, 1995; 19:248

8.

Xiao CZ, et al. An autocontroller system for cell perfusion cultivation on microcarrier.

Chinese Patent, ZL 91208893.1, 1994-09-14 9.

Jia XH, Xiao CZ. MTT method for the esstimation of the number of cells cultivated in microcapsules. Bull Acad Mil Med Sci, 1993; 17:207

10.

Xiao CZ, et al. High density cultivation of a recombinant CD-1 cell line producing prourokinase using a Biosilon microcarrier culture system. Chin Med Sci J,1994;9:203

11.

Xiao CZ, et al. High density cultivation of genetically-engineered CHO cell lines with microcarrier culture systems. Chin Med Sci J, 1994; 9:71

12.

Kamiya K, et al. Subculture for large scale cell culture using macroporoous microcarrier. in: Beuvery EC. et al. eds. Animal Cell Technology: Development towards the 21st Century. Kluwer Acad Publ, The Netherlands, 1995;759

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CELL-SETTLER PERFUSION SYSTEM FOR THE PRODUCTION AND GLYCOSYLATION OF HUMAN INTERFERON- BY CLUMPED CELLS D. LAMOTTE1, J. STRACZEK2 & A. MARC1 1 LSGC-CNRS, BP 451, 54001 Nancy Cedex - France 2 Lab. de Chimie - CHU, 54035 Nancy Cedex - France 1. Introduction The production of recombinant proteins by mammalian cells is commonly performed in batch or fed-batch cultures. Nevertheless, productivity in such a culture can be low. In cell lines where protein synthesis is proportional to the cell division, a variety of means have been designed to increase the maximum cell density. Amongst them are perfusion systems, in which cells are retained whilst spent medium is replaced continuously with fresh medium1,2. Natural aggregation of animal cells has also been widely studied and proven to be an efficient means of culture3 and sedimentation of aggregates in a column allows the efficient retention of cells4. However, we report here the first animal cell perfusion

culture based upon cell aggregate sedimentation. This shape is very convenient for cells sensitive to shear stress that can not be cultivated in classic perfusion systems. Culture conditions may dramatically affect glycosylation and therefore the efficacy of glycoproteins. Because the cell environment could change in perfusion cultures, the glycosylation pattern of the protein of interest may be altered in a long-term culture, and hence the product quality could be compromised. Few studies have reported the protein glycosylation changes in perfusion cultures 5 despite the emergence of cell perfusion systems for industrial purposes6. In this study, we have cultivated the CHO 320 cell line for the production of human interferon- (IFN) in an 'in-house' perfusion system based on cell retention by gravitational sedimentation. The IFN glycosylation was monitored by capillary electrophoresis throughout the culture. 2. Materials & Methods The producing IFN CHO cell line (CHO 320) was supplied by the Wellcome Foundation Laboratories. Cultures were performed with a serum-free medium. We have modified a 2L-stirred-tank bioreactor to allow the perfusion, and it was fitted with an internal glassware tube (diameter: 1 cm, length: 12.5 cm) to provide a cell settling zone. The bottom of the tube was lower than the agitation blades. The supernatant was removed continuously from the vessel through the top of the glassware tube using a peristaltic pump. The working volume in the reactor was 1.1L during the perfusion phase. Prior to perfusion, cells were grown in batch culture for 80 hours. Separations and detections of the IFN glycoforms were performed with a Beckman P/ACE 2100 capillary electrophoresis according to the protocol of James et al.7 395

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 395-397. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

396

3. Results 3.1. KINETICS OF THE CHO 320 PERFUSION CULTURE

Cells were seeded in the reactor with an initial density of After being propagated in batch culture for 80 hours to reach , the perfusion was initiated in standard medium and the dilution rate increased daily from 0.022 to 0.055 until 350 hours of culture. During this phase, the IFN level increased rapidly to reach the mean value of In parallel, initial clumps (100 m diameter) enlarged with cell growth to reach a maximum diameter of 800 m after 550 hours of culture (phase I). Thereafter, the perfusion rate was kept constant leading to the stabilisation of the cell density at . The addition of amino-acids (excepted glutamine) in the standard feeding medium (phase II) gave a transient decrease of the IFN level. Conversely, the addition of vitamins in medium did not have significant effect on the kinetic parameters (phase III). Figure 1 shows that perfusion culture generated a twofold higher IFN accumulation than batch culture. Consequently, the volumetric IFN production (in IU/1/hr) is significantly higher in perfusion culture (x8). These results confirm that perfusion systems are powerful tools to increase productivity of recombinant proteins.

397

3.2. IFN GLYCOSYLATION MACRO-HETEROGENEITY

The IFN glycosylation macro-heterogeneity was monitored during the perfusion culture by use of capillary electrophoresis. The three classes of IFN glycoforms (doubly (2N), singly (IN) and non glycosylated (ON)) were observed and their proportions reported in figure 2. The proportion of fully glycosylated IFN decreased throughout the initial batch phase of culture. Conversely, the glycoform proportions were constant during the four phases of the perfusion culture and similar to those found at the end of the batch culture. The cell-settler system described here allows the good product consistency for more than 1000 hours despite changes in the culture environment and the kinetic parameters. 4. References 1. Goergen J.L., Lourenso da Silva A., Marc A. and Engasser, J.M. (1995) Fast propagation of hybndoma cells in a forced-flow membrane perfusion reactor, in E.G. Beuvery, R.E. Spier and W.P. Zeistemaker (eds.). Animal Cell Technology: Developments towards the 21st century, Kluwer Academic Press. Dordrecht, pp.

705-710. 2. Pinion H., Rabaud J-N., Engasser J.M. and Marc A. (1991) Cytoflow: a new perfusion bioreactor for research and production, BFE 8, 344-347. 3 Chevalot I., Visvikis A., Nabet P., Engasser J.-M. and Marc A (1994) Production of a membrane-bound

protein, the human gamma-glutamyl transferase, by CHO cells cultivated on miccrocarriers, in aggregates and in suspension, Cytotechnology 16 121-129. 4. Moreira J.L., Alves P.M., Rodrigues J.M., Cruz P.E., Aunins J.G. and Carrondo M.J.T. (1995) Growth of BHK aggregates in a 2 liter bioreactor, in E.C. Beuvery, R.E. Spier and W.P. Zeistemaker (eds.). Animal Cell Technology: Developments towards the 21st century, Kluwer academic press, Dordrecht, pp. 805-809 5. Gawlitzek M., Valley U., Nimtz M., Wagner R. and Conradt H.S. (1995) Characterization of changes in the glycosylation pattern of recombinant proteins from BHK-21 cells due to different culture conditions, J. Biotechnol. 42 117-131.

6. Bödecker B.G.D. (1994) Production of recombinant factor VIII from perfusion cultures: 1. large-scale fermentation, in R.E. Spier, J.B. Griffiths and W. Benhold (eds.). Animal Cell Technology: Products of today, prospects for tomorrow, Butterworths, London, pp. 580-583. 7 James D.C., Freedman R.B., Hoare M. and Jenkins N. (1994) High-resolution separation human interferongamma glycoforms by micellar electrokinetic capillary chromatography, Anal. Biochem. 222 315-322.

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DISPOSABLE BIOREACTOR FOR CELL CULTURE USING WAVE-INDUCED AGITATION

Vijay Singh Schering-Plough Research Institute 1011 Morris Avenue Union, NJ 07016 US A email: [email protected]

Abstract This work describes a novel bioreaclor system for the cultivation of animal, insect, or plant cells using

wave agitation induced by a rocking motion. This agitation system provides good nutrient distribution, off-bottom suspension, and excellent oxygen transfer without damaging fluid shear or gas bubbles. Unlike other cell culture systems, such as spinners, hollow-fiber, roller bottles, scale-up is simple, and systems

with up to 10 liter liquid volume have been operated successfully. The device is disposable and therefore requires no cleaning or sterilization. Additions and sampling can be done without the need for a laminar flow cabinet. The unit can be placed in an incubator requiring

minimal controls. All these features dramatically lower the purchase and operating cost of a lab scale cell cultivation system. Results are presented for various model systems: 1) recombinant NS0 cells in suspension; 2) human 293 cells in suspension/adenovirus production; 3) sf9 insect cell/baculovirus system; and 4) human 293 cells

on microcarrier. These examples show the general suitability of the system for suspension cells, anchorage-dependent culture, and virus production. The system is especially well suited to virus and

vaccine production because of the high degree of containment.

Background Technology for the cultivation of animal, plant and insect cells depends on the batch volume required. In the laboratory, devices such as spinner flasks, roller bottles, T-flasks and similar systems are typically used However, these devices can only produce 1 to 2 liters or culture per batch due to inherent oxygen transfer limitations. In particular, spinner flasks are very popular for suspension culture as well for anchorage-dependent systems on microcarriers, but they can only be used for a maximum liquid volume of 1 liter. There is no simple system to make 1 to 10 liters of cell culture which is the volume often needed for protein characterization, inoculum production and pilot production. It is usually necessary to use bioreactors which are typically stirred-tank bacterial fermentors modified to reduce shear forces. These bioreactors are complex, expensive, and do not really provide an optimum environment for cell growth due to high local shear and bubble aeration. Adapting stirred tank technology to cell culture is a futile exercise because this design has intrinsically high local shear rates. Instead it is critical to recognize the special demands of cell culture and design directly to satisfy these needs. It is also essential to make the cultivation system as simple as possible to operate. There have been many designs in the past, for example fluidized bed bioreactors and hollow-fiber systems which have worked well, but were too complex to displace the spinner flask as the workhorse of cell cultivation. The objectives of this work was to develop a new cell culture bioreactor that: 1. Can be used to cultivate 100 ml to 10 liters of cells A high turndown ratio (maximum volume : m i n i m u m volume) is also required to reduce the number of transfers. The poor 2:1 turndown 399 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 399-407. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

400 ratio of a conventional spinner flask makes inoculum scale-up very labor intensive, because the culture

must be transferred from one size spinner to the next larger size many times under sterile conditions. 2. Eliminates sparging of gas. It is clear that hubbies cause damage, so any good cell culture device should

not generate bubbles. Gas diffusion membranes are not satisfactory because of cost, complexity and poor reliability. Also scale-up is limited by the membrane surface available for diffusion. 3. Eliminates mechanical agitation. Mixers generate high local shear. Seals around the mixer shaft are costly and have poor reliability. 4. Guarantees sterility. The bioreactor should be available presterilized and disposable after use. This eliminates the need for heat sterilization and cleaning. A large portion of the cost of a bioreactor is the sterilization system. Cleaning of a bioreactor can take 8-10 hours per run making this a major operating expense. 5. Containment. The unit should operate as a closed system so that it can be operated in the laboratory or plant without the need for a laminar flow cabinet. 6. Simple controls. Like a spinner, the unit should not require complex controls. In cell culture, media can be buffered to control pH and if the aeration system is capable of delivering the maximal oxygen demand

there is no need for DO control. 7. Low cost. A stainless steel bioreactor (10 liters) costs about $ 80,000. It should be possible to develop a cell cultivator for 1/10 of this cost. The lack of moving parts, seals and instrumentation should also significantly reduce operating expenses. 8. Easy to operate. Conventional bioreactors and pump-around systems require extensive training to operate. Even small mistakes can lead to contamination. Just mastering the sterilization and calibration

procedures requires 1 -2 weeks of training. Many scientists just want to grow cells to study the protein being expressed rather than become bioreactor experts. The cell cultivation system should therefore be

simple to learn. The goal was to develop a bioreactor which could be operated by an undergraduate student with 1 hour of instruction.

A bioreactor system was developed that meets all these objectives. It is low cost, simple to operate, and provides an optimal environment for cell and virus culture. This paper summarizes the oxygen transfer capabilities, mixing time and cell cultivation potential of this technology based on wave-induced agitation.

Materials and Methods Bioreactor System and Operation

The bioreactor consists of a presterilized plastic bag that is partially filled with media and inoculated with cells. The remainder of the bag is inflated with air which is also continuously passaged during the cultivation. Mixing and mass transfer are achieved by rocking the bag back and forth. This rocking motion generates waves at the liquid-air interface, greatly enhancing oxygen transfer. The wave motion also promotes bulk m i x i n g and off-bottom suspension of cells and particles such as microcarriers. The bag is discarded after harvest, eliminating any need for cleaning or sterilization since this is the only component in contact with the cells. New bags are delivered guaranteed sterile by gamma radiation. Bags are made of FDA approved biocompatible polyethylene. These bag materials are commonly used for blood collection and biological fluid handling. Special ports have been developed to allow sterile additions to the

bag and to withdraw samples, without the need to place the bag inside a biosafety cabinet.

401

The rocking mechanism was developed by the author. The device as shown in Figure 1 consists of a platform that rotates or rocks in one axis through an angle of 5 to 10°. Pneumatic bellows are used to rock the platform at an adjustable rocking rate between 5 to 40 rocks/minute (rpm). A pre-sterilized bag is

placed on the platform; partially filled with media and then inflated using the sterile inlet gas filter integral to the bag. Air is continuously passaged through the headspace of the bag by a pneumatically driven pump. This provides oxygenation and gas exchange for pH control and CO2 removal. Exhaust air passes through an exit sterilizing filter and a backpressure control valve. The unit requires only compressed air (2 - 3 bar) to operate. No electrical connection is necessary. Temperature and pH control is

achieved by placing the entire unit inside a conventional cell culture CO2 incubator. The rocking platform systems: CellbagTM, including specially designed cultivation bags (2 liter and 20 liter), are available from BioPro

International, Farmingdale, New York, USA ([email protected]).

Oxygen Transfer Measurement

The oxygen transfer capabilities of the system were assessed by measuring the volumetric oxygen transfer coefficient kLa at various rocking rates, liquid volumes and aeration rates. The dynamic method was used for kLa measurement. Here, the liquid in the bag is deoxygenated by passing nitrogen through the

headspace. Once the dissolved oxygen (DO) concentration was near zero, air was introduced in to the headspace and the rise in DO recorded. kLa was calculated from the slope of the following mass balance equation:

For the oxygen transfer studies an oxygen sensitive dye was added to enable non-invasive dissolved oxygen measurement. Selected runs were confirmed using conventional DO probes inserted into the bag. Mixing Time Measurement

Mixing time in the bag at various rocking rates were determined by injecting a fluorescent tracer dye into the bag and videotaping the dispersion of the dye in the bag. UV light was used to enhance the contrast.

402 Time-tagged images were captured from the videotape and the mixing time was determined visually from photographs. Mixing time was defined as the time required after injection to achieve complete homogeneity.

Dissolved Oxygen and Carbon Dioxide Off-line measurement of dissolved oxygen and dissolved were done by sampling the bag using a syringe and analyzing it with a blood gas analyzer (Radiometer ABL5). Cell counts were done by

hemocytometer, and product assays were done by appropriate methods such as ELISA. Results - Oxygen Transfer and Mixing

I. Oxygen Transfer Studies:

The rocking motion of liquid in the bags generates a larger mass transfer surface that static culture which leads to much greater volumetric transfer coefficient Figures 2 and 3 show the rocking and aeration rates in 2 liter bags and 20 liter bags.

The oxygen transfer studies show that a

achieved at various

of 3 hr -1 is possible in a 1000 ml liquid volume (2Lbag).

This is almost three-fold higher than the for 1000ml in a comparable 2L spinner (Table 1). This should result in potentially 3X higher maximal cell densities in the bag compared to a spinner.

The data for spinner flasks in good agreement with published values. Aunins, et al (1989) report a around 1 hr-1 for 500ml liquid in a 500ml spinner. Dorresteijn et al (1994) report a value around 2 hr -1 for 300ml liquid in a 600ml spinner system. For the l0liter scale (in 20L bag) the maximum is around There is no comparable spinner system since the surface to volume ratio decreases substantially as the spinner volume is increased. For example, a 5 liter spinner would have a less than making it essentially useless for cell cultivation. With

of 3 to

possible to grow up to

and assuming a typical oxygen demand of

(Singh, 1996), it is

while maintaining dissolved oxygen concentrations above 10%

saturation.

With this knowledge of oxygen transfer it is possible to guarantee against oxygen depletion at any given

cell density by selecting the appropriate rocking/aeration rate. Unlike stirred tank bioreactors, there is no bubble damage or danger of “overaeration”. stripping can be controlled by adjusting the concentration of the inlet air. This effectively eliminates the need for a dissolved oxygen probe/ control

403 system. Samples may be taken and assayed periodically on a blood gas analyzer, off-line, to verify the DO and levels. II. Mixing Studies: Estimates of mixing time were made by injecting a fluorescent dye and videotaping the dispersion of the dye under various conditions. These experiments showed that the wave-induced motion was very effective in mixing the liquid in the bag. Mixing time (defined as time for complete homogeneity) was typically 5 to 10 seconds. Subsequent studies with microcarrier cultivation showed good off-bottom suspension with some particle gradients in the liquid. However, no significant settling of microcarriers or cells was

observed. The wave-agitation phenomena is the subject of a pending patent application. Results - Biological Systems 1. Monoclonal Antibody Production - 1 liter and 10 liter scale

An important application for cell culture is the production of monoclonal antibodies in vitro. Spinner or shake flasks can be used for 1 to 3 liters or culture, however oxygen transfer limitations in these system preclude further scale-up. For larger volume it is necessary to use complex bioreactors based on stirred

tanks, hollow fiber, or immobilized technology.

The applicability of the Wave Bioreactor system for lab-scale (10 liter) culture was evaluated. The cells used were a NS0 cell line expressing a humanized monoclonal antibody. Culture was performed in serumfree media commonly used for this cell line. Cultivation was started in a 1 liter culture volume with 250ml

of inoculum generated in a 500ml spinner flask. After 96 hours the cell density was cells/ml.

Two liters of fresh media was added to the bag and the cultivation continued. After an additional 96 hours the cell density again reached and 7 liters of fresh media was added to the bag to bring to the final culture volume of 10 liters, and cultivation was continued until zero viability (300 hours). Figure 4 shows the cell counts and monoclonal antibody production. The stepwise dilution technique used demonstrates how the large turndown ratio (maximal volume/minimum volume) of the bag enables large culture volumes without the need for transfers as would be the case using spinner flasks. Culture profile was very consistent with smaller scale spinners. Cell densities exceeded Dissolved oxygen levels remained above 50% saturation. Antibody expression was normal at over 600

mg/l. The system is clearly well suited for suspension cell cultivation and monoclonal production. Inoculum scale-up can be done within the system without the need for splits.

404 2. 293 cells/Adenovirus Production

The potential of the Wave Bioreactor for virus production was tested using a recombinant adenovirus system. A human embryonic kidney cell line (293) grown in suspension was used as the host. Initial seeding density in a 2L bag containing 1000ml of serum-free media was The

unit was placed in a 37°C incubator with a 5% CO2 atmosphere. The culture was grown for 5 days at a rocking rate of 10 rpm and 0.1 vvm aeration. At this stage, the viable cell count was 500 ml of the culture was removed and replaced with 500 ml of fresh media. After an additional 24 hours the culture was infected with an MOI of 30 virus particles/cell. The culture was harvested three days postinfection at which point the viable count was essentially zero (Figure 5). The final virus titer was 10,000 particles/cell. From Figure 5, it is apparent that the system is quite capable of maintaining adequate dissolved oxygen

levels at these cell densities. The pH remains very constant due to the excellent gas exchange as evidenced by the constant until late in the infection when the pH drops rapidly beyond the capability of the buffer system. The system is very easy to use. All additions and sampling were done in the incubator itself. No biosafety hood was required. The standard 0.1 micron exhaust filter was supplemented by a Pall DFA cartridge filter. The integrity of the exhaust filter system was tested by passing the exhaust gases to an uninfected

293 culture. Absence of any infection in the second culture confirmed the effectiveness of the exhaust filtration in containing adenovirus.

3. Baculovirus Production - 10 liter scale

The sF9/Baculovirus system is an excellent method for the rapid expression of large amounts of recombinant proteins. However, the oxygen requirements of sF9 cells are higher than mammalian cells

and this restricts the volume of spinner or flask culture to around 1 liter. Typically, larger volumes than are possible in flasks are required to produce sufficient protein for isolation and complex bioreactors are usually necessary. It was desired to determined if the simple Wave Bioreactor system could be used to scale-up inoculum and produce virus in 10 liter liquid volume. The gene sequence for a recombinant protein was cloned into the pAcHLT-B baculovirus expression

vector, transfected into sf9 cells and a high titer virus slock was generated. For cultivation in the bag, 1 liter of Bio-Whittaker X-Press Insect Cell Media was added to a 20 liter (total volume) bag. This was inoculated with Rocking rate was 20 rpm in a humidified

405 incubator at 25 °C. After 6 days of cultivation the cell count reached At this point 4 liters of fresh media was added to the bag. Within an additional 4 days the cell count reached

in this increased volume. At this point another 4 liters of fresh media was added along with the virus at a MOI of 0.5 bringing the system to a total volume of close to 10 liters. Harvest was done 2 days post infection.

This system produced a titer comparable to 100 ml shake flasks. It demonstrated the ability to increase cell mass by simply added fresh media to the system. No conventional series of flasks or splits were required, reducing labor and potential for contamination. All additions and transfers into the bag were done in the incubator. Figure 6 shows that oxygen transfer and CO2 desorption was not limiting. 4. Microcarrier Culture Human 293 cells were cultivated on Cytodex 3 microcarriers. Growth media was DMEM with 10%

bovine calf serum. Inoculum was collected by trypsinizing microcarrier cultures grown in spinner flasks. 60ml of inoculum was added to 3g Cytodex 3 in 900 ml of media and then transferred to a 2 liter bag. 10 ml of the inoculum was added to a spinner flask with 0.45g Cytodex 3 and 140ml media (250ml Bellco) that was run in parallel at 40rpm. Growth in the bag culture was very similar to the spinner. Cell counts, glucose consumption and lactate formation were equivalent with the exception of slightly higher cell counts in the supernatant from the bag. Cells grew to confluency ( >50 cells/bead) in the bag . Applications 1. Substitute for Spinner Flasks and Roller Bottles

Spinner flasks cannot be used for liquid volumes greater than 1 liter because of insufficient oxygen transfer. The Wave Bioreactor generates much more mass transfer surface because of the unique wave action. Operation to 10 liters is routine with cell densities over

406

The Wave Bioreactor has a greater range of operating volumes. This reduces the number of transfers that

need to be made minimizing handling and potential for contamination.

The Wave Bioreactor is an ideal way to make a few liters of cell culture for lab purification, inoculum propagation and cell bank generation. 2. Ultra Low cost Bioreactor: Traditionally the only way to produce 5 to 20 liters of cell or virus culture was to use expensive and complex bioreactors. The Wave Bioreactor is 1/10 the price of a comparable bioreactor and does not require substantial training. No utilities such as steam, cooling water and process gasses are required. No complex instrumentation is necessary. The unit can be set up in a conventional cell culture incubator. A CO2 controlled incubator can be used if pH control is desired. This makes lab scale cell and virus cultivation possible even in small labs, hospitals and universities.

3. High Containment / GMP Systems: The u n i t is completely closed. Additions and samples can be taken using a hospital-type needleless syringe connector or tube-fuser. These operations do not require a laminar flow cabinet but can be done with the unit inside the incubator. The bag is certified to be sterile by gamma radiation. Air into and out of the bag is sterilized by filters appropriate to the containment level necessary. At the end of cultivation the bag can be removed to another area for processing. The disposable nature of the system makes is very suitable for virus production and BL2/BL3 operations.

407

4. Cell Therapy and Gene Screening: The Wave Bioreactor allows even the smallest lab or clinic to perform cell culture. The simplicity of the

systems enables the scientist to concentrate of the use of the cells rather than learning how to operate complex bioreactor systems. The large cultivation volumes and the ability to grow attachment-dependent lines make it useful for primary cell cultivation for cell therapy applications and the simplicity of the system allows the rapid expression and screening of hundreds of genes needed for gene therapy and human genome elucidation. Conclusions

A novel bioreactor (patent pending) for cell culture has been developed. The system utilizes an inflated disposable plastic bag as the cell cultivation chamber. These specially designed bags are made of biocompatible materials and are delivered sterile, eliminating the need for cleaning and sterilization.

Oxygen transfer and mixing are accomplished by wave-induced agitation caused by rocking the bag back and forth. The rocking mechanism has been optimized in terms of rocking angle, rocking rate, aeration, and mechanical design to provide a kLa for oxygen transfer of 2 to

Based on typical cell respiration

rates, this transfer rate is sufficient to grow up to The bioreactor system, unlike spinner flasks, is not limited by gas-liquid transfer surface and scale-up to 10 liters operating volume has been demonstrated using several typical cell and virus production systems. This simple, low cost wave bioreactor system can be used for animal, insect and plant cell culture. The closed design is very suitable for virus production or other applications requiring high containment. The system requires minimal instrumentation and can be operated inside a laboratory incubator. All handling can be done in the open, eliminating the need for a laminar flow hood. References Aunins. J.G., Woodson, B.A.. Hale, T.K. and Wang, D.I.C.. 1989. Effects of Paddle Impeller Geometry on power Input and Mass Transfer

in Small-Scale Animal Cell Culture Vessels. Biotechnol. Bioeng. 34: 1127-1132. Dorresteijn, R.C., de Gooijer, C.D., Tramper, J. and Beuvery, E.C., 1994. A Simple Dynamic Method for On-line Determination of k L a During Cultivation of Animal Cells. Biotechnol. Techniques. 8, 9 675-680. Singh.V., 1996. On-Line Measurement of Oxygen Uptake in Cell Culture Using the Dynamic Method. Biotechnol. Bioeng. 52: 443-448.

Acknowledgments: *US and European patents are pending for portions of this work. I would like thank my many collaborators especially Nancy Connelly, John McDowall, Edward Glowacki, Raphael Nir, Colleen Brolly, Peter Pappas and Jeffery Mutter.

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USE OF miniPERM SYSTEM FOR AN EFFICIENT PRODUCTION OF MOUSE MONOCLONAL ANTIBODIES

M.L.NOLLI*, R.ROSSI°, A.SOFFIENTINI°, D.ZANETTE°, C.QUARTA° °Biosearch Italia, Via R Lepetit 34, 21040 Gerenzano (VA) Italy *Consultant Biotechnology, Biosearch Italia

ABSTRACT

A rapid and efficient method for the production of monoclonal antibodies has been set up using the miniPERM system. The miniPERM represents a new generation of compact and disposable laboratory bioreactors for the growth of suspension cells. The combination of some important requirements as 1) high surface volume ratio; 2) sterility; 3) small space occupied; 4) monouse; 5) easy to use; 6) possibility to use four modules at the same time makes the system very useful for an efficient production of hundreds of milligram quantities

of MAbs. A mouse hybridoma cell line, from NSO Ag 14 non secreting cells, has been cultivated in miniPERM modules and the corresponding IgG1 MAb was collected after 1 week run. 70-100 mg of MAb/module/week have been produced either in conventional medium (with serum) or in serum free medium. This module productivity permits a production of more than 300 mg of MAb/week using at the same time the four modules. The SDS-PAGE and IEF analysis of the purified IgG1 MAb is presented. 1. INTRODUCTION

At early phases of a biotechnology project, aimed to produce either monoclonal antibodies (MAbs) or recombinant proteins, it is crucial to have available several mg quantities of the protein, possibly in one batch, to set up purification and analytical methods and to carry out preformulation studies. Small disposable bioreactors, specifically designed for high density cell cultures, offer the possibility to easily cultivate either hybridomas or recombinant cells using a high-surface volume ratio concept and produce the needed secreted product. Furthermore, the combination of the use of these flexible bioreactors with appropriate serum free media permits reliable cell cultivation and product generation (1). We describe here the production of a mouse monoclonal antibody from NSO myeloma parent cells in miniPerm bioreactor (2). The production was shown to be very efficient. 70100 mg/module/week of MAb were produced in serum free medium. It was also demonstrated that, when using simultaneously four modules, there is a consistency in the MAb production in terms of number and composition of isoforms and quantity of MAb produced. 409

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 409-415. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

410

2. METHODS 2.1 CELLS The mouse hybridoma, deriving from NSO parent myeloma cells, was adapted to grow in serum free medium

(SFM,Gibco cat. N: 12045-084) using the following protocol: 2.1.1. Cells were plated at in RPMI + 10% FCS + 5% Origen (Igen). 2.1.2. Then they were progressively diluted with SFM, 1:2, 1:4, 1:16, 1:32 and then in SFM alone. Every dilution with SFM, cells were left to grow for several generations, monitoring the cell viability and MAb production. 2.2 BIOREACTOR miniPERM bioreactor (Heraeus Instruments GmbH) is a small size bioreactor made up of two connected modules:

2.2.1. a production module (disposable culture chamber) 2.2.2. a nutrient module (reusable culture chamber) The bioreactor is rolled on a special turning device, specifically designed for a 37 °C

incubator. This roller apparatus permits the simultaneous use of four modules. 2.3 MODE OF CULTURE (FED-BATCH) 2.3.1 .The two miniPERM modules (nutrient and production module) were assembled in sterility following the operating instructions. 2.3.2. , at the end of the log phase, in 35 ml of SFM were then inoculated into the production module. 2.3.3. 350-400 ml of SFM were used to fill the nutrient module. 2.3.4. The apparatus was put onto the roller apparatus and then rolled. 2.3.5. 7-8 days runs were carried out, changing medium twice and taking samples every day. 2.4 .MAb TITRATION

The MAb produced was tested by an antibody capture ELISA set up in house. The antibody quantitation was done by using a standard curve of the purified MAb. The results were calculated with the use of logit-log regression. 2.5. MAb PURIFICATION AND ANALYSIS 2.5.1. The MAbs in the ascitic fluids and miniPERM supematants were purified using

Protein G Sepharose 4 FF (Pharmacia cat N° 17-0618-02). The samples were loaded onto the column with the conductivity and pH of the equilibration buffer (0.02M Na-phosphate pH=7.0). The column was washed with the same buffer. The MAbs were eluted with 0.1M

411

Gly-HCl pH=2.7 and immediately neutralized with 1 .0M Tris-HCl pH=9.0. 2.5.2. SDS PAGE (reduced): The reduced SDS-PAGE was done with a precast NOVEX™ Tris-Glycine gel with a 4-20 % gradient (Cat. N° EC6025). -mercaptoethanol was used as reducing agent. The running time was about 90 min. at a constant voltage of 120 V. At the end of the run the gel was fixed and then stained with Coomassie blue. 2.5.3. Isoelectric Focusing (IEF)(3): The IEF analysis was done using precast NOVEX™ IEF pH 3-7 gels (Cat. N° EC6655B). After running, the gels were fixed and then stained with Coomassie blue. 2.5.4.Densitometric measurements: The densitometric measurements were done with the SHIMADZU CS-9301PC dual wavelength flying spot scanning densitometer.

3. RESULTS AND DISCUSSION The miniPERM is a small modular bioreactor for the production of MAbs for analytical use. The results of the MAb production using this system are reported in Table 1. Four modules were simoultaneously used on the same roller apparatus to carry out one-week runs. The fed-batch mode of culture was used, changing medium twice/week. As shown, the quantity of MAb produced and its concentration in the medium, is comparable in the four modules. In fact the total quantity of MAb is in a range of 80-100 mg/module, while the concentration of the MAb is in the range of 2.5-3 mg/ml in 32 ml volume /module. The MAb production is very efficient. The recovery of MAb in the medium is at a high concentration (2.5-3 mg/ml) and in a limited volume (32 ml). The quantity of MAb, 80-100 mg /module /week, is an important amount for analytical use. In addition, the simultaneous use of the 4 modules makes possible a production of more than 350 mg of MAb in one week. An additional advantage is the production in serum free medium that allows either the use of MAb as it is or for an easier purification. Once purified, the MAb produced in the four modules (A,B, C, D) was analysed by SDSPAGE and by isoelectric focusing along with the densitometric distribution of the glycoforms. The results of these analyses demonstrate: 1) in reduced SDS-PAGE this MAb contains the typical IgGl heavy and light chain bands as those produced by the MAb from the ascitic fluid (fig. 1) and 2) both the IEF and densitometric analyses show the consistency of the glycoform distribution of the MAb produced in the four modules (fig 2 and fig. 3). Isoelectric focusing was also used to compare the respective glycoform distribution of the MAb produced by miniPERM and ascitic fluid(fig.4). The densitometric analysis (fig. 5) of the IEF gels show that the MAbs have the same number of glycoforms and similar percentage distribution for every glycoform.

412

413

414

415 4. CONCLUSION The consistency of the glycoform distribution and the amount of the MAb produced in the miniPERM modules prove the reliability ofthe system for production of hundred mg quantities of Mab in a single batch.

REFERENCES 1) Merck & CO., INC. Large scale production of mammalian cell infective viruses e.g. hepatitis A- in order to produce a commercial vaccine. Pubblication: WO95/04812. 16 February 1995. 2) Falkenberg, F.W., Hengelage, T., Krane, M., Bartels, I., Albrecht, A., Holtmeier, N., and Wuthrich, M.: A simple and inexpensive high density dialysis tubing cell culture system for the in vitro production of monoclonal antibodies in high concentration., J. Immunol. Methods 165 (1993), 193-206. 3) Williamson, A.R.: Isoelectric focusing of immunoglobulins. In " Handbook of Experimental Immunology 1, Blackwell scientific Publication Oxford, 1978.

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PRODUCTION OF ANTI-EGF RECEPTOR hMAB IN HOLLOW FIBER BIOREACTORS FOR IN VIVO DIAGNOSIS.

Arias, M.A., Valdés A., Curbelo D., Morejón O.M., Caballero I., Villán J., Gómez J.A., Fernandéz A., Rodríguez J., Morales A., Boggiano T., Castillo A., Bouzó L., Hermida C., García G., Vitón P., Pérez N., Rodríguez T. Center of Molecular Immunology, P.O.Box 16040, Havana 11600,Cuba. 1. ABSTRACT NSO transfected myeloma producing an anti-EGF receptor humanized MAb was cultivated in hollow fiber bioreactors up to production scale (Acusyst-P3/X). Its production rate and metabolic uptakes were compared with a hybridoma, that secreted the murine variant of this product also produced in hollow fiber. The control strategy of the fermentation is discussed. The purification scheme, mainly based on affinity and ion exchange, was established to obtain a product with a purity higher than 96 %. The freeze dring process was studied to obtain a final reduced product for labeling with for “in vivo” diagnosis. 2. INTRODUCTION The production of an anti-EGF receptor humanized MAb was carried out in the GMP facilities of the Center of Molecular Immunology, which is a Cuban Biotechnology institution devoted to production of proteins from animal cell culture(1). NSO myeloma was transfected to secrete a “reshaped” monoclonal antibody, which recognizes the EGF-receptor (2). It has been demonstrated that this MAb is a very efficient as marker of tumors with high level of anti-EGF receptor (lung, breast, brain, head and neck) (3). 3. RESULTS AND DISCUSSION

417 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 417-419. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

418 3.1 Upstream process

The production process is summarized in Figure. 1. Hollow fiber bioreactors were used to cultivate NSO transfected myeloma up to production scale. The inoculum preparation was done in T flasks and spinners (feed-batch mode) to obtain with viability higher than 90 %, using DMEM/F12 medium plus 5% of FBS. During stationary culture it was demonstrated that the protein production rate increases when the growth rate decreases due to nutrient limitation (4). The protein production rate (PPR) at large scale was stabilize around 280 mg/d (Figure 2). The metabolic profiles don't suggest any limitation in perfusion (Figure 3) and the dissolved oxygen

was kept high enough (data not showed). The PPR could be increased by manipulation of the cell line.

The control strategy for nutrients was designed from kinetic studies in stationary culture (5). It was shown that during the growth phase the glucose concentration had a minimum range between 6 - 7 mM and the lactate concentration has a maximum of 12 mM. Considering the difference between stationary culture and hollow fiber systems, the

main parameters to be controlled during the run were defined as follows. During the growth phase, pH should be 7.2 ; minimum glucose concentration, 8.3 mM and maximum lactate concentration, 11.1 mM. During production phase, pH should be 7.0, minimum glucose concentration, 5.5 mM and maximum lactate concentration, 16.6 mM. The minimum partial pressure of oxygen should be 100 mm Hg during both phases. 3.2 hMAb Purification Process

As it is shown in Figure 4, during anion exchange there are losses of about 20 % of the initial mass, probably due to pH (7,3), which should be 1 unit lower than the isoelectric point of the protein. According to DNA contents of the product, the relation A280/A260

was kept above 1,5 in the different streams after Protein A, implying a low level.

419

3.3 Formulation of freeze-dried product

To enhance the performance and quality during in vivo diagnosis a freeze-dried

formulation is used, which contains the reduced MAb ready for labeling with The reduction process is done by the Schwartz method, Mather modified (3). Then the

product is desalted to nitrogen purged PBS. After that, a reducer agent

is added.

The filled vials are lyophilized to with a first freezing to -45°C, a primary drying to -20°C and a secondary drying to 25°C. The final product is submitted to Quality Control criteria, which is summarized in Table 1. 4. CONCLUSIONS

The process showed produces a freeze-dried formulation of anti-EGF receptor hMAb

with the required quality to be used as an “in vivo” diagnosis drug, in the programmed phase I clinical trial. The production process of this hMAb can be enhanced, mainly during the upstream and purification steps. 5. REFERENCES 1. 2.

3.

Villán J. Et. Al., Strategy for Scale Up of Mammalian Cell Culture in the New GMP Manufacturing Facility of the Cuban Biotechnology Industry. In : Animal Cell Technology. From Vaccines to Genetic Medicine. Proceedings 14th ESACT Meeting. Mateo C., Moreno E., Amour K., Lombardero J., Harris W., Pérez R., (1997). Reshaped of humanized monoclonal antibody to EGF-R with retention of full binding activity. Immunotechnology 3: 71-81

Iznaga N., Morales A., Ducongé J., Caballero I., Fernández E., Gómez J.A., (1998) Pharmacokinetics, biodistribution and dosimetry of anti-human epidermal growth factor receptor humanized monoclonal antibody R3 in rats. Nuclear Medicine & Biology, Vol. 25. (in press).

4. 5.

Boggiano T. (oral communication) Castillo A. J., Fernández A., Boggiano T., (1997), Study of different cell culture conditions for the production of “reshaped” Mab in NSO cells, ESACT Meeting poster.

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OPTIMISATION OF THE PRODUCTION OF VLP'S IN 2 LT STIRRED BIOREACTORS

P. E. Cruz 1 , A. Cunha 1 , C. C. Peixoto1, J. Clemente 1 , J. L. Moreira1, M.J.T. Carrondo 1,2 1 - IBET/ITQB, Apartado 12, P-2780 Oeiras, Portugal 2 - Laboratório de Engenharia Bioquímica, F. C. T./ U. N. L, P-2825 Monte da Caparica, Portugal

1. Abstract In this work the production of virus like particles (VLP’s) in insect cells was optimised for 2 Lt bioreactors. For this purpose the effects of and hydrodynamic conditions on product quality were evaluated. Although the did not show a significant effect upon cell growth in the range from 10 to 50%, cell infection and specific productivity were dramatically affected. The production was optimal at a of 25% and decreased up to two fold when the decreases to 10 or increases to 50%. The highest VLP titre was obtained at 96 hours post infection (hpi) and at of 25%. The effect of overcritical conditions upon productivity was also studied: cell infection is affected by agitation rates above 270 rpm or by aeration rates higher than 0.04 vvm, with a three fold decrease in VLP titre. 2. Introduction Several strategies have been used to design a safe and efficient vaccine for AIDS including whole inactivated virus (Murphey-Corb et al., 1989), recombinant HIV subunits like gp120 (el-Amad et al., 1995) and genetically engineered virus like particles (Rovinsky et al., 1992). In this work, the insect cell (Spodopter a frugiperda, Sf-9) / baculovirus (AcNPV) system expressing Pr55gag particles was used. The Pr55gag is targeted to the plasma membrane and assembles in 100-120nm particles and therefore this product is extracellular. In order to gel high quality product it is essential to ensure that particle formation is performed correctly, since it has been shown that the titre of antibodies after immunisation with virus like particles is significantly higher than after immunisation with cell lysate or recombinant proteins (Vzorov et al., 1991). The cells were infected at several values and product titre and quality were determined for the hydrodynamic conditions previously optimised for this system (270 rpm and 0.04 vvm). 421

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 421-427. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

422

Finally, to confirm that these hydrodynamic conditions cannot be overcome, two experiments were performed: one at higher agitation rate and the other at higher aeration rate; the Pr55gag titre obtained was compared with the ones obtained in the optimal conditions. 3. Materials and Methods 3.1 CELL GROWTH AND INFECTION EXPERIMENTS Spodoptera frugiperda Sf-9 insect cells (ECACC no. 89070101), previously adapted to SF900II (Gibco, Glasgow, UK) (Cruz et al, 1997) were maintained in Wheaton stirred vessels (Wheaton, Millville, USA). Cell growth and infection experiments were performed in a fully controlled stirred bioreactor (B. Braun, Melsungen, Germany) inoculated at Cell concentration was determined as previously described (Cruz et al, 1997). Cells were infected with baculovirus that generate Pr55gag particles, provided by Prof. Polly Roy (IVEM, Oxford, UK), in the mid exponential phase of cell growth with a multiplicity of infection (MOI) of 2 pfu/cell. Samples were taken at several times post infection and the supernatant was collected for VLP quality analysis. The effect of upon cell growth and infection was studied at 10, 25 and 50% controlled by the aeration rate up to a maximum of 0.04 vvm. The agitation rate in these experiments was maintained at 270 rpm. To test the effect of overcritical conditions in VLP titre two experiments were conducted: one to test the effect of an overcritical aeration rale (0.06 vvm) and another to test the effect of an overcritical agitation rate (300 rpm); the was maintained at 25% in both cases. 3.2 PR55GAG CONCENTRATION MEASUREMENTS AND QUALITY ANALYSIS Pr55gag was quantified by using the Innotest HIV Antigen Mab (Innogenetics N.V., Zwijndrecht, Belgium), kindly provided by Innogenetics N.V. (Ghent, Belgium). Since this test uses monoclonal antibodies against p24, related polypeptides containing the recognised p24 epitopes will also be detected (e.g. breakdown products of Pr55gag). In order to determine the fraction of Pr55gag in the form of high molecular weight particles the culture supernatant was injected into a gel filtration column (Pharmacia Biotech, Uppsala, Sweden) connected to a FPLC system (Pharmacia Biotech). Phosphate buffer (10 mM pH7.2) was used as running buffer at a flow rate of 0.4 ml/min. The column was calibrated with high and low molecular weight standards (Bio-Rad, Hercules, USA) and the void volume was determined by using blue dextran 2000 (Bio-Rad). Fractions of 1 ml were collected for 1.5 column volumes and the activity was measured using the ELISA test described above. The fraction of product in the form of particles was determined by calculating the ratio between the activity in the high molecular weight fractions (0.9 to 2.4 MD) and the tolal measured activity.

423 4. Results and Discussion 4.1 INFLUENCE OF

IN CELL GROWTH AND INFECTION

Figure 1 shows the results of three cell growth and infection experiments performed at different values: 10, 25 and 50%. In all cases the agitation rate was maintained at 270 rpm and the was controlled by the sparged air flow up to a maximum of 0.04 vvm (the critical values obtained in previous experiments)(data not shown).

As can be observed in Figure 1A, maximum sustainable cell concentration (obtained at 270 rpm, 0.04 vvm sparged air) increased from to as the was lowered from 50 to 10% resulting from an increase in the oxygen supply. As can also be observed, the specific growth rate was the same in all the experiments (0.031 ) which indicates that cell growth is not significantly affected by in this range. In fact, the optimal values reported for cell growth range from 5 to 100, depending on bioreactor size and configuration, culture medium and inlet gas (Schmid, 1996). The optimal dissolved oxygen tension is known to be different for different proteins (Agathos, 1996); to study the influence of upon cell infection, the variation of normalised cell concentration, (the ratio between the cell concentration at a given time and the cell concentration at infection), with time was obtained for 10, 25 and 50% (Figure IB). As can be observed, cell death caused by virus infection is slightly faster for 25% dissolved oxygen than at 10 or 50%. Since the decrease in viability is related with virus infection, higher titres can be expected for the experiment with 25% From these results it can also be concluded that a MOI of 2 is sufficient for synchronous infection, since the cell concentration did not increase after infection. In addition, since the infection is performed in the mid exponential growth phase, higher MOIs would not give significant enhancement of the final product titre (Neutra et al., 1992).

424 4.2 INFLUENCE OF

IN PRODUCT TITRE AND QUALITY

In order to determine if these differences in infection profiles affect the product and particle titre, ELISA and gel filtration chromatography tests were performed with the samples obtained from each experiment (Figure 2).

In terms of total product (Figure 2A) a much higher titre was obtained when

was

maintained at 25%; higher or lower values decreased the maximum titre to half of the one obtained at the optimal Moreover, the specific productivity at 25% was similar to the one found in small

spinner flasks under no oxygen limitation,

indicating that the cells are not affected by the increase in scale (data not shown). The product quality determined by gel filtration chromatography is defined as the percentage of product in the form of high molecular weight particles; this analysis is depicted in

figure 2B for 10, 25 and 50% By applying these percentages to the total titre (Figure 2A) the particle titre could be obtained (Figure 2C). As can be observed from Figure 2C, also the particle titre was larger at 25% than at 10 or 50%. By comparing the results obtained for 10% and 50% it can be observed that, although the total titre

425

was similar in both cases (Figure 2A), the particle titre at 10% was almost twice the one obtained at 50% due to the higher quality achieved at 10% From Figure 2B, it is possible to conclude that the maximum particle titre is not coincident with the maximum quality, i.e. with the maximum particle percentage. While the maximum particle titre was obtained at 96 hpi (Figure 2C) the best quality was obtained at 48 hpi, independently of the value (Figure 2B). In fact, it is at 48 hpi that the cell lysis caused by baculovirus begins (King and Possee, 1992), indicating that there may be some proteolythic degradation starting at that time, resulting in a decrease in the relative amount of particulate material. From these results it is possible to conclude that the harvest time has to be carefully optimised for each product and for each bioreactor size.

In order to study the extent of the influence of conditions slightly more harsh than the ones at which cells are affected, but less stringent than the ones at which cells are killed two experiments were conducted. In the first experiment the agitation rale was

increased to 300 rpm and in the second case the aeration rate was increased to 0.06 vvm. In order to maintain the a the aeration rate had to be decreased to 0.03 vvm in the first experiment and the agitation rate had to be decreased to 200 rpm in the second experiment. Figure 3 represents the particle titre obtained in these two experiments, compared with the results obtained in the optimal conditions.

As can be observed, the small increase in the shear stress caused a dramatic decrease in the case of higher agitation rate (74%) and a similar decrease in the case of higher aeration rate (77%), when compared with the titre obtained in the previously determined optimal conditions. Since in both cases of overcritical conditions neither a change in morphology due to stress nor an abnormal decrease in cell viability were observed it can be concluded that although the stress is below the cell killing threshold it is already

affecting productivity.

426

5. Conclusions

From this work the following conclusions can be drawn: A) The did not show a significant effect upon cell growth in the range from 10% to 50%. Conversely, cell infection and specific productivity were dramatically affected up to a two fold decrease when the decreases to 10 or increase to 50%, the optimal value being 25%. B) The maximum product quality is not coincident with maximum product titre. Although the best quality product was obtained at 48 hpi, independently on the , the highest particle titre was obtained at 96 hpi and at of 25% (according to the gel filtration chromatography and Western immunoblot results).

C) It was demonstrated that cell infection is affected by agitation rates above 270 rpm and by aeration rates higher than 0.04 vvm, even when the overcritical values are still far from the limit at which cell death starts to occur. These results emphasise the importance of working within the limits where the cells are not affected by the environmental conditions, not only in terms of but also in terms of agitation and aeration rate. ACKNOWLEDGMENTS

The authors acknowledge and appreciate the technical support of Ms. Maria do Rosário Clemente, the financial support received from the European Commission (VALUE CE-CTT-535) and Fundação para a Ciência e Tecnologia - Portugal (EUREKA PUEM/C/EU and BD/4545/94) and the partnership with Innogenetics S.A.. REFERENCES

Agathos, S N. (1996) Insect cell bioreactors. Cytotechnol., 20, 173-189. Cruz, P. E., Moreira, J. L., Carrondo, M. J. T. (1997). Insect cell growth evaluation during serum-free adaptation in stirred suspension cultures. Biotechnol. Techniques 11, 117-120. el-Amad, Z., Murthy, K. K., Higgins, K., Cobb, E. K., Haigwood, N. L., Levy, J. A., Steimer, K. S. (1995)

Resistance of chimpanzees immunized with recombinant gp120SF2 to challenge by H1V-ISF2. AIDS 9, 1313-1322. Murphey-Corb, M., Martin, L. N., Davison-Fairburn, B., Monelaro, R. C., Miller, M., West, M., Ohkawa, S., Baskin, G. B., Zhang, J. Y., Putney, S. D., Allison, A. C., Eppstein, D. A. (1989) A formalin-inactivated whole SIV vaccine confers protection in macaques. Science 246, 1293-1297. Neutra, R., Levi, B.-Z., Shoam, Y. (1992) Optimisation of protein production by the baculovirus expression vector system in shake flasks Appl. Microbiol. Biotechnol. 37, 74-78.

Rovinsky, B., Haynes, J. R., Cao, S. X., James, O., Sia, C., Zolla-Pazner, S., Matthews, T. J., Klein, M H.

(1992) Expression and characterization of genetically engineered human immunodeficiency virus-like particles containing modified envelope glycoproteins: implications for development of a cross-protective AIDS vaccine. J. Virol 66, 4003-4012. Schmid, G. (1996) Insect cell cultivation: growth and kinetics. Cytotechnol. 22, 43-56. Vzorov, A. N., Bukrinsky, M. I., Grigoriev, V. B., Tentsov, Y., Bukrinskaya, A. G. (1991) Highly

immunogenic human immunodeficiency virus-like particles are produced by recombinant vaccinia virusinfected cells. AIDS Res. Hum. Retroviruses 7, 29-36.

427

Discussion

Anon:

What evidence do you have for having capsid particles, other than size occlusion? We have had trouble finding capsid particles that are stable when they are non-enveloped from Maloney Leukaemia virus. You could just have a conglomeration of gag proteins. Do you have EM confirmation?

Cruz:

Yes, we have confirmed the presence of particles. However, the particles are not that stable as after 1 month at 4° C there is a considerable reduction.

Bernard:

I am unsure of your strategy for process control after you had set maximal operating parameters - do you have constant agitation and air gassing and on top oxygen supply, or do you vary to these points?

Cruz:

These are limiting points. So we have done experiments both maintaining agitation and aeration rates, whilst the other variables went to the limit, and the results were similar. If you increase the scale to 10 1 the air is not enough, so we use oxygen-enriched gas and in that case we are already working at the limiting conditions.

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DIRECT CAPTURE OF MONOCLONAL ANTIBODIES WITH A NEW HIGH CAPACITY STREAMLINE SP XL ION EXCHANGER

C. PRIESNER, N. AMESKAMP, D. LÜTKEMEYER, J. LEHMANN Cell Culture Technology, P. O. Box 100131, University of Bielefeld Germany

1. Abstract A new sulphopropyl-based strong cation exchange matrix STREAMLINE®SP XL (Pharmacia Biotech) designed for fluidized bed adsorption was tested in both bench and pilot scale for direct capture of mouse from undiluted and unclarified feedstock containing 500 mg/L human serum albumin (HSA) and 30-50 mg/L MAb. In scouting

experiments in packed bed mode with clarified feedstock the optimal pH for binding of the target protein (pI = 6.3) was found to be 4.6 where co-chromatography of HSA (pI = 4.8) predictably occurred. Frontal adsorption analysis under the same conditions gave enhanced dynamic capacities of more than 6 mg IgG/ml STREAMLINE®SP XL matrix compared to those attainable with conventional STREAMLINE®SP matrix (1.5 mg IgG/ml). These results could not be reproduced in expanded bed mode with unclarified feedstock (0.5 mg IgG/ml matrix) where massive biomass adsorption to the matrix could be observed. By adding glucose in different concentrations to the feedstock and buffers the dynamic capacity increased to 2 mg IgG/mL matrix (200 mM glucose) and 4.5 mg IgG/ml matrix (400 mM glucose), respectively (Table 1). The cochromatography of HSA varied according to the glucose content. In pilot scale no break-through of the antibody could be achieved due to the limiting dynamic capacity.

2. Materials and Methods

2.1. Cultivation Murine hybridoma cells were cultivated in 5-, 20-, and 100-L-bioreactors in split batch

mode using a culture media with 500 mg/L HSA, 10 mg/L bovine insulin and 8 mg/L human transferrin. Except those used in the scouting experiments all feedstocks contained 0.002 % Pluronic F-68® and 0.003 % Antifoam C®. Until the end of each batch the number of viable cells reached up to with a viability higher than 90%. 429 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 429-431. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

430

2.2. Chromatography STREAMLINE®SP and SP XL are strong cation exchangers based on highly crosslinked 6% agarose including an inert quartz core (density 1.2 g/ml, particle size 100 300 m). The sulphonate groups of SP XL are coupled to polydextran spacers to obtain a higher ligand density. Experiments were carried out in packed bed mode with CIO/10 columns / 4 ml adsorbent / FPLC® system, expanded bed mode with a SL®25 column / 75 ml adsorbent / GradiFrac® system (bench scale), and a SL®200 column / 5000 ml adsorbent / pilot system (pilot scale). All chromatographic equipment: Pharmacia Biotech. Buffer A: 20 mM Acetate, 150 mM NaCl, pH 4.6, 0/200/400 mM Glucose; Buffer B: 20 mM Acetate, 1 M NaCl, pH 4.6, 0/200/400 mM Glucose. Equilibration, loading and washing: 300 cm/h, upward flow direction; Elution: 100 cm/h, upward flow direction.

2.3 Analytics Total antibody concentration was measured by ELISA. Particle number and distribution were measured with a Casyl-system (Schärfe) and interpreted according to the definition of Glauner [1].

3. Results

431

4. Summary and conclusion

The new high capacity cation exchange matrix STREAMLINE SP XL has proved its

enhanced capacity compared to STREAMLINE SP. The values for dynamic capacities

for pure substances could not be reached due to the co-chromatography of HSA, other media components, high salt concentration in the feedstock and the biomass adsorption to the matrix. A scale up was successfully performed: 100 L of unclarified hybridoma broth were concentrated with a binding rate of 96 % in less than three hours. Future investigations have to focus on the observed effects of additives as Glucose and surfactants as well as on the cell binding which seems to be the major problem of the expanded bed ion exchange chromatography under the chosen conditions.

5. Acknowledgement

We would like to thank Pharmacia Biotech - especially Dr. P. Girard and Dr. L. Larsson - for kindly supplying the pilot equipment and 5 L of SP XL matrix.

6. References Glauner, B. (1991) Vitalitätskontrolle in der Zellkulturtechnik; BioTec 3(5)

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THE EFFECT OF DIFFERENT PURIFICATION SCHEMES ON THE ACTIVITY OF A MONOCLONAL ANTIBODY D.J. BLACK 1 , J.P. BARFORD 1 , C. HARBOUR 2 , and A. FLETCHER3

Departments of Chemical Engineering1 and Infectious Diseases1, University of Sydney, NSW 2006 and NSW Blood Transfusion Service3, Sydney, NSW 2000, Australia

Abstract Five different chromatographic purification schemes, encompassing affinity, hydrophobic interaction, cation exchange and gel filtration techniques, were developed and used to purify monoclonal antibody from a common supernatant pool produced by human

lymphoblastoid cells grown in batch culture. Comparison of purified samples revealed that the purification scheme could affect both the antigen binding and functional activities of the purified product. Introduction

Advances in genetic engineering and cell culture technology have made it possible to generate an increasing number of biopharmaceuticals in non-traditional ways. Factor VIII for example, a coagulation factor, can now be derived from mammalian cell cultures rather than the traditional source of pooled human plasma. Many of these biopharmaceuticals are glycoproteins and it is recognised that the post-translational modifications involved in the production of complex biological molecules can differ from species to species and thus result in different product profiles when produced in vitro compared to in vivo. Thus the product is often a complex mixture of different glycoforms which may have different activities. It is possible that the use of different purification systems could preferentially select certain glycoforms resulting in final products with different glycoform compositions and hence activities. In order to address this issue a human monoclonal antibody with both antigen binding (Fab-dependent) and functional (Fc-dependent) activities was selected as the model protein for study. The effects of five different purification schemes on both

these activities have been studied and a preliminary report is presented here. Materials and methods Human monoclonals (IgG) with therapeutic potential (anti-D) were produced from

lymphoblastoid cells (generated by Dr A. Fletcher at the NSW BTS) grown in IMDM medium supplemented with 10% fetal calf serum and 2 mM glutamine. A pooled, single batch which was then aliquoted and stored at -20°C until purified using one of five different

purification schemes shown in the tables, i.e., protein G; protein A; anti-human IgGagarose; two stage hydrophobic interaction chromatography (HiC) and combined HiC, ion exchange (Ion-X) and gel filtration (GF) chromatography. Antigen (Fab-dependent) 433 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 433-435.

© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

434

435

binding activity is presented as the anti-D titre determined by haemagglutination performed according to Whitson (1985). The percentage of active antibody was calculated by subtracting the mean antibody content of supernatant contacted with from the mean content of supernatant contacted with and dividing this figure by the latter and multiplying by 100. The antibody content was determined by IgG ELISA after purified anti-D was mixed with either washed cells and incubated at 37°C for 3 hr. The

suspension was centrifuged at 2000 g for 10 min to pellet red cells. Functional (Fcdependent) binding activity was determined by measuring monocyte-mediated antibody dependent cellular cytotoxicity (ADCC) performed according to Kirkwood et al., (1993). In addition resetting with U937 monocyte-like cells was determined as described by Krumpel and Hadley (1990). The analysis of monosaccharide and sialic acid content was performed as described by Cant et al., (1994). Results and discussion The effects of the different purification schemes on antibody yield and purity are shown in Table 1. The antibody yield was clearly highest using the protein G purification scheme but highest purity was achieved using protein A. The Fab and Fc activities of antibodies generated by different purification schemes are shown in Table 2. It was noted that the

purification scheme did affect the amount of antibody in the final purified product which was bound to antigen. The HiC (2 stage) and combined methods produced the highest Fabdependent activity and the anti-human IgG-agarose the lowest activity. In terms of Fcdependent activity rosetting activity was highest in the anti-human IgG-agarose preparation. The increase in functional activity compared to the raw superantant pool implies that a preferential separation was occurring and this most likely reduced the proportion of aglycosyl (and hence functionally inactive) molecules in the purified product. The effect may have been more subtle however, with more active species being preferentially separated from less active ones. The monosaccharide composition of the purified products was then analysed to determine which of these mechanisms was the most likely. It was concluded that the amount of aglycosyl anti-D present was the main determinant of the overall sugar content and was the most likely explanation for the increase sugar content of the combined HiC/lon X/GF-purified anti-D. This scheme also separated certain sugar species over others generating a product with a lower sialic acid content and appeared the most applicable for production scale applications although its cost would be a disadvantage compared to either protein A or G. In conclusion the purification system did affect the Fab and Fc activities and is clearly an issue which must be addressed in designing a manufacturing process. References Cant, D., et al. (1994) Cytotechnology, 14, 223-228.

Kirkwood, J., et al. (1993) Trans. Med. 3, 269-273. Krumpel, B.M. and Hadley, A.G. (1990) Mol. Immunol. 27 (3) 247-256. Whitson, K.J. (1985) Aust. J. Med. Lab. Sci., 6, 106-109.

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INTERFACING OF PROTEIN PRODUCT RECOVERY WITH AN INSECT CELL BACULOVIRUS PRODUCTION SYSTEM

I.A. CARMICHAEL, M. AL-RUBEAI AND A. LYDDIATT Biorecovery Group, Centre for Bioprocess Engineering, School of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K. 1. Abstract Conventional protein recovery requires several unit operations to clarify, concentrate and isolate the product, resulting in high processing costs, extended separation times and reduction in molecular yield. Any foreshortening in this purification process will therefore be beneficial. Interfacing of fluidised bed adsorption with production systems achieves this goal, negating the need for problematic centrifugation and filtration steps. Direct processing of an unclarified insect cell-Baculovirus -galactosidase feedstock with a custom made, fluidisable adsorbent has facilitated successful clarification, 10-fold concentration and purification, and greater than 30% improvement in capture efficiency over conventional purification operations. Keywords: Fluidised Bed, Product Recovery, -galactosidase. 2. Introduction Animal cell culture is commonly used for the production of complex, high-value protein therapeutics, often characterised by extensive post-translational modification. Regulatory approval requires stringent levels of reproducible purity and defined molecular integrity. To achieve such demands, product purification conventionally uses many unit operations to clarify, concentrate and isolate the product. However, this is disadvantaged by high capital, space and time investments1, reduction in yield (due to multistep operations) and concomitant product degradation. Rapid purification would therefore confer benefits of reduced costs and increased product quality. Realisation of this goal has been achieved by replacing centrifugation and microfiltration steps in the purification process with fluidised bed adsorption2. The fluidisable adsorbent employed herein facilitates capture, concentration and purification of product, without the need for centrifugation or filtration of feedstock, which is mandatory for conventional chromatography and contribute to product losses (see Figure 1). 437

O.-W. Merten et al. (eds.). New Developments and New Applications in Animal Cell Technology, 437-439. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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3. Materials and Methods SF9 cells were grown in suspension at using TC-100 + 5% FCS. Infection was achieved by resuspension of cells, at late log phase, in fresh media and addition of recombinant Baculovirus lacZ-ACMPV at a multiplicity of infection (m.o.i.) of 10. Protein concentrations were determined by the method of Bradford3, -galactosidase assays were performed using a modification of the method of Miller4. The fluidisable affinity matrix was constructed by CDI activation of Macrosorb-K4AX (Phase Separations Ltd) and sequential derivatisation with 3,3’-iminobis-propylamine (DADPA), glycidol and the affinity ligand p-aminobenzyl-1-thio -Dgalactopyranoside (ABTG)5. The matrix was designated Macrosorb-ABTG. Fixed bed operation involved loading and elution at a linear flow rate of 0.22cm/hr. Desorption was achieved by a step of 0.1 M borate buffer, pH 10, followed by immediate desalting in Sephadex G50. Fluidised bed operation was achieved by recirculation of unclarified feed upwards through a custom-built fluidised contactor (dimensions 20x2.2cm), containing 7mls of Macrosorb K4AX-ABTG, at 600cm/hr. This yielded a bed expansion of 100%. Bound protein was desorbed as in conventional fixed bed mode. 4. Results Macrosorb-ABTG was constructed with the final chemistries of 10 mole/ml DADPA, 13 mole/ml glycidol modified DADPA and 1 mole/ml ligand. As can be seen in Figure 1, lab scale purification involved losses of 27% in steps which serve only to clarify the feedstock for further processing. Fixed bed affinity chromatography achieved a final concentration factor of 20-fold and purification factor of 10-fold. The final specific activity of the product was ~400units/mg protein (data not shown).

Figures 2 and 3 show the loading and elution data for interfaced recovery of product from unclarified culture. 180mls of unclarified insect cell culture was contacted with Macrosorb-ABTG in a fluidised bed. The matrix removed 85% of enzyme activity, conferring a capacity of 2.5 mg enzyme/ml matrix. Figure 3 shows fixed bed desorption of the saturated matrix, yielding a recovery of >90% of total protein, with an overall concentration of 4.5-fold, of which 30% was in a 0.1 M borate pool (pH10) that retained activity upon desalting (specific activity ~700units/mg protein). Elution of Macrosorb-ABTG, in 3M potassium thiocyanate, recovered the remaining protein.

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5. Conclusions The opportunity for enhancement of the conventional purification process by circumvention of clarification steps is clearly shown in Figure 1. Judicious sizing of the matrix volume allows removal of at least 85% of enzyme activity from unclarified culture. Greater than 90% of total protein can then be recovered in a two stage desorption strategy. The first stage recovers -galactosidase with a purity 40% higher than conventional recovery, achieving an overall purification factor and volume reduction both of ~10 fold, together with clarification of the product stream.

The

remaining bound protein can then be desorbed with 3M KSCN. Although overall recovery of enzyme activity from this system is only ~30%, this is clearly a system specific problem (compare with previous work with yeast and bacteria2,6) concerned with heterogeneous interaction of -galactosidase with the surface chemistries of the Macrosorb-ABTG. In order to increase recovery of activity, (i) manipulation of contact time, (ii) matrix construction and (iii) ligand chemistries are currently under investigation. A logical extension of this work is the use of direct product sequestration (DPS) during fermentation. This will offer the advantages outlined above, coupled with potential to decrease negative feedback of protein synthesis, stabilise product structure and steepen secretion gradients2. 6. References 1) Datar, R., (1986) Process Biochemistry 21 19-26. 2) Morton, P.M. and Lyddiatt, A., (1994) In: Separations for Biotechnology 3. Ed; Pyle, D.L., Elsevier Applied Science pp. 329-325. 3) Bradford, M., (1972) Analytical Biochemistry 72 248-254. 4) Miller, I.M., (1972) In: Experiments in Molecular Genetics, Ed; L. Cold Spring New York, Cold Spring Laboratories pp. 352-355. 5) Hermanson, G.T., Krishna Mallia, A. and Smith, P., (1992) Immobilised Affinity Ligand Techniques. Academic Press Inc, New York. 6) Burns, M.T. and Lyddiatt, A., (1996), Proceedings of the 5th World Congress Of Chemical Engineering 2 633-639. Acknowledgements : IC was funded by a BBSRC quota studentship.

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PREPARATION OF PLASMID DNA USING DISPLACEMENT CHROMATOGRAPHY

R. FREITAG and S. VOGT Laboratoire de biotechnologie cellulaire Institut de Génie Chimique IV ETH Lausanne Switzerland

1. Introduction Large amounts of pure plasmid DNA are increasingly in demand. Possible areas of application include, of course, gene therapy but also the transient transfection of mammalian cells. The plasmids are routinely produced in E. coli and their recovery from the cell lysates may involve precipitation and extraction operations, -density gradient centrifugation or various adsorption techniques. By these operations numerous E coli proteins, other polynucleotides, the toxic lipopolysaccharides (LPS) stemming from the cell membrane, etc. are removed. While extraction and precipitation are easily scaled, the resulting product is often of insufficient quality. The -density gradient centrifugation yields a highly purified product, but is difficult to use at large scale. Adsorption and especially chromatographic techniques combine scalability with high resolution and thus present a possibility for large scale applications. Concentrated DNA-solutions are viscous, however, thus only diluted fractions may be collected from ordinary gradient elution chromatography, Figure 1. Displacement chromatography (DC) is a littleknown alternative to preparative elution chromatography [1]. In DC the substances are focused into consecutive zones of constant concentration by an advancing displacer front. The displacer may be any substance which shows a superior binding to the stationary phase under operating conditions. The concentration in the zones can be controlled via the displacer concentration and thus be kept in its entirety just below the critical value, Figure 2. The overall concentration in the pooled product fractions 441

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 441-443. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

442 may thus be considerably higher than in the elution mode, while smaller columns can be used to give the same amount of product due to a more effective use of the column’s capacity [2]. The results are higher throughputs and easier scale up [3]. 2.

Experimental

Plasmid preparation:: Double-stranded plasmid DNA (DH5 , C2007-1, Lot 56682, Clontech Laboratories Palo Alto, USA) was produced in E. coli. Qiagen kits were used for isolation according to the manufacturer’s instructions. The final preparations were free of protein and non-plasmid DNA, but some of the large scale preparations contained residual LPS [4]. Sample preparation for DC: Samples were prepared by spiking pure plasmid DNA preparations with equal amounts of protein (holo-transferrin, Sigma) and/or E. coli LPS (scrotype 026:B6, Lot 46H4024, Sigma). Displacement chromatography: Investigated were a 2 ml Bio-Scale Q2 strong anion exchanger column (7 x 52 mm, porous 10 m beads, Bio-Rad), a 1.3 ml UNO™ Ql continuous bed strong anion exchanger column (7 x 35 mm, Bio-Rad), and two columns (250 x 4 mm) filled with porous, ceramic hydroxyapatit beads (10 m, type I and type II, Bio-Rad). The carrier was a 0.02 M TRIS/HC1 buffer, pH 8.0. A flow rate of 0.1 ml/min was used throughout. Column regeneration was by a high salt step (2 M NaCl). Fractions were collected twice per minute. Analytical methods: Protein concentrations in solution were measured either by the BCA assay (Pierce) according to the manufacturers instruction or by reversed phase chromatography (RPC) as described previously [2]. Plasmid DNA in the DC fractions was quantified as described by Sten et al. [5]. LPS concentrations were measured either by a kinetic LAL assay or by capillary electrophoresis as described previously [4]. 3.

Results

The purification of plasmid DNA from cell lysates involves the removal of proteins, polynucleotides, and lipopolysaccharides. Residual LPS does present a major problem to both gene therapy and transient transfection. Its maximum residual dosis in parenteralia is 0.5 ng/kg of body mass and LPS has also been known to influence the transfection efficiency in eukaryotic cells [6]. The possibility of separating all three substance classes by displacement chromatography was investigated by us. Mass transfer tends to present a problem in plasmid preparation with columns packed from conventional i.e. porous stationary phase particles. The major part of the adsorptive surface in the pores can only be reached by diffusion, a time consuming task for large biopolymers. Evidence has been presented that within the normal time frame of a chromatographic separation the inner pore surface is not at all accessible to the plasmid molecules and adsorption is restricted to the - small - outer surface of the particles [7]. 3.1

CONVENTIONAL STATIONARY PHASES (POROUS PARTICLES)

Displacement chromatography is based on the displacer enforced competition of the feed components for the stationary phase binding sites. If this is not possible, e.g. because the inner article surface is accessible to the proteins but not to the larger DNA molecules or the equally large LPS aggregates, no separation takes place. This was indeed observed by us in the case of the BioScale Q 2 column. Plasmid DNA and LPS aggregates are of equal size

443 and do compete for binding. Their separation is possible, but the zone overlap (shock layer) between the zones is broad, presumably also due to pronounced mass transfer limitations. 3.2

HYDROXYAPATIT

Hydroxyapatit (HA) is a crystalline calciumphosphate modification with known affinity to molecules containing phosphate groups. For HPLC application two types of ceramic porous particles are available. The type II material is advertised for its DNA binding ability. While the DNA binding capacity of the type I material was indeed unsatisfactory, and the corresponding column had to be abandoned, columns packed with the type II HA showed similar problems as the particulate anion exchanger as far as the DNA / protein separation were concerned. A separation of LPS and DNA was not possible at all with the type II material, both substances were found in all fractions at roughly the same concentration ratio. 3.3

CONTINUOUS BED COLUMNS (UNO™ Q COLUMN)

The mass transfer poses a problem to all types of biopolymer chromatography, not only to displacement. Recently, special stationary phases have been introduced that hope to circumvent the problem. The continuous bed UNO™ column by Bio-Rad is investigated here. The UNO column is a polymer rod prepared in situ by radical polymerisation. It consists of a continuous network of polymer nodules with interconnecting channels. Thus the surface is high, while there is no « intraparticle » surface. Band broadening is small even at high flow rates. In the displacement mode the UNO™ Q column behaves as the comparable BioScale Q anion exchanger from the same manufacturer. Displacers designed for the BioScale Q column [8,9] could also be used with the UNO™ Q. The separation between plasmid DNA and LPS aggregates became sharper, although it was still not possible to collect the pure substances. 4.

Conclusions

The isolation of plasmid DNA at larger scale continuous to present a problem to preparative biotechnologists. Given both its scalabilty and its high resolution, chromatographic operations and especially displacement chromatography may well contribute to the final answer to this problem. 5. References [1] [2] [3] [4] [5] [6] [7] [8] [9]

Freitag R.: In: Analytical and Preparative Separation Methods for Biomolecules. H.Y. Aboul-Enein (Ed.) Marcel Dekker, 1998 Kasper C., Vogt S., Breier J., Freitag R. Bioseparation 6 (1996) 247-262 Gerstner J.A. BioPharm 9 (1) (1996), 30-35 Freitag R., Fix M., Brüggemann O.: Analysis of Endotoxins by Capillary Electrophoresis. Electrophoresis (1997), in press Sten R.G., MacDonald C.G., Weaver A.L., Pitt A.M. Bio Techniques 15(5) (1993) 932-933 Weber M., Möller K., Welzeck M., Schorr J. BioTechniques 19(6) (1995) 930940 Schwarz A., Data presented at the PREP´97, Washington DC, June 1 to 4, 1997 Vogt S., Freitag R. J. Chromatogr., 760 (1996) 125-137 Freitag R., Vogt S., Mödler M.: Customised in affinity and solubility - new protein displacers for ion exchange protein displacement chromatography. (submitted)

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NOVEL METHODS FOR LARGE-SCALE PREPARATION OF NUTRIENT MEDIA AND BUFFERED SALT SOLUTIONS.

David W. Jayme Life Technologies, Inc. 3175 Staley Road, Grand Island, NY 14072 USA

1.

ABSTRACT

Large-scale manufacture of solutions used to feed bioreactors and purify biologicals creates logistical challenges which impact facility footprint, personnel utilization and overall manufacturing cost. In collaboration with large volume users, we have identified novel formulation configurations and reconstitution options for preparation of nutrient media and buffered salt solutions. Some options exploit continuous reconstitution from concentrated intermediates using an automated mixing device. Other options enhance traditional batch reconstitution processes by automatically adjusting pH and osmolality (conductivity) to target ranges in a closed formulation tank or bioreactor. We have also identified hybrid options for campaigned manufacture of similar formulations from common intermediates. These alternatives produce solutions of comparable quality, reduce capital investment in facility and equipment, improve space/time utilization within kitchens, optimize personnel utilization, focus on user core competencies, and reduce the overall cost of biological fluid production.

2.

INTRODUCTION

Traditional approaches to formulate nutrient medium and buffered salt solutions from biochemical constituents or homogeneous powders have been adequate for production requirements of a few thousand liters. Biotechnology industry maturation has escalated required volumes substantially and justified exploration of alternative approaches [1]. 2.1.

The Challenge

Beyond the daunting volumes of batch-scale mammalian cell fermenters ( 10,000 liters) perfusion bioreactor feeding during an extended production campaigns (30-200 days), requiring 1-2 volume exchanges per day, can consume vast quantities of nutrient fluids. A pilot-scale bioreactors (100L) operating over a 30 day campaign will consume 30006000 liters of nutrient medium. By comparison, a production scale bioreactor (1000L) 445 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 445-448. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

446

operating over a 200 day campaign may consume over 400,000 liters. Protein-free supplemental nutrient concentrates, designed to replenish exhausted nutrients in fedbatch mode, have been implemented to reduce medium cost and harvest volume [1-2]. Beyond logistical concerns relating to nutrient medium quantities, downstream product purification typically requires 1-3 times that volume in buffered salt solutions for column equilibration, elution, and regeneration and final product formulation [3]. 2.2.

The Issues

Escalating volumetric demands for nutrient medium or buffer to meet production projections stresses the kitchen facility, equipment and personnel. Balancing capital investment for facility space, formulation and holding tanks, and water systems against risks for a biological undergoing clinical investigation is a challenging task. Various additional factors contribute to the complexity of determining the “preferred” method for preparing large-scale biological fluids: • Technical factors: solubilization time, constituent degradation, endotoxin generation, number and diversity of formulations; • Quality assurance factors: batch-to-batch precision, status of existing facility and equipment, batch-driven testing requirements, project regulatory status; • Human resource factors: recruitment and training, utilization efficiency; • Business factors: storage space, geographic location, distribution logistics, current and projected volumetric scale, accuracy of planning and market projections

3.

THE OPTIONS

Alternatives used or under evaluation by global biotechnology and biopharmaceutical manufacturers for large-scale production of nutrient media and buffers include: 3.1.

Continuous preparation from intermediate concentrates using a mixing device.

Liquid medium or buffer concentrates are purchased in bulk packaging compatible with mixing device connections. A mixing device [4], custom-engineered to user specifications, is resident at the user facility. High precision I X fluid is reconstituted at the user site either in large batches or as a continuous feedstream. Advantages include increased batch size or potential for continuous bioreactor feeding, and reduction in facility footprint, technical labor and overall manufacturing and qualification cost. Considerations include the design and capital cost of the mixing device, requirement for supplemental validation, and maximal benefit derived by commitment to mixing device and concentrate utilization at the design phase. 3.2.

Campaigned production of similar formulations

Campaigning [1] permits manufacture of multiple, similar formulations from a minimal number of common concentrated intermediates, with reconstitution performed either in

447

batch or continuous mode. Examples include growth and perfusion media or column

equilibration, elution and regeneration buffer combinations. Advantages include increased manufacturing flexibility, facilitated inventory management, and more efficient use of kitchen space and formulation/holding tanks. Considerations include the initial design complexity of concentrate sub-groups and formulation-specific limitations. 3.3.

Hybrid of powdered base and liquid concentrated additive

A common example is a complete medium which combines a powdered component with a concentrated liquid supplement of “difficult to solubilize” components. Another option is a “salt-free” concentrated supplement designed to replenish key nutrients which have been metabolically consumed or degraded [1-2]. Advantages include the ability to produce a complex nutrient formulation which may be challenging to solubilize with full potency and biological performance from a single component powder. Bulk powders may be solubilized in a pre-existing kitchen facility. Nutrient replenishment improves bioreactor productivity in terms of space/time utilization and specific product yield. Nutrient supplement composition may vary with bioreactor type and feeding regimen.

3.4.

Batch formulation using either single concentrated component or multiple concentrated sub-groups

The multiple component option formulates IX fluid from 2-5 concentrated sub-groups

[1]. Single component concentrates may be provided in bulk containers with integral filter and recirculation tubing for user dilution [3]. Concentrate formulation accelerates mixing time, personnel utilization, and batch consistency, particularly as reconstitution may be performed in closed vessels without manual adjustment of pH or osmolality (conductivity). Diluting a single component concentrate within a disposable container with an integral filter reduces capital investment in facility and equipment and may be a useful transition strategy pending regulatory agency approval and volumetric scale-up. 3.5.

Internal production vs. out-sourcing

Ready-to-use IX fluids are manufactured per user specification in large batches (1050,000 liters), aseptically dispensed into bulk containers, and released for manufacturing

based on vendor certification. Advantages include forestalling of capital investment in facility, equipment and utilities. Considerations include the aseptic connections to the bioreactor and inventory management based upon consumption rate, batch size and stability and refrigerated storage space. Cost effectiveness will depend upon volumetric requirement and regulatory status and capacity of existing kitchen facility [4]. 4.

SUMMARY

Escalating volume requirements for nutrient media and buffered salt solutions demand novel formulation options to improve quality and consistency, increase batch size,

448 decrease fully-burdened cost and retain manufacturing flexibility. Stable concentrated intermediates have been successfully implemented at pilot and production scales to accelerate media and buffer production time, increase batch size and decrease batchdriven costs, combine with powdered components to improve solubility and performance of complex formulations, replenish consumed nutrients in fed-batch and perfusion bioreactor feeding modes, and facilitate inventory management flexibility. Coupling bulk concentrates with an automated, high precision mixing device provides additional options. Large-volume batches (10-50,000 liters) of liquid medium have been commercially produced from concentrated intermediates using this validated process. Customized mixing devices for continuous reconstitution of concentrated fluids are under active design and qualification for various biotechnology applications.

5.

REFERENCES

1.

Jayme DW, Fike RM, Kubiak JM, Nash CR and Price PJ (1993), "Use of Liquid Medium Concentrates to Enhance Biological Productivity", In Animal Cell Technology: Basic & Applied Aspects. Volume 5, eds. S. Kaminogawa, A. Ametani and S. Hachimura (Kluwer) pp. 215-222.

2.

Jayme D, Kubiak J, Fike R, Rashbaum S, and Smith S (in press), “Costsaving Design and Operational Options for Large-Scale Production of Nutrient Medium and Buffers” (JAACT ‘96 meeting in Yokohama, Japan, September 1996)

3.

Fike R, Kubiak J, Price P and Jayme D, BioPharm (1993) 6(8): 49-54, "Feeding Strategies for Enhanced Hybridoma Productivity: Automated Concentrate Supplementation".

4.

Jayme DW, Kubiak JM, Battistoni, and Cady DJ Cytotechnology (1996) 22: 255-261, “Continuous, High Capacity Reconstitution of Nutrient Media from Concentrated Intermediates.”

LARGE SCALE APPLICATION OF THE SEMLIKI FOREST VIRUS SYSTEM: 5-HT3 RECEPTOR PRODUCTION

H.D. BLASEY, B. BRETHON , R. HOVIUS , K. LUNDSTRÖM , L. REY, H. VOGEL , A.-P. TAIR1 AND A.R. BERNARD Geneva Biomedical Research Institute, 14 Chemin des Aulx, CH 1228 Plan les Ouates, Switzerland EPFL, Lausanne, Switzerland, Institut National des Sciences Appliquées, Toulouse, France, Hoffmann-La Roche, Basel, Switzerland

Abstract After we had applied the Semliki Forest Virus (SFV) expression system to produce large amounts of hCOX-2 in spinners, we succeeded in the first reactor runs with the SFV used and produced the serotonin receptor 5-HT3 in large quantities. We successfully scaled up the SFV technology allowing rapid gene expression at 11.5 litre scale. BHK cells were chosen as host cells for expression. They were grown in a bioreactor, equipped with a Vibromix agitation system providing for mixing and preventing cell aggregation. Medium exchange and subsequent viral infection was followed by a one day expression phase yielding 3 million of serotonin receptors per cell. After the harvest the, receptor protein could be purified in a single step at high yield (50%).

Introduction The Semliki Forest Virus expression system (Liljeström and Garoff, 199la) is a recent system for the expression of heterologous genes in mammalian cells cultures offering distinct advantages over other mammalian systems with respect to expression levels and speed. Co-transfection of in vitro transcribed recombinant and helper RNA into BHK cells leads to the in vivo packaging of only recombinant RNA and the production of infectious, but non replicative, recombinant virus particles at high titer (approximately This virus can infect most mammalian, insect and amphibian cells.. On the other hand, the ability of the virus to infect almost any eukaryotic cell raises a biological safety issue. These concerns however have been met by the production of recombinant virus particles which are only conditionally infectious due to three mutations in the pSFV-Helper2 plasmid (Berglund et al, 1993). Various recombinant proteins (enzymes, receptors, viral proteins and others) have been expressed with the SFV system (reviewed by Bernard and Blasey, in press). Table 1 449 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 449-455. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

450 summarises one specific class of proteins, the receptors, which in addition to the 5-HT3

receptor were expressed with SFV.

Recently we have applied the SFV technology to the expression of human cyclooxygenase-2 in spinners and produced this enzyme at the one litre scale (Blasey et

al., 1997). Already at this scale we could satisfy the demand for large amounts of active cytosolic recombinant protein preparations to run high-throughput compound screens. The 5-HT3 receptor has become of great interest as an object for functional and structural studies due to its important role as a drug target. The serotonin receptor is a ligand-gated cation channel and belongs to the group of nicotinic acetylcholine receptors

which are involved in several CNS disorders and for which drugs are on the market (e.g. against emesis induced by chemotherapy). In order to enable structural studies of the 5HT3 receptor we scaled up receptor expression. To achieve the goal of producing between 10-20mg receptor protein, we had to demonstrate satisfactory protein production at the scale of 11.5 litres using a mechanically mixed bioreactor, and thus showing for the first time the feasibility of using the SFV expression system at large scale.

Materials and methods

Medium, cell maintenance and glucose/ lactate analysis were reported earlier (Blasey et al., 1997). The plasmids pSFV3-LacZ, pSFVHelper-2 have been described elsewhere (Liljestrom and Garoff, 1991; Berglund et al., 1993). The pSFVl-5HT3 plasmid has been described earlier by Werner et al. (1994). However, to facilitate purification, an inframe hexa-histidine tag was introduced at the C-terminus of the 5-HT3 receptor by PCR technique. The amplified 5-HT3 receptor gene was then subcloned into the BAM HI site of the pSFVl vector. Generation of recombinant 5-HT3 SFV. In-vitro RNA transcripts were made from pSFVl-5-HT3 and pSFV Helper plasmids (Lundström et al., 1994) and the RNA products were co-electroporated into BHK cells according to Liljeström and Garoff (1991b). The in-vivo packaging of virus particles was completed 24 hours after

451 clectroporation, virus stocks collected and stored at -80° C after eliminating cell debris by centrifugation (5 min at 200g). Activation of virus stocks generated with pSFV-Helper2 was done with Chymotrypsin

followed by enzyme inactivation with Aprotinin. The virus titre estimation was described earlier (Lundström et al., 1994). 5-HT3 receptor characterisation: The and total binding were determined by binding of [3H]-GR65630 in absence or presence of the 5-HT3 antagonist quipazine. Assays were done in duplicates.

Results and Discussion

After we had found that 5-HT3 receptor expression was highest in BHK (amongst a variety of 10 cell lines, Fig. 1) we had selected BHK cells as host for the production of

5-HT3 receptors. Also, BHK cells are widely used to express a large variety of different recombinant proteins.

Large scale production was carried out in a mechanical mixed reactor from MBR (Switzerland) with a working volume of 11.5 litres. Oxygen was supplied via a silicone tubing (length 28m, 1.2mm, thickness: 0.2mm). Our objective for the scale up of BHK cell culture was to obtain a single cell suspension culture i.e. with no aggregation of cells (cell aggregates prevent optimal virus infection therefore potentially reduce recombinant protein yield, nor shown). Therefore, mixing was carried out with a VIBROMIX (Gerber+ Pfenninger, Switzerland): 2 stainless steel plates of 5.5 cm

diameter, fixed on a vibrating shaft (vibration: 50Hz, amplitude 1.5 mm). The plates carry 20 conical holes each. The oscillation of the plates also induces shear forces which provide conditions for non aggregate, single cell growth of BHK.

452

The culture was grown to Since the medium is widely used up at this stage and because the infected cells have a significantly increased metabolic rate (Blasey et al., 1997), we exchanged about 90% of the medium, using external crossflow filtration device (CellFlow, MICROGON, USA,

The culture was then infected by addition of 330 ml activated virus, containing an estimated virus per ml to yield MOI of 30. The pH at infection was 7.3, but not controlled. The cells were harvested 20 hours post infection by pelleting (250 ml centrifuge tubes, Corning, USA, spun for 5 minutes at 420 g in a RC3B centrifuge Sorvall, USA). Aliquots were frozen at As shown in Table 2, the two reactor runs yielded very high level receptor expression, significantly higher than has been obtained from stable, amplified cell lines.

453

Optimisation pH: a glass spinner (Bellco, USA) was equipped with a pH probe (Ingold, Switzerland) connected to a pH controller (Bioengineering) controlling two peristaltic pumps (Chemap). The pH was adjusted by addition of 5% (w/v) NaOH and 10 % (w/v) acetic acid. BHK cells from T225 flasks cultures were transferred with 270 ml medium into the spinner (at and infected at an MOI of 10. We compared 5-HT3 receptor expression for cultures run at pH 6.9 (optimal for infection) and pH 7.3 (identified to be optimal for SPAP expression by BHK cells after SFV infection, data not shown). Results (from two independent experiments each) showed an advantage by a factor two for pH 7.3 (Table 3) versus pH 6.9.

Purification of the 5-HT3 receptor was performed by exploiting the C-terminally engineered His-tag by immobilized metal-ion chromatography. The final preparation was judged pure by SDS-PAGE: one band was observed at 65 kD corresponding to the glycosylated receptor. After deglycosylation with peptide N-glycosidase F, a band at 49 kD appeared. The specific ligand binding activity was found to be pmol [3H]-GR65630 binding per mg protein. The molecular size of the receptor complex was determined by gel filtration chromatography to be approximately 280 kD, agreeing well with the calculated molecular mass of 270 kD for a pentameric structure. The secondary structure of the purified receptor protein was probed by circular dichroism and appeared to be dominated by helical elements (48 %) and poor in nonregular structure (9 %).

454

Summary This is the first report of the SFV expression system being applied successfully for the expression of recombinant proteins at reactor scale. Previously reported experience with the expression of human Cyclooxygenase-2 using the SFV at the 1 litre spinners allowed to take the step towards large scale application of the virus. BHK cells were grown in suspension using a specially designed reactor system at a scale of 11.5 litres. Medium exchange before infection then allowed the culture to express more than 3 million receptors per cell before harvesting at 24 hours p.i. Studies on pH optimisation revealed the potential for increasing the receptor yield to 8 million receptors per cell when stabilising the pH at 7.3.

Acknowledgement We thank Dr. J. Delamarter (Geneva Biomedical Research Institute) for his continuing support of this project.

References: Berglund P, Sjödberg M, Garoff H, Atkins J.G, Sheahan B.J. and Liljeström P. (1993) Semliki Forest virus expression system: production of conditionally infectious recombinant particles. Bio/Technology 11, 916920. Bernard A.R. and Blasey H.D. (in press) Transient expression systems, in M.C. Flickinger and S.W. Drews

(eds.), The Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis & Bioseparation, John Wiley & Sons. Blasey, H.D., Lundström, K., Tate S. and Bernard, A.R. (1997) Recombinant protein production using the Semliki Forest virus expression system Cytotechnology 24:65-72. Liljeström P and Garoff H. (1991a) A new generation of animal cell expression vectors based on the Semliki Forest virus system Bio/Technology 9, 1356-1361. Liljeström P and Garoff H. (1991b) Current Protocols. Mol. Biol. 2, 16-22. Lundström K, Mills A, Buell G, Allet E. Adami N and Liljeström P. (1994) High-level expression of the

human neurokine-1 receptor in mammalian cell lines using the Semliki Forest virus expression system. Eur. J. Bioehem. 244, 917-921.

Lundström K, Vargas A. and Allet B. (1995) Functional activity of a biotinylated human neurokinin 1 receptor fusion expressed in the Semliki Forest virus system. Biochem. and Biophys. Res. Comm. 208, 1, 260-266.

Lundström, K., Michel, A. Blasey, H., Bernard, A.R. Hovius, R., Vogel, H. and Suprenant, A. (1997) Expression of ligand-gated ion channels with the Semliki Forest virus expression system , J. of Rec. & Signal Transductuction Research 17(1-3), 115-126. Lundström, K., Mills, A. Allet, E., Ceszkowski, K., Agudo, G. Chollet, A. and Liljeström P. (1995) Highlevel expression of G-protein-coupled receptors with the aid of the Semliki Forest virus expression system, J. of Receptor & Signal Transduction Research 15(1 -4), 23-32. Werner P. , Kawashima, E., Reid, J. Hussy, N., Lundström, K., Buell, G., Humbert, Y and Jones, K.A. (1994) Organisation of the mouse 5-HT3 receptor gene and functional expression of two splice variants. Molecular Brain Research 26, 233-241.

455 Discussion

Grammatikos:

A slide comparing CHO and BHK cells showed that when the cells go into suspension, the BHK cells produce more than when attached. However, the CHO cells are producing twice as much in suspension. Why did you conclude, therefore, that BHK cells are more appropriate?

Blasey:

I did the comparisons in spinner culture as well and the expression level in BHK cells is always several factors higher than with CHO. We have found this for other proteins as well.

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FROM APPLIED RESEARCH TO INDUSTRIAL APPLICATION: THE SUCCESS STORY OF MONITORING INTRACELLULAR RIBONUCLEOTIDE POOLS

S. I. GRAMMATIKOS, K. TOBIEN, W. NOÉ and R.G. WERNER Department of Biotech Production Dr. Karl Thomae GmbH (Boehringer Ingelheim) Birkendorfer Str. 65, D-88397 Biberach an der Riss, Germany

Extended abstract of the oral presentation1

The development of industrial cell culture processes for the production of commercially-relevant recombinant proteins is governed by constraints which pertain to

issues such as costs, competitiveness and the meeting of project timelines. Usually, the time allowed for proper process development is rather short and in this time culture conditions and scale-up protocols have to be defined in such a way as to maximize cell productivity and final titers and to minimize process length and overall costs. The developer tries to satisfy these demands by working on various development areas (rapid cell growth, high cell densities, no lags during scale-up, long periods of high cell viability, homogeneous and good product quality, defined culture conditions, straightforward process etc.). The whole excercise is characterized by a constantly urgent need for better and better tools.

Central to this situation are cell growth and viability and the ability to obtain information on changes in cell physiology as early as possible. In this regard, it is important to realize that cell growth and viability are consequences of physiological processes and represent a balance of metabolic, growth-promoting and growthinhibiting events that occur inside the cell. Because of this, information gained from monitoring only cell concentration and viability and a number of other on-line and off-

line extracellular data (glucose, lactate, ammonia, oxygen uptake rates etc.), however useful, suffers from inherent limitations. First and foremost, it comes too late and allows no predictions. This is immediately evident in the case of cell number and viability monitoring. When a sample from a bioreactor is taken and it is determined that, since the previous sample, cell concentration has doubled and viability is at 95%,

nothing can be deduced about the state of the cells at the time of sampling, let alone predicted what the cell number and viability will be 24 hours later. Secondly, all of the conventional on-line and off-line parameters are no help in pinpointing the exact time of physiological change inside the cell. For example, is a nutrient limiting at a concentration which correlates with entry into a stationary phase or with drop in viability from 95 to 75%, or is its limiting concentration truly higher, affecting cell physiology and growth potential already long before reduced growth and viability are noted? One could most certainly not tell by looking at the usual on-line and off-line parameters. Some on-line parameters like the oxygen uptake rate correlate well with

cell concentration and viability and are very useful in the monitoring of already established production processes. Even these parameters, however, are insensitive at 457 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 457-462. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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low cell concentrations. Furthermore, it should be considered that oxygen consumption is only one aspect of cell metabolism and as such it can only be regarded as a rough indicator of cell physiology. Based on these considerations, then, it is logical to expect that the most useful information to complement all of the above-discussed extracellular measurements will be obtained from intracellular physiologic data. Some years ago, Roland Wagner and Thomas Ryll at the GBF in Braunschweig, published a paper in which they described the sensitivity of certain ratios of nucleotides in responding to physiological changes of BHK and hybridoma cells and speculated that these parameters could prove useful in the monitoring of production processes2. These investigators formulated three ratios which they used to describe the physiological condition of cells: The so-called NTP ratio of purine to pyrimidine nucleotide triphosphates increases as cell condition and growth potential worsen, mainly because UTP and CTP decrease as cells enter the reduced exponential and eventually the stationary phase (Fig. 1). The U ratio of UTP to its derivative UDPN-acetylhexosamines is a measure of growth potential versus the accumulation of toxic metabolic by-products such as ammonia which lead to increases of UDP-activated sugars3. The U ratio decreases as cell condition worsens, first because of decreased UTP and further because of possible increases in UDP-activated sugars (Fig. 1). The NTP/U ratio combines the NTP and U ratios in one very sensitive parameter which increases as cell condition, growth potential and eventually viability become worse.

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At THOMAE the sensitivity of the NTP/U ratio and its ability to report on cell status and growth potential were tested. A large number of fermentations with CHO and hybridoma cells producing a variety of products, at scales from 4 to 10000 liters and under various culture modes were analyzed. In this presentation several examples were shown from CHO cells under development and in large-scale production processes, in which it was confirmed that the NTP/U ratio is a very sensitive parameter which allows prediction of the behavior of cells in culture up to two days before any changes are noted by classical cell number and viability measurements (Fig. 2).

This information is used to advantage in the timely development of efficient cell culture processes. An example of the applicability of the method in process development was shown in which nucleotide information was used to select the optimal perfusion starting point of a perfusion culture. Further applications of this method in

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process development include the optimization of feed start and the selection of optimal feeding strategies for fed-batch processes, the optimization of perfusion rates in perfusion processes and the selection of optimal scale-up schedules and inoculum conditions. Finally, an additional advantage of the method, is the insights one obtains on cell metabolism and clues about improving the culture conditions.

References: 1 This 2

work will be published in detail elsewhere.

Ryll, T., Wagner, R. Intracellular ribonucleotide pools as a tool for monitoring the physiological state of in vitro cultivated mammalian cells during production processes. Biotechnol. Bioeng. 40, 934-946, 1992.

3 Ryll,

T., Valley, U., Wagner, R. Biochemistry of growth inhibition by ammonium ions in mammalian cells. Biotechnol. Bioeng. 44, 184-193, 1994.

461

Discussion Aunins:

Grammatikos:

When you showed a slide on scale-up to 10,000 1, you had dislocations on your NTPU ration whenever you had a split. You indicated that the high starting level limited your growth. Do you have an explanation for those dislocations? The NTPU ratio is variable dependent on cell type, medium conditions, and culture mode. In this 10,000 1 reactor, 3 changes

occurred: lower initial cell densities, the medium exchange to go into production conditions, and the new production medium. In this case we reached a new basal level and reflected a slower growth rate of the cells. Barteling:

When you scale-up you see this change all the time. Do you take care to have the medium as well conditioned as possible, in the sense that the temperature is right or that you start at a low volume and then add the medium slowly, as that can make a big difference?

Grammatikos:

The cells entered the 10,000 1 reactor in a good physiological state, so the changes were not there. With tPA these changes are according to the protocol, and there is nothing that can be done to change the process. The only thing is to monitor the process by nucleotides.

Ozturk:

If you control the environment, then you are maybe controlling the cells. This ratio might be controlled by the metabolite concentrations in the medium. If you measure the nutrients such as glucose and glutamine available to the cell, can you predict the nucleotide ratio?

Grammatikos:

We could try to correlate this ratio with certain nutrients in the medium, or certain conditions in the reactor, and then perhaps monitor these instead of the nucleotide ratio. It is possible that in the later stages of this project, when we rely on this parameter, we could make such correlations.

Guan:

You have only correlated this NTPU ratio with cell concentration. Have you correlated it to any other ratios such as productivity or product quality?

462

Grammatikos:

It is not true that we correlate this with cell number and viability. We use this as a mirror of cell physiology, and it so happens that we can predict changes in viability and cell growth with this NTPU ratio before they happen. We could correlate the ratio with other parameters but I think this ratio should be looked at on its own.

Konstantinov:

There is value in defining indices representing physiology. However, I would give higher priority to indices that measure online as you do not need to sample. How does your index correlate with oxygen uptake rate, which is easy to measure and immediate?

Grammatikos:

There could be on-line parameters that could be monitored, and

oxygen is a good one, which may be more suitable for the production setting. In development you need to have information as early as possible on physiological changes and the OUR may still be a late indicator of this change. Ryll:

On-line parameters, which you can correlate and then define, are

much better to control fermentation processes. The power of this ratio technique is that it is very universal for animal cells, and you can use it early on before on-line parameters have been developed. These physiological parameters depend a lot on cell cycle distribution, for example. So it is a direct measurement for the growth rate, etc, so you can use these data for things other than for just controlling fermentation processes.

NEW TECHNOLOGY OF PRODUCTION RECOMBINANT ERYTHROPOIETIN FOR PERORAL ADMINISTRATION

HUMAN

T.D. KOLOKOLTSOVA, N.E. KOSTINA, E.A. NECHAEVA, N.D. YURCHENKO, O.V. SHUMAKOVA, A.B. RIZIKOV, N.A. VARAKSIN, T.G. RYABICHEVA Research Institute of cell cultures, State Research Centre of Virology and Biotechnology Vector, Koltsovo, Novosibirsk region, 633159, RUSSIA

1. Introduction Erythropoietin (EPO) is a glycoprotein hormone that stimulates erythrocyte formation in human blood. At present, EPO is regarded as an effective medicament

for treating patients with anemia caused by renal insufficiency, being a side-effect of treatment for AIDS with azidothymidine (AZT) or other medicines (1,2). Such patients need frequent blood transfusions or EPO hormone injections. Since transfusion and injections entail the possibility of transfer of infecting agents, peroral form of EPO would make treatment more safe and effective.

The present work was aimed at the development of a new technology for production of recombinant human erythropoietin (rhEPO) for peroral administration and testing the new preparation in experiments involving animals. 2. Materials and methods rhEPO substance has been produced in the new strain of cell culture CHOpE . Cell culture CHOpE using methods of genetic and cellular engineering has been developed (3). Cultural media containing rhEPO was centrifugated and concentrated (1:50-1:100). Isolation, concentration, purification and certification of rhEPO substance were performed in accordance with the modern requirements .

The tabletted form of rhEPO was produced under laboratory conditions by mixing erythropoietin substance with stabilizers followed by lyophilization, addition of admixtures and tabletting. Specific activity of the new tabletted form of rhEPO was studied by the method of enzymoimmune assay (EIA method) (4) and in experiments involving animals. Intensity of erythropoesis in mice C57BL strain was registered by measuring reticu463

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 463-465. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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locyte concentration in the animals’ blood. Placebo tablets and EPO for injections

(St. Petersburg, Russia) were used as controls. Control of tablets harmless and toxicity was carried out on laboratory animals. Tablets was dissolved and 5 ml of rhEPO solution (1 tablet) was injected subcutaneously in each uinea pig (weight 300+ 50g) and on 0.2 ml in each mice (weight 1720g.). In each experiment were involved not less than 5 animals. 3. Results and Discussion Erythropoietin is known to be of great importance for practical medicine. The demand for EPO for treatment of patients is rather high. At the same time production of EPO from physiological materials (urine and plasma) is a labourconsuming and expensive procedure. Establishment of rEPO producers by genetic and cell engineering methods and production of recombinant preparation turned out to be most promising.

A strain of CHO cells producing rhEPO using methods of genetic and cellular engineering has been developed in State Research Center of Virology and Biotechnology “Vector” ; the strain was patented and certified in accordance with con-

temporary requirements; saving and working banks of the cell culture have been created and certified in the State Institute of Control and Standardization of Immunological preparations.

As a result of the performed studies conditions on cultivation, isolation, concentration, purification and certification of rhEPO substance have been developed, the effects of conditions of drying the substance are being studied. The results of

this work was represented in the previous publication. The selection of admixtures for stabilization showed that lactose, gelatose and sacharose were the best to be added. The more best mixture of tablets for maintaining of specific activity stability of rhEPO consists of 12-18% gelatose, 12-18% saharose, 25-70% lactose, not more than 1% calcium stearat and 0,2 % vanilin. The tablets produced in this way preserved a stable double-convex form, had the taste and the smell of vanilin and contained EPO in the dose of 100-2000 U. The tablets does not contein patogenic microorganisns was harmless and does not call the toxicity or pyrogenic effect on enimals. The microscopic studies of animal tissue samples does not show necrosis or pathologic process in the places of injections.

Control of specific activity of rhEPO in tablet’s form by the method of enzymoimmune assay demonstrated stability of properties for 10-12 months. Taking into account the obtained data of experimental series, the tabletted form of the preparation supplemented with admixtures for stabilization has been obtained.

Specific activity of the tabletted form of rhEPO was studied in experiments on registration of intensity of reticulocyte formation involving mice strain C57BL. Tablets were pressed in powder and added in the food of mice. Injection form rhEPO was injected intramuscular. Fig. l presents the results of measuring the hormone activity in vivo.

465

Experimental results showed that EPO injected directly into blood promoted erythropoiesis faster than the tabletted form of rhEPO administered per os. However, on day 3 an increase in reticulocyte concentration was observed in murine blood at the level which does not significantly differed from that after EPO injection. According to the date of another autors the effect of EPO significantly depend on the dose and on the way of introduction (5). CONCLUSION

Thus, new technology has been developed for production of human recombinant erythropoietin in the tabletted form for peroral administartion.

The more stable tablet’s form of rhEPO consists of 12-18% gelatose, 12-18% saharose, 25-70% lactose, not more than 1% calcium stearat and 0,2 % vanilin. New form rhEPo for peroral administration demonstrated the specific effect on erythropoiesis in white mice. REFERENCES 1. Besarab, A.., Ross, R.P., Nasca, T.J. (1995) The use of recombinant human erythropoietin in predialysis patients, Curr. Opin. Nephrol. Hypertens 4(2), 155-61, 2. Muirhead, N., Bargman, J., Burgess, E., Jindal, K.K., Levin, A., Nolin, L., Parfrey, P. (1995) Evidence-based recommendations for the clinical use of recombinant human erythropoietin, Am. J. Kidney. Dis., 26(2), 1-24. 3. Patent U S S R - N 1 8 0 1 1 1 8 - 1994. 4. Storring, P.L., Tiplady, R.J., Gaines Das, R.E., Rafferty, B., Mistry, Y.G. (1996) Lectin-binding assays for the isoforms of human erythropoietin: comparison of urinary and four recombinant erythropoietins, J. Endocrinol 150(3), 401-12. 5. Breymann, C., Bauer, C., Major, A., Zimmermann, R., Gautschi, K., Huch, A., Huch, R. (1996) Optimal timing of repeated rh-erythropoietin administration improves its effectiveness in stimulating erythropoiesis in healthy volunteers, Br. J. Haematol 92(2), 295-301.

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GENERAL SAFETY ASPECTS

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SIGNIFICANCE OF MULTIPLE TESTING ON MURINE LEUKEMIA VIRUS OF MOUSE HYBRIDOMAS

E.J.M. AL, T. JORRITSMA, A. BLOK, H.M.G. SILLEKENS AND P.C. VAN MOURIK. CLB Biotechnology Services PO Box 9190, 1006 AD Amsterdam, The Netherlands.

1. Introduction

Monoclonal antibodies derived from mice are known to produce Murine Leukemia

Viruses (MuLV; Ref.l) and if intended for human in vivo use they should be tested for production of these viruses according current guidelines (Refs.2-4). Detection of MuLV can be done in the following ways: - XC-Plaque Assay for infectious ecotropic (i.e. mouse-tropic) MuLV;

- S + L-Focus Assay for infectious xenotropic (i.e. not mouse-tropic) MuLV - Electron Microscopy (EM) studies on retroviral particles.

- Reverse Transcriptase enzyme activity (RT) assay. The objective of this study was to reveal correlation and redundancy of multiple MuLV testing of 5 mouse hybridomas. Furthermore, both cell banks and crude harvests of these hybridomas were studied for determination of the optimal test strategy for MuLV-testing.

2. Test Procedures The XC-Plaque-Assay was performed by inoculation of subconfluent mouse SC-1

fibroblasts and, at confluency, UV-irradiation and subsequent overlay with rat XC-cells (Ref.5). Plaques were detected macroscopically after staining of the cell layers. The Extended XC-Plaque Assay was done by performing 5 passages on SC-1 cells prior to the

direct assay. The S+L-Focus Assay was performed by inoculation of subconfluent (S + L-)- mink lung cells and microscopic detection of foci after reaching confluency up

to 10 days (Ref.5). The Extended S+L-Focus Assay was done by performing 5 passages on prior to the direct assay. Reverse Transcriptase (RT) Assay was performed on polyethylene glycol precipitated culture supernatants, using Poly rA/dT-

template and either

as divalent cation (Ref.6). Positive test results are

subjected to confirmatory testing by using Poly dA/dT template for detection of contaminating DNA-polymerase activity. Electron Microscopy studies (EM) were

subcontracted. Either transmission EM (TEM) on cultured cells or negative staining (NSEM) on viral pellets obtained after ultracentrifugation of crude harvests were performed (Ref.7). 469

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 469-472. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

470 3. Results

Table 1 presents the results of the two types infectivity assays, both direct as well as extended. The results show that four out of the five hybridomas tested were positive in either of the assays; three hybridomas produced ecotropic MuLV and two hybridomas produced xenotropic MuLV. Hybridoma #2 was positive in both infectivity assays indicating both the presence of ecotropic and xenotropic MuLV. This was confirmed by performing XC-Plaque assays with culture supernatants after 5 passages on cells, which assay yielded > 2000 focus-forming units in the S + L-Assay but only 1 plaque in the XC-Assay. In the reciprocal test, culture supernatants after 5 passages on SC-1 cells yielded >200 plaque forming units in the XC-Assay but very low number infectious units in the S + L-Focus Assay. Therefore, we concluded that this hybridoma produced both

types of MuLV.

All tests on infectious MuLV on the bulk harvests derived from the 5 hybridomas yielded

negative results (not shown). Table 2 presents the results of the RT-Assays performed with both cell bank culture supernatants of the cell banks and bulk harvest material obtained after large-scale fermentation. Table 2 shows that RT-activity was detected in cell banks of three hybridomas (#l-#3) using the Poly rA/dT template; however, only in one case (Hybridoma #2) this

RT-activity was confirmed by a low DNA-polymerase activity using the poly dA/dT template. When testing bulk harvest preparations, none of the samples were clearly positive for RT-activity.

471

Table 3 presents the results of EM-studies on both cell banks (by TEM) and crude harvests (by NS-EM).

Table 3 shows that intracellular Type A Retroviral particles were detected in all five cell banks; the percentage of positive cells ranged from 40 to 100%. The production of retroviral particles (Type C) by the Cell Banks, however, was only clearly positive in 4 out of five cell lines. Viral-like particles were also detected in 3 of the 5 Bulk Harvest samples. However, there is no clear correlation of the bulk harvest EM-results with the EM results of the cell banks tested.

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4. Discussion European guidelines (Note for Guidance) differ from US Points to Consider by their description of MuLV testing: whereas the EC guidelines stress the tests for infectious MuLV in cell banks, the US prescribes EM-studies especially in Bulk Harvests. Moreover, since mouse hybridomas are a priori considered to be able to produce MuLV, the US guideline indicate that MuLV testing was not required for murine hybridoma seedlots. The recent international guideline from the ICH includes both infectivity tests, EM and RT testing. Our results show that the most informative results were obtained by infectivity tests on seedlots: 4 out of 5 hybridomas tested were positive in either XC or S + L-Assay. These results were confirmed by EM-testing on Type C Retroviral particles whereas RTtesting yielded only positive results with the hybridoma producing the highest amounts of infectious virus (see Table 1, ). Since EM-studies require specialized equipment and procedures, we promote the application of infectivity tests on cell banks. Analysis of bulk harvests may be compromised by long storage time and probably unfavourable storage conditions: in contrast to seedlot testing, all infectivity tests and RTtest were negative with this material. By EM-studies viral-like particles (VLPs) were detected in bulk harvests by negative stain EM, however, these results did correlate with neither the infectivity tests nor the RT-tests. The hybridoma producing thew highest number of infectious units did not produce detectable levels of VLPs. The absence of a clear relation between MuLV detected in cell banks and the VLPs detected in bulk harvests, questions the relevance of bulk harvest testing for MuLV. References 1. 2. 3. 4. 5. 6. 7.

Weiss RA (1982) Retroviruses produced by hybridomas. N Eng J Med 307:1587. European Commission Note for guidance: Production and quality control of monoclonal antibodies. Ad hoc Working party on Biotechnology/Pharmacy. III/5271/94. Points to consider in the manufacture and testing of monoclonal antibody products for human use (1994). Center for Biologics Evaluation and Research (CBER). Note for Guidance on quality of Biotechnological Products: Viral safety evaluation of biotechnology products derived from cell lines of human or animal origin. CPMP/ICH/295/95. Purification and Assay of Murine Leukemia Viruses. Sherr CJ and Todaro GF (1979). Methods in Enzymology, Vol. LVIII, pages 412-424. Assay of C-type infectivity by measurement of RNA-dependent DNA polymerase activity. Kellof GJ, Hatanaka M and Gilden RV (1972). Virology, Vol.48, pages 266-269. Detection and Identification of Viruses by Electron Microscopy. Miller SE (1986) Journal of Electron Microscopy Technique, Vol. 4, pages 265-301.

MURINE RETROVIRUS DETECTION USING MUS DUNNI CELLS AND CELLS P Seechurn, B Mortimer, P Newton, C Martin Covance Laboratories, Otley Road, Harrogate, North Yorkshire HG3 1PY,

United Kingdom

1. Introduction Regulatory authorities require that murine cells used for the production of biopharmaceuticals and gene therapy vector-banks are evaluated for the presence of murine leukaemia viruses (MuLV). The classification of the four classes of MuLV is based on the ability of the virus to infect and replicate in different cell types. Ecotropic MuLV have a limited host range, only replicating in cells of mouse or rat origin. Xenotropic MuLV infect xenogeneic cells but are not infectious for murine cells. Polytropic retroviruses, which are formed by a recombination between exogenous ecotropic retrovirus and non-ecotropic endogenous proviral DNA sequences, are characterised by their ability to induce focus formation on cultured mink lung cells; hence their designation as mink cell focus forming (MCF) viruses. These viruses can infect murine cells but have limited infectivity to non-murine cells. Amphotropic MuLV differ from MCF in their wider host range for non-rodent cell lines. Lander MR and Chattopadhyay SK (1984) have shown that, with the exception of ecotropic Moloney MuLV, the Mus dunni cell line will detect all four major classes of MuLV when directly inoculated with MuLV. The "Points To Consider in Manufacturing and Testing of Monoclonal Antibody Products for Human Use (1997)" suggests that infectivity assay should be comprised of an amplification step on Mus dunni cells coupled with detection of infectious virus on a suitable cell line. We have evaluated the level of extracellular virus following passage of Mus dunni cells infected with the four classes of MuLV. In addition, we have evaluated the retroviral host range of various S+L- cells. 473

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 473-480. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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2. Method 2.1 MCF AND AMPHOTROPIC VIRAL ASSAYS The method used was essentially as described by Peebles (1975). Briefly S+L- cells were seeded and polybrene treated. Following polybrene treatment, cells were inoculated and refed as necessary with growth medium until foci appeared. 2.2 ECOTROPIC VIRAL ASSAY Two methods were used to detect ecotropic MuLV. Supernatant samples were assayed on murine S+L- cells (FG-10) essentially as described above. Ecotropic viral evaluation was also carried out using the SC-l/XC method as described by Rowe et al (1970). 2.3 INFECTION OF MUS DUNNI CELLS Cells were inoculated as described by Lander & Chattopadhyay (1984). Briefly, the cells were seeded and polybrene treated prior to inoculation with 200 infectious units of each type of MuLV. Once confluent, the supernatant was collected (PO) and the cells passaged twice. The supernatant samples (P1 & P2) were collected each time the cells reached confluence. 3. Results Subconfluent cultures of Mus dunni cells were separately inoculated with xenotropic, ecotropic, polytropic and amphotropic MuLV. When the cells reached confluence, culture supernatant samples were collected. The cells were passaged on two occasions and culture supernatant samples collected each time confluence was reached. The supernatant samples were assayed for infectious MuLV. During the course of the experiment cells were observed for cytopathic effect (CPE). No CPE was observed when Mus dunni cells were inoculated with xenotropic and amphotropic MuLV either following direct inoculation or after passage. Similarly no morphological effects were observed on Mus dunni cells following direct inoculation with ecotropic and polytropic MuLV. After passage, however, CPE was observed with these viruses. The CPE was more pronounced by the second passage (Figures 1-3).

475

476

The results in Table 1 show that for each type of MuLV, there was an increase in viral titre following passage of infected Mus dunni cells. By passage 2 there was a substantial increase in titre values. The results from Table 1 are also expressed graphically in Figure 4.

477

The sensitivity of various indicator cells to the four classes of MuLV was evaluated and the results presented in Tables 2a and 2b also shown graphically in Figures 5 and 6. These results show that feline cells were the most sensitive cell line for the detection of amphotropic MuLV. Mink cells were more suitable for the detection of xenotropic and polytropic MuLV. The SC-l/XC assay and murine cells were the most sensitive indicator system for the detection of ecotropic MuLV. Table 2b also shows that comparable titre values were obtained when ecotropic MuLV was assayed on SC-l/XC cells compared to murine cells.

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4. Conclusion •

Mus dunni cells amplified the four types of MuLV used; the litre increased with passage number

•

Feline cells provided the most sensitive infectious assay system for amphotropic virus

•

Xenotropic and polytropic MuLV were more readily detected on mink cells compared to feline cells.

479

•

Murine cells were the most suitable detection of ecotropic MuLV.

indicator cell line for the

5. Discussion

The data presented here are in disagreement with the findings of Hughes et al (1996) where the authors show that feline cells were more sensitive for the detection of xenotropic MuLV and polytropic MuLV compared to mink cells. In their study the level of detection of xenotropic MuLV on feline was variable when using the same stock, ranging from 2 to 55 times higher titres on feline compared to mink Our findings indicate that the most sensitive cell line for the detection of xenotropic and polytropic MuLV was mink cells. Our observation supports the findings (Hughes et al 1996) that feline cells are the most sensitive indicator cell line for the detection of amphotropic MuLV. This observation is in agreement with those of Dr Janet Hartley (NIH), personal communication. The reason for the differences in observation between the work presented here and that of Hughes et al (1996) is unclear. However, differences in strains of MuLV used may account for these observations. It would appear that in the method employed by Hughes et al (1996) indicator cells were not treated with polybrene. Differences in methodology between these two studies may be a contributing factor. The effect of passage of retrovirally infected Mus dunni cells on supernatant viral titre was evaluated. The viral titre for the four types of retroviruses increased with passage number, suggesting that the maximum level of virus may not have been achieved by the second passage. The findings presented here do not support the observation made by Hughes et al 1996; these authors reported that there were no differences in the titre of supernatant samples from amphotropic infected Mus dunni cells between passage 1 and passage 2. We would recommend that when using Mus dunni to amplify MuLV, the cells should be passaged twice as a minimum.

6. References Hughes, J.V., Messner, K., Burnham, M., Patel, D., White, E.M. (1996) Validation of Retroviral Detection for Rodent Cell-Derived Products and Gene Therapy Applications. Developments in Biological Standardisation 88. 297-304 Lander, M.R., Chattopadhyay, S.K. (1984) A Mus dunni Cell Line that Lacks Sequences Closely Related to Endogenous Murine Leukemia Viruses and Can Be Infected by Ecotropic, Amphotropic, Xenotropic, and Mink Cell Focus-Forming Viruses. Journal of Virology 52 695-698 Peebles, P.T., (1975) An In Vitro Focus-Induction Assay for Xenotropic Murine Leukemia Virus, Feline Leukemia Virus C, and The Feline-Primate Viruses Rd-114/CCC/M-7. Virology 67 288-291

480 Rowe, W.P., Pugh, W.E. and Hartley, J.W. (1970) Plaque Assay Technique for Murine Leukaemia Viruses. Virology 42 1136-1139.

Acknowledgements

Thanks to Karen Heelan and Kay Beddoe for secretarial support. Covance Laboratories Ltd Otley Road, Harrogate, North Yorkshire, HG3 1PY United Kingdom Tel: +44 (0) 1423 500011 Fax: +44 (0) 1423 569595 Website: http:\\www.covance.com

THE REMOVAL OF MODEL VIRUSES DURING THE PURIFICATION OF HUMAN ALBUMIN USING CHROMATOGRAPHIC PROCEDURES

R. CAMERON 1 , C. HARBOUR 1 , Y. COSSART1 and J.P. BARFORD2 Departments of Infectious Diseases1 and Chemical Engineering2, University of Sydney, NSW 2006, Australia

Abstract

The capacity of an ion exchange chromatography system, designed to purify human albumin from plasma, to remove two model viruses, i.e. polio virus type 1 and canine

parvovirus, has been evaluated. The viral removal studies were performed using a scaleddown model of the production scale process which was shown to perform within the limits established for the production process with regard to product purity. An aliquot of either high titre polio virus or canine parvovirus was added to human plasma (supernatant 11 + 111) in a 1:10 ratio and then applied to a DEAE column. This column was then washed and eluted and the eluant applied to a CM column. The latter was also washed and then product eluted. Cell culture assays were then used to establish the levels of both viruses in the different chromatography fractions. Following the two-stage purification procedure which generated high purity albumin (>99%) it was shown that polio virus levels were reduced by 5.8 logs and canine parvovirus 2 logs thus demonstrating that chromatographic procedures can play a role in increasing the safety of biopharmaceuticals with respect to viral transmission. Introduction

Chromatographic procedures are now widely used at some stage during the downstream processing or purification of the majority of recently developed biopharmaceuticals, such

as monoclonal immunoglobulins produced by hybridoma technology and recombinant proteins derived from microbes or cell culture systems. There is also an increasing trend towards the incorporation of some chromatographic steps in the purification of biologicals which have not traditionally employed these techniques for large-scale production including, for example, the manufacture of plasma-derived products such as the coagulation factors, immunoglobulins and albumin. A chromatographic procedure for the purification of human plasma albumin was first developed almost twenty years ago (Curling et al., 1977) but did not gain widespread approval among plasma product manufacturers for a number of reasons but most significantly the excellent safety record of heat-treated albumin produced by the traditional Cohn-ethanol fractionation procedure. There are, however, advantages claimed for the production of plasma proteins such as 481 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 481-483. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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albumin by using chromatographic procedures including increased purity of product and the capacity to recover more proteins and, as a result, some manufacturers have begun to adopt this approach while being mindful of the need to validate their processes for viral elimination. As yet there have only been a few reports which have investigated the capacity of chromatographic processes to reduce viral loads and this project was designed to collect data in this important area. A new process for manufacturing albumin which uses a combination of traditional Cohn fractionation and chromatographic methods, has been investigated for its capacity to remove viruses.

Materials and methods

The source material used in this study, supernatant 11 + 111, was generated from human plasma by Cohn fractionation as described previously (Yap et al., 1993). Ion-exchange chromatography was performed using Pharmacia XK/20 columns containing DEAE

Sepharose Fast Flow and CM Sepharose Fast Flow resins connected in series. Albumin was eluted from the DEAE column and the resulting eluate applied to the CM column which was equilibrated and washed and then proteins eluted. Protein levels of samples were determined by capillary electrophoresis (Applied Biosystems). Viral clearance during the process was assessed following spiking of supernatant 11 + 111 with virus in a 10:1 ratio of supernatant : virus. Polio type 1 (Mahoney vaccine strain) was assayed for cytopathic effect in HeLa cells and canine parvovirus was assayed by haemagglutination of porcine red blood cells following growth in and lysis of feline kidney cells. Titres of both viruses are expressed as log and calculated using the Spearman and Karber method. Viral clearance was calculated as follows Clearance = total virus input spike (calculated from real value) total virus detected in product

Results and discussion

The results shown in table 1 demonstrate the viral clearance factors for three consecutive process runs or cycles. The polio clearance factor was 6.1, 5.5 and 5.9 for cycles 1,2 and 3 respectively while that for canine parvovirus was 3.3, 1.3 and 1.3 respectively. There is an indication that the clearance of polio virus is slightly more efficient in the first cycle than in cycles 2 and 3 but the results are probably within experimental error. A similar trend was observed for canine parvovirus where the clearance factor in the first cycle is clearly greater than that in either cycles 2 and 3. In an attempt to determine whether or not this effect could be reversed by cleaning of the gels between cycles a sanitization regime

was introduced after each cycle and these results are reported elsewhere (Cameron et al., Biologicals, in press). This study has established the capacity of an ion-exchange process used in the production of a human biopharmaceutical to reduce viral load. Non-enveloped viruses were selected

483

for analysis because, unlike enveloped viruses such as hepatitis B and C and HIV, they are not effectively inactivated using solvent/detergent treatments. In addition polio virus serves as a useful model for hepatitis A virus and canine parvovirus for human parvovirus

B19 both of which have been transmitted to patients during treatment with coagulation factors derived from human plasma. The results demonstrate that the ion-exchange process studied can remove reproducibly large amounts of non-enveloped viruses. The levels of clearance which were found using chromatographic processes therefore add to the overall safety of therapeutic proteins and augment other removal/inactivation procedures. The results also show however that viruses can behave quite differently in the same system which emphasises the need to carry out further research in this area so that the complex interactions between viruses and matrices in the presence of different biological materials can be more clearly understood and hence predicted.

References

Curling, J.M., Berglof, J., et al. (1977) A chromatographic procedure for the purification of human albumin, Vox Sang 33,97-107. Yap, M.B. et al. (1993) Development of a process for the preparation of human serum albumin using chromatographic methods, Biotechnology of bloodproducts 227, 143-149.

Acknowledgements The authors would like to thank the Australian Research Council for funding for this project and a scholarship to RC and Arthur Webster Pty. Ltd. for supplies of canine

parvovirus and CSL Pty. Ltd. for helpful advice and super II + I I I .

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INACTIVATION OF HEPATITIS A VIRUS DURING THE PRODUCTION OF NORDIMMUN D W BIRCH* P KAERSGAARD C MARTIN* *Covance Laboratories Ltd Harrogate North Yorkshire, HG3 1PY UK

HemaSure A/S Gentofte Denmark

Introduction

One of the process steps in the manufacture of Nordimmun is a Pasteurisation step (60°C heat treatment for 10 hours). Parenterally administered plasma products should be free of any infectious agents and historically, Pasteurisation has commonly been used to inactivate both enveloped and non enveloped viruses in plasma products. Thermal stabilizers such as sucrose are added to protect the plasma product against thermal inactivation. These stabilizers however, also protect any contaminating viruses present in the material. Additionally, the presence of virus neutralising antibodies in the product may also mask or elevate any virus clearance demonstrated by the process. Concerns regarding the transmission of hepatitis A virus (HAV) in blood products was reported in 1992 with haemophiliacs in Italy contracting the disease after treatment with purified factor VIII [1]. The factor VIII preparation in question had been prepared using a solvent/detergent virus inactivation step, which although effective against lipid enveloped viruses (eg HIV), is ineffective against non-enveloped viruses such as HAV. Poliovirus has previously been used as a virus with some degree of thermal stability. It has been reported that HAV is significantly more heat stable than poliovirus and the use of poliovirus in heat inactivation validations may have overestimated the degree of virus inactivation caused by the process. The strain of HAV we have used is a lytic strain and has been shown to be more thermostable than poliovirus[2]. In a recent paper, Niessen et al [3] demonstrated that the addition of sucrose and BSA stabilisers influenced the stability of infectious viruses under Pasteurising conditions and that HAV showed greater thermostability under such conditions than poliovirus. 485

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 485-490. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

486

Methods

Aliquots from three different batches of purified and concentrated IgG solution (Nordimmun), each with different levels of HAV antibodies were separately spiked with HAV. In addition a placebo solution was also spiked with virus. An appropriate volume of 0.5M HC1 was added to each spiked test solution such that pH values of between 5.4 - 5.5 were obtained. Sucrose was added to each solution. Each spiked sample was then dispensed into 8 appropriately labelled vials.

Six vials from each solution were completely immersed in a water bath maintained at 60°C. At 0.5, 1, 2, 4, 7 and 10 hours after incubation, a vial of each sample was removed and diluted 1 in 4 with ice-cold diluent. This was further diluted 1 in 2.5 with diluent. The samples were serially diluted 10 fold with diluent and assayed. The contents of one of the remaining vials was diluted and assayed immediately. The other remaining vial from each batch was incubated under ambient conditions for 10 hours, whereupon a sample was removed, diluted and assayed.

To assess the effect of the heat treatment alone, HAV was spiked into a portion of virus diluent and incubated at 60°C along with the spiked test solutions. Samples were

withdrawn at the same sampling points as for the spiked test material, diluted 1 in 4 with ice cold virus diluent, followed by a 1 in 2.5 dilution and then further serially diluted and assayed. Virus diluent was spiked with HAV and samples withdrawn and assayed immediately (assay positive control) and after 10 hours incubation under ambient conditions. Virus diluent was used as the assay negative control.

Results In the treatment control, which did not contain IgG or sucrose, infectious virus was not detected beyond 4 hours incubation at 60°C.

After 10 hours incubation at 60°C, the test solution A, containing (w/w) sucrose, IgG but no anti HAV antibodies contained infectious virus.

Test solution B, contained antibodies against HAV and (w/w) sucrose. This solution contained the higher amount of antibody and was detected at the 2 hour sampling point but not at the 4 hour sampling point.

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In the other HAV antibody containing test solution (test solution C), which contained a lower level of anti HAV antibody, virus was present at the 4 hour sampling point but was not detected at the 7 hour sampling point. In test solution D, which contained sucrose but not IgG, virus was detected in the 7 hour sample but not in the 10 hour sample.

488

489

Summary of Sample Titres

Conclusion The Pasteurisation step was effective in removing HAV over a 10 hour incubation period in the presence of sucrose and anti HAV antibodies. It appear's that anti HAV antibodies alone do not appreciably reduce viral titre during incubation under ambient conditions. Heating of HAV to 60°C in test solution D, which did not contain IgG but did contain sucrose, lead to no detectable virus being present at 10 hours incubation. A combination of heating to 60°C in the presence of anti HAV antibodies, lead to an accelerated viral clearance, with no detectable virus present 2 - 4 hours after spiking depending on the level of antibodies present. It is not possible to explain the mechanism by which this phenomenon occurs. Incubation of HAV at 60°C over a 10 hour incubation period, in an IgG solution which did not contain anti HAV antibodies but did contain sucrose (test solution A), resulted in an approximate 4.5 log reduction in viral titre over 10 hours but not total clearance of virus. From a comparison between a treatment control which contained no sucrose or IgG and a placebo solution (test solution D, which contained sucrose but no IgG), it can be

490

inferred that the presence of sucrose imparts a protective effect on HAV during incubation at 60°C. References 1.

Mannucci PM. (1992) Outbreak of Hepatitis A among Italian Patients with Haemophilia. The Lancet, 339, 819

2.

Seechurn, L.P., Birch, D.W., Mortimer, B, Devlia, A., Martin, C. and Loudon, P.T. (1995) Inactivation of Hepatitis A Virus. Animal Cell Technology: Developments Towards the 21st Century, (1995), 625-629

3.

Niessen, E., König, P., Feinstone, S. M. and Pauli, G. (1996) Inactivation of Hepatitis A and other Enteroviruses During Heat Treatment (Pasteurisation). Biologicals, (1996), 24, 339-341

Acknowledgements Thanks to Kay Beddoe and Karen Heelan for secretarial support.

Covance Laboratories Ltd. Otley Road, Harrogate, North Yorkshire, HG3 1PY United Kingdom Tel: +44 (0) 1423 500011 Fax: +44 (0) 1423 569595 Website: http:\\www.covance.com

SESSION ON : VIRAL VECTOR PRODUCTION FOR GENE THERAPY

The application of gene therapy is moving from fundamental research to clinics and questions concerning new production technologies, biodistribution of the transgene and

safety issues become more important. This increasing orientation towards clinical application is reflected by the fact that the ESACT-Meeting was the third one, after the ESACT-Meeting held in Vilamoura in 1996 and the preconference symposium of the ESACT-Meeting in Veldhoven in 1994, during which a gene therapy session was organized. Among the different viral vectors which have been used for gene transfer, three had expanded from research and developmental assessment to gene therapy clinical trials: retroviral, adenoviral, and adeno-associated viral (AAV) vectors. Whereas the two first ones are already used since several years, the interest in AAV vectors is constantly

increasing, due to the fact that they are nonpathogen for humans and that they can transfect dividing and non-dividing cells of almost any tissue. Besides the search for gene transfer efficacy, probably one of the major challenge gene

therapy had to face is linked to the safety and genetic stability of viral vectors. Considerable efforts are spent in the development of new optimal packaging cell lines preferable of human or old world monkey origine, for the production of different viral vectors. Aspects of the scale-up and the optimisation of the production and purification of retroviral and adenoviral vectors were presented. As elsewhere in animal cell technology, the trends in the production are going to <> reactor processes (for retroviruses as well as for adenoviruses) by using serum-free media where feasible. These developments are all in vain, when there is not a validated safety precedure for verifying not only the absence of mycoplasmas and other microorganisms (classical) but more important, proving the absence of replication-compentent viruses.

Other aspects of this session were the use of macrophages as potential shuttles for widespread/systemic targeting in neuromuscular disorders, like Duchenne Muscular Dystrophy, for which also a dog model has been described. Finally, the analysis of biodistribution is of utmost importance in order to verify which percentage of cells of which tissue of treated animals had been transfected. This analysis plays a key role for the evaluation of efficacy and safety of any viral vector. In conclusion, this chapter gives an overview about the actual problems and developments in gene therapy and the production and use of viral vectors for gene therapy.

J.-M. Guillaume, A. Crespo Chairpersons 491

O. - W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 491. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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ADENO-ASSOCIATED VIRAL VECTORS : PRINCIPLES AND IN VIVO USE

OLIVER DANOS Gene Therapy Program. Généthon, CNRS URA 1922, Ibis, rue de l’Internationale, BP 60 91002 Evry, France.

1. Biology of Adeno-Associated Viruses Small non-enveloped viruses with an icosahedral capsid and a single-stranded DNA

genome are grouped in the family of Parvoviridae. The virions are usually around 20 nm in diameter and are extremely resistant to a variety of harsh physical conditions (low pH, heat, presence of detergent). Within this family, Dependoviruses where first

identified as contaminants of human adenovirus preparations and shown to be strictly

dependent on the presence of the helper adenovirus for their replication. These Adenoassociated viruses (AAV) are found in a variety of birds and mammals including

humans. Five human serotypes have been identified, and around 70 % individuals are seropositive in the general population. However, no pathology has been associated

with AAV infection and although the AAV genome is known to integrate the host cell genome under certain conditions, no vertical transmission has been documented. For a complete review on Parvoviruses and AAV, see (1). AAV particles are able to infect a variety of cell type in culture. Viral capsids entering the cytosol are rapidly transported to the nucleus were the single stranded genome is delivered. In the presence of a helper virus, such as Adeno, Herpes or

Vaccinia virus, the genome is actively transcribed and replicated. This step is performed mostly by the cellular DNA and RNA synthesis machineries and requires the presence of AAV proteins encoded by the rep gene. It is most efficiently activated by a co-infecting helper virus, but more generally, it can be seen in response to a variety of cellular stresses (2). Viral capsids assemble in the nucleus, package the replicated genome and virions are liberated upon cell lysis (3). In the absence of helper functions, the AAV genome is not productively transcribed or replicated. The single stranded (ss) DNA genome is converted into a double

stranded (ds) form and eventually integrates into the cellular DNA. When human cells are infected with helper free AAV, tandem arrays of the genome are found at a preferred location on chromosome 19. This site specific integration requires the presence of Rep proteins (4). The latent genomes can be reactivated and rescued by an infection with a helper virus (5).

The 4679 nucleotide genome packaged into AAV virions can be of plus or minus polarity. It contains a 145 nt inverted terminal repeat (ITR) of which the first 125 nt can form a T-shaped hairpin structure. The ITR contains all the information needed in cis for replication, packaging and integration of the genome (6). Viral proteins are 493 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 493-502. © l998 Kluwer Academic Publishers. Printed in the Netherlands.

494 encoded by two genes, rep and cap. Multiple proteins are translated from differentially initiated or spliced transcripts. There are 4 Rep (MW 78, 68, 52 and 40) and 3 Cap proteins (VP1, VP2, VP3). The protein composition of AAV virions is 80% VP3. The smaller Rep proteins are involved in viral RNA maturation and packaging. The two larger Rep proteins display site specific endonuclease, ATPase and helicase activities (7) and bind repeated copies of the motif GAGC on double stranded DNA. Binding sites are found: i) on the A stem of the ITR; ii) within the three AAV promoter regions (8) and iii) at several loci the human genome, including one close to the AAV preferential integration site on chromosome 19 (4 , 9). The current view of the role of the large Rep proteins is that they: i) resolve the closed T-shaped end of the double stranded replication intermediates and allow for the formation of monomeric single stranded genomes ; ii) modulate AAV gene expression through transcriptional regulation (10) ; iii) mediate site specific integration, possibly by bridging the viral and cellular DNA binding sites (11).

2.AAV-derived Vectors

2.1. PRINCIPLE AND METHODS AAV vectors only retain the duplicated 145 nt ITR from the viral genome, the rep and cap genes being removed and replaced by the sequences of interest. Vector particles can theoretically be produced in the absence of wild type AAV, if the rep and cap gene products are provided in trans (6). The initial method to achieve this was to transfect cells with the vector and a rep-cap expression cassette on two separate plasmids (12, 13). Human 293 cells, which are highly permissive to both AAV and adenovirus replication and can be efficiently transfected, are of general use for vector production. Following transfection, helper functions are provided by infecting the cells with Adenovirus. Since the rep and cap sequences are not linked to the AAV ITR, only the recombinant construct is replicated and packaged. Recombinant virions, free of wt AAV can be purified from cell lysates and separated from the Adenovirus particle by density centrifugation. This procedure can be optimized to yield recombinant AAV (rAAV) particles per transfected cell, which is within the range of wild type AAV production (13, 14). Still, the current method presents several limitations : i) a typical prepartion is very work intensive, since it involves the transfection of cells, followed by a week-long purification and assay procedure (13); ii) each viral preparation is made from a new transfection, and reproducibility problems arise ii) a low level contamination of the final stock by the helper adenovirus is unavoidable, following physical separation (14); iii) illegitimate recombination, which may occur during the transfection, or under the influence of Rep results into structures where ITR sequences are linked to rep and cap. These chimeric forms can subsequently evolve into replication competent AAV genomes, through additional recombination (15). Several significant improvements have been made to the transient transfection method. Adenovirus infection can now be replaced by the cotransfection of a cloned Adenovirus genome deleted for the packaging sequence and part of the El region (unpublished observation). This allows for the production of rAAV particles in the

495 absence of functional Adenovirus. Further deletions in the Adenovirus genome, eliminating most of the late (L) genes, have also been shown to provide efficient help for rAAV production (X Xiao and RJ Samulski, personal communication). Following this method, adenoviral capsids are eliminated and cleaner prepartions should be obtained. It was also observed that the level of Rep proteins produced after transfection was critical for obtaining high titer rAAV. Surprisingly, low amounts of rep expression result in higher rAAV production, probably because of the negative influence of Rep on cap expression. Helper (rep-cap) plasmids designed to

express low levels of Rep allow for the production of 5-10 fold more rAAV (16). In achieving high titer preparations, the ability to synthetize large amounts of Cap proteins is critical (14). One obvious goal is to obtain stable packaging systems for the production of rAAV. This would not only simplify the preparation method, but also facilitate the

definition of specifications for the production of vectors to be used in pre-clinical and clinical experiments. The development of a universal packaging line is underway.

Clones stably expressing rep and cap as well as a vector construct have been used to produce helper free rAAV, following Adenovirus infection (17). The isolation of « proto-producer » clones in which rAAV production is triggered by Adenovirus infection, requires more work initially, but they result in permanent sources of rAAV that can be mobilized by Ad infection. This approach is likely to become of general use, once its reproducibility will be established. 2.2.FACTORS INFLUENCING EFFICIENCY

AAV-MEDIATED

GENE

TRANSFER

The understanding of the replication mechanism of AAV is only partly relevant to the situation encountered with vectors. Like the parental wild-type (wt) AAV, recombinant genomes entering the cell nucleus must be converted to a dsDNA in order to be transcribed. In wt AAV the Rep proteins either directly facilitate second strand synthesis, or can trigger the (still undefined) cellular functions involved. On the other hand, vectors do not retain the rep gene and the Rep proteins are not detectably associated with the released viral particles. Thus the recombinant genomes uniquely depend on the cell DNA synthesis machinery for second strand synthesis. This implies that the expression of sequences transferred by an AAV vector will be highly dependent on the presence of an appropriate cellular environment. Different situations have been shown to enhance rAAV-mediated gene transfer by allowing for a more efficient second strand synthesis. One is the expression in the target cell of the open reading frame 6 from the E4 region of Adenovirus (18, 19), a protein that can bind p53 (20), and may therefore interfere with cell cycle signals. More generally, drug or radiation induced genotoxic stresses, have an enhancing effect on rAAV-mediated gene transfer in cultured cells, or in vivo (20-24). Although a unifying explanation for these observations is still lacking, the initiation of unscheduled DNA repair synthesis is probably central for rAAV second strand synthesis.

3.AAV vectors for direct gene transfer in vivo:

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A number a features of rAAV make them well suited for direct gene transfer in vivo. Compared to retroviruses or adenoviruses, the rAAV particles are small and very resistant, and may therefore remain in the circulation or in an injected tissue for longer times, and diffuse more efficiently across some the natural barriers within the organism. In addition, rAAV should, in principle, be able to transduce non-dividing cells, which constitute the overwhelming majority of targets encountered in vivo. Limitations include, the size of the transferred sequences and, in the prospect of human clinical trials, the widespread seropositivity for AAV in human populations. Over the past couple of years, the methods for rAAV production, have become efficient enough to yield preparations with titer and quality compatible with in vivo experimentation. A number of in vivo targets have been explored, including central and peripheral nervous system (23, 25-30, 39), repiratory epithelium (31), vascular, cardiac and skeletal muscle (32-36) and liver (24). Here we discuss our experiments with skeletal muscle and liver (37, 38). 3.1.GENE TRANSFER TO THE SKELETAL MUSCLE Our interest was to further document the remarkable efficiency of rAAV for gene transfer into the skeletal muscle and to analyze the structure of the recombinant genome in the transduced muscle fibers. A secondary goal was to start to analyse the contribution of the post-mitotic environment found in the terminally differentiated muscle fiber, to the high-efficiency of AAV-mediated gene transfer. The efficiency of AAV-mediated gene transfer into the skeletal muscle of mice was evaluated by performing a single injection of purified recombinant AAV (rAAV) particles encoding a modified E. coli ß-galactosidase with a nuclear localization signal, under the control of the CMV promoter. The rAAV preparations were titered by dot blot hybridizationand (10 12 physical particles / ml) and by limiting dilution infections and X-gal staining of 293 cells in the absence of presence of adenovirs co-infection, respectively. The stocks were determined to be free of detectable contamination by adenovirus and by replication competent AAV less than 1wt AAV / 10 9 rAAV). Young adult Balb/c mice were injected into the quadriceps muscle with 30 (m1 of rAAV preparationcontaining gal infectious units and animals were sacrificed between 2 and 28 weeks later. Histological sections of the injected muscles, stained for ß-galactosidase activity revealed between 10 and 70% positive fibers on more than 75% of the muscle length. Two weeks after gene transfer, the muscle had a normal histological aspect, whereas on sections taken at four and eight weeks, infiltrates became conspicuous in the areas containing positive fibers. Regenerated fibers with centrally located nuclei were also noted in the infiltrated areas. Remarkably, these fibers still expressed the transgene and may therefore have arisen through the proliferation and fusion of transduced satellite myoblasts containing the recombinant AAV genome. The cellular response was not observed at later time points. In conclusion, this gene transfer procedure is associated with a transient and limited immune response, without any major impact on the long term genetic modification of muscle cells. We then asked whether an enhancement of gene transfer is observed when muscle satellite cells are induced to proliferate as a consequence of muscle injury. The same gene transfer procedure was applied to animals pretreated 48 hours before by an intra-

497 muscular injection of barium chloride (BaC12) which provoks a rapid necrosis of the muscle fibers, followed by tissue regeneration. In animals analysed 2 weeks after gene transfer, over 85% of the muscle fibers had been regenerated and displayed centrally located nuclei. Surprisingly, most of the galactosidase positive fibers were confined to the remaining, non regenerated, areas and accounted for less than 20% of all fibers. At this time point, cellular infiltrates were observed in the regenerated areas, but not in the intact tissue. At late time points, the infiltrates became much less pronounced, and only limited areas of positive and mostly regenerated fibers remained. We concluded that there is no obvious enhancement of gene transfer when the AAV vector is applied to a regenerating muscle containing proliferating cells. On the contrary, the terminally differentiated, post-mitotic, muscle fibers appear to be preferentially permissive to AAV vectors. Another group of mice received a single intra-muscular injection of a rAAV encoding murine erythropoietin (Epo, the hormone regulating erythopoiesis), under the control of the CMV promoter. Each animal was injected with total vector particles, blood samples were collected over time and Epo production was monitored either directly by a radio-immunoassay on plasma, or indirectly by measuring the hematocrit which reflects the number of circulating erythrocytes. The hematocit of

every animal increased during the first four weeks following gene transfer, and reached a plateau value of 80 to 90%. These high hematocrits were observed until the animals were sacrificed after 2 to 4 months. They corresponded to 10 to 20 fold increase in serum Epo levels. The structure of the rAAV genomes was analyzed by Southern blot on high molecular weight DNA prepared at different time points from the injected muscles. Eight weeks after injection, 1 to 3 copies of rAAV per haploid genome were measured. This material was associated with high molecular weight DNA, under the form of concatemers. No proof of integration, like the characterisation of junction between cellular and vector genome, has been obtained. It is possible that the rAAV genome remains extrachromosomal, under the form of either head-to-tail tandem repeats or interlocked circles. The status of the vector DNA was also analyzed at early time points following injection. After one and two days, a strong signal is found corresponding to the input

ssDNA genome. This signal progressively decreases until 2 weeks. Double stranded monomers of the genome are also observed during the first days following gene transfer. Although this could represent an artefactual reassociation of the complementary strands of DNA delivered by the rAAV particles, it may also reflect the high permissivity of the muscle fiber. This ds monomers progressively disappear during the first two weeks, and are chased into the high molecular weight forms. In conclusion, we observe two successive transformation of the recombinant genome upon entry into the muscle fiber nuclei : a conversion into dsDNA monomer, followed by a concatemerisation. 3.2.GENE TRANSFER INTO THE LIVER The steady state liver contains mostly non dividing cells, 90% of which are hepatocytes. These hepatocytes are normally quiescent but they are induced to divide in response to liver injury.

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The quiescent or regenerating liver can be subjected to gene transfer using a variety of vectors by portal infusion. We have examined the potential of rAAV in this system by administrating a preparation of rAAV carrying the human Factor IX cDNA to adult C57B1/6 mice (37). Factor IX is a component of the blood coagulation cascade, normally produced by the liver. Its deficiency results in

Hemophilia B. Beetween 2 and vector particles were infused in the portal vein and the presence of human Factor IX was periodically measured in plasma samples. The human protein was detectable at low levels during the first week and increased to steady state concentrations of 250 to 2000 ng/ml. The secreted Factor IX was active in coagulation assays and the serum levels were dose dependent and persisted for at least 36 weeks (the duration of the experiment). When extrapolated to a clinical situation, such concentrations would be relevant for the treatment of Hemophilia B. The amount of rAAV genomes present in the infused livers was measured by Southern blot to be between 1 and 4 copies per haploid cellular genome. In situ hybridization showed that human Factor IX mRNA could be found in only 1 to 5 % of the hepatocytes, implying that either a few cells contain and express multiple (30 to 50) copies of the rAAV genome, or that most cells contain vector genomes and only a minority expresses them. Considering the number of target cells in the mouse liver and the estimated amount of infused infectious particles it is unlikely that most cells can be transduced. As a comparison, over: adenoviral particles are needed to transduce most cells in the liver . We therefore favor the hypothesis where the gene transfer procedure would deliver the ssDNA genome to 1 to 5 % of the liver cells, and after conversion into the ds form, an amplification step would take place. 4.

Conclusions

AAV vectors have now proven to be very efficient for stable gene delivery into a

number of in vivo targets. At this time, preparation of high quality and high titer rAAV, although dramatically improved over the past couple of years, remain a significant bottleneck. Yet, specifications for clinical grade material are starting to be

defined and phase I clinical trials involving rAAV as vector are now underway. In animal experiments, high gene transfer efficiency is seen in myotubes and neurones, which are post-mitotic cells, and possibly in a sub-population of hepatocytes for which the cell cycle status is unknown. Not all arrested primary cells, however are permissive to rAAV gene transfer. Bone

marrow cells enriched in non cycling CD34+ hematopoietic progenitors will accumulate rAAV DNA, but mostly fail to express the transfered gene (C.Jordan , personal communication). Understanding the determinants of cellular permissivity to AAV will have important implications for the definitions of optimal gene transfer targets in vivo.

499 5. References

1. Berns, K. I. (1996) in Fields Virology, eds. Fields, B., Knipe, D. & Howley, P. (Lippincott-Raven, Philadelphia), Vol. 2, pp. 2173-2220. 2. Schelehofer, J. R., Matthias, E. & Zur Hausen, H. (1986) Virology 152, 110117. 3. Wistuba, A., Kern, A., Weger, S., Grimm, D. & Kleinschmidt, J. (1997) J. Virol 71, 1341-1352. 4. Linden, R. M., Winocour, E. & Berns, K. I. (1996) Proc. Natl. Acad. Sci.

U.S.A. 93, 7966-7972. 5. McLaughlin, S. K., Collis, P., Hermonat, P. L. & Muzyczka, N. (1988) J. Virol. 62, 1963-1973. 6. Muzyczka, N. (1992) Curr. Top. Microbiol. Immunol. 158, 97-129. 7. Im, D. & Muzycska, N. (1990) Cell 61, 447-457. 8. Me Carty, D. M., Pereira, D., Zolotukhin, I., Zhou, X., Ryan, J. H. & Muzyczka, N. (1994) J. Virol. 68, 4988-4997. 9. Wonderling, R. & Owens, R. (1997) J. Virol. 71, 2528-2534. 10.Pereira, D., McCarty, D. & Muzyczka, N. (1997) J. Virol 71, 1079-1088. 11.Weitzman, M. D., Kyostio, S. R. M., Kotin, R. M. & Owens, R. A. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 5808-5812. 12.Samulski, R. J., Chang, L. S. & Shenk, T. (1989) J. Virol. 63, 3822-3828. 13.Snyder, R., Xiao, X. & Samulski, R. J. (1996) in Current Protocols in Human Genetics, ed. Haines, J., pp. 12.1.1-121.1.23. 14. Vincent, K., Piraino, S. & Wadsworth, S. (1997) J. Virol. 71, 1897-1905. 15. Allien, J., Debelak, D., Reynolds, T. & Miller, A. (1997) J. Virol 71, 68166822. 16.Li, J., Samulski, R. J. & Xiao, X. (1997) J Virol 71, 5236-5243. 17.Clark, K., Voulgaropoulou, F., Fraley, D. & Johnson, P. (1995) Hum. Gene Ther. 6, 1329-1341. 18.Ferrari, F., Samulski, T., Shenk, T. & Samulski, R. (1996) J. Virol. 70, 32273234. 19.Fisher, K. J., Gao, G. P., Weitzman, M. D., De Matteo, R., Burda, J. F. & Wilson, J. M. (1996) J. Virol. 70, 520-532. 20.Dobner, T., Horikoshi, N., Rubenwolf, S. & Shenk, T. (1996) Science 272, 1470-1473. 21. Alexander, L, Russell, D. & Miller, A. (1994) J. Virol. 68 N°12, 8282-8287. 22.Russell, D. W., Alexander, I. E. & Miller, A. D. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 5719-23. 23. Alexander, I., Russell, D., Spence, A. & Miller, A. (1996) Hum. Gene Ther. 7 , 841-850.

24.Koeberl, D. D., Alexander, I. E., Halbert, C. L., Russels, D. W. & Dusty Miller, A. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 1426-1431. 25.Kaplitt, M., Leone, P., Samulski, R., Xiao, X., Pfaff, D., O'Malley, K. & During, M. (1994) Nat. Genet. 8. 26.Ali, R. R., Reichel, M. B., Thrasher, A. J., Levinsky, R. J., Kinnon, C., Kanuga, N., Hunt, D. M. & Battacharya, S. S. (1996) Hum. Mol. Genet. 5 , 591-594.

500 27.Lalwani, A. K., Walsh, B. J., Reilly, P. G., Muzyczka, N. & Mhatre, A. N. (1996) Gene Therapy 3. 28.McCown, T. J., Xiao, X., Li, J., Breese, G. R. & Samulski, R. J. (1996) Brain Res. 713, 99-107.

29.Peel, A., Zolotukhin, S., Schrimsher, G. W., Muzyczka, N. & Reier, P. J. (1997) Gene Therapy 4, 16-24. 30.Flannery, J.G., Sergei Zolotukhin, S., Isabel Vaquero, M., LaVail, M.M., Muzyczka, N. & Hauswirth, W. (1997) Proc. Natl. Acad. Sci. USA 94, 69166921. 31.Afione, S. A., Conrad, C. K., Kearns, W. G., Chunduru, S., Adams, R., Reynolds, T. C., Guggino, W. B., Cutting, G. R., Carter, B. J. & Flotte, T. R. (1996) J.Virol 70, 3235-3241. 32.Kaplitt, M. G., Xiao, X., Samulski, R. J., Li, J., Ojamaa, K., Klein, I. L., Makimura, H., Kaplitt, M. J., Strumpf, R. K. & Diethrich, E. B. (1996) Ann Thorac Surg 62, 1669-1676. 33.Xiao, X., Li, J. & Samulski, R. J. (1996) J. Virol. 70, N°11, 8098-8108. 34.Kessler, P. D., Podsakoff, G. M., Chen, X., McQuiston, S. A., Colosi, P. C., Matelis, L. A., Kurtzman, G. J. & Byrne, B. J. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 14082-14087.

35.Fisher, K. J., Jooss, K., Alston, J., Yiping , Y., Haecker, S. E., High, K., Pathak, R., Raper, S. E. & Wilson, J. M. (1997) Nature Medicine 3, 306-312. 36.Herzog, R., Hagstrom, J., Kung, S., Tai, S., Wilson, J., Fisher, K. & High, K. (1997) Proc. Natl. Acad. Sci. USA 94, 5804-5809. 37.Snyder, R., MIiao, C., Patijn, G., Spratt, K., Danos, O., Nagy, D., Gown, A., Winther, B., Meuse, L., Cohen, L., Thompson, A. & Kay, M. (1997) Nat. Genet. 16, 270-276. 38.Snyder, R., Spratt, S., Lagarde, C., Bohl, D., Kaspar, B., Sloan, B., Cohen, L. & Danos, O. (1997) Hum. Gene Ther. 8, 1891-1900. 39.Jomary, C., Vincent, K.A., Grist, J., Neal, M.J. & Jones, S.E. (1997) Hum. Gene Ther. 4, 683-690.

501 Discussion

Lupker:

What will happen if someone treated with your AAV factors gets a

superinfection by Adenovirus? Danos:

To get a re-mobilisation of the vector, you need to have products of the rev gene. So if the AV preparation is free of rev genes or wild type AV, then nothing will happen. If you have a cell with an integrated recombinant pro-virus and you infect this with an AV,

you do not rescue the recombinant structure. You need the AV functions as well.

Zhang:

It is puzzling that in your slides you showed a gradual decrease in the DNA but at the same time you showed that the virus was stable. Can you comment on this?

Danos:

There is a decrease in the overall DNA signal but it does not disappear. Only a minority of the genomes are being stabilised and converted into whatever form is needed for them to be expressed. So probably only 1% of the input ends up being expressed. At the beginning you see a lot of the input DNA, and it is difficult to see anything on the blots.

Zhang:

What happens if you do a Northern Blot - do you see a stable messenger RNA?

Danos:

We do not know when the expression really starts. One can see the expression going progressively up over time. The number of cells and the intensity of staining is much less. So there is a progressive recruitment of the single stranded DNA into an active

form. The problem we have is to define this active form. One obligative pathway for this active form is double stranded conversion. Ostrove:

A question regarding the

gal experiment. You showed that the

addition of adenovirus increased your transduction efficiency by approximately 2 logs. Can you comment on that observation, and is it true for in vivo as well as in vitro, or are all the data in vitro?

502

Danos:

Most of the data are for in vitro. I discussed adenovirus helper function in terms of production of AV particles. Adenovirus can also help gene expression from the recombinant AV. This has been mapped now in the E4 region of the virus. There is one particle

open-reading frame, a protein, that is central for that. There is debate over transduction with AV because of possible contamination with adenovirus. There is now no problem as you can prepare AV without any adenovirus by using mini-plasmids. Using these preparations you do see transductions in vivo. If you add adenovirus to liver you do see an increase and there is a 10 fold increase in Factor IX production transiently, which then drops totally. This is due to an immune reaction against the adenovirus which eliminates the transduced hepatocytes. Berthold:

A question on the safety aspects:

you described a transient inflammatory response - is this related to non-mouse sequences, or to the virus infection? Also, how do you make sure that other components of your production system are not replicating as well?

Danos:

You do get an immune response against the particles but this is

only seen when you re-administer. It is a cellular immune response seen after 4 weeks. It is not an acute inflammation. We have looked for wild type AV, and wild type adenovirus, or El deleted adenovirus. We do not detect any gross contamination, or anything that can create a significant response in this animal system. If we use this in the clinic we will have to be much more thorough with the characterisation of the preparation.

NOVEL RETROVIRAL PACKAGING CELL LINES: IMPROVED VECTOR PRODUCTION FOR EFFICIENT HEMATOPOIETIC PROGENITOR CELL TRANSDUCTION

Sean Forestall, Richard Rigg, Jonathan Dando, Jingyi Chen, Barbara Joyce, Robert Tushinski, Chris Reading, and Ernst Böhnlein SyStemix Inc., 3155 Porter Drive, Palo Alto, CA 94304, USA

1. Introduction Safe and efficient means to transfer therapeutic genes stably must be developed for successful hematopoietic cell-based gene therapy. Currently, retroviral vectors offer the only practical way to integrate genes stably, although retroviral gene transfer to primary hematopoietic stem and progenitor cells has been low [1-2]. While amphotropic vectors have been used in the majority of studies, gibbon ape leukemia virus (GaLV) enveloped vector has been reported to transduce hematopoietic progenitor cells and peripheral blood lymphocytes (PBL) more efficiently than amphotropic vector [3-4], suggesting that targeting different retroviral receptors may improve gene transfer efficiencies into these lineages. High efficiency gene transfer into human hematopoietic progenitor cells has been achieved with retroviral vector generated from the novel ProPak packaging cell lines. The ProPak cell lines have complementary tropisms producing either murine leukemia virus (MLV) xenotropic (ProPak-X cells) or amphotropic envelope (ProPak-A), and were designed to minimize the risk of replication competent retrovirus generation [56]. Vector supernatants from ProPak or existing packaging cell lines producing different pseudotyped particles (amphotropic MLV, xenotropic MLV, or gibbon ape leukemia virus) were compared for the ability to transduce clinically relevant human hematopoietic cells. All vector types transduced primary human CD34-positive cells regardless of tropism. However, consistently higher transduction of target cells was achieved with ProPak-derived amphotropic vector than with PA317-packaged amphotropic vector. Improved conditions for the production of vector supernatants by the coculture of the complementary ProPak packaging cell lines in a packed-bed bioreactor are presented that result in 100% transduction efficiencies of human hematopoietic progenitor cells.

2. Materials and Methods Packaging and producer cell lines(proPak-A, ProPak-X, PA317, PG13) were grown in Dulbecco's modified Eagle’s medium (high glucose DMEM: JRH Biosciences, 503 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 503-508. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

504

Lenexa, KS, USA) supplemented with fetal bovine serum (FBS: HyClone Laboratories Inc., Logan, UT, USA). Vector supernatants were produced in a 500 ml packed-bed bioreactor with 10 g of Fibra Cell discs (New Brunswick Scientific, Edison, NJ, USA). All supernatants used for gene transfer experiments were free of RCR as determined by the extended S+L- assay on PG4 cells (CRL 2032; ATCC). The vector used in all

experiments (LMiLy: LTR-RevM10-IRES-Lyt2) is described elsewhere [7]. PEL were isolated from peripheral blood mononuclear cells and depleted of

CD8-positive cells. The CD34-positive (CD34+) fraction was isolated from mobilized peripheral blood (MPB) or adult bone marrow (ABM) using a positive selection device. Further purification to obtain the fraction carrying the Thy1 antigen (CD34+/Thy 1+) was achieved by high-speed flow cytometric sorting. Hematopoietic stem and progenitor cells were cultured in a 1:1 mixture of IMDM and RPMI media (JRH Biosciences) containing 10% FBS, interleukin-3 (IL-3) and IL-6 (20 ng/ml each; Sandoz Pharma, Basel, Switzerland), and stem cell factor (SCF; 100 ng/ml) or leukemia inhibitory factor (LIF; Sandoz) (100 ng/ml). Cell lines were inoculated with vector once at unit gravity for 3 h at 37°C. Primary cells were exposed to vector supernatants under centrifugation, termed spinoculation, at 2550 g for 3 or 4 h. Polybrene (8 mg/ml; Sigma) or protamine sulfate (4 mg/ml; Sigma) was added for cell lines or primary cells, respectively.

Transduction efficiencies achieved with Lyt2-encoding vector supernatants were quantitated as the proportion of target cells that expressed the Lyt2 antigen 2 or 3 days

following inoculation. Integration of the RevM10 gene into clonogenic progeny (CFUC) was determined using aDNA PCR assay [8]. At least 92 colonies were analyzed for

each condition to allow precision in experimental comparisons. Colonies were scored positive if both RevM10 and the endogenous beta-globin sequences were detected. 3.

Results

ProPak Packaging Cells

The derivation of the ProPak packaging cell lines has been described elsewhere [5-6]. Briefly, separate minimal env and gag-pol expression plasmids with heterologous

promoters were stably transfected into an MLV-free cell line (human 293). These steps were taken to minimize the chances of recombination which could lead to RCR. Using a vector known to reproducibly give rise to RCR in PA317 producer cells, ProPak cells

were shown to be RCR-free using conditions favorable to RCR formation (ping-pong amplification of the vector by coculture of both the amphotropic and xenotropic producer cells). Further, vector produced by either amphotropic or xenotropic ProPak producer

cells is resistant to inactivation by human serum making it suitable for in-vivo gene transfer applications. Improved Retroviral Vector Supernatant Production in a Bioreactor We have previously shown that a packed-bed bioreactor is applicable to the production of retro viral vector using PA317-based producer cells [9]. We have since

505

determined that the packed-bed bioreactor is suitable for vector production with all 3T3and 293-based producer cell lines tested. In comparison with T-flask cultures, bioreactor supernatants have yielded 2- to 20-fold higher transduction of cell lines (data not shown). Similar to results on cell lines, bioreactor supernatants have yielded approximately 3-fold higher transduction of CD34+ cells than T-flask supernatants as shown in Figure 1.

Primary Cell Transduction with Different Vector Tropisms

Using the packed-bed bioreactor, vector supernatants from PA317, PG13, ProPak-A and ProPak-X producer cell clones, all carrying the LMiLy vector, were produced. These vector supernatants with different tropisms were compared for their ability to transduce CD4+ PBL and primary human hematopoietic progenitor and stem cells (defined as CD34+ or the more highly purified CD34+ Thy1 + expressing cells). Primary cells were exposed to vector once to directly compare transduction efficiencies which are presented as Lyt2 surface marker expression relative to the expression achieved with PA317-packaged vector (Table 1). Regardless of tropism, all vector types successfully transduced CD34-positive cells isolated from ABM or MPB, or CD4+ PBL (Table 1). This implies that functional receptors for all three vector types are expressed on these cells. While no single vector tropism appeared to mediate significantly higher levels of transduction than any other, the highest transduction efficiencies were achieved with ProPak-A or ProPak-X supernatants (Table 1). Most significantly, amphotropic vector supernatants from the human ProPak-A cell lines consistently transduced a higher proportion of target cells than amphotropic vector prepared from PA317-based producer cells.

506 Table 1. Transduction of primary human hematopoietic cells with vector supernatants of different tropisms from LMiLy-based producer cells cultured in the packed-bed

bioreactor. Transduction efficiencies were measured as the proportion of cells expressing Lyt2, and values have been normalized to that achieved with PA317-based supernatants. Duplicate samples from 4 different tissues were inoculated as indicated.

Vector Production in Producer Cell Cocultures Next we attempted to increase transduction efficiency by producing vector supernatants by coculture of the complementary ProPak-X.LMiLy and ProPak-A.LMiLy producer cells in the packed-bed bioreactor. This technique, known as ping-pong amplification, results in higher titers as a result of increased vector copy number [10-12]. The problem that has arisen in the past with coculture production is the generation of RCR [11]. We have already shown that no RCR arises during extended coculture of ProPak-A and ProPak-X cells carrying the BC140revM10 vector [6], an event that is even less likely with the LMiLy vector which contains the shorter psi packaging sequence, and therefore lacks sequences overlapping with the gag/pol structural genes. Using the coculture supernatant, we investigated different cytokine combinations and multiple rounds of inoculation in an effort to optimize gene transfer. CD34+ cells isolated from MPB were spinoculated with PP-A/X.LMiLy vector once, twice on the same day, or once each day on two consecutive days in the presence of IL-3, IL-6, and either LIF or SCF. The results in Table 2 show that while gene transfer was higher in cultures treated with SCF after a single inoculation, gene transfer into CFU-C was 100% for cultures inoculated twice on consecutive days in either LIF or SCF. Furthermore, in both cytokine cocktails the Lyt2 transgene was expressed in up to 40% of the total cell population, and 21% of the total cell population co-expressed the Lyt2 transgene and CD34 antigen as shown in Figure 2. Not surprisingly, the gene transfer efficiency measured by DNA PCR is consistently higher than that measured by transgene expression. This is likely due to the differences in the assays, and the inability of quiescent hematopoietic cells to express the transgene until activated. We are, however, confident that the gene marking is biologically relevant as parallel studies from our group show that transduction of hematopoietic progenitor cells leads to sustained marking and expression in lymphoid and myeloid progeny [8].

507

4. Discussion We have constructed safe, new retroviral vector packaging cell lines which stably produce particles that target distinct receptors on human cells, are resistant to inactivation by human serum, and provide efficient transduction for gene therapy applications. In our experiments, we observed consistently higher transduction efficiencies with ProPak-A-derived particles compared with amphotropic vector packaged in PA317 cells. There are at least two possible explanations for this difference. Other reports have shown that murine cells secrete proteoglycans that inhibit transduction [13], and the possibility that ProPak cells secrete less or no such inhibitor(s) is currently being examined. Our own studies suggest that PA317 vector supernatants contain a viral

508 inhibitor which is envelope-specific [9]. It may be that the ProPak cells, which were

constructed with a split genome, produce a more favorable ratio of Env to Gag components. Intriguingly, in comparison to PA317 supernatants, ProPak-A supernatants contain less Env protein and similar levels of Gag protein (unpublished observations). Using ProPak-A/X vector produced in a packed-bed bioreactor, we achieved 100% marking of CFU-C derived from CD34+ cells purified from MPB, and demonstrated expression of the Lyt2 transgene in 40% of the CD34+ cells 2 days post inoculation. Previous applications of the ping-pong amplification technique were hampered by the generation of RCR, and employed ecotropic producer cell partners, generating particles which cannot transduce human cells. However, the demonstrated safety of the complementary ProPak cell lines allows for RCR-free ping-pong amplification. 5. References

1. Miller, AD. Progress towards human gene therapy. Blood 1990; 76: 271-278. 2. Xu, LC. et al. Poor transduction efficiency of human hematopoietic progenitor cells

by a high-titer retrovirus producer cell clone. J. Virol. 1994; 68: 7634-7636. 3. von Kalle, C. et al. Increased gene transfer into human hematopoietic progenitor cells by extended in vitro exposure to a pseudotyped retroviral vector. Blood 1994; 84: 2890-2897. 4. Bunnell, BA. et al. High-efficiency retroviral-mediated gene transfer into human and nonhuman primate peripheral blood lymphocytes. Proc. Natl. Acad. Sci. USA 1995;

92: 7739-7743. 5. Rigg, RJ. et al. A novel human amphotropic packaging cell line: high titer, complement resistance, and improved safety. Virol. 1996; 218: 290-295. 6. Forestell, SP. et al. Novel retroviral packaging cell lines: complementary tropisms and improved vector production for efficient gene transfer. Gene Ther. 1997; 4: 600. 7. Rigg, RJ. et al. Detection of intracellular HIV-1 Rev protein by flow cytometry. J. Immunol. Meth. 1995; 188: 187-195. 8. Plavec, I. et al. Sustained retroviral gene marking and expression in lymphoid and myeloid cells derived from transduced hematopoietic progenitor cells. Gene Ther. 1996; 3: 717-724.

9. Forestell, SP. et al. Retroviral end-point titer is not predictive of gene transfer efficiency: implications for vector production. Gene Ther. 1995; 2: 723-730. 10. Bestwick, RK., Kozak, SL., Kabat, D. Overcoming interference to retroviral superinfection results in amplified expression and transmission of cloned genes. PNAS USA 1988; 85: 5404-5408. 11. Bodine, DM. et al. Development of a high-liter retrovirus producer cell line capable of gene transfer into rhesus monkey hematopoietic slem cells. Proc. Natl. Acad. Sci. USA 1990; 87: 3738-3742. 12. Cosset, FL. et al. Use of helper cells wilh iwo hosl ranges to generate high-titer retroviral vectors. Virol. 1993; 193: 385-395. 13. Le Doux, JM., Morgan, JR., Snow, RG., Yarmush, ML. Proteoglycans secreted by packaging cell lines inhibit retrovirus infection. J. Virol. 1996; 70: 6468-6473.

SCALE-UP AND OPTIMISATION ISSUES IN GENE THERAPY VIRAL VECTOR PRODUCTION

J.E. BOYD. L. BORLAND. C.E. FISHER

Q-One Biotech Ltd West of Scotland Science Park, Todd Campus, Glasgow G20 0XA

1.

Scale-Up

To produce viral vectors for gene therapy, it is essential, as in conventional biopharmaceutical GMP systems to maximise and maintain production levels. The growth and production characteristics of cells and vector should be investigated in a range of scaleup scenarios e.g., roller bottles, microcarriers and fermenters. However, these systems vary in their potential for manufacturing scale-up. Roller bottles

I can

be used very effectively to yield high cell numbers when bulking of a cell line is required for cell banking or virus/viral vector propagation is required. This system is disposable so no cleaning validation is required. The average cell yield from a roller bottle is viable cells. A large scale production of 100 roller bottles may require up to 15 litres of medium and requires a large number of manipulations upon set up and harvest. Variable cell growth is observed and growth curve evaluation of each cell line should be carried out. There are no dissolved oxygen or pH controls in place in this system. While most surfaces used in cell culture possess a specific density of small charged

molecules to promote attachment and growth of cells, Cytodex 3 microcarriers have a surface layer of denatured collagen covalently bound to a matrix of cross-linked dextran spheres. The microcarriers are prepared by swelling and sterilised by autoclaving and they are disposable. The surface area of cytodex 3 is of microcarriers. It can therefore be calculated that 18.5g of these microcarriers would be equal to 100 roller bottles in 6.2 litres of medium. Technology may be used for production of virus / viral vector which is directly scalable but has no dissolved oxygen or pH control. However, microcarriers may also be considered as a matrix for cellular growth and virus production in the New Brunswick Celligen System using a double screen cell lift impeller. The Celligen system is designed for growth of anchorage dependent ( microcarriers and fibrous polyester disks ) and suspension cells in working volumes of 1.251 to 51. The fibrous polyester disks are disposable with 70g of disks being equivalent to 100 roller bottles but in a volume of 1.41 medium. This system has the advantages of dissolved oxygen and pH control with medium circulation and feeding for optimal growth conditions 509

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 509-513. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

510

in a fully controllable temperature bench top unit. It requires few manipulations once set up and the system is directly scalable. One important factor to consider is the cleaning validation that would be required for use of this system. In conclusion it can be noted that a smaller set up volume is required using the microcarrier and fibrous disk systems promoting a more efficient use of medium, particularly important

if expensive serum-free or selection medium is required. The Celligen system allows easy monitoring of medium exchange, whereas in the roller bottle system medium change is labour intensive and carries the potential risk of contamination. The microcarrier and Celligen systems can readily alter culture conditions e.g., from serum to serum-free medium. 2.

Recovery of Viable Cells After Freezing In Liquid Nitrogen

Cryopreservation is the storage of cells at very low temperatures such that any metabolic / enzymic activity is virtually zero. Genetically manipulated cell lines used in gene therapy

often have altered freezing and recovery properties from that of their parent cell line. In order to investigate freezing and recovery parameters a range of cell lines namely: 293; vero; HeLa; NIH 3T3, 3T3 (retroviral vector producing) derivative cell line and PALidSN cells were cultured in DMEM, 10% FCS/NBCS. These cells were harvested and then frozen down in vapour phase liquid nitrogen at various cell densities using the freezing down medium formulation of 95% complete medium, 5% DMSO.

These studies showed no distinct pattern of recovery. In some cases freezing at

yielded poor results (38% recovery for the 3T3 derivative) whereas for some cell lines , 293, the percentage recovery was consistently good. Freezing at a higher cell density viable cells/4.5ml freezing down medium yielded results ranging

511

from 56% to 111%. Freezing and recovery studies are therefore vital before laying down a Master or Working Cell Bank (MCB/WCB). 3.

Growth Curve Parameters

Mammalian cells can be characterised in culture by four growth phases; Lag, (resting) Log, (exponential growth) Plateau and Decline. The cell lines 293 and Vero were cultured in order to investigate growth curve parameters. The cells were grown in DMEM [4,500mg/l glucose with sodium pyruvate] containing 10% FCS, and growth curves were generated from roller bottles, tissue culture flasks culture systems and thirdly a Cytodex 3 microcarrier system (using spinner vessels containing cultures of 293 cells on 574mg of Cytodex 3 microcarriers).

Generations of exponential growth, doubling time and viable cell yield per can be calculated for each system studied. Analysis of supernatant generated during the culture process for glucose content will give some indication of when the cultures should be fed. All this information is required to determine accurately the requirements for each individual cell line including tissue system to be used, average cell yield and feeding and harvesting patterns.

4.

Retrovirus Production

Retrovirus production schedules have been varied to maximise vector yield but it can be

demonstrated that if long term cultures are used the vector stability, rather than the production rate may be limiting. PALidSN is a cell line which expresses the iduronidase (IDUA) gene in a retroviral vector which contains a neo marker enabling positive selection of cells containing the neo gene to occur using the G418 geneticin selection system. PALidSN cells cultured in DMEM [4,500mg/l glucose], 10% FCS were harvested and roller bottles were initiated and the supernatant harvested either;

512

daily, after four days incubation at then daily or after 7 days incubation and then daily. The PALidSN roller bottle supernatant were titrated on NIH 3T3 cells with

Geneticin selection.

Roller bottle 2 displayed low viral tires for the first 3 days with viral production increasing on day 4. Roller bottle 1 was incubated for four days and then harvested daily. The viral titre on day 4 was the same as that for roller 2 but the highest titre obtained, harvested on

day 5, was double that of roller 2. Leaving the cell culture for 7 days prior to harvest saw the titre drop proving that this viral vector is not stable at for prolonged periods of time and therefore, should be harvested daily after the cells have grown to confluency.

5.

Retrovirus Stability

Stability of the vector and Amphotropic Murine Leukaemia Virus (A-MLV) supernatant were investigated at various temperatures over 24 hours and long term storage over a number of days at Over this time, samples were removed for titration on NIH 3T3 and PG4 respectively. No significant change in viral titre was observed after eight hours storage at each temperature for either virus or vector. However, after 24 hours, a log decrease in titre was observed for vector stored at and A-MLV stored

at and A-MLVincubated at for 24 hours retained only 0.4% of the initial titre, a reduction in units. At vector titre displayed greatest stability, decreasing only by units over 24 hours. Both vector and A-MLV were completely

513

inactivated by storage at 37°C for 3 days. However, vector supernatant was more stable than A-MLV over long term storage at 4°C A-MLV was inactivated after 37 days whereas vector supernatant titre decreased by after 37 days at this temperature. 6.

Downstream Processing

Downstream processing capacity is an important factor particularly for adenoviruses which require separation from inactive particles. Current practices for adenovirus vector purification involve at least two rounds of density gradient centrifugation. This technique separates cellular debris and defective particles from infectious adenovirus in the first round and further separates defective from infective in subsequent rounds of centrifugation. However this technology is not readily scalable and losses may be observed depending on skill and accuracy when removing the bands. De-salting and further purification is carried out using chromatography techniques. Investigation into alternative downstream processes including filtration and chromatography techniques will be carried out in a view to optimising this and other processes.

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COMPARISON OF MANUFACTURING TECHNIQUES FOR ADENOVIRUS PRODUCTION

Jeffrey M. Ostrove* , Paddy Iyer, Jon Marshall and Dominick Vacante MAGENTA Corp.; 9900 Blackwell Road, Rockville MD 20850 and MAGENTA Services, Ltd; Innovation Park, Hillfoots Road, Stirling FK9 4NF Scotland. Keywords: 293 cells, adenovirus, bioreactor, serum-free medium, microcarriers 1. Abstract We have compared three different production methods which may be suitable for the large-scale production of adenovirus vectors for human clinical trials. The procedures compared 293 cells adapted to suspension growth in serum-free medium in a stirred tank bioreactor, 293 cells on microcarriers in serum containing medium in a stirred tank bioreactor, and standard tissue culture plasticware. With a given virus, yields varied between 2000 and 12,000 infectious units (iu)/cell. The stirred tank bioreactor routinely produced between 4000 and 7000 iu/cell when 293 cells were grown on microcarriers. 293 cells adapted to suspension growth in serum free medium in the same stirred tank bioreactor yielded between 2000 and 7000 iu/cell. Yields obtained from standard tissue culture plasticware were up to 12,000 iu/cell. Cell culture conditions were monitored for glucose consumption, oxygen utilization, lactate production, and ammonia accumulation. Oxygen utilization rate, glucose consumption and lactate accumulation correlated well with the cell growth parameters. Ammonia production does not appear to be significant. Based on virus yields, ease of operation and linear scalability, large scale adenovirus production seems feasible using 293 cells (adapted to suspension/serum free medium or on microcarriers in serum containing medium) in a stirred tank bioreactor. 2. Introduction Adenovirus vectors have various applications in the field of gene therapy and as viral oncolytics (1). Methods of adenovirus production for phase I clinical trials have relied on standard tissue culture plasticware for the most part. Apart from being labor intensive, these methods have limited potential for scale-up. We have compared three different production methods for the generation of

adenovirus vectors. Adenovirus production in a stirred tank bioreactor using both anchorage dependent 293 cells and 293 cells adapted to growth in suspension using a proprietary serum free medium were compared to cultures grown on tissue-culture plasticware. 293 cells in bioreactor cultures were infected at a multiplicity of infection (MOI) of 5-10 when the cell density reached cells/ml. At 48 to 72 hours post-inoculation, viral infected cultures were harvested and virus quantitation was performed using standard 50% tissue culture infectious dose * Corresponding author. Tel: (301) 738-3936; FAX: (301) 738-1036; e-mail: [email protected]

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O. - W. Merten el al. (eds.), New Developments and New Applications in Animal Cell Technology, 515-521. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

516 assay (2). Glucose consumption, lactate accumulation and ammonia production from the cultures were monitored using a Kodak metabolite analyzer. 3. Methods

HEK 293 cells obtained from ATCC and grown in fetal bovine serum containing medium (DMEM, 10%FBS) in tissue culture flasks were used to seed microcarrier cultures. 293 cells adapted to growth in suspension using serum-free medium in 100 ml shaker flasks were used to seed suspension bioreactor cultures in serum-free medium. A benchtop stirred tank bioreactor (Artisan Inc., Waltham, MA) with a 4L working volume and height/diameter ratio of 2:1 was used. Oxygen, air and was supplied by headspace aeration and agitation provided by a marine impeller. In the bioreactor, the temperature was controlled at Dissolved Oxygen (D.O.) as a percent of air saturation was controlled at 50% and the was controlled using or 7.5% sodium bicarbonate at 7.3. Total cell densities for microcarrier cultures were determined using nuclei staining method (3), while viable cell densities for suspension cultures were determined by the Trypan Blue dye-exclusion method. Cell densities were determined on days 1, 3, and 6 postseeding and glucose consumption, lactate and ammonia production was monitored using a Kodak metabolite analyzer. When the cell densities reached c/ml, a one volume fed-batch exchange was performed. After the fed-batch exchange, an adenovirus with Ad-5 backbone and E1 A region deleted was introduced into the culture at a MOI of 5-10. After infection, conditions were maintained as above for cell growth. 48 and 72 hours post virus infection, samples were

collected for virus titer assays and glucose, lactate and ammonia analysis. Virus quantitation was

performed using the

assay on the clarified infected cell lysate.

For experiments using plasticware, HEK 293 cells grown in DMEM with 10%FBS in tissue culture flasks were used to seed tissue culture plasticware Day 1 post seeding, an adenovirus with Ad-5 backbone and E1 A region deleted was introduced into the culture at a MOI of 5-10. Samples were collected 48 hours post infection and virus quantitation was performed. 4. Results and Discussion:

HEK 293 cells in serum containing medium were grown in 2L and 4L microcarrier bioreactor cultures. The cells seeded at c/ml typically by day 6 or 7 of the culture. During this time, increased glucose consumption and lactate production was noticed (Figure 1). No trend toward increased ammonia production in the same time period was detected. This seems to indicate that glutamine at levels present in the medium may not be significant as an energy source. A fed-batch one volume exchange on day 6 or 7 of the culture led to a rebound in glucose levels and a drop off in lactate levels (Figure 1). These conditions were considered ideal for the infection phase and yielded between 4000 and 7000 infectious units per cell (Table 1). HEK 293 cells were adapted to suspension growth using a proprietary formulation of serum free medium. Once adapted, these cells have been maintained for more than sixty days and passaged

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518 nine times in 100 ml shaker cultures with consistent cell growth parameters and cell viabilities (Figure 2). 293 cells adapted to suspension growth were used to seed 2L bioreactor suspension cultures. The cells seeded at

reached

by day 6 or 7 of the culture. As in the

serum containing microcarrier cultures, increased glucose consumption and lactate production was noted. Adenovirus added after a one volume fed-batch exchange led to virus yields between 2000 and 7000 infectious units per cell (Table 2). Standard tissue culture plastic ware Nunc trays) provided the highest adenovirus yields. Virus yields from such an optimized system were

upto 12,000 infectious units per cell (based on

assay).

519 5. Conclusion

Standard tissue culture plasticware provided the highest adenovirus yields. However, these methods are labor intensive and scale limited. We have shown that with a stirred tank bioreactor and anchorage dependent HEK. 293 cells, it is possible to produce adenovirus yields that is linearly scaleable. Further, we have successfully adapted 293 cells to grow in suspension culture in serumfree medium in a stirred tank bioreactor. Virus yields from suspension 293 cells in serum-free medium, show promise for large scale adenovirus production given the potential ease of scale-up operation. This also will provide a less complex feedstream for downstream processing. Efforts are ongoing to optimize virus yields for such a system. 6. Acknowledgements

The authors would like to thank the following for technical support: Umme Habiba and Marcia Meseck. 7.

References

1. Crystal, R. Science 270: 404-409, 1995. 2. Schmidt, N. and Emmons, R. in Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections, 18-21, 1989. 3. Nahapetian, A., Thomas, J. and Thilly, W. J. Cell Sci. 81: 65-103, 1986.

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Discussion

Aunins:

If you take your suspension adapted serum-free cells and put them back into a T flask, do you get back your 12,000 infectious units?

Ostrove:

We have not tried this, but it is worth doing.

Southwick:

About the growth of your 293 cells in shaker flasks; you say that you keep them growing for 60 days - do you maintain a single cell suspension during that time, or do you find the cells differ in the way they grow over that period?

Ostrove:

We can maintain fairly good single cell suspensions. 293 cells historically have clumping problems but we have worked out conditions now, in both the shaker flask and bioreactor, to maintain single cell suspension.

Guillaume:

With regard to different productivities with different adenoviruses, do you have any comments on the construct of the virus?

Ostrove:

When we do multiple manufacturing runs of the same virus our

yields are very comparable, within a factor of two. When you look at the 6 different viruses we used, we consistently find that in many cases certain viruses which express certain transgenes have a lower productivity within the cell. Whether this is due to the transgene expression itself or the way the virus was constructed, is unknown. When manufacturing cell lines expressing retroviruses, we recommend that anybody making an adenovirus infectious virus through transfection procedures should pick multiple plaques and try to obtain a virus that has a higher yield. We do see differences due to the recombination events. Singhvi:

You maintained cells for 60 days and then used them in a production run. Did you look at the population doubling levels to

see if it had any impact on your productivity? Were you able to do production runs at different PDL levels without running into

problems?

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Ostrove:

We were concerned about population doublings. The rumour is that as the passage of 293 cells increases, the yield decreases and they even lose the ability to plaque. In our case we start with a Master Cell Bank certified at a passage number of 31. Even after 60 days, the passage level in suspension is relatively low and we did not see an effect on yield. We freeze these cells and bring them back up into suspension to see how far we can take them and whether there is a point that yield does decrease.

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SAFETY CONSIDERATIONS IN THE DEVELOPMENT OF NEW RETROVIRAL AND ADENOVIRAL VECTORS FOR GENE THERAPY

D.MORGAN1, I. FORGIE1, J.OSTROVE2 and M.H. WISHER1 MA BioServices 1 Innovation Park, Stirling, Scotland, FK9 4NF 2 9900 Blackwell Road, Rockville, MD 20850, USA

1. Introduction

As the development of new gene therapy vectors progresses and the number of disorders which may be treated grows, it is crucial that safety is addressed from the development

and preclinical stages, through the manufacturing processes and into clinical trials. This paper will deal with adenoviral and retroviral vectors as they have comprised the

majority of approved clinical trials to date. 2. Historical Perspective

Since regulatory aspects of gene therapy are well-documented [1], it may be questioned whether or not safety issues have in fact been resolved. To illustrate that safety is still an

issue, Table 1 summarises data accumulated over recent years of testing cell banks and their products from research laboratories and production facilities for the presence of

mycoplasma.

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 523-529. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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This shows that a considerable number of cell banks sourced from research laboratories are contaminated with mycoplasmas. This is an important observation because many of the cell lines used in gene therapy have been derived from academic research groups. The lower level of contamination observed in the products of these cell banks may be due to purification processes which eliminate mycoplasmas. The cell banks derived from production facilities are also contaminated with mycoplasma at a significant level. 3. Potential Hazards 3.1 DURING DEVELOPMENT Table 1 illustrated the hazard of mycoplasma contamination arising from cell banks. Other contaminants which may arise from cell banks are bacteria, fungi, viruses, retroviruses and prions. All of these may also arise from media components or operators and are all cause for concern both during development and at later stages.

3.2 DURING PRODUCTION

As cell growth is scaled up to production levels, so the number of hazards increases. The contaminants which are a hazard in development continue to be a risk. Using uncontrolled and/or unvalidated manufacturing procedures may lead to further problems of contamination. Replication competent viruses may be generated at this stage when the producer cell line is expanded for the production of the vector. Problems may also arise if the vector is unstable. A less effective product may result if degradation of the vector takes place. 3.3 DURING CLINICAL USE

Stability of the product continues to be a problem at this stage. Storage conditions should be validated to ensure stability and the buffer formula should be optimised. In addition, the vector may not target the appropriate tissue or it may not be expressed correctly. Incorrect expression may arise from expression in the wrong tissue or lead to the wrong product in the correct tissue. This may be a particular problem with retroviral vectors since retroviruses integrate into the host cell genome in a random fashion. This has the potential to lead to activation of oncogenic genes or inactivation of tumour suppressor genes. Another problem which may occur during clinical use is inappropriate or unexpected immune response at the delivery site. This may lead to a loss of efficacy of the vector. Mobilisation of the vector may occur if target cells are infected with a replication competent virus. Recombination may occur leading to the formation of a replication competent virus carrying the gene from the replication incompetent virus.

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4. Assessing the Risks There are a number of factors to be considered when an assessment of the risk is being

made and decisions required to minimise the risk. Some of these are fairly easily defined such as the phase of development, the patient population and the clinical prognosis of the patients to be treated. For example, the level of safety testing required may be different when a Phase 1 trial is being planned on terminally ill patients compared to that required when a late-phase trial is being planned with patients who are not terminally ill. There are other factors which may also be considered when the risk is being assessed

which are rather less-well defined. For example, the perception of liability may be very different for an academic group when compared to that perceived by a pharmaceutical

company. 4.1. QUESTIONS TO BE ASKED There are a number of questions which should be asked in order to minimise the risks.

How targeted is the gene? Will the gene enter non-target sequences and if so, what will be the consequences?

What are the likely toxic effects of the vector? How long will the gene persist in the cell? How long is the gene likely to be expressed ie. will multiple administration be required?

It is possible that the answers to all these questions may not be available so it is imperative that steps are taken to minimise the risks.

4.2. MINIMISING THE RISKS When viral vectors are being produced, the risk of microbial and viral contamination is

minimised by preparing viral vectors using Good Manufacturing Practice (GMP).

This starts by banking cells under GMP which includes a thorough screening package to test cells and all raw materials for contaminants. Vectors are then produced using a controlled production system with appropriate tests performed on process intermediates and final products. It is important to note that most viral vector production does not include any significant downstream purification, so it is important to detect possible contaminants upstream. Another stage where risk may be minimised is during development when the packaging

cell line is being selected. This is described below.

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5. Replication-Competent Retrovirus (RCR) Testing The current US Food and Drug Administration (FDA) recommendations for the detection of RCR suggest co-cultivation with Mus Dunni Cells testing 1% or cells, whichever is less and 5% of the supernatant at the Master Cell Bank stage. At the Manufacturer’s Working Cell Bank stage, it is recommended to test either supernatant or cells.

6. Replication-Competent Adenovirus (RCA) Testing The rate of detection of RCAs is significantly higher than that of RCRs as is shown in Table 3. It should be noted that these results have been obtained using 293 cells and the development of new packaging cell lines may reduce the number of RCAs.

527

The titre of adenovirus tested is generally dependent on the dosage planned for clinical treatment and as Table 3 shows, the rate of RCAs is in excess of 1 in 3 samples tested when the dosage is greater than 7. Development of Safer Vectors

Factors to be considered during design and development of any vector include ensuring the vector will be well-targeted, that the gene expression will be well-controlled and that the formula ensures a low immune response. The use of purification steps and optimisation of the formulation are also required to ensure a stable vector. 7.1. RETROVIRAL VECTORS

Recombination between the genes encoding the structual proteins of the retrovirus within the packaging cell line and the vector genome leads to the formation of RCRs. Viral sequences endogenous to the murine host cell line ( NIH-3T3) have been shown to participate to form RCR [3][4]. A system that uses human 293 cell lines as the vector producing cell line and therefore eliminates the presence of homologous endogenous retroviral sequences has been developed [5]. This has failed to show any evidence of RCR after stringent testing. However, it has been shown that even in a vector producer line with the lowest theoretical rates of RCR generation, generation of RCR may occur which cannot be explained [6].

7.2 ADENOVIRAL VECTORS RCAs may not be considered to be as high a safety risk as RCRs because they are not integrated into the target cell genome. The principle area of concern with adenoviruses is that since they generally require repeated treatments in order to maintain an effect, the likelihood of an immune response is increased. To reduce the likelihood of adenoviral antigens on the target cell surface, recombinant adenoviruses have been constructed which lack the major late promoter (MLP) and therefore should not express late viral genes [7].

8. Patient Monitoring for RCR Current FDA recommendations for patient monitoring suggest that blood samples should be tested 4-6 weeks after treatment then every three months for the first year and annually thereafter. This testing should comprise serological assays and/or PCR as appropriate taking into consideration the mode of vector administration and the patient

population. The data should be submitted to the FDA with the annual progress report.

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Accumulation of this data by the FDA should lead to a great deal of information on the safety of retroviral vectors. However proposals are under consideration to reduce the follow-up testing to a level which is less stringent. 9. Summary Safety in the gene therapy field is a complex area with many issues which have to be considered. This field is growing very quickly and as data is accumulated, it will become more evident where problems occur. It is essential that everyone in the field works towards safer vectors and new production techniques. It may also be important that testing is standarised and streamlined so that the minimum amount of testing material gives the maximum amount of information. 10. References 1. Wilson, A.C., Ng, T-H. & Miller, A.E. (1997) Evaluation of Recommendations for Replication-Competent Retrovirus Testing Associated with Use of Retroviral Vector, Human Gene Therapy, 8, 869 - 874. 2. Donahue, R.E., Kessler, S.W., Bodine, D., McDonagh, K., Dunbar, C., Goodman,S., Agricola,B., Byrne, E., Raffeld, M., Moen, R., Zsebo, K.M. & Nienhuis, A.W. (1992) Helper Virus Induced T Cell Lymphoma in Non-Human Primates after Mediated Gene Transfer, J. Exp. Med, 176, 1125 - 1135. 3. Otto, E., Jones-Trowler, A., Vanin, E.F., Stambaugh, K., Mueller, S.N., Anderson, W.F. & McGarrity, G.J. (1994) Characterisation of a Replication-Competent Retrovirus Resulting from Recombination of Packaging and Vector Sequences, Human Gene Therapy, 5, 567 - 575. 4. Purcell, D.F.J., Broscuis, C.M., Vanin, E.F., Buckler, C.E., Nienhuis, A.W. & Martin, M.A. (1996) An Array of Murine Leukemia Virus-Related Elements is Transmitted and Expressed in a Primate Recipient of Retroviral Gene Transfer, J. Virol., 70, 887 - 897. 5. Rigg, R.J., Chem, J., Dando, J.S., Forestall, S.P., Plavec, I. & Bohnlein, E. (1996) A Novel Human Amphotropic Packaging Cell Line: High Titer, Complement Resistance, and Improved Safety, Virology, 218, 290 - 295. 6. Chong, H. & Vile, R.G. (1996) Replication-Competent Retrovirus Produced by a “Split-Function” Third Generation Amphotropic Packaging Cell Line, Gene Therapy, 3, 624 - 629. 7. Schneider, S.D., Rusconi, S., Seger, R.A. & Hosslew, J.P. (1997) Adenovirus-Mediated Gene Transfer into Monocyte-Derived Macrophages of Patients X-Linked Chronic Granulomatous Disease: Ex Vivo Correction of Deficient Respiratory Burst, Gene Therapy, 4, 524 - 532.

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Discussion

Goodhall:

Could you briefly outline how you test for your mycoplasma contamination?

Morgan:

We use 2 methods. We co-culture the test article withVero cells and use the Hoescht stain. We also try to grow the mycoplasma directly on suitable media.

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GENE THERAPY FOR NEUROMUSCULAR DISORDERS: MACROPHAGES AS SHUTTLES FOR WIDESPREAD TARGETING E. PARRISH, E. INSERM U421, 8 rue du

AND L. GARCIA 94010

cedex, France

Abstract

Gene therapy as a treatment for neuromuscular diseases is an ever-developing concept based on the use of DNA as the therapeutic agent. In the search for appropriate strategies, however, a bottleneck exists concerning the targeting of the therapeutic gene to all pathologic sites. These diseases are often characterised by multiple widespread lesions spread over a large area, rendering administration by local injection into tissues,

clinically irrelevant. We are therefore confronted with the need to seek a method of targeting which uses the systemic pathway, but nevertheless specifically limits the delivery of the therapeutic agent to the pathologic sites only. To this end, we have proposed that circulating cells which home naturally to inflammatory lesions, could be used as shuttles able to track down every damaged site. Using a murine model of muscular dystrophy (mdx), we have validated the possibility of using engrafted monocyte-macrophages to perform this function. The objective is now to engineer them into ‘monocyte-derived cargo cells’ (MDC-cells) able not only to target, on demand, any pathological site, but also to locally deliver a given therapeutic agent. In muscular dystrophies, particularly DMD, the genetic defect results in the sporadic necrosis of muscle fibres, leading to muscle degeneration. For a period of time, however, a certain plasticity allows the compensation of this degenerative process by at least a partial regeneration, giving us two points of attack to limit muscle wasting. The first is

preservation of the tissue by either protecting the fibres or enhancing regeneration. This would require ‘secreting’ MDC-cells engineered to deliver a factor appropriate to this

purpose. The second aims to mobilise a corrective gene from the MDC-cells into muscle cells, a process of in situ cell to cell gene transfer which could be accomplished using a retroviral vector, since the regeneration process involves the proliferation of muscle precursors before they fuse to form replacement fibres. For this, MDC-cells must be rendered capable of packaging retroviral vectors. Secreting MDC-cells might also be suitable for targeting, via the systemic pathway, the diffuse pathological sites occurring in many CNS diseases and delivering a neuroprotective factor. They could be used not only as ‘patrollers’ summoned in acute reaction to inflammatory episodes, as proposed for the muscular dystrophies, but additionally, as ‘sleepers’, having colonised the CNS from the hematopoietic tissue via the natural turnover of resident macrophages (i.e. microglia). 531

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 531-539. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

532

Introduction Gene therapy as a treatment for genetic disorders is an ever-developing concept based on the use of DNA as the therapeutic agent. For any given disease, the identification of the implicated gene is, in principal, a prerequisite, although only the beginning of a multistep process requiring the successful vectorisation of the curative gene and its efficient targeting to the damaged tissue. Indeed, in the search for appropriate therapeutic strategies, the latter represents a bottleneck common to the therapy of many disorders, in particular, the different muscular dystrophies and degenerative diseases of

the central nervous system. In the case of these neuromuscular disorders we are faced not only with the problem of the widespread and sometimes inaccessible nature of the affected sites, but also with the scale of the territory, as a whole, to be treated. To overcome this difficulty, a targeting strategy needs to be sought, therefore, which is adapted to disseminated lesions and so uses the systemic pathway, but nevertheless specifically limits the delivery of the therapeutic agent to the pathologic sites.

Muscular dystrophies

Gene therapy for muscular dystrophies typically seeks to transfer a healthy or corrective gene into muscle fibres. This transfer has, so far, been achieved through injection of recombinant viral vectors (Quantin et al., 1992; Ragot et al., 1993; Vincent et al., 1993), preparations of naked DNA (Wolff et al., 1990; Acsadi et al., 1991), or lethally processed murine packaging cells (Fassati et al., 1995), directly into the affected tissues. Delivery by local injection is, however, of limited clinical use in such diseases where the sites to be treated are, as already discussed, multiple and disseminated throughout the skeletal muscle as a whole. One way to circumvent this obstacle has been suggested to us by considering the pathological process itself. Basically, a muscle lesion, whether accidental or as the result of a genetic defect, triggers a complex response which can be broken down into three arbitrary phases. First, the fibres degenerate. Second, at each site of damage, there is a subsequent rapid infiltration of mononucleated cells, particularly macrophages. Third, if the muscle plasticity allows, neighbouring satellite cells are activated, proliferate and fuse, giving rise to new replacement muscle fibres (figure 1). In view of the specific recruitment of macrophages to these sites of lesion, we have proposed that this cell type and its blood precursor, the monocyte, could be used as a cell carrier to shuttle a given therapeutic agent to each afflicted area (Parrish et al., 1996). We have demonstrated the possibility of using transplanted immortalised monocytes as naturally homing shuttles able to target multiple disseminated lesions in skeletal muscle diseases. These cells, injected directly, intravenously, into mice, successfully attained experimentally induced necrotic sites in muscle, showing that a one-off administration of cells could rapidly target a given pre-existing muscle injury and probably any inflammatory zone. However, because inherited muscle dystrophies are in fact characterised by the sporadic occurrence of muscle fibre degeneration, throughout the lifespan of the individual, we have also demonstrated the possibility of

533

creating, using bone marrow transplantation, a constitutive reservoir of genetically modified cells able to infiltrate any spontaneous site of muscle damage, as it occurs. The aim now is to modify the exogenous macrophages, or their precursors, to produce circulating ‘monocyte-derived cargo cells’ (MDC-cells), able to work as therapeutic patrollers, targeting 'on demand' any pathological site as it arises. The engineering required for this modification (developed later) depends upon the choice of therapeutic approach. The pathogenic process in muscular dystrophies is summarised in figure 2. The genetic defect results in the sporadic necrosis of muscle fibres leading, eventually, to muscle degeneration. However, the dystrophic muscle maintains, for a period of time, a certain plasticity allowing the compensation of this degenerative process with at least a partial regeneration. This gives us two points of attack to limit muscle wasting. The first is based on the possibility of preserving the muscle tissue by either protecting the fibres, thus slowing the rate of degeneration, or enhancing the

regeneration, thus boosting the compensation. The second deals with the possibility of escaping from this vicious circle by genetically correcting the replacement fibres as they form. Two types of MDC-cell are therefore required. For myopreservation, the macrophage must be engineered to secrete, locally, a factor acting either on the existing fibres themselves, or on their precursors, the satellite cells. This idea remains essentially theoretical however, since no such candidate molecule is currently available. Nonetheless, it is not unreasonable to imagine that in the not too

534

distant future, protection against the lack of dystrophin might perhaps be achieved by factors stimulating alternative related proteins such as utrophin (Deconinck et al., 1997; Grady et al., 1997). In addition, in vitro studies have suggested that macrophages themselves could directly participate in muscle fibre regeneration by exerting mitogenic and chemotactic effects on muscle precursor cells (Robertson et al., 1993; Cantini et al.,

1995). The anticipated identification of the factors responsible may well provide candidate molecules, more appropriate than the non-specific LIF and FGFs, for enhancing the regeneration. For correction, the aim is to mobilise the corrective gene from the blood borne macrophage into target muscle cells, a process of in situ cell to cell gene transfer which could be accomplished using a retroviral vector, since the regeneration process occurring at the site of damage involves the proliferation of muscle precursors before they fuse to form replacement fibres. This then, requires that the macrophage be engineered into a ‘packaging cell’ containing both a replication deficient retrovirus carrying the gene of interest, and an helper genome (gag-pol-env) needed for its packaging and secretion. In this way, monocyte shuttles distributing the therapeutic retrovirus to all widespread sites of necrosis-regeneration, would allow the ‘correction’ of an ever increasing number of fibres as they regenerate. The corrective gene in this strategy could encode either a functional truncated dystrophin (e.g. mini-dystrophin), a trans-activating factor inducing the expression of a dystrophin-related protein (e.g. utrophin), or sequences allowing exon skipping to restore the endogenous dystrophin, in truncated form.

CNS degenerative disorders

As things stand, the application of gene therapy to degenerative diseases of the CNS at first sight poses a problem, since very few of the genes implicated have been identified, and even when this is the case (Lefebvre et al., 1995; Melki et al., 1996), there is not always an obvious solution for delivering a therapeutic gene into the subpopulation of cells concerned. In the face of this difficulty an alternative strategy, based on phenotypic compensation, has emerged from the ability of neurotrophic factors to act on the survival adult neurones. The interest for these molecules in this context, stems from an

535

ever increasing body of evidence demonstrating their ability to exert neuroprotective

effects both on a wide range of neuronal populations and in diverse lesion paradigms (Arakawa et al., 1990; Sendter et al., 1990 et 1992a,b; Oppenheim et al, 1991 et 1995; Martinou et al., 1992; Louis et al., 1993; Mitsumoto et al. 1994; Sariola et al., 1994; Yan et al. 1995). The presence of the blood-brain barrier imposes the administration of these factors directly into the CNS and to date, this has been achieved either by using mini-pumps (Hagg et al., 1992; Anderson et al., 1996) or encapsulated BHK producer cells implanted into the lateral ventricle (Emerich et al., 1996). However, the very nature of most neurodegenerative diseases, progressive and occurring over a protracted period of time, would seem to call for a system of long term delivery. This could be achieved by genetically modifying the cells in the target area, to produce themselves the therapeutic molecule. Most viral vectors, adenovirus (Akli et al., 1993; Le Gal la Salle et al., 1993; Davidson et al., 1993; Bilang-Bleuel et al., 1997), herpes virus (Lawrence et al., 1996), AAV (Peel et al., 1997) and lentivirus (Naldini et al., 1996) have proved to be efficient tools for such a gene transfer in situ in the CNS, when administered by stereotaxic injection. However, this route of local administration is not appropriate for the targeting of the widespread lesions characteristic of most neurodegenerative

diseases, where a large part of the CNS as a whole needs to be treated. As proposed for dystrophic muscle, therefore, ‘secreting MDC-cells’ might also be suitable for targeting,

via the systemic pathway, the diffuse pathological sites occurring in CNS diseases, and delivering, locally, a neuroprotective factor (figure 3). Therapeutic MDC-cells could be used in two ways: as ‘patrollers’, summoned on demand, in acute reaction to inflammatory episodes such as those seen in multiple sclerosis for example, or additionally, as ‘sleepers’, having colonised the target tissue from the hematopoietic tissue, via the natural turn-over of a proportion of the resident macrophages (i.e. microglia) (Krall et al., 1994). The sleepers could act either on demand, by induction of secretion of the therapeutic agent, or chronically by its constitutive production. These two approaches might well determine the use of multiple therapeutic factors, their secretion being governed by the state of differentiation of the MDC-cells.

536 Engineering of monocyte-macrophages

To achieve their purpose, the monocyte-macrophages need, therefore, to be genetically engineered into either ‘secreting’ or ‘packaging’ MDC-cells, depending upon their therapeutic goal. Secreting MDC-cells could originate either from a transplanted classically engineered bone marrow, or from primary macrophages transduced using different defective viral vectors (adenovirus, herpes simplex virus, lentivirus) able to infect post-mitotic cells. In either case, the aim is simply to efficiently introduce, into the cell, a cassette containing sequences coding for a secretable therapeutic factor, under

the control of a specific promoter. In contrast to secreting MDC-cells, whose engineering can be envisaged using a single step transduction procedure, the creation of packaging MDC-cells entails a double modification of the monocyte-macrophages with incorporation into the cell of i) the replication deficient retrovirus carrying the gene of interest, and ii) the helper genome (gag-pol-env) required for packaging and secretion of the retrovirus. This could be achieved in two ways: a multistep transduction using more than one viral vector or a single step transduction using one complex vector (figure 4). The multistep procedure would require sequential transduction with: a) a defective viral vector (matrix vector), able to transduce post-mitotic cells, carrying the sequences

entirely encoding the provirus to be mobilised (which carries the therapeutic gene); b) a

537

defective viral vector (assembling vector), again able to transduce post-mitotic cells, carrying a defective MuLvs gag-pol-env helper genome for transcomplementation of the provirus. In the single step procedure, on the other hand, monocyte-macrophages would

be transduced by a single complex viral vector (master vector), able to transduce postmitotic cells, this time carrying both the provirus and a defective gag-pol-env helper genome, for ciscomplementation of the therapeutic provirus. So far, we have tested the idea of assembling vector driven mobilisation, in vitro, by the double transduction of a monocyte cell line with a defective retrovirus (MoMuLV)

carrying the lacZ reporter gene and an HSV-amplicon carrying the Moloney gag-pol-env sequences (kindly provided by A. Epstein - Lyon), respectively. Mobilisation of

MoMuLV -lacby the resulting engineered monocytes was demonstrated by the successful transduction of gliobastoma cells (of the 9L cell line) using engineered monocyte conditioned medium (figure 5).

Conclusion Our first phase results show that monocyte-macrophages are appropriate cell vectors either to target widespread inflammatory sites on demand, or to colonise a target tissue via a process of natural cell turn-over. In addition, it is possible to extemporaneously engineer these cells into ‘packaging monocyte-macrophages’ able to mobilise defective retroviral vectors. The next phase is now to engineer these cells into ‘secreting’ or

‘packaging’ MDC-cells able to confer a therapeutic benefit in appropriate animal models. Eventually of course, the aim is to specifically restrict the secretion of the therapeutic agent to the target sites. This is a problem which, to a certain extent, could be addressed by a judicious choice of the promoters controlling its expression. It is not unreasonable to envisage the possibility of limiting secretion using appropriate macrophage differentiation-specific promoters, such as CD68, which should restrict expression of the therapeutic substance to sites of inflammation. In addition, since

538

resident macrophages display considerable phenotypic heterogeneity, to such an extent that have been individually defined (e.g. Kupffer cell, alveolar macrophage, osteoclast, microglia), one can speculate that, in the future, their molecular characterisation might also provide us with candidate promoters for tissue specific expression. This targeting strategy, although developed here in the context of neuromuscular diseases, could theoretically be applied to any pathology where the sufferance or death of individual or groups of cells induces the recruitment of blood borne macrophages. In addition to the applications discussed in this paper therefore, it is perhaps also worth considering the possible application of this method of targeting to strategies for the gene therapy of certain cancers, in particular glioblastoma, in which macrophages naturally encircle the tumour. Such a situation might allow the possibility of containing the tumour and limiting its invasion. References Akli S. et al., Nature Gen. 3: 224-228, 1993. Anderson K. et al., Proc.Natl.Acad.Sci.USA 93: 7346-7351, 1996. Arakawa Y. et al., J. Neurosci. 10: 3507-3515, 1990. Acsadi G., et al., Nature. 352: 815-818, 1991. Bilang-Bleuel A. et al., Proc.Natl.Acad.Sci.USA 94: 8818-8823, 1997. Cantini M., & Carraro U. J. Neuropathol. Exp. Neurol. 54: 121-128, 1995. Davidson B. et al., Nature Gen. 3: 219-223, 1993. Deconinck A. et al., Cell. 90: 717-727, 1997. Emerich P. et al., J. Neurosci. 16: 5168-5181, 1996. Fassati A. et al., H.Gene Therapy. 7: 595-602, 1996

Grady R. et al., Cell. 90: 729-738, 1997. Hagg T. et al., Neuron 8: 145-158, 1992.

Krall W. et al., Blood. 83: 2737-2748, 1994. Lawrence et al., J. Neurosci. 16: 486-496, 1996. Lefebvre et al., Cell 80: 155-165, 1995. Le Gal la Salle G. et al., Science. 259: 988-990, 1993. Louis JC. et al., Science. 259: 689-692, 1993.

Martinou JC. et al., Neuron. 6: 737-744, 1992. Melki J. et al., Genomics 32 (3): 479-482, 1996. Mitsumoto H. et al., Science. 265: 1107-1110, 1994. Naldini L. et al., Science. 272: 263-268, 1996. Oppenheim R. et al., Science. 252: 1616-1618, 1991. Oppenheim R. et al., Nature. 373: 344-346, 1995. Parrish E. et al., Gene Therapy, 3: 13-20, 1996 Peel A. et al., Gene Therapy. 4: 16-24, 1997.

Quantin B. et al., Proc. Natl. Acad. Sci. USA, 89: 2581-2584, 1992. Ragot T. et al., Nature. 361: 647-650, 1993. Robertson T. et al., Exp. Cell. Res. 207: 321-331, 1993.

Sariola H. et al., Ann. Med. 26: 355-363, 1994.

Sendtner M. et al., Nature 345: 440-441, 1990. Sendtner M. el al., Nature 358: 502-504, 1992a. Sendtner M. et al., Nature 360: 757-759, 1992b. Vincent N. et al., Nature Genet. 5: 130-134, 1993. Wolff J. et al., Science. 245: 1465-1468, 1990. Yan Q et al., Nature 373: 341-343, 1995.

539

Discussion

Massie:

Since the cells you are trying to engineer are good presenting cells for antigen, are you not concerned about immune reactions if you engineer the cell to express these antigens: for example, packaging function of retroviruses?

Garcia:

Yes, it is a problem. The range of time for these experiments is 2-3 days, so we can expect that the cells were still able to reach their target site and carry out their specific function. We can also control the expression of the alpha genome by using constitutive promotors. We also use differentiation control promotors which give transient expression when they just arrive at the necrotic site, or when they begin to phagocytose. Maybe we can decrease the extent of presentation to some degree.

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GENE TRANSFER INTO DOG MYOBLASTS

S. Braun, C. Thioudellet, C. Escriou*, M-C, Claudepierre, F. -Rouault, E. Jacobs, R. Bischoff, D. Elmlinger, H. Homann, Y. Poitevin, M. Lusky, M. Mehtali, F. Perraud and A. Pavirani

Transgene S.A., Strasbourg, France. *ENV, Alfort, France.

Keywords: Gene transfer, dog skeletal muscle, culture.

1. Introduction

Duchenne Muscular Dystrophy (DMD) is a lethal X-linked genetic disease. The disorder is manifest in early childhood with progressive skeletal muscle weakness and wasting. In a predictable fashion, affected boys loose muscle strength and ability to

walk. Death usually occurs due to respiratory and cardiac complications. DMD muscle degeneration is caused by absence of dystrophin, a large rodlike cytoskeletal protein found at the inner membrane surface of muscle fibers, which is thought to play an important role in maintaining muscle fiber integrity. Gene therapy of DMD aims at the expression of a functional dystrophin gene in skeletal muscles and myocardium. The GRMD dystrophic dog is the animal model which clinically resembles the human disease. Various vectors and strategies of dystrophin or minidystrophin cDNA delivery to muscles need to be evaluated: plasmids, non-viral (synthetic) vectors, adenovirus, retrovirus, engineered satellite cell transplantation. The vectors are being validated in vitro and in vivo and 2. In Vitro tranfection

Among the in vitro models that we have developped, myoblasts cultures of healthy and dystrophic dogs (6 to 8 month old) are being used.The principle of this type of culture is based on the ability of skeletal muscle fibers to regenerate from mononucleated satellite (myoblast) cells. When cells reach confluency, myoblasts fuse and form multinucleated myotubes. Naked plasmid DNA is unable to transfect cells in vitro Using the calcium phosphate precipitation method, up to 50 % dog myoblasts are transfected (LacZ reporter gene).

High levels of luciferase activity (109 RLU/mg protein) were obtained after transfection with plasmids encoding this firefly enzyme. Synthetic vectors were also effective, the polyamines spermidine and spermine isomers-based cationic lipid/DNA complexes being very potent vectors. 541

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 541-543. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

542

Interestingly, the efficiency of the various isomers varies depending on the transfected cell type. Expression of dystrophin was also obtained in GRMD dog myoblasts as demonstrated by Western Blot and immunocytochemistry after plasmid transfection with various synthetic vectors and with the calcium phosphate precipitation method.

Infection of dog myoblasts with an adenovirus carrying the LacZ reporter gene, led to up to 90 % of blue-stained cells (as in the case of other primary cultures, the viral multiplicity of infection had to be elevate, i.e.200 for maximal efficiency).

Retroviruses infect dog myoblast cultures also well ( after 3 cycles of infection 75% of cells stained blue). No differences in infection were seen between healthy and dystrophic dog myoblasts and between types of muscle. 3. Myoblast graft

In vitro transfection or infection can be used in view of myoblast graft.Cells are transduced in vitro before reimplantation into dog muscle. The engrafted cells would allow regeneration of new muscle fibers expressing dystrophin or minidystrophin. In a preliminary assay, we injected retrovirus ( ß-Gal)-transduced autologous myoblasts in two 8 month old litermate dogs (1 healthy and 1 dystrophic dog). During anesthesia, extensor digitory communis muscles were visualized after skin and fascia incision. Three million myoblasts were injected in 3 different sites. Two non-resorbable suturae were left to show the injection sites. Muscle biopsies were retrieved 8 days and 1 month after myoblast graft, respectively, cryosections showed some positive fibers in muscle biopsies in both animals. One month after graft, no positive staining was observed, even though plasmid DNA was detected by PCR.

4. Conclusions Primary dog myoblast cultures represent a useful model for in vitro evaluation of viral and non viral vectors. The cells can also be used for cell grafting with applications in DMD, vaccination or production of circulating proteins. Supported by the Association Francaise contre les Myopathies.

543

Discussion

Petrie:

In the chairman’s overview he mentioned 1,400 gene therapy clinical trials, including AIDS and cystic fibrosis. How may of these give really broad positive effects to the patient?

Crespo:

The development of gene therapy is in a very early stage so only very few clinical trials are reaching phase III. More than 200 clinical protocols have been presented but, unfortunately, very few are in clinical phase II. More time is needed for this new technology with the complexity of manufacturing and safety issues to be developed.

Ostrove:

You showed that in order to have 75% transduction efficiency in

the myoblasts with the adenoviral vector, you needed an MOI between 100 and 800. Are those particles or infectious units, and can you comment on the need for such an MOI, and is there a practical way of getting around this problem? Perraud:

Infectious unit. 100 is minimal for myoblasts as primary cultures are more difficult to infect than routinely used cell lines. Clearly this is a problem for in vivo clinical trials as we are limited by the bulk we can inject into the patient.

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BIODISTRIBUTION ANALYSIS OF A GENE THERAPY VECTOR USING THE POLYMERASE CHAIN REACTION (PCR) TECHNIQUE Joanne Proffitt*, Martha Leibbrandt#, Keri Jarvis* and Carl Martin* *Covance Laboratories Otley Road Harrogate, North Yorkshire,

#Chiron Corporation, Emeryville, California 94608 USA

HG3 1PY

Introduction The gene therapy T7(3)TK plasmid contains the herpes simplex thymidine kinase

(HSV-TK) gene under the control of the T7 RNA polymerase promoter (T7RNAP). Rats were injected with T7(3)TK plasmid, at either low dose (group 2) or high dose (group 3), and the T7 RNA polymerase protein (Table 1). A control group of rats (group 1) were injected with formulation buffer.

The study was performed to determine the biodistribution of HSV-TK originating

from the T7(3)TK plasmid in certain organs and germ line tissue isolated from the treated rats. DNA extracted from tissue samples taken from treated rats was tested by the polymerase chain reaction (PCR) technique (Refs: [1] and [2]) using primers

designed specifically to the HSV-TK DNA sequence. Analysis of a single aliquot of sample which may contain a DNA sequence at extremely low levels potentiates the likelihood of a false negative result. Therefore, to circumvent the possibility of missing a positive signal in a single sample and hence to ensure validity of the results obtained, each sample was analysed in quadruplicate. In addition 10 plasmid copies of T7(3)TK plasmid DNA were used to spike duplicate aliquots of each DNA sample 545 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 545-548.

© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

546

hence providing a positive control specific to each DNA sample. The presence of HSV-TK DNA sequence in any test article DNA samples (identified by PCR analysis) was confirmed by Southern blot hybridisation (Ref: 3) using a labelled HSV-TK specific probe. Assay Regime

DNA was isolated from each tissue sample. Each DNA sample was analysed in replicates of 4 with additional duplicate aliquots spiked with T7(3)TK positive control plasmid containing the HSV-TK sequence (Figure 1).

An example template scheme for PCR is given:

1)

Purified water:

Blank reaction

2)

Purified water:

Sentinel control

3)

Negative control DNA:

0.1 µg DNA

4)

Test DNA:

0.1 µg DNA

5)

Test DNA:

0.1µg DNA

6)

Test DNA:

0.1 µg DNA

547

7)

Test DNA:

0.1 µg DNA

8)

Spiked test DNA:

0.1 µg DNA spiked with 10 plasmid copies

9)

Spiked test DNA:

0.1 µg DNA spiked with 10 plasmid copies

10)

Positive control DNA:

1 plasmid copy

11)

Positive control DNA:

10 plasmid copies

12)

Positive control DNA:

50 plasmid copies

NB One set of assay controls (assay blank, sentinel, negative and positive controls) were used per run of PCR. The positive control samples (1, 10 and 50 plasmid copies) ensured consistency of PCR sensitivity between different runs of PCR. To demonstrate reproducibility of data, PCR was repeated on any samples which were positive in one or more, but not all of the quadruplicate DNA samples. The sentinel control functioned as a monitor for airborne contaminants and therefore remained open during the preparation steps, until initiation of PCR. An aliquot of each PCR reaction was analysed by agarose gel electrophoresis. Results PCR analysis of the positive control T7(3)TK plasmid, established that the sensitivity for HSV-TK detection was routinely 1 plasmid copy. The blank and sentinel controls containing purified water only, and the DNA negative control samples were all negative for the presence of HSV-TK DNA hence demonstrating that no crosscontamination had occurred and that the primers used for PCR were specific to the HSV-TK sequence. Spiking the DNA samples with positive control plasmid provided a “mixed pool” positive control sample specific to each DNA preparation. In addition it ensured that the DNA preparations were free from the presence of any factors capable of inhibiting the amplification process.

Table 2 _____Group____________Negative___________Positive____ 1 42/42 0/42 2 42/42 0/42 _______3________________39/42______________3/42_____ All the DNA samples (0.1 µg DNA) prepared from tissues isolated from animals in group 1 (negative control group) and group 2 (low dose T7(3)TK plasmid) were determined as negative for the presence of HSV-TK DNA (Table 2). Three DNA samples (0.1 µg DNA) prepared from tissues isolated from animals in group 3 (high

548

dose T7(3)TK plasmid) were positive for the presence of HSV-TK DNA sequence (Table 2). The positive samples originated from three different animals, two samples were derived from auxilliary lymph nodes and one from spleen. The signals from all three samples were weak in comparison to the positive control spiked DNA samples indicating the presence of less than 10 plasmid copies in each sample. The weak amplification of the HSV-TK sequence in replicates suggested that the HSV-TK sequence was present at levels close to the level of assay sensitivity. All other DNA samples from group 3 animals were negative for the presence of HSVTK DNA as determined by PCR analysis for the HSV-TK gene sequence. Confirmation of the positive status of the 3 DNA samples from group 3 animals was performed by Southern blot hybridisation using a labelled HSV-TK specific probe. All DNA samples which tested positive by PCR were confirmed as positive for the presence of HSV-TK DNA sequence as determined by Southern blot hybridisation. Conclusion We have demonstrated the ability to determine the biodistribution of a gene therapy plasmid in tissues of treated animals by using a PCR strategy followed by Southern blot hybridisation using primers homologous to the therapeutic gene. Only 3/42 tissues isolated from animals treated with high dose levels of gene therapy plasmid were confirmed as positive for the HSV-TK DNA sequence. Furthermore the target sequence was detected at extremely low levels (< 10 plasmid copies) demonstrating the extreme sensitivity of the methodology employed.

References 1.

Mullis, K.B. and Faloona, F.(1987) Specific synthesis of DNA in vitro via a polymerase- catalysed chain reaction. Methods in Enzymol. 155.335-350.

2.

Saiki, R. K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.G. and Erlich,

H.A.(1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239. 487-491.

3.

Sambrook, J., Fritsch, E. and Maniatis, T. (1989) Molecular cloning: A laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Covance Laboratories Ltd Otley Road, Harrogate, North Yorkshire, HG3 1PY United Kingdom Tel: +44 (0) 1423 500011 Fax: +44 (0) 1423 569595 Website: http:\\www.covance.com

SESSION ON : DEVELOPMENTS FOR IMMUNOLOGICALS AND VACCINES

The vaccine field is the oldest domaine of animal cell technology because this technology started with the use of primary and diploid cells for the production of viral vaccines. Still today many new and important developments are going on in this field. Principally four different subchapters were treated in this session: development of new

classical vaccines, development of subunit vaccines, research on new adjuvant systems, and development and use of new cytokines for modifying and modulating the immune system. In detail, new classical vaccines (= infection of cells with viruses for the production of

viral vacines) are still developed today in those fields, where no vaccines exist actually or where old technologies, like the use of embryonated eggs, are still in use. New developments in the production of influenza virus on MDCK in cell culture and in the use of serum-free media for virus production were presented as well as the optimisation of production methods for HSV-2 and live measles vaccines.

A lot of vaccines cannot be produced in a classical way, wherefore subunit vaccines are produced and use, however, whose efficacy (i.e.: immunogenicity) relies largely on the adjuvant systems used. Another new approach is based on the use of recombinant suicidal DNA/RNA. Upon vaccination the gene(s) encoding for the relevant antigen(s) is transiently expressed within the cells of the host, leading therefore to an activation of the immune system. Animal cell technology can also be used for the production of non-viral vaccines. This can be achieved by infecting animal cells with parasites (e.g. for the production of Cowdria ruminantium on goat endothelial cells) or by a subunit approach where the relevant antigen is produced by a recombinant baculovirus - insect cell culture system. The modulation of the immune system by cytokines is of the same importance as the vaccination approach. This can be achieved by using interferons and gangliosides, for instance. In this context, the in vitro immunization has to be mentioned, which is a very

important step for the development of human monoclonal antibodies which are useful for diagnosis and treatment. In conclusion, although this session contained only about 10% of all papers, it presented a good reflect of the actual research and developments in this field 549

O.-W. Merten et al. (eds,), New Developments and New Applications in Animal Cell Technology, 549. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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THE PRODUCTION OF INFLUENZA VIRUS BY IMMOBILISED MDCK CELLS D . Looby, J . Tree, A . Talukder, K . H a y e s , H . House, G . Stacey 1 2

CAMR, Porton Down, Salisbury, Wiltshire, UK, SP1OJG Department of Biomedical Science, University of Newcastle-upon -Tyne,

INTRODUCTION Currently most human influenza vaccines are produced in embryonated hens eggs, however

there is now considerable interest in the development of tissue culture based processes. The main reasons for this are : 1) ready supply of substrate for virus growth particularly during a

pandemic1, 2) virus passaged in tissue culture is more representative of the natural isolate 2,, and 3) vaccine produced in tissue culture provides better protection than egg derived vaccine 2 . Currently the cell culture systems of choice for large scale manufacturing of influenza

vaccine are batch processes based on either roller bottles or solid microcarriers3, however the production of high titre influenza virus from a continuously perfused solid microcarrier process has also been reported 4. Fixed bed reactors based on the immobilisation of cells within porous carriers are increasingly being used for the production of biologicals from adherant and suspension cells. In this study we describe the production of influenza virus from MDCK cells immobilised in a fixed bed perfusion bioreactor. The potential advantages of this approach are: 1) high immobilised cell density leading to high virus titres, 2) cells are

protected from the effects of fluid mechanical shear thus potentially preventing premature sloughing of infected cells from the carriers which tends to happen in solid microcarrier processes, and 3) the fixed bed system is scaleable e.g. an industrial process for recombinant protein production has been scaled up to 30 litre bed volume without any loss in performance. Data is presented on the kinetics of influenza virus production in the fixed bed system and yields are compared to roller bottle and embryonated hens egg processes.

AIMS

• To establish a fixed bed perfusion process for the production of influenza virus • To compare the performance of the fixed bed process to egg and roller bottle processes MATERIALS AND METHODS

Cell Line: The cell line was MDCK. CCL34 (ATCC) Virus: The virus strain was Influenza A/ PR/8/34 (N1BSC, UK) Media: The cell growth media was High glucose DMEM supplemented with 10% FCS. The virus maintenance media was high glucose DMEM supplemented with trypsin (25 mg/1) and extra glucose (1.5g/L). 551 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 551 -554. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

552

Analysis: Glucose was measured with a YSI Glucose/Lactate analyser. Virus titre was determined by both haemagglutination and plaque assay. Fixed Bed Process: The fixed bed culture system5 consisted of a reactor filled with 100 ml porous borosilicate glass carriers and a media reservoir (1 litre working volume). The fixed bed was inoculated with approximately

cells. The cells were grown until steady state was achieved as

indicated by a levelling off of the glucose consumption rate i.e.after approximately 6 days . The cell growth media was removed and the fixed bed of immobilised cells was washed three times with 100 ml PBS, then infected with virus by filling the bed with 100 ml of virus suspension (MOI 0.05 approximately). 0.9 litre of virus growth media were added to the reservoir, and after an incubation period of two hours (virus adsorption phase) media recirculation was started (20 linear cm per min): for batch virus production the culture was harvested at 50 hours: for continuous perfusion virus production, the virus maintenance media was perfused (after 24 hours) at a rate of 1 litre per day until virus production ceased (120 hours). Samples were taken daily and assayed for glucose and stored at for virus assay. Roller bottle process Confluent roller bottles were washed 3 times with PBS then infected at an MOI of 0.05 per

cell in 10 ml virus growth media. After one hour incubation (to allow the virus to adsorb) the media volume was increased to 100 ml. Samples were taken daily and assayed for glucose and stored at

virus assay.

553

Total harvest volume produced: fixed bed perfusion = 4.75 litres, fixed bed batch = 1.0 litre and roller bottle = 0.1 litre. Typical virus yield in eggs =

• A fixed bed perfusion process producing a total of

PFU from a 100 ml fixed

bed has been established this is approximately equivalent to the production from 10 roller bottles or 200 embryonated hens eggs (Table 1).

554 • Batch virus production in the fixed bed process produced more virus as determined by plaque assay than continuously perfused virus production (fig 1).

• The potential for large scale production of influenza virus from immobilised cells in a fixed

bed perfusion system with batch virus production has been demonstrated.

REFERENCES 1

WHO report: Influenza vaccines: prospects for production from viruses grown in cell culture , Geneva, Switzerland (1995)

2

Robertson, J.S., Cook, P., Attwell, A.M., and Williams, S.P. (1995). Replicative

advantage in tissue culture of egg - adapted influenza virus over tissue culture derived virus: implications for vaccine manufacture.Vaccine. 13 , 1583 -1588, 3

Brands, R., van Scharrenburg, G.J.M. and Palache, A.M. (1997). Production of Influenza virus in cell cultures for vaccine preparation . In: Animal Cell Technology (Eds M.J.T. Carrondo et al.) Kluwer Academic Publishers pp 165-167.

4

Merten, O.W., Hannoun, C., Manuguerra, J.C., Ventre F and Petres. S., (1996).

Development of influenza subunit vaccine produced using mammalian cell technology. In: Novel Srategies in Design and Production of Vaccines (Eds S. Cohen and A. Shafferman) Plenum Press, New York. pp 141 -151 5

Looby, D. and Griffiths, J.B. (1988). Fixed bed porous glass sphere (porosphere)

bioreactors for animal cell culture. Cytotechnology 1, 339 - 346.

Acknowledgements

This work was supported by a grant from the Department of Health, UK.

SERUM-FREE

GROWN

MDCK

CELLS

:

AN

ALTERNATIVE

FOR

INFLUENZA VACCINE VIRUS PRODUCTION

N. KESSLER, G. THOMAS, L. GERENTES, AND M. AYMARD

Laboratoire de Virologie, Faculté de medecine Grange-Blanche, 8 avenue Rockefeller, 69373 Lyon cedex 08, FRANCE

Abstract

Adaptation of MDCK cells to serum-free conditions was performed using UltraMDCK medium (BioWHITTAKER). Growth properties and karyotype stability of MDCK cells were monitored over a one year period of cultivation in Ultra-MDCK . Scaling up of adapted cells to spinner culture was achieved using several porous / non porous microcarriers. Adapted MDCK cells were tested for their suitability to replicate influenza A and B viruses, starting from egg and cell isolated strains and isolates. Replication of influenza viruses in serum-free adapted MDCK cells, under standard conditions (1µg/ml trypsin) was analysed using several tests : hemagglutinin (HA) and neuraminidase (NA) activities , infectious titre, immunofluorescence of infected cells. Replication of influenza viruses in serum-free adapted MDCK cells, in absence of trypsin, was also analysed. All A and B influenza viruses were successfully adapted to replicate to high titres without addition of exogenous proteolytic enzyme, and production of trypsin-independent viruses was obtained as well in static flasks as under agitated conditions.

1. Introduction

Production of influenza vaccine viruses is traditionally performed in embryonated hens’eggs, but several constraints in such a technology generated an urgent need to

develop alternative cell culture systems (WHO memorendum, February 1995). Due to potential contamination of sera with microorganisms, it is essential to cultivate cells in serum-free media (SFM) and to evaluate the effects of such culture conditions on

influenza virus replication. With most of cells, the presence of trypsin is required during influenza virus infection, in order to cleave the hemagglutinin precursor and to induce multicycle virus replication; nevertheless, previous data (Rott et al, 1984) mentioned X-

31 (H3N2) virus multicycle replication in MDCK cells in absence of trypsin. Consequently, in order to eliminate a potential source of microbial contamination in the final product, we analysed the suitability of MDCK cells adapted to serum-free conditions, to replicate influenza viruses in absence of trypsin. 555

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 555-559. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

556

2. Materials and Methods MDCK cells (BioWhittaker) were grown either in FBS supplemented EMEM (abbreviation : EMEM cells) or in serum-free UltraMDCK medium (BioWhittaker), (abbreviation U-MDCK cells). Cell cultivation was performed either in static flasks or in spinner under agitated conditions, using several microcarriers. Karyotype analysis of cells was performed using a modification of the GTG banding procedure described by Seabright (1971). Influenza viruses were selected at once, on the basis of their type/subtype and isolation substrate (embryonated hen’s eggs or MDCK cells). Viruses were as following:

Virus infection conditions were as following : 1) Standard infection conditions in presence of trypsin 2) Trypsin-free conditions Virus production was estimated using the following tests : hemagglutinin and neuraminidase activities, infectious titre in MDCK cells (TCID50/ml), indirect immunofluorescence and hematoxylin-eosin staining of infected cells, plaque assays in MDCK cells with and without trypsin in agar overlay. Virus analysis was performed in terms of PAGE profile and of antigenic profile of the hemagglutinin after introduction of viruses in hemagglutination inhibition test with monoclonal antibodies and specific polyclonal anti-sera.

3. Results and discussion 3.1. MDCK CELL CULTIVATION IN SERUM-FREE ULTRA-MDCK MEDIUM 3.1.1. Static flasks MDCK cells previously grown in FBS supplemented EMEM (EMEM cells) , were transferred directly to Ultra-MDCK medium and then serially cultivated over a more than one year period of time in that environment. Ten serial passages were necessary to cells for a complete adaptation to their new serum-free environment, as shown by a significant decrease (student’s t test) in the doubling time of cells between p1 and p11 post transfer. Mean doubling time of adapted U-MDCK cells was shown identical to control EMEM cells, i.e, day. 3.1.2. Karyotype analysis of U-MDCK cells In order to verify the genetic stability of MDCK cells whih were adapted to grow in Ultra-MDCK medium, chromosome number distribution of U-MDCK cells was

557

monitored at regular intervals of time during their long term cultivation in absence of serum and then compared to control MDCK cells as mentioned in the ATCC catalogue. No significant modification was detected in the chromosome distribution after 75 passages in Ultra-MDCK and the modal number was found similar to control cells : 79 versus 78 chromosomes. 3.1.3. U-MDCK cell cultivation on microcarriers When tested for growth on microcarriers, EMEM cells and U-MDCK cells exhibited significant differences; indeed, EMEM cells were capable to spread and grow on porous uncoated microcarriers while U-MDCK cells could not. Nevertheless, U-MDCK cells were efficiently cultivated on two different kinds of microcarriers : porous, collagen coated microcarriers and non porous, uncoated microcarriers. In both cases, cell confluency was observed four days post seeding, but cell yield was shown consistently higher on non porous , uncoated microcarrier than on porous, collagen coated one , i.e. These results are very interesting, when considering a potential industrial utilization of Ultra-MDCK medium for viral vaccine production.

3.2. INFLUENZA VIRUS PRODUCTION IN U-MDCK CELLS UNDER STANDARD CONDITIONS

Capacity of U-MDCK cells to serve as a potential substrate for influenza virus

production was demonstrated by analyzing virus progeny collected through ten serial passages of A and B viruses, under different MOI (Multiplicity of Infection), in presence of All different influenza viruses replicated to high titre in U-MDCK cells irrespective of : virus type/subtype, isolation substrate, passage number of viruses in U-MDCK cells and strains or isolates. In addition, virus replication was shown 1) consistently higher in U-MDCK cells than in EMEM control cells 2) as efficient in U-MDCK cells as in standard embryonated eggs (Table 1).

Immunofluorescence (anti NP monoclonal antibodies) and hematoxylin-eosin staining

studies confirmed the high sensitivity of U-MDCK cells to influenza virus infection.

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3.3 INFLUENZA VIRUS PRODUCTION IN U-MDCK CELLS UNDER TRYPSINFREE CONDITIONS If considering a potential utilization of U-MDCK cells for influenza vaccine virus production, it would be interesting not to work in presence of a proteolytic enzyme, in order to eliminate a potential source of viral and microbial contamination. 3.3.1. Selection of trypsin-independent influenza viruses Trypsin-independent viruses were selected by serial passages in U-MDCK cells, in absence of trypsin and under selected MOI. Such a selection was performed, starting from three different kinds of viruses : 1) viruses after five passages with trypsin, 2) viruses after ten passages with trypsin and 3) viruses grown in allantoic fluid. All A and B influenza viruses were efficiently selected and serially replicated to high titre in absence of exogenous enzyme; The best results were observed when selection started from Three selection profiles emerged as a function of the easiness with which adaptation to trypsin-free conditions was performed but, with the exception of B viruses which adapted quite immediately, it was impossible to attribute the two other profiles to a specific A subtype. 3.3.2. Biological, structural and antigenic properties of selected trypsin-independent influenza viruses Trypsin-independent influenza viruses replicated efficiently in U-MDCK cells, as shown by to HA ratio analysis of A and B viruses grown in either U-MDCK cells or embryonated eggs ; for example 2.5×103 and 0.7×103 for A/Vic/36/88 (H1N1) viruses grown in U-MDCK and eggs respectively, 2) by comparative plaque efficiency of control viruses and selected trypsin-independent viruses when tested with / without trypsin in agar overlay. In absence of trypsin, control viruses gave small size plaques

(0.5-1 mm in diameter) while selected, trypsin-independent viruses gave plaques whose diameter (2-3 mm) was shown similar to that of control viruses in presence of trypsin. It is interesting to notice, that the hemagglutinin of trypsin-independent viruses was likely cleaved at the stage of entry in cells, since PAGE of all different A and B viruses clearly confirmed a very high amount of HA0 precursor when compared with virus grown in trypsin or produced in eggs. Such a cleavage of influenza virus hemagglutinin at entry in cells, was previously mentioned by Boycott et al (1994) when A/WSN/33 virus was grown in MDBK cells without addition of trypsin. The comparative analysis (figure 1) of antigenic reactivity (HI test) of the hemagglutinin of both, control and trypsin-independent viruses with anti-HA monoclonal antibodies and polyclonal antisera, showed significant differences which were consistent with conformational differences in HA; indeed, HI titre of some MAbs (H240 and H375) differed up to five dilutions when reacted with viruses obtained with/without trypsin.

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3.3.3. Production of trypsin-independent influenza viruses in U-MDCK cells grown on microcarriers Finally, selected trypsin-independent A and B influenza viruses were produced in UMDCK cells grown on microcarriers (porous/non porous) under rod-stirred conditions. Virus harvest was performed after 3-4 days as a function of virus type/subtype and titres were shown consistently higher when using non porous, uncoated microcarriers in comparison with porous, collagen coated ones : 20480-40960 HAU/ml and 2560-5120 HAU/ml respectively as mean values; as a consequence, mean production yield of A and B influenza viruses was 5 to 10 times higher when using the former. Studies are now in progress, in order to correlate this phenomenon with potential differences in the level of differentiation of U -MDCK cells as a function of microcarriers. In view of these data, MDCK cells grown in serum-free Ultra-MDCK medium appeared full of promise as an alternative substrate for influenza virus production in presence as well as in absence of trypsin. Acknowledgements This work was supported by a grant from Boehringer Ingelheim Bioproducts Partnership. 4. References Boycott, R., Klenk, H.D., and Ohuchi, M. (1994) Cell tropism of influenza virus mediated by hemagglutinin activation at the stage of virus entry, Virology 203, 313-319.2 Rott, R., Orlich, M., Klenk, H.D., Wang, M.I., Skehel, J.J., Wiley, D.C.0984) Studies on the adaptation of influenza viruses to MDCK cells, EMBO 3, 3329-3332. Seabright, M.A. (1971) Rapid banding technique for human chromosomes, Lancet ii: 971-972.

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EVALUATION OF THE NEW MEDIUM (MDSS2N), FREE OF SERUM AND ANIMAL PROTEINS, FOR THE PRODUCTION OF BIOLOGICALS.

H. KALLEL1, P. PERRIN2 and O.-W, MERTEN3. 1

Institut Pasteur de Tunis. 13, Place Pasteur. B.P. 74. 1002 Tunis Belvèdère. Tunisie. 2,3 Institut Pasteur. Laboratoire des Lyssavirus2; Laboratoire de Technologie cellulaire3. 25, rue du Docteur Roux. 75 724 Paris Cedex 15. France.

Abstract: The development of media free of serum and animal protein is of utmost importance for increasing the safety of biologicals produced for therapy and vaccination. In order to reduce the risk of contamination, we have modified the serum free medium MDSS2, a very efficient serum free medium for the production of various biologicals including experimental vaccines using different cell lines (Merten et al. Cytotechnology 14 (1994), 47), by replacing the animal derived products by plant

extracts. This new serum and animal protein free medium (MDSS2N), can be efficiently used for biomass production of various cell lines : BHK-21/BRS cells, adapted to MDSS2N medium, grew slightly slower in this medium versus in the old formulation), whereas the growth of Vero cells was not influenced by this modification Cultures of Vero cells on microcarriers in stirred tank reactor did not show any differences with respect to the growth rate when the old and the new formulation of MDSS2 were used. A and were observed when MDSS2 and MDSS2N were

used respectively. The use of MDSS2N for cell culture and the production of various biologicals like rabies virus will be discussed.

1. Introduction The use of serum containing media for the production of biologicals has many

disadvantages like varying quality, high cost, risks of contamination with mycoplasma, virus, BSE agent, etc. Thus today, different serum free media had been formulated and are used for the culture of various cell lines. However, most of these media still contain animal derived proteins.

To overcome this problem, we developed a medium, frée of serum and any animal protein, (MDSS2N). This medium is essentially based on MDSS2, a serum free

medium, which we have previously developed and used for the culture of different cell lines (Merten et al. (1994) ; Perrin et al. (1995)). 561 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 561-568.

© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

562

In this work, we present the evaluation of this new medium, to support the growth of various cells lines, like BHK-21/BRS and Vero cells. In addition, we have evaluated the ability of MDSS2N medium to sustain the production of biologicals. In this context, rabies virus production by BHK-21/BRS cells grown in MDSS2N medium, is studied as a model. 2. Materials and Methods Cell lines : Vero cells and BHK-21/BRS cells, derived from BHK-21 C13 and adapted to suspension growth, were used (Perrin et al. (1995)).

Virus strain : The rabies virus PV-Paris/BHK-21 and PV-Paris/BHK-21/BRS were used to infect the BHK-21/BRS cells (Perrin et al. (1995)). Culture Medium : DMEM (Axcell Biotechnologies 36213 SPM) + 5% FCS (Hyclone A-1115-L), MDSS2 (Axcell Biotechnologies 39615) and MDSS2N (Axcell Biotechnologies 39615N) were used. Growth assay : Cell culture assays were performed at in 25 flasks, at an initial concentration ranging between and cells/ml. Samples were taken daily to determine cell concentration. The assays were performed in duplicate. Virus production : BHK-21/BRS cells were infected by two strains of rabies virus (PVParis/BHK-21 and PV-Paris/BHK-21/BRS) at a cell concentration of cells/ml

with a MOI of 0. 1/cell. Virus production was performed at

in 50 ml agitated

tubes containing 10 ml of the medium to be tested. Samples were taken daily to determine cell concentration, virus titre and glycoprotein content. The assays were performed in duplicate. Bioreactor cultures : The cultures were performed in a 2 litres bioreactor (1.6 1 working volume) equipped with a spin filter fixed on the axes for the retention of cells growing on microcarriers. The following conditions were applied: air-saturation, and agitation =45 rpm. Cells were grown on microcarriers : Superbead (Flow 60-085-12) or Cytodex 1 (Pharmacia 17-0448-01) at a concentration of 2.5 g/1, 5 g/1 and 6.25 g/1. The cultures were performed in perfusion mode. Perfusion was started when the cell density was about

Cell counting : For BHK-21/BRS cells growing in clumps, 0.5 ml of a trypsin/versene solution (50/50 mixture of a 0.1% trypsin-solution in PBS with a 0.04% versenesolution in PBS) was added to 0.5 ml of a cell suspension. After incubation at for 10 minutes, cells were stained with trypan blue (0.2% in PBS) and counted. Vero cells were washed with PBS then trypsinized at for 5 to 10 minutes and counted. For

563

Vero cells grown on microcarriers, they were treated with 0.5 ml 0.1M citric acid containing 0.1% crystal violet then incubated at for at least one hour, the released cell nuclei were counted. Rabies virus titration : Virus titres were determined according to a modified RFFIT method (Smith et al. (1973)) and expressed in Fluorescent Focus Units per ml (FFU/ml).

Glycoprotein titration : rabies glycoprotein content was determined by EL1SA using polyclonal antibodies (Perrin et al. (1990)). 3. RESULTS AND DISCUSSION

3.1. Growth in serum and animal protein free medium : 3.1.1. BHK-21/BRS cells: MDSS2N has been tested for supporting the growth of BHK-21/BRS cells in comparison to MDSS2. Tests have been performed in T-flasks. The results obtained show that the maximal cell density of BHK-21/BRS cells obtained was cells/ml and cells/ml, for MDSS2N and MDSS2, respectively (figure 1). The average specific growth rates were 0.02 and in MDSS2N and MDSS2, respectively. These results indicate that BHK-21/BRS cells exhibit similar growth kinetics in the new protein-free medium MDSS2N as in the previously developed

medium MDSS2.

3.1.2. Vero cells: MDSS2N has been also evaluated for its ability to sustain the growth of Vero cells in static (T-flasks) and agitated (micro-carrier in bioreactor) cultures. After 3 successive subcultures of Vero cells in MDSS2N and MDSS2, the maximal cell density reached was and in MDSS2N and MDSS2,

564

respectively (figure 2). With regard to the average specific rate, we obtained 0.0153 and in MDSS2N and MDSS2, respectively. These results show that Vero cells growth is equal in both media tested. MDSS2N supports Vero cells growth as MDSS2 does.

For the reactor cultures different cell densities have been obtained, when three different media were used. The maximal cell densities were and when DMEM + 5% FCS, MDSS2, and MDSS2N, respectively, were used (figure 3A). These differences were essentially due to the concentration of the microcarriers used. By comparing the average specific growth rates for these three cultures, it becomes evident that the cultures in both serum-free media showed a slightly higher growth rate than the culture done in DMEM + 5% FCS (table 1). In addition, by comparing the cell of microcarriers, it is evident that the differences between the three media used are very low with respect to the The cell number per increased rather parallelly (figure 3B), and the fact that the culture done in MDSS2N stopped earlier was due to the fact that the available surface was limited and saturated at 116 h and that this culture was a conducted as a batch and not as a perfusion culture.

565

3.2. Rabies virus production : To evaluate the capacity of the new medium MDSS2N for the production of biologicals like vaccines, we infected BHK-21/BRS cells (grown in MDSS2N or MDSS2) with two strains of rabies virus : PV-Paris/BHK-21/BRS (a strain adapted to MDSS2) and PVParis/BHK-21 (a strain non adapted to MDSS2). The results obtained shown in figures 4 A and 4B, indicate that the behaviour of BHK21/BRS cells infected with PV-Paris/BHK-21 or PV-Paris/BHK-21/BRS was relatively

566

equal in both media. In both cases, we observed a decrease of the cell density from to 5 days post infection. The maximal virus titres obtained in MDSS2N, after the infection by the strain PV-Paris/BHK-21, was (see table 2). In MDSS2, virus titres were 2.4 times higher. With regard to virus production after infection by PV-Paris/BHK-21/BRS strain, we obtained and in MDSS2N and MDSS2, respectively. Figures 4A and 4B also show that by using the non adapted strain (PV-Paris/BHK-21), the levels of glycoprotein, obtained at the end of the culture, were 330 ng/ml and 960 ng/ml, in MDSS2N and MDSS2, respectively. The specific productivity of glycoprotein was also lower in MDSS2N than in MDSS2 (table 3).

• : The values correspond to the ratio of final glycoprotein concentration cell density before infection ×107 : « immune » glycoprotein level was calculated by subtracting « the soluble » glycoprotein content from total glycoprotein. Soluble glycoprotein content was measured after centrifuging the medium at the end of the culture, at 40 000 rpm at and for 2 hours. Then its glycoprotein content was determined by ELISA.

567

On the other hand, the levels of glycoprotein obtained after the infection of cells by PV-Paris/BHK-21/BRS strain were 1700 and 3800 ng/ml, in MDSS2N and MDSS2, respectively (see figures 4A and 4B). MDSS2N seems to be less favourable for rabies virus production than MDSS2, this is probably due to the non adaptation of the strain PV-Paris/BHK-21/BRS to cells grown in the new serum free MDSS2N. Specific productivity of glycoprotein obtained in MDSS2N was cells whereas the production rate in MDSS2 was cells (see table 3). These values are comparable to the standard roller process and to those obtained by Merten et al. (1994). The normal productivity of the standard roller process ranged from 8 to 12

µg /107 cells.

The comparison of the « immune » glycoprotein levels obtained in the different conditions studied (see table 3) shows that the percentage of the « immune » glycoprotein is slightly higher after the infection with PV-Paris/BHK-21/BRS strain in

568

MDSS2N or MDSS2, than after the infection with the strain PV-Paris/BHK-21. Nevertheless, MDSS2N seems to enhance the soluble glycoprotein production. 4. CONCLUSION

MDSS2N supported BHK-21/BRS cells growth as well as MDSS2 did. Vero cells also exhibited similar growth in MDSS2N and MDSS2. Continuous cultures performed in perfusion mode of Vero cells, have also shown that cell growth was not affected by the medium used. Rabies virus production seems to be influenced by the nature of the medium tested. MDSS2N induces virus and glycoprotein productions but lower than MDSS2 does. This is probably due to the non adaptation of rabies virus strains to cells grown in MDSS2N. However, the results obtained are very encouraging. Further experiments will be run to optimise rabies virus production in MDSS2N in batch and perfusion mode. 5. REFERENCES Merten, O.-W., Kierulff, 1, Castignolles, N. and Perrin, P. (1994) Evaluation of the new serum free medium (MDSS2) for the production of different : use of various cell lines, Cytotechnology 14, 47-59.

Perrin, P., Madhusudana, S., Gontier-Jallet, C., Petres, S., Tordo, N. and Merten, O.W. (1995) An experimental rabies vaccine produced with a new BHK-21 suspension cell culture process : use of serum-free medium and perfusion-reactor system, Vaccine 13, 1244-1250.

Perrin, P., Morgeaux, S. and Sureau, P. (1990) In vitro rabies vaccine potency

appraisal by ELISA : advantages of the immunocapture method with a neutralizing anti-glycoprotein monoclonal antibody, Biologicals 18, 321-330.

Smith, J. S., Yager, P. A. and Baer, G. M. (1973) A rapid tissue culture test for determining rabies neutralizing antibody, in M. M. Kaplan and H. Koprowsky (eds.). Laboratory Techniques in Rabies, WHO, Geneva/CH, pp. 354-357.

PRODUCTION OF HIGH TITRE DISABLED INFECTIOUS SINGLE CYCLE (DISC) HSV-2 FROM A MICROCARRIER CULTURE T.A. Zecchini, R.J. Smith. Cantab Pharmaceutical Research Ltd., 184 Cambridge Science Park, Cambridge. U.K. CB4 4GN

1. Introduction

Disabled Infectious Single Cycle (DISC) HSV, is a genetically inactivated HSV-2 from which the viral glycoprotein H (gH) gene has been deleted (1). Modified in this way, the release of the virus from the cell is not blocked. However, in non-complementing cells, the lack of glycoprotein H prevents the virus from re-infecting new cells, thereby blocking any further viral spread. The DISC

virus can only replicate and be propagated in a gH complementing cell line, which in our case is the modified Vero cell line CR2C9. The aim of this work was to evaluate the production of DISC HSV-2 in a microcarrier based culture system, compared to a roller bottle culture process. Anchorage-dependant cell lines (Vero, MRC5) have been propagated at low cell con-centrations

to produce human viral vaccines (2). Griffiths et at (3) have also demonstrated HSV-2 production from cells cultured on Cytodex microcarriers, yielding virus

titres similar to those expected from a roller bottle system. The work set out to determine the conditions for the optimum growth of cells and ultimately,

production of high titre virus. Production of the virus was initially determined in small-scale

(1 litre) cultures. Scale-up of the production system was then demonstrated at the 15 litre scale. 2. Small scale production – 1 litre scale 2.1 METHODS After extensive investigation, Cytodex 1 was selected as the most suitable microcarrier for DISC HSV-2 production. The growth medium used was Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 5% Foetal Bovine Serum. Cultures were inoculated at approximately 10 CR2C9 cells per microcarrier (Fig 1) and were maintained at 37°C throughout the growth period. Cell growth was monitored by counting nuclei released from a sample incubated in 0.1M citric acid containing 0.1% (w/v) crystal violet (4). Glucose and lactate concentrations were controlled by partial media changes on days 2 and 3 of the growth period. Prior to infection with DISC HSV-2 the cell cultures were washed with complete Dulbecco’s PBS to reduce contaminating FBS levels. The culture medium for the infection stage was serum free (DMEM). Confluent microcarriers (Fig 2), approximately 100 hours post inoculation, were infected with DISC HSV-2 at an MOI of 0.01. The temperature during the infection period was decreased and maintained at 34°C Infected cells (Fig 3) were harvested from the microcarriers approximately 60-72 hours post infection by removal of the media

followed by the addition of hypotonic saline. The detached cells were separated from Cytodex 1 by filtration through a sterilisable 569 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 569-571.

© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

570 The DISC HSV-2 virus was then released from the cells using a low pressure disruption technique (Fig 4). Virus titres were calculated using an in-house TCID 5 0 assay with subsequent conversion to 2.2 CULTURE RESULTS Typically we have achieved

total pfu from a 1 litre

culture, with approximately a 20 hour period during which the culture can be harvested without loss of titre achieved. From an equivalent number of 25 roller bottles we routinely achieve total pfu (Table 1). Therefore the two systems yield similar DISC HSV-2 titres. 3. Large scale production – 15 litre scale

3.1 METHODS

Cultures were inoculated at a density of approximately 10 CR2C9 cells per microcarrier and 5g of The growth period was maintained at a temperature of The growth medium used was DMEM with 25mM HEPES (5% FBS). Cell growth was monitored by counting released nuclei as for the small scale cultures. Glucose and

lactate concentrations were controlled by partial media changes on days 2,3,4 and 5 of the growth period. The culture was maintained for an additional 48 hour period compared to the small scale cultures in order to provide a maximal cell density at the point of infection.

Prior to infection with DISC HSV-2 the cell cultures were washed with complete Dulbecco’s PBS to reduce contaminating FBS levels. The culture medium for the infection stage was changed to serum free DMEM and the temperature was decreased to Cultures were infected with DISC HSV-2 after approximately 140-160 hours at an MOI of 0.01 The i n f e c t e d cells were harvested from the m i c r o c a r r i e r s approximately 60-72 hours post infection with DISC HSV-2 by the addition of hypotonic saline. The detached cells were separated from the microcarriers by filtration through a sterilisable mesh. The

low pressure disruption technique (Fig 4) was again used to release DISC HSV-2 virus from the cells. Virus titres were calculated using an in-house assay with subsequent conversion to

571 3.2 CULTURE RESULTS From this size culture we would expect to achieve approximately total DISC HSV-2 pfu (Table 1). However, this culture produced total DISC HSV-2 pfu. This high titre was able to be recovered over a sustained period of at least 9 hours. Our results, therefore, compare very favourably with the expectations from roller bottle cultures and the results achieved from the 1 litre cultures.

4. Summary

Cytodex 1 microcarriers can be used for the production of our DISC HSV-2 virus. Yields achieved were comparable to those obtained in roller bottle culture systems.

The process can be scaled up demonstrating increased yields in the 15 litre culture compared to those obtained in a I litre culture. DISC HSV-2 titres for the 15 litre culture exceeded the

anticipated titre for the equivalent number of roller bottles. A 15 litre culture has been shown to produce an equivalent amount of virus as roller bottles. This reduces the labour necessary, minimising costs of materials and process time. The virus is produced in a single batch culture with one set of aseptic manipulations. References 1.

M.E.G. Boursnell, C. Entwisle. D. Blakeley, C. Roberts, I.A. Duncan, S.E. Chisholm, G.M. Martin, R. Jennings, D.Ni Challanain, I. Sobek and C.S. McLean (1997). A genectically inactivated Herpes Simplex virus type 2 (HSV-2) vaccine provides effective protection against primary and recurrent HSV-2 disease. J. Infect. Dis; 175: 16-25.

2.

L. Fabry, B. Baijot, E. D'Hondt, M. Duchene (1989). High density microctirrier cell culture for viral vaccine production. Advances in animal cell biology and technology for bioprocesses; 361 - 365.

3.

B. Griffiths, B. Thornton, 1. McEntee (1980). Production of Herpes viruses in microcarrier cultures of human diploid and primary cluck fibroblast cells. Eur. J. Cell Biol; 22: 606,

4.

K.K. Sanford, W.R. Earle, V.J Evans et al (1951). J. Nat. Cancer Inst; 11 : 773 - 795.

5.

Glas-Col Apparatus Company, 711 Hulman Street, P.O. Box 2128, Terre Haute, IN 47802, USA

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NEW FORM OF ADMINISTRATION

THE

LIVE

MEASLES

VACCINE

FOR

ORAL

E.A. NECHAEVA. E.A. KASHENTSEVA, A.P. AGAFONOV, N.A. VARAKSIN, T.G. RYABICHEVA, A.P. KONSTANTINOV, V.N. BONDARENKO, T.D. KOLOKOLTSOVA, I.V. KITS, T.YU. SEN’KINA, N.V. ZHILINA

Research Institute of cell cultures. State Research Centre of Virology and Biotechnology , Koltzovo, Novosibirsk region, Russia

1. Introduction The live measles vaccine based on measles virus strain Leningrad-16 (L-16) and Japanese quails embryonic fibroblasts are now used in Russia for the prophylaxis of measles. The vaccine possesses high immunogenicity and epidemiological efficacy, low reactogenicity, and meets the WHO’s requirements to live measles vaccines. However, the primary cell culture that lacks standard biological and genetic characteristics is used in the production of this vaccine, and its administration does not exclude a possibility of HIV and hepatitis infection during vaccination. In addition, production of the vaccine requires considerable expenses at the stages of preparation and sterilization of ampules, pouring of the preparation, sealing, preparation and control of the solvent. In this connection, the development of oral forms of the measles vaccine is a topical problem, since oral immunization is the most simple, sparing, untraumatic, and physiologically based method of population vaccination. Besides, penetration of the antigen in lymphoid tissue of the digestive tract and formation of specific secretory immunoglobulins, which arc an additional protective factor at the level of infection “entrance”, are characteristic of oral immunization.

The goal of this work was to study the immunogenicity and innocuousness of encapsulated form of the live measles vaccine in measles virus-sensitive model, rhesus monkeys. 2. Materials and methods Measles virus strain L-16, isolated in Russia in 1960 from the blood of a measlesinfected child [1], was used for vaccine production. The Japanese quails embryonic 573

O.-W. Merten et at. (eds.), New Developments and New Applications in Animal Cell Technology, 573-575. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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fibroblasts (JQEF) culture from lcukosis-free farm of the SRC VB “Vector” were used as cell substrates. JQEF culture were multiply infected with suspension of the measles virus at a dose of 0.01-0.05 1g TCID50/cell. Sterile virus-containing liquid from different bottles with specific activity not less than 5.0 1g TCE50/0.5 ml was united, supplemented with stabilizer, and lyophilized. The dry virus-containing material was placed in solid gelatine capsules, the capsules were then covered with acid-resistant layer.

In accordance with the requirements of the State Pharmacopoeia, the vaccine samples were certified according to their physical-chemical characteristics, innocuousness for laboratory animals, and contents of foreign microorganisms [2]. Specific activity of the measles virus was determined by its cytopathic effect in Vero cell culture; contents of foreign viruses, mycoplasmas. and mycobacteria was estimated with techniques approved by the Pharmacopoeial Committee of Russia [3]. Immunogenic characteristics of the vaccine were tested in monkeys (rhesus macaques) by oral introduction of the encapsulated form of the vaccine at a dose of 100,000 TCE50 of measles virus and administration of injection form of the vaccine at a dose of 20,000 TCID50 of measles virus. Blood was collected form the animals on days 0, 1, 3,

5, 7, 9, 14, 21, 28, and 48 post immunization. Humoral immunity was assayed by hemagglutination retardation reaction (HARR) with 2 AU of purified measles virus and EL1SA [4, 5]. Virus-specific IgA in monkey saliva was determined by technique described in [6] using modified EL1SA.

3. Results and discussion Experimental batches of encapsulated form of the live measles vaccine were produced by the technique which we developed. Specific activity of the vaccine batches amounted to 3.5-4.5 1g TCID50 of measles virus per capsule. The vaccine samples did not contain any pathogenic bacteria, mycoplasmas, cytopathogenic and hemoadsorbing viruses. Capsules with the vaccine with covering did not dissolve in an acid medium over and dissolved in a weakly alkaline medium over 1-1.5h.

Trials of the encapsulated vaccine in monkeys demonstrated marked specific changes of immune system in the experimental animals. The data on virusemia and humoral and secretory immunity in monkeys immunized with different forms of measles vaccine are listed in Table 1. Measles virus was revealed in blood mononuclears on days 7-9 post immunization. While estimating secretory immunity, specific IgA at titers 1:8-1:16 were detected in saliva samples of all monkeys immunized with the capsulated form of the vaccine and were absent in animals vaccinated with the measles vaccine subcutaneously. Application of the vaccine also resulted in the induction of the specific antibodies in all animals, as determined by HARR and ELISA. The increase in antibody titers was recorded starling from day 21 post immunization; 8-16-fold growth in antibody tilers, by days 28-48. Increased proliferative activity of lymphocytes after their stimulation with measles virus antigen was recorded in all animals; by days 28-48, this index

575

significantly exceeded the initial level of lymphocyte activity (day 0). Thus, a clone of the cells that responded with increased proliferation to the encounter with the measles antigen was formed in these animals.

Thus, the studies performed demonstrated that the encapsulated form of the live measles vaccine caused stimulation of humoral, secretory, and cell-mediated immunity in the vaccinated animals, which manifested itself in the induction of antibodies in blood serum and saliva and formation of the clone of memory cells which acquired the ability to transform into blastic forms on the repeated in vitro encounter with the antigen. 4. Conclusion The technology developed in the SRC VB “Vector” made it possible to create the encapsulated form of the live measles vaccine for oral administration. The vaccine no contains pathogenic bacteria, alien viruses, mycoplasmas, and in the studied parameters meets the requirements of the national control institution of Russia to measles vaccines. Immunization of monkeys with the encapsulated form of the vaccine of measles viruses results in the induction of humoral, secretory, and cell-mediated immunity.

5. References 1 2. .1 4.

5. 6.

Taros, Yu. L. (1967) An attempt of direct isolation of measles virus from monolayer cultures of guinea-pig kidney tissue and chicken embryonic fibroblasts, Voprosy Virusologii 4, 399-402 The State Pharmacopoeia oft he USSR (1987), Moscow. Pharmacopoeial Article 42-179 VS-94 (1987), Dry cultural live vaccine. Voller A., Bidwell, D., and Bartlett, A. (1976) Microplate enzyme immunoassays for the immunodiagnosis of virus infections. In: Rose, N.R. and Friedman, II. (eds.). Manual of clinical immunology Washington, pp. 506-512. Norrby, E. and Gollmar, Y. (1975) Identification of measles virus-specific hemolysis inhibiting antibodies separate from hemagglutinin-inhihiting antibodies, J. Infection and Immunity 11, 231-239. Fridman M. (1982) Radioimmunoassay for detection of virus-specific IgA antibodies in saliva, J. Immunol. Methods 54, 203-205.

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STUDY OF LENINGRAD-16 VACCINE STRAIN OF MEASLES VIRUS REPRODUCTION IN CELL CULTURES PERSPECTIVE FOR BIOTECHNOLOGY

E.A. NECHAEVA, T.N. GETMANOVA, T.Y. SEN’KINA, N.D. YURCHENKO Research Institute of cell cultures, State Research Centre of Virology and Biotechnology Koltzovo, Novosibirsk region, Russia

1. Introduction L-16 strain measles virus and primary cell culture of Japanese quail embryo fibroblasts (QEF) are used in Russia for live measles vaccine production. Some heterogeneity of this culture and the possibility of contamination during the embryo

tripsinization gave rise for search of another more safe and uniform cell substrate. During the last few decades much attention was paid to the use of human diploid cell cultures in immunologicals production, because they are characterized by normal karyotypes and uniformity, have no tumorogenecity, can be subjected to kryoconservation [1-3]. Human diploid cell culture L-68 could be just the same one to improve the technique of live measles vaccine production. The aims of this study were to compare the main characteristics of virus production and morphogenesis in infected cell cultures which are the most promising for biotechnology, using equal conditions of cell cultivation and infection; to estimate the sensibility of L-68 diploid cell to measle virus infection depending on the passage level number; to receive data allowing to choose the most promising cell substrates for improving the current measles vaccine technology.

2. Materials and methods.

Cell cultures. Vero cells (an African green monkey continuous cell line) was obtained from Flow Laboratories. Continuous cell line 4647 produced from the kidney of an Afrikan green monkey, was obtained from the Institute of Virology of Russian Academy of Medical Sciences. Primary cell culture of quail embryo fibroblasts (QEF) was produced from 10-day-old embryos of Japanese quails certified by the Slate Institute of Standardization and Control of Medical and Biologi-

cal Preparations (SISC). Diploid cell culture L-6H, produced from the lung of 11577

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week-old human embryos, was obtained from the Research Institute of Viral

Preparations of Russian Academy of Medical Sciences, then certified according to the WHO system by SISC and used at the 21st, 23rd and 29th passage level. Measles virus. Vaccine strain Leningrad-16 measles virus, certified as a working bank for live measles vaccine production by SISC, was used. Confluent cell monolayers grown on glass cover slips were inoculated at a multiplicity of infection of 0,001 TCD50\cell. Pared control and infected samples were prepared for electron microscopy daily from 1 to 10-13 days post infection. Cultural liquid samples were tested for virus specific activity by titration on Vero cell culture. 3. Results and discussion Electron microscopy data revealed that all cell cultures under study were free from any contaminants.

After inoculation an active virus reproduction took place in the cell cytoplasm with development of typical changes in cell ultrastructure (4). The surface compartments of the infected cell cytoplasm contained the short, brunched and diluted cysternas of endoplasmatic reticulum. The density of the ground substances of the

cytoplasm was increased by diffuse implantation of ribosomes. Within the infected cells numerous viral nucleocapsids assembled and formed inclusions; sometimes nucleocapsids were found in cell nucleus, too. Cytoplasmic nucleocapsids were thicker and covered by "fuzzy" coat, while those located in the nucleus had a "smooth" surface. Giant multinuclear cells and syncytia were scattered over the monolayer. In Vero cell culture the infection had the most active character: syncytia were detected in 3 days post infection and reached the maximum size. In QEF culture the infected cells were revealed more later (in 5-6 days), multinuclear cells were formed in 7 days, syncytia and viroplasma had smaller sizes. Cell culture L-68 studied at different passage had no differences in virus morphogenesis.

Measles virus released from the cell surface by budding, accumulated in intracellular space, revealed the typical form and structure and ranged from 150 to 300 nm in diameter.

Specific virus activity measurement data were in accordance with results of virogenesis ultrastructural study. All investigated cultures were characterized by high values of the virus output, which confirmed their high sensitivity level. At the same lime they differed from

each other in terms of determination of the time of reaching the maximum titres and the time of keeping them steady (Table), The earliest virus cytopathic effect and long period of the titers keeping steady were observed in the studied continuous and diploid cell cultures. Diploid cell culture L-68 was able to produce high amounts of measles virus on passages 21, 23 and 29 and to keep its output steady for a long period even in the experimental conditions without the cultural medium replacing. Taking into account the

genetic uniformity, lack of contaminants, availability for mass production and safety of using L-68 cell culture as the diploid one, the results obtained indicate the promising character of the diploid culture studied.

579

4. Conclusion Cell cultures under study promising for biotechnology provided the high virus output hut differed from each other by the moment of reaching the maximum titers and the time of keeping them steady at the same conditions of cultivation and inoculation. Human diploid cell culture L-68 possessed high productivity and sensitivity to measles virus infection, and could be used to improve the technique of live measles vaccine production. There were no differences in the measles virus morphogenesis and production level between various passages of the human diploid cell culture L-68, which allowed to use this culture in the passage level range recommended by the WHO for immunologicals production. 5. References 1.

Hayflick, L. (1989) History of cell substrates used for humanbiologicals, Develop. Biol. Stand. 70, 143146

2.

3.

Mirchamsy, B.H., Shafyi, A., Nazari, P., Ashtiani, M.P., Sassani, A. (1988) Evaluation of live measles vaccine prepared in human diploid cells for reimmunization, Epidemiol. and Infect. 101, 431-443.

Wood, D. and Minor, P . D . (1990) Use of human diploid cells in vaccine production, Biologicals 18, 143-146.

4.

Dubois-Dulg, N., Holmes, H.V., Rentier, B. (1984) Assembly of Enveloped RNA Viruses, Springer-

Verlag, Wien.

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RECENT ADVANCES WITH NEW VACCINE ADJUVANTS: PRECLINICAL TO CLINICAL DEVELOPMENT

FROM

T. VOSS SmithKline Beecham Pharmaceuticals Rue de 1’Institut 89 B-1330 Rixensart, Belgium

Abstract: Subunit, and recombinant subunit approaches to vaccine design have long been recognised for their potential safety. However, their efficacy (i.e.: immunogenicity) largely relies on the adjuvant systems used. The recent understanding of the role of effector cell mediated immunity in disease prevention and control have opened the way to an unprecedented search for novel adjuvant systems able to induce such cellular responses. This presentation will describe novel adjuvant systems developed at SmithKline Beecham Biologicals, and their use in preclinical as well as clinical development of vaccines against genital herpes, RSV, malaria and other diseases, as well as for the immunotherapeutic treatment of chronic diseases such as chronic HBV or cancer. Discussion

Aunins:

You said your GD antigen is produced in CHO cells and it is glycosylated. You have stressed the adjuvants, but I was wondering if you had seen any effects of the glycosylation structure on the antigenicity of the GD itself?

Voss:

We characterised the immunogenicity of our truncated glycosylated GD with monoclonal antibodies. We are quite sure, compared to the native glycoprotein present on the virus, that we have not seen differences using this technology.

Aunins:

You have stressed CMI response, yet one of your criteria for clinical trial inclusion was sero-negativity. Why did you pick seronegativity as opposed to an indicator of pre-existing cellular immunity?

Voss:

Sero-negativity was chosen because you want to be able to assess in detail the CMI responses induced by the vaccine. So in the phase II trial we had sero-positive and sero-negative individuals, but you will see a clear induction of this response only in the sero-negatives. For the efficacy trial it is clear because you need sero-negative subjects to be potentially infected during the course of the study 581

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Vazquez:

Do you have any experience with your adjuvants to obtain responses against tumour associated antigen of a carbohydrate nature?

Voss:

We have an on-going cancer programme, but I cannot discuss the results here. With carbohydrate antigens we do have data indicating that the adjuvants do work with that type of antigen.

Vazquez:

The big problem is to obtain these antigens with helper because they do not produce a memory.

Voss:

So you may want to couple these antigens to carriers to provide

help. Keck:

The field of adoptive immunotherapy has been around for manyyears, particularly the tumour infiltration lymphocyte, or CTL. In most of the cases, particularly melanoma and ovarian carcinoma, the results have not been that satisfactory. A number of laboratories, including NIH and Anderson, are getting into antigen presentation approach to this. Have you done any work on the in vitro or in vivo expansion of TIL cells or other immune cells?

Voss:

No. Our strategy is focused on adjuvant with sub-unit approaches.

Wurm:

A question about your strategy in the phase III trials. You have already vaccinated some 10,000 patients, or candidates, in phases I and II. What is the rate of infection when you have a partnership between HSV positive and negative individuals? Also, what is the time frame during which normal infection would occur in this instance, and what are the numbers that you need to vaccinate to get statistically relevant data?

Voss:

We are expecting efficacy results next year. The study was initiated

in 1995 which gives us a period of 3 years. We have incorporated about 800 couples in the multi-centre study. I cannot tell you the exact rate of infection, but our statisticians told us that we need to include that number of patients, and that time period, to get a statistically relevant outcome.

VACCINATION WITH RECOMBINANT SUICIDAL DNA/RNA P. BERGLUND1 M. FLEETON1, C. SMERDOU1, I. TUBULEKAS2, B.J. SHEAHAN3, G.J. ATKINS4 AND P. LILJESTRÖM1,5 Microbiology and Tumorbiology Center1 and Center for Biotechnology2, Karolinska Institute, Stockholm, Sweden. Department of Veterinary Pathology, University College3 and Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College4, Dublin, Ireland Department of Vaccine Research, Swedish Institute for Infectious Disease Control, Stockholm, Sweden 5

1. Introduction Efficient protection from viral disease has traditionally relied on the use of live attenuated vaccines, such vaccines being potent in generating broad humoral and cellular immunity. However, use of these vaccines has been hampered for reasons of biosafety. In contrast, killed or subunit vaccines are safe, but usually inadequate with regard to efficacy. Simplistically, design of novel vaccines should combine the efficacy of live or attenuated vaccines and the safety of subunit vaccines. A vaccine resulting in antigen presentation which mimics that during natural infection by the cognate pathogen would appear have the highest probability of success. The strategy is to achieve (i) effective protective immunity, (ii) a high population response, (iii) prolonged duration of immunity, (iv) high level of safety, (v) stable vaccine preparations which easily can be produced on large scale at low cost, (vi) easy to administer. During the last few years, the mechanisms by which antigens are presented by the major histocompatibility complex (MHC) class I and class II pathways (7, 11, 12, 15, 29), the role of natural killer (NK) cells (13, 14, 22) and the roles of cytokines and co-stimulatory factors in determining the balance of the immune response (10, 24, 30), have become better characterized. This knowledge has suggested ways whereby design of new generation vaccines may be approached, and many of these approaches have turned to use gene technology and viral vectors. Many different viral vectors have been used to express antigens in vivo in pursuit of new vaccines (8, 25, 27, 28). However, a problem is the expression 583

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of a large number of vector derived antigens which may negatively influence the specific immune response, and safety still remains an issue with many of these vectors. To circumvent these problems, immunization using naked DNA has recently been tested by a number of groups (9, 23). We have used a novel strategy of vaccine design and here describe vectors which are based on a self-replicating, suicidal, recombinant RNA, originating from Semliki Forest virus (SFV). In this system, genes encoding relevant antigens are cloned into vectors which upon vaccination will transiently express the antigen within the cells of the host. The SFV system allows efficient delivery of vaccines as infectious virus particles or as naked

nucleic acids (NA).

2. A Vector System Based On An Alphaviral Replicon

Alphaviruses such as Semliki Forest virus (SFV) are positive strand RNA viruses which have a very broad host range and infect mammalian, insect, reptile and even fish cells (31). Due to the organization of the RNA genome (Fig. 1), the viral RNA is on its own able to initiate productive replication when introduced into a cell, be it by normal infection or by transfection. The viral replicase will produce both new full-length RNA molecules but also a subgenomic RNA species which encodes for the structural proteins of the virus. Within a few days, the replication of the viral RNA will lead to cell death.

585

2.1 BASIC EXPRESSION VECTORS

For creating a general expression vector a cDNA copy of the SFV genomic RNA was modified by deleting the region for the structural genes and by replacing it by a polylinker for insertion of foreign sequences (20, 21). Later, other modifications of this strategy has allowed use of a variety of different strategies for expressi,ng genes or gene fragments (Fig. 2) (1, 6, 1619, 32). RNA produced in vitro by the SP6 RNA polymerase can be directly transfected into cells for expression of the cloned sequences.

2.2 A HELPER SYSTEM

Since transfection of cells is a rather inefficient process, a helper vector system was developed in which the structural genes of SFV were provided in trans (4, 20). When co-transfected with the formed recombinant vector RNA, co-replication resulted in production of infectious recombinant SFV (Fig. 2). Such particles only carry recombinant RNA molecules, since the very stringent packaging sequence of the virus is absent from the helper molecules. In consequence, infection by such recombinant SFV will result in only one round of virus RNA replication and expression of the foreign

sequences, while new virions cannot be formed in the absence of the structural genes of the virus.

2.3 DNA/RNA VECTORS To allow direct injection of DNA into host cells, yet another vector system was created. In this case the SP6 RNA polymerase promoter was changed to

the cytomegalovirus (CMV) immediate early promoter. Thus, when the plasmid is introduced into a cell, it will be transported to the nucleus where it will be transcribed by the host RNA polymerase II (Fig. 2). After transport to the cytoplasm this positive-strand RNA will be translated to produce the viral replicase which then drives the further replication of the RNA molecule and eventual production of the messenger encoding the foreign sequences. As is the case in all three strategies, replication and expression of the foreign sequences is transient and leads to cell death within a few days.

586

587

3. Vaccination The properties of the SFV replicon makes it potentially very promising for vaccine design: (i)Vigorous RNA replication combined with efficient translation leads to production of large amounts of antigen; (ii) The SFV

RNA functions directly as a mRNA, which allows naked nucleic acid delivery; (iii) The RNA is cytoplasmically self-replicating, with no risk of integration into the chromosome. Replication leads to cell death which further underlies the safety of the system and circumvents possible problems

related to tolerance; (iv) Large and multiple genes can be expressed simultaneously. This multisubunit, multiepitope expression possibility alleviates the need to define epitopes and/or haplotypes; (v) SFV has a broad host range and infects all animal cell types, including non-replicating cells; (vi) SFV is not wide spread, and therefore there is no preexisting immunity against the vector; (vii) Vector structural proteins are not made escaping an immune response against the vector itself.

3.1 INFLUENZA MODEL The SFV vaccine approach has been most extensively tested in the influenza model. The first experiments utilized injection of naked RNA encoding the

nucleoprotein of influenza A virus. Although naked RNA is expected to be quite unstable, good immune responses could be generated (33). From a more practical point of view, recombinant SFV particles or the SFV DNA/RNA vector approach would be of better value for vaccination. Both modalities of immunization have been tested in mice and shown that high

titers of antibodies to influenza HA and NP can be generated and that the antibody levels stay high for very long times. Similarly, strong cellular

immunity is also achieved with prolonged memory (2, 34). When comparing conventional DNA vaccine vectors to the SFV DNA/RNA vectors, we found that to achieve a 100% take significantly less DNA was required when using the SFV vector that using the conventional one. Moreover, despite the transient expression of the antigen from the SFV vector, frequency of

precursor CTLs were much higher than when conventional DNA vectors were used. The reason for this is not known at present, but may be due to induction of when the SFV RNA replicates in the host tissue (5). When mice vaccinated with either recombinant SFV or with SFV DNA/RNA vectors were challenged with influenza A virus, protection against death and disease was observed

588

3.2 FLAVIVIRUS MODEL Recombinant SFV particles expressing Louping ill (LIV) flavivirus antigens prM, E (spike proteins) and NS1 (nonstructural protein) were also used to

immunize mice. Again, strong humoral and cellular responses could be measured, including IgA, when a mucosal (intranasal) route was used. When mice immunized either intraperitoneally or intranasally were challenged with lethal doses of LIV, protection was observed. Interestingly, the best protection was obtained when only the NS1 gene was expressed, indicating that cellular immunity plays a significant role in protection from flavivirus infection. Most importantly, neuronal degeneration, present in all challenged mice that showed clinical signs, were absent in all mice that survived challenge.

3.3 HIV/SIV MODEL In a previous study employing recombinant SFV for vaccination of pigtail macaques, expression of the SIV envelope gp160 protein of PBj14 showed induction of Env-specific antibodies. When the macaques were challenged with a lethal dose of PBj14 SIV virus, the highly virulent character of this isolate resulted in killing of 75% of the control (unimmunized) animals within two weeks, whereas the animals which had received the SFV vaccine were completely protected (26). In a subsequent monkey trial animals were immunized with recombinant SFV expressing the Env of HIV-1. After vaccination and boosts the animals were challenged with an extremely high dose (10.000 MID100) of SHIV-4, a hybrid virus expressing the Env from HIV and the Gag-Pol from SIV. This model only allows measurement of viremia and cannot lead to AIDS. While all animals were infected the vaccinated animal group showed significant reduction in viral load, despite the high challenge dose (3). 4. References 1. Atkins, G. J., B. J. Sheahan, and P. Liljeström. 1996. Manipulation of the Semliki Forest virus genome and its potential for vaccine construction. Mol. Biotechnol. 5:33-

38. 2.

Berglund, P., M. Fleeton, and P. Liljeström. 1997. Immunization with recombinant Semliki Forest virus generates long-term cytotoxic T lymphocyte and humoral responses and protective immunity. Submitted. 3. Berglund, P., M. Quesada-Rolander, P. Putkonen, G. Biberfeld, R. Thorstensson, and P.

Liljeström. 1997. Outcome of immunization of cynomolgus monkeys with recombinant

589 Semliki Forest virus encoding human immunodeficiency virus type 1 envelope protein

and challenged with a high dose of SHIV-4 virus. AIDS Res.Hum. Retroviruses, In press. 4. Berglund, P., M. Sjöberg, H. Garoff, G. J. Atkins, B. J. Sheahan, and P. Liljeström. 1993. Semliki Forest virus expression system: production of conditionally infectious recombinant particles. Nature Biotechnol. 11:916-920. 5. Berglund, P., C. Smerdou, M. Fleeton, I. Tubulekas, and P. Liljeström. 1997. Vaccination with self-amplifying suicidal DNA induces protection against influenza. Submitted. 6. Berglund, P., I. Tubulekas, and P. Liljeström. 1996. Alphaviruses as vectors for gene delivery. Trends Biotechnol. 14:130-134. 7. Busch, R., and E. D. Mellins. 1996. Developing and shedding inhibitions: how MHC

class II molecules reach maturity. Curr. Opin. Immunol. 8:51-58. 8. Caruso, M., K. Pham-Nguyen, Y. L. Kwong, B. Xu, K. I. Kosai, M. Finegold, S. L. Woo, and S. H. Chen. 1996. Adenovirus-mediated interleukin-12 gene therapy for metastatic

colon carcinoma. Proc. Natl. Acad. Sci. USA. 93:11302-11306. 9. Donnelly, J. J., J. B. Ulmer, J. W. Shiver, and M. A. Liu. 1997. DNA vaccines. Annu.

Rev. Immunol. 15:617-648. 10. Grewal, I. S., and R. A. Flavell. 1996. A central role of CD40 ligand in the regulation of T-cell responses. Immunol. Today. 17:410-414. 11. Groettrup, M., A. Soza, U. Kuckelkorn, and P.-M. Kloetzel. 1996. Peptide antigen production by the proteasome: complexity provides efficiency. Immunol. Today. 17:429-435. 12. Koopmann, J.-O., G. J. Hämmerling, and F. Momburg. 1997. Generation, intracellular transport and loading of peptides associated with MHC class I molecules. Curr. Opin. Immunol. 9:80-88. 13. Kos, F. J., and E. G. Engleman. 1996. Immune regulation: a critical link between NK cells and CTLs. Immunol. Today. 17:174-176. 14. Lanier, L. L. 1997. Natural killer cell receptors and MHC class I interactions. Curr. Opin.

Immunol. 9:126-131. 15. Lehner, P. J., and P. Cresswell. 1996. Processing and delivery of peptides presented by MHC class I molecules. Curr. Opin. Immunol. 8:59-67. 16. Liljeström, P. 1994. Alphavirus expression systems. Curr. Opin. Biotechnol. 5:495500.

17. Liljeström, P. 1996. Alphavirus Vectors for Gene Delivery. In I. D. Dubé and M. Cantley (ed.), Gene Delivery Systems - An OECD Review, 109-118. 18. Liljeström, P. 1995. Recombinant self-replicating RNA vaccines, p. 173-180. In G. Gregoriadis, B. McCormack, and A. C. Allison (ed.), Vaccines: New Generation Immunological Adjuvants, vol. 282. Plenum Press, New York. 19. Liljeström, P., and H. Garoff. 1994. Expression of proteins using Semliki Forest virus vectors, p. 16.20.1-16.20.16. In F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. A. Smith, J. G. Seidman, and K. Struhl (ed.), Current Protocols in Molecular Biology, vol. 2. Greene Publishing Associates and Wiley Interscience, New York. 20. Liljeström, P., and H. Garoff. 1991. A new generation of animal cell expression vectors

based on the Semliki Forest virus replicon. Nature Biotechnol. 9:1356-1361. 21. Liljeström, P., S. Lusa, D. Huylebroeck, and H. Garoff. 1991. In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the 6,000-molecular-weight membrane protein modulates virus release. J. Virol. 65:4107-4113.

22. Lopez-Botet, M., L. Moretta, and J. Strominger. 1996. NK-cell receptors and recognition of MHC class I molecules. Immunol. Today. 17:212-214.

590 23 . McClements, W. L., M. E. Armstrong, R. D. Keys, and M. A. Liu. 1996. Immunization with DNA vaccines encoding glycoprotein D or glycoprotein B, alone or in combination, induces protective immunity in animal models of herpes simplex virus-2 disease. Proc. Natl. Acad. Sci. USA. 93:11414-11420. 24. Mosmann, T. R., and S. Sad. 1996. The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol. Today. 17:138-146. 25. Moss, B. 1996. Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety. Proc. Natl. Acad. Sci. USA. 93:11341-1138. 26. Mossman, S., F. Bex, P. Berglund, J. Arthos, S. P. O'Neil, D. Riley, D. H. Maul, C. Bruck, P. Momin, A. Burny, P. N. Fultz, J. I. Mullins, P. Liljeström, and E. A. Hoover. 1996. Protection against lethal SIVsmmPBj14 disease by a recombinant Semliki Forest virus gp160 vaccine and by gp 120 subunit vaccine. J. Virol. 70:1953-1960. 27. Palese, P., H. Zheng, O. G. Engelhardt, S. Pleschka, and A. Garcia-Sastre. 1996. Negative-strand RNA viruses: genetic engineering and applications. Proc. Natl. Acad. Sci. USA 93:11354-11358. 28. Paoletti, E. 1996. Applications of pox virus vectors to vaccination: an update. Proc. Natl. Acad. Sci. USA. 93:11349-11353. 29. Pieters, J. 1997. MHC class II restricted antigen presentation. Curr. Opin. Immunol. 9:89-96. 30. Shearer, G. M., and M. Clerici. 1997. Vaccine strategies: selective elicitation of cellular or humoral immunity? Trends Biotechnol. 15:106-109. 31. Strauss, J. H., and E. G. Strauss. 1994. The alphaviruses: Gene expression, replication, and evolution. Microbiol. Revs. 58:491-562. 32. Tubulekas, I., P. Berglund, M. Fleeton, and P. Liljeström. 1997. Alphavirus expression vectors and their use as recombinant vaccines - a minireview. Gene. 190:191-195. 33. Zhou, X., P. Berglund, G. Rhodes, S. E. Parker, M. Jondal, and P. Liljeström. 1994. Selfreplicating Semliki Forest virus RNA as recombinant vaccine. Vaccine. 12:1510-1514. 34. Zhou, X., P. Berglund, H. Zhao, P. Liljeström, and M. Jondal. 1995. Generation of cytotoxic and humoral immune responses by nonreplicative recombinant Semliki Forest virus. Proc. Natl. Acad. Sci. USA. 92:3009-3013.

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Discussion Hauser:

You mentioned the translational enhancer. Could you briefly state where it is located and the functional mechanism?

Liljeström:

The enhancer is actually overlapping the first 34 amino acid residues of the capsid gene. The structure proteins of the alpha viruses are made as a polyprotein. It is one open-reading frame which then self-cleaves into individual sub-units. It is a secondary structure with a stem loop and we think it stores the ribosomes. You cannot transfer it to a normal cDNA gene to enhance it. It has to be in the context of the alpha virus RNA.

Wurm:

You mentioned, as an advantage of the viral approach, the suicide mechanism. Is it something you think about as a FDA requirement, or what is your real argument about this?

Liljeström:

The argument about the conventional DNA vaccine is that you may have two disadvantages, which have not turned out to be true. One is induction of tolerance. Because of long term low expression levels of the antigen, you would induce tolerance against the vaccine. The other is that you would integrate the DNA into the chromosome. In cell culture we know that we can easily make stable cell lines and certainly DNA goes into the chromosomes, but does it have a carcinogenic effect? We do not know. I do not think that it will be a problem in conventional DNA vaccines. However, who can tell whether in 40 years someone will get a cancer from a paediatric vaccine. So I am trying to sell my model by the fact that we get rid of the nucleic acids.

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GROWTH OF GOAT ENDOTHELIAL CELLS FOR THE PRODUCTION OF A VETERINARY VACCINE MIRANDA P. M.1 MOREIRA J.L.1, CARRONDO M. J. T.1,2 1 - Inst. Biol. Exp. Tecn. /Inst. Tecn. Quim. Biol, Ap. 12, 2780 Oeiras, Portugal 2 - Lab. Eng. Bioq., FCT/UNL, 2825 Monte da Caparica, Portugal Abstract Goat jugular vein endothelial cells (cje), involved on the in vivo regulation of a large number of important physiological processes, are essential for the in vitro production of candidate heartwater vaccines based on its infection with Rickettsia Cowdria ruminantium. The main purpose of this work is the definition of the best culture system and its operational conditions for mass production of cje cells.The scale up of monolayer cultures was performed in cell growth surfaces from 9.6 to 6320 cm.2. The optimal inoculum concentration was 2*104 cells/cm2, leading to a maximum cellular density of 6 *104 viable cells/cm2 and specific growth rate of 0.014 h-1, independent upon the scale. These cells were also grown in 250 cm3 spinner flasks operated as batch cultures and several operational variables were studied: agitation rate, inoculum concentration, support type and concentration. The maximum cell concentration 9.5* 105 cells/cm3 was achieved at 40 rpm (with a specific growth rate of 0.023 h-1). 1. Introduction As described by many authors, the culture of endothelial cells often present changes in morphology [1,2,4], microstructure, size [6, 7] and biochemical activities [6] depending on the culture system and particular shear stress. The bacteria Rickettsia Cowdria ruminantium parasites in vivo the vascular endothelial cells of wild and domestic ruminants, provoking a disease (Heartwater) that is endemic in Sub-Saharan Africa and Caribbean [5]. Since a vaccine against the disease requires the production of the bacteria and this is, in vitro, a specific parasite of endothelial cells [3], the problem associated with the man production of these cells has to be overcome. Thus, the main goal of this work was the cultivation of endothelial cells under different culture systems and its comparison regarding cell man production. Other problems are the poor ability for the cells to proliferate in vitro and an uncontrollable phenotypic variability that led to many difficulties for culturing endothelial cells. 2. Materials and Methods Cje cells isolated from goat were obtained from Dr. Dominique Martinez (CIRAD/EMVT, Guadeloupe, France). Tissue cultured flasks, 6 well plates, Triple T-flasks, and Cell Fáctories were supplied by Nunc (Roskilde, Denmark) and Roller Bottles from Corning Costar (Badhoevedor, The Netherlands). The cells were maintained in Dulbecco’s Modified Eagle’s medium (DMEM), supplemented with 10% (v/v) foetal bovine serum (FBS), 2 mM L-Glutamine, 1% (v/v) Streptomycin / 593 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 593-595. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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Penicillin (all final concentrations and from Life technologies, Glasgow, UK). Bioreaction studies were performed in 250 spinner-flasks (Wheaton). Cell number was evaluated using the Trypan Blue dye exclusion method and a hemacytometer (Brand, Wertheim/Main, Germany). 3. Stirred Tank Studies

The growth of cje 102 cells was performed in different types of microcarriers (Figure 1) with an inoculum concentration of 105 cells/cm3. Cytodex 3 was the microcarrier that led to the highest cellular concentration, Cultispher G also

led to a good cell growth (but lower than cytodex 3) when

comparing with the other microcarriers tested. The growth of cje cells in all types of supports, specially the non porous, is limited by an aggregation process induced on the surface before confluency, leading to cell death since these cells are strictly anchorage dependent. The strength of this phenomena seems to be cell line dependent (data not shown). During the development of this work the cell growth was highly dependent upon both the inoculum concentration and the support concentration. In order to investigate the optimal operational conditions, a two step approach was used by varying one variable (inoculum or support concentration) maintaining the other constant.

The best ratio is 2 grams/l of Cytodex 3 per of inoculum (data not shown). In a second step the inoculum concentration was varied from maintaining the ratio (i.e.; The Cytodex 3 concentration was

concomitantly varied from 2 to The results are presented on figure 2. The maximum cell concentration was obtained with 6

g/l

of cytodex 3 and of inoculum.

The specific growth rate was , and the maximum cell concentration achieved was (viability always higher than 95%). Similar values were obtained for other cje cells with the some origin (data not shown). An alternative to stirred

595

tanks and the cell immobilised on the surface of supports are static cultures, which are operated in the absence of physical stresses. This work was performed in a large range of culture growing areas, from 10 to 6320 The initial part ofthe work involve the optimisation of the culture conditions: the optimal inoculum concentration of this leading to a maximum cell density of viable and a specific growth rate of (data not shown). 900 Roller bottles were also tested, the optimal operational conditions being 100 ml of liquid media, an inoculum of rotational rate of 12 rph.

and a

4. Scale-up in different culture systems As presented in figure 3, the maximum cell concentration achieved in the different culture systems is quite similar (average of 5 to Also the specific cell growth rate is independent upon the scale 5. Conclusions It is possible to grow goat jugular vein endothelial cells both in static and stirred cultures. In static the optimal inoculum concentration is this leading to 5 to 6 and a specific growth rate of not depending on the scale In stirred tanks (250 the optimal culture conditions are: 40rpm, of Cytodex 3 and an inoculum of leading to at a specific growth rate of 6. References 1. Ballerman B.J. & Ott M.J. (1995) Adhesion and differentiation of endothelial cells by exposure to chronic shear stress: A vascular graft model. Blood Purif; 13:125-134 2. Barbee K. A. (1995) Changes in surface topography in endothelial monolayers with time at confluence: influence on subcellular shear stress distribution due to flow. Biochem. Cell Biol. 73: 501-505 3. Bezuidenhout J.D. (1987) The present state of Cowdria ruminantium cultivation in cell lines. Onderstepoort J. Vet. Res., 54: 205-210 4. Davies P.F., Remuzzi A , Gordon E.J., Dewey C.F., Gimbrone M.A. (1986) Turbulent fluid sheai stress induces vascularendothelialturnover in vitro. Proc Natl. Acad. Sci. USA 83: 2114-2117 5. Kobold A.M., Martinez D, Camus E., Jongejan F. (1992) Distribution of Heartwater in the Caribbean determined on the basis of detection of antibodies to the conserved 32-Kilodalton Protein of Cowdria ruminantium J. Clin. Microbiology 1870-1873 6. Levesque M.J., Sprague E.A., Schwartz C.J., Nerem R.M. (1989) The influence of shear stress on cultured vascular endothelial cells: the stress response of an anchorage-dependent mammalian cell Biotec. Progress 5, 1: 1-8 7. Ott, M. J., Ballermann, B. J. (1995) Shear stress-conditionated, endothelial cell-seeded vascular grafts: improved cell adherence in response to in vitro shear stress. Surgery 3 : 334-339.

7. Acknowledgements The authors acknowledge the financial support from the European Union (CTT-634, DG XIII, VALUE) and PED1P Medida 5.3, Acção C (Projecto JT1) The authors are grateful to Dr. D. Martinez - CIRAD/EMVT, (Guadaloupe), Dr. Paul Bensaid - CIRAD/EMVT, (Montepelier) for the supply of the cells and Ms. Rosário Clemente (1BET) for technical support

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EXPRESSION IN INSECT CELLS OF THE MAJOR PARASITE ANTIGEN ASSOCIATED WITH HUMAN RESISTANCE TO SCHISTOSOMIASIS Argiro L.(1),Doerig C.(1),Liabeuf S.(2),Bourgois A.(1), Romette J.L.(2)* (1) U399 INSERM, UFR médecine La Timone, F 13385 Marseille Cedex 5 (2) DISP/UPR 9039 CNRS, CESB Case 925, F 13288 Marseille Cedex 9

Introduction Glyceraldehyde-3-Phosphate Dehydrogenase (G3PDH) is a key enzyme in the glycolytic metabolism and the production of energy . This probably explains why G3PDH was evidenced as a major therapeutical target in several parasitic diseases ; either as a vaccine candidate or as a target for chemotherapeutic treatments . Schistosoma mansoni G3PDH (Sm 37-G3PDH) is one of the main schistosome vaccine candidate (Wright et al ,1991 ; Bergquist, 1995) . The production of a recombinant Sm37-G3PDH has been performed to evaluate if this molecule is able to induce a protective immunity in animals and eventually in humans . The cDNA coding for Sm37-G3PDH has been cloned and sequenced (Dessein et al , 1988)(Goudot-Crozel et al, 1989) . In addition five B-cell and two T-cell epitopes have been localized on the molecule among which a major B-cell epitope has been evidenced. Different expression systems have been evaluated in respect with the production yield and the recombinant protein quality . Most of them have led to either a high production of insoluble material (Bacteria) or to an inactive enzyme (Yeast). A large amount of soluble rSm37-G3PDH with an excellent bio-reactivity have been obtained using the baculovirus-insect cell system . Construction of the transfer vector. Sm37-G3PDH cDNA was PCR-amplified from Sm37-lgt 11 clone prepared as previously described (Goudot-Crozel et al. 1989) with primers containing restriction sites: -Eco RI: 5’-CCGAATTCATGTCGAGAGCAAAG-3’ -Not I: 5’-GCGGCCGCTTATGCATGGTCGAC-3’) The amplified products were run on a 1.2% agarose gel electrophoresis and the 1.1 kB band was purified with the QiaQuick gel extraction kit (Qiagen). Ligation products were transfected in JM109 competent bacteria (Promega) and recombinant plasmide was extracted from positive colony by Wizard maxipreps (Promega). The purified fragment encoding for Sm37-G3PDH was digested with Eco RI and Not I and inserted into the pAcHLT-A baculovirus transfer vector (Pharmingen, BaculoGold System) This vector is a derivative of the pAcSGl vectors family which contain a six histidine tag to express recombinant protein as a polyhistidine-containing fusion protein . The presence of this N terminus tag allows the protein to be purified from a cleared cell lysate based on the high affinity of the nickel-nitrilo triacetic acid resin ( Qiagen NiNTA) for the protein carrying the 6X His tag . The protein bound to the resin can be eluted under very mild conditions. 597 O.-W. Merten el al. (eds.), New Developments and New Applications in Animal Cell Technology, 597-600. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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This pAcHLT-A /Sm37 plasmide was entirely sequenced on a Pharmacia Biotech apparatus. Production of recombinant baculovirus The insect cell line Spodoptera frugiperda , Sf9 , was cultured in TNM-FH medium (Sigma) supplemented with 10 % feotal calf serum (Sigma) at 27 °C . Following the BaculoGold system protocole the insect cells were co-transfected with the BaculoGold DNA and the recombinant transfer vector . BaculoGold DNA is a modified wild baculovirus DNA which contains a lethal deletion and cannot develop into viable virus by itself . Recombination between the flanking regions of the polyhedrin gene from the transfer vector and modified wild type baculovirus DNA therefore results in 100 % recombinant baculovirus DNA.

The recombinant virus stock was amplified by a single large culture of Sf 9 insect cells . A high titer virus stock was harvested rG3PDH Expression Comparative studies were achieved using the multiplicity of infection (MOI) and the host cell density at the time of the infection as variables. The results obtained demonstrated a significant impact of those parameters on the cell productivity. The highest level of expression was found with an high cell density and a low MOI. (5 to 10 times more than with high MOI and low cell density) Similar results have been recently confirmed by J D Yang and co-workers (JD Yang et al 1996). The insect cells derived from Trichoplusia ni , BTI-TN-5B1-4 were cultivated in suspension and in serum free medium EXCELL 405 (JRH Sciences) . An inoculum was produced in 300 mL spinner flasks (INTEGRA Biosciences) up to a cell density of 2.6X10E6 cells per mL . Then the inoculum was infected with the recombinant baculovirus , MOI = 0.1 , and incubated under low stirring speed condition during 3 hrs . After incubation the infected cell culture was transferred in a 3 liter working volume bioreactor (Cytoflow ,INCELTECH - France) and diluted with fresh medium down to a cell density of 5X10E5 cells per mL . In order to prevent the cells to aggregate 0.1% V/V Pluronic P68(Sigma) was added to the medium. The rSm37-G3PDH production was followed in real time evaluating the enzyme activity in the 3 mL samples collected from the bulk solution . An average of 130 mg / L of rSm37-G3PDH was routinely obtained.

Product concentration was evaluated by following the G3PDH activity according to the Ferdinand method (1964)

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Biochemical characterizations The open reading frame encodes a protein of 338 amino acids. The translation product of

the gene has a deducted molecular mass of 36,589 Da. The purified protein synthesized in baculovirus-insect cells system migrated in denaturing conditions at a position,relative to standard markers of approximatively 37 kDa, indicating that the full length protein has been produced. G3PDH isolated from others organisms has been described as a tetrameric assembly where each monomer has its own active site. The purified rSm37-G3PDH was eluted from a gel filtration column together with protein standards showing a molecular weight of approximatively 150 kDa indicating that most likely a tetrameric form of the protein has been extracted. This will be confirmed by cristallographic studies under investigation. The optimum pH was found at pH 9.2 using the DL-glyceraldehyde-3-phosphate as a substrate. Specific activity of the purified enzyme of 25 unit/mg was obtained. Immunological characterizations The fraction corresponding to the purified rSm37-G3PDH was clearly identified in western blot by an Alkaline phosphatase-NiNTA conjugate and by an antibody antiSm37-5 antibody mouse serum followed by an anti-mouse IgG-AP conjugate. Sm37-5 contains the major Sm37 B cell epitope. These results confirm that the purified molecule is the rSm37-G3PDH

In addition, rSm37-G3PDH was recognized by sera from S.mansoni infected subjects and its enzymatic activity was strongly inhibited by them: -the anti-Sm37-G3PDH IgG levels in sera from 77 adolescents living in a S.mansoni endemic area were evaluated. Most sera (63) contained anti-Sm37-G3PDH IgG(mean=9.8 microg ;S.E.M.=1.4). -the capacity of these sera to specifically inhibit the enzimatic activity of the purified rSm37-G3PDH was checked. A strong inhibition (78% as an average) was observed.

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Conclusion We have produced and purified a functional and active recombinant S.mansoni G3PDH . To the best of our knowledge, this report is the first to describe the quantitative production of biologically active rS.mansoni G3PDH . Given that IgG antibodies to the S.mansoni G3PDH are associated with resistance to infection in human, the biologically active rSm37-G3PDH may prove important as a component of an anti S.mansoni vaccine. Recombinant antigens isolated under nondenaturating conditions which retain biological activity as in this case should resemble the natural parasite antigen on the opposite to inactive or denaturated forms. With the availability of biologically active recombinant antigen, we are now in a position to test the effectiveness of rSm37-G3PDH in vaccination and protection experiments as well as to examine the type of immune responses stimulated by this antigen, alone or in combination with other molecule, in animal models.

Bibliography Balaban, N., Waithaka, H. K., Njogu, A. R. and Goldman R. (1995) J . Infectious Diseases, 172, 845850.

Bergquist, N. R., (1995) Parasitology Today, 11, 191-194.

Dessein, A. J., Begley, M., Demeure, C., Caillol D., Fueri, J., Galvao dos Reis M., Andrade, Z. A., Prata, A. and Bina, J. C. (1988) J. Immunol., 140, 2727-2736.

Callens, M. and Hannaert, V. (1995) Ann . Trop. Med. Parasitol., 89, 23-30. Goudot-Crozel, V., Caillol, D., Djabali, M. and Dessein, A. J. (1989) J. Exp. Med., 170, 2065-2080.

Waine, G. J., Becker, M., Yang, W., Kalinna, B. and Mc Manus, D. P. (1993) Infection and Immunity, 61, 4716-4723.

Wierenga, R. K., Swinkels, B., Michels, P. M. A., Osinga, K., Misset, O., Van Beeumen, J., Gibson,

W. C., Postma, J. P. M., Borst, P., Opperdoes, F. R. and Hol, W. G. J. (1987) EMBO J., 6, 215-221.

Wright, M. D., Davern, K. M. and Mitchel, G. F. (1991) Parasitology Today, 7, 56-58. Yang, J.D., Gecik, P., Collins, A., Czarnecki, S., Hsu, H.H., Lasdun, A., Sundaram, R., Muthukumar, G. and Silberklang, M. (1996) Biotechnol. Bioeng., 52, 696-706.

MODULATION OF CD4 EXPRESSION ON HELPER T LYMPHOCYTES AND U937 CELLS BY GANGLIOSIDE GM3 AND ITS DERIVATIVES D. HEITMANN, P. BUDDE*, J. FREY*, J. LEHMANN AND J. MÜTHING Institute of Cell Culture Technology, University of Bielefeld, P.O. Box 100131, 33501 Bielefeld, Germany *Department of Biochemistry, University of Bielefeld

Abstract

Ganglioside GM3 and a variety of chemically modified derivatives (e.g. lyso-GM3, de-Nacetyl-GM3, GM3-amides) have been assayed for their potential upon CD4 modulation on human peripheral blood lymphocytes and cells from the monocytic cell line U937. The data presented show specific ganglioside-mediated CD4 down-regulation from minor effects of about 14% fluorescence reduction to strong effects with a maximum reduction of

92% depending on oligosaccharide and ceramide composition of the assayed compounds. 1. Introduction

Gangliosides, sialylated glycosphingolipids (GSLs), are ubiquitous compounds of mammalian cell membranes. Gangliosides and some of their derivatives are known to exert various important biological functions [1-3].

The CD4 antigen is a 55 kD transmembrane glycoprotein present on T cells and cells of the monocyte/macrophage lineage. CD4 participates in the interaction with MHC class II molecules and is known as the receptor for HIV [4]. Recently, it was shown that gangliosides reduce surface expression of CD4 by inducing CD4-internalization and degradation [5]. In this study, HPLC purified ganglioside GM3 (the major GSL of human serum and lymphocyte membranes), a variety of its natural descendants (GM1, GD3) and a panel of chemically modified derivatives (Fig. 1) were employed to gain some insight into the structure-function relationship underlying ganglioside-induced CD4 internalization on T lymphocytes and U937 cells.

2. Materials and Methods

Natural gangliosides, sialic acids, neutral GSL GM3 from CHO cells, GM1 from human brain and LacCer from human granulocytes were isolated and purified by standard procedures [6]. GM3(NeuAc) and GM3(NeuGc) were separated by anion exchange chromatography using TMAE (trimethylaminoethyl)-Fracto601 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 601-605. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

602 gel (Merck, Germany) [7]. Bovine GD3 was purchased from Pallmann GmbH (München,

Germany), NeuAc and NeuGc from Sigma (Deisenhofen, Germany). Chemically modified GM3 GM3-ester and -amides were prepared from GM3(NeuAc) according to Lanne et al. [8], and GMS-derivatives Dl to D5 according to Nores et al. [9]. Permethylation of GM3(NeuGc) was carried out as previously described [10] and compounds were structurally characterized by FAB-MS [11]. Lymphocytes. U937 cell cultures Human peripheral blood lymphocytes were isolated by Lymphoprep (Nycomed Pharma, Oslo, Norway) density gradient centrifugation of whole blood. The CD4-positive human monocytic cell line U937 was cultivated in RPMI 1640 medium containing 10% FCS. Prior to incubation with gangliosides, cells were washed three times and resuspended in serum-free RPMI 1640. Antibodies Murine monoclonal anti-human CD4 antibody was purchased from DAKO (Hamburg, Germany); DTAF-conjugated goat anti-mouse antibody was obtained from Dianova (Hamburg, Germany). Flow cytometry

Prior to labeling with anti-CD4 antibody at +4 °C for 45 min, human peripheral blood lymphocytes and U937 cells were incubated with natural gangliosides, derivatives and related compounds (Fig. 1) with concentrations of 100 µM and 400 µM in

serum-free RPMI 1640 at 37 °C for 60 min. After staining with DTAF-conjugated secondary antibody at +4 °C for 30 min, fluorescence intensity was quantified by FACSanalysis (FacSORT, Becton Dickinson, Heidelberg, Germany).

3. Results and Discussion

Highly purified gangliosides, GM3 derivatives and related compounds (Fig. 1) were proved for their potential upon down-modulation of CD4 expression on U937 cells (Fig. 2)

and T lymphocytes (Fig. 3). Low CD4 down-regulation Only minor effects (max. 14%) were observed employing GM3 precursor LacCer, derivatives with modified sialic acid (GM3 derivative D1, GM3-methylester, GM3-amide), permethylated GM3 and sole sialic acids. Moderate CD4 down-modulation

CD4 down-modulation ranging from 25% to 65% on U937 cells and from 13% to 40% on T lymphocytes was observed after treatment of cells with native monosialogangliosides. The type of sialic acid (NeuAc, NeuGc) as well as the oligosaccharide moiety chain length seem to be of minor importance with regard to modulatory capacity. Strong CD4 down-modulation Strong effects concerning CD4 down-regulation occured by employment of disialoganglio-

side GD3 and GM3 derivatives D2, D3, D4 and D5 with a maximum reduction of 92% on U937 cells after incubation with D5, and 31% and 60% on T lymphocytes after incubation

with D5 and GD3, respectively. Structural requirements for these effects seem to be either a disialic character (GD3) or the loss of the fatty acid residue (GM3 derivatives D2, D3,

603 D4 and D5). In the latter case, N-acetylation of the sialic acid and/or the sphingosine moiety do not markedly influence the modulatory efficacy.

604

Suggested structure-Function relationship Results concerning compounds with structural modifications in the oligosaccharide moiety

(chain length, type, number and modifications of sialic acids) indicate the importance of

605 the structural integrity of at least one sialic acid molecule. These findings would fit with the hypothesis that gangliosides act by inducing conformational changes in the CD4 molecule [5] prior to its internalization and degradation. Due to their probable potential to pass the plasma membrane, the strong effects of GM3 derivatives D2, D3, D4 and D5 (see Fig. 1) lacking long chain fatty acids suggest the existence of an alternative until yet unknown mechanism of CD4 down-modulation.

4. References [I]

Igarashi, Y., Nojiri, H., Hanai, N., and Hakomori, S.-I. (1989) Gangliosides that modulate membrane protein function, Methods Enzymol. 179, 521-540. [2] Hakomori, S.-I. (1990) Bifunctional role of glycosphingolipids, J. Biol. Chem. 265, 18713-18716. [3] Zeller, C.B., and Marchase, R.B. (1992) Gangliosides as modulators of cell function, Am. J. Physiol. 262, C1341-C1355. [4] Sattentau, Q. J., and Weiss, R. A. (1988) The CD4 antigen: physiological ligand and HIV receptor, Cell 52, 631-633. [5] Saggioro, D., Sorio, C., Calderazzo, F., Callegaro, L., Panozzo, M., Bertons, G., and Chieco-Bianchi, L. (1993) Mechanism of action of the monosialoganglioside GM1 as a modulator of CD4 expression, J. Biol. Chem. 268, 1368-1375. [6] Ledeen, R. W., and Yu, R. K. (1982) Gangliosides: structure, isolation and analysis, Methods Enzymol.. 83, 139-191. [7] Müthing, J., and Unland, F. (1994) Improved separation of isomeric gangliosides by anion-exchange highperformance liquid chromatography, J. Chromatogr. 658, 39-45. [8] Lanne, B., Uggla, L., Stenhagen, G., and Karlsson, K.-A. (1995) Enhanced binding of enterotoxigenic Escherichia coli K99 to amide derivatives of the receptor ganglioside NeuGc-GM3, Biochemistry 34, 1845-1850. [9] Nores, G. A., Hanai, N., Levery, S. B., Eaton, H. L., Salyan, M. E. K., and Hakomori, S.-I. (1989) Synthesis and characterization of ganglioside GM3 derivatives, Methods Enzymol. 179, 242-253. [10] Ciucanu, I., and Kerek, F. (1984) Rapid and simultaneous methylation of fatty acids and hydroxy fatty acids for gas-liquid chromatographic analysis, Carbohydr. Res. 131, 209-218. [11] J., and Egge, H. (1990) Desorption mass spectrometry of glycosphingolipids, Methods Enzymol. 193, 713-733.

5. Acknowledgements This work was financed by the Deutsche Forschungsgemeinschaft SFB 223. The participation on the 15th ESACT Meeting was supported by the European Commission Directorat General XII.

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HUMAN SERUM IN LEUKOCYTE CULTURES PRODUCING HUMAN INTERFERON ALPHA

P.Mattana, L. Scapol, S. Silvestri, and G. C. Viscomi. Biotech.&Immunol. Dept., Alfa Wassermann SpA, Via Ragazzi del ‘99, 5 I-40133 Bologna, Italy Keywords: leukocyte alpha interferon, human agamma serum. ABSTRACT Interferon Alpha (IFNα) is an antiviral, antiproliferative and immunostimulant cytokine world-wide applied in the treatment of several viral and oncologic diseases. Among the different preparations of , namely leukocyte lymphoblastoid and recombinant LEis particularly relevant, since it is better tolerated and it does not induce any antigenic reaction to the administred drug. LEis produced through the induction by viruses, such as Sendai virus, of human purified leukocyte cultures, whose media contain human agamma serum (HAS). Any attempt to omit the use of HAS caused poor yield of LETherefore, the role of HAS was investigated testing different preparations, performing dose (HAS concentration)/response curves, and evaluating the importance of the presence of HAS during the time of the culture. No differences in production

were detected based on the different HAS preparations. It was evident the dependence of LEyield on the HAS concentration, even if some differences could be detected between different preparations. Optimal amount of HAS was depended on the cell concentrations. Reduction of HAS concentration was possible only after having started the culture at the optimized conditions. It could be concluded that an optimal concentration of HAS per cell can be defined and the presence of HAS is critical in particular at the beginning of leukocyte cultures, even before the addition of Sendai virus, suggesting that the main role of HSA could be to restore the capacity of purified leukocytes to produce rather than to support the expression during the culture. INTRODUCTION Alpha interferon a complex family of several proteins, is one of the most physiologically important defenses of leukocytes against foreign agents such as viruses and Several studies have been published indicating that pharmaceutical preparations, i.e. recombinant, lymphoblastoid and leukocyte interferon, LYand respectively) offer therapeutic benefits in patients with viral (1,2) diseases, hematologic and solid tumors. Among the pharmaceutical preparations '. does not induce antibodies to during therapy, since it is the most similar to the physiologically expressed while the other do. Furthermore, is effective in restoring response in patients who lost response following production of 607 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 607-612. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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The genes are transiently expressed only under appropriate stimuli, such as the addition of viruses, by primary leukocytes harvested from healthy donor blood donations and cultured in a Minimum Essential Medium (MEM) supplemented with gamma globulin-free human serum (human agamma serum, HAS). After six hours from infection reach a maximun steady state and decline in the remaining time of the culture, and in the meantime proteins are released in the culture supernatants.(3 ,4) The optimization of leukocyte culture parameters is critical to maximize the yields, since even minute changes could have significant effects. In this respect the role of HAS was investigated by testing different preparations, performing dose (HAS concentration)/response curves, and checking the HAS concentration with respect to the age and concentration of the leukocytes, and the culture time. MATERIALS AND METHODS HAS preparation - The tested HAS preparations were prepared by combining described procedures.(5 ,6) They were prepared starting from different plasma sources: fresh frozen (FF) plasma, plasma harvested from buffy coat pools and plasma collected from single buffy coats. In all the cases the sera were obtained by freezing/thawing the plasma or by adding 0.7 mg/ml of calcium chloride to the plasmas at 4°C over night. The removal of gamma globulins from the sera was obtained by precipitation at 4°C over night with

polyethylenglycol 6000 (PEG) at the concentration of 6% (w/v) or with 40% (v/v) of saturated ammonium sulfate. The precipitated gamma globulins were removed by centrifugation and when ammonium sulfate was used the solutions were also dialyzed. All the HAS preparations, after sterile filtration, were inactivated at 56°C for 30 min, stored at -20° C and thawed at 4° C just before use. The HAS preparations were tested on the basis of production following the Cantell’s procedure.(5) Human leukocytes, obtained from 24-hour-old or 48-hour-old buffy coats (BCs) after lysis of erythrocytes by 0.83% ammonium chloride treatment, were cultured at two different cell concentrations: 4x106 and 10x10 6 cells/ml at 37°C in a MEM containing HAS of different preparations and at the concentrations ranging from 0.2 to 2.2 mg/ml, according to what is specified in each experiment. The leukocyte cultures (each of 100 ml in round-bottom flask) were induced to produce by adding 100-200 IU/ml of as primer and after 2 hours by adding 150 haemmaglutinating units per milliliter (HAU/ml) of Sendai virus. After 4.5 hours the temperature was lowered to 30° C. The cultures were stopped after 18-20 hours and the produced was collected by centrifugation. Leukocyte cultures were also performed in the absence of HAS and by replacing HAS with 2.0 mg/ml of human serum albumin. During the culture time, changes in the cell concentration alone or in combination with modifications of HAS concentration were carried out by properly diluting the cultures with prewarmed MEM with and without added HAS. Changes in HAS concentration alone were performed by centrifuging the leukocyte cultures and resuspending the pellets in media with the new HAS concentration to be tested. concentrations were determined by evaluating the antiviral activity through the reduction of the cytopathic effect assay.(7 ,8) Statistical Analysis - The Student’s t test was applied. A p value was considered significant.

609

RESULTS The effect on the yields of the different HAS preparations are provided in Table 1, where the specific preparation procedures of each HAS lot can be deduced from inspection of column 1-5.

The reported yields show differences, which, however, are not statistically significant, therefore the remaining part of the experimentation was performed using only HAS-1 and HAS-2, which provided the highest titers. These two HAS lots, prepared with the same procedure, were analyzed by performing a dose/response experiment. The experimental points were fitted with the function where y represents concentration; x the HAS concentration; a,b,c, and d are constants (Figure 1). The results indicate that the interpolated curves of HAS-1 and HAS-2 had similar shapes; nevertheless, since the plateaus were reached at different concentrations, 1.5 and 3.53 mg/ml, respectively, the curves did not overlap in the range from 0.5 to 1.8 mg/ml of HAS.

Based on these curves, the concentrations of 1.5 and 2.0 mg/ml for HAS-1 and HAS-2, respectively, were chosen among those possible, since they are the closest values which allow . yields higher than 90% of the plateaus for both the HAS preparation. When the addition of HAS to the leukocyte cultures was omitted and when HAS was replaced by identical amounts of human albumin, significant reduction of the yields, and respectively, was obtained and these values

610

correspond to approximately 20% of the productivity in the presence of optimal amounts of HAS. The figure 2 shows the examination of the yields of induced leukocyte cultures, coming from 24-hour-old BCs (Figure 2A) and 48-hour-old BCs (Figure 2B) performed at and at cells/ml, in which HAS-2 was used at the concentration of 2.0 mg/ml and at 0.8 mg/ml, where the later value was obtained by reducing 2.0 mg/ml by the same factor used to diluted the cell concentration from

In the case of 24-hour-old BCs the results indicate that by keeping the ratio of HAS amount per cell constant at 0.2 ng/cell or increasing it at 0.5 ng/cell, a similar productivity of per cell can be achieved even at different absolute values of HAS. When this ratio was lowered to 0.08 ng/cell, as in the case of cells/ml cultures at 0.8 mg/ml, statistically significant reduction of yields were obtained (Figure 2A). A different behaviour in 48-hour-old leukocyte cultures can be appreciated, since at the ratio of HAS/cell of 0.5 ng/cell at the cell concentration of cells/ml a significant increase of yield respect to those obtained with 0.2 ng/cell both at and cells/ml was reached (Figure 2B).

When cells/ml cultures, whose leukocytes were obtained from 24- and 48-hourold BCs, were diluted down to cells/ml, after 4.5 hour from the beginning, and the HAS concentration was either maintained (1.5 mg/ml, since HAS-1 was used) or reduced proportionally to the cell concentration (0.6 mg/ml), the obtained yields did not show any significant differences, even when compared with the productivity

611

of a culture, whose HAS-1 and cell concentrations (1,5 mg/ml and cells/ml, respectively) were kept constant (Table 2). Reductions of HAS concentration under the optimal value of 2.0 mg/ml were performed during the culture to assess the importance of HAS concentrations in relation

to culture time (Table 3).

As controls of the conditions 2,3 and 4 leukocyte cultures were centrifuged at the same

time and resuspended in the initial HAS concentration. The following yields were obtained: 9,268 ± 2,352, 8,765 ± 2,964 and 8,125 ± 1,812 IU/million cells, respectively. The analysis of the results reveals that respect to the culture kept at the suboptimal HAS concentration for all the time differences in the yields are detectable even in those leukocyte cultures whose HAS concentration was at the optimal value for only 1 hour, even before the addiction of the stimulus to induce .. However the

differences became significant after 3 hours. DISCUSSION

synthesis by primary leukocytes under viral stimuli is a transient event, which does not take more than 6 hours from infection. After this point a downregulation mechanism takes place which strongly reduces any further

synthesis.(3,9) Therefore, since no leukocyte growth is expected in the described culture conditions, the production can be considered as a unique and irreversible event. In vitro synthesis does not require any specific medium component,

since has been also produced in absence of sera, amino acids and vitamins. (5,10) Nevertheless, when an optimization process is carried out to maximize the LEproduction by human leukocytes, several components of the culture medium

became important. In the present work the contribution of HAS to the improvement of the yield was studied. Even though the specific HAS components which play a relevant role in production are unknown, it can still be concluded from the data of Table 1 that the

choice of the precipitation agent, PEG or ammonium sulfate, and that the source of the plasma, fresh-frozen plasma, plasma from BC pools or plasma from single BCs, did not introduce any relevant differences in those HAS components, which could be essential for the production. While some of the differeces detected from lot to lot should be attributed to the quality of the plasma (donors, anticoagulants, storage conditions, transport) used for the HAS preparation.

612

This consideration was confirmed by the dose/response curves in Figure 1, where the yields are correlated with the quality of HAS preparations obtained by applying the same preparation procedure, thus allowing for each HAS lot the definition of the concentrations at which production is maximized at the minimal protein concentration in the leukocyte cultures. The main role of HAS in improving the yield cannot be attributed either to an enhancement of stability or to a protection of from aspecific proteolysis, since the yields obtained with human albumin in place of HAS were similar to those obtained in absence of any protein stabilizer. The optimal HAS concentration is related to the cell concentration and to the leukocyte age. It could be proportionally reduced, if more diluted cells are used and it has to be optimized on the basis of BC age, since leukocyte cultures coming from 48-hour-old BCs seems to require higher HAS concentration than that of 24-hour-old BC to maximize the yields (Figure 2 and Table 2). The importance of the use of HAS at the optimal concentration seems to be restricted to the early hours of the culture (Table 3). This result is conceivable, since, as was mentioned before, the mRNA expression in leukocytes is a fast event, which reaches the maximun in only six hours. In conclusion, production by human leukocytes is a unique and transient event, which is affected by even minute changes of the culture conditions. Among them the concentration and the quality of HAS, the age of BCs, and the cell concentration resulted to be important. From the data reported, it could stated that an optimal concentration of HAS amount per cell can be defined to maximize production depending on the HAS preparation and the age of BCs. Furthermore, the presence of HAS is critical at the beginning of leukocyte cultures, even before the addition of any stimulus to produce suggesting that the main role of HAS could be to restore the capacity of purified leukocytes to produce rather than to support the expression during the culture. REFERENCES 1

In Interferon, Priciples and Medical Applications, Baron, S., Coppenhaver, D.H., Dianzani, F., Fleishmmann, Jr, W.R., Hughes, Jr, T.K., Kimpel, G.R., Niesel, D.W., Stanton, G.J. and Tyring, S.K. Eds., The University of Texas Medical Branch of Galveston, Department of Microbiology, Galveston , TX, 1992. 2 Viscomi, G.C., Grimaldi, M., Palazzini, E., and Silvestri, S. (1995) Human leukocyte interferon alpha: structure, pharmacology, and therapeutic application, Medicinal Research Reviews 15, 445-478. 3 Hiscott, J., Cantell, K., Weissmann, C. (1984) Differential expression of human interferon genes, Nucleic. Acids. Res. 12, 3727-3746. 4 Pitha, P.M., Au, W.C. (1995) Iduction of interferon alpha genes expression, Semin. Virol 6, 151-159. 5 Cantell, K., H i r v o n e n , S., Kauppinen, K.L., Myllyla, G. (1981)Production of interferon in human leukocytes from normal donors with the use of sendai virus, Methods in Enzymol. 78, 29-38. 6 Macleod, A.J. (1991) Serum and its fractionation. In M.Butler (ed.), Mammalian Cell Biotechnology, IRL Press New York, pp 27-37. 7 Armstrong, J.A. (1981) Cytopatic effect inhibition assay for interferon: microculture plaque assay, Methods in Enzymol. 78, 381-387. 8 Berg, K., Hansen, M.B., Nielsen, S.E. (1990) A new sensitive bioassay for precise quantification of interferon activity as measured via the mitocondrial deydrogenase function in cells (MTT-METHODS), AMPIS. 98, 156-162 9 Tovell, D., Cantell, K . , (1971) Kinetics of interferon production in human leukocyte suspensions, J.Gen. Virol. 13, 484-489. 10 Goore, M.Y, Dickson, J.H., Lipkin, S., and DiCuollo, C.J. (1973). The production of Human Leukocyte Interferon in a Serum-free Medium, Proc.Soc.Exp.Biol. Med. 142, 46-49.

THE NEW TYPE OF IMMUNOMODULATOR

M.V.MEZENTSEVA*, V.A.MOZGOVOI, L.Yu.MOZGOVAYA, R.Ya.PODCHERNYAEVA. *N.F.Gamaleya Institute of Epidemiology and Microbiology, D.I.Ivanovsky Institute of Virolology Moscow, Russia

1. Introduction There is quite a number of substances (both synthetic or of plant origin) capable of modulating the

immune system of human beings and animals, either enhancing, or decreasing the interferons (IFN) and other cytokines production [3]. All of them are introduced s/c, i.v., i.p., per os or per

rectum. Interferons of different types

are synthesized by different categories of

immune competent cells. Earlier studies using monoclonal antibodies revealed that lymphocytes

and macrophages produced mostly type I IFN, while T-cells produced type II IFN [2]. It was exciting to study the production of endogenous IFN by normal murine cells belonging to different populations following Applicator usage. The ability to produce IFN may be used for assessment of the immune cell functional activity [5]. 2. Materials and methods

Applicator [4] is a disk (45-50 mm in diameter, 7 mm thick), made out of the biopolimer with biologically active metal suspension (Cu, Fe, Zn, Al, Co, Ni, Na, Li, Mg) with variable valency in the ionized high-spin condition. The structure of the metallic ingredients has similarity with that of the microelements in the active center of proteins.

A study was made using CBA mice and hybrids. Applicator was applied from the distance of 15 cm from mice or in the direct contact. Time of application varied from 15 to 40 min daily during the 5-day period. The murine immune cells were isolated from thymus (mostly T-cells), bone marrow (mostly B-cells), and spleen - cell mixture, 30% of which are active macrophages, and the remaining 70% - lymhocytes, from which T-cells comprise 60%, and B-cells - 40% [2]. IFN litres were estimated in mice blood and immune cells using standard procedure [1, 2]. 3. Results and discussion

The IFN-inducing ability of Applicator was studied in mice during the 5-day period. Serum IFN titers were estimated daily. Fig. 1 shows that Applicator usage enhanced the IFN production 5-32fold.

613 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 613-615. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

614

The main cells producing IFN both in men and animals are cells of the immune system. Substances of different classes are able to stimulate the production of antigenically different IFNs in different kinds of cells [2]. As the IFN producing locally is known to play an important role in the pathology of different infections, it was interesting to study the influence of Applicator on the IFN production in the thymus, bone marrow and spleen cells. Fig.2 depicts the ability of Applicator to modulate the IFN production in thymus cells, mostly consisting of T-cells, following its application. Usually thymocytes produce while synthesis of does not occur. But we managed to show that Applicator usage oppressed the synthesis of by the thymus cells, while was produced by them at a high titer.

Bone marrow cells, consisting mostly of B-lymphocytes, in contrast to thymocytes, in response to various inducers stimulation normally produce We shown that Applicator usage completely suppressed the synthesis (Fig.3).

615 The production of in splenic cells (mixture of T-cells, B-cells and macrophages) is shown in Fig.4. The splenocytes of mice that did not have a contact with Applicator synthesized in the quantuty 80 IU/ml. Following the contact with Applicator the ability of splenocytes to produce

increased 16-fold.

The data shown above prove that Applicator used possess a high IFN-inducing activiy. In normal mice it is shown to induce the production of endogenous

Meanwhile Applicator is

probably able to modify the immune competent cells in the unusual way, somehow turning on their concealed reserves. 4. Conclusions

1. Applicator usage is shown to induce the IFN-synthesis in the murine organism in the quantities high enough to control different infectious pathology. 2. Applicator is probably able to modify the immune competent cells of mice in the unusual way, somehow turning on their concealed reserves. 3. Applicator possess high IFN-inducing activity. It is shown to enhance the IFN synthesis up to

the titer of 320 IU/ml. 4. Applicator might be used for therapeutical aims, as it used outwardly. The course of the treatment is 1-10 days. 5. References

1. Campbell, I., Greenber, T., Kochman, M.A. (1975) A microplaque reduction assay human and mouse interferon, Can. J. Microbiol. 21, 1247 - 1253. 2. Ershov, F.I. (1996) The Interferon System in Normal State and in Case of Pathology,

Medicine, Moscow. 3. Ershov, F.I., Chizhov, N.P., Tazulakhova, E.B. (1993) Antiviral medicines, Ingoda, SPeterburg. 4. Mozgovoi, V.A. and Mozgovaya, L.Yu. (1995) Applicator, Patent N 2040248, Russia. 5. Stewart II, W.E. (1979) The Interferon System, Springer-Verlag, New York.

for

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IN VITRO IMMUNIZATION OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES WITH CHOLERA TOXIN B SUBUNIT A. ICHIKAWA, Y. KATAKURA, T. KAWAHARA, S. HASHIZUME* and S. SHIRAHATA Graduate School of Genetic Resources Technology, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812, Japan *Morinaga Institute of Biological Science, 2-2-1 Shimosueyoshi, Tsurumi-ku, Yokohama 230, Japan

Abstract In vitro immunization (I VI) techniques have a great potential in the production of human monoclonal antibodies (MAbs) against various antigens. An IVI method of human peripheral blood lymphocytes (PBL) has been developed with a human lung adenocarcinoma cell line in our laboratory. We have succeeded in generating several cancer specific human MAbs using this IVI method. Because this IVI method was not available for soluble antigens, we improved it for soluble antigens. IVI with soluble antigens was effectively caused by the addition of muramyl dipeptides, interleukin-2 (IL-2) and interleukin-4 (IL-4). It was found that the difference of sensitivity of lymphocytes depending upon donors could be overcome to find the optimal concentrations of IL-2 and IL-4. IVI of human PBL was performed with cholera toxin B subunit (CTB) and the immunized B cells were transformed by Epstein-Barr virus. Anti-CTB antibody was detected using an indirect ELISA. Clones producing anti-CTB antibodies were directly cloned by a soft agar cloning method. 1. Introduction Human monoclonal antibodies are useful for diagnosis and treatment of various diseases. However, in order to obtain the human monoclonal antibodies, passive immunization, such as injection of dangerous antigens in human bodies, should not be permitted for ethical and moral reasons. One of the mothods to solve this problem is the in vitro immunization method which is a primary activation of antigen-specific B lymphocytes. In vitro immunization techniques have great potential in the production of human monoclonal antibodies against various antigens. We have developed an in vitro immunization method against cultured lung adenocarcinoma cell line1) and succeeded in generating several cancer specific humanmonoclonal antibodies2), 3). However,this method could notbe available for immunization against soluble antigens. The purpose of this work is to improve this in vitro immunization method for immunization against soluble antigens and to generate human monoclonal antibodies against cholera toxin B subunit with new in vitro immunization method. 617 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 617-623. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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2. Materials and Methods 2.1. Antigens and reagent

Keyhole lympet hemocyanin (KLH) was obtained from Calbiochem-Novabiochem Corporation (LaJolla, CA, USA). Ovalbumin was obtained from Chemicon international Inc. (Temecula, CA, USA). Cholera toxin B subunit was obtained from List Biological Laboratories, Inc. (Campbell, CA, USA). Recombinant human IL-2, IL-6 and IL-10 were purchased from Genzyme corporation (Cambridge, MA, USA). Recombinant human IL4 was purchased from Pepro Tech EC LTD. (London, England). Recombinant human IL-5 was obtained from R&D systems (Minneapolis, MN, USA). Muramyl dipeptide (MDP) was purchased from Chemicon international Inc. (Temecula, CA, USA). L-Leucyl-leucin methyl ester was obtained from Boehringer GmbH (Mannheim, Germany). 2.2. Isolation

of human lymphocytes

Human peripheral blood lymphocytes (PBL) were separated by density-gradient centrifugation from several healthy donors. In brief, 25 ml of peripheral blood was layered on 20 ml of lymphocyte separation medium (LSM; Organon Teknika, Durham, NC, USA)

and was centrifugcd at 400g for 30 min. The stratum of lymphocytes were harvested and washed three times with ERDF medium. Lymphocytes from peripheral blood were treated with 0.25 mM Leu-Leu-OMe to remove the cytotoxic T cells, CD8+ suppressor T cells, and natural killer cells before use. 2.3. In vitro

immunization

In vitro immunization of the human PBL was performed in 24 well culture plates (Becton

Dickinson). The Leu-Leu-OMe-treated PBL were cultured for 7 days in ERDF medium containing 10% heat inactivated fetal bovine serum, MDP (10µg/ml), IL-2 (10 units/ml), IL-4 (10 ng/ml), 2-mercaptoethanol (20 µM) and antigens. 2.4. Enzyme-linked

immuno sorbent assay

Ninety-six well microtiter plates were coated with CTB or BSA in 0.1 M Na-carbonate buffer (pH9.6) at concentration of 0.2 µg/well and incubated at 4°C overnight. The plate were washed three times with PBS/0.05% Tween 20 (PBS-T), the supernatants of EBV

transformed B cells were diluted 1:2 in PBS-T, added in , aliquots per well, and incubated at 4°C overnight. The wells washed three times with PBS-T and incubated for 2 hours at 37°C with l/well horseradish peroxidase-conjugated goat antibodies against human IgM. After washing three times with PBS-T, l/well of substrate solution [0.1 M citrate buffer (pH4.0) containing 0.003% and 0.3 mg/ml p-2.2’-azino-di (3ethylbenzothiazoline-6-sulfonic acid) diammonium salt] were added, and after 20 minutes, the absorbance at 405 nm was measured by using an ELISA reader. 2.5. EBV transformation

After in vitro immunization, human B cell were Epstein-Barr virus (EBV) infected using

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supenatants from the B95-8 marmoset cell line. EBV-transformedcells were washed with ERDF medium containing 20% FBS and seeded into 96 well culture plates at cells/well. 2.6. Cloning

EBV-B cells producing antibodies to choleratoxin B subunit were cloned by limiting dilution at density of 1 cell/well in 96 well culture plates or by soft agarose colony forming method. The supenatants from the positive wells were screened by ELISA. 3. Results and Discussion

3.1. Establishment antigens

of protocol for in vitro immunization

with soluble

We examined the effect of various recombinant human cytokines which are concerned with the proliferation and differentiation of B lymphocytes. Peripheral blood lymphocytes (PBL) derived from healthy donors were incubated with various combinations of IL-2, IL-4, IL5, IL-6 and IL-10 in the presence of KLH, as an antigen and MDP for 7 days. Table 1 summarizes the effects of various cytokines on in vitro immunization against soluble antigen. It was found that IgM production and cell proliferation were greatly increased in the combination of IL-2 and IL-4. Similarly, we could observe high production of IgM reactive to KLH. These results indicated that the protocol using the combination of IL-2 and IL-4

was the most effective for in vitro immunization against soluble antigens.

We established the new protocol of in vitro immunization for soluble antigens. Fig. 1 shows a scheme of the new protocol. Major difference of the protocol for soluble antigens from that for cancer cell antigens was the use of IL-4 instead of IL-6 in the culture medium for in vitro immunization of lymphocytes.

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3.2. In vitro immunization

with cholera toxin B subunit (CTB)

To illustrate the effect of the new protocol, we actually performed in vitro immunization of human PBL with CTB as an example of soluble antigens. Fig. 2 shows reactivity of antibodies induced by in vitro immunization of PBL with CTB as an antigen. We examined the effects of several combinations of IL-2, IL - 4 and IL-6 on in vitro immunization with CTB. Although there were not much differences on immunoglobulin secretions among every combination, the combination of IL-2 and IL-4 was most effective on the level of CTB-specific antibodies. Exogenous addition of IL-6, which was effective for in vitro

621

immunization with cultured cell antigen, had no effect on immunization with soluble

antigens.

3.3. Individual differences of lymphocytes

on in vitro immunization

All examinations mentioned above were carried out using PBL derived from single donor. We examined whether this protocol was also effective for lymphocytes derived from different donors or not. Fig. 3 shows results of in vitro immunization of lymphocytes derived from three different donors. We could detect the increase of antigen specific IgM antibody secreted by lymphocytes derived from donor A, but not on donor B and C. This result seems to suggest the possibility that each lymphocytes derived from different donors demand their own optimum concentrations of IL-2 and IL-4.

Then we tried to optimize the concentrations of IL-2 and IL-4 on in vitro immunization of lymphocytes derived from different donors. Table 2 shows the optimum concentrations of IL-2 and IL-4 in the culture of in vitro immunization of lymphocytes derived from different donors. It was found that each lymphocytes from different donors demanded their own optimum concentrations of IL-2 and IL-4. For this reason, if we can find optimum concentrations of IL-2 and IL-4 for each lymphocytes beforehand, the effective immunization will be caused by this protocol using the combination of IL-2 and IL-4.

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3.4. Acquisition

of EBV-B cell lines secreting

CTB-specific

MAbs

To immortalize the B lymphocytes after in vitro immunization with CTB, we adopted the Epstein-Barr virus (EBV) transformation. Fig. 4 shows screening of EBV-B lymphocytes supernatants in ELISA. Several EBV-B lymphocytes secreted antibodies reacting strongly with CTB. But most supernatants also reacted to BSA as a control. Lymphocytes cultured in the well no. 13C had the highest specificity to CTB of all the wells. Positive EBV-B lymphocytes in 13C were amplified and cloned by soft agar cloning method. We could obtain 4 clones secreting IgM antibodies reactive to CTB among 280 clones picked up. However, only one clone, 13C/C7, had a specificity to CTB (Fig. 5).

4. Acknowledgment

This work is supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. A. I. is a research fellow of the Japan Society for the Promotion of Science. 5. References 1. Kawahara, H., Shirahata, S., Tachibana, H. and Murakami, H. (1992) In vitro immunization of human lymphocytes with human lung cancer cell line A549. Hum. Antibod. Hybridomas 3 , 8-13.

2. Shoji, M., Kawamoto, S., Sato, S., Kamei, M., Kato, M., Hashizume, S., Seki, K., Yasumoto, K., Nagashima, A., Nakanishi, H., Suzuki, T., Imai, T., Nomoto, K. and Murakami, H. (1994) Specific reactivity of human monoclonal antibody AE6F4 against cancer cells in tissues and sputa from lung cancer patients. Hum. Antibod. Hybridomas 5 , 116-122. 3. Shoji, M., Kawamoto, S., Setoguchi, Y., Mochizuki, K., Honjoh, T., Kato, M.,

Hashizume, S., Hanagiri, T., Yoshimatsu, T., Nakanishi, K., Yasumoto, K., Nagashima, A., Nakahashi, H., Suzuki, T., Imai, T., Nomoto, K. and Murakami, H. (1994) The 14-33 protein as the antigen for lung cancer-associated human monoclonal antibody AE6F4.

Hum. Antibod. Hybridomas 5 , 123-130.

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Discussion Barteling:

Do you have any idea what makes the individual background of the cells that you use? What gives the difference in dependency on

IL-2 and IL-4? Is it receptors, or efficiency of uptake, maybe?

Ichikawa:

We think that the ratio of B and T cells and other accessory cells in our in vitro immunisation system will affect immunisation efficiency and cytokine sensitivity of each cell population which are maybe different depending upon donors. Determining the optimum concentration of R2 and RS4 is useful to overcome the problem of donor difference.

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SESSION ON : NEW TECHNOLOGIES FOR HEALTH CARE PRODUCTS

The use of ex vivo grown animal cells for cell therapy or for the establishment of bioartificial organs needs coculture techniques and a deep view into the metabolic interactions between cells, cell and matrices and cells within a tissue. The different aspects are covered by studying the extension of human hematopoietic progenitor cells in coculture with stroma cells or by studying the metabolic competence of primary porcine hepatocytes in the presence of endothelial cells and Kupfer cells. The animal imaging and spectroscopy studies in hollow fibre bioreactros offers an excellent possibility to measure the interactions of cells under tissue-like densities. The capability of cells to migrate within the tissue is difficult to measure. Therefore, new methods for its determination are of high interest.

J. Lehmann, E. T. Papoutsakis Chairpersons

625 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 625. 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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ANALYSIS OF CELL GROWTH IN A FIXED BED BIOREACTOR USING MAGNETIC RESONANCE SPECTROSCOPY AND IMAGING

PETER E. THELWALL, MARIA L. ANTHONY, DIETER KEVIN M. BRINDLE University of Cambridge Department of Biochemistry, 80 Tennis Court Road, Cambridge CB2 1GA, U.K. ‡ Techinical University of Hamburg-Harburg Bioprocess and Biochemical Engineering Denicke Str. 15, 21071 Hamburg, Germany

1. Introduction Intensive bioreactors, in particular the hollow-fibre bioreactors (HFBR), are widely used

for the production of proteins from mammalian cells [1-3] and for the growth of human and animal cells ex vivo for cellular therapies, biohybrid artificial organs and tissue engineering applications [4-6]. The principle problem with intensive reactor systems is the formation of nutrient and waste product gradients [1]. These could have a negative effect on cell growth and also, potentially, the quality of a protein product in terms of its homogeneity. For example culture pH has been shown to affect the glycosylation patterns of a secreted protein [7]. Perturbation of cellular metabolism by these gradients could be particularly important in bioartificial organs, such as the bioartificial liver, where the metabolic activity of the tissue is intimately related to its function. Improvements in the design and operation of these systems require techniques for monitoring the levels of nutrients and waste products and the cell distribution in specific regions of the reactor. The closed nature of the reactors make them intrinsically inaccessible to conventional monitoring techniques. For example studies of oxygen distribution, which is frequently a limiting nutrient in these systems, have relied on indirect methods or invasive electrode measurements [8-10], which have the potential to disrupt reactor function. There is a need, therefore, to develop non-invasive techniques for monitoring reactor performance. Magnetic resonance imaging (MRI) and spectroscopy (MRS) are non-invasive techniques which have already been used to monitor cellular metabolism [11,12], cell density [13] and medium flow rates [14] within HFBRs. We have shown that diffusion-weighted imaging techniques can be used to map cell distribution in an HFBR [15] and that MR relaxation time imaging measurements, on a perfluorocarbon probe molecule, can be used to map oxygen distribution [16]. We show here that diffusion-weighted imaging and spectroscopy can be used to monitor cell distribution and cell volume respectively in a fixed bed bioreactor composed of macroporous carriers. 627 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 627-633. 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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2. Materials and Methods

2.1. MATERIALS

Enzymes, cell culture media and foetal calf serum were obtained from Sigma Chemical Company (Poole, Dorset, U.K.). 2.2. CELL CULTURE

Chinese hamster ovary cells (CHO K1) were cultured in DMEMcontaining glucose supplemented with 10% foetal calf serum, 2 mM glutamine, 100 units penicillin and 100 streptomycin. The bioreactor system consisted of a stirred tank fermenter (FT Applikon, Tewkesbury, U.K.), containing 1.25 l of medium, which was pumped via water-jacketed tubing at

to the bioreactor positioned in the NMR

magnet. The bioreactor consisted of a 25 ml volume fixed bed, composed of carriers (Biomaterials Co., Japan). To innoculate the bioreactor, cells were harvested from dishes and suspended in 200 ml medium. This was pumped through the reactor until the majority of the cells had adhered. The seeding density was thus approximately The reactor was then perfused with medium from the fermenter at a flow rate of 25 ml

Medium in the

fermenter was replaced by a continous bleed and feed system, with a maximum rate of 300 ml per day. The medium was gassed with by passing these gases through silicone rubber tubing wound on a former surrounding the impeller blade of the fermenter. The of the medium was monitored using a polarographic oxygen electrode (Mettler Toledo, Leicester, U.K.) and the pH using a glass electrode (Broadley James Co., Santa Ana, CA.). Oxygen tension and pH were controlled by regulating the composition of the gas stream. The pH was maintained at 7.35 and the oxygen tension at 100%. The temperature was maintained at 37°C. 2.3 NMR METHODS

Experiments were performed using a Varian Unity Plus 400 MHz wide-bore spectrometer equipped with an unshielded gradient set. spectra and images were

acquired using a Varian 25 mm 1H imaging probe and spectra using a Bruker 25 mm probe. Conventional spin echo images (TE = 20 ms; TR = 3 s) were acquired from transverse slices of the bioreactor using 256 phase encode increments. The in-plane resolution was 0.1 x 0.1 mm and the slice thickness 2 mm. Diffusionweighted images were acquired using a stimulated echo (STEAM) sequence [15], which included a pair of pulsed magnetic field gradients of 0.2 T and 2.5 ms duration in a TE period of 40 ms. The mixing time (TM) was 0.3 s. Sixty four phase encode increments, with 128 transients per increment, were acquired with a repetition time of 2 s. The slice thickness was 2 mm and the in-plane resolution was 0.1 x 0.4 mm. Diffusion-weighted spectroscopy was performed using a STEAM sequence with a pair of pulsed magnetic field gradients of 2.5 ms duration in a TE period of 40 ms. The mixing time was 0.3 s. Fifteen gradient strengths between 0.02 and 0.3 T were used. The intracellular and extracellular water fractions were calculated as described in [17].

629

NMR spectra were acquired using a 40 µsec, 90° pulse and a pulse repetition time of 1.4 s. The spectra, which were the sum of 2000 transients, were acquired into 8000 data points, with a spectral width of 10 kHz. Chemical shifts and signal intensities were referenced to the resonance of methylene diphosphonate (MDP), which was contained in a capillary tube within the fixed bed reactor.

3. Results Figure 1 shows a MR spectrum from the fixed bed bioreactor, 14 days after cell seeding. The chemical shift or frequency of the inorganic phosphate peak, which was predominantly due to extracellular phosphate, can be used to estimate extracellular pH within the reactor [12]. This indicated that the reactor pH was 7.04, which was signficantly lower than the pH in the feed vessel, which was determined to be 7.35, using a glass electrode.

Figure 2 shows the increase, with time, of the volume of a water fraction in the reactor which has a low apparent diffusion coefficient (ADC). This fraction with low ADC has been assigned previously to intracellular water [15,17]. The volume of this fraction was determined using a diffusion-weighted MR spectroscopy experiment [17] on a region of the reactor which was similar to that interrogated in the MR experiment. The increase in cell volume, as determined from this increase in intracellular water fraction, showed a good correlation with the MR measurements of ATP content (fig. 3). On day 10 of the culture the media exchange rate was increased from 150 mL to 300 mL . This resulted in an increase in cell growth which was apparent in the ATP measurements. The figure quoted for the percentage of

630

intracellular water in the reactor is only approximate since its calculation depends on knowing the and relaxation times of intra- and extracellular water. These have not been determined and have been assumed to be equal for intra- and extracellular water.

Figure 4 shows a conventional

spin echo image and a diffusion-weighted

image of water protons from the same transverse slice through the reactor. The position of the carriers can be clearly identified in the conventional image. The diffusion-weighted image shows water with a low ADC, i.e. intracellular water [15]. High signal intensity in this image corresponds to high cell density. The image shows that cell growth in the reactor is very heterogeneous and maximal at the periphery of the carriers.

631

4.

Discussion

is a relatively new macroporous cellulose carrier which can support the growth of adherent and non-adherent cell lines. The surface has been modified with polyethyleneimine (PEI), which imparts a slight positive charge that enhances the attachment of non-adherent cells, such as hybridoma [18]. The pores are larger than 100 µM and this enhances mass transfer of oxygen and other nutrients into the carrier. The large size of the carriers used in this study ( 5 x 5 x 5 mm) allowed packing of the

carriers without the aggregation and channelling of medium flow that we have experienced with other, smaller, carriers. The diffusion-weighted images (figure 4) showed that cells grew to a depth of

~ 1 m m from the surface of the carriers and that the cell density was increased around the cavities between the carriers. These cavities are assumed to contain regions of increased

medium flow. Although not determined in this study, this can easily be investigated using flow-sensitive imaging methods [14]. An important parameter to determine in metabolic studies of intensive bioreactor systems is the viable cell concentration. A non-invasive technique is required as sampling the reactor would be difficult and involve considerable disruption of the

culture. Previous studies have made estimates based on nutrient uptake rates [1]. However this assumes constant specific uptake rates, which may not be maintained when conditions in the reactor are changed (discussed in [13]). An NMR-based method has been described in which MR was used to measure total sodium ion concentration in an HFBR [13]. As the cells grew and filled the extracapillary space of the reactor, the sodium NMR signal declined as the intracellular concentration (~ 7 mM) was very much less than the extracellular concentration (158 mM). This increase in cell volume in the reactor showed a good correlation with the ATP content determined by MR. However the problem with this experiment is that it involves measuring a relatively small decrease in intensity of the sodium signal and will not be very good, therefore, at detecting cells present in low numbers. The cell volume determination experiment described here, in which the intracellular volume in the reactor was determined by measuring the fraction of water with low apparent diffusion coefficient (ADC) is, in principle, more sensitive. The main problem with the experiment is that there is a small fraction of water, which is not intracellular, but nevertheless has a low ADC. However this fraction, which is presumably water trapped within the carrier matrix, can readily be determined by making measurements on the reactor prior to cell loading (see fig. 2). The increase in the apparent intracellular volume in the reactor (fig. 2) shows a good correlation, under these reactor conditions, with the increase in ATP concentration determined by MR measurements (compare figs. 2 and 3). MR spectroscopy and imaging are clearly useful tools for investigating intensive bioreactor performance. Conversely, intensive reactors are invaluable to those wishing to use MR spectroscopy to investigate the metabolism of cultured cells. Cultured cells, derived from specific tissues e.g. brain, tumours etc. have been used by MR spectroscopists to generate relatively homogeneous and well defined tissue models. MR studies on these systems can then be used to increase our biochemical understanding of the MR data generated from studies on human tissues in the clinic. Although short term studies on immobilised cell systems have been used, intensive reactor systems, such as the HFBR, have the distinct advantage that they allow MR

632

measurements to be made on actively growing cells and are therefore likely to be more physiologically relevant. The fixed bed reactor described in this study is particularly attractive as it is easy to construct and can be sterilised by autoclaving. Hollow-fibre reactors, on the other hand, require specialist skills to fabricate, are expensive to purchase in a configuration which is compatible with use in a high field NMR instrument and may require more sophisticated sterilisation techniques. 5. Acknowledgements The work was supported by the European Community Framework IV Programme (Biotechnology 950207). PET thanks the MRC for a studentship. Thanks to Biomaterials Co. for providing the carriers.

6. References 1.

2.

Chresand, T. J., Gillies, R. J., and Dale, B. E.: Optimum fiber spacing in a hollow fiber bioreactor, Biotech. Bioeng. 32 (1988), 983-992.

Knazek, R. A., Gullino, P. M, Kohler, P. O., and Dedrick, R. L.: Cell culture on artificial capillaries: An approach to tissue growth in vitro., Science 178 (1972), 65-67.

3. 4. 5.

Knight, P.: Hollow fiber bioreactors for mammalian cell culture, Bio/Technology 7 (1989), 459-461. Hubbell, J. A., and Langer, R.: Tissue engineering, Chem. Eng. News 73 (1995), 42-54.

Jauregui, H. O., Chowdhury, N. R., and Chowdhury, J. R.: Use of mammalian liver cells for artificial

liver support. Cell Transplantation 5 (1996), 353-367.

6.

Langer, R., and Vacanti, J. P.: Tissue engineering, Science 260 (1993), 920-926.

7.

Borys, M. C., Linzer, D. I. H., and Papoutsakis, E. T.: Culture pH affects expression rates and glycosylation of recombinant mouse placental lactogen proteins by Chinese Hamster ovary (CHO)

cells, Bio/Technology 11 (1993), 720-724. 8. 9.

Drury, D. D., Dale, B. E., and Gillies, R. J.: Oxygen transfer properties of a bioreactor for use within a nuclear magnetic resonance spectrometer, Biotech. Bioeng. 32 (1988), 966-974. Piret, J. M., and Cooney, C. L.: Model of oxygen transport limitations in hollow fiber bioreactors,

Biotech. Bioeng. 37 (1990), 80-92. 10. Wiesmann, R., Maier, S. T., Marx, U., and Buchholz, R.: Characterization of oxygen transfer in a membrane-aerated hollow-fibre bioreactor using modified microcoaxial needle electrodes, Appl.

Microbiol. Biotechnol. 41 (1994), 531-536. 11. Gillies, R. J., MacKenzie, N. E., and Dale, B. E.: Analyses of bioreactor performance by nuclear magnetic resonance spectroscopy, Bio/Technology 7 (1989), 50-54. 12. Gillies, R. J., Scherer, P. G., Raghunand, N., Okerlund, L. S., Martinez-Zaguilan, R., Hesterberg, L., and Dale, B. E.: Iteration of hybridoma cell growth and productivity in hollow fiber bioreactors using 31 P NMR, Magn. Reson. Med. 18 (1991), 181-192. 13. Mancuso, A., Fernandez, E. J., Blanch, H. W., and Clark, D. S.: A nuclear magnetic resonance technique for determining hybridoma cell concentration in hollow fiber bioreactors, Bio/Technology 8

(1990), 1282-1285. 14. Hammer, B. E., Heath, C. A., Mirer, S. D., and Belfort, G.: Quantitative flow measurements in bioreactors by nuclear magnetic resonance imaging, Bio/Technology 8 (1990), 327-330. 15. Callies, R., Jackson, M. E., and Brindle, K. M.: Measurements of the growth and distribution of

mammalian cells in a hollow-fiber bioreactor using nuclear magnetic resonance imaging, Bio/Technology 12 (1994), 75-78. 16. Williams, S. N. O., Rainer, R. M., and Brindle, K. M.: Mapping of oxygen tension and cell distribution

in a hollow fiber bioreactor using magnetic resonance imaging, Biotech. Bioeng. 56 (1997), 56-61. 17. van Zijl, P. C. M., Moonen, C. T. W., Faustino, P., Pekar, J., Kaplan, O., and Cohen, J. S.: Complete separation of intracellular and extracellular information in NMR spectra of perfused cells by diffusion-weighted spectroscopy, Proc. Natl. Acad. Sci. U.S.A. 88 (1991), 3228-3232. 18. Ong, C. P., Portner, R., Markl, H., Yamazaki, Y., Yasuda, K., and Matsumura, M.: High density cultivation of hybridoma in charged porous carriers, J. Biotechnol. 34 (1994), 259-268.

633

Discussion

Noé: Brindle:

What is the interpretation of the peaks on your last slide? The interesting ones are from phosphomonoester compounds which are membrane precursors which change in various disease states, and also when the cells are proliferating there are characteristic increases in their levels.

Al-Rubeai:

It is interesting to see the relationship between apoptosis and acidification, and that when cells are subjected to hypoxia they undergo acidification.

Brindle:

They do that anyway. I would not say it is due to apoptosis. With agents such a Cerimide, which induce apoptosis, you do see acidification.

Al-Rubeai:

Could not this be due to changing the cell cycle as cells in G1 are more acid than cells in S and G2 where you have alkalisation before

cell division? Brindel:

We have tried to synchronise cells but with little success.

The

interesting part was that a fraction of the cells apparently died and the rest survived in anoxic conditions.

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EXPANSION OF HUMAN HEMATOPOIETIC PROGENITOR CELLS IN A FIXED BED BIOREACTOR

P. MEISSNER, P.WERNER°, B. SCHRÖDER*, C. HERFURTH, C. WANDREY, M. BISELLI Institute of Biotechnology, Forschungszentrum Jülich, °Stem Cell Bank, Heinrich-Heine-Universität, *ASTA Medica, Frankfurt, Germany.

Abstract: The ex vivo expansion of hematopoietic progenitor cells is very important for a variety of clinical applications, e.g. bone marrow transplantation or gene therapy Therefore it is of general interest to develop a culture system able to mimic the in vivo hematopoietic environment. We have modified a continuously perfused bioreactor originally developed for the production of monoclonal antibodies. In a fixed bed reactor we immobilized stromal cells (human primary stromal cells or the murine stromal cell line M2-10B4) in porous

glass carriers and inoculated human hematopoietic progenitor cells two days later and cocultivated them for several weeks. At different times after inoculation of mononuclear cells (MNCs) derived from umbilical cord blood or peripheral blood stem cells both adherent and non adherent cells were harvested and analyzed by flowcytometry, short-term colony assays, and LTC-IC analysis. During the cultivation there was a permanent production of progenitor cells and mature blood cells derived from the immobilized cells in the carriers. We could demonstrate the expansion of CFU-GM (7-fold), BFU-E (1.8-fold), and CFU-GEMM (4.2-fold) in contrast to the maintenance of late progenitors (CFU-E). Additionally we could observe the immobilization and 4.8-fold expansion of LTC-ICs. Discussion

Hatzfeld:

When you say you have expansion of CFU-GM cells did you really check that the CFU-GM cells at day 1 provide the same size colony as the CFU-GM you observe at day 8?

Meissner:

The distribution of the colony size was the same on day 0,1 and day 8, and therefore we have a real expansion

Hatzfeld:

Are you sure the cells were CFU-GM and not CFU-E? Have you checked for granulocytes, etc?

Meissner:

Our colonies have no CFU-E cells at the periphery. There are white cells, monocytes and granulocytes at the periphery 635

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 635-636.

1998 Kluwer Academic Publishers. Printed in the Netherlands.

636 Ostrove:

Regarding the starling material - you mentioned immobilised cells what was the actual starting material in the bioreactor and were they

CD34 selected? Meissner:

We started only with mononuclear cells.

Ostrove:

Do you have any evidence that CD34 will grow in the reactor?

Meissner:

We can show immobilisation of CD34 positive cells but there is no selection as cells are in the medium as well.

Scheirer:

How do your results compare with standard monolayer cultures in bottles with feeder layers?

Meissner:

You cannot compare these expansion results with normal co-cultures

because it is a perfusion system so the media change rate is not the same. Also we did not add cytokines.

Anon:

Did you also look at the more primitive CD34 and CD38 cells?

Meissner:

No.

Lowagie:

Did you try other immobilisation methods - microcarriers for example?

Meissner:

No.

Lehmann:

Did you check the medium requirements for different cells and then

design the medium, or was it just by chance? Meissner:

We used the typical Dexter type medium only to see if the reactor system was OK. This was because we used this medium before for co-cultivation of the stroma cells with CD34 positive hemopoetic cells, so we did not change the medium or add any cytokines.

Scharfenberg:

How homogeneous is your fixed bed - is it homogeneous the whole

time, or for a number of weeks? Meissner:

Yes, it is homogeneous because the circulation pump gives a

permanent medium flow. Scharfenberg:

To clarify the question - is it homogeneous with respect to the

different cell types?

Meissner:

When we take samples it is only from the bottom of the bead bed, so I cannot comment on your question.

A NOVEL ASSAY TO DETERMINE AND QUANTIFY THE REGULATION OF CELL MOTILITY AND MIGRATION DEMONSTRATED ON HEMATOPOIETIC CELLS D.Möbest1),2), S. Ries1), R.Mertelsmann1), R. Henschler1)

1) Dept. of Hematology/Oncology, University Medical Center, Freiburg, FRG 2) Dept. of Biology, University of Freiburg, Freiburg, FRG

Hematopoiesis occurs in the bone marrow and is supported by a complex network of extracellular matrix (ECM) and stromal cells (1). Hematopoietic cells including developing progenitors have been shown to specificially interact with matrix proteins and adhesion molecules on fibroblasts and other stromal cells. Progenitor-stromal cell interactions may occur through the vascular cell adhesion molecule (V-CAM) expressed on fibroblasts with the integrin receptor on hematopoietic cells, or through the interstitial (I)-CAM 1 molecule on macrophages with integrins (2,3). The extracellular matrix protein fibronectin (FN), through various domains, can bind to integrins expressed on hematopoietic progenitor cells, whereas laminin (Lam) may bind

progenitors through the receptor (2,4). In long-term bone marrow cultures (LTBMC), very primitive progenitor cells selectively bind to fibronectin and thus avidly adhere to the stromal matrix (5). Moreover, monoclonal antibodies against the

integrin, when applied to non-human primates, induce hematopoietic progenitor cells to leave the bone marrow and circulate in blood (6). Blocking antibodies against the CD44 (Hermes) antigen resulted in decreased lympho-hematopoiesis within LTBMC (7). Levesque et al. (8) showed that cytokines rapidly upregulate the avidity of the integrins on hematopoietic progenitors, resulting in their adhesion to FN or Lam. Since

during the homing of the hematopoietic stem and progenitor cells, e.g. after bone marrow transplantation in patients, the recolonizing cells have to traverse the endothelial barrier 637 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 637-643. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

638

and subsequently the stromal interstitial space within the bone marrow, cell migration on extracellular matrix proteins is very likely to be intricately involved in the reinitiation of blood cell formation after stem cell transplantation (2,3). We therefore investigated possibilities to quantify and further characterize the behaviour, especially the migration and motility, of hematopoietic progenitors on ECM by studying migration on tilted, matrix-coated cell culture plates.

Resistance or shear forces, which occur in vivo and which have to be overcome by circulating blood cells, have been previously introduced into in vitro culture systems: Transwell pores require active movements of cells through artificial lumina; flow chambers allow to study the initial adhesion steps e.g. of selectin binding (9). We

analyzed migration on ECM coated plates using photography or video microscopy. For the assays, FDCP-mix cells (Factor dependent cells Paterson-mixed potential (10)) were seeded at 10.000 per well on FN or Lam- precoated (10 µg/cm2) 96 well plates in IMDM and 20% horse serum. As shown in Fig.1, cells were allowed to accumulate at one end of

the well by tilting the plates at an 80° angle for 24 h.

639

Subsequently, at timepoint 0 h of experiments, cells were allowed to move after lowering the tilt angle to 15°. It was noticed that the progenitors spread uphill from about 6 h after start of the experiment, reaching plateau levels of cell migration after 15-24h (Fig.1). Video microscopy revealed that cell movements occured at random directions. As a first

endpoint, the number of cells that had moved away from the upper edge of the coherent cell mass that located at the bottom of the wells was quantified. As a second endpoint, the velocity of individual cells was determined. A variety of cytokines and chemokines was investigated showing stimulatory, inhibitory and indifferent effects (Table 1). A dose response of hematopoietic cytokines granulocyte macrophage-colony stimulating factor (GM-CSF) and Interleukin (IL)-3 could be shown. Variation of ECM concentration and inclination angle on FN, Lam resulted in dosedependent effects, respectively. An optimal resolution of these effects could be seen at a coating concentration of 10 and an inclination of (Table 1). Anti-integrin antibodies against the and the chain abrogated migration on FN, and anti or antion Lam (Tab.l). The activation time of migration was dependent on exposure

time to gravity force, but not on the exposure time of IL-3. Migration speed depended on FN, but not on IL-3 concentration. A sudden incrase of the IL-3 concentration resulted in an sharp decrease of the motility of cells, which was reversible over time. The net

potential energy, which could be achieved by migrating cells was higher on FN coated

surfaces as compared to BSA coated surfaces. We observed that our migration assay was able to detect and quantify the migrational behaviour of hematopoietic progenitor cells, which was mainly influenced by cytokines

and ECM. In particular, this assay induces a restraint force onto the cells by simple tilting

of the plates. One main result was that the kinetics determined in this migration assay are different from the induction of adhesion or induction of migration on even surfaces (11). The delay observed may be due to the requirement of intracellular reorganization of the cytoskeleton for example, posttranslational modifications which may have to take place before a coordinated migration against gravity force is inducable. In our assay the

"antigravity" migration was dependent on

integrins, whereas migration on even

surfaces was not (11). Interestingly, a restraint force on fibroblasts by withholding FN coated beads that bound to

integrins has been induced in integrins with an optical trap

(12,13). Possibly, in both situations an extracellular force is required to induce a coordinated activation of focal adhesion complexes, or related structures, which are

neccessary to induce migrational movements.

640

641

This assay has been used to make visible the transition of a cellular integrin-mediated migrational into an adhesional state (Tab.1). To our knowledge, this has so far not been possible with conventional assays. This method will therefore be of advantage to possibly dissect the role of certain G-protein families, such as rho, rac, or cdc42, GTP-ase in these phenomena (14). An advantage of this assay is that it uses the same culture equipment that can be applied in classical adhesion assays, thus allowing additional comparison to

established technology. Compared to the flow chamber assay, our assay is technically relatively more simple. To address the question how progenitor cells find their appropriate niche within the bone marrow stroma, this assay may be more suitable, whereas the flow chamber assay has originally been designed to imitate the situation in blood stream. Therefore, this system allows screening experiments, especially if it is

combined with automated cell scoring systems. In addition it is a very open system, allowing quick qualitative answers, but can also be used to achieve quantitative results

e.g. regarding motility of cells, energy of cells used for migration, or the effect of external force on the dynamics of cells.

References 1.

Dexter, T.M., Allen, T.D., and L.G. Lajtha.: Conditions controlling the proliferation

2.

of hematopoietic cells in vitro. J. Cell. Physiol. 91:335-344, 1977 Hynes, R.O.: Integrins: versatility, modulation, and signalling in cell adhesion. Cell

3.

69:11-25, 1992 Springer, T.M.: Traffic signals on endothelium for lymphocyte recirculation and

leukocyte emigration. Annu. Rev. Physiol. 57:827-872, 1995 4. Williams, D.A., Rios, M., Stephens C., Patel V.P.: Fibronectin and VLA-4 in haematopoietic stem cell-microenvironment interactions. Nature 352:438-441, 1991

5. Verfaillie, C., Blakolmer, K., McGlave, P.: Purified primitive human hematopoietic progenitor cells with long term repopulating capacity adhere selectively to irradiated bone marrow stroma. J. Exp. Med. 172:509-520, 1990 6. Papayannopoulou, T., Nakamoto, B.: Peripheralization of hemopoietic progenitors in primates treated with anti-VLA4 integrin. Proc. Natl. Acad. Sci. U.S.A. 90:9374-9378, 1993

7. Miyake, K., Medina K.L., Hayashi S.I., Ono, S., Hamaoka, T., Kincade, P.W.: Monoclonal antibodies to Pgp-1/CD44 block lympho-hematopoiesis in long-term bone marrow cultures. J. Exp. Med. 171:477-488, 1990

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8. Levesque, J.-P., Leavesley, D.I., Niutta, S., Vadas, M., Simmonds, P.J.: Cytokines increase human hematopoietic cell adhesiveness by activation of very late antigen (VLA)-4 and VLA-5 integrins. J. Exp. Med. 181:1805-1815, 1995 9. Chen, S., Alon, R., Fuhlbrigge, R.C., Springer, T.A.: Rolling and transient tethering of leucocytes on antibodies reveal specializations of selectins. Proc. Natl. Acad. Sci. U.S.A. 94(7):3172-3177, 1997 10. Spooncer, E., Boettiger, D., Dexter, T.M.: Isolation and culture of factor-dependent haemopoietic cell lines, in N.G. Testa, and G. Molineux (eds): Haemopoiesis - a practical approach. Oxford (UK), Oxford University Press, 1992, p 106-121 11. Strobel, E.-S., Möbest, D., von Kleist, S., Dangel, M., Mertelsmann, R., Henschler, R.: Adhesion and migration are differentially regulated in hematopoietic progenitor cells by cytokines and extracellular matrix. Blood 90:3524-3532, 1997. 12. Felsenfeld, D.P., Choquet, D., Sheetz, M. P.: Ligand binding regulates the directed movement of integrins on fibroblasts. Nature 393:438-440, 1996 13. Choquet, D., Felsenfeld, D. P., Sheetz, M. P.: Extracellular matrix rigidity causes strengthening of integrin-cytoskeleton linkages. Cell 88:39-48, 1997 14. Nobes, C.D., Hall A.: Rho, rac, and cdc42 GTPases regulate the assembly of

multimolecular focal complexes associated with actin stress fibres, lamellipodia, and filopodia. Cell 81:53-62, 1995

643

Discussion

Papoutsakis:

What was quantitatively different between the cell line and the CD34 positive cells which you saw at the end?

Möbest:

So far we have not found any differences but all the results have not yet been analysed.

Papoutsakis:

Would you expect to see any differences?

Möbest:

The CD34 cells look very similar but there may be some differences in the sub-populations.

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IMMORTALIZATION OF DIFFERENTIATED HEPATOCYTES GARY S. JENNINGS* AND MICHAEL STRAUSS§ *HepaVec AG and §Humboldt University of Berlin Robert-Roessle-Strasse 10, D-13122 Berlin, Germany

1. Introduction 1.1.

IMMORTALIZED HEPATOCYTES

The potential uses for continuously proliferating hepatocyte-derived cells have mutiplied in the last thirty years but with this the demand for ever more differentiated functions has grown louder. Amongst the most vociferous lobbies are those of xenobiotic toxicology and recombinant protein technology, two fields where human liver-specific metabolism and polypeptide processing would be advantageous. The development of a bioartificial liver using hepatocyte cell lines instead of primary cells is another application which especially interests us. While current ex vivo liver-support devices containing porcine hepatocytes have been shown to maintain some degree of the complexity of liver functions [1], the low proliferative capacity and rapid loss of specific enzyme activities in primary culture limits their use to the very short-term [2]. Moreover, in the longer term the use of non-human hepatocytes may not confer the required specificity of metabolism. In seeking a cell line suitable for clinical application we decided to avoid those stemming from hepatoma material, several of which produce infectious hepatitis B virus [3-7], and others derived from cells expressing immortalizing viral antigens, such as the large T-antigen of SV40 [8,9] or the E1 antigens of human adenoviruses (Table 1). In both cases, despite the maintenance of many liver-specific activities, levels of most are decimated [2]. Furthermore, foetal antigens such as -fetoprotein may be reinduced indicating a dedifferentiated phenotype and indeed, many of these cell lines are tumorigenic [9]. However, analysis of changes in two tumour suppressor genes involved in cell division gives us a clue as to how we might stimulate proliferation in normal human hepatocytes in a controlled fashion. The product of the retinoblastoma susceptibility gene (RB1) is a critical regulator of cell cycle entry and the protein is involved in gating cells for apoptosis. In the majority of hepatoma cell lines and in all cell lines expressing immortalizing viral antigens one or both of the tumour suppressors is deregulated (Table 1). 645 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 645-655. © 1998 Khiwer Academic Publishers. Printed in the Netherlands.

646

1.2. THE RETINOBLASTOMA PROTEIN IN CELL CYCLE ENTRY E2F-1 transcription factor is synthesized in the midphase of the cell cycle and is required for induction of genes in lateand S phase. Levels of free E2F-1 are regulated through sequestration by the retinoblastoma protein [12,13], which thereby prevents transcription from E2F-1 binding promoters and also enters into an active

suppressor complex with specificity for a different class of promoters [14,15]. In this way pRb coordinates induction of genes during to S transition (Fig. 1). The pRb checkpoint is also the site at which positive and negative growth signals are integrated and the decision taken to cross the restriction (R) point [14,15]. Mitogenic signalling results in the induction of D-type cyclins [16], the regulatory subunits of cyclin-

dependent kinase 4 (cdk4). The active kinase hyperphosphorylates and inactivates pRb, causing release of free E2F-1 (Fig. 1). Another class of proteins, the cyclin-dependent

kinase inhibitors (CKI), can bind to and block the kinase activity of the cdks and one

647

such inhibitor, has cdk4 as its sole target [17]. Expression of appears to be repressed by the pRb-E2F-l complex and this is relieved by hyperphosphorylation of pRb [18]. In normal cells, cyclin Dl becomes degraded in leaving an inactive complex which survives until cyclin D1 is resynthesized in the next phase (Fig. 1). In most tumour cell lines the pRb checkpoint is deregulated, either by functional inactivation of pRb or , or by overexpression of cyclin D1 or, occasionally, cdk4 [14,19]. This enables cells to enter S phase with reduced growth factor requirement and importantly removes a checkpoint essential for normal growth control. While permanent changes in the pRb checkpoint may signal the initiation of neoplasia it is also possible to generate hyperplasia in the liver. Stimuli which act to compromise liver function, such as partial hepatectomy, induce the remaining cells to enter cell division. New cells differentiate and assume the role of fully functioning hepatocytes but retain the ability to proliferate when necessary [20]. By transiently modulating expression of cell cycle regulators in vitro, we attempted to recreate hepatocyte hyperplasia and enable the outgrowth of hepatocyte cell lines with intact and S phase checkpoints.

2. Transient neutralization of the retinoblastoma checkpoint Previous work has shown that a transient reduction in the level of pRb in human embryonic lung (HEL) fibroblasts leads to stimulation of proliferation in vitro (Fig. 2) [21]. This was accomplished by transfection of phosphorothioate-based antisense

oligodeoxyribonucleotides, eighteen bases in length, with a sequence directed against

648

the human RB1 message. However, the high concentration of oligonucleotide required

and lower level of uptake by cultured human hepatocytes forced us to consider an altenative strategy. We constructed a plasmid with a 500-nucleotide antisense sequence from the human RB1 gene which covered the initiation codon, under control of an albumin promoter/enhancer element (Fig. 3) which should be expressed when

transfected into adult hepatocytes. Additionally, plasmids containing cDNA sequences

for the human cyclin Dl and E2F-1 genes driven by modified viral promoters were made (Fig. 3) and these were transfected singly or in combination with the antisenseRB1 plasmid. Cultures of human hepatocytes transfected with the described plasmids were maintained without selection in a serum containing medium. Non-transfected cells ceased to divide after one week and at 6 weeks most cells were dead and had detached from the plate. Over the following 2 weeks the outgrowth of epitheloid colonies was observed (Fig. 4a) the number of these being greater in cotransfection experiments than in single plasmid transfections. However, without exception, the colonies underwent a wave of cell death at 8 to 12 weeks after transfection. In each case cell-cell contact was lost and cell size decreased. Subsequently, nuclear condensation and fragmentation and later plamsa membrane blebbing was evident (Fig. 4b), all of which are consistent with an apoptotic phenotype.

3. Inhibition of p53-dependent apoptosis Apoptosis is one consequence of contradictory growth signals in cells unable to arrest in

[22]. As discussed above, the majority of hepatoma cell lines and cells harbouring immortalizing viral antigens also target the p53 protein for inactivation. Other viral antigens, the E1B 19 kDa protein for example, are homologues of cellular anti-apoptotic

649

proteins [23]. It appears then that removal of the pRb-checkpoint necessitates suppression of apoptosis to secure continuous proliferation. To this end we constructed a fourth plasmid in which a 500-nucleotide antisense sequence from the human p53 gene was placed downstream of the albumin promoter/enhancer element (Fig. 3). This

plasmid was cotransfected with one or more of the previous three plasmids into human hepatocytes as before. Similar numbers of colonies were obtained as with earlier transfections however, several colonies continued to expand after the majority had died during the apoptotic stage. One of these colonies could be stably passaged and was established as the cell line, HepZ (Fig. 4c).

4. Characterization of the immortalized hepatocyte line, HepZ

4.1. CELL CYCLE ENTRY

The HepZ cell line has been maintained for over 40 passages (approximately 120 population doublings) with no observable changes. Cells display a polygonal shape and intercellular spaces reminiscent of canaliculi in primary hepatocyte culture (Fig. 4c). At confluence, cell-cell contacts are remarkably strong whilst adherence to plastic dishes is very weak and HepZ was routinely plated on collagen I-coated dishes. Survival in culture depends on the presence of serum and growth rate is proportional to serum content between 0.5-10% FBS. This indicates that the growth factor sensitive checkpoint regulated by pRb is intact. Western blots of protein extracts from logarithmic phase

cultures show that pRb is amply present and exists primarily in the hyperphosphorylated form (Fig. 5a). Cyclin D1 is also expressed at similar levels as in other proliferating hepatocyte cell lines (Fig. 5b). These data are consistent with the effects in of mitogenic signalling and not of genomic integration of antisense-RB1 or cyclin

650

plasmids. E2F-1 protein, on the other hand, is present at highly elevated levels when compared to other immortalized cells or with regenerating mouse liver (Fig. 5c) and this may be due to expression from an exogenous integrated plasmid. The fourth protein whose synthesis might have been influenced by our intervention was p53. We performed immunoprecipitations with mouse monoclonal anti-p53 antibodies specific for wild-type or mutant protein conformations followed by Western analysis using a rabbit polyclonal antibody. No protein was recognized by the wild-type specific antibody (data not

shown) but a strong band was produced with antibody specific for the mutant form (Fig. 5d). Western blotting of other to S phase proteins, cdk4, cdk2, cyclin E, cyclin A, cdc25A and PCNA, and of the revealed no abnormalities in expression (data not shown). 4.2. HEPATOCYTE SPECIFIC ACTIVITIES Because of our interest in the use of viral vectors in gene therapy we next examined the efficacy and specificity of infection of HepZ by adenovirus and baculovirus. Adenovirus efficiently infects hepatocytes [24] and Fig. 6a shows that HepZ is also efficiently infected by adenovirus carrying a -galactosidase gene. Recently we reported specific infection of hepatocytes and hepatocyte-derived cell lines by baculovirus [25]. Infection of HepZ by baculovirus (Fig. 6b) though not as efficient as that by adenovirus is at least

651

as high as in primary hepatocyte cultures [25]. We conclude that HepZ cells maintain at least some viral specific cell-surface proteins present in hepatocytes.

Hepatocytes respond to glucocorticoids by increasing gluconeogenesis. The switch in metabolic emphasis away from protein and DNA synthesis results in a slower

proliferation rate in cultured cells [26]. HepZ responded to addition of 0.1 µM dexamethasone to the medium with a reduction in growth to around 40% of controls

(Fig 7). In further immunohistochemical characterization HepZ was found to produce albumin but not -fetoprotein and to express several isoforms of cytochrome P450 (data not shown). 5. Summary

The majority of hepatocyte cell lines are derivatives of neoplasias and contain growth deregulating genetic abnormalities. Whilst some liver specific activities are still detectable, their low levels make it unlikely that these cell lines could substitute for hepatocytes in bioartificial liver systems hence new immortalized differentiated hepatocyte lines are needed. The liver is capable of mounting a rapid proliferative response to a loss of cell mass, either through degenerative disease or surgical

intervention. Hepatocytes produced during this compensatory hyperplasia later differentiate but maintain the capacity to enter cell division. We attempted to induce a similar hyperplasia in cultured primary hepatocytes by repressing tumour suppressor protein action in the transition from to S phase. Plasmids bearing antisense-RB1 and antisense-p53 sequences as well as plasmids encoding E2F-1 and cyclin D1 were

transfected into primary human hepatocytes in an attempt to create a transient environment where cell cycle entry would be ameliorated. In transfections omitting the antisense-p53 plasmid, all colonies produced underwent rapid cell death after 8 weeks

652

but in those including antisense-p53 a small number continued to grow, indicating that

transient reduction of p53 allows primary hepatocytes an extended proliferative phase. One colony was passaged and established as the cell line HepZ. HepZ cells maintain a hepatocyte-Iike phenotype judged by several criteria and may be useful in the development of a liver support device incorperating immortalized polymer

cells. In this respect it has already been demostrated that the cells are able to grow on beads (Jürgen Lehmann, personal communication) a feature enabling their culture at high cell density, a prerequisie for a bioartificial liver system. Western blotting shows that HepZ expresses elevated amounts of E2F-1 protein and

has a mutant p53. The former probably allows the cells rapid transit through the checkpoint controlled by pRb and the latter escape from programmed cell death. This is resemblant of cell lines derived from hepatomas or transformed with immortalizing viral antigens in which the same control points are usurped. In these experiments no selection was applied with the result that the fastest growing cells were isolated and this is likely the reason for the acquisition of a mutation in the p53 gene. In future experiments a selection pressure to maintain the integrity of checkpoints will be applied.

6. References 1. Gerlach, J.C.: Development of a hybrid liver support system: a review, Int. J. Artif. Organs 19 (1996),

2. 3.

4.

645-654. Rogiers, V., and Vercruysse, A.: Rat hepatocyte cultures and co-cultures in biotransformation studies of xenobiotics, Toxicology 82 (1993), 193-208. McNab, G.M., Alexander, J.J., Lecatas, G., Bey, E.M., and Urbanowicz, J.M.: Hepatitis B surface antigen produced by a human hepatoma cell line, Br. J. Cancer 34 (1976), 509-515. Aden, D.P., Fogel, A., Plotkin, S., Damjanov, I., and Knowles, B.B.: Controlled synthesis of HBsAg in a differentiated human liver carcinoma-derived cell line, Nature (London) 282 (1979), 615-616.

5.

Nakabayashi, I I . , Taketa, K., Miyano, K., Yamane, T., and Sato, J.: Growth of human hepatoma cell

lines with differentiated functions in chemically defined medium, Cancer Res. 42 (1982), 3858-3863.

653 6. He, L., Isselbacher, K.J., Wands, J.R., Goodman, H.M., Shih, C., and Quaroni, A.: Establishment and

characterization of a new human hepatocellular carcinoma cell line, In vitro 20 (1984), 493-504. 7.

Alexander, J.J.. In vitro studies of human hepatocellular carcinoma cell lines. Adv. Hepatitis Res. (1984), 190-195.

8. Pfeifer, A.M.A., Cole, K.E., Smoot, D.T., Weston, A., Groopman, J.D., Shields, P.G., Vignaud, J-M, Juillerat, M., Lipsky, M.M., Trump, B.F., Lechner, J.F., and Harris, C.C. Simian virus 40 large tumor antigen-immortalized normal human liver epithelial cells express hepatocyte characteristics and metabolize chemical carcinogens, Proc. Natl. Acad. Sci. USA 90 (1993), 5123-5127. 9. Woodworth, C.D., Kreider, J.W., Mengel, L, Miller, T., Meng, Y.L., and Isom, H.C.: Tumorigenicity of simian virus 40-hepatocyte cell lines: effect of in vitro and in vivo passage on expression of liver-specific genes and oncogenes, Mol. Cell. Biol. 8 (1988), 4492-4501.

10. Puisieux, A., Galvin, K., Troalen, F., Bresac, B., Marcais, C., Galun, E., Ponchel, F., Yakicier, C., Ji, J., and Ozturk, M.: Retinoblastoma and p53 tumor suppressor genes in human hepatoma cell lines, FASEB J. 7 (1993), 1407-1413. 11. Hsu, I.C., Tokiwa, T., Bennett, W., Metcalf, R.A., Welsh, J.A., Sun, T., and Harris, C.C.: p53 gene mutation and integrated hepatitis B viral sequences in human liver cancer cell lines, Carcinogenesis 14 (1993), 987-992.

12. Qin, X.Q., Livingston, D.M., Ewen, M., Sellers, W.R., Arany, Z., and Kaelin, W.G. Jr.: The transcription

factor E2F-1 is a downstream target of RB action. Mol. Cell. Biol. 15 (1995), 742-755. 13. Sellers, W.R., and Kaelin, W.G.: pRB as a modulator of transcription, Biochim. Biophys. Acta. 1288 (1996), M1-5.

14. Weinberg, R.A.: The retinoblastoma protein and cell cycle control, Cell 81 (1995), 323-330. 15. Herwig, S. and Strauss, M.: The retinoblastoma protein: a master regulator of cell cycle, differentiation and apoptosis, Eur. J. Biochem. 246 (1997), 581-601.

16. Lukas, J., Bartkova, J., and Bartek, J.: Convergence of mitogenic signalling cascades from diverse classes of receptors at the cyclin D-cyclin-dependent kinase-pRb-controlled Gl checkpoint, Mol. Cell. Biol. 16 (1996), 6917-6925 17. Serrano, M., Hannon, G., and Beach, D.: A new regulatory motif in cell cycle control causing specific

inhibition of cyclin D/CDK4, Nature 366 (1993), 704-707. 18. Lukas, J., Parry, D., Aagaard, L., Mann, D.J., Bartkova, J., Strauss, M., Peters, G., and Bartek J.: Retinoblastoma-protein-dependent cell-cycle inhibition by the tumor suppressor p16, Nature (London) 375 (1995), 503-506. 19. Strauss, M., Lukas, J., and Bartek, J.: Unrestricted cell cycling and cancer, Nature Med. 1 (1995), 12541246. 20. Fausto, N., and Weber, E.M.: Liver regeneration, in I.M. Arias, J.L. Boyer, N. Fausto, W.B. Jakoby, D.A. Schachter and D.A. Shafritz, (eds.), The Liver: Biology and Pathobiology, third edition, Raven Press Ltd., New York, (1994), pp. 1059-1084. 21. Strauss, M., Hering, S., Lubbe, L., and Griffen, B.E.: Immortalization and transformation of human fibroblasts by regulated expression of polyoma virus T antigens, Oncogene 5 (1990), 1223-1229.

22. Yonish-Rouach, E., Grunwald, D., Wilder, S., Kimchi, A., May, E., Lawrence, J.J., May, P., and Oren, M.: p53-mediated cell death: relationship to cell cycle control, Mol. Cell. Biol. 13 (1993), 1415-1423. 23. Rao, L., Debbas, M., Sabbatini, P., Hockenbery, D., Korsmeyer, S., and White, E.: The adenovirus E1A proteins induce apoptosis, which is inhibited by the E1B 19-kDa and Bcl-2 proteins, Proc. Natl. Acad.

Sci. USA 89 (1992), 7742-7746. 24. Cristiano, R.J., Smith, L.C., Kay, M.A., Brinkley, B.R., and Woo, S.L.: Hepatic gene therapy, efficient

gene delivery and expression in primary hepatocytes utilizing a conjugated adenovirus-DNA complex, Proc. Natl. Acad. Sci. USA 90 (1993), 11548-11552. 25. Hofmann, C., Sandig, V., Jennings, G.S., Rudolph, M., Schlag, P., and Strauss, M.: Efficient gene transfer into human hepatocytes by baculovirus vectors, Proc. Natl. Acad. Sci. USA 92 (1995), 1009910103. 26. Vintermyr, O.K., and Doskeland, S.O.: Characterization of the inhibitory effect of glucocorticoids on the DNA replication of adult rat hepatocytes growing at various cell densities, J. Cell. Physiol. 138 (1989), 29-37.

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Discussion Singhvi:

By using anti-sense to inactivate tumour suppressor genes, like p53 or Rb, are you not making this potentially tumourigenic?

Jennings:

The growth rate increases but if you passage them long enough, then the anti-sense RG is degraded and they go back to the same growth rate. It is this transient effect that we are trying to use to promote hepatocyte growth. Within the hepatocyte outgrowth from a primary culture we can then use selection methods like collogen gel sandwich, etc, and hopefully develop a cell line. The gel sandwich technique has been shown to work well.

Noé:

Could you give more information on the status of the biochemical characterisation of these cells and their potential in artificial livers?

Jennings:

Various P450’s have been identified with 3a4 being highly expressed. They express glucourinyl transferase but at what levels and specificity, I am unsure. The cells were selected only on the basis of growth, so the next step will be to select them on specificity.

MacDonald:

How stable is the expression of the P450’s?

Jennings:

The cells have been passaged 50 times and there was no difference between passage 20 and 40.

Grammalikos:

A question on semantics. What do you mean by a non-transformed cell line?

Jennings:

We are thinking in terms of clinical aspects and thus we want to avoid tumourigenic cell lines. Transformation can result in a reduction of hepatocyte characteristics. Transformation by Tantigen has all sorts of other effects which reduce hepatocyte specific function but promote a general protein synthesis, which is not wanted either. We require a proliferating, hepatocyte specific, non-malignant cell line which is immortalised.

Hauser:

A question of how you transform these cells. You have a transient application of your transforming genes and suddenly you have a stable cell line. What is your understanding at the molecular level above this phase and is it reproducible?

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Jennings:

People may get upset by the fact that this cell line has an integrated E2F-1 plasmid. You have to do something to the cells to make them grow. If we reduce the cell cycle blockers pRb53 low enough for them to proliferate for a long time, or to use extra special medium, somewhere along the line they will probably fix a mutation and become immortalised. If this mutation is not deleterious to our requirements, then it is OK for us. If the mutation is in ras for instance and the whole cell cycle is intact, then we would not be able to use this for clinical applications. Ultimately, in a combination of very complicated selection factors, we may be able to get the cells to proliferate without integrating mutations. In regenerating mouse liver there are no mutations but all the cells proliferate and then switch off when it gets to the right size. We would like to do this in culture.

Singhvi:

Your comment on liver coming to the right size, is this not regulated on a cellular level? The size of the cells regulates

whether it is differentiated or growing. Jennings:

If you transplant a mouse liver into a rat, the liver gets bigger but the cells do not. If you transplant a rat liver to a mouse, the cells

remain the same size but the liver shrinks by apoptosis. This is a systemic regulation and many factors control proliferation and differentiation.

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FIXED-BED REACTORS FOR ANIMAL CELL CULTIVATION: AN APPROACH TO ARTIFICIAL ORGANS R. PÖRTNER1, S. RÖSSING1, J. STANGE2, D. FASSNACHT1 1 Technische Universität Hamburg-Harburg, Bioprozeß- und Bioverfahrenstechnik, Denicke Str. 15, D-21071 Hamburg, Germany 2 Universität Rostock, Klinik und Poliklinik für Innere Medizin, Ernst-Heydemann-Str. 6,D-18055Rostock, Germany

1. Abstract In this study the immortalised mouse hepatocyte line mHep-R1 was used for cultivation. The cells were first grown in culture flasks and in a small-scale fixed-bed

system in order to determine growth characteristics and a suitable carrier type. The cell line was then cultivated in a 40 ml fixed-bed reactor over a period of 75 days at a perfusion rate of 6.25 ml medium per ml fixed-bed and day. A cell density of approx. cells per ml carrier was reached at the end of the experiment proving that a stable cultivation was possible over a long period of time with constant consumption and production rates. 2. Introduction The immobilisation of animal cells in fixed-bed systems proved to be of advantage in many ways. By immobilising the cells on porous carriers, cells are retained in the system during continuous operation. This is of significance when cultivating cells, which are adherent, have a high demand of substrate and slow growth-rates. This is also accomplished by hollow-fibre modules or systems were cells are immobilised on or into microcarriers, but these systems proved to be difficult in scale-up, a factor which is especially important for the development of artificial organs, where a large amount of cells is needed. A scale-up is easily achieved for the fixed-bed system by pumping the medium radial through the fixed-bed instead of axial and therefore preventing the risk of an oxygen limitation. Due to the development of new immortalised hepatocyte lines that still show considerable Cytochrome P450 activity, an approach to an artificial liver support system might be accomplished with the aid of a bioreactor containing these cell lines instead of primary hepatocytes which do not proliferate in such a system. 657 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 657-659. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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3. Cell Line and Culture Conditions The immortalised mouse hepatocyte line mHep-R1 was used for cultivation. It shows in comparison to primary hepatocytes lower but still significant P450 activity of the subenzymes CYP1A2/3A4 and CYP2C (Stange et al., 1995). For growth low glucose Dulbecco’s MEM (Life Technologies, Germany) was used as basal medium. This medium contains 25 mmol HEPES, 4 mmol L-glutamine and 5.5 mmol glucose and was supplemented with 5% (v/w) foetal calf serum. The glucose level was increased in the 40 ml fixed-bed system after the first week by addition of glucose, because growth in preliminary experiments proved to be clearly glucose limited. For the batch experiments cultivation took place in 25 T-flasks. Growth curves were obtained by analysing a doublet of flasks each day. The 40 ml fixed-bed reactor, a modified reactor from meredos (Germany), contained two DO sensors, automatic pH and temperature control ( Pörtner et al., 1997). Cellulose carriers (Cellsnow, Biomaterials, Japan) with a diameter of approx. 5 mm were used to immobilise the cells. 4. Results 4.1. BATCH EXPERIMENTS The cells grew to a maximum cell density of cells after 72 hours with a membrane intact index of approx. 95%. The reason for cell death in the following death phase was clearly due to glucose limitation. 4.2. FIXED-BED REACTOR Continuous operation of the fixed-bed system was initiated 2 days after inoculation. The dilution rate was kept constant at 6.25 ml medium per ml fixedbed and day throughout the 75 days of operation. Glucose was again the growth limiting factor during the first few days (Fig. 1). To improve growth conditions, the glucose concentration in the feed was raised to 16 mmol on the day, resulting in increased glucose consumption

and lactate production rates as well as a higher glucose concentration. Several steady-states with different glucose

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concentrations in the feed were obtained in the following 60 days. A limitation of glucose can be excluded for feed concentrations over 16 mmol because no further increase in the glucose consumption rate was observed. With a high concentration of approx. 1.5 mmol , glutamine was also not limited (Fig. 2). We assume that a substance in the serum is the cause for limited growth because the glucose consumption and lactate production rates decreased rapidly when the serum level was reduced to 4% in the feed on the day (Fig. 1) and started to recover on the day after returning to the serum level of 5%. The average production rate was approx. 0.6 mmol for ammonia and 0.08 mmol for urea. 4.6 mmol ammonia th was added to the feed on the 30 day (Fig. 2) to estimate the stability of the production rates at higher ammonia levels. The ammonia production rate decreased to and the urea production rate to approx. 0.03 mmol Several carriers were removed at the end of the experiment, and the total cell density was determined with the crystal violet method to be cells per ml fixed-bed. This corresponds to cells per ml carrier with an external porosity of 40%. 5. Scale-up considerations For an adult human approx. 300 g of full functional hepatocytes (or 20% of the average liver mass) are able to maintain sufficient function for detoxification. If we assume that a permanent cell line still shows 100% of the function of primary hepatocytes, an extracorporeal liver support system would need a fixed-bed volume of approx. 6.6 litres. Large fixedbed reactors are operated by pumping the medium in a radial way through the bed rather than axially because of a possible oxygen limitation in fixed-beds over 15 cm of height. References We thank Prof. Strauß of the Max Delbrück Center for Molecular Medicine, Berlin, Germany, for the donation of the mHep-R1 cell line and Biomaterials, Japan for the supply of the Cellsnow carrier.

Acknowledgement Pörtner, R., Rössing, S., Koop, M., Lüdemann, I. 1997. Kinetic studies on hybridoma cells immobilised in fixed bed reactors. Biotech. Bioeng. 55: 535-541 Stange, J., Milzner, S., Strauß, M., Fischer, U., Lindemann, S., Peters, E., Holtz, M., Drewelow, B., Schmidt, R. 1995. Primary or established liver cells for a hybrid liver? Comparison of metabolic features. ASAIO J (United States) 43 (3):M10-M315

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HIGH DENSITY PERFUSION CULTURE OF PRIMARY RAT HEPATOCYTES FOR POTENTIAL USE AS A BIOARTIFICIAL LIVER DEVICE K. BRATCH, A.J. STRAIN & M. AL-RUBEAI School of Chemical Engineering and Liver research laboratory, University ofBirmingham, Edgbaston, Birmingham B15 2TT, U.K.

1. Abstract Primary rat hepatocytes were cultured in a perfusion bioreactor system in an attempt to develop a bioartificial liver support device. The bioreactor is based on immobilisation of cells attached to microporous carriers packed into a stationary bed reactor. The bioreactor is perfused with media and oxygenated independently thereby allowing better homogeneity through optimum control of nutrient and oxygen transfer. Hepatocytes which are essentially non-dividing cells when cultured in vitro, are required to be maintained for prolonged periods with minimum loss of cell viability and function. Attachment to microcarriers was optimised by investigating the influence of various parameters including temperature, serum levels, pH and cell density. Keywords: tissue engineering, hepatocytes, packed bed reactor, bioartificial liver. 2. Introduction

Fulminant hepatitis remains a highly lethal liver disease with the mortality rate remaining very high in the absence of transplantation. Due to a shortage of donors many of the patients will die while awaiting liver transplantation, therefore there is a need to develop a bioartificial liver system to help keep these patients alive until either an organ becomes available or the liver recovers from injury. The major problem in the development of an artificial liver has been the complex biochemical nature of the organ. An artificial support system should be capable of carrying out the complex synthetic and metabolic functions of the liver as well as detoxification and excretion. The most important functioning cells constituting the liver are hepatocytes; in the case of bioartificial livers isolated hepatocytes are employed as the biological material. We are currently using primary rat hepatocytes as the biological component for a bioartificial device. Hepatocytes are anchorage dependant cells and the attachment of hepatocytes to an extracellular matrix is essential for maintenance of function. Hepatocytes are usually cultured as monolayers on collagen coated surfaces. In order to 661 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 661-663.

© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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obtain the high numbers of cells that are required to constitute a bioartificial liver it would be advantageous to immobilise hepatocytes on microporous carriers. This would provide a high surface to volume ratio thereby achieving a larger cell attachment area. Unlike cell lines primary rat hepatocytes have a limited capacity to divide in vitro, therefore large numbers of cells must initially attach to the microcarriers if they are to be utilised in a liver assist device. As a result our work is aimed at investigating the attachment of hepatocytes to microporous carriers (fibracell discs) and parameters which may influence this are under investigation. 3. Materials and Methods

Hepatocytes are obtained from male Wistar rats by standard perfusion of the liver via the hepatic portal vein with collagenase.The bioreactor consists of a glass cylinder packed with microporous carriers (fibracell discs). Oxygen is supplied via a central core of coiled silicone tubing held in position by a glass support. The fibracell discs consist of a 50:50 mixture of polyester and polypropylene, with dimensions of 6mm in diameter and 2mm in thickness.

Experiments to investigate various factors effecting the attachment of cells to fibracell discs were carried out in duran bottles containing magnetic stirrers. The discs and cells suspended in DMEM were stirred at 72rpm in a 37°C incubator. MTT assays using MTT at a concentration of 5mg/ml were carried out on samples of

the discs and quantitative data was obtained by solubilising the formazan product with 0.04M HC1 in propan-2-ol and measuring absorbance at 570nm using a spectrophotometer. 4. Results The two plots in Figure 1 represent data from two separate experiments investigating the effect of incubation time on cell attachment to fibracell discs. Greatest attachment in both cases was observed after shorter incubation times. When comparing attachment in the presence and absence of serum, the presence of serum markedly increases cell attachment to the discs. However, it was apparent that cell attachment was as efficient at low serum levels as higher serum levels (Figure 2).

The larger the ratio of cells to discs the more cell attachment observed (Figure 3), but this did not appear to be linear, further experiments are being carried out to investigate the saturation point of the discs. The effect of pH on the attachment of cells to the microcarrier is shown in Figure 4. The results indicate that greater attachment appears to be achieved at pH 7.3 with decreasing attachment at higher pH.

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5. Conclusion

Preliminary data suggests that factors such as pH, serum levels, cell to disc ratio and incubation period do have an effect on the levels of cell attachment to the microcarriers. As hepatocytes are essentially non-dividing cells it is important to achieve high initial cell attachment if these microcarriers are to be utilised in a bioartificial liver system where high numbers of cells are needed.

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METABOLIC COMPETENCE AND HORMONAL REGULATION OF PRIMARY PORCINE HEPATOCYTES IN A 3-D SANDWICH CONFIGURAITON 1

A. Bader, 1D. Rocker, 1A. Acígköz, S. 1Schwintek, 1Jarosch von Schweder M. Maringka, 3V. Armstrong, 2R. Wagner, 1Steinhoff G, 1Haverich A 1 Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Division of Cardiovascular Surgery, MHH, 2GBF, Braunschweig, Klinische Chemie, University of Göttingen, Germany 1

The necessary culture conditions for maintenance of metabolic competence such as benzodiazepine metabolism or lidocaine biotransformation in primary hepatocyte cultures are a matter of intense debate. We therefore investigated and developed novel culture conditions with respect to tissue culture requirements such as hormonal additives, medium exchange rates, matrix geometry, collagen additives, nutrient and trace element composition. These results finally enabled us to achieve a fully serum free long-term culture of primary porcine hepatocytes maintaining oxidative biotransformation and protein secretion for at least 8 weeks in culture. This cell culture technology is crucial for a bioartificial liver based on primary cells.

665 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 665-667. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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Discussion Noé:

I anticipate your system to be used for liver transplant as an intermediate step. How far are you from the application of your system in patients?

Bader:

Scale-up has been difficult and we have lost a lot of time. However, we start in vivo experiments 2 weeks from now. We have to do safety studies, then studies on a hepactectomised model we have been using for the past year. Everything is ready for human trials once we get permission from the ethical committee.

Noé:

What type of animal studies have you done?

Bader:

A hepactectomised model of pigs is used.

Handa-Corrigan: In a recent clinical trial in London of hollow fibre bioreactors all the patients were successfully taken on to transplantation. However, the cells were non-viable after a day or two, so it is a dying population. What was the viability in your bioreactor?

Bader:

A plateau of viability of over 90% is maintained based on cell specific activity during in vivo use.

Keck:

I think your data showing expansion of primary porcine hepatocytes is a tremendous advance for the bioartificial liver field. What was the medium used and have you done any work on primary human hepatocytes?

Bader:

We are in the process of developing human cells expanding from 10 and 20,000 cm2 to 150,00 cm2. The culture medium is important and has well known growth factors such as EGF, but it is a very complex picture and needs foetal calf serum.

Bernard:

What are the advantages of your system compared with in vitro P450’s, and if you use such a system what would be the through-put in terms of numbers of compounds tested per unit time?

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Bader:

It is a complementary picture rather than an advantage. We have carried out in vitro and in vivo correlations comparing metabolic patterns. Using an isolated P450 system you can identify individual peaks on an HLPC chromatogram or mass spectrometry to correlate it with the P450 enzyme family. We can identity metabolites generated by a specific type of enzyme. In primary hepatocytes we

get a full pattern. We have compared various culture configurations and find an inversion of metabolite patterns between rats, and humans, and the same was found in vitro with rat and human primary hepatocyte sandwich cultures. It has been used against a range of compounds including cyclosporin, a range of Bayer antibiotics, and for predictive evaluation before going to the clinical phase. The main advantage of a primary hepatocyte system is to find metabolite patterns. This can also be done using a liver slice. The advantage of a culture system is you can study drug interaction to look for P450 induction. This is a useful model for this.

Miltenburger:

How large has your device to be for use as a human liver, and how many cells do you need?

Bader:

100 g of cells to replace a human liver will detoxify plasma in a patient whilst

regeneration

occurs over 2 weeks,

surface area is needed.

Lehmann:

If you harvest cells from the pig you may not get a culture of single hepatocytes. What procedure do you use to separate Kupffer cells, endothelial cells, etc, from the cell mixture obtained from the disintegration of the liver?

Bader:

We do not separate the cells but just co-culture the mixture of cells. It is useful to keep them because of cellular interactions. Also pig cells are isolated as aggregates, so separation would be difficult compared with the rat.

Lehmann:

So you do not get overgrowth from fibroblasts?

Bader:

Overgrowth of fibroblasts has not been seen over 8 weeks.

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NOVEL MINI-BIOREACTORS FOR ISLET CELL CULTURE

A. HANDA-CORRIGAN, I.C. GREEN*, J. MABLEY*, S. HAYAVI, G.N. KASS, R.H. HINTON, L.M. MORGAN and J. WRIGHT University of Surrey, UK. and * University of Sussex, UK

Abstract

A brief summary of whole islet transplantation and the use of implants or Bioartificial Pancreases (BAPs) for the treatment of diabetes is presented. We have recently developed an alternative BAP design using a macro-porous carrier called Porocell (Porvair Sciences, UK) for the culture of islet cells. The three dimensional organisations of islets in Porocell closely resemble whole islets in vivo. We have demonstrated that rat islet cells are able to survive, secrete insulin and respond to glucose when cultured in Porocell. Introduction

Diabetes mellitus comprises a heterogeneous group of disorders in which the regulatory mechanism of blood glucose control by insulin is impaired. Treatment with insulin results in a reduction in glucose levels but cannot achieve the degree of regulation of blood glucose seen in non-diabetic subjects. An ideal long-term treatment is to transplant

islets of Langerhans cells to restore insulin production for maintaining glucose within normal limits. Since 1990 approximately 150 Type 1 diabetic subjects world-wide have

received islet transplants from cadavers. The islets in most patients were unable to control blood glucose or had completely lost their activity after 3 years or less [1]. The alternative to transplantation is to use an implant or Bioartificial Pancreas (BAP) containing whole or dispersed islets . The salient objective in BAP design is to achieve long-term blood glucose control with viable & functional islets that do not stimulate the recipient's immune system. Various types of BAP devices have been developed over the years. Diffusion chambers in the shape of discs, hollow fibres and tubular membranes have been tested. Discshaped chambers induce fibrosis and islet necrosis has been reported in hollow fibre membranes [2]. A wide-bore, tubular membrane diffusion chamber has been shown to restore normoglycaemia in strepozotocin induced diabetic rats for 150 days, without

immunosuppression [3]. Vascular shunts have direct access to arterial circulation and are easier to remove after implantation. However implant failure due to vascular thrombosis is a major disadvantage of a device of this kind [4]. The encapsulation of islet cells in capsules or gels surrounded by semi-permeable membranes is one of the more recent developments in BAP design. The semi-permeable membrane protects the cells from immunorejection and prevents islet cell leakage in the recipient animal. Recent examples of encapsulated BAPs include modified poly (L-lysine) capsules which have shown to support islet al lografts in diabetic rodents for up to one year [5]. Jain et al (1996) have demonstrated restoration of normoglycaemia for over 100 days by implanting 669 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 669-672. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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agarose/collagen macrobeads in streptozotocin-induced diabetic mice [6]. Although encapsulation offers immune protection to the cells, islet dysfunction and cell necrosis caused by poor oxygen and nutrient supply remain problematic with this technology. In addition, evidence is presented here that clearly demonstrates that islets require a technology which enables them to be anchored in a spatial arrangement which can not be offered by encapsulation technology. Techniques For Investigating Rat Islet Survival In Porocell The isolation and culture of islet cells is both difficult and labour intensive. Poor yields, viability and purity of islets can be expected unless highly skilled and experienced staff are dedicated for the isolation and preparative stages. In this study, islet cell viabilities were in the range of 85-95%, as assessed by the trypan blue exclusion method. Each Porocell disc (6.2mm x 2mm) was inoculated with whole islets, islet clusters or single cells. The discs were left undisturbed in an incubator for 1-2 hours prior to addition of fresh medium. The following techniques were adapted for Porocell - islet studies:

1. SCANNING ELECTRON MICROSCOPY (SEM) This technique has proved to be invaluable for microscopic examination of islet morphology and Porocell-islet interactions. Porocell inoculated discs were prepared for SEM by fixation in glutaraldehyde and subsequent dehydration in ethanol solutions. The final stage of dehydration in acetone was minimised to 5 minutes because prolonged acetone exposure caused microscopic “bubbling and pitting” of the Porocell surface. Using SEM, we were able to show that rat islet preparations of single cells, islet clusters and whole islets all formed cytoplasmic adhesion extensions directly onto Porocell (Fig. 1). The islet populations in all cases appeared healthy and had securely adhered to Porocell. Another consistent observation was that of islet adhesion to the underlying monolayer of fibroblasts (Fig. 2).

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2.

FLOURESCENCE MICROSCOPY

Many combinations of fluorescence stains normally suitable for use with animal cells could not be used on Porocell because of problems with auto and induced fluorescence.

We have successfully developed a rapid fluorescent staining assay for islet cells on Porocell which uses a combination of Hoechst 33342 and propidium iodide stains. The method was carefully optimised so that cut sections of Porocell were first rapidly stained with propidium iodide to detect dead cells, then each section was fixed in formaldehyde and stained with Hoechst 33342 to stain the total cell mass. This combination of stains could rapidly differentiate between live and dead cells on Porocell.

3.

CONFOCAL MICROSCOPY

This technique was invaluable for islet cell measurements and for 3-D reconstruction of islets in Porocell (Fig. 3). It appears that the best islet population for inoculation into Porocell are single cells containing a mixed population of A, B and D cells. These single

cells re-aggregate on Porocell to form islet-like structures similar to those in vivo.

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4.

INTRACELLULAR INSULIN EXTRACTION FROM ISLET CELLS IN POROCELL

In this study we have established the assay techniques for measuring intracellular insulin from islet cells cultured in Porocell. Two methods were developed: (a) Extraction for 18 hours in acid/ethanol mixture and (b) Sonication of each disc for three 20 second bursts, with a pause of 5 seconds in between each burst. The total insulin extraction method is an excellent method for estimating the number of B cells retained in Porocell discs because:

The total extracted insulin from Porocell discs, 8 days after inoculation with a mixed suspension of 1 x 105 single cells was 129.4 ±13.5 pmol insulin/disc. The calculated number of B cells on the disc is therefore 6.9 ± 0.7 x 104 cells per disc. Therefore, using this total extracted insulin method, we estimated that 70% of the initial inoculum was a B cell type and that this cell type was retained in Porocell for 8 days after inoculation. Since the B cell type usually accounts for 70-80% of the total islet cell population we conclude that most of these cells were retained in Porocell. 5.

INSULIN SECRETION AND RESPONSE TO GLUCOSE

The secretion of insulin in response to challenges with glucose and secretagogues is the most important quantitative test that determined islet survival and function in Porocell: Porocell discs float on the medium surface and can therefore be cultured in simple designed vessels, equipped with filters for gas exchange and magnetic bars for mixing. We have demonstrated that rat islet cells cultured in Porocell responded to 12 mM challenges of glucose, by secreting more insulin than that in basal medium supplemented with a glucose concentration of 2.5 mM. (Fig. 4) Conclusions

We have demonstrated that the macro-porous material, Porocell can be used for in vitro culture of rat islet cells. Islet cells adhere directly to Porocell and to fibroblast monolayers growing on Porocell. Our preliminary investigations show that islet cells cultured on Porocell in stirred, surface aerated flasks are able to respond to elevated concentrations of glucose, by secreting more insulin into the culture medium. The future development of Porocell as an implantable device will require a combination of cell and material engineering and remodelling. In its present form, the Porocell-islet bioreactor provides an invaluable tool for research and drug evaluation studies in vitro. References 1. Lacy et al. Scientific American, July 1995, p 40 2. Altman et al. In Islet-pancreas transplantation and artificial Pancreas, Federlink K, Pfeiffer E, Raptis, Eds. New York, Theme-Stratton, 1982 3. Lanza et al. Transplant proc 1992, 24, p669-71 4. Maki et al. Diabetes, March 1996, 45, p342-347 5. Jain et al. Transplantation 1996, 61 (4), p532-536 6. Tun et al. Cell Transplantation 1996, 5 (5S1), pS59-S63

We wish to thank BBSRC for funding this study (Grant ref. no. 90/TO6356)

CULTIVATION OF SKIN CELLS SUITABLE FOR RECOVERY OF BURN WOUNDS

T.D. KOLOKOLTSOVA, N.D. YURCHENKO, N.G.KOLOSOV*, O.V. SHUMAKOVA, E.A. NECHAEVA Research Institute of cell culture State Research Centre of Virology and Biotechnology Vector, Koltsovo, Novosibirsk region, RUSSIA * Sibirian Military Hospital N 133, Novosibirsk , RUSSIA

1. Introduction Thermal injuries are-still among urgent problems of surgery, due to the increase in

number of burn patients in the world, especially in industrially developed countries. At present, for treatment and healing of burn wounds a cleaved autoplast perforation is most extensively employed, helping to cover the surface of granulated wounds. But the problem of deficiency of donors skin always exists. That is why researches aimed at the development of new, less traumatic methods of recovery of missing skin integument are of high importance. The method of growing skin cells in vitro with their consequent transplantation to the wound seems the most promising. Recently it has been shown that not only auto- but also allokeratinocytes can be used to restore the skin on a burn wound [1]. At the same time the Russian scientists Sarkisov et al. developed an entirely new method of treating burns by transplantation of cultivated fibroblasts to the wound surface [2]. The present work seeks to produce, to cultivate and to use of scin cells suitable

for recovery of bum wounds. 2. Materials and methods More than 50 human skin speciaments as material for researchs were obtained

from different sources (burn victims, foreskins, aborted material, cadavers). Fibroblasts were obtained according to standard methods of fragmentation and enzymatic treatment of tissue pieces with trypsin solutions. The cell cultivation was carried out in Eagle MEM nutrient medium supplemented with 10% FBS. Firbroblast cells were passaged in 3-5 days at 1:2-1:4 seeding coefficient. Keratinocytes were selected and cultivated accoding to modified Rheinwald and Green's method [4]; keratinocytes were cultivated in a mixture of DMEM and F673 O.-W. Merten et al. (eds.). New Developments and New Applications in Animal Cell Technology, 673-675.

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12 media supplemented with 5-10% of FBS, insulin, hydrocortison and epidermal growth factor . 3. Results and discussion

Production and cultivation of keratinocytes. During the last two years of work 12 strains of human epidermal cells were produced and cultivated from 20 skin samples taken from donors of different age and slate of disease. In two cases when the skin was taken from strongly burnt patients the cell suspension appeared to be low viable, althow the percentage of live cells being 80%; the cells attached to the culture vessel surface badly and practically did not proliferate. In 8 cases the cells were evenly distributed on the glass and formed colonies. At regular change of the medium in 3-4 days they formed either a mono- or a multilayer by clay 20 of in vitro cultivation. Further passage of the cells appeared to be not a success. The cells grew badly and then died. Production and cultivation of fibroblasts. In the course of work about 60 strains of human diploid fibroblasts were produced. 5 cell strains were produced from skin pieces taken from adults, and 25 strains of fibroblasts were produced from embrional dermomuscular and lung tissues. 4 cell strains were rejected after bacterial or mycoplasma contamination was revealed. In spite of preliminary medical control of patients after obtaining the primary cell culture fibroblasts, one case of human hepatitis B virus infection and one case of chlamidial infection were revealed. In the latter case the cells demonstrated good cultural potential and preserved normal morphology. These data demonstrate the importance of testing the obtained cell cultures for contaminantion.

The more suitable way of initiation of fibroblasts from adult's derma was the method of tissue explants. This method allous us to obtain fibroblasts when there is limited quantity of donor's materials. In this case the cells started to migrate out of the skin's explants on day 7-10. As fibroblasts produced from derma

of adults did not show a high proliferative activity in all the cases and required the nutrient medium to be enriched and, besides, had a limited life time (up to 20-25 passages in vitro), we used them only for autotransplantation and did not study their possible use in the future. Embryo fibroblasts possessed a better growth potentiality as compared with fibroblasts of adults’ derma, the proliferation index being 2.0-3.0; the cells

formed an even monolayer on day 2 . As the experience of our work showed, diploid cells preserved good cultural properties up to passages 45-50 in vitro. Embryonal fibroblasts cell cultures which had been preliminarily tested for contaminants and possessed good cultural characteristics were deposited for storage as initial cell culture banks. The use of keratinocytes and fibroblasts for recovery of burn wound. Transplantation of autokeratinocytes was carried out in 5 cases. Unfortunately, the experiments were unsuccessful. Cells badly attached on the wound surface and died after 5-10 days.

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On the other hand , according to literary data, the use of fibroblasts as an alternative source of transplanted cells for skin restoration gives a number of advantages. Fibroblasts are shown to stimutale by synthesis of components of extracellular matrix both adhesion of keratinocytes and proliferation of the latter followed by differentiation. In this connection transplants of cultivated fibroblasts can be used in most cases when there is no total lesion of the skin and stimulation of proliferation of the organism’s own epidermocytes in preserved foci without transplantation of epidermal layers is possible [2]. On the other hand, it was shown that fetal fibroblasts promote healing of wounds without scar [3]. Lately it has been shown that embryo fibroblasts are not only efficient with respect to epidermocyte stimulation but also actively migrate into the zones of mechanic lesion of the cell layer unlike analogous cells of adults [5]. In connection with the above it was interesting to study the possibility of use of human embryo fibroblasts for recovery of burn wounds. For this purpose a monolayer of fibroblasts grown on a sublayer was transplanted to the wound after a burn. In patient B., 55 years old, with a flame burn of 8% (3%) of the body and extremities the wounds of donor sites after autodermoplastics did not close up and suppurated for a long time. On day 3 after transplantation of allofibroblasts the wound clearance of the pus was registered, and complete epithelization was observed on day 7. Histologic examination proved the wound close up with epithelium. The obtained data confirmed the supposition of D.S. Sarkisov et al. [2] concerning the possibility of use of allofibroblasts in treatment of wounds. Besides, they demonstrated the high efficiency of application of human embryo fibroblasts. As fibroblasts differ from keratinocytes by a higher proliferative activity and a simpler composition of the nutrient media required for their growth and support in vitro, we consider it expedient to create a bank of certified human fibroblast cell cultures suitable for wide application in medicine. Creation of a cell cultures bank will allow to provide medical studies with a cell material standardized in biological and genetical properties. References 1. Wood, E.J. and Raxworthy, M.J. (1994) In vitro reconstruction of human skin, Biochemistry 1, 3 -7 2. Sarkisov, D.S., Fyodorov.V.D., Glushchenko, E.V., Alekseev, A.A. et al. (1995) Use of cultured fibroblasts for reconstruction of scin’s cover in strongly burnt patients, Byulleten experimentalnoy biologii i medicini 6, 566-570 (rus.) 3. Rheinwald, J.G. and Green, II. (1975) Serial cultivation of human epidermal keratinocytes: the formation of keratinizing colonies from single cells, Cell 6, 331-344 4. Sullivan, K.M., Meuli, M., MacGillivray, T.E., Adzick, N.S. (1995) An adult-fetal skin interface heals without scar formation in sheep, Surgery 118(1), 82-86 5. Kondo Hiroshi, Matsuda Rei, Yonezawa Yumiko (1993) Autonomous migration of human fetal skin fibroblasts into denuded area in a cell monolayer is mediated by basic fibroblast growth factor and collagen, In Vitro Cell. and Dev. Biol. Anim. 12, 929-935

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TISSUE THERAPY FOR TREATMENT OF PRIMARY MYODYSTROPHIES

Krokhina T.B., Raevskaja G.B., Shishkin S.S.. Research Center for Medical Genetics, Moscow, Russia

1. Introduction. Myoblast transfer therapy (MTT) is an universal treatment for inhereted muscular disorders with defect of dystrofin gene - Duchenne and Becker dystrophies. The main ideas of MTT are: 1) the establishment of primary lines of myoblasts derived from byopsies from healthy donors; 2) the large-scale cultivation of donor's myoblasts (MB); 3) injecting of normal donor's MB into patient's muscles. Donor's MB and patient's myofibers after injection fuse together and form myofibers with normal dystrophin genes [3]. As a result of MTT normal dystrophin appears in patient's muscles. This effect was registrated by immunohistochemical assay of tangential muscle sections from patient's biopsy. Muscle is composed of a mixture of cell types present in variable ration. When muscle is dissociated and the cells plated in culture, both myoblasts and fibroblasts (FB) are obtained and the rate of proliferation of FB frequently exceeds that of MB. After several passages of such culture quantity of myoblasts is decreased [1]. Hight per cent of injected FB ( more than 10%) leads to immunological regection reaction. That is why the obtaining of pure noncontaminated by FB myogenic culture is one of the main problems of cell transfer therapy of myodystrophies. One possibility to resolve this problem is using of special selective conditions for preferential growth of MB and at the same time inhibition of FB proliferation. 2. Materials and Methods Muscle samples were obtained from volonteers during surgical treatment for orthopedic nonmuscle problems. To isolate cells from biopsy (1g) muscle was dissected to remove connective tissue and treated with trypsin (0.25%) and 0.2% collagenase. Dissociated cells were plated on tissue culture dishes or flasks ("Nunc") in growth medium. After 3-6 day in culture, the medium containing the debris of dissociated myofibers was removed. The growth medium contained Ham's nutrient mixture F-10 with 15% fetal calf serum, MOPS, vitamins, sodium pyruvate and antibiotics. Cell were grown at 37°C and 5% For identification of myoblasts in the mixture we used a standart cytochemical test for alkaline phosphotase activity (APA). 677 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 677-679. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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3. Results and Discussion

During the cultivation morphology of human muscle cells changes. Three or five days old culture consists of proliferating mononucleated cells. Then MB begin to align in preparation for fusion; early MB fusion forms immature multinucleated myotubes interspersed with mononuclead cells and aligning MB and at the end multinucleated myotubes are formed. It is one of the best evidenses of myogenic nature of our culture. To improve selective growth of MB we used fibroblast growth factor (FGF) and epidermal growth factor (EGF) - 5 ng/ml. The growth factors were in the growth medium constantly. MB were cultivated on flasks. Cell number was counted for some flasks and thier APA was tested every week. The results of this experiment are presented on Fig. 1,2. As we can see, EGF stimulates proliferation of our culture more effective then FGF. Moreover, EGF leads to increasing of per cent of real MB upto 50% compared to control. That is why we can use EGF in the combinative selective medium in our next investigations.

Then we tried other chemical selective agent - sodium butyrate (SB). SB delays the expression of differentiation in L6 cell line culture. SB (3mM) reversibly inhibits the formation of myotubes without affecting the normal program the formation of differentiation. An almost complete careful arrest in thymidine incorporation was observed in the presence of 2 mM SB for fibroblasts and 5 mM for HeLa cells after 24 h exposure [2,4]. We used these data and treated MB and FB cultures from our cell bank by SB (2,4,8 mM). Like in the experiment with growth factors we cultivated cells on

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flasks with SB. After 4,9,14 days the amount of cells was counted and tested for APA. SB depressed FB proliferation stronger then proliferation of MB. That is why the amount of MB in the mixed culture increases in 1,5 2,0 times. We suppose that sodium butyrate would be very useful for selective medium becouse of SB can decrease not only FB proliferation but can inhibite MB differentiation. It is very important for large-scaile cultivation of MB. Acknowledgments. We thank European Society for Animal Cell Technology and Russian Foundation for Fundamental Research for the financial support of participation of Dr.T.Krokhina in The 15-th Meeting of ESACT. 4. References. 1. Blau, H.M., Webster C., Pavlath G.K. (1991) Purification and proliferation of human myoblasts isolated with fluorescence activated cell sorting, in R.C. Griggs and G. Karpati (eds.),

Myoblast transfer therapy, Plenum Press, New York and London, pp. 97-100. 2. Kruh, J. (1982) Effects of sodium butyrate, a new pharmacological agent, on cells in culture, Molecular and Cellular Biochemistry, 42, 65-82. 3. Law, P.K. (1994) Myoblast Transfer: Gene Therapy for Muscular Dystrophy, R.G. Landes Company Austin, Medical Intelligence Unit. 4. Prasar, K,N. (1980) Butiryc acid: a small fatty acid with livers biological functions, Life Sciences, 27, 1351-1358.

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POSSIBILITY OF APPLICATION OF THYROID ORGAN CULTURE FOR THE TREATMENT OF PERSISTENT HYPOTHYROIDISM

I.P. PASTEUR, N.D. TRONKO, E.N. GORBAN, V.I. KRAVCHENKO Institute of Endocrinology and Metabolism, Acad. Med. Sci. Ukraine Vyshgorodska Str. 69, 254114 Kyiv, Ukraine

1. Introduction Persistent hypothyroidism is rather a frequent after-effect of an excessive surgical

intervention on the thyroid gland, and it requires an additional treatment with LThyroxine for a long period of time, and even for life [1]. Hormonal therapy of persistent hypothyroidism using transplantation of thyroid tissue represents an

attractive alternative, because it may deliver such patients from a long-term pharmacotherapy [1-3]. Reports already exist on clinical use of autotransplantation of cryopreserved thyroid gland in patients with postoperative hypothyroidism [1-3].

However, all other forms of hypothyroidism (besides postoperative one) exclude the possibility of autotransplantation of thyroid gland. Moreover, transplantation as a method of treatment faces today an important and increasing problem: that of shortage of donor organs and tissue [4]. A number of investigators try to resolve this problem, considering that xenotransplantation combined with modern immunosuppressive therapy is a quite realistic way out of this situation

[4]. Porcine tissue is considered as one of the most suitable donor material. The antigenie affinity between human and porcine tissue proteins and blood proteins makes these animals quite convenient donors, and a preliminary organ culturing allows to significantly decrease the immunogenic properties of transplantation material [5]. 2. Materials and Methods Newly removed newborn pig thyroid gland were cut into pieces (less than washed and cultured in medium 199 with a 10% calf serum (“Sigma”, USA) at 37 °C. By the end of each term of culturing, aliquots of medium were selected for quantitative determination of thyroxine and triiodothyronine in them by radioimmunoassay. After this, 74 kBq of 131-I were introduced in each sample for 90 minutes in order to assess the level of iodine uptake by thyroid tissue using radiometric method. Statistical processing of experimental results was performed using methods of analysis of variance. 681 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 681-683. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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3. Results The investigations carried out by the authors showed a capacity of newborn pig thyroid organ culture to actively uptake iodine from cultural medium during a long period of time (Table). A high functional activity of thyroid tissue in vitro has also been confirmed by the results of a quantitative determination of thyroid hormones (thyroxine and triiodothyronine) in cultural medium. The levels of these hormones were inversely proportional to the terms of culturing. Contrary to this, T4/T3 ratio increased from (on day 3 of culturing) to (on day 20).

4. Discussion Thyroid transplantation requires a functioning thyroid follicle, whose structure includes follicular cells, follicular colloid, basement membrane and capillaries, as a minimum unity ensuring thyroid function [3]. For synthesis and secretion of hormones into an appropriate medium, thyroid tissue of a maximum size of pieces up to 1 (such a size prevents central necrosis in each tissue sample) is quite sufficient and it guarantees good results in transplantation of thyroid tissue [6,7]. Moreover, thyroid tissue transplantation instead of thyrocyte transplantation is a more simple process from technical point of view [1,3]. Iodine uptake by thyrocytes represents the first phase of thyroid hormones´ biosynthesis and has a diagnostic impact, since iodine accumulation at the transplantation site confirms, in addition, the viability of thyroid tissue graft [1-3,8]. The radioisotopic criteria of the viability of thyroid tissue graft are the following: scintigraphically radioisotopc accumulation at the transplantation site [1-3], radiometrically - a more than 4-fold relationship between its accumulation by transplantation region and control one (i.e. symmetric part of body) [5,8]. The amount of radioiodine accumulation is considered as a qualitative index of graft viability [5]. Our results also confirmed a report on the capacity of thyroid cells of pig in vitro to actively produce thyroid hormones [9].

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When using results of such investigations in experimental and clinical transplantology, it should be taken into account that the degree of hormonal compensation of hypothyroidism and real amount of radioiodine absorbed by a thyroid graft depend on its functional activity at the moment of study, on the amount of transplanted tissue and level of thyrotropic hormone which circulates in the blood of the recipient [5]. Though functioning of an additional source of thyroid hormones is recognized as the main mechanism of therapeutic effect of transplantation in case of hypothyroidism [1-3], one may not exclude a possible stimulating effect on the recipient’s thyroid gland [2]. So, a study of iodine-accumulating function of a thyroid gland stump in patients with postoperative hypothyroidism showed that, after autoimplantation of cryopreserved thyroid parenchyma, a progressive increase of the capacity for iodine accumulation was observed [2]. An increase in T4/T3 ratio in the process of culturing, in the presence of a progressive decrease of indices of both hormones’ secretion, points out the capacity of thyroid tissue, under unusual conditions for itself, to maintain on a higher level (in the first place) thyroxine secretion as the main thyroid hormone, what is evidence of its high adaptive properties. Therefore, the newborn pig thyroid organ culture preserves its functional activity for a long period of time and it may be used in transplantology. 5. References

1. Shimizu, K., Nagahama, M, Kitamura, Y., et al.: Improvement of thyroid function after autotransplantation of cryopreserved thyroid tissues in rats: clinical application of the procedure to patients with persistent hypothyroid Graves’ disease after thyroidectomy, Thyroidol Clm. Exp., 8 (1996), 55-62. 2. Puchkar, N.S., Makedonskaya, V.A., Utevsky, A.M., et al.: Autoimplantation of cryopreserved

(-196°C) thyroid parenchyma as a method of treatment of post-operative hypothyroidism, Problemy Endokrinologii (Problems of Endocrinology) (Moscow), 30 (1984), 42-46. 3. Shimizu, K., Nagahama, M., Kitamura,Y., et al.: Autotransplantation of cryopreserved thyroid tissues for the treatment of irreversible postoperative hypothyroid Graves’ disease. Report of the first case, Thyroidol. Clin. Exp., 9 (1997), 23-26.

4. Kemp, E.: Xenotransplantation, J. Intern. Med. 239 (1996), 287-297. 5. Lafferty, K.J., Cooley, M.A., Woolnough, J., and Walker, K.Z.: Thyroid allograft immunogenicity is reduced after a period in organ culture, Science, 188 (1975), 259-261. 6. Bauer, M.F., and Herzog, V.: Mini organ culture of thyroid tissue: a new technique for maintaining the structural and functional integrity of thyroid tissue in vitro, Lab. Invest., 59 (1988), 281-291. 7. Kitamura, Y., Shimizu, K., Nagahama, M., and Shoji, T.: Cryopreservation of thyroid pieces - Optimal

freezing condition and recovery, J. Jpn. Surg. Soc., 95 (1994), 14-20. 8. Fischel, R.J., King, N.J., Boyle, E.M., et al.: Evaluation of xenogeneic thyroid transplants as a model of cell-

mediated response, Transplant. Proc., 24 (1992), 535-536. 9. Gruffat, D., Venot, N., Marriq, C., and Chabaud, O.: Thyroid hormone synthesis in thyroglobulin secreted by porcine thyroid cells cultured on porous bottom chambers. Effect of iodide, Endocrinology, 131 (1992), 2921-2927

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HUMAN CELLS AS THERAPEUTIC AGENTS

H. GREEN Harvard Medical School Boston, MA 02115, USA

Abstract: Cell culture in the modem sense is about fifty years old and has been indispensable for the development of modern biology. In addition, cultured cells have been used for the production of some of the proteins encoded by recombinant DNA and in this way have acted as synthetic machinery for the manufacture of therapeutic proteins. Of more recent history is the use of cultured cells themselves as therapeutic agents. There is a wide gulf in thinking and in practice between these applications. Examples will be described of the cultivation and grafting or implantation of cells to treat disease. Discussion

Aunins:

I was surprised in a recent talk by Advanced Tissue Sciences on their skin graft product, when they said that they freeze down their

product without cryopreservatives, and that most of the cells in

their first generation product are dead. Can you comment on those grafts, and why they are successful?

Green:

That product contains fibroblasts; there are no epidermal cells, and it is mainly designed for use as a temporary replacement for the surface before conventional grafts are added later. So, it is not a similar product. The epidermal cells are also not capable of multiplication after cryopreservation but the products they put out are obviously stable enough to carry out the desired objective.

Partridge:

When corneal grafts are done, do the cells repopulate the limbus?

Green:

Yes, they must do so. After they have regenerated the corneal epithelium by a graft, they are sometimes obliged to do a corneal transplant because the corneal stroma has been damaged. So they remove the whole cornea and put a new one down and that gets reepithelialised by normal corneal epithelial cells The only place that

they could come from would be the perimeter and that is a reestablishment of the limbus. Hatzfeld:

Do you know anything about the stem cells of keratinocytes, and about ageing during the many divisions which occur when you do these grafts? If you have ageing, as seen in many other types of 685

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cells, does it have any influence on the quality of the skin? I have heard that some of the burns patients with grafts cannot go into the sun, and that the skin is very fragile. Can you improve this by purifying stem cells and using early progenitors?

Green:

It is not easy to answer those questions. The stem cells are present in the basal layer and, if the cultures are properly done, you will preserve them for a long period because the culture life is of the order of 150 cell generations. If the cultures are not in a good condition when they are applied, they may take but give rise to an atrophic epidermis. That is avoidable by having the best possible cultures. As far as sun exposure is concerned, if the cultures are performed fresh, ie not cryopreserved, the melanocytes will repopulate and so should not be sunlight sensitive. Unfortunately the

melanocytes do not survive cryopreservation, so there would not be many in a preserved culture. Perhaps what you are referring to is heat intolerance, which is a different matter. This is because they lack sweat glands. In third degree burns, sweat glands are destroyed and there is no way of restoring them, even by conventional grafting. Handa-Corrigan:

We have recently started culturing keratinocytes in perfusion culture. At the moment we are obtaining fibroblasts from cadavers. Would you advise me to change this procedure?

Green:

There are additional problems of cells from this source imposed by safety. The human keratinocytes do better on 3T3 cells, than on human fibroblasts, because they are not so firmly attached to the dish. It is necessary when the keratinocyte colonies expand to excavate the 3T3 cells from the surface - they are displaced, so at confluency there are virtually no 3T3 cells left. Human fibroblasts prevent colony expansion to a degree.

Grammatikos:

How long can you keep a patient alive without grafting, and what other treatments can a hospital use?

Green:

The first thing that kills is dehydration and loss of electrolytes, but this is now an exact science and patients are protected from this.

They may also die if the lungs are burnt. The other factor is infection later on, which always occurs, but here antibiotics play an important role. Cultured epidermal cells in large enough quantities are usually not available for 3 weeks, but it takes a good deal of that time to stabilise the patient sufficiently for surgery to be undertaken. In good hospitals patients are easily kept alive for that period but the longer it goes on, the more that infection becomes a problem.

SESSION O N :

USE OF ANIMAL CELLS FOR IN VITRO TESTING Increasing links between animal cell technology and biomolecular screening were reflected by numerous contributions which have been submitted, finally leading to a session dedicated to the use of cells for screening in Vilamoura, in 1996, and a subsequent one in Tours, in 1997, which is featured in this chapter. Biomolecular screening is, like animal cell technology, a highly interdisciplinary and technology-oriented discipline. It integrates well in biological hazard assessment, pharmacology and pharmaceutical lead finding. The use of animal cell cultures within these screening assays is steadily increasing and, therefore, the obvious common interests

and problems suggest the exploration of synergies in both fields. With related goals in mind, both disciplines are using organ resembling and other functional celluler models to investigate and manipulate cellular transport as well as membrane functions and cellular signalling/regulation. One group is focusing on using cells for the production of biomolecules whilst the other is utilizing the cellular product as a reporter signal. The techniques applied for process analytics by the cell technologists and for analyzing biologically active substances by the screening people are also very similar. Both apply related staining methods in combination with microoptical devices as well as physical methods including electrical, optical and others for similar sensors despite their different geometry. Today, cell technologists are probably ahead when controlling the physiological status of cultured cells in order to optimize their processes whereas screening people may be

advanced in observing cellular responses for examining physiological effects of their substances. Each group can learn from the other and cross-fertilize the generation of ideas, which again will hopefully promote the technology development within both fields. This session, dedicated to the use of animal cells for in vitro testing, was designed to give an wide overview on the field covering new cell lines for routine in vitro screening of pharmacological functions, toxicology, and tissue-resembling culture systems. Several examples of the above mentioned numerous technological improvements were presented in this or in other sessions; this attribution was performed in essential where the contents fitted better to the session. G. Barlovatz-Meimon, W. Scheirer Chairpersons

687 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 687. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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EMBRYONIC STEM CELL DIFFERENTIATION MODELS: CARDIO-

VASCULAR, MYOGENIC AND NEUROGENIC DEVELOPMENT IN VITRO

A.M.WOBUS, K. GUAN, J. ROHWEDEL, C. STRÜBING* and M. DRAB #

In Vitro Differentiation Group, IPK Gatersleben, D-06466 Gatersleben, *Institute #

of Pharmacology, FU Berlin, D-14195 Berlin,

Franz-Volhard Clinic, MDC, D-13122 Berlin-Buch,

Germany

Abstract

Embryonic stem (ES) cells, totipotent cells of the early mouse embryo, established as

permanent cell lines, provide one of the most important developmental systems for the introduction of preselected genes into mice by using the gene targeting technology. ES

cells when cultivated as embryo-like aggregates, so-called 'embryoid bodies', are able to

differentiate into derivatives of all primary germ layers, endoderm, ectoderm and mesoderm. 689 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 689-703. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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Differentiation protocols for the in vitro development of ES cells into cardiomyocytes, skeletal muscle, neuronal and vascular smooth muscle cells were established, and a developmentally controlled expression of genes, proteins, ion channels, receptors and action potentials was observed. The in vitro differentiation system is now being used for genetic analyses by 'gain of function' and 'loss of function' approaches and for studying

regulation of differentiation by exogenous compounds.

1. Introduction

Embryonic stem (ES) cells, totipotent cells of the early mouse embryo were established

as permanent lines which retain their developmental capacity in vitro (Fig. 1; Martin,

1991; Evans and Kaufman, 1981; Wobus et al., 1984; Doetschman et al., 1985; Wobus et al., 1991). After retransfer of ES cells into the mouse blastocyst, ES cells in vivo are able to generate cells of all lineages including the germ line (Bradley et al., 1984).

Therefore, ES cells provide an important cellular system for introducing preselected genes into mice by the gene targeting technology: Mutations introduced into ES cells by homologous recombination are transferred into the germ line and result in the generation of mice strains containing specific genetic defects (Thomas and Capecchi,

1987; see Fig. 1). In addition, ES cells differentiating in vitro via embryo-like aggregates, the so-called 'embryoid bodies', develop into cellular derivatives of all three primary germ layers of endodermal, ectodermal and mesodermal origin. Undifferentiated ES cells develop from early stages into terminally differentiated and specialized cells of the cardiogenic,

myogenic and neurogenic lineage. During this differentiation we found a

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developmentally controlled expression of genes, ion channels and receptors (Maltsev et al., 1993; 1994; Rohwedel et al., 1994; Strübing et al., 1995). Recently, we also

described epithelial differentiation and the in vitro formation of vascular smooth muscle cells (Bagutti et al., 1996; Drab et al., 1997). Other laboratories established the differentiation of ES cells into haematopoietic and endothelial cells (Risau et al., 1988;

Wiles and Keller, 1991; Weiss et al., 1994). This controlled in vitro developmental pattern may be modulated by exogenous factors, for example, retinoic acid (Wobus et al., 1994; 1997b), or by genetic means: the

targeted inactivation of genes (= "loss of function", Fässler et al., 1996) or the overexpression of genes (= "gain of function", Rohwedel et al., 1995).

2. In vitro differentiation of ES cells

2.1. DIFFERENTIATION PROTOCOLS

The ES cell differentiation protocol is shown in Fig. 2 and the details have been

described in detail elsewhere (Wobus et al., 1991; Maltsev et al., 1993; Wobus et al.,

1997a). In principal, ES cells are cultivated in hanging drops for two days, and after transfer into bacteriological plates, cultivated in suspension for additional two to five days. During this process the ES cells differentiate in the embryoid body (EB) and build

up differentiated cellular structures of endodermal, ectodermal and mesodermal origin. The EBs are plated at day 4 (neuronal cells), 5 (cardiac or myogenic cells) or 7 (cardiac

or vascular smooth muscle cells) and the differentiated cells are growing out (for details

692 see Wobus et al., 1991; Rohwedel et al., 1994; Maltsev et al., 1994; Strübing et al., 1995; Drab et al., 1997).

During in vitro development of EBs the expression of cardiac-, muscle-, neuron-, epithelial and vascular smooth muscle-specific genes and ion channels was analysed by

RT-PCR and by patch clamp analysis, respectively (reviewed by Wobus et al., 1997a). The formation of proteins was estimated by immunofluorescence on differentiated cells of embryoid body outgrowths.

2.2. FACTORS THAT INFLUENCE IN VITRO DEVELOPMENT

The differentiation efficiency of ES cells is determined by several parameters; (i) the

number of cells differentiating in the embryoid body, (ii) culture conditions, media and additives, the quality of fetal calf serum, and (iii) the time of plating the EBs. In

addition, different ES cell lines show a different developmental pattern in vitro. To obtain maximal differentiation of ES cells into defined cell types specific differentiation conditions and cell lines were used (see Wobus et al., 1997a).

3. ES cells differentiate into cardiogenic, myogenic, neurogenic, epithelial and

vascular smooth muscle cells in vitro

In the following chapter a short overview about the characterization of ES cell-derived phenotypes is given. The specific genes, proteins and ion channels are explained in the legend to Figure 3.

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Cardiogenesis: ES cells of several lines (D3, Doetschman et al., 1985, R1, Nagy et al., 1991, and CCE, Wiles and Keller, 1991) differentiate via EBs into clusters of

spontaneously beating cardiomyocytes. During differentiation, ES cells develop into mesodermal progenitor cells and early cardiomyocytes which further specialize into

cardiac cells representing atrium-, ventricle- and pacemaker-like cells (Maltsev et al., 1993). They are characterized by developmentally controlled gene expression, formation of proteins and receptors as well as cardiac-specific ion channels (Fig. 3; Miller-Hance et al., 1993; Maltsev et al., 1993; 1994; Wobus et al., 1997a; Hescheler et

al., 1997). Furthermore, we found that the differentiated cardiomyocytes respond with characteristic chronotropic responses to cardiotropic drugs (Wobus et al., 1991).

Therefore, in vitro differentiated cardiomyocytes were used to establish a semiautomatic computer-assisted imaging system for a routine screening of chronotropic cardioactive drugs (Pich et al., submitted).

Myogenesis: Myogenic differentiation of ES cells into skeletal muscle cells is shown by the formation of myoblasts which during terminal differentiation fuse into

multinucleated myotubes. During myogenic development, muscle-specific genes, proteins and ion channels are time-dependently expressed. The data obtained from ES

cell line BLC6 are summarized in Fig. 3 (Rohwedel et al., 1994; Rohwedel et al., 1995; K. Guan, unpublished data).

Neurogenesis: ES cells of line BLC6 differentiate after induction with retinoic acid

(RA) into neuronal cells which express neuron-specific genes and possess the complex

electrophysiological and immunocytochemical properties of postmitotic nerve cells

(Strübing et al., 1995; Bain et al., 1995). Data about gene expression, formation of neurofilament proteins and synaptic vesicle proteins (synaptophysin), voltage-

694

dependent ion currents and neuron-specific receptor-operated ion channels are presented in Fig. 3. In addition to the expression of specific neuronal receptors neuronal

cells generate Na+-driven action potentials and are functionally coupled by inhibitory (GABAergic) and excitatory (glutamatergic) synapses as revealed by measurements of postsynaptic currents (Strübing et al., 1995; Wobus et al., 1997a). Epithelial cell differentiation: One of the most prominent cell types in the ES cell-

derived embryoid body outgrowths are epithelial cells. Genes and proteins characteristic for early (K8, K18, K19), intermediate (K14) and terminal (involucrin)

differentiation of epithelial cells are expressed (Fig. 3; Bagutti et al., 1995).

Vascular smooth muscle cell differentiation: A complex cell type which differentiate

from ES cells, are spontaneously contracting vascular smooth muscle (VSM). We established a specific differentiation protocol by using RA and db-cAMP for the

induction of VSM cells (Drab et al., 1997). RT-PCR and immunostaining confirmed the expression of VSM cell-specific transcripts and VSM-specific MHC proteins. In addition, smooth muscle cells expressed typical ion channels and responded to specific agonists with an increased intracellular

release (Fig. 3; Drab et al., 1997).

4. Summary and conclusions

The establishment of ES cell differentiation models allowed to study cellular

differentiation processes during early embryonic development in vitro. The developmental systems permit the analysis of differentiation of early embryonic cells

695 via progenitor cells into highly differentiated and specialized cells of the

cardiovascular, myogenic and neurogenic lineages. With respect to cardiogenic development, the ES cell differentiation system is suitable for a routine screening of pharmacological functions on differentiated cardiomyocytes (Wobus et al., 1991; Wobus et al., 1997b; Pich et al., submitted). Furthermore, the

effects of growth and differentiation factors or extracellular matrix proteins may be investigated (Wobus et al., 1994; 1997b; Johansson and Wiles, 1995).

In addition, the differentiation of genetically modified cells by "gain of function"

(Rohwedel et al., 1995) and "loss of function" (Fässler et al., 1996; Wobus and Guan,

submitted) of totipotent ES cells in vitro is an excellent alternative and substitute to in vivo studies with transgenic animals to analyse the phenotypes of mutant cells during

early embryogenesis.

Acknowledgements

We wish to thank the Deutsche Forschungsgemeinschaft (Wo 503/1-3, SFB 366/YE1) and Fonds der Chemischen Industrie for financial support of our research projects. Jürgen Rohwedel present adress: Institute of Medical Molecular Biology, University of Lübeck.

696

Permanent ES cell lines were cultivated from the inner cell mass (ICM) of mouse

blastocysts. These pluripotent ES cells are able to differentiate via embryoid bodies into derivatives of the endodermal, ectodermal and mesodermal lineage. In addition, ES cells were used to inactivate genes by homologous recombination (Thomas and

Capecchi, 1987). After retransfer of these mutant ES cells into the blastocyst, the cells can colonize the ICM and, after transplantation into pseudopregnant foster mothers result in chimaeric animals showing different phenotypes. If the mutation results in early embryonic death (i.e.,

integrin-deficient embryos die around day 5 of

pregnancy, see Fässler et al., 1996), the in vitro "loss of function" approach is an alternative to analyze effects of the specific mutation on cellular differentiation.

697

ES cells (ESC) were differentiated as embryoid bodies (EBs, schematical picture) in

hanging drops for two days, and after further cultivation in suspension, plated between day 5 or 7 (depending on the differentiation into the specific cell lineage). EBs attach to tissue culture plates and differentiated cells develop in the EB outgrowths. The cells are

characterized by a controlled expression of genes and proteins as well as functional properties.

698

699

Fig. 3: Developmentally controlled expression of tissue-specific genes, proteins and ion channels after differentiation of embryonic stem cells into cardiac, skeletal muscle, neuronal, epithelial and vascular smooth muscle cells in vitro.

Cardiogenesis:

,

ANF (atrial natriuretic factor), MLC-2v (ventricular

isoform 2 of myosin light chain), MHC (myosin heavy chain); cardiac-specific ion currents:

Myogenesis: Myogenic determination genes Myf5, Myogenin, MyoD and Myf6, nicotinic acetylcholine receptors

skeletal muscle-specific L-type and

channels Neurogenesis: NFL (68 kDa), NFM (160 kDa) and NFM (200 kDa) neurofilament proteins; neuron-specific voltage-dependent ion currents: neuron-specific receptors:

Gly (Glycin), Kai (Kainate), NMDA (N-methyl-D-aspartate), Epithelial differentiation: K8, K10, K14, K18, K19 (keratins 8, 10, 14, 18, 19), inv

(involucrin),

Vascular smooth muscle cell differentiation: V-SM-MHC-A (vascular smooth muscle myosin heavy chain A), I-SM-MHC-B (intestinal smooth muscle myosin heavy chain B),

(smooth muscle

(platelet-derived growth factor AB),

), A II-R (angiotensin II-receptor), PDGF AB

700 References

Bagutti, C, Wobus, A.M., Fässler, R. and Walt, F. (1996) Differentiation of embryonal stem cells into keratinocytes: Comparison of wild-type and

integrin-deficient cells, Dev. Biol. 179, 184-196.

Bain, G., Kitchens, D., Yao, M., Huettner, J.E. and Gottlieb, D.I. (1995) Embryonic stem cells express neuronal properties in vitro. Dev. Biol. 168, 342-357. Bradley, A., Evans, M., Kaufman, M.H., Robertson, E. (1984) Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines, Nature 309, 255-256.

Doetschman, T.C., Eistetter, H.R., Katz, M., Schmidt, W. and Kemler, R. (1985) The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and

myocardium. J. Embryol. Exp. Morphol. 87:27-45. Drab, M., Haller, H., Bychkow, R., Erdmann, B., Lindschau, C., Haase, H., Luft, C. and Wobus, A.M. (1997) From totipotent embryonic stem cells to spontaneously contracting vascular smooth muscle cells: a retinoic acid and db-cAMP in vitro differentiation model, FASEB J. (in press).

Evans, M.J. and Kaufman, MH (1981) Establishment in culture of pluripotential stem cells from mouse embryos. Nature 291, 154-156. Fässler, R., Rohwedel, J., Maltsev, V., Bloch, W., Lentini, S., Guan, K., Gullberg, D., Hescheler, J.,

Addicks, K.and Wobus, A.M. (1996) Differentiation and integrity of cardiac muscle cells are impaired

in the absence of

integrin. J Cell Sci 109, 2989-2999.

Hescheler, J., Fleischmann, B.K., Lentini, S., Maltsev, V.A., Rohwedel, J., Wobus, A.M., Addicks, K.: Embryonic stem cells: a model to study structural and functional properties in cardiomyogenesis. Cardiovasc. Res. (in press).

Johansson, B.M. and Wiles, M.W. (1995) Evidence for involvement of Activin A and bone morphogenetic

protein 4 in mammalian mesoderm and hematopoietic development. Mol. Cell. Biol. 15, 141-151.

701 Maltsev, V.A., Rohwedel, J., Hescheler, J. and Wobus, A.M. (1993) Embryonic stem cells differentiate in vitro into cardiomyocytes representing sinusnodal, atrial and ventricular cell types. Mech. Dev. 44, 41-

50.

Maltsev, V.A., Wobus, A.M., Rohwedel, J., Bader, M. and Hescheler, J. (1994) Cardiomyocytes differentiated in vitro from embryonic stem cells developmentally express cardiac-specific genes and

ionic currents. Circ. Res. 75, 233-244. Martin, G. (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma cells. Proc. Nat. Acad. Sci. USA 78, 7634-7638. Miller-Hance, W.C., LaCorbiere, M., Fuller, S.J., Evans, S.M., Lyons, G., Schmidt, C., Robbins, J. and Chien, K.R. (1993) In vitro chamber specification during embryonic stem cell cardiogenesis. J. Biol. Chem. 268, 25244-25252. Nagy, A., Rossant, J., Nagy, W., Abramow-Newerly, Roder, J.C. (1993) Derivation of completely cell

culture-derived mice from early passage embryonic stem cells. Proc. Natl. Acad. Sci USA 90, 84248428.

Pich, U., Pütz, D. and Wobus, A.M. Ein neues Screeningverfahren zur Messung chronotroper Effekte an Herzzellen (A new screening method for the measurement of chronotropic effects on cardiac cells)

(submitted). Risau, W., Sariola, H., Zerwes, H.-G., Sasse, J., Ekblom, P., Kemler, R. and Doetschman, T. (1988) Vasculogenesis and angiogenesis in embryonic stem cell-derived embryoid bodies. Development 102, 471-478 Rohwedel, J., Maltsev, V., Bober, E., Arnold, H.-H., Hescheler, J. and Wobus, A.M. (1994) Muscle cell

differentiation of embryonic stem cells reflects myogenesis in vivo: Developmentally regulated expression of myogenic determination genes and functional expression of ionic currents. Dev. Biol. 164, 87-101.

702 Rohwedel, J., Horak, V., Hebrok, M., Füchtbauer, E.-M. and Wobus, A.M. (1995) M-twist expression inhibits mouse embryonic stem cell-derived myogenic differentiation in vitro. Exp. Cell Res. 220, 92100. Strübing, C., Ahnert-Hilger, G., Jin, S., Wiedenmann, B., Hescheler, J. and Wobus, A.M. (1995) Differentiation of pluripotent embryonic stem cells into the neuronal lineage in vitro gives rise to

mature inhibitory and excitatory neurons. Mech Dev 53, 275-287. Thomas, K.R. and Capecchi, M.R. (1987) Site-directed mutagenesis by gene targeting in mouse embryoderived stem cells. Cell 51, 503-512. Weiss, M., Keller, G. and Orkin, S. (1994) Novel insights into erythroid development revealed through in vitro differentiation of GATA-1-embryonic stem cells. Genes Dev. 371, 221-226.

Wiles, M.V. and Keller, G. (1991) Multiple hematopoietic lineages develop from embryonic stem (ES) cells in culture. Development 1 1 1 , 259-267.

Wobus, A.M., Holzhausen, H., Jäkel, P. and Schöneich, J. (1984) Characterization of a pluripotent stem cell line derived from a mouse embryo. Exp. Cell Res 152, 212-219.

Wobus, A.M., Wallukat, G. and Hescheler, J. (1991) Pluripotent mouse embryonic stem cells are able to

differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca 2+ channel blockers. Differentiation 48, 173-182. Wobus, A.M., Rohwedel, J., Maltsev, V. and Hescheler, J. (1994) In vitro differentiation of embryonic stem cells into cardiomyocytes or skeletal muscle cells is specifically modulated by retinoic acid. Roux's Arch. Dev. Biol. 204, 36-45.

Wobus, A:, Rohwedel, J., Strübing, C., Jin Shan, Adler, K., Maltsev, V. and Hescheler J. (1997a) In vitro

differentiation of embryonic stem cells. In: Klug, E and Thiel R. (Eds.) Methods in Developmental Toxicology and Biology. Blackwell Science Berlin Vienna, p. 1-17.

703 Wobus, A. M., Guan, K ., Shan. J., Wellner. M.-C., Rohewdel, J.., Ji G., Fleischmann, B., Katus, H.A., Hescheler J., and Franz, W.-M. (1997b) Retinoic acid accelerates embryonic stem cell-derived cardiac differentiation

and enhances development of ventricular cardiomyocytes. J. Mol. Cell. Cardiol. 29, 1525-1539. Wobus. A.M. and Guan, K, Embryonic stem cell-derived cardiac differentiation. Modulation of differentiation and 'loss of function' analysis in vitro (submitted).

Discussion

Bader:

How do you get the smooth muscle cells to contract spontaneously?

Wobus:

The embryonic bodies were differentiated from a totipotent stem cell line and specifically induced by retinoic acid and dibutyrl c AMP in a very defined window of embryonic development. You can induce different cell types with retinoic acid but it depends upon the concentration and the time during development on how the retinoic acid acts. We use very defined protocols of medium, number of cells, etc. which are specific for cardiac, or neuronal, cells.

Singhvi:

What sub-stratum was used for cell out-growth, and was it different for the different types of cells?

Wobus:

It is not different for the different lineages. We use gelatin (0.01%) coated tissue culture plates just to get an efficient adhesion of cells. The main characteristic of the embryonic stem cell is totipotency, the capacity to differentiate into cells of different lineages.

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RESPONSES

OF

HUMAN

LUNG

EPITHELIAL

CELLS

(A549)

TO

PATHOGENIC INFECTION BY MYCOPLASMA PNEUMONIAE A549 cells and Mycoplasma pneumoniae.

GOODMAN J., MORLEY K., PACKER P., BATTLE T. CAMR, Porton Down, Salisbury, Wiltshire, SP4 0JG, UK. 1. Abstract

Mycoplasma pneumonia have been shown to be infectious to the human respiratory system. The cells were grown in 'Transwells' to investigate whether they could be used in a new in vitro model of mycoplasmal infection. Trans-Epithelial Electrical Resistance (TEER) of M. pneumoniae-infected A549 cells, laser-beam counting (FACS) and PCR were used for monitoring infection. Mycoplasma orale served as a non-pathogenic control. Three hours after inoculation with M. pneumoniae, TEER Values showed a marked disruption of the A549 monolayers combined with an increase in the number of mycoplasma. In comparison M. orale caused no significant change to the monolayer.

The number of mycoplasma was calculated from standard curves (FACS). Elimination of external M. pneumoniae and PCR of the infected A549 cells, produced specific bands

of amplified DNA. Thus suggesting that the cells become penetrated. 2. Introduction

Mycoplasma are the smallest free living, self-replicating type of bacteria known. An individual cell can range from in diameter and a colony varies between 10in diameter (Kenny,G. 1985). M. pneumoniae is pathogenic to humans. It causes primary atypical pneumonia, accounting for 10% of all X-ray proven pneumonia. It is also responsible for other minor respiratory illnesses such as pharyngitis. As a consequence it carries much scientific interest. The main aim of this project was to investigate an in vitro test for mycoplasma infection using M. pneumoniae and human lung epithelial cells (A549) with M. orale as a non-pathogenic control. The cells were

grown on Transwell systems (Costar). The cells grew on a collagen-coated, porous membrane that was suspended 1mm above the base of the well, creating compartments above and below the cell monolayer. The responses of individual cell monolayers to infection were measured by Trans-Epithelial Electrical Resistance (TEER). The untreated confluent cell monolayer has a specific resistance if this monolayer is altered in any way then the resistance will change. Counting mycoplasma is difficult and time consuming as they can only be seen through a light microscope when they are grown on agar plates. The use of Fluorescent Activated Cell Sorter (FACS) was therefore

investigated. An invasion assay aimed to determine the location of M. pneumoniae during infection. 705 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 705-711. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

706

3. Materials and Methods

A549 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) purchased from Sigma chemical co., which was supplemented with 10% foetal calf serum and 1% L-Glutamine. Mycoplasma were grown in mycoplasma broth prepared in the Culture Collection. 3.1 TRANSWELLS

The bottom compartment was filled with 0.5 ml of DMEM. A volume of 0.5ml of cell suspension was grown on each membrane until reaching confluency, in 5%

atmosphere at 37°C. Mycoplasma were added to the cells at a concentration of in their exponential growth phase. 3.2 TEER

Using a Millicell-ERS system, three Transwells were measured between 30-330 minutes after infection and each individual Transwell was measured in triplicate to ascertain the

accuracy of the measurements. The changes in resistance were compared to the original resistance of the same cells before infection. 3.3 FACS

Standard curves were set up from a serial dilution of mycoplasma of known concentration (standard curves). The number of counts in 60 seconds against the Log of concentration was plotted. M. pneumoniae was inactivated by 0.6% v/v formaldehyde, before use in the FACS machine. Samples were taken, in duplicate, from above and below the cell monolayer at each time interval and counted in the FACS machine.

3.4 INVASION ASSAY The external mycoplasma were eliminated by kanamycin in DMEM. The solution was removed after two hours and the cells washed three times with phosphate buffered saline (PBS). The cells were then lysed by 0.1% TritonX-100. The DNA was

extracted using Qiagen QIAamp blood kit. A polymerase chain reaction was carried out on the extracted DNA sample using M. pneumoniae specific primers. 4. Results

The stability of uninfected A549 cells over time was ascertained. The Ohms. were calculated using the surface area of the Transwells The values for the untreated A549 cells were stable between 30 and 330 minutes; the resistance did not increase above (Graph 1). The mean TEER profile of three experiments of M. pneumoniae infected A549 cells showed a change in resistance. The resistance

707

decreased gradually after infection and reached the lowest value of at 210 minutes (Graph 1). This can be compared to the control profile of A549 cells which displays around at 180 minutes.

The experiment was carried out on M. orale, a non-invasive mycoplasma, in order to confirm that the TEER profile of M. pneumoniae was due to infection. The profile of M. orale was similar to that of untreated A549 cells.

The values were stable and the

resistance did not fall more than -30 Ohms.cm2 the profile being very close to zero

at 180 minutes (Graph 2).

i

708

4.1 FACS COUNTS The concentrations of the mycoplasma samples were calculated from the standard curves. Two separate counts experiments showed a similar pattern for M. pneumoniae over time. The numbers in the top compartment in the first experiment began to increase at 90 minutes, reaching a peak of at 150 minutes. The numbers decreased and levelled out at 180 minutes. The amount in the lower compartment showed no large change over the infection time apart from a small rise at 210 minutes (Graph 3).

709

4.2 INVASION ASSAY

The primers used in the PCR experiments were tested for their specificity. A control PCR carried out on different strains of M. pneumoniae proved that the primers were specific. M. orale, M. genitalium and A549 cell DNA were also tested on the primers and none of their DNA was amplified. The invasion assay experiment allowed for a long infection time, in order to maximise the chance of invasion. The cells were left to infect the cells overnight for 16.5 hours. The primers amplified DNA that formed bands at 345bp suggesting that M. pneumoniae DNA was detected inside of the cells. 5. Discussion

TEER has been shown to be a sensitive and reliable method for measuring small changes to the confluent A549 cell monolayers over a period of time. The profiles of pattern of change over time produced are important rather than the absolute values. The TEER experiment on untreated A549 cells demonstrated the stability of the TEER device. The TEER of M. pneumoniae infected cells portrayed a definite decrease in resistance, indicating an alteration in the cell monolayer. The subsequent increase in resistance suggests that the cells recovered and the monolayer is restored. It is not clear whether the disruption is due to the M. pneumoniae penetrating, attaching to, or passing through the gap-junctions of the cells. The recovery of the monolayer implies that M pneumoniae cause only temporary alteration to the cells. The profile of M. orale was similar in shape to that of the untreated A549 cells, indicating that the addition of M orale does not physically damage or disrupt the A549 cell monolayer between 30 and 330 minutes. The large difference between the profiles of the two types of mycoplasma implies that they are behaving differently. The two FACS counts experiments on M. pneumoniae showed no significant change in the numbers below the cell monolayer between 30 and 330 minutes. It therefore seems

710

unlikely that they were passing through the gap junctions into the lower compartment. The peak above the monolayer that occurs in both of the separate experiments has no obvious explanation. The increase in numbers may simply be due to the growth of M. pneumoniae. The subsequent decrease in numbers could be due to either the attachment to or penetration of the cells or to the death of M. pneumoniae. The M. orale FACS counts experiment showed no peak in the numbers above the cell monolayer. The lower and upper compartments contained similar amounts that remained stable throughout the time period (30 and 330 minutes). Since M. orale were also added to the cells in the same growth phase, it seems unlikely that the rise in numbers of M. pneumoniae were due to rapid growth. The invasion assays aimed to determine the location of the M. pneumoniae during infection. The results obtained suggest that the M. pneumoniae do penetrate the cells. Without photographic evidence such as electron microscopy it is hard to determine their exact location. Further studies in this area are needed before any definite conclusions can be drawn about the process of M. pneumoniae infection. The results obtained from this study

show that M. pneumoniae, a pathogen, temporarily disrupts A549 cell monolayers between 30 and 330 minutes after infection. It behaves differently to M. orale, a nonpathogen and comes into contact with the A549 cells.

6. Conclusion The Transwell systems provided an effective method for studying mycoplasmal infection of cell monolayers in vitro. The TEER is a sensitive method for monitoring changes in cell monolayers. M. pneumoniae temporarily disrupts the cell monolayer, whereas M. orale has no apparent effect. The FACS analysis is an effective way of monitoring the pattern of infection of the mycoplasma. M. pneumoniae acts differently to M. orale. The invasion assay experiments suggested that M. pneumoniae either enter the cells or become firmly attached to the cell membrane. 7. References

Kenny, G. (1985) The manual of clinical microbiology, 4th edition, American Society of Microbiology, Washington DC,USA. 8. Acknowledgements

This work was in part sponsored by the Department of Health.

711

Discussion

Onadipe:

Did you have any visual confirmation of the disruption of the mono layer?

Goodman:

No, it is very difficult to see them breaking apart. That is why we use TEER as it is very sensitive.

Noé:

Can this be used as a very quick test for detection of mycoplasma infection?

Goodman:

The TEER? Yes, although with M. orali there was no difference with TEER, so only pathogenic mycoplasma can be used.

Noé:

Can you state the time-span to perform the test?

Goodman:

330 min.

Shirahata:

Why did you choose the A549 cell line? Can any other cell line derived from lung be used?

Goodman:

We chose a human cell line because it is a human respiratory

pathogen and the A549 was convenient to use at ECACC. Shirahata:

Have you tried other cell lines?

Goodman:

No.

Bernard:

Did you check whether the amplitude of your TEER response was dependant on the number of mycoplasma particles?

Goodman:

We added mycoplasma to each transwell. We did not try other concentrations.

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POLAR LIPID PROFILING OF MYCOPLASMA PNEUMONIAE-INFECTED HUMAN LUNG EPITHELIAL CELLS.

Polar lipids and Mycoplasma pneumoniae infection.

GOODMAN J., WAIT R., BATTLE T. CAMR, Porton Down, Salisbury, Wiltshire, SP4 0JG, UK.

1. Abstract

Some mycoplasma are known to contain novel glycolipids which have no counterparts in eukaryotic cells. Profiling of polar lipids is thus a potential strategy for the detection of mycoplasmal contamination of cell lines. Polar lipids were isolated by Bligh-Dyer extraction, and were analysed by positive and negative ion fast atom bombardment

mass spectrometry (FAB-MS). Individual phospholipid molecular species were identified by helium collisional activation and linked scanning techniques. Polar lipid profiles were determined from A549 lung cells, Mycoplasma pneumoniae-infected A549 cells and pure cultures of M. pneumoniae. Uninfected A549 cell extracts

contained hexadecanoate- and octadecenoate substituted phosphatidylcholines, whereas the major phospholipids of M. pneumoniae were sphingomyelin, phosphatidylglycerol and phosphatidylcholines, the latter apparently derived from the culture medium. 2. Introduction

Mycoplasmal contamination is a common problem in experimental cell culture [1], M. orale, M. arginini, M. hyorhinis, M. fermentans, M. salivarum and Acholeplasma laidlawii accounting for the majority of instances. Infection of eukaryotic cells by mycoplasma may have subtle and unpredictable effects, including growth inhibition and altered physiology and metabolism [1]. Moreover, since several mycoplasma are pathogenic to humans, contamination of cell lines or cell-derived therapeutic products

poses a potential health hazard. Mycoplasma species have been shown to contain unusual phospholipids [2], some of which have no counterparts in eukaryotic cells. Profiling of polar lipids might thus provide a potential strategy for the detection of mycoplasmal contamination of cultured cell lines, and could possibly also aid diagnosis of human infections, as current methods of clinical diagnosis are relatively unsatisfactory. Since fast atom bombardment mass spectrometry (FAB MS) is one of

the most sensitive and powerful methods available for the characterization of polar lipids, we have used it to investigate the influence M. pneumoniae infection on the phospholipid composition of a human bronchial epithelial cell line, A549. 713 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 713-715. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

714 3. Materials and Methods

A549 cells were grown in Dulbecco’s modified Eagle medium (DMEM) until confluent, trypsinized, harvested by centrifugation and freeze-dried. M. peumoniae was cultured in Mycoplasma broth at for 3-4 days until exponential growth phase, recovered by centrifugation and lyophilized. For infection experiments A549 cells were incubated with M. pneumoniae, and harvested as described after 2 or 5 days growth. Polar lipids were extracted from the freeze-dried biomass by the procedure of Bligh and Dyer [3]. Fast atom bombardment mass spectra were recorded with a Kratos MS80 RFA spectrometer. Spectra of phospholipids were recorded in both the positive and negative ion modes using 3-nitrobenzyl alcohol (3-NBA) as liquid matrix. 4. Results

In the positive ion FAB mass spectrum of a Bligh-Dyer extract of A549 cells, protonated molecules are observed at m/z 732, 760, 786 and 788. An abundant ion at

m/z 184 was consistent with the presence of phosphocholine. Collisional activation experiments indicated that this ion was a daughter fragment of the protonated molecules at m/z 732, 760 and 786/788, suggesting that the major species present were phosphatidylcholines with differing fatty acid substitution patterns. In the corresponding negative ion spectra carboxylate anions were observed at m/z 253, 255, 281 and 283, attributable to the presence of hexadecenoate (16:1), hexadecanoate (16:0), octadecanoate (18:0) and octadecenoate (18:1) substituents. Thus the major phospholipids are phosphatidylcholines substituted with 16:0/16:1 (m/z 732), 16:0/18:1 (m/z 760) and 18:1/18:1 (m/z 786). Minor signals at m/z 718 and 746 were identified as phosphatidyl ethanolamines by means of constant neutral loss scan for 141, the characteristic mass of the ethanolamine phosphate head group. Confirmation of these assignments was provided by analysis of an authentic standard of 1-hexadecenoyl-2octadecenoyl-sn-glycero-3-phosphocholine. The predominant high mass signal in the positive ion FAB spectrum of the lipids extracted from a pure culture of M. peumoniae was observed at m/z 703, with less abundant ions at m/z 731, 758 and 786. A fragment at m/z 184 indicated the presence of phosphocholine containing components; however the protonated molecules at m/z 758, 786 and 808, although consistent with the protonated molecules of phosphatidyl cholines, are too weak to account for its high abundance. Moreover, in the corresponding negative ion spectrum the major carboxylate anions were observed at m/z 255 and 283, suggesting that the predominant fatty acyl groups were hexadecanoate and octadecanoate, rather than octadecenoate substituents originating from phosphatidyl choline. The major deprotonated molecules in the negative ion spectrum were observed at m/z 747 and 775, corresponding to phosphatidly glycerols substituted 16:0/18:1 and 18:0/18:1.

715 The unidentified component at m/z 703 was identified by collisional activation and B/E linked scans. The major daughter ion was observed at m/z 183.9, consistent with a facile loss of phosphocholine, which suggested that the ion at m/z 703 is the protonated molecule of a hexadecanoate-containing sphingomyelin. The species at m/z 731 is

presumably an octadecanoate-containing homologue. This observation is consistent with previous reports of sphingomyelins in the membranes of mycoplasma, including M. pneumoniae [5]. The phosphatidylcholine composition of mycoplasma broth was very similar to that of the M. pneumoniae cells, suggesting that exogenous PC was being directly incorporated rather than synthesised de novo. We did not observe the modification of endogenous PC to a disaturated species which has been reported in M. gallisepticum, M. pulmonis and M. pneumoniae [6]. Only trace amounts of sphingomyelin were detected in the broth, suggesting either that this material is synthesised de novo, or, alternatively that M. pneumoniae is able to scavenge it with

high efficiency.

After two days infection of A549 cells with M. pneumoniae there was little change in polar lipid profile compared to uninfected cells. There appears to be no significant difference in the proportion of sphingomyelin, phosphatidylglycerol, or of fully

unsaturated phosphatidylcholines, suggesting that the population of mycoplasma remains relatively low, and causes no significant disruption of cellular phospholipid metabolism.

5. Conclusions. The polar lipid profiles of A549 cells as determined by FAB MS are stable and reproducible, and are predominantly comprised of phosphatidylcholines. M. pneumoniae, when grown in mycoplasma broth, exhibits a polar lipid profile characterized by phosphatidyl glycerol, sphingomyelins and di-unsaturated phosphatidylcholines. Co-culture of A459 cells with M. pneumoniae for two days does not result in any significant change in cellular phospholipid profiles. 6. References. [1] Hay, R. J., M. L. Macy, and T. R. Chen. 1989. Mycoplasma infection of cultured cells. Nature 339:487488. (2] Matsuda, K., T Kasama, I. Ishizuka, S. Handa, N. Yamamoto, and T. Taki. 1994. Structure of a novel phosphocholine-containing glycoglycerolipid from Mycoplasma fermentans. J. Biol. Chem. 269:33123-

33128.

[3] Bligh, E. G., and W. J. Dyer. 1959. A rapid method for total lipid extraction and purification. Can. J. Biochem Physiol. 37:911-917 [4] Jensen, N. J., and M. L. Gross. 1988. A comparison of mass spectrometry methods for structural determination and analysis of phospholipids. Mass Spectrom. Rev. 7:41-69.

[5] Razin, S., S. Kutner, H. Efrati, and S. Rottem. 1980. Phospholipid and cholesterol uptake by mycoplasma cells and membranes. Biochim. Biophys. Acta. 598:628-640. [6] Rottem, S., L. Adar, Z. Gross, Z. Ne'Eman, and P. J. Davis. 1986. Incorporation and modification of exogenous phosphatidylcholines by mycoplasmas. J. Bacteriol. 167:299-304

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COMPARISON OF THREE DIMENSIONAL (3-D) RAT HEPATOCYTE CULTURES IN SIMULATED MICROGRAVITY CONDITIONS

MAGUIRE T., MOULSDALE H.J., STACEY G., BATTLE T. CAMR, Porton Down, Salisbury, Wiltshire, SP4 0JG, UK.

1. Abstract

Amongst the existing 3-D culture models, the Rotary Cell Culture System (RCCS) enables 3-D growth in a unique environment (increased mass transfer and minimal shear forces). We investigated whether these conditions could generate improved hepatocyte 3-D models as potential tools for in vitro toxicological assays. Cells were seeded into

three types of culture vessel which rotate on a horizontal axis and differ in their respective chamber dimensions.

Primary and immortalised cells were both observed to respond differently in the alternative culture vessels. Non-uniform large lobular aggregates (0.5cm diameter) of primary cells were observed in the High Aspect Ratio Vessel (HARV), whereas the Disposable Cell Culture Vessel and the Cylindrical Cell Culture Vessel (CCCV) tended to yield homogenous suspensions of numerous smaller spheroid-like aggregates

provided the appropriate rotational speed was used. Preliminary studies with immortalised hepatocytes showed variation in the degree of vacuolation in cellular aggregates between the HARV and the larger CCCV.

2. Introduction

The Rotary Cell Culture System was originally designed by NASA to create a microgravity environment for the transport of cellular samples during space flight. However the RCCS is also unique in that it allows for large scale tissue culture with extremely low fluid shear stress and turbulence, spatial freedom and high mass transfer. The combination of these factors has subsequently allowed for the culture of many cell

lines in 3-D, including cells which previously did not culture in large scale (Chantret et al 1988). Hepatocytes cultured as tightly packed freely suspended 3-D spheroids have previously proved their usefulness for in vitro toxicology. We investigated whether primary rat hepatocytes could be cultured in the RCCS in order to create reproducible 3-D models for toxicological assays. We also attempted to

evaluate the factors which govern tissue morphogenesis in the RCCS. Pilot studies with immortalised cells were also performed. 717 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 717-719. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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3. Materials and Methods Three different types of culture vessel were used with the RCCS. Primary rat

hepatocytes were obtained form a two step collagenase perfusion of Wistar rats (200300g) for culture in; The Cylindrical Cell Culture Vessel (CCCV), The High Aspect Ratio Vessel (HARV) and the Disposable Cell Culture Vessel. The effect of different seeding densities, between cells per ml and rotational speed on aggregate formation were also examined. Media samples were taken twice daily to monitor cell growth using glucose/lactate analysis. The cell culture media used was William’s E supplemented with albumin 1g/1, insulin 5mg/L and 10% FCS. An immortalised rat hepatocyte cell line was established by transfection of primary rat hepatocytes with SV40 large T antigen using strontium phosphate. Its culture within the RCCS at a seeding density of cells per ml, was also examined. 4. Results

4.1 PRIMARY RAT HEPATOCYTES In all circumstances aggregate formation was seen within eight hours after seeding. The type of vessel used and the rotational speed proved to have a profound effect on 3-D aggregate formation within the RCCS. There was no correlation between seeding density and aggregate formation. Primary rat hepatocytes, when cultured in the HARV at the lowest rotational speed (8 rpm) for 24 hours, formed a single giant lobular shaped aggregate (0.5 cm2 diameter) Khaoustov et al 1995). Increasing the rotational speed did not prevent the lobular aggregate from forming. Primary rat hepatocytes cultured in the CCCV and Disposable Cell Culture Vessel formed loosely assembled aggregates after 24 hours. After 36 hours, smooth surfaced spheroids of approximately were present However a high rotational speed was

essential to prevent spheroids from fusing together. Glucose/Lactate growth curves for spheroids and giant lobular structure were similar. Histological examination of the spheroids after four days of culture, revealed a small necrotic zone at the centre. 4.2 IMMORTALISED RAT HEPATOCYTES

Immortalised hepatocytes cultured well within the HARV and CCCV although aggregates were not visible for 48 hours and did not form very smooth aggregates as seen with primary cells. The aggregates (0.05-0.1 mm in diameter) were maintained in culture for five days. Variation occurred in the degree of vacuolation in the cellular

aggregates cultured in the HARV (large vacuoles) and the CCCV (tightly packed).

719

5. Discussion A microgravity environment is created within the RCCS irrespective of the vessel used. However, it is clear that due to the particular dimensions of each vessel, different results may be seen. Interestingly the HARV (55ml volume) was originally designed for the culture of suspension cell lines. It would appear that in our circumstances the CCCV (110ml volume) and the Disposable Cell Culture Vessel (50ml volume) are better suited for the culture of attachment dependant cells. Primary rat hepatoctyes created in the RCCS are better suited for toxicological model than the giant aggregate as they provide a greater exposed surfaced area. Necrotic zones forming at the centre of spheroids may reflect the poorer availability of oxygen and/or nutrients to deeper cells. It is hoped that future experiments will elucidate the time when spheroids are at an optimum i.e. tightly packed with a smooth surface and no necrotic zone. Glucose/Lactate analysis suggests this time may be before 30 hours of culture, the point at which glucose consumption and lactate secretion stabilise. Previously the culture of spheroids in William's E medium has proved troublesome due to the fusion of many spheroids together. Our study has shown that a homogenous culture of hepatocyte spheroids is possible in William’s E medium in conjunction with the RCCS. Nevertheless it must be determined if spheroids cultured in the RCCS have enhanced liver specific function notably the increased secretion of albumin and expression of cytochrome P450 enzymes. The use of an immortalised cell line for in vitro toxicology has many obvious advantages such as increased feasibility and reproducibility and avoiding recurrent sacrifice of animals. Consequently must work is required to characterise the cell line

established.

6. Conclusion

The culture of individual hepatocyte spheroids is possible within the RCCS. The type of vessel and the rotational speed are fundamental in determining the subsequent tissue morphology. The RCCS will no doubt provide a useful tool for the generation of spheroids and for in vitro toxicology.

7. References Chantret, I., Barbat, A., Dussaulx, E., Brattain, M.G., and Zweibaum, A. (1988) Epitheial polarity, villin

expression, and entererocytic differentiation of cultured human colon carcinoma cells: Asurvey of twenty cell lines. Cancer. 48:1936- 1942.

Khaoustov, V.I., Darlington, G.J., Soriano, H.E., Krishnan, B.,Risin, D., Pellis, N.R., and Yoffe, B. (1995) Establishment of 3-dimensiomal primary hepatocyte cultures in microgravity environment .Hepathology Vol 22 No.4 Pt.2

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HIGH DENSITY CULTURE OF THE HUMAN HEPATOMA CELL LINE HepG2: LONG-TERM CULTURE FOR IN VITRO TOXICOLOGY

A. HANDA-CORRIGAN, R.M TRAYNOR, I. ADAMOPOULOS AND J. SALWAY, University of Surrey, UK.

Abstract One of the major limitations to the use of cultured mammalian cells for in vitro toxicological applications is the lack of robust, long term test systems that represent in vivo responses. Recent advances in maintaining differentiated hepatocyte cultures in fed-batch and perfused bioreactors offers an important means of extended toxicological testing in vitro. However, the lack of sufficient animal and human material limits the possibilities of using hepatocyte culture on an extensive scale for such applications. The human hepatoma cell line, HepG2 retains many of the differentiated properties seen in hepatocytes and has been used for many years as an alternative to primary hepatocytes for in vitro toxicology studies. Despite its increasing use, the growth, functional status and response of this cell line in high density cell cultures have not been well characterised in the literature. In this paper we describe the morphological, growth and biochemical responses of HepG2 cells to the glucocorticoid dexamethasone. Transmission and scanning electron microscopy were used to define the morphological and utrastructural status of the cells. Finally, batch and perfused mini-bioreactors were used for culture of cells at high densities on a novel porous matrix, Porocell. In HepG2 cells cultured at high densities for periods of up to 29 days in Porocell, the glucocortidosteroid dexamethasone was capable of inducing the production and secretion of glucose. This response is comparable to the in-vivo effect of the drug.

Introduction The HepG2 (human hepatoma) cell line maintains some differentiated, morphological and biochemical characteristics of normal hepatocytes [1,2]. In this study, HepG2 cells were challenged with the glucocorticosteroid, dexamethasone. Dexamethasone is prescribed as an anti-emetic to help relieve nausea and vomiting caused by

chemotherapeutic and cytotoxic agents such as cisplatin and digoxin. Other clinical uses include replacement therapy, anti-inflammatory therapy and immunosupression [3]. Dexamethasone has a relatively high affinity for the glucocorticoid receptor and is

clinically thirty times more potent as an anti-inflammatory agent than hydrocortisone. The duration of action of the drug is between 36 to 72 hour post oral administration [3]. The long-term effects of dexamethasone dosing in cell cultures have not previously been

investigated. In this paper, HepG2 cells cultured on a novel porous matrix (Porocell, Porvair Sciences, U K ) were challenged with dexamethasone in static, stirred and packed

721 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 721 -724. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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bed bioreactors. The morphological, growth and biochemical responses of cells challenged with dexamethasone were investigated.

Materials and Methods Cell cultures: HepG2 cells were cultured in medium supplemented with 5% foetal calf serum. Cells were adapted for one month in medium supplemented with dexamethasone at concentrations of Where stated cells were also challenged with dexamethasone at concentrations ranging from Porocell disc culture: Monolayer cultures of HepG2 cells were trypsinized, washed and concentrated to a density of Batches of six Porocell discs were transferred to 6 well plates and 0.025ml of the cell suspension was carefully pipetted into each disc. The discs were incubated at 36.5°C and subsequently transferred to stirred and minipacked bed bioreactors. The stirred cultures (50ml working volume) were surfaceaerated and fresh medium was periodically added to the culture. The packed bed bioreactors were micro-bubble aerated and perfused continuously with fresh medium. Further details of Porocell culture and microscopical analyses are described by HandaCorrigan et al, 1997 [5].

Results and Discussion 1. EFFECT OF DEXAMETHASONE ON HepG2 CELL MORPHOLOGY HepG2 cells cultured on plastic showed two distinct morphologies, as observed by light microscopy (Figures 1a and 1b). During the exponential growth phase, the cell population consisted of (a) flattened cells which formed close contact with each other and (b) small clumps of rounded cells which were attached to each other and to the underlying flat cell sheet. During the death phase, the majority of cells had adopted a rounded morphology and large clumps of cells were observed in culture. Adaptation of cells to dexamethasone at concentrations of did not appear to affect the morphology of this cell line. Transmission electron microscopy showed that dexamethasone adapted HepG2 cells had a similar utlrastructure to cells cultured in medium without dexamethasone (Figures 2a - 2d).

723

M=Mitochondrion, S=smooth ER, G=glycogen rosettes, R=rough ER, N=Nucleus, Nu= nucleolus, L= lipid droplet 2. HIGH DENSITY CULTURE IN STIRRED AND PACKED BED BIOREACTORS

HepG2 cells were inoculated into batches of 6 Porocell discs (Fig 3a) which were subsequently transferred to surface-aerated, stirred cultures or into mini packed-bed bioreactors. Two weeks of culture in stirred and packed-bed bioreactors resulted in smooth, multi-layered rounded cells, growing deep into and over the porous structure of

Porocell (Fig. 3b). In both bioreactors, the cells were in such close proximity to each other that cell-cell boundaries were difficult to identify by SEM. Extended culture for 28 days in the packed bed bioreactors showed that the cells had ‘fused’ to form tissue-like sheets (Fig. 3c).

724 In high density, stirred and perfused cultures of HepG2 cells, the cells did not show any significant biochemical and morphological changes when challenged with dexamethasone at concentrations of (Fig. 4a). However, during repeated, half-hourly exposures to concentrations of dexamethasone, the glucose

uptake rate by the cells was significantly reduced (Fig. 4a) compared with the control at 350 hr. During long-term exposure to dexamethasone HepG2 cells no longer consumed glucose but were able to synthesize glucose by gluconeogenesis and secrete it into the culture medium (Fig. 4b). •

Conclusions The origin of hepatic glucose production in vivo is either from glycogenolysis or gluconeogenesis. Dexamethasone induces the regulatory enzymes of gluconeogenesis, namely: pyruvate carboxylase, phophoenolpyruvate carboxykinase, fructose 1,6bisphosphatase, and glucose 6-phosphatase [6]. It is therefore most probable that the dexamethasone stimulated production of glucose is due to enhanced gluconeogenesis from substrates in the medium such as glutamine. The ATP needed for gluconeogenesis is likely to be provided by oxidative phosphorylation coupled to of fatty acids bound to the serum albumin included in the medium. The vigorous induction of gluconeogenesis in response to dexamethasone is evidence that all the biochemical processes are functioning in HepG2 cells cultured in Porocell. References 1. 2.

Wolff, M., Fandrey, J. and Jelkman, W. (1993). Am J. Physiol. 265, p 1266-1270. Knowles,B.B., Howe, C.C. and Aden, D.P. (1980). Science, 209, p 497-499.

3.

Rang, H.P. and Dale, M.M. (1991). Pharmacology 2nd ed, Churchill Livingstone, N.Y.

4. 5.

Newsholme, E.A. and Leech, A.R. (1983). Biochemistry for the Medical Sciences, J. Wiley and Sons, Chichester. Handa-Corrigan, A., Hayavi, S., Ghebeh, H., Mussa, N.A. and Chadd, M. (accepted). Novel porous matrix and bioreactors for high density cultures of insulinoma cell lines: Insulin secretion and response to glucose. J. Chem. Technol. &

6.

Biotechnol. (December 1997 issue). Mayes, P.A. (1996) in Murray, R.K., Granner, D.K., Mayes, P.A, and Rodwell, V.W. (eds.) Harper’s Biochemistry, 24th ed., Appleton & Lange.

APPLICATION OF PRIMARY CULTURES OF RAT FETAL NEURONS TO THE STUDY OF NEUROTROPHIC ACTION OF PEPTIDES O.V. DOLOTOV, I.A. GRIVENNIKOV

Institute of Molecular Genetics, Russian Academy of Sciencies, Moscow, Russia, 123642 Kurchatov sq., fax: 7(095)196-0221

1. Introduction Primary cultures of neurons are extensively used in neurobiological studies and testing in vitro of compounds for neurotrophic/neurotoxic action. The effect on neuronal survival may depend on conditions of isolations and cultivation of neurons. However these factors are not as yet properly investigated. The subject of our investigations is the study of neuroactive properties of

some new stable analogues of N-terminal fragment of ACTH(4-10) [1, 2]. One of these was synthesized in the Institute of Molecular Genetics and named “Semax” [2]. This peptide has the structure: Met-Glu-His-Phe-Pro-Gly-Pro.

Semax penetrates into the brain after intravenous injection [3] or intranasal application [2]. As of now, no toxic effects of Semax have been reported. The Russian Pharmacological Committee gave permission for Semax to be used as a

remedy for some mental disorders accompanying brain injury and to improve the consolidation of memory and the adaptation to hypoxia and ischemia. To test the hypothesis that Semax might exhibit neurotrophic effects on nervous tissue, we tried to examine Semax for enhancement of fetal basal forebrain neurons survival during the cultivation. Because of ambiguity of results of using of primary neuronal cultures for testing of neurotrophic effects in

literature and from our experience, we tried to reveal factors which might influence on magnitude of the effects. Part of this investigation is presented here.

2. Methods

1. Cell culture Fetal (E16-18) rat brain primary cultures were established as described [4],

with some modifications. Cells were plated at 1000 - 2000 cells per mm2.

Composition of culture medium was standard [5]. The tested compounds were added after cell plating. NGF was applied in concentration 100 ng/ml.

2. Estimation of cell survival Semi-quantitative estimation of number of living cells was carried out on the base of determination of cells with neurite length at least equal to the cell body 725 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 725-727.

© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

726

diameter [6].Measurement of cellular MTT reduction (which reflects number of living cells) was carried out as described [7], with minor modifications. Cholinergic neurons were identified using cytochemical visualization of actylcholinesterase (AChE) as described [4].

3. Results Initial examination of Semax for neurotrophic activity was quite successful. Injection of Semax into culture medium enhanced survival of neurons of the rat basal forebrain by a factor of 1.5 by a week of cultivation. Injection of Semax at day of cultivation other than first had no effect on neuronal survival. More recent similar experiments have failed since effect of Semax and NGF (as positive control) on neuronal survival was not revealed. In attempt to tregger neuronal death we tried to prolong time period between isolation of brains and cell plating. Isolated brains were incubated at 5°C (24 h) and 37°C (30 min) in the solution of isolation and number of living cells were estimated 7 and 3 days later, respectively, after cells plating. Both NGF and Semax extremely increased number of living cells (Figure 1). In the case of immediate isolation of basal forebrains and cell plating Semax and NGF did not exhibit survival effect (Figure 1).

4. Discussion Although primary neuronal cultures is widely applied for investigation of neurotrophins, there is some ambiguity of results. In particular, a number of investigators have described the survival effect of well-known classic neurotrophic factor, NGF, on survival of rat fetal basal forebrain neurons in vitro while others have not revealed this effect (for review see [8]). On the other hand, there is the set of data describing neurotrophic effects of NGF in vivo [9]. Data presented indicate that cellular processes happening in all stages of isolation and cultivation of neurons must be given proper weight in designing of investigations of neurotrophic compounds. We believe that speed and temperature of isolation are no less important than culture conditions and must be controlled because they dictates the value of survival effect of the compound

727

of interest. Moreover, there is the risk of musking of action of a neurotrophic/ neuroprotective compound under good isolation conditions. On the other hand we hope that our data may serve as base for development of novel model of the pathological state of brain - hypoxia/ischemia which might occupy place intermediate between recently used models of this state in vivo and

in vitro because it allows to investigate on the cell culture level processes initiated on the level of the fetal brain. It counts in favour of this speculations that there is correlation between our data and clinical anti-insult results obtained for Semax [10]. We believe that there is common base for these effects. It is assumed that when insult is occurred, a region of apoptotic neurons is formed. However, a possibility of the reversing of these neurons to normal state with the help of neurotrophic factor exists. When described here manipulations with fetal brain are carried out, apoptosis is triggered and neurotrophic factors may prevent cultivated neurons from activation and/or progression of this process. We recognize that our data are not sufficient for a clear understanding of described processes but hope that further study in this direction may be useful.

5. Acknowledgments Supported by INTAS-RFBR N 95–1246. The presentation of poster was sponsored by ESACT.

6. References 1. Verhoef, J. and Witter, A.: In vivo fate of behaviorally active ACTH(4-9) analog in rats after systemic administration, Pharmacol. Biochem. Behav. 4 (1976), 583-590. 2. Potaman, V.N., Alfeeva L.Y., Kamensky A.A., Levitzkaya N.G., and Nezavibatko V.N.: N-terminal degradation of ACTH(4–10) and its synthetic analog semax by the rat blood enzymes, Biochem. Biophys. Res. Commun. 176 (1991), 741-746. 3. Potaman V.N., Antonova L.V., Dubynin V.A., Zaitzev D.A., Kamensky A.A.,

Myasoedov N.F., and Nezavibatko V.N.: Entry of the synthetic ACTH(4–10) analogue into the rat brain following intravenous injection, Neurosci. Lett. 127 (1991), 133–136. 4. Hefti F., Hartikka J., and Sanchez-Ramos J.: Dissociated cholinergic neurons of the basal forebrain in culture, in A. Shahar, J. de Vellis, A. Vernadakis, and B. Haber (eds.), A Dissection and Tissue Culture Manual of the Nervous System, Wiley-Liss, New York, 1989, pp. 172-182. 5. di-Porzio U., Daguet M.C., Glowinski J., and Prochiantz A.: Effect of striatal cells on in vitro maturation of mesencephalic dopaminergic neurones grown in serum-free conditions, Nature 288 (1980), 370-373. 6. Veprintsev B.N., Viktorov I.V., and Vilner B.J.: A Manual on Cultivation of the Neural Tissue, Nauka, Moscow, 1988, pp. 172-174. 7. Hansen M.B. Nielsen S.E., and Berg K.: Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill, J. Immunol. Methods 119 (1989), 203-210. 8. Nonner D., Barrett E.F., and Barrett J.N.: Neurotrophin effects on survival and expression of cholinergic properties in cultured rat septal neurons under normal and stress conditions, J. Neurosci. 16 (1996), 6665-6675. 9. Hefti, F.: Nerve growth factor promotes survival of septal cholinergic neurons after fimbrial transections, J. Neurosci. 6 (1986), 2155-2162. 10. Gusev E.I., Skvortsova V.I., Myasoedov N.F., Nezavibatko V.N., Zhuravleva E.Yu., and Vanichkin A.V.: Semax efficiency in acute period of hemispheral ischemic stroke, Korsakov Zhurn. Nevrol. Psikh. 6 (1997), 26.

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DEVELOPMENT AND VALIDATION OF AN IMAGE ANALYSIS SYSTEM FOR SINGLE CELL CHARACTERIZATION IN CELL MONOLAYERS

D. KAISER, M.A.FREYBERG, G. von Wichert, P. Marenbach, H. Tolle and P. Friedl, Technical University of Darmstadt, Petersenstraße 22, D-64287

Darmstadt, tel: +49 (6151) 163655, fax: +49 (6151) 164759, e-mail: dh 7y@pop. tu-darmstadt. de

1. Subject The characterization of single cells in a monolayer is a necessary and helpful tool for a variety of applications: determination of cell proliferation, apoptosis and vitality as well as questions concerning the presence or absence of various cell surface molecules. Most of these techniques are based on the use of fluorescent dyes or labeled antibodies. The direct microscopic morphological determination of labeled cells is a very inconvenient, elaborate, time consuming procedure and cumbersome for quantitative analysis. Unfortunately currently availale image-analysing systems are very expensive and so we developed an economically reasonable alternative able to identify in an endothelial cell-monolayer proliferating and apoptotic cells. It could also be used for counting living and dead cells after Trypanlue staining. The presented image analysis system is based on a conventional fluorescence microscope (Nikon Diaphot). The only additional equippment is a CCD-camera (about $ 1000) that is interfaced to a Matrox framegrabber board (about $ 500) and a newly

developed image analysis software that offers fundamental data analysis modes and works Windows 3.11 and Windows 95.

2. Methods 2.1

DETERMINATION OF APOPTOTIC CELLS

The analysis of the nuclear morphology is used to determine apoptotic endothelial cells (1, 2): The supernatant of the culture dish is carefully aspirated and the cells are immediately fixed with 0.1 ml 10% (v/v) formalin solution on ice for 15 min. The dish is once washed with PBS, the DNA stained for 15 min by addition of

4',6' Diamidine-2-phenylindoledihydrochloride (DAPI) in methanol and after a final wash with PBS the nuclear morphology is evaluated by fluorescence microscopy with a 20x objective. Fragmented nuclei show fine granules, brightly stained with DAPI throughout the entire nucleus and are counted as apoptotic. 2.2

DETERMINATION OF THE PROLIFERATIVE INDEX OF A CELL MONOLAYER

The cell monolaye, is incubated with a culture medium containing BrdU. Incorporated

729 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 729-731. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

730 BrdU is detected with a specific first antibody (3, 4): All incubation steps have to be done in the dark because BrdU is light-sensitive. The BrdU/Deoxycytidin stock solution (15 mM/15 mM) is mixed with culture medium 1:1000. The cell monolayer is incubated with the modified medium for 60 minutes and washed two times with PBS. The cells are fixed with 70% (v/v) ethanol for 30 minutes at 4°C and washed again three times with PBS. The following steps are performed on a shaker. For denaturation of DNA the cells are incubated with 3 N HCI for 20 minutes. To neutralise the acid, the cells are washed five times with PBS. The antibody against BrdU (0.25 mg/ml) is diluted 1:1000 in PBS with 0.5% (v/v) Tween 20 (PBT) and 0.5% (w/v) bovine serum albumine (BSA) are added. After 30 minutes incubation the cells are washed five times for three minutes with PBT. For negative control another cell layer is treated with unspecific at the same concentration as the monoclonal antibody. The secondary antibody, a Rabbit Anti-Mouse Anti-IG (H+L) (1 mg/ml protein) is diluted 1:3000 in PBT with 0.5% BSA. After two hours incubation the cells are washed five times with PBT followed by the addition of conjugated streptavidin for 60 minutes. After a final wash with PBT the labeled cells are counted. 3. Results

3.1

SHELL OF THE PROGRAMM

The picture shows the shell of the programm while analysing the nuclear morphology of DAPI stained endothelial cells (20x objective). [A] Grabbed picture: The grabbed picture may be stored and saved for later analysis or print. [B] Segmented picture: The processing of the grabbed picture results in the segmented picture: positive cell counts i. e. apoptotic cells in a cell monolayer are marked with a margin. [C] Analysis site: The segmentation specifications are defined in this area: the different signal intensities are

731 separated by their characteristic grey values. A free choosable size limit discriminates positive signals from debris. [D] Calculation Site: The counting result of each picture is displayed in this area; it may be added to a data file until a given number of events has been counted. 3.2

DETERMINATION OF APOPTOTIC CELLS

The counitng results are based on the evaluation of the one randomly choosen visual field shown as the grabbed picture on the shell of the programm:

3.3

DETERMINATION OF THE PROLIFERATIVE INDEX OF A CELL MONOLAYER

The counting results are based on the evaluation of the one randomly choosen visual field:

4. Conclusions The results received either by counting via eye sight or by the use of the image analysis system coincided very well as demonstrated with the determination of the apoptotic and proliferative index of an endothelial cell monolayer. The presented system offers the possibility of rapid and reliable quantitative analysis: higher cell numbers are scanned in shorter times, counting results saved as Excel-files and processed for further data analysis or presentation. Outlook: The system will further be tested for the analysis of the cell size. 5. References 1) Göhde, W., Schumann, J. and Zante, J. (1978) in Pulse Cytophotometry (Lutz, D., Ed), pp. 229-232, European Press, Ghent 2) Kaiser, D., Freyberg, M.A., Friedl, P. (1997) Biochem. Biophys. Res. Commun. 231, 586-590. 3) Klöppinger, M. (1987) Zeitschr. d. GUM 4, 10-12. 4) Gratzner, H. G., Leif, R.C., Ingram, D. J. and Castro, A. (1975) Exptl. Cell Res. 95, 88-94. 6. Acknowledgement Presentation of the poster and participation of D. Kaiser at the meeting was supported by the European

Commission Directorat General XII.

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DEVELOPMENT OF AN OPTICALLY ACCESSIBLE PERFUSION CHAMBER FOR IN SITU ASSAYS AND FOR LONG-TERM CULTIVATION OF MAMMALIAN CELLS M. A. FREYBERG and P. Friedl, Technical University of Darmstadt, Petersenstraße D-64287 Darmstadt, tel: +49 (6151) 163655, fax: +49 (6151)164759, e-mail: [email protected] 1. Subject In vitro cells are usually grown in artificial culture media, in tissue culture flasks and in a batch mode and thus are exposed to an unphysiological situation. An improved culture system should provide the cells with an environment of constant composition. We have developped an alternative culture system based on a conventional tissue culture plate (3.5 cm diameter) which is changed into a closed perfusion chamber. The system can be scaled up from one to several chambers. The shape and the size of the area of cell growth is defined by silicone sheets and can be designed to individual demands. The culture chamber is optically accessible, so cell growth and morphology can be evaluated by light and fluorescence microscopy. Furthermore the cellular physiology can be characterised by any fluorimetric assay using a bottom type fluorescence reader. A peristaltic pump sustains a constant medium flow through the chamber thus creating true homeostasis. The use of HPLC-valves and connectors allows the switching between different media or assay solutions. Thus it is possible to perform in situ assays also measuring transient effects. 2. Methods 2.1

CELL CULTURE

HUVEC are isolated from umbilical cord veins by a modification of the previously published method of Gimbrone et al. (4) and maintained in culture as described (3). Ea.hy926 cells are kindly provided by Dr. Edgell et al. (2). For use in the perfusion system the media are modified: 25 mM HEPES is substituted for the usual NaCI is added for maintaining the osmotic balance. 3. Results 3.1

PERFUSION SYSTEM

733 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 733-736. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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3.2

Perfusion Chamber

3.3

CELL NUMBER CORRELATION

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3.4 LONG-TERM CULTIVATION

3.5 DETERMINATION OF CRITICAL OXYGEN CONCENTRATION

736

4. Conclusions

We have developed a perfusion system on the basis of normal cell culture plates. The cultivation of various cell lines is possible under more physiological conditions than in static culture. Futhermore adherent cells are cultured under defined shear stress conditions. The working system is accessible for fluorescence measurements and microscopical observations so the cells can be characterised by any fluorimetric assay. The wide range of fluorescence indicators allows the examination of many parameters of cell physiology under perfusion mode. 5. References 1) DePaola N, Gimbrone MA Jr, Davies PF, Dewey CF Jr (1992) Vascular endothelium responds to fluid

shear stress gradients. Arteriosclerosis and Thrombosis 12,11: 1254-1257 2) Edgell CJS, Mc Donald CC, Graham JB (1983) Permanent cell line expressing human factor VIII related antigen established by hybridisation. Proc Nat Acad Sci USA 80: 3734-3737 3) Friedl P, Tatje D, Czapala R (1989) An optimized culture medium for human vascular endothelial cells from umilical cord veins. Cytotechnology 2: 171-175

4) Gimbrone MA, Shefton EJ Jr, Cruise SA (1978) Isolation and primary culture of endothelial cells from human umbilical vessels. Tissue Culture Association Manual 4: 813-817 5) Kjellström BT.Örtenwall P, Risberg B (1987) Comparison of oxidative metabolism in vitro in endothelial cells from different species and vessels. J of Cellular Physiology 132: 578-580

6. Acknowledgement Presentation of the poster and participation of M.A.Freyberg at the meeting was supported by the European Commission Directorat General XII.

ANALYSIS OF MITOGENIC ACTIVITY OF PROTEINS AFTER SEPARATION

BY GEL ELECTROPHORESIS

O. HOHENWARTER, G. MARZBAN, E. JISA and H. KATINGER Institute of Applied Microbiology, University of Agricultural Sciences Vienna, Austria

1. Introduction

Complex mixtures of proteins may be separated rapidly by Phast system gel electrophoresis and tested subsequently in cell culture. Using transwell inserts in microwell plates, gel slices were eluted directly into the cell culture supernatant and the

mitogenic effect was evaluated by incorporation (Kuo et al., 1991). We used a modification of this procedure in conventional slab gels to be able to apply sample volumes up to 0,1 ml. In order to avoid the use of radioactivity enzymatic assays for the mitogenic effect were applied. Two examples we used to evaluate the feasibility of the method:

– a purified recombinant fusion protein of human superoxid dismutase and interleukin 2 (SOD-IL2) – an extract from bovine pituitaries The activity of the fusion protein SOD-IL2 was tested with cell line CTLL-2 which is strictly dependent on the presence of IL2 in the culture medium (Gillis et al., 1978). Human umbilical vein endothelial cells were used to evaluate the mitogenic response of pituitary extract.

2. Materials and methods

Preparation and cultivation of human endothelial cells has been described (Hohenwarter et al., 1992). CTLL-2 cells (ATCC No TIB 214) were cultivated as described (VorauerUhl, 1993). Frozen bovine pituitaries were pulverized and 1 g was dissolved in 15 ml 0,1 M solution by stirring for 1h at 4°C. Unsoluble material was separated by centrifugation. The protein content of the extract was 30 mg/ml. Purified SOD-IL2 (Vorauer-Uhl, 1993) and pituitary extract were separated by isoelectric focusing (IEF) or native electrophoresis (pH 5,5 or pH 8,9) on polyacrylamid gels (Clean gel from Pharmacia) according to the instructions of Pharmacia. The gels were washed thoroughly before use in destilled water. After separation one lane was cut into equal pices. The slices were transferred into tissue culture inserts (8 well 737 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 737-739. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

738

strip inserts from Nunc) which contained culture medium (Fig. 1). The inserts were placed in 96-well microplates which contained the test cells (CTLL-2: 3000 cells/well, Endothelium: 1500 cells/well). For the bioassays culture medium with antibiotics was used. In parallel wells 3 ng/ml SOD-IL2 or EGGS were added as positve control. After 24 h the inserts were removed. After 72 h the the mitogenic activity on CTLL-2 cells was evaluated by an MTT test procedure (details described by Hohenwarter et al., 1996). The effect on human endothelial cell was evaluated after 96 h using the acidic phosphatase assay (Connolly et al., 1986). The results correlated with microscopic observation. Parallel lanes of the gel were stained by silver staining.

3. Results 100 ng purified SOD-IL2 with a known isoelectric point of 5,85 were focused (pI 3-10). The protein has serveral isoforms which are active in the bioassay (Fig 2). pituitary extract were separated by isoelectric focusing (pI 5-8). In the bioassay of the gel slices a broad region with mitogenic activity was found (Fig. 3). The most active fraction lies between pI 5,3 and 6,2.

of pituitary extract were separated by native gel electrophoresis either under acidic (pH 5,5) or basic (pH 8,9) conditions.

In both gels mitogenic fractions could be identified (data not shown).

739

4. Discussion

The combination of gel electrophoresis and a cell culture assay in microwell plates is a rapid method to screen for mitogenic fractions. Only small amounts of material are needed. Since most growth factors are active in very low concentrations, the cell culture assay allows the detection of proteins in the gel which cannot be visualized by the most sensitive staining techniques. The use of transwell inserts is essential to separate the gel slices from the cells to avoid inhibitory effects. Sometimes we observed toxic effects which could be minimized by extensive washing of the gels before electrophoresis. We have shown that the recombinant fusion protein SOD-IL2 remains biologically active during the isoelectric focusing procedure. Furthermore the activity of single isoforms could be determined.

The growth promoting activity of bovine pituitary extract on endothelial cells shows a broad peak after isoelectric focusing. This could be due to different proteins or different isoforms of one protein. The pituitary is a source of many mitogenic peptides for example FGFs, EGF, TGFs and IGFs (Houben and Denef, 1994). The separation in narrow pI ranges will allow a more detailed characterization of the mitogenic proteins.

6. References

Connolly, D.T., Knight, M.B., Harakas, N.K., Wittwer, A.J. and Feder, J. (1986) Determination of the number of endothelial cells in culture using an acid phosphatase assay. Anal. Biochem. 152, 136-140. Gillis, S., Ferm, M.M., Ou, W. and Smith, K.A. (1978) T cell growth factor: parameters for production and a quantitative microassay for activity. J. Immunol. 120, 2027-2032. Hohenwarter, O., Schmatz, C. and Katinger, H. (1992) Stability of Von Willebrand Factor production in different human endothelial hybrid cell lines. Cytotechnology 8, 3137. Hohenwarter, O., Waltenberger, A. and Katinger, H. (1996) An in vitro test system for

thyroid hormone action. Anal. Biochem. 234, 56-59. Houben, H. and Denef, C. (1994) Bioactive peptides in anterior pituitary cells. Peptides 15, 547-582.

Kou, K., Yeh, H., Chu, D.Z.J. and Yeh, Y. (1991) Separation and microanalysis of growth factors by Phast system gel electrophoresis and by DNA synthesis in cell culture. J. Chromatography 543, 463-470. Vorauer, K. (1993) Expression, Reinigung und Charakterisierung eines SOD-IL2Fusionsproteins. Thesis, University of Agricultural Sciences, Vienna.

Acknowledgements We thank Karola Vorauer-Uhl for generous gifts of purified SOD-IL2 protein.

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WORKSHOP ON :

THE USE OF ANIMAL CELLS VERSUS THE USE OF TRANSGENIC ANIMALS FOR THE PRODUCTION OF RECOMBINANT PROTEINS

The moderator of the workshop, Dr. Simon Barteling, expressed in his introduction the back to the future feeling of ESACT members, who build their careers on animal cells grown in fermenters or other sophisticated high-tech systems. Now, the animal has to do the job. Presentations were given by Dr. L. M. Houdebine, Dr. H. Meade, Dr. I. Garner, Dr. H. Yoshida, and Dr. R. Werner. They discussed the and more general aspects. Dr. Houdebine (INRA, France) explained what enabled the production of proteins in the milk of transgenic animals: knowledge of milk protein gene promoters, targeting of gene constructs into the casein locus, in vitro fertilization and embryo development, and the identification of transgenic embryos before implantation into the mother animals. The quality of the proteins must be evaluated for glycosylation, carboxylation, and cleavage. Dr. Meade (Genzyme Transgenics) described the production of proteins in the milk of several animal species. Some of the proteins are very difficult to produce in animal cell

cultures. Yields of 1 mg of active protein per ml of milk are typical, which is at least 10

times as much as obtained from cell culture. Dr. Garner (PPL, Roslin, Scotland) described how by prescreening of cells for the expression of the milk promoter driven transgene, the highest expressing line could be detected and used for the cloning of highly productive animals (e.g. Sheep Dolly). Dr. Werner (Dr. Karl Thomae) discussed the pros and cons of the production of proteins

in transgenic animals versus the production in animal cell cultures. In general, the production in animals seems to be more economical than in animal cells, however, the animals are a less defined system, which might hamper the registration of the products.

Also, production of certain pharmaceuticals (e.g. insulin) might, by leakage into the blood, impair the health of the animals. Production of such unhealthy transgenic animals should be prohibited. Technical, economical, and ethical aspects of the use of transgenic animals were vividly discussed for almost an hour.

741 O. - W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 741. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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SECRETION OF FUSION PROTEINS INTO MILK BY TRANSGENIC MOUSE MAMMARY EPITHELIUM D. POLLOK, L. CHEN, H. LIEM, B. W1LBURN, J. WILLIAMS, M. HARRINGTON, Y. ECHELARD, H. MEADE Genzyme Transgenics Corporation 1, Mountain Road Framingham, MA 01701-9322, USA

Abstract: Mammary epithelial cell processing may be more efficient than that found in traditional cell cultures. Certain fusion proteins were not efficiently secreted from traditional cell culture, which includes COS and BHK cells. Yields of the proteins were very low and sometimes undetectable. The same proteins were secreted into milk by animals transgenic for the constructs at typical levels of 1 mg per ml active protein. The fusion proteins are antibodies fused to enzymes and heterologous proteins, as well as protein fusions to Transgenics has enabled the secretion of proteins not normally secreted and has permitted the secretion of complex proteins that remain a significant challenge for cell culture. The economic viability of these fusion proteins to be used therapeutically is thereby enabled, whereas a cell culture process could either be technically impossible and/or economically not feasible. The economics of the production of these proteins will be also discussed. Discussion Hauser:

You suggest that the secretion system in the uterus is completely different from other mammalian cells. Could you comment on this; for instance, why is the protein stuck in the CHO cell and not in the others?

Meade:

I have no explanation but can make a comment. It was in an experiment, that totally surprised me, when the protein was able to get out of the memory cell. When I think, back early on when we got monoclonals to try, one which was humanised was put into the mouse system and only got 0.1 mg/ml. We subsequently found that that cell did not secrete any antibody in tissue culture. I do not know whether the memory gland is more efficient or it does not just edit as well. As we test the proteins, the monoclonal binding and enzyme activity are OK.

Hauser:

You are saying that the memory gland has different properties in the secretion system. Did you also test different mammalian cell lines because it could be due to differentiation?

Meade:

We have not tested different cell lines. We only used COS cells because they are easy to handle. 743

O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 743. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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THE PRODUCTION OF PROTEINS IN THE MILK OF TRANSGENIC LIVESTOCK: A COMPARISON OF MICROINJECTION AND NUCLEAR TRANSFER I. GARNER PPL Therapeutics, Roslin,

Edinburgh, EH 25 9PP, UK

1. Introduction

The milk of transgenic livestock offers a commercially viable source for the production of pharmaceutical and nutraceutically valuable proteins and peptides. High-level expression of the required protein in the mammary gland of transgenic animals can be achieved and complex post-translational modification is accommodated. The technique has been successful in producing human proteins in the milk of a variety of species including mice, rabbits, pigs, goats, sheep and cattle. The high expression levels achieved make the transgenic route an attractive alternative to mammalian cell culture which can produce some complex proteins but at large scale can be costly and yields can be low. However, even in cases where large amounts of complex proteins are required, the commercial viability of projects could be improved through developments in transgenic technology, which has remained largely unchanged for over a decade. This paper summarises the use of transgenic livestock in the production of two medically important proteins, (AAT) and fibrinogen. It also focuses on the potential of nuclear transfer technology to improve the efficiency and range of transgenic technology.

2. The core technology - microinjection The basic elements of transgenic protein production in livestock for commercial use have remained largely unaltered since 1982 (James, 1993). However practical advances have been made, such as the use of interference contrast microscopy and centrifugation of the eggs to make pro-nuclei more easily visible. Techniques for obtaining supplies of fertilised eggs at the right stage of development from supér-ovulated donors and for the re-implantation of injected eggs into pseudopregnant recipients have also been

developed. While these have increased the ease with which the method can be performed the fundamental strategy remains the same. 745 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 745-750. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

746

A few hundred molecules of the DNA construct are injected, via a microscopically small glass needle, into one of the pro-nuclei of a fertilised egg prior to fusion. The embryos are then implanted into recipient animals which act as surrogate mothers. The presence

of the transgene in the resultant offspring can be determined, and expression of the protein detected, using standard techniques such as PCR, Southern blot analysis and Western blot analysis. In most cases, it is desirable to have the protein present in the milk of the animal, although for some applications, expression in the blood is employed. In order to direct expression of the transgene to the mammary gland, consideration must be given to the nature of the DNA construct. Milk-specific expression is effected by fusing the target gene downstream of the regulatory sequence from any of a number of milk-protein genes including murine whey acidic protein, ovine bovine -lactalbumin, bovine and caprine With the exception of whey acidic protein-driven genes in pigs, the regulatory sequences have been shown to be approximately equivalent. However, the configuration of the target gene is of importance. Experiments in mice have shown that genomic DNA is almost always superior to cDNA. This trend has also been shown in the production of -antitrypsin in sheep (Wright et al, 1991; Carver et al, 1993). The choice of species for a commercial transgenic project is influenced by a number of factors including generation time, disease status of the animals, number of offspring and the volume of milk produced (Colman, 1996). Thus for pilot studies, mice are routinely used. The time from birth to milk production for mice is approximately 3.25 months. Where higher levels of expression are required, a larger species would be necessary such as sheep, goats or cattle. However, in these species

generation time has traditionally presented a limitation for transgenic programmes as no indication of expression level is possible until first lactation which, even when hormonally induced, takes place after 9 months for sheep or goats and 12 months for cattle. Only then can selection of suitable animals for flock or herd development take place.

Purification of proteins such as AAT from the milk of transgenic livestock has not presented considerable hurdles (Colman, 1996). Of crucial importance is the removal of lipid which is achieved by low-speed centrifugation, differential precipitation and chromatography. Challenges in separating highly homologous proteins remain but these are common to other production systems.

3. Transgenic production of

-antitrypsin

One of the first commercial targets for transgenic protein production was AAT. antitrypsin is a 394 amino acid, single chain glycoprotein which is normally present at 2 grams per litre in plasma. It is a serine protease inhibitor secreted from hepatocytes and mononuclear phagocytes and its major substrate is thought to be neutrophil elastase. A deficiency in AAT is a comparatively common human genetic disorder. A certain proportion of sufferers develop breathing problems leading to emphysema and resulting in premature death. In addition, in other diseases of the lung such as cystic fibrosis (CF) there is an imbalance between the concentrations of AAT and elastase in the lung. The

747

latter is an enzyme involved in the maintenance of the tissue lining of the lung. It acts by cleaving bonds adjacent to neutral amino acids in the protein elastin which is present in this lining. An overabundance of elastase is thought to cause lung damage in diseases such as CF. In addition, elastase can act in the degradation of immunoglobulin which may hinder the immune response to lung infections in CF patients. Thus, if the balance between AAT and elastase could be restored, the progress of these lung disorders could be slowed and the quality of patient life improved. A plasma-derived form of AAT is

currently available commercially but supplies are limited. The protein was thus a suitable candidate for production in the milk of transgenic animals. High level expression of active human AAT (hAAT) in the milk of transgenic sheep was achieved using a hybrid ovine AAT gene (Wright, 1991). The transgene was shown to be present in several lambs by Southern blot analysis. A comparison of band intensities suggested that the number of copies incorporated varied between these lambs. Human AAT expression was demonstrated using radial immunodiffusion assays and ELISA. The protein level from one of the animals reached 63 grams per litre hAAT in week one of lactation and stabilised at 35 grams per litre. This remains the highest reported expression level for a protein in the milk of a transgenic animal. Analysis of the milk from the founder animals by SDSPAGE indicated a novel band of apparent 54 kDa molecular weight. The mobility of this band corresponded with that of native plasma-derived AAT. Cleavage with Nglycosidase F indicated that the milk-derived protein was glycosylated. The amino terminal sequence of AAT purified from the milk of these animals was shown to be identical to that predicted for the human protein (Carver et al, 1992). Isoelectric

focussing showed some minor differences between plasma and milk derived hAAT.

However, the biological activity of the protein from the two sources was shown to be indistinguishable (Wright et al, 1991). A production flock for human AAT has been established and the transgenically-derived protein is currently in Phase II clinical trials in the UK.

4. Fibrinogen

The work described above established the viability of transgenic protein production for

a single chain protein. The versatility of the technique has since been confirmed through the production of human fibrinogen in the milk of transgenic mice and sheep. Fibrinogen is a complex plasma protein required for blood clotting. The protein is a heterodimer made up of 2 sets of 3 different polypeptide chains, with molecular weights of 66.0, 54.0 and 48.5 respectively. The protein is synthesised in liver cells where the six chains are assembled and linked by 29 disulfide bonds. The assembled molecule is then secreted into the bloodstream. During the coagulation process, thrombin acts enzymatically to remove amino-terminal peptides

from fibrinogen forming fibrin monomers which polymerise into insoluble fibrin clots. Factor XIII acts to cross link adjacent fibrin strands and stabilise the clot. While various mammalian cell culture systems have produced apparently functional human fibrinogen, high level expression has not been achieved. Fibrinogen could have a widespread use

748

in the development of tissue sealants for surgical applications but only if larger amounts can be economically produced than seems feasible with conventional expression systems. High-level expression of recombinant human fibrinogen has been achieved in the milk of transgenic mice (Prunkard et al, 1996). Genomic sequences of the were placed under the control of the sheep promoter. An equimolar mix of the three constructs was introduced into mouse embryos by microinjection. The presence of the transgenic sequences in founder animals was detected by PCR. Southern blot analysis was used to identify animals carrying all three fibrinogen genes. Fibrinogen protein was detected in the milk of transgenic animals by Western blot analysis. Assembly of the protein subunits was analysed using a monoclonal antibody (ZG177.4.1.2.3) which specifically recognises the assembled fibrinogen hexamer. The percentage of total fibrinogen that existed as a fully assembled hexamer was established by comparisons between reducing and non-reducing gels. Figures from the highest producing animal suggested that up to 100% of the fibrinogen was present as the fully polymerised form. The functional activity of transgenic fibrinogen was investigated using clotting reactions. The addition of thrombin to recombinant fibrinogen in mouse milk resulted in an insoluble clot which was collected

by centrifugation.

Western blot analysis of this material indicated that both

fibrinopeptides had been removed by thrombin and cross-linking had occured. The addition of Factor XIII enhanced the reaction to produce complete cross-linking. Electron microscopy has shown that clots made from transgenically-derived fibrinogen display the expected structure. Having established that functional fibrinogen could be produced in transgenic mice, work was initiated to produce the protein in the milk of sheep. Expression levels of 5 grammes per litre have been achieved and several founder animals have been produced with the aim of creating a production flock to generate material for clinical trials.

5. Nuclear Transfer

The examples given above indicate the commercial potential of transgenic protein production using microinjection. Complex proteins can be produced at high levels and purification carried out to a degree sufficient to allow regulatory approval for clinical trials. However, the technique has several important limitations. Using microinjection,

only a small number (aproximately 5%) of the animals born are transgenic. The site of integration is random and this results in variations in levels of transgene expression. To generate a production flock, conventional breeding is necessary, often taking several

years depending on the sex of the founder. A further limitation is that microinjection is suitable only for the addition of genes. Gene deletion or replacement (gene targeting) cannot be carried out. Thus an alternative approach to generating transgenic animals would be advantageous. Embryonic stem cells offer one avenue. Genetic manipulation of these cells in culture, followed by reintroduction to a recipient embryo can give rise to

749

transgenic animals in which genes have been deleted, modified, replaced or added. This has allowed the development of a number of disease models in mice. However, the use of embryonic stem cells has yet to be successful in the generation of transgenic livestock. An alternative approach is nuclear transfer which has been shown to be successful in sheep (Campbell et al, 1996; Wilmut et al, 1997). In these experiments, cells were taken from a number of tissues and cultured (Campbell et al, 1996). The cell cycle was arrested in G0 by the reduction of foetal calf scrum in the culture medium from 10% to 0.5%. These quiescent cells were then fused with oocytes recovered from ewes between 28 and 33 hours after injection of gonadotropin-relcasing hormone (GnRH) and subsequently enucleated. Fusion of the donor cell and enucleated oocyte and activation of the oocyte was induced using electrical pulses. Reconstructed embyros were cultured in the ligated oviducts of sheep or in a chemically-defined medium. Embryos which developed to morula or blastocyst after six days of culture were transferred to recipient ewes and allowed to develop to term. The technique has been successful in producing sheep from embryonic, foetal and adult tissues (Wilmut et al, 1997) and led to the much-publicised birth of Dolly, produced from mammary epithelial cells taken from a 6-year-old ewe. Applied to the production of transgenic livestock, nuclear transfer offers solutions to a number of the shortfalls inherent in microinjection. The cell culture step provides an opportunity for gene targeting so that genes can not only be added but also deleted or replaced. The use of suitable selectable markers can ensure that all nuclear donor cells contain the desired construct and thus all transplanted embryos are

transgenic. It may also be possible to pre-select for high expression at the cell culture stage. Furthermore, it would be possible to produce a production flock in a single generation rather than through conventional breeding as is the case for microinjection. The technique has already shown the potential to be more efficient than

microinjection in terms of the numbers of transgenic lambs born per recipient ewe used. Between 1993-1996 our microinjection programme resulted in 49 live births of transgenic lambs from a total of 2875 sheep used. The 1996 nuclear transfer programme

resulted in 5 live births from 207 sheep. Expressed as a ratio of sheep/lamb, these figures are 58.6 for microinjection and 41.4 for nuclear transfer. Assuming that all lambs born following nuclear transfer would be transgenic, this represents a promising increase in efficiency. For commercial use, a protocol for the addition of a transgene using nuclear transfer would involve the transfection of a suitable cell type with a construct containing the gene for the protein of interest. Of the cell types so far used successfully for nuclear transfer in livestock species, foetal fibroblasts are the most promising, possessing a

number of advantages. Large numbers of cells can be derived from a single foetus and primary cultures can be readily established and maintained under standard culture conditions. This cell type is also amenable to the introduction of exogenous DNA via transfection. The production of transgenic livestock (sheep) through nuclear transfer of transfected foetal fibroblasts has now been achieved with an efficiency of 1 transgenic

lamb per 27 sheep used. (Schnieke, unpublished results). Thus nuclear transfer has distinct advantages in terms of both time scale and efficiencies.

750

6. Conclusions The potential of transgenic protein production in the milk of livestock has been clearly demonstrated. Complex therapeutic proteins, such as fibrinogen, can be obtained at higher yields than would be possible through mammalian cell culture. Purification has been shown to be reasonably simple and cost effective for a number of proteins. The issue of regulatory compliance has also been addressed and products derived from the milk of transgenic livestock, such as AAT, have entered clinical trials. However, despite these successes, for microinjection, nuclear transfer promises, and has begun to deliver, marked improvements over the technique. Instant flocks or herds could be generated from a transfected cell line greatly reducing the time for development of a product. Cell culture prior to nuclear transfer offers the opportunity for more sophisticated genetic manipulation than the simple addition of genes, including gene removal, modification or replacement. There is also the potential to exploit the technique to increase the yields of proteins obtained from livestock. Nuclear transfer thus offers a significant advance in the application of transgenic technology to the production of proteins and peptides. 7. References

James, R. (1993) Human therapeutic proteins generated in animals, The Genetic Engineer and Biotechnologist 13, 189-197. Wright, G., Carver. A., Cottom, D., Reeves, D., Scott, A., Simmons, P., Wilmut, 1., Garner, I., and Colman,

A. (1991) High level expression of active human alpha-1-antitrypsin in the milk of transgenic sheep, Bio/Technology 9, 830-834.

Carver, A., Dalrymple, M., Wright, G., Cottom, D., Reeves, D., Gibson, Y., Keenan, J., Barrass, J., Scott, A., Colman, A., and Garner, I. (1993) Transgenic livestock as bioreactors: stable expression of human alpha-1 antitrypsin by a flock of sheep, Bio/Technology, 11, 1263-1270.

Colman, A. (1996) Production of proteins in the milk of transgenic livestock: problems, solutions and successes, Am. J. Clin. Nutr., 63, 639S-645S.

Carver, A., Wright, G., Cottom, D., Cooper, J., Dalrymple, M., Temperley, S., Udell, M., Reeves, D., Percy, J., Scott, A., Barrass, D., Gibson, Y., Jeffrey, Y., Samuel, C., Colman, A. and Garner, I. (1992) Expression of human antitrypsin in transgenic sheep, Cytotechnology, 9, 77-84. Prunkard, D., Cottingham, I., Garner, I., Bruce, S., Dalrymple, M., Lasser, G., Bishop, P., and Foster, D.

(1996) High-level expression of recombinant human fibrinogen in the milk of transgenic mice, Nature Biotechnology, 14, 867-871. Campbell, K., McWhir, J., Ritchie, W., and Wilmut, I. (1996) Sheep cloned by nuclear transfer from a cultured cell line. Nature, 380, 24-25. Wilmut, I., Schnieke, A., McWhir, J., Kind, A., and Campbell, K. (1997) Viable offspring derived from fetal and adult mammalian cells, Nature, 385, 810-813.

CREATION OF MICE EXPRESSING HUMAN ANTIBODY BY INTRODUCTION

OF A HUMAN CHROMOSOME H. YOSHIDA1, K. TOMIZUKA1, H. UEJIMA2, H. KUGOH2, K. SATOH1,

A. OHGUMA1, M. HAYASAKA3, K. HANAOKA3, M. OSHIMURA2 and I. ISHIDA1 1

Central Laboratories for Key Technology, Kirin Brewery Co., LTD., 1-13-5,

Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236, Japan 2

Department of Molecular and Cell Genetics, School of Life Sciences, Tottori

University, Nishimachi 86, Yonago, Tottori 683, Japan 3

Laboratory of Molecular embryology, Department of Bioscience, Kitasato University School of Science, 1-15-1, Kitasato, Sagamihara, Kanagawa 228, Japan

ABSTRACT. A human chromosome or its subchromosomal fragment (SCF) derived from normal fibroblasts was introduced into mouse embryonic stem (ES) cells via microcell-

mediated chromosome transfer (MMCT) and viable chimaeric mice were produced from them. Serum antibodies with each human Ig were detected and various V segments were

identified in human Ig H,

and

transcripts. Upon immunization of the chimaeras with

HSA, HSA-specific antibodies with specific human antibody with

were detected in the sera. A HSA-

producing hybridoma was obtained from fusion of murine

myelomas with spleen cells from the chimaeric mouse . 1. Introduction Monoclonal antibodies are not only used in human diagnostics, but also in human therapy.

People cannot be immunized in vivo with any kind of antigen, and many challenges have been done to obtain human monoclonal antibodies for therapeutic use. Transgenic approach is useful to generate high affinity monoclonal antibodies. But only a limited

amount of DNA can be transferred using standard techniques. We have developed a novel procedure to introduce foreign genetic material into mice by using a chromosome itself as a "vector". Human chromosome(hChr.) 14,22,or hChr.2-derived fragment including Ig heavy, lambda or kappa genes was transferred into mouse ES cells via microcell-mediated

751 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 751-756.

© 1998 Kluwer Academic Publishers. Printed in the Netherlands.

752 chromosome transfer(MMCT) [1].

In this study we immunized the chimaeric mice by

human serum albumin(HSA) and obtained a hybridoma expressing specific antibody to HSA comprised human

chain from chimaeric mouse containing hChr. 14-fragment.

2. Material and Methods 2.1. CONSTRUCTION OF MH(ES) CELLS AND CHIMERA PRODUCTION

Our strategy to introduce human chromosomes into mice is outlined in Fig. 1. MMCT was utilized to introduce a human chromosome tagged with G418-resistance gene into mouse

ES cells. First, we constructed the libraries of mouse A9 cells containing a human chromosome tagged with pSTneoB suitable for conferring G418-resistance to mouse ES cells. Human primary embryonic fibroblasts were used as a source of human chromosomes. About 3,000 independent G418-resistant colonies of HFL-1 transformed with pSTneoB

were divided into 30 pools and then each pool was fused with mouse A9 cells. G418- and

ouabine- double resistant human-mouse hybrids were used to prepare microcells for the next fusion experiments with mouse A9 cells. Finally, we cloned about 700 independent

G418-resistant human-mouse microcell-hybrids. Mouse ES cells (TT2: 40,XY) were fused with the microcells prepared from these donor hybrid A9 clones and selected with G418 or puromycin. The drug-resistant MH(ES) clones were analyzed by PCR. Representative microcell hybrids, MH(ES)2-1 (containing a hChr.2-SCF), MH(ES)22-1 (containing an

intact hChr.22), MH(ES)14-4 and MH(ES)14-5 (containing an intact hChr.14), MH(ES)14and MH(ES)

(containing a gamma-ray irradiated hChr. 14) were used to produce

chimeras. The SCF(2-W23) was further tagged with puromycin resistant marker was used

as a microcell donor. One of the resultant puromycin-resistant MH(ES) clone, MH(ES)221, was confirmed to retain SCF(2-W23) by the PCR and the FISH analysis (data not shown) and used to produce chimeras. From ten to twenty MH(ES) cells from each cell line or wild type TT2 were injected into a 8-cell stage embryo derived from Jcl:MCH(ICR) mice. Injected embryos were then transplanted to the uteri or oviducts of pseudo pregnant recipients and allowed to

proceed to term. Chimerism in a resulting offspring was determined by extent of coat

pigmentation. The TT2 line, derived from C57BL/6xCBA-F1 embryo, gives an agouti coat color in an albino MCH(ICR) background.

753

2.2. HYBRIDOMAS

The spleen was removed from the chimaeric mouse bearing human chromosome #14 immunized human serum albumin. The spleen cells were fused with a P3X63-Ag8.653 myelomas using PEG. After a fusion, the cells were seeded in 96-well plates in HAT supplemented medium. After HAT selection, G418 was supplemented to select human

chromosome containing clones. The number of wells positive for hybridoma growth was determined visually and the human antibody-secreting hybridomas were screened by ELISA.

2.3. ASSAYS

Human immunogloblin were assayed using anti-human antibodies immobilized on

ELISAplates and detected with peroxidase-conjugated anti-human immunogloblin antibodies as described before [1]. Anti-HSA human immunogloblin

were assayed using

HSA immobilized on ELISAplates and detected with peroxidase-conjugated anti-human immunogloblin antibodies using human IgM for the negative control. The samples, standard and antibody conjugates were diluted with mouse serum supplemented PBS.

Absorbance was measured using a spectrophotometer .

754 3. Results and Discussion

3.1. ANTIBODIES WITH HUMAN IMMUNOGLOBULINS IN CHIMAERIC MICE

We assessed human immunogloblin expression in the sera of chimaeric mice. Human Igs in the sera from non-immunized chimaeric mice were identified by ELISA assays using

anti-human Ig antibodies (Table 1). All the tested chimaeric mice (14/14) derived from MH(ES)14-4, 14-5,

produced

polypeptides in their

sera. Further analysis revealed the production of all four

in

chimera C14-15. Chimaeric mice derived from MH(ES)2-1 (15/19) and from MH(ES)22-1 (5/5) also produced hk and h1 polypeptides, respectively. Upon immunization of these chimeras with human serum albumin (HSA), HSAspecific antibodies with each human (data not shown).

were readily detected in their sera

755 3.2. CREATION OF HYBRIDOMAS FROM THE CHIMERAS

The spleen was removed from chimera C14-1 and were fused with a P3X63-Ag8.653 myelomas.

The number of growth positive wells in HAT medium was 670. The

number of growth positive wells in G418 medium, it was supposed to contain human chromosome, was 140. The frequency of G418 resistant clones was similar to that

estimated from coat color of mouse. Six human antibody positive clones was obtained. The anti-HSA human

positive wells were cloned by limiting dilution culture. The

clone was named H4B7 (Table 2).

756 The amino acid sequences were deduced from variable region of human antibody cDNA derived from clone H4B7. It was revealed that the H4B7 hybridomas contained a combination of genes for VH4 family and JH2. These results show that the chimaeric

mouse retaining human #14 partial fragment produced a functional human antibody heavy chain protein (data not shown). The clone H4B7 was cultured and the culture supernatant was diluted, followed by ELISA using HSA as an antigen with peroxydase-labeled anti-human IgM goat antibody. As a result, the absorbance decreased with the increase in the dilution of the culture solution. Two

of human

showed low absorbance regardless of dilution

ratios. This suggests that the antibody produced by hybridoma H4B7 had a specificity to HSA (Fig .2).

4. Conclusions 1. Human chromosome or its fragment into mouse ES cells by MMCT and viable chimaeric

mice were produced from them. In the case of #2-fg., the fragment was transmitted to the offspring. 2. Serum antibodies with each human Ig were detected.

3. A HSA-specific human antibody with hm producing hybridoma was obtained from fusion of murine myelomas with spleen cells from the chimaeric mouse.

5. Acknowledgements

We thank M. Kato, A. Kurimasa and M. Shimizu for technical advice and valuable

discussions; T. Kato and N. Inoue for giving facility to use Gamma Cell 40 in Yokohama City University; A. Fujiyama and J. Hourov for efforts in early stages of the project; H. Kondoh for giving pSTneoB; S. Watanabe, T. Yagi and P.W. Laird for giving pPGKpuro.

6. References

1. TOMIZUKA, K., YOSHIDA, H., UEJIMA,H., KUGOH, H., SATOH, K., OHGUMA, A., HAYASAKA, M., HANAOKA, .K. , OSHIMURA, M. and ISHIDA, I. (1997) Functional expression and germline transmission of a human chromosome fragment in chimaeric mice, Nature Genet., 16, 133-143

TRANSGENIC TECHNOLOGY-A CHALLENGE FOR MAMMALIAN CELL CULTURE PRODUCTION SYSTEMS

ROLF G. WERNER

Boehringer Ingelheim Pharma Germany, 88397 Biberach an der Riss

Due to price restrictions of the health care system, innovative biopharmaceutical

products have to compete with low price products on the market or have to demonstrate higher efficiency and safety, which add value to the

biopharmaceutical product. Thus, as well as product safety, optimizing the economy of biopharmaceutical manufacturing processes is the main goal of process development. This can be

achieved by more efficient expression systems, shorter generation time of the host cell, media optimization such as use of serum free or protein free media with corresponding feeding strategies as well as improved automatization and sterile technology to increase the overall success rate of the manufacturing process. An optimized combination of these factors will improve the productivity during the fermentation process. For improvement of the yield in the downstream processing, the efficiency of

the purification steps and the number of the purification steps are decisive. In

addition, loss during sterile filling and lyophilisation contribute to the overall yield of the finished product. 757 O.-W. Merten et al. (eds.), New Developments and New Applications in Animal Cell Technology, 757-763. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

758

One of the most efficient expression vectors in mammalian cell culture is pEE 14 in NSO cells, regulated by CMV promoter and SV 40 termination region. The gene of interest is expressed up to 1 gram protein per liter. In

addition the glutamine synthesis mini-gene provides a detoxification of ammonium during the mammalian cell culture process. Although the productivity can be increased significantly with this expression vector in

comparison to the expression of the desired gene in the pPA DHFR vector system, which is controlled by a SV 40 promoter and the hepatitis surface antigen termination region, glycosilation of the glycoprotein is different in the N-acetyl neuraminic acid content. Whereas the CHO cell glycosilates the proteins with a N-acetyl neuraminic acid content of about 20 mole N-acetyl neuraminic acid per mole protein in the NSO expression system, the N-acetyl neuraminic acid content is almost zero. However, the N-glycolyl neuraminic

acid content in the NSO expression system is increased to the level of 10 mol N-glycolyl neuraminic acid per mole protein, whereas in the CHO expression system N-glycolyl neuraminic acid is not present. Since normal human tissue cells do not glycosilate proteins with N-glycolyl neuraminic acid it can be expected that glycoproteins expressed in the NSO expression system might be antigenic.

An alternative expression system is the Neospla-vector where the gene of interest is spliced into an impaired neomycin resistance marker. With this weak selection marker, clones are selected which are inserted into highly active

regions of the chromosome. Titers which are obtained in this expression system for monoclonal antibodies, are in the range of 1 to 2 g/l.

Although these expression titers are enormous, expression systems in transgenic animals are more productive by one order of magnitude. Transgenic expression systems make use of the bovine lactoglobulin promoter or the

759

promoter. Both are proteins which are highly expressed in the mammary gland. The gene of interest is the genomic DNA which provides higher titer than the

c-DNA. In the

expression vector the gene of interest is inserted

between the exon 2 and exon 7. Proteins which have been expressed by the expression vector in mice are summarized in the following table.

Proteins which are further developed and are expressed by the

expression system in goat or by the are presented in the following table.

expression system in sheep

760

These data demonstrate that recombinant DNA derived proteins can be expressed in sheep in the case of

antitrypsin with a titer of up to 30 g/1 and

monoclonal antibodies like BR 96 expressed by the

vector system in

goat with a titer up to 14 g/1. These high titer in the milk of transgenic animals implicate low manufacturing cost. However, economic manufacturing in

transgenic animals require a high annual output of the purified protein. The transgenic approach becomes superior to the mammalian cell culture fermentation systems at a range of 20 and 50 kg of purified protein per year.

With an annual output of 100 kg protein in CHO cells or in goat milk, cost of goods per gram protein are estimated for the CHO cell fermentation systems in the range of US $ 300 to 3.000 depending on the growth conditions and the economy of scale and are estimated to be US $ 105 per gram protein in goat milk. At these quantities, the production of recombinant DNA derived proteins certainly is superior using transgenic animal technology.

The downstream processing has to be designed in a way that it can handle variations in total protein, in casein, whey protein, fat and lactose during the

lactation period or during different feeding of the animals in order to obtain consistent product quality from animal to animal and from batch to batch.

761

As far as glycosilation of the protein is concerned, there are only very few

examples where mammalian cell culture systems have been compared to transgenic animals. One example is interferon gamma which is glycosilated at

two sites, asparagin 25 and asparagin 97. In Chinese hamster ovary cells,

interferon gamma is glycosilated as a complex biantennary type core focusylated at asparagin 25 and complex triantennary type non-core focusylated at

asparagin 97. However, in transgenic mice, interferon gamma at asparagin 25 is a complex biantinnary type core focusylated and at asparagin 97, oligomannose hybride and complex N-glycans are present and the molecule is non-core glycosilated. This implicates that it is not very likely to change from a mammalian cell culture system to a transgenic animal expression system, without changing the glycosilation of the protein.

If product safety aspects are considered there are a number of issues that have to be addressed. Raw materials in transgenic technology are less defined and more heterogeneous than in biotechnology. In biotechnology, the basis for the

fermentation process is a well characterized Master Cell Bank (MCB). Transgenic technology usually uses genomic DNA for the expression of the desired gene in order to obtain higher expression levels. The definition of the MCB in transgenic technology is not uniform. Whereas biotechnology uses a single well characterized cell clone and validated fermentation processes, transgenic technology uses multiple individual animals. In the case of mammalian cell culture, the absence of viral contaminants in the MCB as well as in biological raw materials can be analyzed. In the case of

transgenic technology this is not realistic for infectious agents such as scrapies or bovine spongiform encephalitis. The trust in a reliable source of animals has

762

to replace the proof of absence of such prions. Testing for the absence of prions in the transgenic animals would take more than one year for the final product. A non issue for biotechnology is the sickness of animals. What happens if one

or more of the individuals of a flock become sick? Does this have any influence on product quality or on the impurity profiles? Are the analytical tools

appropriate to address changes? Can the batch be released?

The Commission of the European Communities has some answers in their guideline: "Use of transgenic animals in the manufacture of biological

pharmaceutical products for human use (1993)." For transgenic technology these guidelines require the characterization of the

genomic DNA vector construct, expression of the gene in the appropriate tissue,

description of the measure for creation of transgenic animals, determination of the gene stability during breading and production and demonstration of similar

productivity in different individuals. In comparison to the authorization of a MCB, in the transgenic approach market authorization for a single individual can be given or market authorization for a number of identified individuals can

be provided. Compared to sterile fermentation or closed systems, specific pathogen-free conditions have to be provided and no antibiotics or hormones have to be given in order to prevent infections. The health status of the animals

have to be evaluated and infected animals can not be used for production. The purification process has to be validated to handle a wide range of compound

variations. The specification for product activity has to be given per unit of non

fat dry solid. Removal of unknown adventitious agents have to be demonstrated by validation procedures. For product purity the same requirements are valid as

for biopharmaceutical derived from mammalian cell cultures. However special attention has to be given to product processing, homologous proteins,

763

glycosilation of the protein and lipid association to the protein. Immunogeneity

aspects of trace impurities might have an higher impact in the manufacturing of proteins derived from transgenic animals since these proteins usually are used in high dosage forms or for chronic diseases. In conclusion, transgenic technology is an economic challenge for biopharmaceutical manufacturing of proteins or glycoproteins, especially if high dosages of the active ingredients are required or a high annual capacity for the

proteins in the upper kilogram range is needed. Drawbacks for the expression of

proteins can be the tolerance of the synthesized biopharmaceuticals to the

animals. In respect to product quality and safety, issues have to be addressed such as genetic stability in different individuals, variation in productivity and impurity profile during lactation period, product processing in milk and variable biological active contaminants which are usually a non issue in biotechnology. If these safety issues can be addressed properly and if high volume of the biopharmaceutical have to be produced on a constant annual basis, transgenic technology will be the choice for such products.

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Workshop Discussion

Hoppie:

We hear about mad cow disease and scrapie in sheep but is there a similar disease in goats?

Panel:

Goats can get scrapie and they get sick, and you can see that they are sick. There have been 4 cases in the USA since 1947.

Massie:

With respect to economics, there was a factor of 3 or 4 in the cost of monoclonal antibodies per gram. When you look at production in transgenic animals the yield is much higher, so what other factors contribute to the cost?

Panel:

The problem is that we have not been able to make the advances in purification that we have in production. A great deal of the cost is purification.

Massie:

Do you take into account the time that it takes to get the animals ready for production?

Panel:

I cannot compare the time that it takes to derive a cell line, to optimise the bioreactor and to produce the monoclonal antibodies, with preparing an animal. The slow part is getting your processes ready.

Berthold:

Can we have more information on the new transfection technology which we have heard about? There is the possibility to have

plasmids that define single genes, then you take chromosomes; now you take nuclei. What is the next extension? Panel:

The use of cloning techniques to transfer genes is just at the

beginning. It will be possible to replace the gene, but we are just at the beginning, and it will take us more than 5 years before it becomes routine. Berthold:

My question concerns the fact that the elegance of molecular biology is using a single gene. With a chromosome, or nucleus, you have a lot of non-translated material, ie it is complex with a lot of unknowns. 765

766

Panel:

Whether we are using a CHO cell line or an embryo, we are introducing a single gene so the system is basically the same.

Naveh:

Did you look at sero-conversion, antigenicity and antibody

formation for AT3? Panel:

Yes, but these were for only 1 injection and there are no antibodies

after just 1 injection. Whether there will be sero-conversion after repeated injections, will be known after the next trial. Naveh:

Especially in light of the differences in glycosylation you see?

Panel:

Yes, we will be focusing on these differences but then we all realise

there are differences with the human plasma proteins, including proteins from CHO cells.

Naveh:

By serendipity, it just so happens that proteins made by CHO cells show very little sero-conversion.

Aunins:

Some of the issues between transgenics and cell culture were discussed by Dr Werner. Could you tell us what you have done as process validation on the goats or sheep?

Panel:

We have spiked at various points with specific viruses.

Aunins:

I was referring to the animals themselves.

Panel:

We have a closed flock of sheep from New Zealand that are scrapie free. We have a monitoring system and four vets to examine their health status. Sick animals are removed from the flock until they are better and batches are held from such cases until they are screened. We have an array of ELISA’s for product consistency against common milk contaminants, eg caseins and beta lacto globulins. We look at the product the same way as for CHO cells MWt, amino acid composition, etc.

Builder:

The time taken to get herds ready is a major barrier to the use of

transgenics. Can someone comment on the use of transomatics?

767

Panel:

Infection by retrovirus, or adenovirus, is not very high or efficient. Remember, the gene is submitted to a formidable number of events from the first cell stage to the differentiated stage. If you introduce the gene into the differentiated mammary gland, it is just like a transfection of a cell in a culture system.

Panel:

If you want a quick answer to how would your protein look, you

can use retrovirus packaged in a cell line which is put into the mammary gland. The problem is that the yields are very low. Another system uses a cell line which mimics the goat or cow mammary gland. Having a transient system in the mammary gland gives you a quick look but sometimes it takes as long to make the test as to get the animal ready. Anon:

If you test the construct in a mouse, can you predict how it would be in a goat?

Panel:

With AT3 the mouse product is more heavily sialyated, but it is similar to monoclonal antibodies.

Panel:

The mouse does not process in the same way as a sheep does. The mouse has trouble in gamma carboxylating some proteins.

Hauser:

We have heard that certain proteins, which are not secreted by CHO cells, are secreted by the mammary gland. A thought about why they are not secreted is that there is some sort of control

system. Mis-folded and incompleted proteins are held back. Is the mammary gland a more relaxed system as this sort of control is not so important? Did you look for the amount of proteins that are mis-folded and compare it to an alternative system, such as mammalian cells? Panel:

No, we have not tried this although it would be an interesting experiment. I should imagine that the mammary glad is much more efficient in secretion. We have found that protein sitting in COS cells is not degraded but just remained inside. We are also more

selective in using proteins that we know can be secreted. Szperalski:

As every milk drinker knows, taste and quality is dependent upon the season and kind of food which the animal gets. What is your experience of these points for the quality of the product?

768

Panel:

The sheep’s rations do affect the quality of the milk but we have not seen any major differences in consistency.

Panel:

Throughout lactation, glycosylation is very consistent between animals. In fact, it is probably more consistent than in cell cultures where glycosylation is often very variable.

Panel:

The only major difference we have found was when the sheep were

given salt licks as this changed the electrolyte composition of the milk.

Sasaki:

A comment on proteins secreted from mammary epithelial cells but not CHO cells. Casein would be secreted as a micelle, not as a single protein, and other proteins would be in the micelle.

Panel:

There are proteins in milk which are not bound to micelles, such as beta lacto globulin. So it is not certain that a micelle would help these whey proteins to be secreted.

Sasaki:

If the protein has an affinity for the casein, that would be included in the micelle form.

Panel:

Most of the proteins we work with are in the whey fraction but this

fraction seemed to be associated with the caseins, so this is an interesting observation. Panel: Miller:

BHK and HepG2 cells are very poor at secreting fibrinogen which is not associated with micelles when it comes out in the milk. Importing your sheep from New Zealand does not protect you from prions suddenly appearing - no-one knows how these appear and it maybe that some part of the genome escapes control. My response is that the patient should be able to choose whether he receives the protein from transgenics or from biotech. The figures

you gave should be evaluated independently of the costs. It does not mean that biotech is dead as there is still much basic science that can be done to improve yields. The cost figures were impressive but I would not like to be led by financial considerations alone. Panel:

If you take industry as independent, we recalculated all these figures. Manufacturing costs are cheaper but there is a need to produce a large amount of material - above 20 to 30 Kg per year.

769

Panel:

A comment on prions. In Scotland we operate a closed flock on

arable land which has never had sheep on it. We have not had a case of scrapie in the years we have used this site. If it was a spontaneous event, as suggested in the question, we would have had a case by now. Anyway, our spiking experiments show that we have 10 to 20 logs of clearance over and above what we would need to clear anything found in milk. Prion particles have never been found in milk. WHO categorises milk as a category 4 substance which is as safe as you can get. Panel:

The patient may not get a choice because some proteins can only be produced by transgenic technology.

Lupker:

There are not that many therapeutic proteins that have to be produced in ton quantities. Has anyone looked at producing other than therapeutic proteins in transgenic animals, and how does the production compare to that in micro-organisms?

Panel:

We think that we can make a protein for the same price as in E. coli. The advantage is that it is properly folded.

Merten:

When you compared costs, was it only the production costs or was it validation and regulatory costs as well?

Panel:

The $105 included everything.

Rhyll:

I would be interested in more details of your prion validation studies, and what contingency plans have you for a herd getting sick?

Panel:

Prion validation is done by outside contractors so I cannot tell you what the strain was. We have monitored it for 18 months and the clearance rates have been better than expected. Contingency plans for infected herds include having multiple production sites, and

back-up animals. Panel:

We also have a semen bank.

770

Julien:

In the presentations some differences were mentioned between cell culture processes and transgenic processes in terms of downstream processing. I noticed that there was a one log reduction factor of viral safety. Do you pay special attention to viral safety in the downstream processing? Are there any technological differences between the two processes?

Panel:

The short answer is no, there are no differences. We have viral inactivation and removal in transgenic processes using the same technology with the same result. The actual viral load in milk is very low.

Panel:

In the AT3 process we have cross-flow filtration which is a great viral removal step and which is not usually used in cell culture

processes. Aunins:

Transgenics is a great technological achievement but, as far as a production vehicle goes, what exactly is the big deal and what are your concerns? If you look at licensed products, you have influenza vaccines made in embryonic eggs, anti-venoms from snakes and horses, and OKT3 from mice. What is fundamentally different about transgenics?

Panel:

As long as you require low-dosage proteins there is no economical advantage in transgenics, and because of the well characterised status of proteins from cell cultures this is the preferred process. However, if you need 50 Kg or more of proteins, then transgenics is a good choice.

Panel:

Another example is plasma proteins from humans but the fact is

that none of these examples are cloned. There is a new set of regulations because you have cloned genes, as in cell culture. Grammatikos:

Can you tell us about the conditions that the animals are kept in?

Panel:

We have 3,000 sheep in Scotland which are well looked after with 4 full-time vets - in fact a higher vet to sheep ratio than GPs to humans! Animals are kept until they are 7-9 years old. Sheep in Scotland are kept outside in winter but we have housing for 75% of ours. They get out into the fields and graze normally. They have a specific diet.

771

Miller:

If there is an alternative, we should not use animals to satisfy human needs. Pressure has stopped animal testing of cosmetics. I

am strongly against this example of animal eugenics which is purely for financial reasons. Panel:

The animals, as I have described, are well looked after and some of the products cannot be made by other means in the necessary quantities. We are making over a metric ton of AAP a year, and many tons of human serum albumin. If you can show me another way of producing this, I would be happy to try it.

Panel:

Society has to decide whether it is worth spending the money on medicines. If it decided to pay 100 times more to produce it in tissue culture, then many people will not receive the medicine.

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The trade-fair-sponsors of the 15th ESACT-Meeting: Adi Biotech Sarl (Applikon) Aber Instruments Ltd. B. Braun Biotech International Bayer Diagnostics France Bibby Sterilin Ltd. Biolnvent Production AB Bioprocessing Boehringer Ingelheim Bioproducts Partnership CanSera International Inc. Cellex Biosciences Inc. Cellon Sarl Compex B.V. Corning Costar Corp. Covance Laboratories Ltd. Dr. Karl Thomae GmbH ECACC-ESACT Secretary CAMR Genespan Corp. Genetic Engineering News Greiner Labortechnik GmbH Heraeus Instruments GmbH Hyclone Europe S.A. Inceltech France SA Institut Pasteur Relations Industrielles Integra Biosciences Inveresk Research 773

JRH Biosciences Life Technologies Lonza Biologies plc LSL-Biolafitte SA Medi-Cult A/S Microbiological Associates MicroSafe BV Molecular Devices GmbH New Brunswick Scientific Sa Nunc A/S PAA Laboratories GmbH Pall Europe Ltd. Pharmacia Biotech AB PPL Therapeutics Q-One Biotech Ltd. Quest International B.V. Sarstedt Schärfe System GmbH Selborne Sigma-Aldriche-Chimie Sorvall Ltd. Spectrum Europe Stedim SA TC Tech Corporation TCS Biologicals Ltd. The Automation Partnership Unisyn Technologies

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INDEX

A549 cells, 705 A549 lung cells, 713 acoustic perfusion, 379 action potentials, 690 activators, 158 active hydrogen, 94 active oxygen, 93 Adeno-associated virus, 493 adenovirus, 513 agitation, 201, 285, 399 AIDS, 588 2,6-sialyltransferase, 131, 185 1 -antitrypsin, 745 alphaviruses, 584 amino acid consumption, 277 amino acids, 235 ammonia, 135 ammonium, 157, 281 animal protein free medium (MDSS2N), 561 annexin-V, 259 antigen expression, 285 antigenicity, 163 antigens, 583 antisense, 247 antisense glutamine synthetase, 168 antisense RNA, 191 antisense RNA expression, 157 apoptosis, 227, 231–233, 235, 243, 247, 255, 259, 643, 729 apoptosis-resistant, 247 applicator, 613 arrested in the G1 phase, 227 arrhenins, 363 artificial organs, 657 artificial skin, 673 ATP content, 629 ATPase activity, 39 atrium-like cells, 693 bacitracin, 59 baculovirus, 35 baculovirus infection, 153, 277, 329 baculovirus-insect cell expression, 303 baculovirus-insect cell system, 597 baculovirus-Sf9 insect cell system, 39 bag, 399 Bcl-2, 235

322 437 153 BHK, 215 BHK cell, 219 BHK metabolism, 223 BHK-21, 157, 209 BHK-21/BRS cells, 561 BHK-21A, 185 bioartificial liver, 661 biodistribution analysis, 545 biologicals, 561 biopharmaceuticals, 433, 481 bioprocess effects, 5 bioreactor, 117, 399, 409, 513 biosafety testing, 469 biosensor, 357 bone marrow, 613 BrdU-incorporation, 729 buffered salt solutions, 445 buffers, 445 burn wounds, 673

293 cells, 121, 127, 293, 513 4647 cells, 577 calcium phosphate, 122, 125 calorimetry, 355 capacitance, 322 carbon dioxide, 135 cardiogenesis, 693 cardiomyocytes, 690 CD4 modulation, 601 cell, 637 cell counter, 329 cell counting, 729 cell culture, 399 cell cycle, 77, 247, 643 cell cycle distribution analysis, 228 cell density, 324, 630 cell distribution, 627 cell factories, 101 cell growth, 94, 627 cell metabolism, 277, 458 cell physiology, 457 cells, 321 cell-settler, 395 cell shuttles, 531 cell size, 324, 329

775

776 cell substrate, 577

DNA, 333

cell technology, 463

dog skeletal muscle, 541

cell volume, 627 cellular immunity, 583 centrifugal elutriation, 77

downstream processing, 513, 757 Drosophila melanogaster, 29

characterize animal cells, 317

economy, 757 electrolyzed reduced water, 93 embryoid bodies, 689 embryonic stem (ES) cells, 689, 751 emphysema, 746 endoplasmic reticulum, 101 endoprotease, 69 endothelial cells, 593 endothelium, 733 energetic status, 223

Chinese Hamster Ovary (CHO), 59, 77, 81, 141, 205, 227, 293, 321, 459

CHO cells, 131, 181, 310, 359 cholera toxin B subunit, 617 chromatography, 441, 481 c-jun, 247 clonal variability, 81 cloning, 175

clumped cells, 395 CNAH, 191 concentrates, 445

conductive electronical cell count, 333 conductivity, 324 continuous culture, 351 core

181

co-stimulatory factors, 583 count, 317

cryopreservation, 510 CTP, 458 culture, 541 culture conditions, 460 culture systems, 595 cultures, 542 cystic fibrosis, 746

cytochrome P450 isoenzymes, 97 Cytodex, 570 cytokines, 583, 637

cytomegalovirus (CMV) immediate early

promoter, 585 2D microcarriers, 378 3-D, 665 3-D culture, 717

dead cells, 333 derivatives, 601 development of safer vectors, 527 dexamethasone, 721 dhfr, 81 dialysis, 347 dielectric spectroscopy, 321, 355

dielectrophoresis, 369 differential gene expression, 97 differentiation, 690 diploid cell culture, 577

direct capture, 429 DISC HSV-2, 569 displacement chromatography, 441 disposable, 399 DMSO, 29

enolase, 168 environmental stress, 243 epithelial differentiation, 691 erythropoietin, 463 estradiol, 215 EX-CELL ™293-S, 293, 302, 420 EX-CELL ™ Vero SF, 293 excitotoxicity, 255

expression hosts, 5 expression of genes, 690

expression systems, 757 expressions, 209 extracellular matrix, 637 Factor X, 69

fatty acids, 198, 200 fed-batch, 347 fed-batch and continuous cultivations, 141 fed batch cultures, 267

fed-batch process, 460 FIA, 343 fibrinogen, 745 fixed-bed, 243, 657

fixed bed bioreactor, 627, 635 flow chamber, 733 flow cytometry, 77, 259 fluidized bed, 381, 385, 437 fluidized bed adsorption, 429 fluidized-bed bioreactor, 281 fluorescence, 231–233, 733

flux, 351 freeze drying, 417 fructose-6-phosphate, 157 fully automated PC-based image analysis system, 317 Furin, 69

ganglioside GM3, 601

777 gel electrophoresis, 737 gene expression, 94, 101, 105 gene therapy, 442, 503, 531

gene therapy vector, 545 genetic manipulation, 1 gene transfer, 495, 541 gluconeogenesis, 267

glucosamine-6-phosphate, 157 glucosamine-6-phosphate isomerase, 157 glucosamine-6-phosphate synthase, 158

glucose, 669 glucose and glutamine levels, 219 glucose transporter, 168 glucose uptake and lactate production, 223 glucose-6-phosphate, 158

high fire cells, 277 high five cells, 153, 277, 329 high-spin condition, 613 high titre virus, 569 hippocampus, 255 histidine tagged, 73 HIV-1, 588 hollow fibre, 417 hollow fibre cultures, 43 hollow-fibre bioreactors, 627 homing, 531 homologous recombination, 690 hormonal regulation, 665 host system, 163

human agamma serum, 607 human cell lines, 273

glutamate, 255

human cells, 685

glutamine, 158 glutamine synthetase, 43

human chromosomes, 751 human EPO, 185 human GM-CSF, 163

glyceraldehyde-3-phosphate dehydrogenase (G3PDH), 597 glycoforms, 5, 411, 433

human hematopoietic progenitor cells,

glycoproteins, 158, 175, 191

635 humanized MAb, 417 human monoclonal antibodies, 617, 751 human multidrug transporter (MDR1), 39

glycoprotein structure-function

humoral, 583

relationships, 5 glycosylation, 35, 135, 149, 157, 181, 395 glycosylation engineering, 5 glycosylation pattern, 141 green fluorescent protein (GFP), 125 growth-controllable cell line, 247 growth curves, 511 growth deprivation, 247 growth equation, 355

hybridoma, 197, 235, 243, 281, 351, 459, 469

glycolipids, 158 glycoprotein production, 359

growth factors, 273 growth inhibition, 215 growth potential, 457 growth regulation, 209 GST-sialidase fusion protein, 178

hybridoma cells, 167, 347

hydroxyapatit, 443 IDE, 59 IEF, 310

IEF analysis, 409 image analysis, 729 immortalised hepatocyte, 97

immortalised mouse hepatocyte, 657 immortalization, 643 immune glycoprotein, 566 immune system, 613 immunofluorescence, 85

immunoglobulin A, 149 5-HT3 receptor production, 449 heat flow rate, 355 heat flux, 355 HEK293, 117 HEK 293 cells, 105, 381

hematopoietic, 637 hematopoietic progenitor cells, 503 hemicellulose, 274

hepatitis C, 35 hepatocytes, 643, 661, 665, 717

hepatoma, 721 HIA, 345 high cell density, 377

immunomodulator, 613 in vitro development, 690 in vitro immunization, 617 inactivation of hepatitis A virus, 485 influenza, 551, 555, 587 inhibitor, 158 insect cell culture, 153, 277, 329 insect cells, 29 insulin, 669 insulin-degrading enzymes, 59 Interferon Regulatory Factor 1, 209 interferons, 613 interleukin, 617

778 intracellular assay, 85 intracellular physiologic data, 458 intracellular proteins, 101 intralipid, 205 ion channels, 690 ion exchanger, 429 IRF-1, 209, 215 ischemia, 725 islet, 669

metabolic flox, 357 metabolic network analysis, 351 metabolic rates, 243, 356, 657

JQEF cells, 573, 577

metallothionein, 29 method of Vindeløv, 228 mHep-R l cell line, 657

keratinocytes, 673

microcalorimeter, 355

L-68 cells, 577

lactate, 281 lactate and ammonia, 219 LDH, 333 Leningrad-Ib-vaccine strain, 573, 577 leukocyte alpha interferon, 607

linoleic, 197, 199 linoleic acid, 197, 198, 200, 201 lipid, 197, 200 lipid supplements, 205 lipopolysaccharides (LPS), 441 long-term culture, 81

loss of function, 690 Louping iLL, 588

lyophilisation, 757 lysophosphatidic acid, 205 Mab, 197 macrophages, 531 macroporous, 385 macroporous carriers, 322, 627

macroporous microcarriers, 51, 381 magnetic resonance imaging, 627 magnetic resonance spectroscopy, 627 major histocompatibility complex (MHC) class I, 583 major histocompatibility complex (MHC) class II, 583 mammalian cell, 43 mammalian cell culture, 757 mammalian genome, 1 mannose, 158

mannose-6-phosphate, 158 mass, 149 mass spectrometry, 713 mathematical model, 359

MDCK, 551, 555 MDR l-ATPase, 40 measles vaccine, 573, 577 media optimization, 757

membrane-bound polysomes, 102 metabolic activity, 355 metabolic engineering, 157, 168

metabolic shift, 219 metabolism, 219

metal suspension, 613

microcarriers, 385, 513, 561 microcell-mediated chromosome transfer (MMCT), 751

microgravity, 231–233 microinjection, 745 migration, 637 milk, 743 Milk-specific expression, 746

mitogens, 737

mixing device, 445 monitoring, 457 monkey, 573

monoclonal antibodies, 55, 85, 197, 389, 409, 429 monolayer cultures, 593 morphogenesis, 577 mRNA retargeting, 101 multinucleated myotubes, 693 murine leukemia virus, 469 murine retrovirus, 473 mus dunni cells, 473 mycoplasma, 705, 713 Mycoplasma pneumoniae, 705, 713

mycoplasma testing, 523 myoblasts, 677 myoblast transfer therapy, 677 myogenesis, 693 6-N-acetyl-D-glycosaminyltransferase, 181 Na-butyrate, 131 N-acetyl-glucosamine-6-phosphate, 158

N-glycans, 157 naked DNA, 584 natural killer (NK), 583 necrosis, 231-233 neurogenesis, 693

neuromuscular disorders, 531 neuronal, 690, 725 neuronal culture, 255 neurotrophic, 725 new drug form, 463

779 process control, 347 process development, 459

new form, 573 newborn pig thyroid organ culture, 682 NGF, 726 NMR spectroscopy, 223 NSO myeloma, 51 NTP/U ratio, 458 nucellin-Zn, 227 nuclear transfer, 745 nucleoprotein, 587 nucleotides, 458 nutrient medium, 445

product recovery, 437 product safety, 757 production medium, 55 production processes, 457 production schedules, 511 productivity, 77, 363

nutrient supply, 385

progenitor, 637

oleic, 197-199 oleic acid, 197, 200

oligosaccharides, 5, 175

proliferation control, 209 propeptide, 70, 73 protein expression, 29 protein glycosylation, 153

on line monitoring, 223 on-line substrate, 343

protein production, 745

optical density using laser light, 321 optimisation, 509 oral administration, 573 oxygen demand, 277 oxygen uptake rate (OUR), 347 P-glycoprotein, 39 P53,

643

pacemaker-like cells, 693 packed bed reactor, 661 Pasteurisation, 485

patient monitoring for RCR, 527 peptide, 725 perfused bioreactors, 721

perfusion, 733 perfusion cultures, 56, 235, 369, 459

perfusion system, 395 persistent hypothyroidism, 681 pH, 285 pHi, 259 phosphorylation mutants, 39 pilot scale, 429

pituitary extract, 737 plasmid DNA, 441 plasmid production, 113

plasmid purification, 113 polar lipids, 713 polymerase chain reaction (PCR) technique, 545 polysaccharides, 274 porous carrier, 669 porous microcarriers, 281, 389 post-transcriptional control, 101 postmitotic nerve cells, 693 primary cultures, 725 primary porcine, 665

processing, 69 process monitoring, 329 product monitoring, 343

protein processing, 153 protein secretion, 153 proteinase, 309 proteinase inhibitors, 304 proteolytic activity, 306 prourokinase, 389 purification, 417, 433, 442 rabies virus, 561

RAP-PCR, 97 rCHO, 85, 463 reactor pH, 629 receptors, 690 recombinant, 309 recombinant cells, 357 recombinant CHO cells, 389 recombinant insulin, 227 recombinant protein, 29 recombinant RNA, 584 recombinant soluble human PSGL-1, 181 reconstitution, 445 reduced conditions, 51 regulation-friendly media, 293 replication-competent adenovirus (RCA) testing, 526 replication-competent retrovirus (RCR) testing, 526 reproducible, 317

retinoblastoma, 643 retroviral packaging cell, 503 rFurin, 70, 71 rFX, 73 RNA, 168 Rotary Cell Culture System, 717 routine, 317 routine screening of pharmacological functions, 695 RTPCR, 97

780 cells, 473

S2 cells, 29 scale-up, 509, 595 scale-up schedules, 460 schistosomiasis, 597

transgenic mouse mammary epithelium,

743 transgenic technology, 750 transient expression, 121, 125 transient protein expression, 36

secretion, 101, 121

transient transfection, 105, 113, 117, 442

semicontinuous process, 55 Semliki Forest Virus (SFV), 449, 584 serum concentration, 285

TUNEL assay, 227

serum-free, 105, 197, 199

serum-free culture, 117, 273 serum-free media, 513, 555

SF-9, 35, 293 SF21, 293

Sf21 cells, 153, 329 sialic acids, 175, 191

sialidase, 175 sialylation, 131, 135 signal peptide, 101 simulated microgravity, 717 SIV envelope gp 160, 588

skeletal muscle, 690 skin fibroblasts, 673

SP6 RNA polymerase, 585 specific antibody productivity, 81 specific productivity, 141 spectrometry, 149 spleen, 613 Spodoptera frugiperda, 231–233 stability, 81,512 stirred tank, 594 stoichiometric coefficients, 356

streamline, 429

3'untranslated region, 101 UDP-activated N-acetyl hexosamines, 157 UDP-activated sugars, 458 UDP-N-acetylhexosamines, 458 DTP, 458 vaccination, 583 vaccine adjuvants, 581 vaccines, 561, 583 variable valency, 613 vascular smooth muscle cells, 690 vectors, 493, 584 ventricle-like cells, 693 Vero cell line CR2C9, 569 Vero cells, 561, 577 viability, 333 Viable Cell Monitor, 321 viral disease, 583 viral removal, 481

viral vectors, 583 volume fraction of viable cells, 324 von Willebrand Factor, 69 vWF, 70

substrates, 36, 351 suicidal DNA/RNA, 583

wave, 399

suspension growth, 562 SV40 early promoter, 159

xenotransplantation, 681

synapses, 694

zymography, 313

T-cell receptor, 121 TB/C3-pEF, 243 TBK3-bcl2, 243 temperature, 363

therapeutic agents, 685 THOMAE, 459 thymus, 613 tissue engineering, 661 total virus input spike, 482 toxicology, 721 trans-epithelial electrical resistance, 705 transfectants, 43

transfection, 255 transgenic animals, 745 transgenic livestock, 745