Mammalian Cell Biotechnology in Protein Production

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ISBN: 9783110134032 | 3110134039
Cover: Hardcover
Copyright: 6/1/1997

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Supplemental Materials

Hauser and Wagner have presented the new possibilities of Mammalian Cell Biology in a very informative and stimulating manner. Prof. Dr. Hans Fritz, Ludwig-Maximilians-University Munich

1 Genetic Manipulation of Mammalian Cells

(190)

1.1 Hererologous Expression of Genes in Mammalian Cells

(30)

Hansjorg Hauser

1.1.1 Introduction

(1)

1.1.2 Optimizing the Level of Gene Expression

(16)

1.1.2.1 Transcription Determining Elements

(11)

1.1.2.1.1 Transcription Initiation

(2)

1.1.2.1.2 3' End Formation and Polyadenylation

(1)

1.1.2.1.3 Chromatin

(3)

1.1.2.1.4 Selection of General and Tissue Specific Gene Transcription Elements

(1)

1.1.2.1.5 Synthetic Promoters

(1)

1.1.2.1.6 Regulatable Transcription

(3)

1.1.2.1.7 Influence of Chromosomal State and Position of Transferred Genes

(1)

1.1.2.2 Post-Transcriptional Considerations of Gene Expression

(2)

1.1.2.2.1 Capping

(1)

1.1.2.2.2 Introns

(1)

1.1.2.2.3 mRNA Stability

(1)

1.1.2.3 Translational Control

(2)

1.1.2.3.1 Internal Translation Initiation

(1)

1.1.2.3.2 Regulation of Mammalian mRNA Translation

(1)

1.1.3 Protein Sorting, Modification and Degradation

(3)

1.1.3.1 Protein Sorting

(2)

1.1.3.2 Post-translational Modifications of Proteins

(1)

1.1.3.3 Degradation of Proteins

(1)

1.1.4 Choice and Improvement of Mammalian Cell Lines

(10)

1.1.4.1 Cell Lines for Biotechnological Use

(3)

1.1.4.2 Expression Vectors

(1)

1.1.4.3 Cell Growth and Apoptosis

(1)

1.1.4.4 Improvement of Cellular Properties

(6)

1.2 Permanent Gene Expression in Mammalian Cells: Gene Transfer and Selection

(32)

Uwe Schlokat

Michele Himmelspach

Falko G. Falkner

Fiedrich Dorner

1.2.1 Transfer of Foreign DNA into Mammalian Cell Lines

(6)

1.2.1.1 Calcium Phosphate Coprecipitation Method

(1)

1.2.1.2 DEAE-Dextran Mediated Gene Transfer

(1)

1.2.1.3 Polybrene-DMSO Mediated Gene Transfer

(1)

1.2.1.4 Electroporation

(1)

1.2.1.5 Protoplast Fusion

(1)

1.2.1.6 Lipid Mediated Gene Transfer (Lipofection)

(1)

1.2.1.7 Receptor Mediated Gene Delivery

(1)

1.2.1.8 Microinjection

(1)

1.2.1.9 Particle Bombardment (Biolistics)

(1)

1.2.1.10 Retrovirus Mediated Gene Transfer

(1)

1.2.1.11 Laser Poring

(1)

1.2.1.12 Erythrocyte Ghost Fusion

(1)

1.2.2 Selection and Amplification Markers

(7)

1.2.2.1 Dominant Antibiotic Selection Markers

(1)

1.2.2.2 Dominant Biosynthetic Selection Markers

(1)

1.2.2.3 Recessive Biosynthetic Selection Markers

(1)

1.2.2.4 Recessive Amplification Markers

(1)

1.2.2.5 Dominant Amplification Markers

(2)

1.2.3 Selection and Amplification Strategies

(2)

1.2.4 Future Prospects

(13)

1.3 Vectors for Gene Transfer and Expression in Animal Cells

(22)

Volker Sandig

Andre Lieber

Michael Strauss

1.3.1 Introduction

(1)

1.3.2 Papova Viral Vectors

(2)

1.3.3 Vectors Derived from Papilloma Viruses

(1)

1.3.4 Vectors Derived from Epstein-Barr Virus

(2)

1.3.5 Adenoviral Vectors

(2)

1.3.6 Retroviral Vectors

(1)

1.3.7 Vaccinia Virus Vectors

(2)

1.3.8 Alphavirus Vectors

(1)

1.3.9 Baculovirus Vectors

(1)

1.3.10 Comparison of Vector Systems

(1)

1.3.11 Conclusions

(9)

1.4 Aspects of Gene Transfer and Gene Amplification in Recombinant Mammalian Cells

(34)

Florian M. Wurm

1.4.1 Introduction

(1)

1.4.2 History of CHO Cells as Hosts for the Production of Recombinant Proteins

(2)

1.4.3 The DHFR Methotrexate CHO Expression System: A Multilayer Selection System for High Level Expression of Recombinant Genes

(2)

1.4.4 Linearization of Plasmid DNA is a Prerequisite for Integration

(5)

1.4.5 Chromosome Segments with Plasmid Sequences are Excised, Circularize and Form Replicating Episomes which Finally Reintegrate into the Host Genome

(4)

1.4.6 CHO Cells with Highly Amplified, Chromosomally Localized DNA Sequences are the Result of Long-Term Exposure to Incrementally Increased Concentrations of Methotrexate

101

(6)

1.4.7 The Presence or Absence of Methotrexate in the Culture Media Affects the Overall Copy Number of Amplified Sequences Within a Cell Population

107

(1)

1.4.8 Unique Patterns of Chromosomally Integrated Amplified Sequences in Clonal Recombinant Cell Lines

108

(1)

1.4.9 MTX Induced Heterogeneity of Amplified Sequences

109

(3)

1.4.10 Does Chromosomal Heterogeneity of Integrated Plasmid Sequences Within Cell Populations Grown in the Absence of Selection Result in Gradual Loss of Productivity (due to Overgrowth by "Low Producer" Cells)?

112

(2)

1.4.11 Summary and Conclusions

114

(7)

1.5 Isolation of Recombinant Cell Clones Exhibiting High-Level Expression of the Introduced Gene

121

(18)

Manfred Wirth

1.5.1 Introduction

121

(1)

1.5.2 Direct Screening Methods

122

(5)

1.5.2.1 Methods Which Estimate Directly the Expression of the Product

122

(1)

1.5.2.2 The Filter Assay for Protein-Secreting Cells

123

(1)

1.5.2.3 FACS-Analysis for Cells Expressing the Protein Intracellularly or on the Cell Surface

124

(3)

1.5.3 Indirect Screening Methods

127

(6)

1.5.3.1 Double Selection

128

(1)

1.5.3.2 Screening with the Help of Bicistronic Expression Vectors

129

(4)

1.5.4 Prospects

133

(6)

1.6 Genetic Engineering of Antibodies and Derivatives from Mammalian Cells

139

(32)

Michaela Schaffner

Brigitte Kaluza

Ulrich H. Weidle

1.6.1 Introduction

139

(5)

1.6.2 Cloning of V Regions

144

(2)

1.6.3 Expression Systems

146

(4)

1.6.3.1 Baculovirus-System

147

(1)

1.6.3.2 Antibody Expression in Non-Lymphoid Cells

147

(1)

1.6.3.3 Antibody Expression in Lymphoid Cells

148

(1)

1.6.3.4 Transgenic Animals

149

(1)

1.6.4 Manipulation of Antibodies by Gene Targeting

150

(5)

1.6.5 Chimeric Antibodies

155

(2)

1.6.6 Humanized Antibodies

157

(2)

1.6.7 Bifunctional Antibodies

159

(1)

1.6.8 Antibody Fusion Proteins

159

(12)

1.7 Safety Evaluation of Products Derived From Mammalian Cell Lines

171

(20)

David Onions

1.7.1 Introduction

171

(1)

1.7.2 The Regulatory Framework

171

(1)

1.7.3 Characterisation of the Cell Banks

172

(2)

1.7.3.1 Identity of the Cells

173

(1)

1.7.3.2 Validating the Gene Insert

173

(1)

1.7.4 Tests for Adventitious Agents

174

(10)

1.7.4.1 Mycoplasma Screening

175

(1)

1.7.4.2 In Vivo Assays the MAP, and HAP Tests

175

(2)

1.7.4.3 Assays for Bovine Viruses and Other Viruses of Animal Origin

177

(1)

1.7.4.3.1 BVD Virus

178

(1)

1.7.4.3.2 The Bovine Polyomavirus

178

(1)

1.7.4.3.3 BSE and Scrapie

178

(1)

1.7.4.4 In Vitro Assays for Viruses

179

(1)

1.7.4.5 Retrovirus Assays

180

(4)

1.7.5 Integrating Testing with the Production Cycle

184

(4)

1.7.5.1 The Bulk Harvest

184

(3)

1.7.5.2 The Purified Bulk Final Lot Bulk

187

(1)

1.7.5.3 The Final Filled Product or Dosage Preparation

187

(1)

1.7.6 Conclusion

188

(3)

2 Biological Aspects of Animal Cells

191

(86)

2.1 Metabolic Control of Animal Cell Culture Processes

193

(40)

Roland Wagner

2.1.1 Introduction

194

(1)

2.1.2 Carbon Sources

194

(5)

2.1.2.1 Aerobic Glycolysis is the Result of a Misfunction Cascade

196

(1)

2.1.2.2 Mammalian and Insect Cells Differ in their Metabolism

197

(1)

2.1.2.3 The Pentose Phosphate Cycle Competes with the High Glycolytic Rate for Glucose

197

(2)

2.1.3 Glutamine Metabolism

199

(4)

2.1.3.1 Glutamine is the Main Energy Source in Vitro Cultivated Continuous Mammalian Cells

199

(1)

2.1.3.2 Glycolysis and Glutaminolysis are Reciprocally Synchronized

199

(2)

2.1.3.3 ATP Productivity

201

(1)

2.1.3.4 Metabolic Network

202

(1)

2.1.4 Oxygen and Carbon Dioxide

203

(3)

2.1.4.1 Carbon Dioxide

203

(1)

2.1.4.2 Oxygen

203

(3)

2.1.4.3 Respiratory Quotient

206

(1)

2.1.5 End Products

206

(8)

2.1.5.1 Lactate

206

(1)

2.1.5.2 Ammonia

206

(6)

2.1.5.2.1 Uptake of Ammonia and its Toxicity

209

(1)

2.1.5.2.2 Ammonia Increases the Intracellular Concentrations of Activated Sugars

210

(1)

2.1.5.2.3 Ammonia Can Effect Protein Glycosylation

210

(2)

2.1.5.3 Reduction of Ammonium Concentration

212

(1)

2.1.5.4 Maintenance Energy

212

(2)

2.1.5.4.1 Ion Gradients

213

(1)

2.1.5.4.2 Futile Cycles

213

(1)

2.1.6 Control of Growth Dependent Production Processes

214

(8)

2.1.6.1 Environmental Control

215

(1)

2.1.6.2 Intracellular Control

215

(4)

2.1.6.2.1 NMR Offers the Accessibility to Special Metabolites

215

(1)

2.1.6.2.2 Flow Cytometry Distinguishes Between the Proliferative and the Non-Proliferative State of Growth

215

(2)

2.1.6.2.3 The Intracellular Ribonucleotide Pool Generates Robust Control Parameters

217

(1)

2.1.6.2.4 NTP and U Ratio During Growth Cycle

218

(1)

2.1.6.3 NTP to U-Plot

219

(1)

2.1.6.4 Extracellular Nucleotides Act as Effectors of the Cell Proliferation and Productivity

220

(1)

2.1.6.5 Turn Over Rates

221

(1)

2.1.7 Cells Secrete Proteases

222

(1)

2.1.7.1 Proteolytic Activities Derived from Cell Culture Media

222

(1)

2.1.7.2 Proteolytic Activities Derived from the Cells

222

(1)

2.1.8 Productivity and Proliferation

223

(1)

2.1.9 Conclusion

224

(9)

2.2 Glycosylation: A Post-Translational Modification

233

(44)

Angela Savage

2.2.1 Introduction

233

(1)

2.2.1.1 Glycosylation and Glycoforms

234

(1)

2.2.2 Structures and Conformations of Oligosaccharides

234

(5)

2.2.2.1 Structures of N-Linked Oligosaccharides

236

(2)

2.2.2.2 Structures of O-Linked Oligosaccharides

238

(1)

2.2.2.3 Conformation of Oligosaccharides

238

(1)

2.2.3 Intracellular Biosynthesis

239

(5)

2.2.3.1 Glycosylation Sites

239

(1)

2.2.3.2 Biosynthesis of N-Glycans

240

(1)

2.2.3.3 Biosynthesis of O-Glycans

241

(1)

2.2.3.4 Factors Affecting Glycosylation

242

(2)

2.2.3.4.1 N-Glycosylation

242

(1)

2.2.3.4.2 O-Glycosylation

243

(1)

2.2.4 Glycosylation Potential

244

(5)

2.2.4.1 Bacterial Cells

244

(1)

2.2.4.2 Yeast Cells

244

(1)

2.2.4.3 Insect Cells

245

(1)

2.2.4.4 Mammalian Cells

245

(3)

2.2.4.5 Influence of Cell Culture Conditions

248

(1)

2.2.4.5.1 Cell Culture Methodology

248

(1)

2.2.4.5.2 Specific Culture Variables

248

(1)

2.2.5 Biological Function and Therapeutical Significance

249

(6)

2.2.5.1 Physicochemical Roles

250

(1)

2.2.5.2 Molecular Interaction Role

251

(1)

2.2.5.3 Glycosylation of Recombinant Therapeutics

252

(3)

2.2.5.3.1 Tissue Plasminogen Activator (t-PA)

252

(2)

2.2.5.3.2 Erythropoietin (EPO)

254

(1)

2.2.6 Glycosylation Analysis

255

(14)

2.2.6.1 Is the Protein Glycosylated?

257

(1)

2.2.6.2 Does the Glycoprotein Contain N- and or O-Linked Oligosaccharides?

257

(1)

2.2.6.3 How Do I Release Oligosaccharides for Structural Analysis?

257

(4)

2.2.6.3.1 Chemical Release of Oligosaccharides

258

(1)

2.2.6.3.2 Enzymatic Release of Oligosaccharides

259

(1)

2.2.6.3.3 Which to Use: Chemical or Enzymatic Methods?

260

(1)

2.2.6.4 Fractionation, Purification and Mapping of Oligosaccharides

261

(1)

2.2.6.5 Structure Determination of Oligosaccharides

262

(4)

2.2.6.5.1 Monosaccharide Analysis

262

(1)

2.2.6.5.2 Linkage Analysis

262

(1)

2.2.6.5.3 Nuclear Magnetic Resonance Spectroscopy (NMR)

263

(2)

2.2.6.5.4 Mass Spectrometry

265

(1)

2.2.6.5.5 Exoglycosidase Digestion

266

(1)

2.2.6.6 Oligosaccharide Mapping

266

(1)

2.2.6.7 Which Oligosaccharides Are Present at Each Glycosylation Site?

267

(2)

2.2.6.7.1 Chemical Method

267

(1)

2.2.6.7.2 Enzymatic Method

267

(2)

2.2.7 Conclusion

269

(8)

3 Cell Cultivation Technology

277

(134)

3.1 Bioreactors Designed for Animal Cells

279

(40)

Michiyuki Tokashiki

Seiichi Yokoyama

3.1.1 Introduction

279

(1)

3.1.2 Environments Needed for Large-Scale Culture

280

(2)

3.1.3 Cell Culture Processes

282

(2)

3.1.3.1 Batch Culture

282

(1)

3.1.3.2 Continuous Culture

282

(2)

3.1.4 Bioreactors

284

(25)

3.1.4.1 Suspended-Cell Bioreactors

284

(15)

3.1.4.1.1 Types of the Reactors

284

(1)

3.1.4.1.2 Suspended-Cell Batch Culture

285

(1)

3.1.4.1.3 Suspended-Cell Perfusion Culture

285

(14)

3.1.4.2 Immobilized-Cell Bioreactors

299

(10)

3.1.4.2.1 Cell-Immobilizing Carriers

300

(1)

3.1.4.2.2 Immobilized-Cell Culture

301

(8)

3.1.5 Selection and Design for Bioreactors

309

(2)

3.1.6 Conclusion

311

(8)

3.2 Hydrodynamic Properties in Bioreactors

319

(26)

Devamita Chattopadhyay

Miguel Garcia-Briones

Raghavan Venkat

Jeffrey J. Chalmers

3.2.1 Introduction

319

(1)

3.2.2 The Hydrodynamics of Suspension Cell Culture

319

(8)

3.2.3 The Hydrodynamics of Anchorage Dependent Cell Culture

327

(5)

3.2.3.1 History Introduction

327

(1)

3.2.3.2 Agitation Requirements

328

(1)

3.2.3.3 Lethal Effects

329

(3)

3.2.3.3.1 Correlation Studies

329

(1)

3.2.3.3.2 Kolmogoroff Eddy Length Model

329

(2)

3.2.3.3.3 Limitations of the Eddy-Length Scale Model

331

(1)

3.2.3.4 Effects of Silmultaneous Agitation and Sparging of Microcarrier Cultures

332

(1)

3.2.4 Protective Additives

332

(5)

3.2.4.1 Introduction

332

(1)

3.2.4.2 Hypotheses for the Mechanism of Protection

333

(2)

3.2.4.3 The Thermodynamics of Cell Adhesion

335

(2)

3.2.5 Non-Lethal Effects of Hydrodynamic Forces on Cells

337

(1)

3.2.6 Conclusion

338

(7)

3.3 Kinetics and Simulation of Animal Cell Processes

345

(28)

Jean-Louis Goergen

Annie Marc

Jean-Marc Engasser

3.3.1 Introduction

345

(1)

3.3.2 Mammalian Cell Kinetics

345

(13)

3.3.2.1 Kinetics of Cells in Batch Cultures

346

(6)

3.3.2.1.1 Time Variation of the Concentration of Cells, Nutrients and Metabolites

346

(1)

3.3.2.1.2 Influence of Inoculum Age

347

(1)

3.3.2.1.3 Variation of Cell Morphology and Intracellular Content

348

(1)

3.3.2.1.4 Determination of Cell Population Heterogeneity

349

(1)

3.3.2.1.5 Time Variation of the Excreted Protein Composition

349

(1)

3.3.2.1.6 Specific Rates of Cell Growth, Death and Metabolism

350

(1)

3.3.2.1.7 Relationships Between the Specific Rates of Growth and Metabolism

351

(1)

3.3.2.2 Cell Kinetics in Continuous Perfusion Cultures

352

(4)

3.3.2.2.1 Interests of Continuous Cultures

352

(1)

3.3.2.2.2 Time Variation of the Concentration of Cells, Nutrients and Metabolites

352

(2)

3.3.2.2.3 Evaluation of Actual Cell Death and Growth Rates

354

(1)

3.3.2.2.4 Specific Rates of Cell Metabolism in Continuous Cultures

355

(1)

3.3.2.3 Influence of Medium Environment on Cell Kinetics

356

(2)

3.3.2.3.1 Influence of the Medium Composition

356

(1)

3.3.2.3.2 Influence of the Physico-Chemical Parameters

357

(1)

3.3.3 Models for Kinetic Simulations

358

(8)

3.3.3.1 Macroscopic Models for Cell Kinetics

358

(7)

3.3.3.1.1 Kinetic Rate Expressions

359

(1)

3.3.3.1.2 Simulation Capacities of the Model

360

(2)

3.3.3.1.3 Application of Models for Kinetic Analysis

362

(3)

3.3.3.2 Structured and Intracellular Models for Cell Kinetics

365

(1)

3.3.4 Perspectives

366

(7)

3.4 On-line and Off-line Process Analysis

373

(38)

Thomas Scheper

Ruth Freitag

Friedrich Srienc

3.4.1 Introduction

373

(1)

3.4.2 Aseptic Sampling Biotechnology

374

(7)

3.4.3 Optical Sensors for Cell Characterization and Concentration Estimation

381

(4)

3.4.4 On-line Monitoring of the Cell Environment

385

(6)

3.4.5 Process Analysis with Flow Cytometry

391

(11)

3.4.6 Conclusions

402

(9)

4 Down-Stream Processing

411

(72)

4.1 Principles of Product Extraction from Cell Culture and Purification for Pharmaceutical Proteins

413

(40)

Alois Jungbauer

4.1.1 Purity Criteria and Consequences for Downstream Processing

413

(2)

4.1.2 General Strategy of Purification Processes for Biologics

415

(2)

4.1.3 Removal of Cells, Cell Debris Initial Purification

417

(7)

4.1.3.1 Microfiltration

418

(1)

4.1.3.2 Centrifugation

419

(4)

4.1.3.3 Ultrafiltration Diafiltration

423

(1)

4.1.4 Precipitation

424

(2)

4.1.5 Liquid-Liquid Extraction

426

(1)

4.1.6 Conventional Chromatographic Methods

427

(13)

4.1.6.1 Ion-Exchange Chromatography

429

(3)

4.1.6.2 Adsorption Chromatography

432

(1)

4.1.6.3 HIC and Thiophilic Adsorption

432

(1)

4.1.6.4 Reversed Phase Chromatography

433

(1)

4.1.6.5 Gel-Chromatography

434

(1)

4.1.6.6 Affinity Chromatography and Pseudoaffinity Chromatography

435

(3)

4.1.6.7 Displacement Chromatography

438

(1)

4.1.6.8 Design of Generic Purification Methods

438

(1)

4.1.6.9 Productivity Concerns of Chromatography

438

(2)

4.1.6.10 Expanded Bed Operations

440

(1)

4.1.7 Continuous and Semicontinuous Chromatographic Processes

440

(1)

4.1.8 Scale-Up Strategies for Purification of Biologics Using Adsorption Desorption Techniques

441

(2)

4.1.9 Electrophoretic Methods and Scale Up

443

(1)

4.1.10 Removal of Adventitious Agents

444

(1)

4.1.11 Conclusion and Future Prospects

444

(9)

4.2 Validation of Downstream Processes

453

(30)

Joachim Walter

Hermann Allgaier

4.2.1 Introduction

453

(1)

4.2.2 Master Validation Plan

453

(1)

4.2.3 Manufacturing Plant Qualification

454

(1)

4.2.4 Validation of the Manufacturing Process

455

(28)

4.2.4.1 Raw Materials Used for Downstream Processing

455

(1)

4.2.4.2 Removal of Contaminants

456

(17)

4.2.4.2.1 Features of Protein Product and Contaminations from Different Organisms

457

(1)

4.2.4.2.2 Media Derived Contaminants

458

(4)

4.2.4.2.3 Host Cell Derived Contaminants

462

(11)

4.2.4.3 Regeneration and Storage of Chromatographic Matrices and Membranes

473

(1)

4.2.4.4 Validation of Product Changeover Procedures

474

(9)

4.2.4.4.1 Multi-Use Manufacturing Facilities for Biologics

474

(1)

4.2.4.4.2 Cleaning Validation

475

(2)

4.2.4.4.3 Product Changeover Procedures

477

(6)

Index

483

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Mammalian Cell Biotechnology in Protein Production

Summary

Table of Contents

Supplemental Materials