Preface ....................................................... iii
Contributors ................................................... xi
1. Cell Culture Technology — An Overview ....................... 1
Sadettin S. Ozturk
Introduction ................................................ 1
A Brief History of Cell Culture Technology .................. 2
Products from Cell Culture Technology ....................... 6
Future Prospects ........................................... 11
References ................................................. 13
2. Recombinant DNA Technology and Cell Line Development ....... 15
Amy Y. Shen, Jana Van de Goor, Lisa Zheng, Arthur
E. Reyes, and Lynne A. Krummen
Cell Line Development Overview ............................. 15
Cell Source and Host Cell Line Selection ................... 17
Vectors for Expression in Mammalian Cells .................. 19
High Efficiency Selection of Stable Cell Lines ............. 22
Transfection: Introduction of Plasmid DNA into Mammalian
Cells ...................................................... 27
Amplification of Transfected Sequences to Enhance
Production ................................................. 28
Screening and Development of Production Cell Lines ......... 29
Genetic Engineering of Host Cell Lines to Improve
Production Characteristics ................................. 33
Conclusions ................................................ 34
References ................................................. 35
3. Medium Development ......................................... 41
A. Burgener and M. Butler
Introduction ............................................... 41
Culture Media .............................................. 42
Serum-Free Media ........................................... 54
Growth Factors ............................................. 60
Lipids ..................................................... 63
Carrier Proteins ........................................... 64
ECM Proteins ............................................... 64
Choice of Supplements for Serum-Free Media Formulations .... 65
Production of Biologically Active Substances
by Serum-Free Cultures ..................................... 67
Protein-Free Media ......................................... 68
Strategies for the Development of Serum-Free Media ......... 69
Future Prospects ........................................... 73
References ................................................. 73
4. Cell Metabolism ............................................ 81
Francesc Godia and Jordi Joan Cairo Introduction ........ 81
Carbon and Energy Source Metabolism ........................ 82
The Role of Oxygen and C02 in Mammalian Cell Metabolism ... 100
The Main Metabolic End Products and Their Effects:
Ammonium and Lactate ...................................... 102
Redistribution of Cell Metabolism: Toward a More
Efficient Cell Behavior in Culture ........................ 103
Conclusion ................................................ 106
References ................................................ 106
5. Protein Glycosylation ..................................... 113
Sarah W. Harcum
Introduction .............................................. 113
Biological Function and Therapeutic Significance .......... 114
Structures and Conformations of Oligosaccharides .......... 115
Intracellular Biosynthesis ................................ 118
Glycosylation Potential of Various Expression Systems ..... 122
Environmental Effects on Recombinant Therapeutic
Glycoproteins ............................................. 126
Glycosylation Analysis .................................... 132
Conclusions ............................................... 145
References ................................................ 145
6. Cell Culture Bioreactors .................................. 155
Christel Fenge and Elke Lüllau
Introduction .............................................. 155
Bioreactors for Suspension Cell Cultures .................. 156
Bioreactors for Anchorage-Dependent Cell Cultures ......... 174
Bioreactor Operation Modes ................................ 185
Selection and Design of Bioreactors ....................... 190
Conclusion ................................................ 196
References ................................................ 197
7. Aeration, Mixing and Hydrodynamics in Bioreactors ......... 225
Ningning Ma, Mike Mollet, and Jeffrey J. Chalmers
Introduction .............................................. 225
Aeration for Cell Culture Bioreactors ..................... 226
Mixing and Shear Stress ................................... 235
References ................................................ 242
8. Instrumentation and Process Control ....................... 249
Mark Riley
Introduction .............................................. 249
Monitoring and Control of Cell Environment ................ 254
Aseptic Sampling .......................................... 275
Estimation of Rates and Metabolic Ratios from Online
Measurements .............................................. 276
Application of Online Monitoring of the Cell
Environment ............................................... 281
Summary ................................................... 289
References ................................................ 289
9. Cell Culture Kinetics and Modeling ........................ 299
An-Ping Zeng and Jing-xiu Bi
Introduction .............................................. 299
Kinetic Characterization of Cell Culture .................. 300
Influences of Environmental and Physiological Conditions
and Rate Equations ........................................ 311
Models for Simulation of Cell Culture ..................... 331
Concluding Remarks ........................................ 337
References ................................................ 338
10. Fed-Batch Cultivation of Mammalian Cells for the
Production of Recombinant Proteins ........................ 349
Liangzhi Xie and Weichang Zhou
Introduction .............................................. 349
Cell Metabolism ........................................... 351
Design of Culture Media and Feeding Solutions ............. 357
Monitoring of Critical Process Parameters ................. 362
Dynamic Nutrient Feeding Strategies ....................... 365
Examples of Fed-Batch Cultures ............................ 366
Conclusion ................................................ 377
References ................................................ 378
11. Optimization of High Cell Density Perfusion
Bioreactors ............................................... 387
Dhinakar S. Kompala and Sadettin S. Ozturk
Introduction .... 387
Cell Retention Systems .................................... 389
Control and Operation of Perfusion Bioreactors ............ 404
Optimization of Perfusion Bioreactors ..................... 406
Conclusions ............................................... 410
References ................................................ 411
12. Cell Separation and Product Capture ....................... 417
Thomas Seewoester
Introduction .............................................. 417
Removal of Cells and Cell Debris .......................... 417
Product Capture and Primary Purification .................. 424
Conclusions ............................................... 430
References ................................................ 431
13. Downstream Processing ..................................... 439
Ron Bates
Introduction .............................................. 439
Purity Requirements for Biological Purification
Processes ................................................. 440
Potential Product Contaminants Derived from Animal Cell
Culture Processes .... 440
General Principles for the Selection and Sequence of
Downstream Processing Steps ............................... 442
Initial Product Conditioning .............................. 445
Conventional Chromatographic Methods ...................... 452
Nonconventional Chromatographic Methods ................... 468
Design of Purification Protocols .......................... 469
Scale-Up Strategies for Chromatographic Purification
of Biologies .............................................. 472
References ................................................ 473
14. Formulation, Filling and Packaging ........................ 483
M. E. M. Cromwell
Introduction .............................................. 483
Degradation/Inactivation .................................. 484
Formulation Development ................................... 493
Drug Delivery ............................................. 505
Stability Studies ......................................... 509
Methodology for Assessing Protein Stability ............... 510
Filling, Finishing and Packaging .......................... 515
Future Prospects .......................................... 517
References ................................................ 517
15. Validation of Cell Culture-Based Processes and
Qualification of Associated Equipment and Facility ........ 523
Chandra M. Dwivedi
Introduction .............................................. 523
Approach and Rationale .................................... 524
Process Development ....................................... 526
Validation of the Manufacturing Processes and Associated
Equipment Qualification ................................... 538
Manufacturing Plant Qualification ......................... 546
Summary ................................................... 550
References ................................................ 550
16. Facility Design ........................................... 553
Kim Nelson
Introduction .............................................. 553
GMP and Regulatory Considerations ......................... 553
Process Technologies and Functional Area Requirements
Design and Operational Paradigms .......................... 574
Equipment and Sterile Piping Design ....................... 594
Process Utility Systems ................................... 599
References ................................................ 602
17. Production of Proteins by Transient Expression ............ 605
Alain R. Bernard
Introduction .............................................. 605
Stable vs. Transient Expression ........................... 606
Cell Hosts ................................................ 606
Vectors for Transient Expression .......................... 608
Plasmid DNA-Mediated Expression ........................... 612
Virus-Mediated Expression ................................. 617
Conclusions ............................................... 621
References ................................................ 621
18. Principles and Applications of the Insect
Cell-Baculovirus Expression Vector System ................. 627
Laura A. Palomares, Sandino Estrada-Mondaca, and
Octavio T. Ramirez
Introduction .............................................. 627
Insect Cell Lines ......................................... 628
Baculovirus ............................................... 635
Insect Cell Culture ....................................... 642
Bioengineering Issues in Insect Cell Culture .............. 648
Posttranslational Modifications ........................... 658
Larval Production and Stable Expression Systems ........... 665
Products and Applications of IC-BEVS ...................... 669
References ................................................ 676
19. Advances in Adult Stem Cell Culture ....................... 693
E. S. Tzanakakis and C. M. Verfaillie
Introduction .............................................. 693
Somatic Stem Cells ........................................ 693
Adult Stem Cell Culture ................................... 698
Quality Control in Adult Stem Cell Culture ................ 702
Phenotypic Characterization During in Vitro
Differentiation of Stem Cells ............................. 704
Large-Scale Stem Cell Culture Systems ..................... 704
Recent Issues in Adult Stem Cell Research ................. 708
Potential Therapeutic Applications ........................ 710
References ................................................ 713
20. Ex Vivo Culture of Hematopoietic and Mesenchymal Stem
Cells for Tissue Engineering and Cell-Based Therapies ..... 723
A. Mantalaris and J.H.D. Wu
Introduction .............................................. 723
Hematopoiesis and Bone Marrow ............................. 723
Hematopoietic Stem/Progenitor Cell Assays ................. 725
Conclusions ............................................... 736
References ................................................ 736
Index ......................................................... 745
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