Case 1:18-cv-00924-CFC Document 376-1 Filed 09/27/19 Page 1 of 95 PageID #: 28679
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`C.A. No. 17-1407-CFC
`(CONSOLIDATED)
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`IN THE UNITED STATES DISTRICT COURT
`FOR THE DISTRICT OF DELAWARE
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`GENENTECH, INC. and CITY OF HOPE, )
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`AMGEN INC.,
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`Defendant.
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`GENENTECH, INC.,
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`Plaintiff and
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`AMGEN INC.,
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`Defendant and
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`Counterclaim Plaintiff.
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`C.A. No. 18-924-CFC
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`APPENDIX TO GENENTECH’S LETTER-BRIEF CONCERNING
`CONSTRUCTION OF “FOLLOWING FERMENTATION” AND
`SUPPORTING DECLARATION OF DR. HANSJÖRG HAUSER
`
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`Case 1:18-cv-00924-CFC Document 376-1 Filed 09/27/19 Page 2 of 95 PageID #: 28680
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`U.S. Patent No. 8,574,869
`Bruce Alberts et al., MOLECULAR BIOLOGY OF
`THE CELL, Chapter 3 (4th Ed. 2002) (“Molecular Biology of
`the Cell”)
`Mullan et al., Disulphide bond reduction of a
`therapeutic monoclonal antibody during cell culture
`manufacturing operations, BMC Proc., 22(5) Suppl
`8:P110 (2011) (“Mullan 2011”)
`Trexler-Schmidt et al., Identification and Prevention of
`Antibody Disulfide Bond Reduction During Cell Culture
`Manufacturing, Biotechnol Bioeng., 106(3):452-61 (2010)
`(“Trexler-Schmidt 2010”)
`Kao et al., Mechanism of Antibody Reduction in Cell
`Culture Production Processes, Biotechnol Bioeng.,
`107(4):622-32 (2010) (“Kao 2010”)
`Mun et al., Air Sparging for Prevention of Antibody
`Disulfide Bond Reduction in Harvested CHO Cell
`Culture Fluid, Biotechnol Bioeng., 112(4):734-42
`(2015) (“Mun 2015”)
`Hutterer et al., Monoclonal Antibody Disulfide
`Reduction During Manufacturing, MAbs., 5(4):608-13
`(2013) (“Hutterer 2013”)
`Chung et al., Effects of Antibody Disulfide Bond
`Reduction on Purification Process Performance and
`Final Drug Substance Stability, Biotechnol Bioeng.,
`114(6):1264-1274 (2017) (“Chung 2017”)
`Fahrner et al., Industrial Purification of Pharmaceutical
`Antibodies: Development, Operation, and Validation of
`Chromatography Processes, Biotechnology and Genetic
`Engineering Reviews, 18:1, 301-327 (2001) (“Fahrner 2001”)
`Birch and Racher, Antibody production, Advanced Drug
`Delivery Reviews 58(5-6):671-85 (2006)
`Webster’s Dictionary, “Fermentation”
`FDA Biotechnology Inspection Guide (excerpts)
`Persson et al., Mammalian Cell Fermentation, Production of
`Biologicals from Animal Cells in Culture (1991) (“Persson
`1991”)
`
`Appx1
`Appx96
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`Appx127
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`Appx130
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`Appx140
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`Appx151
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`Appx160
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`Appx166
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`Appx177
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`Appx205
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`Appx220
`Appx223
`Appx228
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`2
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`Case 1:18-cv-00924-CFC Document 376-1 Filed 09/27/19 Page 3 of 95 PageID #: 28681
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`Bödeker et al., Production of recombinant factor VIII from
`perfusion cultures: I. Large-scale fermentation, Animal
`Cell Technology (1994) (“Bödeker 1994”)
`Ozturk et al., Real-time Monitoring of Protein Secretion in
`Mammalian Cell Fermentation: Measurement of Monoclonal
`Antibodies Using a Computer-Controlled HPLC System
`(BioCad/RPM), Biotechnol Bioeng., 48:201-206 (1995)
`(“Ozturk 1995”)
`Kemp G., O’Neil P., Large-Scale Production of Therapeutic
`Antibodies: Considerations for Optimizing Product Capture
`and Purification, in: Subramanian G. (eds) Antibodies (2004)
`(“Kemp 2004”)
`Dwivedi, Validation of Cell Culture-Based Processes and
`Qualification of Associated Equipment and Facility, in:
`Ozturk, Cell Culture Technology for Pharmaceutical and Cell-
`Based Therapies (“Dwivedi 2006”)
`US 2007/0141687 (Porro)
`Kaufmann et al., Influence of low temperature on productivity,
`proteome and protein phosphorylation of CHO cells,
`Biotechnol Bioeng., 63(5): 573-582 (1999)
`Matijasevic et al., Hypothermia causes a reversible, p53-
`mediated cell cyle arrest in cultured fibroblasts, Oncol Res.,
`10(11-12): 605-610 (1998)
`Roobol et al., ATR (ataxia telangiectasia mutated- and Rad3-
`related kinase) is activated by mild hypothermia in
`mammalian cells and subsequently activates p53, Biochem J.,
`435(2):499-508 (2011)
`Hunt et al., Low-Temperature Pausing of Cultivated
`Mammalian Cells, Biotechnol Bioeng., 89(2):157-63, (2004)
`Yoon et al., Effect of Low Culture Temperature on Specific
`Productivity and Transcription Level of Anti-4-1BB Antibody
`in Recombinant Chinese Hamster Ovary Cells, Biotechnol
`Prog., 19(4):1383-6 (2003)
`Roobol et al., Biochemical insights into the mechanisms
`central to the response of mammalian cells to cold stress and
`subsequent rewarming, FEBS J., 276(1):286-302 (2009)
`
`Appx234
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`Appx240
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`Appx246
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`Appx272
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`Appx307
`Appx344
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`Appx354
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`Appx360
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`Appx374
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`Appx381
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`Appx385
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`3
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`Case 1:18-cv-00924-CFC Document 376-1 Filed 09/27/19 Page 4 of 95 PageID #: 28682
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`Supplement to Roobol et al., Biochemical insights into the
`mechanisms central to the response of mammalian cells to
`cold stress and subsequent rewarming, FEBS J., 276(1):286-
`302 (2009)
`McGraw-Hill Dictionary of Scientific and Technical Terms
`(6th ed.), “Fermentation”
`Deposition of Jeffrey John Chalmers (excerpts)
`Amgen 2011 Annual Report and Financial Summary
`(excerpts)
`Deposition of Stuart Watt (excerpts)
`
`Appx402
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`Appx408
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`Appx411
`Appx448
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`Appx450
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`4
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`Case 1:18-cv-00924-CFC Document 376-1 Filed 09/27/19 Page 5 of 95 PageID #: 28683
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`CELL CULTURE TECHNOLOGY
`
`FOR PHARMACEUTICAL
`
`AND CELL-BASED THERAPIES
`
`
`
`edited by
`Sudeflin S. Ozlurk
`
`Wei-Shall flu
`
`Appx272
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`Appx272
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`Case 1:18-cv-00924-CFC Document 376-1 Filed 09/27/19 Page 6 of 95 PageID #: 28684
`
`Contents
`
`iii
`Preface . . . .
`Contributors . . . . xi
`
`1. Cell Culture Technology—An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . 1
`Sadettin S. Ozturk
`1
`Introduction . . . .
`A Brief History of Cell Culture Technology . . . .
`Products from Cell Culture Technology . . . .
`6
`Future Prospects . . . .
`11
`References . . . .
`13
`
`2
`
`. . . . . . . . . . . 15
`2. Recombinant DNA Technology and Cell Line Development
`Amy Y. Shen, Jana Van de Goor, Lisa Zheng, Arthur E. Reyes, and
`Lynne A. Krummen
`15
`Cell Line Development Overview . . . .
`17
`Cell Source and Host Cell Line Selection . . . .
`19
`Vectors for Expression in Mammalian Cells . . . .
`22
`High Efficiency Selection of Stable Cell Lines . . . .
`Transfection: Introduction of Plasmid DNA into Mammalian Cells . . . .
`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
`
`27
`
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
`3. Medium Development
`A. Burgener and M. Butler
`Introduction . . . .
`41
`Culture Media . . . .
`42
`Serum-Free Media . . . .
`Growth Factors . . . .
`60
`Lipids . . . .
`63
`64
`Carrier Proteins . . . .
`64
`ECM Proteins . . . .
`Choice of Supplements for Serum-Free Media
`Formulations . . . .
`65
`
`54
`
`v
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`Appx273
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`vi
`
`Contents
`
`Production of Biologically Active Substances
`by Serum-Free Cultures . . . .
`67
`Protein-Free Media . . . .
`68
`Strategies for the Development of Serum-Free Media . . . .
`Future Prospects . . . .
`73
`References . . . .
`73
`
`69
`
`4. Cell Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
`Francesc Go´ dia and Jordi Joan Cairo´
`Introduction . . . .
`81
`82
`Carbon and Energy Source Metabolism . . . .
`The Role of Oxygen and CO2 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
`
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
`
`5. Protein Glycosylation
`Sarah W. Harcum
`113
`Introduction . . . .
`Biological Function and Therapeutic Significance . . . .
`Structures and Conformations of Oligosaccharides . . . .
`Intracellular Biosynthesis . . . .
`118
`Glycosylation Potential of Various Expression Systems . . . .
`Environmental Effects on Recombinant Therapeutic
`Glycoproteins . . . .
`126
`Glycosylation Analysis . . . .
`Conclusions . . . .
`145
`References . . . .
`145
`
`114
`115
`
`122
`
`132
`
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
`6. Cell Culture Bioreactors
`Christel Fenge and Elke Lu¨ llau
`Introduction . . . .
`155
`156
`Bioreactors for Suspension Cell Cultures . . . .
`Bioreactors for Anchorage-Dependent Cell Cultures . . . .
`Bioreactor Operation Modes . . . .
`185
`Selection and Design of Bioreactors . . . .
`Conclusion . . . .
`196
`References . . . .
`197
`
`190
`
`174
`
`7. Aeration, Mixing and Hydrodynamics
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
`in Bioreactors
`Ningning Ma, Mike Mollet, and Jeffrey J. Chalmers
`Introduction . . . .
`225
`Aeration for Cell Culture Bioreactors . . . .
`Mixing and Shear Stress . . . .
`235
`References . . . .
`242
`
`226
`
`Appx274
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`Case 1:18-cv-00924-CFC Document 376-1 Filed 09/27/19 Page 8 of 95 PageID #: 28686
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`Contents
`
`vii
`
`. . . . . . . . . . . . . . . . . . . . . . . . . . 249
`
`8. Instrumentation and Process Control
`Mark Riley
`249
`Introduction . . . .
`Monitoring and Control of Cell Environment . . . .
`Aseptic Sampling . . . .
`275
`Estimation of Rates and Metabolic Ratios from Online
`Measurements . . . .
`276
`Application of Online Monitoring of the Cell Environment . . . .
`Summary . . . .
`289
`References . . . .
`289
`
`254
`
`281
`
`9. Cell Culture Kinetics and Modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
`An-Ping Zeng and Jing-xiu Bi
`Introduction . . . .
`299
`300
`Kinetic Characterization of Cell Culture . . . .
`Influences of Environmental and Physiological Conditions and Rate
`Equations . . . .
`311
`Models for Simulation of Cell Culture . . . .
`Concluding Remarks . . . .
`337
`References . . . .
`338
`
`331
`
`10. Fed-Batch Cultivation of Mammalian Cells for the Production
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
`of Recombinant Proteins
`Liangzhi Xie and Weichang Zhou
`Introduction . . . .
`349
`351
`Cell Metabolism . . . .
`Design of Culture Media and Feeding Solutions . . . .
`Monitoring of Critical Process Parameters . . . .
`362
`Dynamic Nutrient Feeding Strategies . . . .
`365
`Examples of Fed-Batch Cultures . . . .
`366
`Conclusion . . . .
`377
`References . . . .
`378
`
`357
`
`11. Optimization of High Cell Density Perfusion Bioreactors . . . . . . . . . . . . 387
`Dhinakar S. Kompala and Sadettin S. Ozturk
`Introduction . . . .
`387
`389
`Cell Retention Systems . . . .
`Control and Operation of Perfusion Bioreactors . . . .
`Optimization of Perfusion Bioreactors . . . .
`406
`Conclusions . . . .
`410
`References . . . .
`411
`
`404
`
`. . . . . . . . . . . . . . . . . . . . . . . . . 417
`
`12. Cell Separation and Product Capture
`Thomas Seewoester
`417
`Introduction . . . .
`417
`Removal of Cells and Cell Debris . . . .
`Product Capture and Primary Purification . . . .
`Conclusions . . . .
`430
`References . . . .
`431
`
`424
`
`Appx275
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`viii
`
`Contents
`
`13. Downstream Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439
`Ron Bates
`439
`Introduction . . . .
`Purity Requirements for Biological Purification Processes . . . .
`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
`452
`Conventional Chromatographic Methods . . . .
`Nonconventional Chromatographic Methods . . . .
`Design of Purification Protocols . . . .
`469
`Scale-Up Strategies for Chromatographic
`Purification of Biologics . . . .
`472
`References . . . .
`473
`
`440
`
`468
`
`14. Formulation, Filling and Packaging . . . . . . . . . . . . . . . . . . . . . . . . . . . 483
`M. E. M. Cromwell
`483
`Introduction . . . .
`Degradation=Inactivation . . . .
`Formulation Development . . . .
`Drug Delivery . . . .
`505
`Stability Studies . . . .
`509
`Methodology for Assessing Protein Stability . . . .
`Filling, Finishing and Packaging . . . .
`515
`Future Prospects . . . .
`517
`References . . . .
`517
`
`484
`493
`
`510
`
`15. Validation of Cell Culture-Based Processes and Qualification
`. . . . . . . . . . . . . . . . . . . . . . . . . 523
`of Associated Equipment and Facility
`Chandra M. Dwivedi
`523
`Introduction . . . .
`Approach and Rationale . . . .
`Process Development . . . .
`526
`Validation of the Manufacturing Processes and Associated
`Equipment Qualification . . . .
`538
`Manufacturing Plant Qualification . . . .
`Summary . . . .
`550
`References . . . .
`550
`
`524
`
`546
`
`16. Facility Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 553
`Kim Nelson
`553
`Introduction . . . .
`553
`GMP and Regulatory Considerations . . . .
`Process Technologies and Functional Area Requirements . . . .
`Design and Operational Paradigms . . . .
`574
`Equipment and Sterile Piping Design . . . .
`594
`Process Utility Systems . . . .
`599
`References . . . .
`602
`
`561
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`Appx276
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`Contents
`
`ix
`
`. . . . . . . . . . . . . . . . . . 605
`
`17. Production of Proteins by Transient Expression
`Alain R. Bernard
`605
`Introduction . . . .
`Stable vs. Transient Expression . . . .
`Cell Hosts . . . .
`606
`608
`Vectors for Transient Expression . . . .
`Plasmid DNA-Mediated Expression . . . .
`612
`Virus-Mediated Expression . . . .
`617
`Conclusions . . . .
`621
`References . . . .
`621
`
`606
`
`628
`
`18. Principles and Applications of the Insect Cell-Baculovirus
`Expression Vector System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 627
`Laura A. Palomares, Sandino Estrada-Mondaca, and
`Octavio T. Ramı´rez
`627
`Introduction . . . .
`Insect Cell Lines . . . .
`Baculovirus . . . .
`635
`642
`Insect Cell Culture . . . .
`Bioengineering Issues in Insect Cell Culture . . . .
`Posttranslational Modifications . . . .
`658
`Larval Production and Stable Expression Systems . . . .
`Products and Applications of IC-BEVS . . . .
`669
`References . . . .
`676
`
`648
`
`665
`
`19. Advances in Adult Stem Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . 693
`E. S. Tzanakakis and C. M. Verfaillie
`Introduction . . . .
`693
`693
`Somatic Stem Cells . . . .
`698
`Adult Stem Cell Culture . . . .
`702
`Quality Control in Adult Stem Cell Culture . . . .
`Phenotypic Characterization During in Vitro Differentiation
`of Stem Cells . . . .
`704
`704
`Large-Scale Stem Cell Culture Systems . . . .
`Recent Issues in Adult Stem Cell Research . . . .
`Potential Therapeutic Applications . . . .
`710
`References . . . .
`713
`
`708
`
`20. Ex Vivo Culture of Hematopoietic and Mesenchymal Stem Cells
`. . . . . . . . . . . . . . . . . 723
`for Tissue Engineering and Cell-Based Therapies
`A. Mantalaris and J. H. D. Wu
`Introduction . . . .
`723
`723
`Hematopoiesis and Bone Marrow . . . .
`Hematopoietic Stem=Progenitor Cell Assays . . . .
`Conclusions . . . .
`736
`References . . . .
`736
`
`725
`
`Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 745
`
`Appx277
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`15
`Validation of Cell Culture-Based Processes
`and Qualification of Associated Equipment
`and Facility
`
`Chandra M. Dwivedi
`Bayer Corporation, Berkeley, California, U.S.A.
`
`INTRODUCTION
`
`Why validate? Though validation is a well-accepted and recognized cGMP require-
`ment in today’s Pharma business, this question is often posed during the product or
`process development (PD) activities in a start-up or even in an established company.
`In a nutshell, validation is not only a regulatory requirement, but it makes ‘‘good
`business sense.’’ Validated processes assure production of quality product, batch
`after batch, and ultimately result in fewer headaches down the road in terms of fewer
`deviations during production, quality assurance (QA) discrepancy investigations,
`adverse events from the field, and regulatory observations (483s and its global
`equivalent) during regulatory inspections. In addition, they improve cost effective-
`ness in terms of preventing process failures, lot rejections, re-processing of salvage-
`able lots, and attaining maximum plant capacity. Moreover, a sound and thorough
`validation strategy not only assures the production of top quality products, but also
`builds confidence and provides peace of mind to its customers. It also boosts the
`morale of the company employees and help build a sound and trustworthy relation-
`ship and track record with the regulatory agencies. The latter may come as a blessing
`for a company’s future dealings with the regulatory agencies.
`The term process validation originated in 1983 when the Food and Drug
`Administration (FDA) expanded the cGMP guidelines to cover demonstration of
`process consistency=reproducibility, but the guidelines were not finalized until
`1987 (1). These guidelines were originally intended to be adopted by all drug product
`and biological manufacturers, but were later extended to the medical device and
`diagnostic manufacturers and to the blood collection=distribution=users and blood
`product manufacturers (2,3). Though originally intended only for the finished drug
`
` Currently at BIOGEN IDEC, Inc., Cambridge, MA, U.S.A.
`
`523
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`524
`
`Dwivedi
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`product, these regulations have been recently extended to bulk drugs and bulk
`biologicals (4).
`The original definition of the term process validation was described by the
`FDA as ‘‘Establishing a documented evidence which provides a high degree of assur-
`ance that a specific process will consistently produce a product meeting its predeter-
`mined specifications and quality attributes.’’
`In practice, process validation (process performance qualification, PPQ) is more
`complicated than the simple definition stated above and is only one element of the
`overall validation process. It is a culmination of all other validation studies, such as
`equipment qualification (installation qualification, IQ; operational qualification,
`OQ; and performance qualification, PQ), computer qualification (IQ and OQ), utilities
`and facilities qualification (IQ, OQ, and PQ) cleaning validation (PQ), environmental
`qualification (PQ), and analytical qualification (PQ), all covered under a validation
`master plan (VMP) written for each new technology, process, or a product.
`The invention of recombinant DNA technology in the late 1970s and its wide-
`spread application to eukaryotic and prokaryotic cells for developing unique medical
`applications=treatments resulted in the establishment of a new field known as
`‘‘Genetic Engineering’’ today (5,6). These developments opened the floodgates for
`innovation that resulted in the establishment of many biotechnology companies
`worldwide. Of these, more than 50% of the biotechnology companies are working
`on cell culture technology for producing pharmaceutical and cellular therapies.
`Due to continued innovation in this field the application of process validation
`concepts and guidelines are becoming increasingly complex, challenging, and difficult
`to understand by technical professionals, regulatory auditors, and cGMP compliance
`enforcers working in the pharmaceutical and biotechnology-related organizations.
`Since it is impossible to cover all aspects of process validation for the numerous bio-
`technology-derived products in this chapter, an attempt will be made only to provide a
`simplified version of the regulatory requirements that are needed for licensing cell cul-
`ture-derived pharmaceuticals and cellular therapies. This chapter is intended to pro-
`vide a bird’s-eye view of the regulatory requirements for process validation to
`entrepreneurs before they plan for building a new manufacturing plant and expect
`to obtain licensure for a product (Product License Application, PLA) or a biologic
`(Biologic License Application, BLA) or a drug (New Drug Application, NDA) from
`regulatory agencies. This chapter is expected to prepare them well before they begin
`that challenging, eventful, exhausting, memorable, and ultimately rewarding journey.
`
`APPROACH AND RATIONALE
`
`The innumerable amount of research and development studies conducted on a large
`number of medical products has enabled us to understand that the quality attributes
`for any given product are not an unexpected output. But, are largely dependent on
`the process parameters used during their production. Therefore, the control of qual-
`ity attributes for any biological or pharmaceutical product is in our hands; and with
`the development of new technologies, quality attributes for the new products can
`now be built into the manufacturing process. In this respect, the process design in
`relation to the respective product quality attributes has become crucial for the devel-
`opment and licensing of the medical and pharmaceutical products (7–10).
`Since the breakthrough in genetic engineering a few decades ago, numerous
`medical, biological, pharmaceutical, and diagnostic products and applications
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`based on cell culture technology have been invented. They are based on microbial
`fermentation (eukaryotic and prokaryotic), hybridoma technology, and tissue regen-
`eration. Even plant cell technology is being evaluated to produce medical and
`therapeutic products for human use. The examples of the cells used for this purpose
`are: bacteria (Escherichia coli), fungi (Aspergillus, Saccharomyces), mammalian cells
`(CHO, BHK, myeloma, melanoma, hybridoma, etc.), insect cells (Drosophila), and
`plant cells (tobacco, spinach, etc.). The majority of the products are secreted by
`the cells in the spent medium (harvest) by applying the rDNA technologies and
`manipulation of respective genes in the cells. The examples of recombinant products
`derived from these technologies are: erythropoietin (rEPO), anti-hemophilic factor
`(rFVIII), tissue plasminogen activator (rTPA), growth factors (EGF, TGF, PDGF,
`TNF, etc.), hormones (Insulin, LH, FSH, etc.), interferons (IF-1, IF-2, etc.), inter-
`leukins (IL-2, IL-4, IL-6, etc.), monoclonal antibodies (mAbs), and other enzymes
`and proteins (cerezyme, galactosidase, etc.). Some of the products are expressed in
`the inclusion bodies within cells and the cells therefore must be lysed to extract
`the products out (insulin, EGF, etc.). Epithelial cells, neuroblastoma, osteoblastoma
`and cartilage cells are being grown in laboratories and used as medical devices for a
`number of treatments (burns, tissue implant, tissue regeneration, etc.). A number of
`monoclonal antibodies are being generated from bacterial, mammalian, and plant
`cell technologies for the treatment of cancer, autoimmune diseases, and other
`immunological disorders.
`A general approach to streamline validation concepts and policies has been
`evolving over the last number of years. These efforts have resulted in better under-
`standing of the requirements for the validation by the industry professionals. For the
`purposes of clarity and better understanding this article will employ the newly emer-
`ging approach on validation concepts (11,12). Accordingly, qualification of all
`equipment and systems (design qualification, DQ; installation qualification, IQ;
`operational qualification, OQ; and performance qualification, PQ) will be referred
`as ‘‘Equipment Qualification’’ and not as ‘‘Validation.’’ The term ‘‘Validation’’
`will be used only for ‘‘Process Validation’’ studies that are related with the studies
`(with or without active ingredient) at the small-scale (lab-scale) or full production
`scale (process validation, PV or PPQ).
`The variety of cell culture technologies and many different approaches to use
`them as pharmaceutical products or medical devices makes the task of building the
`quality attributes in the manufacturing process very challenging. This also makes
`the task of process validation more difficult as generic models of process validation
`cannot be used, and every process validation study needs to be devised from scratch
`based on the technology being used. For example, the level of impurities (DNA, host
`cell contaminating proteins, etc.) may be substantially less in the starting material
`where the product is secreted out in the spent medium (harvest) as compared to the
`product that is expressed intracellularly such as in the inclusion bodies. Therefore,
`the design of the manufacturing process and the resultant process validation studies
`would be very different for the two approaches to isolate and purify the product(s).
`The possible impurities and contaminants in a cell culture-based product are:
`intact cells, adventitious agents [bacteria, fungi, mycoplasma, viruses, transmissible
`spongiform encephalitis (TSE)=bovine spongiform encephalitis (BSE)], endogenous
`retroviruses, host cell nucleic acids and proteins, foreign proteins (from raw materi-
`als and microbial contaminations), endotoxins, and contaminating process chemicals
`(13). A validated process, therefore, must demonstrate effective removal, inactiva-
`tion, or reduction of these impurities and contaminants to acceptable levels.
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`Though it is preferable to perform process validation studies at full-scale
`operational level, it is not always possible to perform them at manufacturing scale
`due to practical limitations (e.g., virus and nucleic acid reduction studies may require
`huge amounts of model viruses and nucleic acids). In such cases, scaled-down bench-
`level studies are acceptable as long as all process input parameters are kept the same
`as in the full-scale and the output parameters are comparable to the full-scale (14).
`Whenever this approach is used, demonstration and justification of the acceptability
`of the scaled-down model should be performed prior to formal process validation.
`
`PROCESS DEVELOPMENT
`
`Development of a Defined Process
`
`The critical steps for the development of a defined process are outlined in Fig. 1. We
`will examine below the requirements for developing a reliable and reproducible pro-
`cess for a cell culture derived product. The definition of a defined process may be sum-
`marized as ‘‘a process that provides a high degree of assurance that it will consistently
`produce a product meeting its predetermined specifications and quality attributes.’’
`This definition seems simple and doable (in the beginning phase of a project) but
`becomes difficult to achieve when all the details for a cell culture-based product are
`brought into consideration. Adequate confidence must be built by doing sufficient
`experimentation and development work to demonstrate that the process can consis-
`tently produce a product of pre-specified quality. Range finding (feed stream) studies
`should be performed for every critical and noncritical process parameter (15), and
`operational set-points must be established after completion of the range finding stu-
`dies. Worst-case studies (upper and lower ranges) should be performed during the
`development phase, (as it is much easier to do them during development than during
`actual production). Alert and action levels (limits) for out-put parameters (test results
`and specifications) must be established with adequate justification. In-process and final
`product specifications (acceptance criteria) must be defined clearly with sound scientific
`justifications.
`The success of a well executed project depends on a well written process devel-
`opment (PD) report with sufficient details for every aspect of the process and a well
`executed transfer of technology from the R&D department to the operations depart-
`ment. The R&D personnel not only adequately transfer the technology, but must
`provide training to production personnel in every aspect of the process. The role
`of the R&D personnel does not end here, they should actively monitor the process
`after successful process validation by applying the statistical tools such as statistical
`process control. Post-validation process data must be analyzed to ensure that the
`process performs within the established boundaries. Process capability (Cpk) calcu-
`lations must be performed on the post-validation process data to evaluate process
`performance. The process data should also be analyzed by applying other statistical
`tests, such as Student’s t-test, to determine confidence intervals on process perfor-
`mance. A 95% confidence interval is generally acceptable for process validation stu-
`dies. Many companies, however, run their production processes at 98% confidence
`interval or up to 6 SD of the validated process parameters. These analyses demon-
`strate whether the process is in control and build confidence for running the process
`on a consistent basis.
`The importance and relevance of good PD work that eventually pays off many
`fold must be emphasized here. It is generally acknowledged that many pharmaceutical
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`Figure 1 Critical steps for developing a defined process.
`
`and biotechnology organizations shy away from doing comprehensive PD work as
`they are in a rush to reach the marketplace. In our competitive world of today, timing
`is key for making or breaking of an organization. Often what we do not realize is that
`there are no short cuts and eventually (sooner or later) we have to do the required PD
`work. The smart approach, therefore, would be to perform all required PD work
`before process validation, rather than during process validation or after completion
`of a process validation project. In the latter case, the validation projects generally
`become confusing, cost a great deal of money, and delay project completion (16).
`A poorly developed process will typically allow only narrow ranges for opera-
`tional parameters and may result in the rejection of large amounts of otherwise good
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`in-process material produced slightly outside the narrow process ranges developed.
`Extension of the process ranges or scale-up of manufacturing processes after initial
`validation requires time consuming regulatory review and approvals, repetition of
`all the work performed previously, and almost always turns into a costly validation
`project. It prevents pioneering organizations from taking leadership positions in the
`marketplace due to limited product supplies. It may lead and encourage competitors
`to enter the field and snatch the leadership position from the organizations that devel-
`oped the product at the first place. It is a lesson many organizations learn, albeit late.
`
`Process Development Report
`
`The importance of well-executed PD work and a well-written process development
`report (PDR) cannot be emphasized enough. PD and PDRs are the key components
`of a successful technology transfer from R&D to manufacturing (8). The success or
`failure of a process validation project greatly depends on the quality and details of
`the PD work performed and the quality of PDRs in terms of their content, clarity,
`and completeness. Poorly written reports often cause a great deal of frustration for
`all involved, result in unnecessary delays, impact project schedule, and even lead to
`ultimate failure of a project. Many organizations perform excellent PD work, but
`lack in writing clear and complete reports. Ideal PDRs should contain the following
`information in as much detail as possible:
` Objective and definition of a process=product
` Scope and rationale
` Process description
` Process flow chart
` Materials and methods
` Equipment and facilities
` Utilities and accessories
` HVAC and environmental requirements
` Process input and output parameters (critical and noncritical)
` In-process testing and acceptance criteria
` Product specifications
` Calibration and preventive maintenance
` Other process requirements
` Result and discussion
` Conclusion
` References
`
`Process Parameters
`
`It is paramount that all process operating parameters (input parameters) that affect
`product quality attributes (output parameters) ar