Cell Culture and Upstream Processing

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ISBN: 9780415399692 | 0415399696
Cover: Nonspecific Binding
Copyright: 5/25/2007

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Upstream processing refers to the production of proteins by cells genetically engineered to contain the human gene which will express the protein of interest. The demand for large quantities of specific proteins is increasing the pressure to boost cell culture productivity, and optimizing bioreactor output has become a primary concern for most pharmaceutical companies. Each chapter in Cell Culture and Upstream Processingis taken from presentations at the highly acclaimed IBC conferences as well as meetings of the European Society for Animal Cell Technology (ESACT)and Protein Expression in Animal Cells (PEACe)and describes how to improve yield and optimize the cell culture production process for biopharmaceuticals, by focusing on safety, quality, economics and operability and productivity issues. Cell Culture and Upstream Processingwill appeal to a wide scientific audience, both professional practitioners of animal cell technology as well as students of biochemical engineering or biotechnology in graduate or high level undergraduate courses at university.

Contributors	p. ix
Abbreviations	p. xi
Preface	p. xiii
Overview on mammalian cell culture
Cell line development and culture strategies: future prospects to improve yields	p. 3
Introduction	p. 3
Cell line transfection and selection	p. 5
Increase in efficiency in selecting a producer cell line	p. 6
Stability of gene expression	p. 8
Optimization of the fermentation process	p. 9
Apoptosis	p. 11
Bioreactors	p. 11
The capacity crunch	p. 12
Acknowledgment	p. 13
References	p. 13
The producer cell line
Use of DNA insulator elements and scaffold/matrix-attached regions for enhanced recombinant protein expression	p. 19
Introduction	p. 19
The position effect	p. 20
Use of insulators and S/MARs can reduce the effects of heterochromatin on transgene expression	p. 20
DNA insulator elements	p. 22
The scaffold/matrix-attachment regions	p. 23
Binding proteins for DNA insulators and S/MARs	p. 25
DNA insulators or S/MARs can be incorporated into expression vectors	p. 26
DNA insulators and S/MARs act in a context-dependent manner	p. 30
Conclusion	p. 31
Acknowledgements	p. 32
References	p. 32
Targeted gene insertion to enhance protein production from cell lines	p. 37
Introduction	p. 37
Identification of genomic 'hot spot' loci	p. 39
Recombinase-mediated site-specific gene insertion	p. 39
Cre, Flp, and [phiv]C31 recombinase systems	p. 40
Recombinase-mediated cassette exchange	p. 40
Gene insertion at native 'pseudo' recombinase sites	p. 43
Modification of recombinases and their target sites	p. 43
Emerging technologies for targeted gene insertion	p. 44
Homing endonucleases in HDR-mediated targeted gene insertion	p. 46
Targeted gene insertion into native loci by zinc finger, nuclease-mediated, high-frequency, homologous recombination	p. 47
Perspective	p. 50
References	p. 52
Recombinant human IgG production from myeloma and Chinese hamster ovary cells	p. 57
Introduction	p. 57
The need for recombinant human antibodies	p. 57
Recombinant antibodies	p. 58
Decoupling antibody isolation and production	p. 58
Choice of host cells	p. 59
Chinese hamster ovary cells	p. 60
Rodent myeloma cells	p. 60
The glutamine synthetase system	p. 60
Cell line stability	p. 61
Bioreactor process strategies	p. 62
IgG supply during antibody development	p. 62
Strategies for cell line engineering during clinical development	p. 63
Cost of goods and intellectual property	p. 64
Recombinant human IgG production from myeloma and CHO cells	p. 64
Creation of CHO and NS0 cell lines expressing IgG	p. 64
Cell expansion, subculture and production reactor experiments	p. 65
Northern and western blotting	p. 65
Comparison of results of transfections from GS-NS0 and GS-CHO	p. 65
Dilution cloning and analysis of clonal heterogeneity	p. 66
Analysis of instability of a GS-NS0 cell line	p. 67
Output of transfections of GS-NS0 and GS-CHO	p. 68
IgG production stability of candidate GS-NS0 clones	p. 69
IgG production stability of GS-CHO transfectants	p. 70
Fed-batch bioreactor process for GS-NS0 and GS-CHO	p. 71
Analysis of IgG quality produced from GS-CHO and GS-NS0 bioreactor processes	p. 71
Comparative yield of different human IgGs produced from CHO or NS0 cells	p. 74
Summary	p. 74
Acknowledgments	p. 76
References	p. 76
Media development
Cell culture media development: customization of animal origin-free components and supplements	p. 81
Introduction	p. 81
Types of cell culture media	p. 82
Components of animal origin	p. 83
Segregate	p. 85
Mitigate	p. 87
Replace	p. 88
Summary and considerations for the future	p. 95
Acknowledgments	p. 98
References	p. 98
Glycosylated proteins
Post-translational modification of recombinant antibody proteins	p. 103
Introduction	p. 103
Common post-translational modifications	p. 104
Recombinant antibody therapeutics	p. 105
Structural and functional characteristics of human antibodies	p. 106
The human IgG subclasses: Options for antibody therapeutics	p. 106
The structure of human IgG antibodies	p. 108
IgG-Fc glycosylation	p. 110
IgG-Fab glycosylation	p. 112
Cell engineering to influence glycoform profiles	p. 115
IgG glycoforms and Fc effector functions	p. 116
Glycosylation engineering	p. 118
Pharmacokinetics and placental transport	p. 118
Antibody therapeutics of the IgA class	p. 119
Non-antibody recombinant (glyco)protein therapeutics, 'biosimilar' and 'follow-on' biologics	p. 120
Erythropoietin	p. 121
Tissue-type plasminogen activator	p. 122
Granulocyte-macrophage colony stimulating factor (GM-CSF)	p. 122
Granulocyte-colony stimulating factor	p. 122
Activated protein C	p. 122
Conclusions	p. 123
References	p. 123
Metabolic engineering to control glycosylation	p. 131
Introduction	p. 131
Manipulation of fucose content using RNAi technology in CHO cells	p. 132
Metabolic engineering of fucose content with an existing antibody production line	p. 132
Metabolic engineering of fucose content with simultaneous new stable cell line generation	p. 136
Effect of fucosylation levels on Fc[Gamma]R binding	p. 140
Effects of fucose content on antibody-dependent cellular cytotoxicity	p. 143
Discussion	p. 143
Acknowledgments	p. 146
References	p. 146
An alternative approach: Humanization of N-glycosylation pathways in yeast	p. 149
Introduction	p. 149
Yeast as host for recombinant protein expression	p. 152
N-linked glycosylation overview: Fungal versus mammalian	p. 152
A brief history of efforts to humanize N-linked glycosylation in fungal systems	p. 154
Sequential targeting of glycosylation enzymes is a key factor	p. 155
Replication of human-like glycosylation in the methylotrophic yeast	p. 157
A library of [Alpha]-1,2 mannosidases	p. 157
Transfer of N-acetylglucosamine	p. 158
Two independent approaches towards complex N-glycans: How to eliminate more mannoses	p. 159
Some metabolic engineering: Transfer of galactose	p. 161
More metabolic engineering: Sialic acid transfer. The final step	p. 162
Glyco-engineered yeast as a host for production of therapeutic glycoproteins	p. 162
N-linked glycans and pharmacokinetics of therapeutic glycoproteins	p. 164
N-glycans and their role in tissue targeting of glycoproteins	p. 164
N-glycans can modulate the biological activity of therapeutic glycoproteins	p. 165
Control of N-glycosylation offers advantages	p. 165
Conclusions	p. 166
References	p. 166
The Bioprocess
Perfusion or fed-batch? A matter of perspective	p. 173
Introduction	p. 173
Factors affecting the decision on choosing the manufacturing technology	p. 175
Technology expertise	p. 175
Facility design and scope (product dedicated versus multi-product)	p. 179
Impact of switching from perfusion to fed-batch	p. 180
Personnel requirements	p. 180
Liquid handling	p. 181
Equipment	p. 182
Manufacturing space	p. 182
Decrease in cycle time	p. 182
Direct costs of manufacturing	p. 182
Productivity and morale	p. 183
Conclusions	p. 183
Acknowledgments	p. 184
References	p. 184
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Cell Culture and Upstream Processing

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