Cover image for Cell culture and upstream processing
Title:
Cell culture and upstream processing
Publication Information:
London : Taylor & Francis, 2007
Physical Description:
xiv, 187 p. : ill. ; 25 cm.
ISBN:
9780415399692

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30000010209973 TP248.25.C44 C444 2007 Open Access Book Book
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Summary

Summary

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 Processing is 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 Processing will 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.


Author Notes

Michael Butler-Associate Dean of Science; Professor of Cell Technology, Department of Microbiology, School of Science, University of Manitoba, Winnipeg, Canada


Table of Contents

Michael ButlerHelen KimTrevor N. Collingwood and Fyodor D. UrnovRay FieldStephen GorfienRoy JeffriesAmy Shen and Domingos Ng and John Joly and Brad Snedecor and Yanmei Lu and Gloria Meng and Gerald Nakamura and Lynne KrummenStefan Wildt and Thomas PotgieterPichia PastorisMarco Cacciuttolo
Contributorsp. ix
Abbreviationsp. xi
Prefacep. xiii
Overview on mammalian cell culture
1 Cell line development and culture strategies: future prospects to improve yieldsp. 3
1.1 Introductionp. 3
1.2 Cell line transfection and selectionp. 5
1.3 Increase in efficiency in selecting a producer cell linep. 6
1.4 Stability of gene expressionp. 8
1.5 Optimization of the fermentation processp. 9
1.6 Apoptosisp. 11
1.7 Bioreactorsp. 11
1.8 The capacity crunchp. 12
Acknowledgmentp. 13
Referencesp. 13
The producer cell line
2 Use of DNA insulator elements and scaffold/matrix-attached regions for enhanced recombinant protein expressionp. 19
2.1 Introductionp. 19
2.2 The position effectp. 20
2.3 Use of insulators and S/MARs can reduce the effects of heterochromatin on transgene expressionp. 20
2.4 DNA insulator elementsp. 22
2.5 The scaffold/matrix-attachment regionsp. 23
2.6 Binding proteins for DNA insulators and S/MARsp. 25
2.7 DNA insulators or S/MARs can be incorporated into expression vectorsp. 26
2.8 DNA insulators and S/MARs act in a context-dependent mannerp. 30
2.9 Conclusionp. 31
Acknowledgementsp. 32
Referencesp. 32
3 Targeted gene insertion to enhance protein production from cell linesp. 37
3.1 Introductionp. 37
3.2 Identification of genomic 'hot spot' locip. 39
3.3 Recombinase-mediated site-specific gene insertionp. 39
3.3.1 Cre, Flp, and [phiv]C31 recombinase systemsp. 40
3.3.2 Recombinase-mediated cassette exchangep. 40
3.3.3 Gene insertion at native 'pseudo' recombinase sitesp. 43
3.3.4 Modification of recombinases and their target sitesp. 43
3.4 Emerging technologies for targeted gene insertionp. 44
3.4.1 Homing endonucleases in HDR-mediated targeted gene insertionp. 46
3.4.2 Targeted gene insertion into native loci by zinc finger, nuclease-mediated, high-frequency, homologous recombinationp. 47
3.5 Perspectivep. 50
Referencesp. 52
4 Recombinant human IgG production from myeloma and Chinese hamster ovary cellsp. 57
4.1 Introductionp. 57
4.2 The need for recombinant human antibodiesp. 57
4.3 Recombinant antibodiesp. 58
4.4 Decoupling antibody isolation and productionp. 58
4.5 Choice of host cellsp. 59
4.5.1 Chinese hamster ovary cellsp. 60
4.5.2 Rodent myeloma cellsp. 60
4.6 The glutamine synthetase systemp. 60
4.7 Cell line stabilityp. 61
4.8 Bioreactor process strategiesp. 62
4.9 IgG supply during antibody developmentp. 62
4.10 Strategies for cell line engineering during clinical developmentp. 63
4.11 Cost of goods and intellectual propertyp. 64
4.12 Recombinant human IgG production from myeloma and CHO cellsp. 64
4.12.1 Creation of CHO and NS0 cell lines expressing IgGp. 64
4.12.2 Cell expansion, subculture and production reactor experimentsp. 65
4.12.3 Northern and western blottingp. 65
4.12.4 Comparison of results of transfections from GS-NS0 and GS-CHOp. 65
4.12.5 Dilution cloning and analysis of clonal heterogeneityp. 66
4.12.6 Analysis of instability of a GS-NS0 cell linep. 67
4.12.7 Output of transfections of GS-NS0 and GS-CHOp. 68
4.12.8 IgG production stability of candidate GS-NS0 clonesp. 69
4.12.9 IgG production stability of GS-CHO transfectantsp. 70
4.12.10 Fed-batch bioreactor process for GS-NS0 and GS-CHOp. 71
4.12.11 Analysis of IgG quality produced from GS-CHO and GS-NS0 bioreactor processesp. 71
4.12.12 Comparative yield of different human IgGs produced from CHO or NS0 cellsp. 74
4.13 Summaryp. 74
Acknowledgmentsp. 76
Referencesp. 76
Media development
5 Cell culture media development: customization of animal origin-free components and supplementsp. 81
5.1 Introductionp. 81
5.2 Types of cell culture mediap. 82
5.3 Components of animal originp. 83
5.3.1 Segregatep. 85
5.3.2 Mitigatep. 87
5.3.3 Replacep. 88
5.4 Summary and considerations for the futurep. 95
Acknowledgmentsp. 98
Referencesp. 98
Glycosylated proteins
6 Post-translational modification of recombinant antibody proteinsp. 103
6.1 Introductionp. 103
6.2 Common post-translational modificationsp. 104
6.3 Recombinant antibody therapeuticsp. 105
6.4 Structural and functional characteristics of human antibodiesp. 106
6.5 The human IgG subclasses: Options for antibody therapeuticsp. 106
6.6 The structure of human IgG antibodiesp. 108
6.7 IgG-Fc glycosylationp. 110
6.8 IgG-Fab glycosylationp. 112
6.9 Cell engineering to influence glycoform profilesp. 115
6.10 IgG glycoforms and Fc effector functionsp. 116
6.11 Glycosylation engineeringp. 118
6.12 Pharmacokinetics and placental transportp. 118
6.13 Antibody therapeutics of the IgA classp. 119
6.14 Non-antibody recombinant (glyco)protein therapeutics, 'biosimilar' and 'follow-on' biologicsp. 120
6.14.1 Erythropoietinp. 121
6.14.2 Tissue-type plasminogen activatorp. 122
6.14.3 Granulocyte-macrophage colony stimulating factor (GM-CSF)p. 122
6.14.4 Granulocyte-colony stimulating factorp. 122
6.14.5 Activated protein Cp. 122
6.15 Conclusionsp. 123
Referencesp. 123
7 Metabolic engineering to control glycosylationp. 131
7.1 Introductionp. 131
7.2 Manipulation of fucose content using RNAi technology in CHO cellsp. 132
7.2.1 Metabolic engineering of fucose content with an existing antibody production linep. 132
7.2.2 Metabolic engineering of fucose content with simultaneous new stable cell line generationp. 136
7.2.3 Effect of fucosylation levels on Fc[Gamma]R bindingp. 140
7.2.4 Effects of fucose content on antibody-dependent cellular cytotoxicityp. 143
7.3 Discussionp. 143
Acknowledgmentsp. 146
Referencesp. 146
8 An alternative approach: Humanization of N-glycosylation pathways in yeastp. 149
8.1 Introductionp. 149
8.2 Yeast as host for recombinant protein expressionp. 152
8.3 N-linked glycosylation overview: Fungal versus mammalianp. 152
8.4 A brief history of efforts to humanize N-linked glycosylation in fungal systemsp. 154
8.5 Sequential targeting of glycosylation enzymes is a key factorp. 155
8.6 Replication of human-like glycosylation in the methylotrophic yeastp. 157
8.7 A library of [Alpha]-1,2 mannosidasesp. 157
8.8 Transfer of N-acetylglucosaminep. 158
8.9 Two independent approaches towards complex N-glycans: How to eliminate more mannosesp. 159
8.10 Some metabolic engineering: Transfer of galactosep. 161
8.11 More metabolic engineering: Sialic acid transfer. The final stepp. 162
8.12 Glyco-engineered yeast as a host for production of therapeutic glycoproteinsp. 162
8.13 N-linked glycans and pharmacokinetics of therapeutic glycoproteinsp. 164
8.14 N-glycans and their role in tissue targeting of glycoproteinsp. 164
8.15 N-glycans can modulate the biological activity of therapeutic glycoproteinsp. 165
8.16 Control of N-glycosylation offers advantagesp. 165
8.17 Conclusionsp. 166
Referencesp. 166
The Bioprocess
9 Perfusion or fed-batch? A matter of perspectivep. 173
9.1 Introductionp. 173
9.2 Factors affecting the decision on choosing the manufacturing technologyp. 175
9.2.1 Technology expertisep. 175
9.2.2 Facility design and scope (product dedicated versus multi-product)p. 179
9.3 Impact of switching from perfusion to fed-batchp. 180
9.3.1 Personnel requirementsp. 180
9.3.2 Liquid handlingp. 181
9.3.3 Equipmentp. 182
9.3.4 Manufacturing spacep. 182
9.3.5 Decrease in cycle timep. 182
9.3.6 Direct costs of manufacturingp. 182
9.3.7 Productivity and moralep. 183
9.4 Conclusionsp. 183
Acknowledgmentsp. 184
Referencesp. 184