Noll Cells and Culture

Proceedings of the 20th ESACT Meeting, Dresden, Germany, June 17-20, 2007
1. Auflage 2010
ISBN: 978-90-481-3419-9
Verlag: Springer Netherlands
Format: PDF
Kopierschutz: 1 - PDF Watermark

E-Book, Englisch, Band 4, 800 Seiten

Reihe: ESACT Proceedings

Regeneration of tissue to replace damaged or injured tissue is the goal of t- sue engineering. Biomaterials like polyglycolic acid, collagen and small-intestinal submuscosa provide a temporary scaffold to guide new tissue growth and or- nization. Typically, they need to be biodegradable, showing good cell atta- ment and proliferation and they should possess appropriate mechanical properties (Kim et al. , 2000). Synthetic polymers ful ll most of these requirements but lack cell-adhesion peptides on their surface to enhance cell attachment. Ce- adhesion peptides are present in ECM proteins like collagen and elastin. Thus a synthetic polymer coated with ECM proteins would result in a scaffold that mimics the natural cellular environment with enhanced cell attachment and p- liferation. The new bioactive scaffold will be made by combining a synthetic polymer coated with a layer of recombinant ECM proteins produced by CHO cells. The rst step consists of identifying polymers that give best results in terms of CHO cell attachment and growth. Classical techniques to determine biomass are inappropriate to evaluate 3-D structures. Thus a screening system based on stable GFP expressing CHO cells was used to compare the different scaffolds. Simple uorescent measurement after cell lysis allows determining cell attachment and p- liferation on synthetic polymers. Finally CHO cells producing human recombinant collagen I and elastin were generated. We showed that both proteins are expressed and secreted by CHO DG44 cells. 2 Materials and Methods 2.

Autoren/Hrsg.

Noll, Thomas

Inhaltsverzeichnis

1;Contents;5
2;Contributors;20
3;Part I Single-Cell Analysis and Engineering;54
3.1;Targeted Gene Knockdown Effects on Recombinant Protein Secretion in CHO Cells;55
3.1.1;1 Introduction;55
3.1.2;2 Materials and Methods;56
3.1.2.1;2.1 Cell Culture Maintenance and siRNA Transfection Assays;56
3.1.2.2;2.2 Cell XpressTM Analysis;56
3.1.3;3 Results and Discussion;57
3.1.3.1;3.1 siRNA Design and Validation;57
3.1.3.2;3.2 Cell XpressTM Analysis of CHO Cells Transfected with IgG siRNAs;57
3.1.3.3;3.3 Cell XpressTM Analysis of CHO Cells Transfected with Biomarker siRNAs;59
3.1.4;4 Conclusion;60
3.1.5;Reference;60
3.2;RNA Silencing Suppressors Boost the Production of Recombinant Proteins and Viruses;61
3.2.1;1 Introduction;61
3.2.2;2 RNA Silencing Suppressors;62
3.2.3;3 RSSs Boost Recombinant Protein Production;63
3.2.4;4 RSSs Boost Virus Production;63
3.2.5;References;63
3.3;Towards the Use of CHO Produced Recombinant Extracellular Matrix Proteins as Bioactive Elements in a 3-D Scaffold for Tissue Engineering;65
3.3.1;1 Introduction;66
3.3.2;2 Materials and Methods;66
3.3.2.1;2.1 Polymer Screening;66
3.3.2.2;2.2 Generation of Cell Lines Expressing Recombinant ECM Proteins;67
3.3.3;3 Results and Discussion;67
3.3.3.1;3.1 Polymer Screening;67
3.3.3.2;3.2 Generation of Cell Lines Expressing Recombinant ECM Proteins;69
3.3.4;References;70
3.4;Transient Gene Expression in Chinese Hamster Ovary Cells at Low Temperature The Effects of Cold-Induced Proteins and an mRNA Regulatory Element;71
3.4.1;1 Introduction;71
3.4.2;2 Materials and Methods;72
3.4.3;3 Results and Discussion;72
3.4.4;References;74
3.5;Single-Cell Approach in Influenza Vaccine Production: Apoptosis and Virus Protein Production;76
3.5.1;1 Introduction;76
3.5.2;2 Materials and Methods;77
3.5.3;3 Results and Discussion;77
3.5.4;References;79
3.6;Chondrogenic Differentiation of Human Mesenchymal Stem Cells During Multiple Subcultivation;80
3.6.1;1 Introduction;81
3.6.2;2 Materials and Methods;82
3.6.2.1;2.1 Cultivation and Passaging;82
3.6.2.2;2.2 Differentiation;82
3.6.2.3;2.3 MIA-ELISA;82
3.6.3;3 Results;83
3.6.4;4 Conclusion;84
3.6.5;References;84
3.7;Cell Xpress-Assisted Analysis of Clone Stability in Recombinant Chinese Hamster Ovary Cells;86
3.7.1;1 Introduction;86
3.7.2;2 Materials and Methods;87
3.7.2.1;2.1 Cell XpressTM Clone Selection and Analysis;87
3.7.2.2;2.2 Cell Culture Maintenance and Growth and Productivity Characterization;87
3.7.2.3;2.3 Molecular Characterization of Cell XpressTM Clones;87
3.7.3;3 Results and Discussion;88
3.7.3.1;3.1 Cell Xpress Clone Generation and Stability Characterization;88
3.7.3.2;3.2 Molecular Characterization of Stable and Unstable CHO Clonal Lines;89
3.8;An Evaluation of the Intrinsic IgG Production Capabilities of Different Chinese Hamster Ovary Parental Cell Lines;91
3.8.1;1 Introduction;91
3.8.2;2 Results and Discussion;92
3.8.2.1;2.1 Analysis of Transient GFP Production in Different Parental CHO Cell Lines;92
3.8.2.2;2.2 Analysis of Transient IgG Production and Secretion in Different Parental CHO Cell Lines;92
3.8.3;3 Conclusions;94
3.8.4;References;94
3.9;Cell Xpress Technology Facilitates High-Producing Chinese Hamster Ovary Cell Line Generation Using Glutamine Synthetase Gene Expression System;95
3.9.1;1 Introduction;95
3.9.2;2 Results and Discussion;96
3.9.2.1;2.1 Secretion Intensity and Population Heterogeneity Analysis Using Cell XpressTM;96
3.9.2.2;2.2 Secretion Dynamics During Single-Cell Clone Expansion;97
3.9.3;3 Conclusions;97
3.9.4;References;98
3.10;CHO-DG44 Cell Line Development by FLP-Targeting High Level Glycoprotein Expression with Significantly Decreased Time Lines;99
3.10.1;1 Introduction;100
3.10.2;2 Results and Discussion;100
3.10.2.1;2.1 The Targeting Strategy;100
3.10.2.2;2.2 Establishment of RMCE Target Clones;100
3.10.2.3;2.3 Case Study -- RMCE Vs. Classical Cell Line Development;103
3.10.3;3 Conclusions;104
3.10.4;References;104
3.11;Transgene Copy Number Impact on Clone Performance;106
3.11.1;1 Introduction;106
3.11.2;2 Forced Integration of Multiple Copies by RMCE;107
3.11.3;3 Hypothesis;108
3.11.4;4 Generation and Characterization of Pharmaceutical Cell Lines;108
3.11.5;5 Reduction of Copy Number in Preestablished Clones;109
3.11.6;6 Conclusions;110
3.11.7;References;111
3.12;Single Use Bioreactors: Expressing Protein in Mammalian Cell Suspension;112
3.12.1;1 Industry Challenges;112
3.12.2;2 In-Vessel Operations;113
3.12.3;3 A New Approach No Shaking, Stirring, Rocking or Rolling;113
3.12.4;4 Experimental Evidence;114
3.12.5;5 Conclusion;117
4;Part II Applied Integrative Physiology;119
4.1;Gene Modified Hematopoietic Stem Cells for the Treatment of Primary Immunodeficiency Diseases;120
4.1.1;1 Text;121
4.1.1.1;1.1 Principles of Hematopoietic Stem Cell Gene Therapy;121
4.1.1.2;1.2 Retroviral Vectors and Transduction Protocols;121
4.1.1.3;1.3 Progress in Gene Therapy for Primary Immunodeficiencies;121
4.1.2;2 Conclusions;123
4.1.3;References;124
4.2;An Avian Cell Line for Production of Highly Attenuated Viral Vectors;126
4.2.1;1 Introduction;127
4.2.2;2 Methods;128
4.2.2.1;2.1 Plasmids;128
4.2.2.2;2.2 Isolation and Culture of Primary Cells;128
4.2.2.3;2.3 Transfection and Focus Recovery;129
4.2.2.4;2.4 Immunofluorescence;129
4.2.2.5;2.5 Virus Propagation and Isolation;130
4.2.3;3 Results and Discussion;130
4.2.4;4 Summary;134
4.2.5;References;134
4.3;Microelectronic Cellular Vitality Monitoring;136
4.3.1;1 Introduction;136
4.3.2;2 Methods and Materials;137
4.3.3;3 Results;139
4.3.4;4 Discussion;140
4.3.5;References;140
4.4;IFN- Glycosylation Macroheterogeneity, Energetic Cell Status and Medium Composition During CHO Cell Cultures;141
4.4.1;1 Introduction;141
4.4.2;2 Materials and Methods;142
4.4.3;3 Results;142
4.4.3.1;3.1 IFN- Production and Energetic Status OF CHO Cells Grown in Enriched Batch Processes;142
4.4.3.2;3.2 IFN- Glycosylation and Energetic Status of CHO Cells Grown in Fed-Batch Process;142
4.4.4;4 Conclusion;145
4.4.5;References;145
4.5;Modelling of Neural Metabolism Using 13C-NMR Spectroscopy and Metabolic Flux Analysis;146
4.5.1;1 Introduction;146
4.5.2;2 Materials and Methods;147
4.5.2.1;2.1 Cells and Culture Conditions;147
4.5.2.2;2.2 Incubation of Astrocytes with [1- 13 C]glucose;147
4.5.2.3;2.3 Metabolic Flux Analysis;147
4.5.3;3 Results;149
4.5.3.1;3.1 Hypoglycemia;149
4.5.3.2;3.2 Anoxia;149
4.5.4;4 Conclusions;150
4.5.5;References;150
4.6;Influence of Glucose and Glutamine Concentration on Metabolism of rCHO Cells;152
4.6.1;1 Introduction;152
4.6.2;2 Materials and Method;153
4.6.3;3 Results and Discussion;153
4.6.4;4 Conclusion;155
4.6.5;References;156
5;Part III Speed and Intensification in Bioprocess Development;157
5.1;Delivery of Biomolecules with Non-Viral Vectors;158
5.1.1;1 Introduction;158
5.1.2;2 The PEI Mechanism;159
5.1.3;3 Synthetic Polymer-Mediated Transient Transfection for Biomanufacturing;160
5.1.4;4 Conclusions and Perspectives;161
5.1.5;References;162
5.2;Circumventing the Pay Now or Pay Later Dilemma: Strategies for Achieving Process Development with Speed and Long-Term Potential;163
5.2.1;1 Introduction;163
5.2.2;2 Process Development Strategy;164
5.2.3;3 Incorporating QbD During Development;166
5.2.4;4 Components of the Manufacturability Assessment;167
5.2.5;5 Application of the Manufacturability Assessment;167
5.2.6;6 Looking Ahead;169
5.3;Recombinant Human Antibody Therapeutics: Supply Strategies for Early and Clinical Development from CHO Cells;171
5.3.1;1 Introduction;172
5.3.1.1;1.1 Choice of Host Cells;172
5.3.1.2;1.2 The GS SystemTM;172
5.3.1.3;1.3 IgG Supply During Antibody Development;173
5.3.2;2 IgG Supply Strategies;174
5.3.2.1;2.1 Supply of IgG from Transient Expression for Early Development Studies;174
5.3.2.2;2.2 Rapid Multi-Gram Production of IgG from GS-CHO Pools;175
5.3.2.3;2.3 Scale Up of IgG Supply for Clinical Trials;177
5.3.3;3 Summary;177
5.3.4;References;178
5.4;Automated Screening of High Producer HEK293F Clones and Analysis of Post-Translational Modifications of Secreted Proteins;179
5.4.1;1 Introduction;179
5.4.2;2 Methods;180
5.4.2.1;2.1 Transfection of HEK293F Cells and Generation of Stable Clones;180
5.4.2.2;2.2 Automated Selection of Cell Lines with Highest Productivity;181
5.4.2.3;2.3 Parallel Analyses of Secreted Clotting Factor Quantity and PTMs Using Two Antibodies;181
5.4.2.4;2.4 Quantification of Protein Secretion and of PTM Levels;181
5.4.3;3 Results and Discussion;182
5.4.4;4 Conclusions and Outlook;186
5.4.5;References;186
5.5;Transcriptomic and Proteomic Analysis of Antibody Producing NS0 Cells Cultivated at Different Cell Densities in Perfusion Culture;187
5.5.1;1 Introduction;187
5.5.2;2 Materials and Methods;188
5.5.2.1;2.1 Cell Line;188
5.5.2.2;2.2 Microarray Analysis;188
5.5.2.3;2.3 2D-PAGE Analysis;188
5.5.3;3 Results and Discussion;189
5.5.4;4 Conclusions;190
5.5.5;References;191
5.6;New Disposable Fixed-Bed Bioreactor for Cell Culture and Virus Production Based on a Proprietary Agitation and Aeration System;192
5.6.1;1 Introduction;192
5.6.2;2 Material and Methods;193
5.6.3;3 Results;194
5.6.4;4 Conclusion and Perspectives;196
5.7;Transcriptomic Analysis of Antibody Producing NS0 Cell Line Under Hypothermic and Hypoxic Conditions;197
5.7.1;1 Introduction;197
5.7.2;2 Materials and Methods;198
5.7.3;3 Results;200
5.7.4;References;200
5.8;Semi-Continuous Cultures as a Tool for Cell Line Characterization During Process Development;201
5.8.1;1 Introduction;201
5.8.2;2 Material and Methods;201
5.8.3;3 Results;202
5.8.3.1;3.1 Semicontinuous Culture, Performance;202
5.8.3.2;3.2 Use of Semicontinuous Cultures for Cell Characterization;203
5.8.4;References;204
5.9;Behaviour of GS-CHO Cell Lines in a Selection Strategy;205
5.9.1;1 Introduction;206
5.9.2;2 Methods;206
5.9.3;3 Results and Discussion;207
5.9.4;4 Summary and the Future;208
5.9.5;References;209
5.10;3D Cultures: Effect on the Hepatocytes Functionality;210
5.10.1;1 Introduction;211
5.10.2;2 Material and Methods;211
5.10.3;3 Results and Discussion;212
5.10.3.1;3.1 Aggregates as 3D Structures;212
5.10.3.2;3.2 Effect of Inoculum Concentration and Impeller Type;212
5.10.3.3;3.3 Effect of Media Composition;213
5.10.4;4 Conclusions;214
5.10.5;References;214
5.11;Differential Protein Expression Induced by c-Myc Over-Expression: Proteomic Analysis of a CHO Cell Line with Increased Proliferation Capacity;216
5.11.1;1 Introduction;217
5.11.2;2 Results and Discussion;217
5.11.3;3 Conclusion;219
5.11.4;References;220
5.12;Development of Pilot-Scale Orbital Shake Bioreactors: Ideal for Cost-Effective and Efficient Transient Gene Expression;221
5.12.1;1 Introduction;221
5.12.2;2 Materials and Methods;221
5.12.3;3 Results and Discussion;222
5.12.4;4 Conclusion;223
5.12.5;References;223
5.13;Helical Tracks in Shaken Cylindrical Bioreactors Improve Oxygen Transfer and Increase Maximum Cell Density Obtainable for Suspension Cultures of Mammalian Cells;224
5.13.1;1 Introduction;224
5.13.2;2 Materials and Methods;225
5.13.2.1;2.1 Cell Culture and Growth Assessment;225
5.13.2.2;2.2 DO Measurement and k L a Determination;226
5.13.3;3 Results and Discussion;226
5.13.3.1;3.1 Determination of k L a for Helical Track Bottles;226
5.13.3.2;3.2 Assessment of Cell Growth in Helical Track Vessels;226
5.13.4;4 Conclusions;228
5.13.5;References;228
5.14;Dynamic Optimisation of CHO-IFN Cell Culture Fed-Batch Time-Profile;230
5.14.1;1 Introduction;230
5.14.2;2 Model Overview;231
5.14.3;3 Model Structure;231
5.14.4;4 Global Parameter Sensitivity Analysis;232
5.14.5;5 Simulation and Optimisation Results;233
5.14.6;6 Conclusions;235
5.14.7;Notations;235
5.14.8;References;236
5.15;Long-Term 3D-Culture of HEP G2 Cell Line on Macroporous Ceramic Carriers;237
5.15.1;1 Introduction;237
5.15.2;2 Bioreactor Systems and Carriers;238
5.15.3;3 Results;238
5.15.4;References;240
5.16;An Integrated Production Process for Human Growth Hormone;241
5.16.1;1 Introduction;242
5.16.2;2 Materials and Methods;242
5.16.3;3 Results;242
5.16.4;4 Conclusions;244
5.17;Efficient Production of Human Monoclonal Antibodies by an Improved Fructose-Based Human Cell Culture;245
5.17.1;1 Introduction;245
5.17.2;2 Materials and Methods;246
5.17.3;3 Results and Discussion;247
5.17.4;References;248
5.18;Coupling Between Cell Kinetics and CFD to Establish Physio-Hydrodynamic Correlations in Various Stirred Culture Systems;249
5.18.1;1 Introduction;249
5.18.2;2 Materials and Methods;250
5.18.2.1;2.1 Cell Cultures;250
5.18.2.2;2.2 Numerical Simulations;250
5.18.3;3 Results;250
5.18.3.1;3.1 Culture Kinetics;250
5.18.3.2;3.2 Physio-Hydrodynamic Correlations;251
5.18.4;4 Conclusions;253
5.19;Confocal Microscopy Observation of CHO Cells Cultivated in Presence of Fluorescent Labelled Rapeseed Peptides;254
5.19.1;1 Introduction;254
5.19.2;2 Material and Methods;255
5.19.3;3 Results;255
5.19.3.1;3.1 Labelling of Rapeseed Peptides;255
5.19.3.2;3.2 Incubation of CHO Cells in Presence of Labelled-Peptides;256
5.19.4;References;257
5.20;Growth and Production Characteristics of Four Mammalian Cell Lines on a Cost-Effective Serum-Free Medium;258
5.20.1;1 Materials and Methods;258
5.20.2;2 Results;259
5.20.2.1;2.1 Growth Characteristics of SP2/0, CHO, EBNA 293 and Hybridoma Cells in Serum-Free Media;259
5.20.2.2;2.2 Production of Monoclonal Antibodies by Hybridomas;259
5.20.2.3;2.3 Transient Transfection Optimisation and Recombinant Protein Expression;261
5.20.3;3 Conclusions;262
5.20.4;References;262
5.21;A Serum-Free, Transient Transfection System for Enhancing Production of Recombinant Antibodies in Mammalian Cells;263
5.21.1;1 Introduction;263
5.21.2;2 Materials and Methods;264
5.21.2.1;2.1 Cell Culture;264
5.21.2.2;2.2 Transfection;264
5.21.3;3 Results and Discussion;264
5.21.4;References;266
5.22;CFD Study of the Fluid and Particle Dynamics in a Spin-Filter Perfusion Bioreactor;267
5.22.1;1 Introduction;267
5.22.2;2 Aims of the Work;268
5.22.3;3 Methodology;268
5.22.4;4 Results;268
5.22.5;References;272
5.23;High-Yielding CHO Cell Pools for Rapid Production of Recombinant Antibodies;273
5.23.1;1 Introduction;273
5.23.2;2 Isolation of High-Producing Pools;274
5.23.3;3 Scale-Up, Stability and Compatability with Disposables Technology;274
5.23.4;4 Product Quality;275
5.23.5;5 Robust Production Process;277
5.23.6;6 Cloning Out from Pools;278
5.23.7;7 Summary;278
5.23.8;8 Methods;278
5.24;Increasing Upstream Process Development Efficiency by Implementing Platform Glutamine Synthetase Cell Culture Processes;279
5.24.1;1 Introduction;280
5.24.2;2 Methods and Materials;280
5.24.3;3 Results and Discussion;280
5.24.3.1;3.1 Initial Analysis of Model GS-CHO Cell Line (1G5);281
5.24.3.2;3.2 First Iteration of the Fed-Batch Process;283
5.24.3.3;3.3 Second and Third Iteration of the Fed-Batch Process;284
5.24.3.4;3.4 Fed Batch v.4 with Multiple IgG-Expressing GS-Cell Lines;285
5.25;Implementation of High Throughput Systems for Media and Process Development;286
5.25.1;1 Introduction;286
5.25.2;2 Observations;287
5.25.3;3 Conclusions;288
5.26;Comparison of Cell Culture Methods for Obtainingof rHU-EPO to Large Scale;289
5.26.1;1 Introduction;289
5.26.2;2 Material and Methods;289
5.26.3;3 Results;290
5.26.4;4 Conclusions;291
5.26.5;References;291
5.27;Optimization and Comparison of Different DNA Methyl Transferase and Histone Deacetylase Inhibitors for Enhancing Transient Protein Expression;293
5.27.1;1 Introduction;293
5.27.2;2 Materials and Methods;294
5.27.2.1;2.1 Cell Culture;294
5.27.2.2;2.2 Transfection;294
5.27.3;3 Results and Discussion;294
5.27.4;References;296
5.28;Proteomic Characterisation of a Glucose-Limited CHO Perfusion ProcessAnalysis of Metabolic Changes and Increase in Productivity;297
5.28.1;1 Introduction;297
5.28.2;2 Materials and Methods;298
5.28.2.1;2.1 Perfusion Cultivation;298
5.28.2.2;2.2 Proteome Analysis;298
5.28.3;3 Results;299
5.28.3.1;3.1 Glucose-Limited Perfusion Cultivation of CHO-MUC1-IgG2a;299
5.28.3.2;3.2 Proteome Analysis;299
5.28.4;Reference;301
5.29;Evaluation of Alternative Signal Sequences;302
5.29.1;1 Methods;302
5.29.2;2 Results and Discussion;303
5.29.3;3 Summary and the Future;304
5.29.4;References;305
5.30;Process Development for the GMP Productionof N-Acetylgalactosamine-6-Sulfate Sulfatase (GALNS) Expressed by CHO Cells;306
5.30.1;1 Introduction;306
5.30.2;2 Results and Discussion;308
5.30.3;3 Conclusion;310
5.30.4;References;310
5.31;Improvement of a CHO Fed-Batch Process by Fortifying with Plant Peptones;311
5.31.1;1 Materials and Methods;311
5.31.2;2 Results;312
5.31.2.1;2.1 Experiment 1: Neutralization of the Toxic Effect from Amino Acid Over-Feeding by Peptones Addition;312
5.31.2.2;2.2 Experiment 2: Dose Study in 50 ml Filtered Tubes;312
5.31.3;3 Conclusion;314
5.32;O-Glycans on Recombinant MUC1 Produced in CHO K1 Cells Become Less Sialylated with Increased Protein Productivity, as Determined by LC-ESI MS;315
5.32.1;1 Introduction;316
5.32.2;2 Materials and Methods;316
5.32.3;3 Results and Discussion;317
5.32.4;4 Conclusions;318
5.32.5;References;318
5.33;A Multiple Minibioreactor Platform for Parallel and Automated Mammalian Cell Culture;319
5.33.1;1 Introduction;319
5.33.2;2 Materials and Methods;320
5.33.2.1;2.1 Culture System;320
5.33.2.2;2.2 Analytical Systems;320
5.33.2.3;2.3 System Operation;320
5.33.3;3 Results and Discussion;320
5.33.3.1;3.1 Hybridoma Cells Growth at Various 0 FCS;320
5.33.3.2;3.2 Toxicicy of Neomycin on Vero Cells;322
5.33.4;4 Conclusions;323
5.34;Cultivation of Adherent-Dependent Animal Cells on Microcarriers in a New Disposable Reactor;324
5.34.1;1 Introduction;324
5.34.2;2 Material and Methods;325
5.34.3;3 Results;326
5.34.4;4 Conclusion and Perspectives;328
5.35;CHO Cells Cultivation and Antibodies Production in a New Disposable Bioreactor Based on Magnetic Driven Centrifugal Pump;329
5.35.1;1 Introduction;329
5.35.2;2 Material and Methods;330
5.35.3;3 Results;330
5.35.4;4 Conclusion and Perspectives;332
5.36;Stability and Productivity of CHO Pools with Respect to Culture Age, Cryopreservation and 20 L Bioreactor Cultivation;334
5.36.1;1 Introduction;334
5.36.2;2 Material and Methods;335
5.36.3;3 Results and Discussion;336
5.36.4;4 Conclusion and Outlook;338
5.36.5;References;338
5.37;In Vitro Disassembly and Reassembly of Triple-Layered Rotavirus-Like Particles;339
5.37.1;1 Introduction;339
5.37.2;2 Materials and Methods;340
5.37.2.1;2.1 Production of RLPs;340
5.37.2.2;2.2 Kinetics of TLP Disassembly;340
5.37.2.3;2.3 Kinetics of TLP Reassembly;341
5.37.3;3 Results and Discussion;341
5.37.3.1;3.1 Kinetics of TLP Disassembly;341
5.37.3.2;3.2 Kinetics of TLP Reassembly;342
5.37.4;4 Conclusion;343
5.37.5;References;344
5.38;Influence of Culture Conditions on Insect Cell Growthand Protein Production -- Comparison of Wave Bioreactorand Shake Flasks;345
5.38.1;1 Materials and Methods;345
5.38.2;2 Results and Discussion;346
5.38.2.1;2.1 Insect Cell Growth in Shake Flasks;346
5.38.2.2;2.2 Insect Cell Growth in Wave Bioreactor;347
5.38.2.3;2.3 Protein Production in Shake Flasks;348
5.38.2.4;2.4 Protein Production in Wave Bioreactor;349
5.38.3;3 Conclusion;350
5.39;Process Intensification Based on Nano-Structured Carbon Carrier Materials and Disposable Devices;351
5.39.1;1 Introduction;352
5.39.2;2 Materials and Methods;352
5.39.3;3 Results;353
5.39.3.1;3.1 Product Yield and Medium Consumption;353
5.39.3.2;3.2 Cell Numbers and pH Value;353
5.39.4;4 Conclusions;355
5.39.5;5 Outlook;356
5.39.6;References;356
5.40;Accelerating Fed-Batch Process Development Using Rationally Designed Feed Media;357
5.40.1;1 Methods;357
5.40.1.1;1.1 Project Phases;357
5.40.1.2;1.2 Cell Culture;358
5.40.1.3;1.3 Spent Media Analysis;358
5.40.2;2 Feed Medium Development;358
5.40.3;3 Development of Hydrolysate and Integration with Feed for Optimal Performance;358
5.40.4;4 Process Development of Feed Volume and Timing;360
5.40.5;5 Conclusions;361
5.40.6;6 Summary;361
5.40.7;References;362
5.41;Development of a Robust Small-Scale Production Format that Is Predictive of Bioreactor Performance;363
5.41.1;1 Introduction;363
5.41.2;2 Methods;364
5.41.3;3 Results;365
5.41.4;4 Conclusion;368
5.41.5;References;368
5.42;Biomass Monitoring and CHO Cell Culture Optimization Using Capacitance Spectroscopy;369
5.42.1;1 Introduction;369
5.42.2;2 Aims of the Work;371
5.42.3;3 Methodology;371
5.42.4;4 Results;372
5.42.5;References;374
5.43;Characterizing Physiology and Metabolism of High-Density CHO Cell Perfusion Cultures Using 2D-NMR Spectroscopy;375
5.43.1;1 Introduction;376
5.43.2;2 Materials and Methods;376
5.43.2.1;2.1 Cell Line Culture Medium and Bioreactor Operation;376
5.43.2.2;2.2 Analytical Methods;377
5.43.2.3;2.3 Sample Preparation for NMR Analysis;377
5.43.2.4;2.4 2D-NMR Analysis;377
5.43.2.5;2.5 Biochemical Network and Metabolic Flux Analysis;378
5.43.3;3 Results;378
5.43.3.1;3.1 Cell Density and Viability;378
5.43.3.2;3.2 Glucose, Glutamine, Lactate, and Ammonium;379
5.43.3.3;3.3 Metabolic Fluxes;381
5.43.4;4 Discussion;382
5.43.4.1;4.1 Pentose Phosphate Pathway;382
5.43.4.2;4.2 Pyruvate Carboxylase Flux;382
5.43.4.3;4.3 Implications for Bioprocess Development;382
5.43.5;5 Conclusions;382
5.43.6;References;383
5.44;BI HEX Platform for Fast Track Generation of High Producer Cell Lines Leading to High-Titer Processes for Production of Therapeutic Proteins from Mammalian Cells;384
5.44.1;1 Introduction;385
5.44.2;2 Molecular Biology;385
5.44.3;3 Cell Biology;385
5.44.4;4 Cell Culture Technology;387
5.44.5;5 Conclusion;387
5.44.6;References;388
5.45;Management of Handling, Long-Term Stability and Shipping of Human T-Lymphocytes Bags for Clinical Studies;389
5.45.1;1 Introduction;389
5.45.2;2 Methods;390
5.45.3;3 Results and Discussion;391
5.45.3.1;3.1 Biological Activity After Long Term Storage;391
5.45.3.2;3.2 Long Distance Shipping;391
5.45.4;4 Conclusions;392
5.45.5;References;393
5.46;Mass Transfer in the CELL-tainer Disposable Bioreactor;394
5.46.1;1 Introduction;394
5.46.2;2 Results;394
5.46.3;3 Conclusion;396
5.47;Efficient Production of Human Monoclonal Antibodies by Combining In Vitro Immunization and Phage Display Methods;397
5.47.1;1 Introduction;397
5.47.2;2 Methods;398
5.47.2.1;2.1 In Vitro Immunization;398
5.47.2.2;2.2 Construction of Phage Antibody Library;398
5.47.2.3;2.3 Detection and Production of ME-Specific Antibody;398
5.47.3;3 Results and Discussion;399
5.47.4;References;399
5.48;Measurement and Control of Viable Cell Density in a Mammalian Cell Bioprocessing Facility: Validation of the Software;400
5.48.1;1 Materials and methods;401
5.48.2;2 Results;402
5.48.3;3 Discussion;402
5.48.4;4 Conclusions;404
5.48.5;References;405
5.49;Development, Validation, and Application of a Fully Integrated Automation System for Screening and Selection of High Yielding Production Cell Lines;406
5.49.1;1 System Overview;406
5.49.2;2 Preparation Stage: Automated Liquid Handling;407
5.49.3;3 Stage 1: High Throughput ELISA Screening;407
5.49.4;4 Stage 2: Digital Imaging to Visualize Cells Secreting Antibody;407
5.49.5;5 Stage 3: Transfer and Expansion of High Producing Cell Lines;408
5.49.6;6 V-Prep Validation;408
5.49.7;7 Tecan Spectrafluor Reader Validation;408
5.49.8;8 Tecan Genesis and Nikon Microscope Validation;408
5.49.9;9 Validation of the ELISA Function;409
5.49.10;10 Comparison of ELISA Data Generated by Automation System and Standard Method;409
5.49.11;11 Comparison of Cell Lines Selected by Automation and Standard Methods;410
5.49.12;12 Summary;410
5.50;Monitoring of Cell Activity: Online Oxygen Uptake Rates in Pulsed Aerated Cell Culture;411
5.50.1;1 Introduction;411
5.50.2;2 Material and Methods;412
5.50.3;3 Results and Discussion;412
5.50.4;4 Conclusions;412
5.50.5;References;413
6;Part IV Systems Biotechnology;414
6.1;Metabolite Analysis in Mammalian Cells: How to Generate Reliable Data Sets?;415
6.1.1;1 Introduction;416
6.1.2;2 Materials and Methods;416
6.1.3;3 Results and Discussion;418
6.1.3.1;3.1 Extraction Screening;418
6.1.3.2;3.2 Optimization of the MeOH/CHCl 3 Extraction Method;419
6.1.3.3;3.3 Determination of Recovery;423
6.1.3.4;3.4 Starvation Experiments;424
6.1.4;4 Conclusions;424
6.1.5;References;424
6.2;Cell-to-Cell Communication for Cell Density-Controlled Bioprocesses;425
6.2.1;1 Cell-to-Cell Communication Systems Cell Phones;426
6.2.2;2 Multi-Step Multi-Species Communication Systems;427
6.2.3;3 Conclusion;429
6.2.4;References;429
6.3;Metabolome Analysis in Mammalian Cells: Development and Application of a Sampling Technique;431
6.3.1;1 Introduction;432
6.3.2;2 Materials and Methods;433
6.3.2.1;2.1 Cell Line and Cultivation;433
6.3.2.2;2.2 Sampling;433
6.3.2.3;2.3 Cell Disruption and Cell Counting;433
6.3.2.4;2.4 LCMS and LCMS-MS Analytics;434
6.3.3;3 Results and Discussion;434
6.3.4;References;438
6.4;Differential Expression Profiling of Industrially Relevant CHO Cell Phenotypes Using a Proprietary CHO-Specific Microarray and Proteomics Technology Platforms;439
6.4.1;1 Introduction;439
6.4.2;2 Collaboration Overview;440
6.4.3;3 Data Analysis;440
6.4.4;4 Target Validation;441
6.5;Towards a Systems-Level Understanding of Increased Specific Productivity in Proliferation Arrested Myeloma NS0 Cells;442
6.5.1;1 Introduction;442
6.5.2;2 Results and Discussion;443
6.5.3;3 Conclusion;445
6.5.4;References;445
6.6;Proteomic Analysis of Influenza a Virus Infected Mammalian Cells by 2D-DIGE;446
6.6.1;1 Introduction;446
6.6.2;2 Materials and Methods;447
6.6.3;3 Results and Discussion;447
6.6.4;References;449
6.7;Involvement of SRC- and MAP Kinase-Signalings in the Effect of Sericin on Cellular Proliferation and Survival;450
6.7.1;1 Introduction;450
6.7.2;2 Materials and Methods;451
6.7.2.1;2.1 Cell Line and Culture Conditions;451
6.7.2.2;2.2 Two-Dimensional Electrophoresis;451
6.7.3;3 Results;451
6.7.3.1;3.1 The Effect of Sericin on Proliferation of Hybridoma Cell Line 2E3-O;451
6.7.3.2;3.2 Proteome Analysis Using 2-DE;452
6.8;The Unfolded Protein Response and Recombinant Protein Production from In Vitro Cultured Mammalian Cells;453
6.8.1;1 Introduction;453
6.8.2;2 Materials and Methods;454
6.8.2.1;2.1 Reagents;454
6.8.2.2;2.2 Cell Lines;454
6.8.2.3;2.3 Growth Profiles of CHO and NS0 Upon UPR Induction;454
6.8.3;3 Results;454
6.8.4;4 Conclusions;456
6.8.5;References;456
6.9;A Scalable Process for Helper-Dependent Adenoviral Vector Production Using PEI-Derived Transfection Strategy in Suspension Culture;457
6.9.1;1 Introduction;457
6.9.2;2 Materials and Methods;458
6.9.2.1;2.1 Cells and Viruses;458
6.9.2.2;2.2 PEI-Adenofection;458
6.9.2.3;2.3 Amplification Step Via Co-Infection;458
6.9.3;3 Results and Discussion;459
6.9.4;4 Conclusion;461
6.9.5;References;461
6.10;Functional Analysis of ER Stress Pathway Genes for Apoptosis of NS/0 Cell Line Using RNAi Methods;462
6.10.1;1 Introduction;462
6.10.2;2 Methods;463
6.10.3;3 Results;463
6.10.4;4 Conclusions;465
6.10.5;References;466
6.11;Identification of New Protein Substrate Candidates of Transglutaminase in Rat Liver Extracts: Use of 5-(Biotinamido) Pentylamine as a Probe;468
6.11.1;1 Introduction;468
6.11.2;2 Materials and Methods;469
6.11.2.1;2.1 Preparation of Rat Liver Extract;469
6.11.2.2;2.2 In Vitro TG-Mediated Biotin Labeling;469
6.11.2.3;2.3 Isolation of Biotinylated Proteins with Avidin-Affinity Column;469
6.11.2.4;2.4 Amino Acid Sequencing Analyses;470
6.11.2.5;2.5 Identification of TG Substrates by Immunoblotting;471
6.11.3;3 Results and Discussion;471
6.11.4;References;473
6.12;Metabolic Flux Distributions of Adherently Growing MDCK Cells in Different Media;474
6.12.1;1 Introduction;474
6.12.2;2 Phase Model;475
6.12.3;3 Metabolic Network Model;476
6.12.4;4 Results and Conclusions;476
6.12.5;References;477
6.13;Expression of Dermal Extracellular Matrix Proteins in a Newly Developed Full-Thickness Skin Model;478
6.13.1;1 Introduction;478
6.13.2;2 Materials and Methods;479
6.13.2.1;2.1 Production of the Skin Model;479
6.13.2.2;2.2 Immunohistochemistry;479
6.13.2.3;2.3 Gene Expression Analysis;479
6.13.3;3 Results;479
6.13.4;4 Summary and Conclusions;481
6.13.5;References;482
6.14;Development of Preparations with Antiviral and Immunostimutory Effects from Extracts of Green Parts of Coniferous Plants;483
6.14.1;1 Introduction;484
6.14.2;2 Materials and Methods;484
6.14.2.1;2.1 Cell Culture--MDCK from Cell Culture Collection of SRC VB VECTOR;484
6.14.3;3 Results;487
6.15;Serum-Free Transfection of CHO Cells with Chemically Defined Transfection Systems to Generate Transient and Stable Cell Lines;488
6.15.1;1 Introduction;488
6.15.2;2 Overview;489
6.15.3;3 Preparation and Transfection Studies;489
6.15.4;4 Conclusion;489
6.15.5;References;491
6.16;Glycomics: Development and Characterization of Glycan-Based Biotechnological Products;492
6.17;Development of a Cell-Culture-Based Platform for Viral Vaccine Production;494
6.17.1;1 Introduction;495
6.17.2;2 Materials and Methods;495
6.17.2.1;2.1 Cell Lines;495
6.17.2.2;2.2 Virus Production;495
6.17.3;3 Results;495
6.17.4;4 Conclusions;497
6.17.5;References;497
6.18;Screening of Natural Compounds Affecting Type I Interferon Signalling;498
6.18.1;1 Introduction;498
6.18.2;2 Results;499
6.18.3;3 Conclusions;500
6.18.4;Reference;500
7;Part V Competing and Complementary Approaches to Animal Cell Technologies;501
7.1;Therapeutic Proteins from Transgenic Cows Milk;502
7.1.1;1 Introduction;502
7.1.2;2 Methodology;503
7.1.2.1;2.1 Obtention of the Founder Animal;503
7.1.2.2;2.2 Expansion of the Transgenic Herd;504
7.1.2.3;2.3 Lactation and Bioactive Protein Levels;504
7.1.3;3 Results;505
7.1.4;4 Bovines Transgenic for Human Insulin;506
7.1.5;5 Conclusion;506
7.1.6;References;506
7.2;Development of Edible Plant Vaccines;508
7.2.1;References;511
7.3;Human Cell Lines for Production of Biopharmaceuticals;513
7.3.1;1 Introduction;514
7.3.2;2 Materials and Methods;515
7.3.3;3 Results and Discussion;516
7.3.4;4 Expression of Reference Proteins in Adherent CAP Cells;517
7.3.5;References;520
7.4;Concentration and Purification of Densonucleosis Virus by Tangential Membrane Filtration and by Ion Exchange Membranes;522
7.4.1;1 Introduction;522
7.4.2;2 Materials and Methods;523
7.4.2.1;2.1 Production of AeDNV Particles;523
7.4.2.2;2.2 Virus Titer Determination;523
7.4.2.3;2.3 Tangential Flow Filtration;523
7.4.2.4;2.4 Ion Exchange Experiments;524
7.4.3;3 Results and Conclusion;524
7.4.4;References;525
7.5;Effect of ManNAc and its Derivatives on Glycosylation to Proteins Produced by Mammalian Cell Culture;526
7.5.1;1 Introduction;526
7.5.2;2 Materials and Methods;527
7.5.2.1;2.1 Cell Line and Culture Condition;527
7.5.2.2;2.2 Two-Dimensional Electrophoresis;527
7.5.3;3 Results and Discussion;527
7.5.4;4 Conclusion;528
7.6;Purification of a Chimeric Simian Human Immunodeficiency Virus-Like Nanoparticle from HEK293 Cell Culture;529
7.6.1;1 Introduction;530
7.6.2;2 Experimental;531
7.6.2.1;2.1 Expression of the Chimeric VLP;531
7.6.2.2;2.2 VLP Purification from Culture Media;531
7.6.2.3;2.3 SDS-PAGE and Immunoblot Analysis;531
7.6.2.4;2.4 Determination of Protein Concentration;532
7.6.3;3 Results;532
7.6.4;4 Conclusion;534
7.6.5;References;534
7.7;Effects of Plant Peptones Supplemented Medium on CHO Cells;536
7.7.1;1 Introduction;536
7.7.2;2 Materials and Methods;537
7.7.2.1;2.1 Cell Culture;537
7.7.2.2;2.2 Peptones;537
7.7.2.3;2.3 Apoptosis Assays;537
7.7.3;3 Results;538
7.7.4;4 Conclusions;539
7.7.5;References;539
7.8;Synthetic Low Density Lipoprotein a Novel Biomimetic Lipid Supplement for Serum Free Tissue Culture;540
7.8.1;1 Introduction;540
7.8.2;2 Synthetic Low Density Lipoprotein;541
7.8.3;3 Cellular Uptake;541
7.8.4;4 Cellular Proliferation;543
7.8.5;5 Conclusion;543
7.8.6;References;544
8;Part VI Solutions and Applications;545
8.1;Scale-Down Approach for Animal-Free Polio Vaccine Production;546
8.1.1;1 Introduction;546
8.1.2;2 Materials and Methods;547
8.1.2.1;2.1 Vero Cells and Polio Virus;547
8.1.2.2;2.2 Polio Production Process;547
8.1.2.3;2.3 The Data-Set and its Organisation;548
8.1.2.4;2.4 Multivariate Data Analysis (MVA);548
8.1.2.5;2.5 Scale-Down Experiments and Animal-Component Free Media;549
8.1.3;3 Results and Discussion;549
8.1.3.1;3.1 Process Analysis by PCA;549
8.1.3.2;3.2 Performance Analysis of Cell Culture at 350-L Scale;550
8.1.3.3;3.3 Scale-Down and Animal-Component Free Culture;551
8.1.3.4;3.4 Performance Analysis of Virus Culture and Scale-Down;553
8.1.4;4 Conclusions;554
8.1.5;References;554
8.2;Novel Scaffolds for Tissue Engineering Nano-Structured Surfaces Promote Selective Cell Attachment and Cell Differentiation;556
8.2.1;1 Introduction;556
8.2.2;2 Materials and Methods;557
8.2.3;3 Results and Discussion;558
8.2.3.1;3.1 Bone Cell Differentiation;558
8.2.3.2;3.2 Cell Selective Surfaces;560
8.2.4;4 Summary and Conclusions;562
8.2.5;References;562
8.3;Unconventional Experimental Concepts Enabling High Speed High Performance Media/Process Development;563
8.3.1;1 Introduction;563
8.3.2;2 Material and Methods;564
8.3.3;3 Results and Discussions;564
8.3.3.1;3.1 Concept 1;564
8.3.3.2;3.2 Concept 2;566
8.3.4;Conclusions;569
8.3.5;References;569
8.4;A Study on Bioscaffolds of Polysialic Acid and -Glucan for Cell Culture and Tissue Engineering Applications;570
8.4.1;1 Introduction;570
8.4.2;2 Materials and Methods;571
8.4.3;3 Results and Discussion;572
8.4.4;4 Conclusions;572
8.4.5;References;575
8.5;A Preliminary Study on Spider Silk as Biomaterial for Peripheral Nerve Regeneration;576
8.5.1;1 Introduction;576
8.5.2;2 Materials and Methods;577
8.5.2.1;2.1 Materials;577
8.5.2.2;2.2 PC-12;577
8.5.2.3;2.3 Immortalized Schwann cells (ISC);577
8.5.2.4;2.4 Methods;578
8.5.3;3 Results and Discussion;578
8.5.4;4 Conclusion and Outlook;580
8.5.5;References;581
8.6;Investigation of the Effect of Mechanical Strain on the Osteogenic Differentiation of Mesenchymal Stem Cells;582
8.6.1;1 Introduction;582
8.6.2;2 Materials and Methods;583
8.6.2.1;2.1 AdMSC Isolation and Cultivation;583
8.6.2.2;2.2 Scaffold Materials;583
8.6.2.3;2.3 Strain Experiments;584
8.6.2.4;2.4 Alkaline Phosphatase Activity Test;584
8.6.2.5;2.5 MTT Assay;585
8.6.2.6;2.6 RT-PCR;585
8.6.2.7;2.7 Fabrication of Flexible Microelectrode Dishes;585
8.6.2.8;2.8 Electric Cell-Substrate Impedance Sensing (ECIS);586
8.6.3;3 Results and Conclusions;587
8.6.4;4 Summary;592
8.6.5;References;592
8.7;Biofunctional Polymer-Mineral Composites as Scaffolds for Bone Tissue Engineering;593
8.7.1;1 Introduction;594
8.7.2;2 Materials and Methods;594
8.7.2.1;2.1 Preparations of Composites;594
8.7.2.2;2.2 Cell Culture;595
8.7.3;3 Results;595
8.7.3.1;3.1 Preparation of Conjugates;595
8.7.3.2;3.2 Cell Culture;597
8.7.4;4 Conclusions and Outlook;597
8.7.5;References;599
8.8;New Water-Soluble Polymers for Construction of Biofunctionalized Scaffolds for Bone Tissue Engineering: Synthesis and Adsorption Study;600
8.8.1;1 Introduction;601
8.8.2;2 Results and Discussion;601
8.8.2.1;2.1 Polymer Synthesis;601
8.8.2.2;2.2 Adsorption;602
8.8.3;3 Conclusions;606
8.8.4;References;606
8.9;Selection of High-Producing Cells Via Cell Sorting Using an Affinity Matrix;607
8.9.1;1 Introduction;607
8.9.2;2 Materials and Methods;608
8.9.2.1;2.1 Affinity Matrix Staining;608
8.9.2.2;2.2 Gating During the Sorts;608
8.9.3;3 Results;610
8.9.4;4 Conclusion;611
8.9.5;References;611
8.10;The Effects of Medium Supplement on High-Level Production of Recombinant Human Factor IX in CHO Cell;612
8.10.1;1 Introduction;612
8.10.2;2 Materials and Methods;613
8.10.3;3 Result and Discussion;614
8.10.4;References;617
8.11;Case Study Large Scale Cell Culture Facility;618
8.11.1;1 A Biotech Landmark in the Heart of Europe;618
8.11.2;2 Conclusion;619
8.12;Glycosylation of Influenza A Virus Hemagglutinin;620
8.12.1;1 Introduction;620
8.12.2;2 Materials and Methods;621
8.12.3;3 Results and Discussion;621
8.12.4;References;622
8.13;Production of Retroviral Pseudotype Vectors in Fixed Bed Reactors for Use in Gene Therapy;623
8.13.1;1 Introduction;623
8.13.2;2 Materials and Methods;624
8.13.3;3 Results;625
8.13.4;4 Conclusion;625
8.13.5;References;626
8.14;Swellscreen -- Rapid Baculovirus Titration Methodin Microplate Format;627
8.14.1;1 Introduction;627
8.14.2;2 Experimental Set-Up;628
8.14.3;3 Results;628
8.14.3.1;3.1 Scale-Down;628
8.14.3.2;3.2 Validation with BacPAK;629
8.14.4;4 Discussion;629
8.14.5;5 Conclusion;630
8.14.6;References ;631
8.15;Comparing Vero and MDCK Cells for Influenza A Virus Production in Microcarrier Systems;632
8.15.1;1 Introduction;632
8.15.2;2 Materials and Methods;633
8.15.3;3 Results and Discussion;633
8.15.4;References;635
8.16;Expansion of Human Articular Chondrocytes on Microcarriers Enhances the Production of Cartilage Specific Matrix Components;636
8.16.1;1 Introduction;637
8.16.2;2 Methods;637
8.16.3;3 Results;638
8.16.4;4 Conclusions;640
8.16.5;References;640
8.17;Automated Cell Culture Handling Characteristics and Procedures;641
8.18;Bioreactor Satellite Culture Experiments in the Start-Up of a Cell Culture Technical Support Lab;643
8.18.1;1 Set-Up of the Technical Support Lab;644
8.18.2;2 Satellite Culture Principles and CCTS Lab Conditions;644
8.18.3;3 Bioreactor Comparison (Small Scale Vs. Large Scale);645
8.18.4;4 Culture Performance: Growth and MAb Production;645
8.18.5;5 Culture Performance: Metabolites;648
8.18.6;6 Conclusion and Perspectives;650
8.19;Advantages of Hydrodynamic Cell Separation in Industrial Cell Culture Processes;652
8.19.1;1 Introduction;652
8.19.2;2 Materials and Methods;654
8.19.3;3 Results;654
8.19.4;4 Conclusion;658
8.19.5;References;658
8.20;Optimization and Characterization of the Process for Large Volume Cell Banking in Bags;660
8.20.1;1 Introduction;660
8.20.2;2 Materials and Methods;661
8.20.3;3 Results and Discussion;661
8.20.4;References;663
8.21;Development of Screening Method for IgA-Promoting Factors Derived from Food Extracts Using a Human Myeloma Cell Line;664
8.21.1;1 Introduction;664
8.21.2;2 Materials and Methods;665
8.21.3;3 Results and Discussion;666
8.21.3.1;3.1 Characteristics of CEC-1LA Cell Line;666
8.21.4;References;669
8.22;Cryopreservative Solution Using Sericin;670
8.22.1;1 Introduction;670
8.22.2;2 Materials and Methods;670
8.22.3;3 Results;670
8.22.3.1;3.1 Influence of Autoclave on Sericin Solution;670
8.22.3.2;3.2 Influence of Autoclave on DMSO Solution;671
8.22.4;Reference;673
8.23;Fructan as a Novel Effective Factor for Mammalian Cell Culture;674
8.23.1;1 Introduction;674
8.23.2;2 Materials and Methods;675
8.23.3;3 Results;675
8.23.4;References;677
8.24;Online Determination of Oxygen Uptake and Carbon Dioxide Production Rates in Mammalian Cell Culture Using Mass Spectrometry;678
8.24.1;1 Introduction;679
8.24.2;2 Materials and Methods;679
8.24.3;3 Results and Discussion;680
8.24.4;4 Conclusion;681
8.24.5;References;682
8.25;Optimisation of mAb Concentration in Microcarrier Based Perfusion Compared to Batch/Fed-Batch Cultivation;683
8.25.1;1 Cell Cultivation and Reactor Setups;683
8.25.2;2 Results;684
8.25.3;3 Conclusion;686
8.26;Characterization of the Novel Human AGE1hn Cell Line for Production of Recombinant Proteins;687
8.26.1;1 Materials and Methods;687
8.26.2;2 Results;688
8.26.3;3 Conclusions;690
8.27;Rapid Selection of Optimal Formulations for Divergent Clones Through Screening Chinese Hamster Ovary Media Library;692
8.27.1;1 Introduction;692
8.27.2;2 Materials and Methods;693
8.27.2.1;2.1 Cell Lines and Media;693
8.27.2.2;2.2 Productivity Assay;694
8.27.3;3 Results;694
8.27.4;4 Conclusions;695
8.28;Development of a Vaccine Candidate Against Heartwater;696
8.28.1;1 Introduction;697
8.28.2;2 Materials and Methods;697
8.28.3;3 Results and Discussion;698
8.28.3.1;3.1 Mass Production of Endothelial Cells;698
8.28.3.2;3.2 Ehrlichia ruminantium Production Under Stirring Conditions;698
8.28.3.2.1;3.2.1 E. ruminantium Life Cycle;698
8.28.3.2.2;3.2.2 Production of E. ruminantium ;699
8.28.3.3;3.3 E. ruminantium Purification;700
8.28.3.4;3.4 Ehrlichia ruminantium Storage and Vaccine Efficacy;701
8.28.3.5;References;702
8.29;Animal Component Free T-Cell Culture;704
8.29.1;1 Introduction;704
8.29.2;2 Materials and Methods;705
8.29.3;3 Results;705
8.29.4;4 Discussion;709
8.29.5;References;709
8.30;Characterization of Cholesterol-Independent GS-NS/0 Recombinant Antibody Cell Lines;710
8.30.1;1 Introduction;710
8.30.2;2 Methods;711
8.30.3;3 Results;711
8.30.3.1;3.1 Cell Growth;711
8.30.3.2;3.2 Titers and qP;711
8.30.3.3;3.3 Product Quality;713
8.30.4;4 Conclusions;714
8.30.5;References;714
8.31;Development of a Fed-Batch Process for the Production of a Recombinant Protein X in CHO-GS System Case Study from the Cell to Reactor Process Ready for Pilot Scale Cultivation;715
8.31.1;1 Results;715
8.31.2;2 Discussion and Conclusion;716
8.32;Scaffolds for Articular Joint Tissue Engineering;718
8.32.1;1 Introduction;718
8.32.2;2 Materials and Methods;719
8.32.3;3 Results and Discussion;720
8.32.3.1;3.1 Scaffold Selection and Characterization;720
8.32.3.2;3.2 Colonization;721
8.32.3.3;3.3 Cell Proliferation and Material Degradation;722
8.32.3.4;3.4 Genetic Profile;723
8.32.4;4 Conclusion;723
8.32.5;References;724
8.33;Effect of Tunisian Aromatic Plant Extracts on Melanogenesis;725
8.33.1;1 Materials and Methods;726
8.33.1.1;1.1 Measurement of Melanin Content and Cell Viability;726
8.33.1.2;1.2 Western Blotting;726
8.33.2;2 Results and Discussion;726
8.33.2.1;2.1 C. spinosa and E. multiflora Extract have Stimulative Effect on Melanogenesis;726
8.33.2.2;2.2 T. hirsuta Extract have Inhibitive Effect on Melanogenesis;727
8.33.3;References;728
8.34;Effects of Capsaicin on Energy Metabolism by Human Intestinal Epithelial Cell Line Caco-2;729
8.34.1;1 Introduction;729
8.34.2;2 Results and Discussion;730
8.34.3;References;732
8.35;Human A1AT Production Propagating a Newly Developed Human Cell Line in a Novel Disposable Perfusion Bioreactor;733
8.35.1;1 Introduction;733
8.35.2;2 The Three Columns of the Process: Protein, Cell Line, Bioreactor;734
8.35.3;3 Process Data of a 31-Day Production Process of A1AT with AGE1.hn in the G-System;735
8.35.4;4 Summary and Conclusions;737
8.36;Characterization of Diffusion and Flow Relations in the Novel Membrane Based Perfusion Bioreactor;738
8.36.1;1 Introduction;738
8.36.2;2 Design and Working Principle of the Membrane Based Bioreactor;739
8.36.3;3 Permeability of the Membrane;740
8.36.4;4 Mixing of the Perfusion Flow into the Medium of the Membrane Surrounding Supply Space;741
8.36.5;5 Summary and Conclusions;742
8.37;Protective Effect of Di-O-Caffeoylquinic Acid on Human-Derived Neurotypic SH-SY5Y Cells Against Alzheimer's Disease Amyloid-Beta-Induced Toxicity;743
8.38;Flow Through Ceramic Foams A Future Cell Culture Challenge;746
8.38.1;1 Introduction;746
8.38.2;2 Experimental Procedures;747
8.38.3;3 Results;749
8.38.4;4 Conclusion;751
8.38.5;References;751
8.39;Measles Virus Production in MRC-5 Cells Grown on Microcarriers in a Stirred Bioreactor;752
8.39.1;1 Material and Methods;753
8.39.2;2 Results;753
8.39.2.1;2.1 Measles Virus Production in Spinner Flask Cultures;753
8.39.2.2;2.2 Bioreactor Cultures;754
8.39.2.2.1;2.2.1 BelloCell500 Culture;754
8.39.2.2.2;2.2.2 2-L Stirred Bioreactor Culture;755
8.39.3;References;755
8.40;A New System for the Enrichment of Cell Subclones Secreting High Levels of IgG Using Magnetic Cell Sorting (MACS Technology);757
8.40.1;1 Introduction;757
8.40.2;2 Material and Methods;758
8.40.2.1;2.1 Separation of Target Cells from Heterogeneous Populations Using MACS ® Technology;758
8.40.2.2;2.2 Subcloning;758
8.40.2.3;2.3 Specific Production Rate (SPR) Determination of Subclones;759
8.40.3;3 Results;759
8.40.3.1;3.1 Analysis of the Effect of Upstream Antibody-Producing Hybridoma Cell Enrichment on the SPR Diversification Pattern Within Cell Populations;759
8.40.3.2;3.2 Analysis of the Effect of Sequential Separation Steps on the SPRs of Different Hybridoma Populations;760
8.40.4;4 Conclusions;761
8.41;Soy Hydrolysate Optimization for Cell Culture Applications;762
8.41.1;1 Introduction;763
8.41.2;2 Materials and Methods;763
8.41.2.1;2.1 Hydrolysate Manufacturing;763
8.41.2.2;2.2 Cell Culture;763
8.41.2.3;2.3 Chemical Composition;765
8.41.3;3 Results and Discussion;765
8.41.3.1;3.1 Cell Culture;765
8.41.4;4 Conclusions;768
8.42;The Effect of Bioreactor pH and Temperature on Protein Glycosylation in Perfusion Cultures of Mammalian Cells;769
8.42.1;1 Introduction;769
8.42.2;2 Materials and Methods;770
8.42.3;3 Results and Discussion;770
8.42.4;Reference;772
8.43;Evaluation of Cell Growth Characteristics on Chitosan-Alginate Membranes to Assess Their Potential Application on Highly Exuding Skin Lesions and In Vivo Evaluation in Wounded Cat;773
8.43.1;1 Introduction;773
8.43.2;2 Material and Methods;774
8.43.2.1;2.1 Membranes Production;774
8.43.2.2;2.2 In Vitro and In Vivo tests;775
8.43.3;3 Results;775
8.43.4;4 Conclusions;777
8.43.5;References;778
8.44;Vaccine Production Utilizing the Potential of Microcarriers in Disposable Bioreactor;779
8.44.1;1 Introduction;780
8.44.2;2 Materials and Methods;780
8.44.3;3 Results;780
8.44.3.1;3.1 Effect of Pluronic F-68 on Vero Cell Growth in the Wave Bioreactor;780
8.44.3.2;3.2 Reproducibility of Wave Cultures and Comparison with Stirred Tank Bioreactor;782
8.44.3.3;3.3 Scale-Up to the 20 L Wave Bioreactor;783
8.44.3.4;3.4 Animal Component Free;783
8.44.3.5;3.5 Polio Virus Production;784
8.44.4;4 Conclusions;785
8.44.5;References;785
8.45;Characterization of an Anti-Idiotypic Antibody Blocking the Capacity of the HIV-1 Specific nMAb 2F5;786
8.45.1;1 Introduction;786
8.45.2;2 Expression of Ab2/3H6 Variants;786
8.45.3;3 Characterisation of Ab2/3H6 Variants;787
8.45.4;4 Conclusions;788
8.45.5;References;789
8.46;Cultivation of PER.C6 Cells in the Novel CELL-TainerTM High-Performance Disposable Bioreactor;790
8.46.1;1 Introduction;790
8.46.2;2 Materials and Methods;790
8.46.3;3 Results and Discussion;791
8.46.4;4 Conclusions;791
8.47;Scale-Up of a PER.C6 Fed-Batch Process in 50 and 250 L Hyclone Single Use Bioreactors Compared to 50 and 250 L Stainless Steel Bioreactors;792
8.47.1;1 Introduction;792
8.47.2;2 Materials and Methods;792
8.47.3;3 Results and Discussion;793
8.47.4;4 Conclusions;793
8.48;Capacitance Sensor as a Robust Tool for Cell Culture Monitoring in Process Development and Manufacturing;794
8.48.1;1 Introduction;794
8.48.2;2 Theory;795
8.48.3;3 Materials and Methods;796
8.48.4;4 Results;796
8.48.5;5 Conclusions;800
8.48.6;Reference;800
8.49;Effect of Different Cell Culture Medium Surfactants on Cell Growth and Viability;801
8.49.1;1 Introduction;801
8.49.2;2 Material and Methods;802
8.49.2.1;2.1 Rationale for Chosen Surfactant Concentrations;802
8.49.3;3 Results;802
8.49.4;4 Conclusion and Next Step;803
8.49.5;References;804
8.50;Conventional Stirred Bioreactor Control System for Monitoring and Controlling pH and DO in a Wave Bioreactor;805
8.50.1;1 Materials and Methods;805
8.50.1.1;1.1 Cell Cultivation;805
8.50.1.2;1.2 pH and DO Analysis;806
8.50.2;2 Results;807
8.50.2.1;2.1 Measurement in Cell Free Medium;807
8.50.2.2;2.2 Measurement During Cultivation;807
8.50.3;3 Discussion and Conclusion;809
8.51;On-Line Monitoring of Vero Cells Cultures During the Growth and Rabies Virus Process Using Biomass Spectrometer;811
8.51.1;1 Material and Methods;812
8.51.2;2 Results;812
8.51.3;References;814
8.52;Inducing of Human IgE Antibodies by In Vitro Immunization;815
8.52.1;1 Introduction;815
8.52.2;2 Methods;816
8.52.3;3 Results and Discussion;816
8.52.3.1;3.1 Establishing Human In Vitro IgE Antibody-Inducing System;816
8.52.3.2;3.2 Effects of the IgE Antibody-Inducing System on Human Plasma;816
8.52.3.3;3.3 Effects of Food Ingredients on the Induction of IgE Antibodies Against Cedar Pollens;818
8.52.4;References;818
8.53;Oxygen Uptake Rate (OUR) Estimation in High Density Mammalian Cell Perfusion Cultures;819
8.53.1;1 Introduction;819
8.53.2;2 Methods;820
8.53.2.1;2.1 Global Mass Balance;820
8.53.2.2;2.2 Dynamic Method;821
8.53.3;3 Results;821
8.53.4;References;823
8.54;Use of Yeast Derived Nutrients for Cell Culture in Serum-Free Media;824
8.54.1;1 Introduction;824
8.54.2;2 Materials and Methods;825
8.54.3;3 Results;825
8.54.3.1;3.1 YDN Screening in Microplates;825
8.54.3.2;3.2 Growth Kinetics and Metabolism in Spinners;826
8.54.4;4 Conclusion;827
8.54.5;References;827
8.55;A Novel Approach to the Production of Plant-Derived Hydrolysates Yields Medium Supplements with Enhanced Performance in Cell Culture Systems;828
8.55.1;1 Introduction;828
8.55.2;2 A Brief Summary of the Novel Process;829
8.55.3;3 Materials and Methods;829
8.55.3.1;3.1 CHO-K1 Cell Culture;829
8.55.3.2;3.2 Pilot Plant Lots of UltraPep Soy;829
8.55.4;4 Results;830
8.55.5;5 Summary;833
8.56;Monitoring the Cell Size Distribution of Mammalian Cell Cultures Using On-Line Capacitance Measurements;834
8.56.1;1 Introduction;835
8.56.2;2 Material and Methods;835
8.56.3;3 Results;836
8.56.4;4 Conclusions;839
8.56.5;References;840
8.57;Biosimilarity of Recombinant Human EPO Products from CHO Cell Lines: A Carbohydrate Structural View;841
8.57.1;1 Introduction;841
8.57.2;2 Material and Methods;842
8.57.2.1;2.1 EPO Preparations;842
8.57.2.2;2.2 Release of Sialic Acids and N-Linked Oligosaccharides;842
8.57.2.3;2.3 Mass Spectrometric Analysis;842
8.57.2.4;2.4 Purification of Released N-Linked Oligosaccharides by Anion Exchange Chromatography;842
8.57.3;3 Results and Discussion;843
8.57.4;4 Conclusions;845
8.57.5;References;845
8.58;Quantitative N-Glycan Mapping of Glycoprotein Therapeutics by HPAEC-PAD: Glycosylation Characteristics of Different Recombinant Human EPO Products;846
8.58.1;1 Introduction;847
8.58.2;2 Results;847
8.58.3;3 Conclusion;847
8.58.4;References;850