Cells and Culture (eBook)

Proceedings of the 20th ESACT Meeting, Dresden, Germany, June 17-20, 2007

Thomas Noll (Herausgeber)

eBook Download: PDF

2010 | 2010
LIV, 800 Seiten
Springer Netherland (Verlag)
978-90-481-3419-9 (ISBN)

Infos

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.

Inhalt

Contents 5
Contributors 20
Part I Single-Cell Analysis and Engineering 54
Targeted Gene Knockdown Effects on Recombinant Protein Secretion in CHO Cells 55
1 Introduction 55
2 Materials and Methods 56
2.1 Cell Culture Maintenance and siRNA Transfection Assays 56
2.2 Cell XpressTM Analysis 56
3 Results and Discussion 57
3.1 siRNA Design and Validation 57
3.2 Cell XpressTM Analysis of CHO Cells Transfected with IgG siRNAs 57
3.3 Cell XpressTM Analysis of CHO Cells Transfected with Biomarker siRNAs 59
4 Conclusion 60
Reference 60
RNA Silencing Suppressors Boost the Production of Recombinant Proteins and Viruses 61
1 Introduction 61
2 RNA Silencing Suppressors 62
3 RSSs Boost Recombinant Protein Production 63
4 RSSs Boost Virus Production 63
References 63
Towards the Use of CHO Produced Recombinant Extracellular Matrix Proteins as Bioactive Elements in a 3-D Scaffold for Tissue Engineering 65
1 Introduction 66
2 Materials and Methods 66
2.1 Polymer Screening 66
2.2 Generation of Cell Lines Expressing Recombinant ECM Proteins 67
3 Results and Discussion 67
3.1 Polymer Screening 67
3.2 Generation of Cell Lines Expressing Recombinant ECM Proteins 69
References 70
Transient Gene Expression in Chinese Hamster Ovary Cells at Low Temperature The Effects of Cold-Induced Proteins and an mRNA Regulatory Element 71
1 Introduction 71
2 Materials and Methods 72
3 Results and Discussion 72
References 74
Single-Cell Approach in Influenza Vaccine Production: Apoptosis and Virus Protein Production 76
1 Introduction 76
2 Materials and Methods 77
3 Results and Discussion 77
References 79
Chondrogenic Differentiation of Human Mesenchymal Stem Cells During Multiple Subcultivation 80
1 Introduction 81
2 Materials and Methods 82
2.1 Cultivation and Passaging 82
2.2 Differentiation 82
2.3 MIA-ELISA 82
3 Results 83
4 Conclusion 84
References 84
Cell Xpress-Assisted Analysis of Clone Stability in Recombinant Chinese Hamster Ovary Cells 86
1 Introduction 86
2 Materials and Methods 87
2.1 Cell XpressTM Clone Selection and Analysis 87
2.2 Cell Culture Maintenance and Growth and Productivity Characterization 87
2.3 Molecular Characterization of Cell XpressTM Clones 87
3 Results and Discussion 88
3.1 Cell Xpress Clone Generation and Stability Characterization 88
3.2 Molecular Characterization of Stable and Unstable CHO Clonal Lines 89
An Evaluation of the Intrinsic IgG Production Capabilities of Different Chinese Hamster Ovary Parental Cell Lines 91
1 Introduction 91
2 Results and Discussion 92
2.1 Analysis of Transient GFP Production in Different Parental CHO Cell Lines 92
2.2 Analysis of Transient IgG Production and Secretion in Different Parental CHO Cell Lines 92
3 Conclusions 94
References 94
Cell Xpress Technology Facilitates High-Producing Chinese Hamster Ovary Cell Line Generation Using Glutamine Synthetase Gene Expression System 95
1 Introduction 95
2 Results and Discussion 96
2.1 Secretion Intensity and Population Heterogeneity Analysis Using Cell XpressTM 96
2.2 Secretion Dynamics During Single-Cell Clone Expansion 97
3 Conclusions 97
References 98
CHO-DG44 Cell Line Development by FLP-Targeting High Level Glycoprotein Expression with Significantly Decreased Time Lines 99
1 Introduction 100
2 Results and Discussion 100
2.1 The Targeting Strategy 100
2.2 Establishment of RMCE Target Clones 100
2.3 Case Study -- RMCE Vs. Classical Cell Line Development 103
3 Conclusions 104
References 104
Transgene Copy Number Impact on Clone Performance 106
1 Introduction 106
2 Forced Integration of Multiple Copies by RMCE 107
3 Hypothesis 108
4 Generation and Characterization of Pharmaceutical Cell Lines 108
5 Reduction of Copy Number in Preestablished Clones 109
6 Conclusions 110
References 111
Single Use Bioreactors: Expressing Protein in Mammalian Cell Suspension 112
1 Industry Challenges 112
2 In-Vessel Operations 113
3 A New Approach No Shaking, Stirring, Rocking or Rolling 113
4 Experimental Evidence 114
5 Conclusion 117
Part II Applied Integrative Physiology 119
Gene Modified Hematopoietic Stem Cells for the Treatment of Primary Immunodeficiency Diseases 120
1 Text 121
1.1 Principles of Hematopoietic Stem Cell Gene Therapy 121
1.2 Retroviral Vectors and Transduction Protocols 121
1.3 Progress in Gene Therapy for Primary Immunodeficiencies 121
2 Conclusions 123
References 124
An Avian Cell Line for Production of Highly Attenuated Viral Vectors 126
1 Introduction 127
2 Methods 128
2.1 Plasmids 128
2.2 Isolation and Culture of Primary Cells 128
2.3 Transfection and Focus Recovery 129
2.4 Immunofluorescence 129
2.5 Virus Propagation and Isolation 130
3 Results and Discussion 130
4 Summary 134
References 134
Microelectronic Cellular Vitality Monitoring 136
1 Introduction 136
2 Methods and Materials 137
3 Results 139
4 Discussion 140
References 140
IFN- Glycosylation Macroheterogeneity, Energetic Cell Status and Medium Composition During CHO Cell Cultures 141
1 Introduction 141
2 Materials and Methods 142
3 Results 142
3.1 IFN- Production and Energetic Status OF CHO Cells Grown in Enriched Batch Processes 142
3.2 IFN- Glycosylation and Energetic Status of CHO Cells Grown in Fed-Batch Process 142
4 Conclusion 145
References 145
Modelling of Neural Metabolism Using 13C-NMR Spectroscopy and Metabolic Flux Analysis 146
1 Introduction 146
2 Materials and Methods 147
2.1 Cells and Culture Conditions 147
2.2 Incubation of Astrocytes with [1- 13 C]glucose 147
2.3 Metabolic Flux Analysis 147
3 Results 149
3.1 Hypoglycemia 149
3.2 Anoxia 149
4 Conclusions 150
References 150
Influence of Glucose and Glutamine Concentration on Metabolism of rCHO Cells 152
1 Introduction 152
2 Materials and Method 153
3 Results and Discussion 153
4 Conclusion 155
References 156
Part III Speed and Intensification in Bioprocess Development 157
Delivery of Biomolecules with Non-Viral Vectors 158
1 Introduction 158
2 The PEI Mechanism 159
3 Synthetic Polymer-Mediated Transient Transfection for Biomanufacturing 160
4 Conclusions and Perspectives 161
References 162
Circumventing the Pay Now or Pay Later Dilemma: Strategies for Achieving Process Development with Speed and Long-Term Potential 163
1 Introduction 163
2 Process Development Strategy 164
3 Incorporating QbD During Development 166
4 Components of the Manufacturability Assessment 167
5 Application of the Manufacturability Assessment 167
6 Looking Ahead 169
Recombinant Human Antibody Therapeutics: Supply Strategies for Early and Clinical Development from CHO Cells 171
1 Introduction 172
1.1 Choice of Host Cells 172
1.2 The GS SystemTM 172
1.3 IgG Supply During Antibody Development 173
2 IgG Supply Strategies 174
2.1 Supply of IgG from Transient Expression for Early Development Studies 174
2.2 Rapid Multi-Gram Production of IgG from GS-CHO Pools 175
2.3 Scale Up of IgG Supply for Clinical Trials 177
3 Summary 177
References 178
Automated Screening of High Producer HEK293F Clones and Analysis of Post-Translational Modifications of Secreted Proteins 179
1 Introduction 179
2 Methods 180
2.1 Transfection of HEK293F Cells and Generation of Stable Clones 180
2.2 Automated Selection of Cell Lines with Highest Productivity 181
2.3 Parallel Analyses of Secreted Clotting Factor Quantity and PTMs Using Two Antibodies 181
2.4 Quantification of Protein Secretion and of PTM Levels 181
3 Results and Discussion 182
4 Conclusions and Outlook 186
References 186
Transcriptomic and Proteomic Analysis of Antibody Producing NS0 Cells Cultivated at Different Cell Densities in Perfusion Culture 187
1 Introduction 187
2 Materials and Methods 188
2.1 Cell Line 188
2.2 Microarray Analysis 188
2.3 2D-PAGE Analysis 188
3 Results and Discussion 189
4 Conclusions 190
References 191
New Disposable Fixed-Bed Bioreactor for Cell Culture and Virus Production Based on a Proprietary Agitation and Aeration System 192
1 Introduction 192
2 Material and Methods 193
3 Results 194
4 Conclusion and Perspectives 196
Transcriptomic Analysis of Antibody Producing NS0 Cell Line Under Hypothermic and Hypoxic Conditions 197
1 Introduction 197
2 Materials and Methods 198
3 Results 200
References 200
Semi-Continuous Cultures as a Tool for Cell Line Characterization During Process Development 201
1 Introduction 201
2 Material and Methods 201
3 Results 202
3.1 Semicontinuous Culture, Performance 202
3.2 Use of Semicontinuous Cultures for Cell Characterization 203
References 204
Behaviour of GS-CHO Cell Lines in a Selection Strategy 205
1 Introduction 206
2 Methods 206
3 Results and Discussion 207
4 Summary and the Future 208
References 209
3D Cultures: Effect on the Hepatocytes Functionality 210
1 Introduction 211
2 Material and Methods 211
3 Results and Discussion 212
3.1 Aggregates as 3D Structures 212
3.2 Effect of Inoculum Concentration and Impeller Type 212
3.3 Effect of Media Composition 213
4 Conclusions 214
References 214
Differential Protein Expression Induced by c-Myc Over-Expression: Proteomic Analysis of a CHO Cell Line with Increased Proliferation Capacity 216
1 Introduction 217
2 Results and Discussion 217
3 Conclusion 219
References 220
Development of Pilot-Scale Orbital Shake Bioreactors: Ideal for Cost-Effective and Efficient Transient Gene Expression 221
1 Introduction 221
2 Materials and Methods 221
3 Results and Discussion 222
4 Conclusion 223
References 223
Helical Tracks in Shaken Cylindrical Bioreactors Improve Oxygen Transfer and Increase Maximum Cell Density Obtainable for Suspension Cultures of Mammalian Cells 224
1 Introduction 224
2 Materials and Methods 225
2.1 Cell Culture and Growth Assessment 225
2.2 DO Measurement and k L a Determination 226
3 Results and Discussion 226
3.1 Determination of k L a for Helical Track Bottles 226
3.2 Assessment of Cell Growth in Helical Track Vessels 226
4 Conclusions 228
References 228
Dynamic Optimisation of CHO-IFN Cell Culture Fed-Batch Time-Profile 230
1 Introduction 230
2 Model Overview 231
3 Model Structure 231
4 Global Parameter Sensitivity Analysis 232
5 Simulation and Optimisation Results 233
6 Conclusions 235
Notations 235
References 236
Long-Term 3D-Culture of HEP G2 Cell Line on Macroporous Ceramic Carriers 237
1 Introduction 237
2 Bioreactor Systems and Carriers 238
3 Results 238
References 240
An Integrated Production Process for Human Growth Hormone 241
1 Introduction 242
2 Materials and Methods 242
3 Results 242
4 Conclusions 244
Efficient Production of Human Monoclonal Antibodies by an Improved Fructose-Based Human Cell Culture 245
1 Introduction 245
2 Materials and Methods 246
3 Results and Discussion 247
References 248
Coupling Between Cell Kinetics and CFD to Establish Physio-Hydrodynamic Correlations in Various Stirred Culture Systems 249
1 Introduction 249
2 Materials and Methods 250
2.1 Cell Cultures 250
2.2 Numerical Simulations 250
3 Results 250
3.1 Culture Kinetics 250
3.2 Physio-Hydrodynamic Correlations 251
4 Conclusions 253
Confocal Microscopy Observation of CHO Cells Cultivated in Presence of Fluorescent Labelled Rapeseed Peptides 254
1 Introduction 254
2 Material and Methods 255
3 Results 255
3.1 Labelling of Rapeseed Peptides 255
3.2 Incubation of CHO Cells in Presence of Labelled-Peptides 256
References 257
Growth and Production Characteristics of Four Mammalian Cell Lines on a Cost-Effective Serum-Free Medium 258
1 Materials and Methods 258
2 Results 259
2.1 Growth Characteristics of SP2/0, CHO, EBNA 293 and Hybridoma Cells in Serum-Free Media 259
2.2 Production of Monoclonal Antibodies by Hybridomas 259
2.3 Transient Transfection Optimisation and Recombinant Protein Expression 261
3 Conclusions 262
References 262
A Serum-Free, Transient Transfection System for Enhancing Production of Recombinant Antibodies in Mammalian Cells 263
1 Introduction 263
2 Materials and Methods 264
2.1 Cell Culture 264
2.2 Transfection 264
3 Results and Discussion 264
References 266
CFD Study of the Fluid and Particle Dynamics in a Spin-Filter Perfusion Bioreactor 267
1 Introduction 267
2 Aims of the Work 268
3 Methodology 268
4 Results 268
References 272
High-Yielding CHO Cell Pools for Rapid Production of Recombinant Antibodies 273
1 Introduction 273
2 Isolation of High-Producing Pools 274
3 Scale-Up, Stability and Compatability with Disposables Technology 274
4 Product Quality 275
5 Robust Production Process 277
6 Cloning Out from Pools 278
7 Summary 278
8 Methods 278
Increasing Upstream Process Development Efficiency by Implementing Platform Glutamine Synthetase Cell Culture Processes 279
1 Introduction 280
2 Methods and Materials 280
3 Results and Discussion 280
3.1 Initial Analysis of Model GS-CHO Cell Line (1G5) 281
3.2 First Iteration of the Fed-Batch Process 283
3.3 Second and Third Iteration of the Fed-Batch Process 284
3.4 Fed Batch v.4 with Multiple IgG-Expressing GS-Cell Lines 285
Implementation of High Throughput Systems for Media and Process Development 286
1 Introduction 286
2 Observations 287
3 Conclusions 288
Comparison of Cell Culture Methods for Obtainingof rHU-EPO to Large Scale 289
1 Introduction 289
2 Material and Methods 289
3 Results 290
4 Conclusions 291
References 291
Optimization and Comparison of Different DNA Methyl Transferase and Histone Deacetylase Inhibitors for Enhancing Transient Protein Expression 293
1 Introduction 293
2 Materials and Methods 294
2.1 Cell Culture 294
2.2 Transfection 294
3 Results and Discussion 294
References 296
Proteomic Characterisation of a Glucose-Limited CHO Perfusion ProcessAnalysis of Metabolic Changes and Increase in Productivity 297
1 Introduction 297
2 Materials and Methods 298
2.1 Perfusion Cultivation 298
2.2 Proteome Analysis 298
3 Results 299
3.1 Glucose-Limited Perfusion Cultivation of CHO-MUC1-IgG2a 299
3.2 Proteome Analysis 299
Reference 301
Evaluation of Alternative Signal Sequences 302
1 Methods 302
2 Results and Discussion 303
3 Summary and the Future 304
References 305
Process Development for the GMP Productionof N-Acetylgalactosamine-6-Sulfate Sulfatase (GALNS) Expressed by CHO Cells 306
1 Introduction 306
2 Results and Discussion 308
3 Conclusion 310
References 310
Improvement of a CHO Fed-Batch Process by Fortifying with Plant Peptones 311
1 Materials and Methods 311
2 Results 312
2.1 Experiment 1: Neutralization of the Toxic Effect from Amino Acid Over-Feeding by Peptones Addition 312
2.2 Experiment 2: Dose Study in 50 ml Filtered Tubes 312
3 Conclusion 314
O-Glycans on Recombinant MUC1 Produced in CHO K1 Cells Become Less Sialylated with Increased Protein Productivity, as Determined by LC-ESI MS 315
1 Introduction 316
2 Materials and Methods 316
3 Results and Discussion 317
4 Conclusions 318
References 318
A Multiple Minibioreactor Platform for Parallel and Automated Mammalian Cell Culture 319
1 Introduction 319
2 Materials and Methods 320
2.1 Culture System 320
2.2 Analytical Systems 320
2.3 System Operation 320
3 Results and Discussion 320
3.1 Hybridoma Cells Growth at Various 0 FCS 320
3.2 Toxicicy of Neomycin on Vero Cells 322
4 Conclusions 323
Cultivation of Adherent-Dependent Animal Cells on Microcarriers in a New Disposable Reactor 324
1 Introduction 324
2 Material and Methods 325
3 Results 326
4 Conclusion and Perspectives 328
CHO Cells Cultivation and Antibodies Production in a New Disposable Bioreactor Based on Magnetic Driven Centrifugal Pump 329
1 Introduction 329
2 Material and Methods 330
3 Results 330
4 Conclusion and Perspectives 332
Stability and Productivity of CHO Pools with Respect to Culture Age, Cryopreservation and 20 L Bioreactor Cultivation 334
1 Introduction 334
2 Material and Methods 335
3 Results and Discussion 336
4 Conclusion and Outlook 338
References 338
In Vitro Disassembly and Reassembly of Triple-Layered Rotavirus-Like Particles 339
1 Introduction 339
2 Materials and Methods 340
2.1 Production of RLPs 340
2.2 Kinetics of TLP Disassembly 340
2.3 Kinetics of TLP Reassembly 341
3 Results and Discussion 341
3.1 Kinetics of TLP Disassembly 341
3.2 Kinetics of TLP Reassembly 342
4 Conclusion 343
References 344
Influence of Culture Conditions on Insect Cell Growthand Protein Production -- Comparison of Wave Bioreactorand Shake Flasks 345
1 Materials and Methods 345
2 Results and Discussion 346
2.1 Insect Cell Growth in Shake Flasks 346
2.2 Insect Cell Growth in Wave Bioreactor 347
2.3 Protein Production in Shake Flasks 348
2.4 Protein Production in Wave Bioreactor 349
3 Conclusion 350
Process Intensification Based on Nano-Structured Carbon Carrier Materials and Disposable Devices 351
1 Introduction 352
2 Materials and Methods 352
3 Results 353
3.1 Product Yield and Medium Consumption 353
3.2 Cell Numbers and pH Value 353
4 Conclusions 355
5 Outlook 356
References 356
Accelerating Fed-Batch Process Development Using Rationally Designed Feed Media 357
1 Methods 357
1.1 Project Phases 357
1.2 Cell Culture 358
1.3 Spent Media Analysis 358
2 Feed Medium Development 358
3 Development of Hydrolysate and Integration with Feed for Optimal Performance 358
4 Process Development of Feed Volume and Timing 360
5 Conclusions 361
6 Summary 361
References 362
Development of a Robust Small-Scale Production Format that Is Predictive of Bioreactor Performance 363
1 Introduction 363
2 Methods 364
3 Results 365
4 Conclusion 368
References 368
Biomass Monitoring and CHO Cell Culture Optimization Using Capacitance Spectroscopy 369
1 Introduction 369
2 Aims of the Work 371
3 Methodology 371
4 Results 372
References 374
Characterizing Physiology and Metabolism of High-Density CHO Cell Perfusion Cultures Using 2D-NMR Spectroscopy 375
1 Introduction 376
2 Materials and Methods 376
2.1 Cell Line Culture Medium and Bioreactor Operation 376
2.2 Analytical Methods 377
2.3 Sample Preparation for NMR Analysis 377
2.4 2D-NMR Analysis 377
2.5 Biochemical Network and Metabolic Flux Analysis 378
3 Results 378
3.1 Cell Density and Viability 378
3.2 Glucose, Glutamine, Lactate, and Ammonium 379
3.3 Metabolic Fluxes 381
4 Discussion 382
4.1 Pentose Phosphate Pathway 382
4.2 Pyruvate Carboxylase Flux 382
4.3 Implications for Bioprocess Development 382
5 Conclusions 382
References 383
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
1 Introduction 385
2 Molecular Biology 385
3 Cell Biology 385
4 Cell Culture Technology 387
5 Conclusion 387
References 388
Management of Handling, Long-Term Stability and Shipping of Human T-Lymphocytes Bags for Clinical Studies 389
1 Introduction 389
2 Methods 390
3 Results and Discussion 391
3.1 Biological Activity After Long Term Storage 391
3.2 Long Distance Shipping 391
4 Conclusions 392
References 393
Mass Transfer in the CELL-tainer Disposable Bioreactor 394
1 Introduction 394
2 Results 394
3 Conclusion 396
Efficient Production of Human Monoclonal Antibodies by Combining In Vitro Immunization and Phage Display Methods 397
1 Introduction 397
2 Methods 398
2.1 In Vitro Immunization 398
2.2 Construction of Phage Antibody Library 398
2.3 Detection and Production of ME-Specific Antibody 398
3 Results and Discussion 399
References 399
Measurement and Control of Viable Cell Density in a Mammalian Cell Bioprocessing Facility: Validation of the Software 400
1 Materials and methods 401
2 Results 402
3 Discussion 402
4 Conclusions 404
References 405
Development, Validation, and Application of a Fully Integrated Automation System for Screening and Selection of High Yielding Production Cell Lines 406
1 System Overview 406
2 Preparation Stage: Automated Liquid Handling 407
3 Stage 1: High Throughput ELISA Screening 407
4 Stage 2: Digital Imaging to Visualize Cells Secreting Antibody 407
5 Stage 3: Transfer and Expansion of High Producing Cell Lines 408
6 V-Prep Validation 408
7 Tecan Spectrafluor Reader Validation 408
8 Tecan Genesis and Nikon Microscope Validation 408
9 Validation of the ELISA Function 409
10 Comparison of ELISA Data Generated by Automation System and Standard Method 409
11 Comparison of Cell Lines Selected by Automation and Standard Methods 410
12 Summary 410
Monitoring of Cell Activity: Online Oxygen Uptake Rates in Pulsed Aerated Cell Culture 411
1 Introduction 411
2 Material and Methods 412
3 Results and Discussion 412
4 Conclusions 412
References 413
Part IV Systems Biotechnology 414
Metabolite Analysis in Mammalian Cells: How to Generate Reliable Data Sets? 415
1 Introduction 416
2 Materials and Methods 416
3 Results and Discussion 418
3.1 Extraction Screening 418
3.2 Optimization of the MeOH/CHCl 3 Extraction Method 419
3.3 Determination of Recovery 423
3.4 Starvation Experiments 424
4 Conclusions 424
References 424
Cell-to-Cell Communication for Cell Density-Controlled Bioprocesses 425
1 Cell-to-Cell Communication Systems Cell Phones 426
2 Multi-Step Multi-Species Communication Systems 427
3 Conclusion 429
References 429
Metabolome Analysis in Mammalian Cells: Development and Application of a Sampling Technique 431
1 Introduction 432
2 Materials and Methods 433
2.1 Cell Line and Cultivation 433
2.2 Sampling 433
2.3 Cell Disruption and Cell Counting 433
2.4 LCMS and LCMS-MS Analytics 434
3 Results and Discussion 434
References 438
Differential Expression Profiling of Industrially Relevant CHO Cell Phenotypes Using a Proprietary CHO-Specific Microarray and Proteomics Technology Platforms 439
1 Introduction 439
2 Collaboration Overview 440
3 Data Analysis 440
4 Target Validation 441
Towards a Systems-Level Understanding of Increased Specific Productivity in Proliferation Arrested Myeloma NS0 Cells 442
1 Introduction 442
2 Results and Discussion 443
3 Conclusion 445
References 445
Proteomic Analysis of Influenza a Virus Infected Mammalian Cells by 2D-DIGE 446
1 Introduction 446
2 Materials and Methods 447
3 Results and Discussion 447
References 449
Involvement of SRC- and MAP Kinase-Signalings in the Effect of Sericin on Cellular Proliferation and Survival 450
1 Introduction 450
2 Materials and Methods 451
2.1 Cell Line and Culture Conditions 451
2.2 Two-Dimensional Electrophoresis 451
3 Results 451
3.1 The Effect of Sericin on Proliferation of Hybridoma Cell Line 2E3-O 451
3.2 Proteome Analysis Using 2-DE 452
The Unfolded Protein Response and Recombinant Protein Production from In Vitro Cultured Mammalian Cells 453
1 Introduction 453
2 Materials and Methods 454
2.1 Reagents 454
2.2 Cell Lines 454
2.3 Growth Profiles of CHO and NS0 Upon UPR Induction 454
3 Results 454
4 Conclusions 456
References 456
A Scalable Process for Helper-Dependent Adenoviral Vector Production Using PEI-Derived Transfection Strategy in Suspension Culture 457
1 Introduction 457
2 Materials and Methods 458
2.1 Cells and Viruses 458
2.2 PEI-Adenofection 458
2.3 Amplification Step Via Co-Infection 458
3 Results and Discussion 459
4 Conclusion 461
References 461
Functional Analysis of ER Stress Pathway Genes for Apoptosis of NS/0 Cell Line Using RNAi Methods 462
1 Introduction 462
2 Methods 463
3 Results 463
4 Conclusions 465
References 466
Identification of New Protein Substrate Candidates of Transglutaminase in Rat Liver Extracts: Use of 5-(Biotinamido) Pentylamine as a Probe 468
1 Introduction 468
2 Materials and Methods 469
2.1 Preparation of Rat Liver Extract 469
2.2 In Vitro TG-Mediated Biotin Labeling 469
2.3 Isolation of Biotinylated Proteins with Avidin-Affinity Column 469
2.4 Amino Acid Sequencing Analyses 470
2.5 Identification of TG Substrates by Immunoblotting 471
3 Results and Discussion 471
References 473
Metabolic Flux Distributions of Adherently Growing MDCK Cells in Different Media 474
1 Introduction 474
2 Phase Model 475
3 Metabolic Network Model 476
4 Results and Conclusions 476
References 477
Expression of Dermal Extracellular Matrix Proteins in a Newly Developed Full-Thickness Skin Model 478
1 Introduction 478
2 Materials and Methods 479
2.1 Production of the Skin Model 479
2.2 Immunohistochemistry 479
2.3 Gene Expression Analysis 479
3 Results 479
4 Summary and Conclusions 481
References 482
Development of Preparations with Antiviral and Immunostimutory Effects from Extracts of Green Parts of Coniferous Plants 483
1 Introduction 484
2 Materials and Methods 484
2.1 Cell Culture--MDCK from Cell Culture Collection of SRC VB VECTOR 484
3 Results 487
Serum-Free Transfection of CHO Cells with Chemically Defined Transfection Systems to Generate Transient and Stable Cell Lines 488
1 Introduction 488
2 Overview 489
3 Preparation and Transfection Studies 489
4 Conclusion 489
References 491
Glycomics: Development and Characterization of Glycan-Based Biotechnological Products 492
Development of a Cell-Culture-Based Platform for Viral Vaccine Production 494
1 Introduction 495
2 Materials and Methods 495
2.1 Cell Lines 495
2.2 Virus Production 495
3 Results 495
4 Conclusions 497
References 497
Screening of Natural Compounds Affecting Type I Interferon Signalling 498
1 Introduction 498
2 Results 499
3 Conclusions 500
Reference 500
Part V Competing and Complementary Approaches to Animal Cell Technologies 501
Therapeutic Proteins from Transgenic Cows Milk 502
1 Introduction 502
2 Methodology 503
2.1 Obtention of the Founder Animal 503
2.2 Expansion of the Transgenic Herd 504
2.3 Lactation and Bioactive Protein Levels 504
3 Results 505
4 Bovines Transgenic for Human Insulin 506
5 Conclusion 506
References 506
Development of Edible Plant Vaccines 508
References 511
Human Cell Lines for Production of Biopharmaceuticals 513
1 Introduction 514
2 Materials and Methods 515
3 Results and Discussion 516
4 Expression of Reference Proteins in Adherent CAP Cells 517
References 520
Concentration and Purification of Densonucleosis Virus by Tangential Membrane Filtration and by Ion Exchange Membranes 522
1 Introduction 522
2 Materials and Methods 523
2.1 Production of AeDNV Particles 523
2.2 Virus Titer Determination 523
2.3 Tangential Flow Filtration 523
2.4 Ion Exchange Experiments 524
3 Results and Conclusion 524
References 525
Effect of ManNAc and its Derivatives on Glycosylation to Proteins Produced by Mammalian Cell Culture 526
1 Introduction 526
2 Materials and Methods 527
2.1 Cell Line and Culture Condition 527
2.2 Two-Dimensional Electrophoresis 527
3 Results and Discussion 527
4 Conclusion 528
Purification of a Chimeric Simian Human Immunodeficiency Virus-Like Nanoparticle from HEK293 Cell Culture 529
1 Introduction 530
2 Experimental 531
2.1 Expression of the Chimeric VLP 531
2.2 VLP Purification from Culture Media 531
2.3 SDS-PAGE and Immunoblot Analysis 531
2.4 Determination of Protein Concentration 532
3 Results 532
4 Conclusion 534
References 534
Effects of Plant Peptones Supplemented Medium on CHO Cells 536
1 Introduction 536
2 Materials and Methods 537
2.1 Cell Culture 537
2.2 Peptones 537
2.3 Apoptosis Assays 537
3 Results 538
4 Conclusions 539
References 539
Synthetic Low Density Lipoprotein a Novel Biomimetic Lipid Supplement for Serum Free Tissue Culture 540
1 Introduction 540
2 Synthetic Low Density Lipoprotein 541
3 Cellular Uptake 541
4 Cellular Proliferation 543
5 Conclusion 543
References 544
Part VI Solutions and Applications 545
Scale-Down Approach for Animal-Free Polio Vaccine Production 546
1 Introduction 546
2 Materials and Methods 547
2.1 Vero Cells and Polio Virus 547
2.2 Polio Production Process 547
2.3 The Data-Set and its Organisation 548
2.4 Multivariate Data Analysis (MVA) 548
2.5 Scale-Down Experiments and Animal-Component Free Media 549
3 Results and Discussion 549
3.1 Process Analysis by PCA 549
3.2 Performance Analysis of Cell Culture at 350-L Scale 550
3.3 Scale-Down and Animal-Component Free Culture 551
3.4 Performance Analysis of Virus Culture and Scale-Down 553
4 Conclusions 554
References 554
Novel Scaffolds for Tissue Engineering Nano-Structured Surfaces Promote Selective Cell Attachment and Cell Differentiation 556
1 Introduction 556
2 Materials and Methods 557
3 Results and Discussion 558
3.1 Bone Cell Differentiation 558
3.2 Cell Selective Surfaces 560
4 Summary and Conclusions 562
References 562
Unconventional Experimental Concepts Enabling High Speed High Performance Media/Process Development 563
1 Introduction 563
2 Material and Methods 564
3 Results and Discussions 564
3.1 Concept 1 564
3.2 Concept 2 566
Conclusions 569
References 569
A Study on Bioscaffolds of Polysialic Acid and -Glucan for Cell Culture and Tissue Engineering Applications 570
1 Introduction 570
2 Materials and Methods 571
3 Results and Discussion 572
4 Conclusions 572
References 575
A Preliminary Study on Spider Silk as Biomaterial for Peripheral Nerve Regeneration 576
1 Introduction 576
2 Materials and Methods 577
2.1 Materials 577
2.2 PC-12 577
2.3 Immortalized Schwann cells (ISC) 577
2.4 Methods 578
3 Results and Discussion 578
4 Conclusion and Outlook 580
References 581
Investigation of the Effect of Mechanical Strain on the Osteogenic Differentiation of Mesenchymal Stem Cells 582
1 Introduction 582
2 Materials and Methods 583
2.1 AdMSC Isolation and Cultivation 583
2.2 Scaffold Materials 583
2.3 Strain Experiments 584
2.4 Alkaline Phosphatase Activity Test 584
2.5 MTT Assay 585
2.6 RT-PCR 585
2.7 Fabrication of Flexible Microelectrode Dishes 585
2.8 Electric Cell-Substrate Impedance Sensing (ECIS) 586
3 Results and Conclusions 587
4 Summary 592
References 592
Biofunctional Polymer-Mineral Composites as Scaffolds for Bone Tissue Engineering 593
1 Introduction 594
2 Materials and Methods 594
2.1 Preparations of Composites 594
2.2 Cell Culture 595
3 Results 595
3.1 Preparation of Conjugates 595
3.2 Cell Culture 597
4 Conclusions and Outlook 597
References 599
New Water-Soluble Polymers for Construction of Biofunctionalized Scaffolds for Bone Tissue Engineering: Synthesis and Adsorption Study 600
1 Introduction 601
2 Results and Discussion 601
2.1 Polymer Synthesis 601
2.2 Adsorption 602
3 Conclusions 606
References 606
Selection of High-Producing Cells Via Cell Sorting Using an Affinity Matrix 607
1 Introduction 607
2 Materials and Methods 608
2.1 Affinity Matrix Staining 608
2.2 Gating During the Sorts 608
3 Results 610
4 Conclusion 611
References 611
The Effects of Medium Supplement on High-Level Production of Recombinant Human Factor IX in CHO Cell 612
1 Introduction 612
2 Materials and Methods 613
3 Result and Discussion 614
References 617
Case Study Large Scale Cell Culture Facility 618
1 A Biotech Landmark in the Heart of Europe 618
2 Conclusion 619
Glycosylation of Influenza A Virus Hemagglutinin 620
1 Introduction 620
2 Materials and Methods 621
3 Results and Discussion 621
References 622
Production of Retroviral Pseudotype Vectors in Fixed Bed Reactors for Use in Gene Therapy 623
1 Introduction 623
2 Materials and Methods 624
3 Results 625
4 Conclusion 625
References 626
Swellscreen -- Rapid Baculovirus Titration Methodin Microplate Format 627
1 Introduction 627
2 Experimental Set-Up 628
3 Results 628
3.1 Scale-Down 628
3.2 Validation with BacPAK 629
4 Discussion 629
5 Conclusion 630
References 631
Comparing Vero and MDCK Cells for Influenza A Virus Production in Microcarrier Systems 632
1 Introduction 632
2 Materials and Methods 633
3 Results and Discussion 633
References 635
Expansion of Human Articular Chondrocytes on Microcarriers Enhances the Production of Cartilage Specific Matrix Components 636
1 Introduction 637
2 Methods 637
3 Results 638
4 Conclusions 640
References 640
Automated Cell Culture Handling Characteristics and Procedures 641
Bioreactor Satellite Culture Experiments in the Start-Up of a Cell Culture Technical Support Lab 643
1 Set-Up of the Technical Support Lab 644
2 Satellite Culture Principles and CCTS Lab Conditions 644
3 Bioreactor Comparison (Small Scale Vs. Large Scale) 645
4 Culture Performance: Growth and MAb Production 645
5 Culture Performance: Metabolites 648
6 Conclusion and Perspectives 650
Advantages of Hydrodynamic Cell Separation in Industrial Cell Culture Processes 652
1 Introduction 652
2 Materials and Methods 654
3 Results 654
4 Conclusion 658
References 658
Optimization and Characterization of the Process for Large Volume Cell Banking in Bags 660
1 Introduction 660
2 Materials and Methods 661
3 Results and Discussion 661
References 663
Development of Screening Method for IgA-Promoting Factors Derived from Food Extracts Using a Human Myeloma Cell Line 664
1 Introduction 664
2 Materials and Methods 665
3 Results and Discussion 666
3.1 Characteristics of CEC-1LA Cell Line 666
References 669
Cryopreservative Solution Using Sericin 670
1 Introduction 670
2 Materials and Methods 670
3 Results 670
3.1 Influence of Autoclave on Sericin Solution 670
3.2 Influence of Autoclave on DMSO Solution 671
Reference 673
Fructan as a Novel Effective Factor for Mammalian Cell Culture 674
1 Introduction 674
2 Materials and Methods 675
3 Results 675
References 677
Online Determination of Oxygen Uptake and Carbon Dioxide Production Rates in Mammalian Cell Culture Using Mass Spectrometry 678
1 Introduction 679
2 Materials and Methods 679
3 Results and Discussion 680
4 Conclusion 681
References 682
Optimisation of mAb Concentration in Microcarrier Based Perfusion Compared to Batch/Fed-Batch Cultivation 683
1 Cell Cultivation and Reactor Setups 683
2 Results 684
3 Conclusion 686
Characterization of the Novel Human AGE1hn Cell Line for Production of Recombinant Proteins 687
1 Materials and Methods 687
2 Results 688
3 Conclusions 690
Rapid Selection of Optimal Formulations for Divergent Clones Through Screening Chinese Hamster Ovary Media Library 692
1 Introduction 692
2 Materials and Methods 693
2.1 Cell Lines and Media 693
2.2 Productivity Assay 694
3 Results 694
4 Conclusions 695
Development of a Vaccine Candidate Against Heartwater 696
1 Introduction 697
2 Materials and Methods 697
3 Results and Discussion 698
3.1 Mass Production of Endothelial Cells 698
3.2 Ehrlichia ruminantium Production Under Stirring Conditions 698
3.2.1 E. ruminantium Life Cycle 698
3.2.2 Production of E. ruminantium 699
3.3 E. ruminantium Purification 700
3.4 Ehrlichia ruminantium Storage and Vaccine Efficacy 701
References 702
Animal Component Free T-Cell Culture 704
1 Introduction 704
2 Materials and Methods 705
3 Results 705
4 Discussion 709
References 709
Characterization of Cholesterol-Independent GS-NS/0 Recombinant Antibody Cell Lines 710
1 Introduction 710
2 Methods 711
3 Results 711
3.1 Cell Growth 711
3.2 Titers and qP 711
3.3 Product Quality 713
4 Conclusions 714
References 714
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
1 Results 715
2 Discussion and Conclusion 716
Scaffolds for Articular Joint Tissue Engineering 718
1 Introduction 718
2 Materials and Methods 719
3 Results and Discussion 720
3.1 Scaffold Selection and Characterization 720
3.2 Colonization 721
3.3 Cell Proliferation and Material Degradation 722
3.4 Genetic Profile 723
4 Conclusion 723
References 724
Effect of Tunisian Aromatic Plant Extracts on Melanogenesis 725
1 Materials and Methods 726
1.1 Measurement of Melanin Content and Cell Viability 726
1.2 Western Blotting 726
2 Results and Discussion 726
2.1 C. spinosa and E. multiflora Extract have Stimulative Effect on Melanogenesis 726
2.2 T. hirsuta Extract have Inhibitive Effect on Melanogenesis 727
References 728
Effects of Capsaicin on Energy Metabolism by Human Intestinal Epithelial Cell Line Caco-2 729
1 Introduction 729
2 Results and Discussion 730
References 732
Human A1AT Production Propagating a Newly Developed Human Cell Line in a Novel Disposable Perfusion Bioreactor 733
1 Introduction 733
2 The Three Columns of the Process: Protein, Cell Line, Bioreactor 734
3 Process Data of a 31-Day Production Process of A1AT with AGE1.hn in the G-System 735
4 Summary and Conclusions 737
Characterization of Diffusion and Flow Relations in the Novel Membrane Based Perfusion Bioreactor 738
1 Introduction 738
2 Design and Working Principle of the Membrane Based Bioreactor 739
3 Permeability of the Membrane 740
4 Mixing of the Perfusion Flow into the Medium of the Membrane Surrounding Supply Space 741
5 Summary and Conclusions 742
Protective Effect of Di-O-Caffeoylquinic Acid on Human-Derived Neurotypic SH-SY5Y Cells Against Alzheimer's Disease Amyloid-Beta-Induced Toxicity 743
Flow Through Ceramic Foams A Future Cell Culture Challenge 746
1 Introduction 746
2 Experimental Procedures 747
3 Results 749
4 Conclusion 751
References 751
Measles Virus Production in MRC-5 Cells Grown on Microcarriers in a Stirred Bioreactor 752
1 Material and Methods 753
2 Results 753
2.1 Measles Virus Production in Spinner Flask Cultures 753
2.2 Bioreactor Cultures 754
2.2.1 BelloCell500 Culture 754
2.2.2 2-L Stirred Bioreactor Culture 755
References 755
A New System for the Enrichment of Cell Subclones Secreting High Levels of IgG Using Magnetic Cell Sorting (MACS Technology) 757
1 Introduction 757
2 Material and Methods 758
2.1 Separation of Target Cells from Heterogeneous Populations Using MACS ® Technology 758
2.2 Subcloning 758
2.3 Specific Production Rate (SPR) Determination of Subclones 759
3 Results 759
3.1 Analysis of the Effect of Upstream Antibody-Producing Hybridoma Cell Enrichment on the SPR Diversification Pattern Within Cell Populations 759
3.2 Analysis of the Effect of Sequential Separation Steps on the SPRs of Different Hybridoma Populations 760
4 Conclusions 761
Soy Hydrolysate Optimization for Cell Culture Applications 762
1 Introduction 763
2 Materials and Methods 763
2.1 Hydrolysate Manufacturing 763
2.2 Cell Culture 763
2.3 Chemical Composition 765
3 Results and Discussion 765
3.1 Cell Culture 765
4 Conclusions 768
The Effect of Bioreactor pH and Temperature on Protein Glycosylation in Perfusion Cultures of Mammalian Cells 769
1 Introduction 769
2 Materials and Methods 770
3 Results and Discussion 770
Reference 772
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
1 Introduction 773
2 Material and Methods 774
2.1 Membranes Production 774
2.2 In Vitro and In Vivo tests 775
3 Results 775
4 Conclusions 777
References 778
Vaccine Production Utilizing the Potential of Microcarriers in Disposable Bioreactor 779
1 Introduction 780
2 Materials and Methods 780
3 Results 780
3.1 Effect of Pluronic F-68 on Vero Cell Growth in the Wave Bioreactor 780
3.2 Reproducibility of Wave Cultures and Comparison with Stirred Tank Bioreactor 782
3.3 Scale-Up to the 20 L Wave Bioreactor 783
3.4 Animal Component Free 783
3.5 Polio Virus Production 784
4 Conclusions 785
References 785
Characterization of an Anti-Idiotypic Antibody Blocking the Capacity of the HIV-1 Specific nMAb 2F5 786
1 Introduction 786
2 Expression of Ab2/3H6 Variants 786
3 Characterisation of Ab2/3H6 Variants 787
4 Conclusions 788
References 789
Cultivation of PER.C6 Cells in the Novel CELL-TainerTM High-Performance Disposable Bioreactor 790
1 Introduction 790
2 Materials and Methods 790
3 Results and Discussion 791
4 Conclusions 791
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
1 Introduction 792
2 Materials and Methods 792
3 Results and Discussion 793
4 Conclusions 793
Capacitance Sensor as a Robust Tool for Cell Culture Monitoring in Process Development and Manufacturing 794
1 Introduction 794
2 Theory 795
3 Materials and Methods 796
4 Results 796
5 Conclusions 800
Reference 800
Effect of Different Cell Culture Medium Surfactants on Cell Growth and Viability 801
1 Introduction 801
2 Material and Methods 802
2.1 Rationale for Chosen Surfactant Concentrations 802
3 Results 802
4 Conclusion and Next Step 803
References 804
Conventional Stirred Bioreactor Control System for Monitoring and Controlling pH and DO in a Wave Bioreactor 805
1 Materials and Methods 805
1.1 Cell Cultivation 805
1.2 pH and DO Analysis 806
2 Results 807
2.1 Measurement in Cell Free Medium 807
2.2 Measurement During Cultivation 807
3 Discussion and Conclusion 809
On-Line Monitoring of Vero Cells Cultures During the Growth and Rabies Virus Process Using Biomass Spectrometer 811
1 Material and Methods 812
2 Results 812
References 814
Inducing of Human IgE Antibodies by In Vitro Immunization 815
1 Introduction 815
2 Methods 816
3 Results and Discussion 816
3.1 Establishing Human In Vitro IgE Antibody-Inducing System 816
3.2 Effects of the IgE Antibody-Inducing System on Human Plasma 816
3.3 Effects of Food Ingredients on the Induction of IgE Antibodies Against Cedar Pollens 818
References 818
Oxygen Uptake Rate (OUR) Estimation in High Density Mammalian Cell Perfusion Cultures 819
1 Introduction 819
2 Methods 820
2.1 Global Mass Balance 820
2.2 Dynamic Method 821
3 Results 821
References 823
Use of Yeast Derived Nutrients for Cell Culture in Serum-Free Media 824
1 Introduction 824
2 Materials and Methods 825
3 Results 825
3.1 YDN Screening in Microplates 825
3.2 Growth Kinetics and Metabolism in Spinners 826
4 Conclusion 827
References 827
A Novel Approach to the Production of Plant-Derived Hydrolysates Yields Medium Supplements with Enhanced Performance in Cell Culture Systems 828
1 Introduction 828
2 A Brief Summary of the Novel Process 829
3 Materials and Methods 829
3.1 CHO-K1 Cell Culture 829
3.2 Pilot Plant Lots of UltraPep Soy 829
4 Results 830
5 Summary 833
Monitoring the Cell Size Distribution of Mammalian Cell Cultures Using On-Line Capacitance Measurements 834
1 Introduction 835
2 Material and Methods 835
3 Results 836
4 Conclusions 839
References 840
Biosimilarity of Recombinant Human EPO Products from CHO Cell Lines: A Carbohydrate Structural View 841
1 Introduction 841
2 Material and Methods 842
2.1 EPO Preparations 842
2.2 Release of Sialic Acids and N-Linked Oligosaccharides 842
2.3 Mass Spectrometric Analysis 842
2.4 Purification of Released N-Linked Oligosaccharides by Anion Exchange Chromatography 842
3 Results and Discussion 843
4 Conclusions 845
References 845
Quantitative N-Glycan Mapping of Glycoprotein Therapeutics by HPAEC-PAD: Glycosylation Characteristics of Different Recombinant Human EPO Products 846
1 Introduction 847
2 Results 847
3 Conclusion 847
References 850

Produkt-Details

• Erscheint lt. Verlag: 17.7.2010
• Reihe/Serie: ESACT Proceedings
• Reihe/Serie: ESACT Proceedings
• Zusatzinfo: LIV, 800 p.
• Verlagsort: Dordrecht
• Sprache: englisch
• Themenwelt: Studium ► 1. Studienabschnitt (Vorklinik) ► Biochemie / Molekularbiologie
• Schlagworte: Bioinformatics • Biotechnology • Cell • Cell Culture • glycoprotein • microarray • Physiology • Protein • proteins • Proteomics
• ISBN-10: 90-481-3419-6 / 9048134196
• ISBN-13: 978-90-481-3419-9 / 9789048134199