In Cell Culture Engineering: Recombinant Protein Production, editors Gyun Min Lee and Helene Faustrup Kildegaard assemble top class authors to present expert coverage of topics such as: cell line development for therapeutic protein production; development of a transient gene expression upstream platform; and CHO synthetic biology. They provide readers with everything they need to know about enhancing product and bioprocess attributes using genome-scale models of CHO metabolism; omics data and mammalian systems biotechnology; perfusion culture; and much more.
This all-new, up-to-date reference covers all of the important aspects of cell culture engineering, including cell engineering, system biology approaches, and processing technology. It describes the challenges in cell line development and cell engineering, e.g. via gene editing tools like CRISPR/Cas9 and with the aim to engineer glycosylation patterns. Furthermore, it gives an overview about synthetic biology approaches applied to cell culture engineering and elaborates the use of CHO cells as common cell line for protein production. In addition, the book discusses the most important aspects of production processes, including cell culture media, batch, fed-batch, and perfusion processes as well as process analytical technology, quality by design, and scale down models.
-Covers key elements of cell culture engineering applied to the production of recombinant proteins for therapeutic use
-Focuses on mammalian and animal cells to help highlight synthetic and systems biology approaches to cell culture engineering, exemplified by the widely used CHO cell line
-Part of the renowned "Advanced Biotechnology" book series
Cell Culture Engineering: Recombinant Protein Production will appeal to biotechnologists, bioengineers, life scientists, chemical engineers, and PhD students in the life sciences.
Table of Contents
About the Series Editors xvii
1 Platform Technology for Therapeutic Protein Production 1
Tae Kwang Ha, Jae Seong Lee, and Gyun Min Lee
1.1 Introduction 1
1.2 Overall Trend Analysis 3
1.2.1 Mammalian Cell Lines 3
1.2.2 Brief Introduction of Advances and Techniques 5
1.3 General Guidelines for Recombinant Cell Line Development 6
1.3.1 Host Selection 6
1.3.2 Expression Vector 7
1.3.3 Transfection/Selection 7
1.3.4 Clone Selection 8
1.3.4.1 Primary Parameters During Clone Selection 8
1.3.4.2 Clone Screening Technologies 9
1.4 Process Development 9
1.4.1 Media Development 10
1.4.2 Culture Environment 10
1.4.3 Culture Mode (Operation) 10
1.4.4 Scale-up and Single-Use Bioreactor 11
1.4.5 Quality Analysis 12
1.5 Downstream Process Development 12
1.5.1 Purification 12
1.5.2 Quality by Design (QbD) 13
1.6 Trends in Platform Technology Development 14
1.6.1 Rational Strategies for Cell Line and Process Development 14
1.6.2 Hybrid Culture Mode and Continuous System 15
1.6.3 Recombinant Human Cell Line Development for Therapeutic Protein Production 16
1.7 Conclusion 17
Acknowledgment 17
Conflict of Interest 17
References 17
2 Cell Line Development for Therapeutic Protein Production 23
Soo Min Noh, Seunghyeon Shin, and Gyun Min Lee
2.1 Introduction 23
2.2 Mammalian Host Cell Lines for Therapeutic Protein Production 25
2.2.1 CHO Cell Lines 25
2.2.2 Human Cell Lines 26
2.2.3 Other Mammalian Cell Lines 27
2.3 Development of Recombinant CHO Cell Lines 27
2.3.1 Expression Systems for CHO Cells 28
2.3.2 Cell Line Development Process Using CHO Cells Based on Random Integration 28
2.3.2.1 Vector Construction 29
2.3.2.2 Transfection and Selection 30
2.3.2.3 Gene Amplification 30
2.3.2.4 Clone Selection 31
2.3.3 Cell Line Development Process Using CHO Cells Based on Site-Specific Integration 32
2.4 Development of Recombinant Human Cell Lines 34
2.4.1 Necessity for Human Cell Lines 34
2.4.2 Stable Cell Line Development Process Using Human Cell Lines 35
2.5 Important Consideration for Cell Line Development 36
2.5.1 Clonality 36
2.5.2 Stability 36
2.5.3 Quality of Therapeutic Proteins 37
2.6 Conclusion 38
References 38
3 Transient Gene Expression-Based Protein Production in Recombinant Mammalian Cells 49
Joo-Hyoung Lee, Henning G. Hansen, Sun-Hye Park, Jong-Ho Park, and Yeon-Gu Kim
3.1 Introduction 49
3.2 Gene Delivery: Transient Transfection Methods 50
3.2.1 Calcium Phosphate-Based Transient Transfection 50
3.2.2 Electroporation 51
3.2.3 Polyethylenimine-Based Transient Transfection 52
3.2.4 Liposome-Based Transient Transfection 52
3.3 Expression Vectors 53
3.3.1 Expression Vector Composition and Preparation 53
3.3.2 Episomal Replication 53
3.3.3 Coexpression Strategies 54
3.4 Mammalian Cell Lines 54
3.4.1 HEK293 Cell-Based TGE Platforms 55
3.4.2 CHO Cell-Based TGE Platforms 56
3.4.3 TGE Platforms Using Other Cell Lines 58
3.5 Cell Culture Strategies 58
3.5.1 Culture Media for TGE 58
3.5.2 Optimization of Cell Culture Processes for TGE 59
3.5.3 qp-Enhancing Factors in TGE-Based Culture Processes 59
3.5.4 Culture Longevity-Enhancing Factors in TGE-Based Culture Processes 59
3.6 Large-Scale TGE-Based Protein Production 60
3.7 Concluding Remarks 62
References 62
4 Enhancing Product and Bioprocess Attributes Using Genome-Scale Models of CHO Metabolism 73
Shangzhong Li, Anne Richelle, and Nathan E. Lewis
4.1 Introduction 73
4.1.1 Cell Line Optimization 73
4.1.2 CHO Genome 75
4.1.2.1 Development of Genomic Resources of CHO 75
4.1.2.2 Development of Transcriptomics and Proteomics Resources of CHO 75
4.2 Genome-Scale Metabolic Model 76
4.2.1 What Is a Genome-Scale Metabolic Model 76
4.2.2 Reconstruction of GEMs 77
4.2.2.1 Knowledge-Based Construction 77
4.2.2.2 Draft Reconstruction 77
4.2.2.3 Curation of the Reconstruction 77
4.2.2.4 Conversion to a Computational Format 79
4.2.2.5 Model Validation and Evaluation 79
4.3 GEM Application 80
4.3.1 Common Usage and Prediction Capacities of Genome-Scale Models 82
4.3.2 GEMs as a Platform for Omics Data Integration, Linking Genotype to Phenotype 83
4.3.3 Predicting Nutrient Consumption and Controlling Phenotype 84
4.3.4 Enhancing Protein Production and Bioprocesses 85
4.3.5 Case Studies 86
4.4 Conclusion 86
Acknowledgments 88
References 88
5 Genome Variation, the Epigenome and Cellular Phenotypes 97
Martina Baumann, Gerald Klanert, Sabine Vcelar,Marcus Weinguny, Nicolas Marx, and Nicole Borth
5.1 Phenotypic Instability in the Context of Mammalian Production Cell Lines 97
5.2 Genomic Instability 99
5.3 Epigenetics 101
5.3.1 DNA Methylation 102
5.3.2 Histone Modifications 102
5.3.3 Downstream Effectors 104
5.3.4 Noncoding RNAs 104
5.4 Control of CHO Cell Phenotype by the Epigenome 105
5.5 Manipulating the Epigenome 107
5.5.1 Global Epigenetic Modification 107
5.5.1.1 Manipulating Global DNA Methylation 107
5.5.1.2 Manipulating Global Histone Acetylation 108
5.5.2 Targeted Epigenetic Modification 109
5.5.2.1 Targeted Histone Modification 110
5.5.2.2 Targeted DNA Methylation 112
5.6 Conclusion and Outlook 113
References 114
6 Adaption of Generic Metabolic Models to Specific Cell Lines for Improved Modeling of Biopharmaceutical Production and Prediction of Processes 127
Calmels Cyrielle, Chintan Joshi, Nathan E. Lewis, Malphettes Laetitia, and Mikael R. Andersen
6.1 Introduction 127
6.1.1 Constraint-Based Models 127
6.1.2 Limitations of Flux Balance Analysis 131
6.1.2.1 Thermodynamically Infeasible Cycles 131
6.1.2.2 Genetic Regulation 131
6.1.2.3 Limitation of Intracellular Space 132
6.1.2.4 Multiple States in the Solution 132
6.1.2.5 Biological Objective Function 133
6.1.2.6 Kinetics and Metabolite Concentrations 133
6.2 Main Source of Optimization Issues with Large Genome-Scale Models: Thermodynamically Infeasible Cycles 134
6.2.1 Definition of Thermodynamically Infeasible Fluxes 134
6.2.2 Loops Involving External Exchange Reactions 134
6.2.2.1 Reversible Passive Transporters from Major Facilitator Superfamily (MFS) 135
6.2.2.2 Reversible Passive Antiporters from Amino Acid-Polyamine-organoCation (APC) Superfamily 136
6.2.2.3 Na+-linked Transporters 136
6.2.2.4 Transport via Proton Symport 137
6.2.3 Tools to Identify Thermodynamically Infeasible Cycles 138
6.2.3.1 Visualizing Fluxes on a Network Map 138
6.2.3.2 Algorithms Developed 138
6.2.4 Methods Available to Remove Thermodynamically Infeasible Cycles 139
6.2.4.1 Manual Curation 139
6.2.4.2 Software and Algorithms Developed for the Removal of Thermodynamically Infeasible Loops from Flux Distributions 140
6.3 Consideration of Additional Biological Cellular Constraints 144
6.3.1 Genetic Regulation 144
6.3.1.1 Advantages of Considering Gene Regulation in Genome-Scale Modeling 144
6.3.1.2 Methods Developed to Take into Account a Feedback of FBA on the Regulatory Network 145
6.3.2 Context Specificity 146
6.3.2.1 What Are Context-Specific Models (CSMs)? 146
6.3.2.2 Methods and Algorithms Developed to Reconstruct Context-Specific Models (CSMs) 146
6.3.2.3 Performance of CSMs 148
6.3.2.4 Cautions About CSMs 149
6.3.3 Molecular Crowding 150
6.3.3.1 Consequences on the Predictions 150
6.3.3.2 Methods Developed to Account for a Total Enzymatic Capacity into the FBA Framework 151
6.4 Conclusion 152
References 153
7 Toward Integrated Multi-omics Analysis for Improving CHO Cell Bioprocessing 163
Kok Siong Ang, Jongkwang Hong, Meiyappan Lakshmanan, and Dong-Yup Lee
7.1 Introduction 163
7.2 High-Throughput Omics Technologies 165
7.2.1 Sequencing-Based Omics Technologies 165
7.2.1.1 Historical Developments of Nucleotide Sequencing Techniques 165
7.2.1.2 Genome Sequencing of CHO Cells 166
7.2.1.3 Transcriptomics of CHO Cells 167
7.2.1.4 Epigenomics of CHO Cells 168
7.2.2 Mass Spectrometry-Based Omics Technologies 168
7.2.2.1 Mass Spectrometry Techniques 168
7.2.2.2 Proteomics of CHO Cells 170
7.2.2.3 Metabolomics/Lipidomics of CHO Cells 171
7.2.2.4 Glycomics of CHO Cells 172
7.3 Current CHO Multi-omics Applications 172
7.3.1 Bioprocess Optimization 174
7.3.2 Cell Line Characterization 174
7.3.3 Engineering Target Identification 176
7.4 Future Prospects 177
References 178
8 CRISPR Toolbox for Mammalian Cell Engineering 185
Daria Sergeeva, Karen Julie la Cour Karottki, Jae Seong Lee, and Helene Faustrup Kildegaard
8.1 Introduction 185
8.2 Mechanism of CRISPR/Cas9 Genome Editing 186
8.3 Variants of CRISPR-RNA-guided Endonucleases 187
8.3.1 Diversity of CRISPR/Cas Systems 187
8.3.2 Engineered Cas9 Variants 188
8.4 Experimental Design for CRISPR-mediated Genome Editing 188
8.4.1 Target Site Selection and Design of gRNAs 189
8.4.2 Delivery of CRISPR/Cas9 Components 191
8.5 Development of CRISPR/Cas9 Tools 192
8.5.1 CRISPR/Cas9-mediated Gene Editing 192
8.5.1.1 Gene Knockout 192
8.5.1.2 Site-Specific Gene Integration 194
8.5.2 CRISPR/Cas9-mediated Genome Modification 195
8.5.2.1 Transcriptional Regulation 195
8.5.2.2 Epigenetic Modification 196
8.5.3 RNA Targeting 196
8.6 Genome-Scale CRISPR Screening 197
8.7 Applications of CRISPR/Cas9 for CHO Cell Engineering 197
8.8 Conclusion 199
Acknowledgment 200
References 200
9 CHO Cell Engineering for Improved Process Performance and Product Quality 207
Simon Fischer and Kerstin Otte
9.1 CHO Cell Engineering 207
9.2 Methods in Cell Line Engineering 208
9.2.1 Overexpression of Engineering Genes 208
9.2.2 Gene Knockout 209
9.2.3 Noncoding RNA-mediated Gene Silencing 209
9.3 Applications of Cell Line Engineering Approaches in CHO Cells 211
9.3.1 Enhancing Recombinant Protein Production 211
9.3.2 Repression of Cell Death and Acceleration of Growth 221
9.3.3 Modulation of Posttranslational Modifications to Improve Protein Quality 227
9.4 Conclusions 233
References 234
10 Metabolite Profiling of Mammalian Cells 251
Claire E. Gaffney, Alan J. Dickson, and Mark Elvin
10.1 Value of Metabolic Data for the Enhancement of Recombinant Protein Production 251
10.2 Technologies Used in the Generation of Metabolic Data Sets 252
10.2.1 Targeted and Untargeted Metabolic Analysis 253
10.2.2 Analytical Technologies Used in the Generation of Metabolite Profiles 253
10.2.2.1 Nuclear Magnetic Resonance 254
10.2.2.2 Mass Spectrometry 255
10.2.3 Metabolite Sample Preparation 256
10.2.3.1 Extracellular Sample Preparation 257
10.2.3.2 Quenching of Intracellular Metabolite Samples 257
10.2.3.3 Metabolite Extraction from Quenched Cells 257
10.2.3.4 Metabolic Flux Analysis 257
10.3 Approaches for Metabolic Data Analysis 257
10.3.1 Data Processing 258
10.3.2 Data Analysis 258
10.3.3 Data Interpretation and Integration 260
10.4 Implementation of Metabolic Data in Bioprocessing 261
10.4.1 Relationship Between Growth Phase and Metabolism 261
10.4.2 Identification of Metabolic Indicators Associated with High Cell-Specific Productivity 263
10.4.3 Utilizing Metabolic Data to Improve Biomass and Recombinant Protein Yield 263
10.4.4 Utilizing Metabolic Understanding to Improve Product Quality 265
10.4.5 Cell Line Engineering to Redirect Metabolic Pathways 265
10.5 Future Perspectives 266
Acknowledgments 267
References 267
11 Current Considerations and Future Advances in Chemically Defined Medium Development for the Production of Protein Therapeutics in CHO Cells 279
Wai Lam W. Ling
11.1 Introduction 279
11.2 Traditional Approach to Medium Development 279
11.2.1 Cell Line Selection 279
11.2.2 Design and Optimization 280
11.2.3 Process Consideration 282
11.2.4 Additional Considerations in Medium Development 284
11.3 Future Perspectives for Medium Development 284
11.3.1 Systems Biology and Synthetic Biology 284
Acknowledgment 288
Conflict of Interest 288
References 288
12 Host Cell Proteins During Biomanufacturing 295
Jong Youn Baik, Jing Guo, and Kelvin H. Lee
12.1 Introduction 295
12.2 Removal of HCP Impurities 295
12.2.1 Antibody Product 296
12.2.2 Non-antibody Protein Product 297
12.2.3 Difficult-to-Remove HCPs 298
12.3 Impacts of Residual HCPs 298
12.3.1 Drug Efficacy, Quality, and Shelf Life 298
12.3.2 Immunogenicity 299
12.3.3 Biological Activity 299
12.4 HCP Detection and Monitoring Methods 300
12.4.1 Anti-HCP Antiserum and Enzyme-Linked Immunosorbent Assay (ELISA) 300
12.4.2 Proteomics Approaches as Orthogonal Methods 302
12.5 Efforts for HCP Control 302
12.5.1 Upstream Efforts 303
12.5.2 Downstream Efforts 304
12.5.3 HCP Risk Assessment in CHO Cells 305
12.6 Future Directions 305
Acknowledgments 306
References 306
13 Mammalian Fed-batch Cell Culture for Biopharmaceuticals 313
William C. Yang
13.1 Introduction 313
13.2 Objectives of Cell Culture Process Development 314
13.2.1 Yield and Product Quality 314
13.2.2 Glycosylation 314
13.2.3 Charge Heterogeneity 315
13.2.4 Aggregation 316
13.3 Cells and Cell Culture Formats 316
13.3.1 Adherent Cells 316
13.3.2 Suspended Cells 316
13.3.3 Batch Cultures 317
13.4 Fed-batch Cultures 317
13.5 Cell Culture Media 319
13.5.1 Basal Media 319
13.5.2 Feed Media 320
13.6 Feeding Strategies 321
13.6.1 Metabolite Based 321
13.6.2 Respiration Based 323
13.7 Feed Media Design 323
13.8 Process Variable Design 325
13.8.1 Temperature 325
13.8.2 pH and pCO2 325
13.8.3 Dissolved Oxygen 326
13.8.4 Culture Duration 327
13.9 Cell Culture Supplements 327
13.9.1 Yield 328
13.9.2 Glycosylation 328
13.10 New and Emerging Technologies 329
13.10.1 Analytical Technologies 329
13.10.2 Bioreactor Technologies 331
13.11 Future Directions 332
References 333
14 Continuous Biomanufacturing 347
Sadettin S. Ozturk
14.1 Introduction 347
14.2 Continuous Upstream (Cell Culture) Processes 347
14.2.1 Continuous Culture without Cell Retention (Chemostat) 348
14.2.2 Continuous Culture with Cell Retention (Perfusion) 348
14.2.2.1 Cell Retention by Immobilization or Entrapment 349
14.2.2.2 Cell Retention by Cell Retention Device 350
14.2.3 Semicontinuous Culture 351
14.3 Advantages of Continuous Perfusion 351
14.3.1 Higher Volumetric Productivities 351
14.3.2 Better Utilization of Biomanufacturing Facilities 352
14.3.3 Better Product Quality and Consistency 352
14.3.4 Scale-up and Commercial Production 353
14.4 Cell Retention Systems for Continuous Perfusion 354
14.4.1 Cell Retention Devices 354
14.4.1.1 Filtration-Based Devices 354
14.4.1.2 Spin Filters 355
14.4.1.3 Continuous Centrifugation 356
14.4.1.4 Settler 356
14.4.1.5 BioSep Device 357
14.4.1.6 Hydrocyclones 358
14.5 Operation and Control of Continuous Perfusion Bioreactors 358
14.5.1 Feed and Harvest Flow and Volume Control 358
14.5.2 Circulation or Return Pump 359
14.5.3 Control of Perfusion Rate and Cell Density 359
14.5.3.1 Cell Build-up Phase 359
14.5.3.2 Production Phase 360
14.5.3.3 Cell Bleed or Purge 360
14.6 Current Status of Continuous Perfusion 360
14.7 Conclusions 362
Acknowledgment 362
References 363
15 Process Analytical Technology and Quality by Design for Animal Cell Culture 365
Hae-Woo Lee, Hemlata Bhatia, Seo-Young Park, Mark-Henry Kamga, Thomas Reimonn, Sha Sha, Zhuangrong Huang, Shaun Galbraith, Huolong Liu, and Seongkyu Yoon
15.1 PAT and QbD - US FDA’s Regulatory Initiatives 365
15.2 PAT and QbD - Challenges 365
15.3 PAT and QbD Implementations 366
15.3.1 NIR Spectroscopy 366
15.3.2 Mid-Infrared (MIR) Spectroscopy 367
15.3.3 Raman Spectroscopy 367
15.3.4 Fluorescence Spectroscopy 368
15.3.5 Chromatographic Techniques 368
15.3.6 Other Useful Techniques 369
15.3.7 Data Analysis and Modeling Tools 369
15.4 Case Studies 370
15.4.1 Estimation of Raw Material Performance in Mammalian Cell Culture Using Near-Infrared Spectra Combined with Chemometrics Approaches 370
15.4.2 Design Space Exploration for Control of Critical Quality Attributes of mAb 372
15.4.3 Quantification of Protein Mixture in Chromatographic Separation Using Multiwavelength UV Spectra 372
15.4.4 Characterization of Mammalian Cell Culture Raw Materials by Combining Spectroscopy and Chemometrics 374
15.4.5 Effect of Amino Acid Supplementation on Titer and Glycosylation Distribution in Hybridoma Cell Cultures 375
15.4.6 Metabolic Responses and Pathway Changes of Mammalian Cells Under Different Culture Conditions with Media Supplementations 377
15.4.7 Estimation and Control of N-Linked Glycoform Profiles of Monoclonal Antibody with Extracellular Metabolites and Two-Step Intracellular Models 378
15.4.8 Quantitative Intracellular Flux Modeling and Applications in Biotherapeutic Development and Production Using CHO Cell Cultures 381
15.5 Conclusion 383
References 383
16 Development and Qualification of a Cell Culture Scale-Down Model 391
Sarwat Khattak and Valerie Pferdeort
16.1 Purpose of the Scale-Down Model 391
16.1.1 Development Challenges 391
16.2 Types of Scale-Down Models 392
16.2.1 Power/Volume (P/V) and Air velocity 392
16.2.2 Oxygen Transfer Coefficient (kLa) 392
16.2.3 Gas Entrance Velocity (GEV) 393
16.2.4 Oxygen Transfer Rate (OTR) 393
16.2.5 Model Refinement Workflow 395
16.3 Evaluation of a Scale-Down Model 395
16.3.1 Univariate Analysis 395
16.3.2 Multivariate Analysis 396
16.3.2.1 Statistical Background 396
16.3.2.2 Qualification Data Set 396
16.3.2.3 Observation Level Analysis 397
16.3.2.4 Batch-Level Analysis 397
16.3.2.5 Scores Contribution Plots 398
16.3.3 Equivalence Testing 399
16.3.3.1 Statistical Background 399
16.3.3.2 Considerations for Evaluation and Test Data Sets 399
16.3.3.3 Types of Analysis Outcomes 400
16.4 Conclusions and Perspectives 401
References 402
Index 407
