Cell Culture Engineering

Recombinant Protein Production

; Jens Nielsen ; Gregory Stephanopoulos ; Gyun Min Lee (Redaktør) ; Helene Faustrup Kildegaard (Redaktør)

Offers a comprehensive overview of cell culture engineering, providing insight into cell engineering, systems biology approaches and processing technology





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. Les mer
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Offers a comprehensive overview of cell culture engineering, providing insight into cell engineering, systems biology approaches and processing technology





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.

Fakta

Innholdsfortegnelse

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

Om forfatteren

Gyun Min Lee, PhD, is Professor at the Department of Biological Sciences at KAIST, South Korea, and heads the Animal Cell Engineering Laboratory. He is also Scientific Director at the Novo Nordisk Foundation Center for Biosustainability at the Technical University of Denmark.





Helene Faustrup Kildegaard, PhD, is a senior researcher and Co-PI for the CHO Cell Line Engineering and Design section at the Novo Nordisk Foundation Center for Biosustainability at the Technical University of Denmark (DTU).