Table of Contents

Foreword v


Preface xxi


List of Contributors xxiii


The History and Economic Relevance of Industrial Scale Suspension Culture of Living Cells 1
Hans-Peter Meyer and Diego R. Schmidhalter


1 Introduction 2


2 A Short History of Suspension Culture (Fermentation) 2


2.1 Ethanol, Organic Acids, and Solvents, the Beginning 2


2.2 Vitamins Fermentation Takes a Long Time to Develop 4


2.3 Steroids, the First Large-Scale Biocatalysis Processes 5


2.4 Antibiotics, a US-Lead Turning Point in Fermentation Technology 5


2.5 Amino Acids, a Japanese Fermentation Success Story 8


2.6 Enzymes, a European Fermentation Success Story 9


2.7 Single Cell Proteins, an Economic Flop 9


2.8 Biofuels are Controversial Story 10


2.9 Recombinant DNA Technology Based Products (Monoclonal Antibodies and Other Recombinant Proteins), Setting off an Avalanche of New Products 11


3 The Contemporary Situation 11


3.1 How Long Can the USA Keep its Leading Role? 11


3.2 China and India Become Global Forces in Fermentation 12


4 The Future of Suspension Culture 13


4.1 New Frontiers 14


4.2 Yet “Uncultured” Cells and Organisms? 15


5 Economic and Market Considerations 16


5.1 The Pharmaceutical Market 19


5.2 Personal Care Products 25


5.3 Chemicals, Industrial and Technical Enzymes 27


5.4 Food, Dietary Supplements (Functional Food, Nutraceuticals), and Feed Products 27


6 Conclusions 32


References 34


Part I Suspension Culture of Bacteria, Yeasts, and Filamentous Fungi 39


1 Bacterial Suspension Cultures 41
Patrick Sagmeister, Mohammadhadi Jazini, Joachim Klein, and Christoph Herwig


1.1 Introduction 41


1.2 Organisms, Cells, and their Products 42


1.2.1 Bacteria as Production Platform for Various Products 42


1.2.2 Historical Outline for Escherichia coli 44


1.2.3 Industrial Aspects of Bacterial Expression Systems 45


1.3 Bioprocess Design Aspects for Recombinant Products 51


1.3.1 Bacterial Cultivation Processes 51


1.3.2 Gram Negative Cell Factory: Cellular Compartments and Transport across Membranes 52


1.3.3 Industrial Strategies: Quality, Folding State, and Location of Recombinant Protein Products 53


1.3.4 Approaches towards Bioprocess Design, Optimization, and Manufacturing 55


1.3.5 Bacterial Bioprocess Design 56


1.3.5.1 Technical and Physiological Constraints for Bacterial Bioprocess Design 56


1.3.5.2 Media Design 57


1.3.5.3 Product Titer is Determined by the Biomass Concentration and the Specific Productivity qp 58


1.3.6 Industrial Production Strategy by Two-Step Cultivation 59


1.3.6.1 Batch Phase for the Accumulation of Biomass 60


1.3.6.2 Structured Approach Towards Batch Design 60


1.3.6.3 Fed-Batch Phase Process Design from Scratch 61


1.3.6.4 Induction Phase: Product Formation Characteristics 63


1.3.6.5 Process Parameters Impacting Recombinant Product Formation 64


1.3.6.6 Concept of Time–Space Yield 65


1.4 Basic Bioreactor Design Aspects 66


1.4.1 Introduction 66


1.4.2 Vessel Design and Construction 67


1.4.3 Dimensioning 67


1.4.3.1 Materials of Construction 67


1.4.3.2 Surface Quality and Welding 69


1.4.3.3 Nozzles and Ports 70


1.4.4 Mass Transfer 70


1.4.5 Cleaning in Place 72


1.4.6 Steaming in Place 73


1.4.7 Monitoring and Control of Bioprocesses 73


1.4.7.1 Standard Instrumentation – Measuring and Control of Process Parameters 73


1.4.7.2 Challenges with Bioreactor Standard Sensors 74


1.4.7.3 Advanced Bioprocess Analytics: Real-Time Monitoring of Process Variables 74


1.5 Single Use Bioreactors for Microbial Cultivation 76


1.5.1 Multi-use or Single Use? 76


1.5.2 Challenges for the Use of Single Use Bioreactors in Microbial Bioprocesses 77


1.5.3 Microbial Bioprocess Development Using Single Use Bioreactors 77


1.5.4 Applications for Single Use Bioreactors in Microbial Suspension Cultures 79


1.6 Quality by Design: Vision or Threat for Twenty-First Century Pharmaceutical Manufacturing 79


1.6.1 Regulatory Drive towards the Implementation of QbD 80


1.6.2 Process Development along QbD Principles 82


1.6.3 Entry Points to QbD for Manufacturers 84


1.6.4 Challenges for Putting QbD Into Practice 84


1.6.5 Process Understanding for Biopharmaceutical Processes 85


1.6.5.1 Quality by Design – Opportunity or Threat for the Pharmaceutical Industry? 86


1.7 Process Economics 87


1.7.1 Optimization of Overall Productivity and Capital Expenses of the Production Facility 87


1.7.2 Further Economic Effects by Intracellular Product Location 88


1.7.3 Comparison of Product Yields, Intracellular Versus Extracellular 88


References 90


2 Yeast Suspension Culture 95
Diethard Mattanovich, Carmen Jungo, Jana Wenger, Michal Dabros, and Michael Maurer


2.1 Introduction 95


2.2 Yeast Species Used in Biotechnology and their Products 96


2.2.1 Expression Systems 98


2.3 Basic Process Design Aspects 98


2.3.1 Process History 98


2.3.2 Yeast Fermentation Processes 99


2.3.3 Process Design for Ethanol Production 106


2.3.4 High Cell Density Fermentations – A Downstream Processing Challenge and a Yield Problem? 106


2.4 Basic Bioreactor Design Aspects 107


2.4.1 Bioreactors for Yeast Cultivation 107


2.4.2 Methanol, Safety and Explosion-Proof (Ex-proof) Measures 108


2.4.2.1 Concerns Regarding Methanol Use 108


2.4.2.2 Safety Issues and Equipment Design 108


2.4.2.3 Risk Assessment 109


2.4.2.4 Regulations 110


2.4.3 Process Monitoring and Control Solutions 111


2.4.3.1 The Classical Four 111


2.4.3.2 Advanced Bioprocess Monitoring and Control 112


2.5 Key Factors Related to Process Economics 114


2.5.1 Equipment Requirements 114


2.5.1.1 Upstream 114


2.5.1.2 Primary Recovery 114


2.5.1.3 Downstream Processing 115


2.5.2 Key Factors Related to Process Economics of the Fermentation Process 116


2.5.2.1 Raw Materials 116


2.5.2.2 Cycle Time 117


2.5.2.3 Formation of By-Products 118


2.5.3 Expression System and Its Impact on the Cost of Primary Recovery and Purification 118


2.5.4 Influence of the Expression System on the Analytical Scope in the Production 119


2.5.4.1 In-Process Controls (IPCs) 119


2.5.4.2 Final Release Analysis 119


2.5.4.3 Additional Costs 119


2.5.5 Estimation of Cost of Goods with Model Simulations 119


2.6 Regulatory Aspects 120


2.6.1 Food Ingredients 121


2.6.2 Pharmaceuticals 121


2.6.2.1 Regulatory Aspects to Consider/Guidelines 122


2.6.2.2 Construction and Quality of Cell Banks (Part of ICHQ5) 122


2.6.2.3 Manufacturing of APIs by Cell Culture or Microbial Fermentation (ICHQ7) 124


2.6.2.4 Test Procedures and Acceptance Criteria for Biotechnological Products (Part of ICHQ6) 125


2.6.2.5 Comparability of Biotechnological Products after Process Changes (Part of ICHQ5) 125


2.7 Summary and Outlook 126


References 127


3 Filamentous Fungi Fermentation 131
Anders Nørregaard, Stuart M. Stocks, John M. Woodley, and Krist V. Gernaey


3.1 Introduction 131


3.2 Products and Organisms in the Industry 132


3.2.1 Background 132


3.2.2 Secondary Metabolites 133


3.2.3 Organic Acids 134


3.2.4 Proteins 134


3.3 Filamentous Fungi as a Production Platform 135


3.3.1 Expression Systems 135


3.3.2 Morphology 136


3.3.3 Genomic Tools 137


3.3.4 Sequencing and Genome Annotation 138


3.4 Fermentation of Filamentous Organisms 140


3.4.1 Fermentation Platforms 140


3.4.2 Reactor Design 141


3.4.3 Agitation and Aeration 141


3.4.4 Mass Transfer 143


3.4.5 Reactor Control 146


3.4.6 Rheology 147


3.4.7 Mixing Time and Cavern Formation 151


3.4.8 Correlation between Viscosity and kLa 151


3.5 Process Scaling 152


3.5.1 Dimensionless Numbers 153


3.5.2 Power Draw 154


3.5.3 Modeling Oxygen Mass Transfer 155


3.6 Regulatory Aspects 156


3.7 Economic Aspects 157


3.8 Conclusions and Perspectives 157


References 158


Part II Suspension Culture of Algae and Plant Cells 163


4 Microalgae Grown under Heterotrophic and Mixotrophic Conditions 165
Karin Kovar, Pavel P?ibyl, and Markus Wyss


4.1 Eco-physiology and Genetics of Biotechnologically Relevant Species 165


4.1.1 Taxonomy 166


4.1.2 Access to Axenic Cultures and Screening for Bioactivities 167


4.1.3 Biotechnologically Relevant Species and their Genetic Improvement 168


4.2 Products from Microalgae Grown in the Absence of Light 172


4.3 Bioreactor Design 174


4.4 Process Design: Culture Media and Process Control Strategies 174


4.5 Process Economics 176


4.6 Commercialization of Microalgae-Derived Products and Regulatory Aspects 176


References 178


5 Recombinant Protein Production with Microalgae 187
Alexandre Lejeune, Rémy Michel, and Aude Carlier


5.1 Organisms, Cells, Expression Systems, Products 187


5.2 Production of Recombinant Therapeutics in Microalgae: Process Design Aspects 189


5.2.1 Overall Process Overview: From Genetic Transformation to Cell Banking 189


5.2.2 Basic Aspects of Cultivation of Microalgal Cells for Production of Recombinant Therapeutic Proteins 190


5.3 Regulatory Aspects 192


5.4 Summary and Outlook 194


References 195


6 Suspension Culture of Microorganisms (Algae and Cyanobacteria) Under Phototrophic Conditions 199
Peter Bergmann, Astrid Nissen, Lars Beyer, Peter Ripplinger, and Walter Trösch


6.1 Introduction 199


6.1.1 Photosynthetic Microorganisms (Algae and Cyanobacteria) in General 200


6.1.2 Microalgal Evolution and Taxonomy 201


6.1.3 Microalgae in Biotechnology 201


6.1.4 Industrial Microalgae Biotechnology – A Brief History 202


6.2 Basic Process Design Aspects 203


6.3 Large-Scale Cultivation Systems 206


6.3.1 Open Ponds – Technology Overview 207


6.3.2 Open Ponds – Production Sites 208


6.3.3 Open Ponds – Performance 209


6.3.4 Open Ponds – Energy Consumption 210


6.4 Photobioreactors – Technology Overview 211


6.4.1 Photobioreactors – Tubular 212


6.4.1.1 Tubular Photobioreactors – Production Sites 213


6.4.1.2 Tubular Photobioreactors – Performance 213


6.4.1.3 Tubular Photobioreactors – Energy Consumption 214


6.4.2 Photobioreactors – Flat-Plate 215


6.4.2.1 Flat-Plate Photobioreactors – Production Sites 217


6.4.2.2 Flat-Plate Photobioreactors – Performance 217


6.4.2.3 Flat-Plate Photobioreactors – Energy Consumption 217


6.5 Conclusion/Outlook 218


References 219


7 Suspension Culture of Plant Cells Under Heterotrophic Conditions 225
Nicole Imseng, Stefan Schillberg, Cornelia Schürch, Daniel Schmid, Kai Schütte, Gilbert Gorr, Dieter Eibl, and Regine Eibl


7.1 Introduction 225


7.2 In Vitro Initiation and Maintenance of Plant Cell Suspension Cultures 229


7.2.1 General Procedure 229


7.2.2 Plant Stem Cells 231


7.2.3 Non-transformed and Genetically Modified Plant Cell Suspensions 233


7.3 Characteristics of Heterotrophic Plant Suspension Cells and Resulting Process Design 235


7.3.1 Culture Characteristics and Typical Cultivation Parameters 235


7.3.2 Primary Cell Metabolism and Culture Media 236


7.3.3 Process Mode 237


7.4 Suitable Bioreactors 238


7.4.1 Categorization Approach 238


7.4.2 Most Often Used Bioreactors Types 240


7.5 Commercial Manufacture of Plant Cell-Derived Cosmetics and Therapeutics under Additional Consideration of Economic and Regulatory Aspects 243


7.5.1 Case Study: PhytoCellTecTM Malus domestica 243


7.5.1.1 Production Process 243


7.5.1.2 Effectiveness of the Bioactive Ingredients Produced In Vitro 244


7.5.2 Case Study: Paclitaxel 248


7.5.2.1 Introduction 248


7.5.2.2 Cell Line Development and Cryopreservation 249


7.5.2.3 PCFTM Process Conditions 250


7.5.2.4 Summary 252


7.6 Conclusion 252


References 252


8 Suspension Culture of Plant Cells Under Phototrophic Conditions 261
Holger Niederkrüger, Paulina Dabrowska-Schlepp, and Andreas Schaaf


8.1 Introduction 261


8.2 BryoTechnologyTM: Production of Biologics with Moss (Physcomitrella patens) 262


8.2.1 Characteristics of BryoTechnologyTM 262


8.2.1.1 Moss Taxonomy and Natural Habitats 262


8.2.1.2 Life Cycle and Physiology 262


8.2.1.3 Homologous Recombination 263


8.2.1.4 Recombinant Protein Production 265


8.2.2 Basic Process Design Aspects 265


8.2.2.1 Transformation 265


8.2.2.2 Expression Vectors 266


8.2.2.3 Strain Development 266


8.2.2.4 Cell Banking 267


8.2.2.5 Upstream Process 267


8.2.2.6 Harvest 269


8.2.2.7 Downstream 271


8.2.2.8 Timelines of Process Development 272


8.2.3 Basic Bioreactor Design Aspects 272


8.2.3.1 Illumination 275


8.2.3.2 Biomass Handling 275


8.2.3.3 IPC 276


8.2.3.4 Process Scale-Up 276


8.2.3.5 Current Limitations 277


8.2.4 Summary and Outlook 279


8.3 The LEX-System: Production of Biologics with Duckweed (Lemna minor) 280


8.3.1 Characteristics of the LEX-System 280


8.3.1.1 Duckweed Taxonomy, Physiology and Morphology 280


8.3.1.2 Biotechnological Aspects of Duckweed 280


8.3.1.3 Timelines of Process Development 281


8.3.2 Basic Process Design Aspects 281


8.3.2.1 Expression Vectors 281


8.3.2.2 Strain Development 282


8.3.2.3 Master-Plant Banking 282


8.3.2.4 Upstream Process 282


8.3.2.5 Downstream 283


8.3.3 Basic Bioreactor Design Aspects 284


8.3.4 Summary and Outlook 285


8.4 Key Factors Related to Process Economics 285


8.5 Regulatory Aspects 286


References 288


Part III Suspension Culture of Protozoa, Insect Cells, Avian Cells, and Mammalian Cells 293


9 Suspension Culture of Protozoan Organisms 295
Marcus W.W. Hartmann and Reinhard Breitling


9.1 Introduction 295


9.2 Ciliates 296


9.2.1 Specific Features of Ciliates 296


9.2.2 Suspension Culture of Ciliates 299


9.2.3 Strengths of the Ciliate Tetrahymena thermophila 304


9.2.3.1 Mass Cultivation, Scalability, and Usability of Market Standard Fermentation Equipment 304


9.2.3.2 Reliable High-Efficiency Transformation Protocols 305


9.2.3.3 Established Expression Vectors 305


9.2.3.4 Serum-Free Complex- and Chemically-Defined Media 307


9.2.3.5 Consistent and Advantageous N-Glycosylation with Lack of Fucose 309


9.2.4 Challenges for using Tetrahymena in Production of Recombinant Proteins 310


9.2.4.1 Lack of Terminal Sialylation and g-Carboxylation as Post-translational Modifications 310


9.2.5 Big Lines to Classes of Products and Main Markets 311


9.2.5.1 Tetrahymena as New Production Platform Technology 311


9.2.6 Basic Process Design Aspects for Tetrahymena Suspension Culture 313


9.2.6.1 Principal Bioreactor Set Up for Tetrahymena Suspension Culture 313


9.2.6.2 Inoculation Titer, Cell Counting and Dry Mass 314


9.2.6.3 Agitation Rate and Shear Stress 315


9.2.6.4 Aeration, Dissolved Oxygen Concentration, and Antifoam Reagents 315


9.2.6.5 Mucocyst Material 316


9.2.7 Basic Bioreactor Design Aspects for Tetrahymena Suspension Culture 316


9.2.8 Key Factors in Process Economics 317


9.2.8.1 Investment Costs 317


9.2.8.2 Cost of Goods for Fermentation 318


9.2.8.3 Other Costs 318


9.3 Flagellates 319


9.3.1 Specific Features of Flagellates 319


9.3.2 Suspension Culture of Hemoflagellates 322


9.3.3 Strengths of the Hemoflagellate Leishmania tarentolae 324


9.3.4 Challenges for the Application of the Hemoflagellate Leishmania tarentolae 326


9.3.5 Big Lines to Classes of Products and Main Markets 327


9.3.6 Basic Process Design Aspects for Leishmania Suspension Culture 328


9.3.7 Basic Bioreactor Design Aspects for Leishmania Suspension Culture 331


9.3.8 Key Factors for Process Economics 332


9.4 Regulatory Aspects of Protozoan Production Organism 334


9.5 Summary and Outlook 335


References 336


10 Industrial Large Scale of Suspension Culture of Insect Cells 349
António Roldão, Manon Cox, Paula Alves, Manuel Carrondo, and Tiago Vicente


10.1 History 349


10.2 Concepts in Insect Cell Culture 351


10.2.1 Cell Types, Expression Systems, and Products 351


10.2.2 Maintaining Insect Cells in Culture – Requirements of the Bioreactor Design 358


10.2.3 Insect Cell Metabolism: A Brief Overview 364


10.2.4 A Bottom-Up Approach for Industrial Insect Cell-Based Cultures 366


10.2.4.1 Upstream Process Development Strategies 367


10.2.4.2 Downstream Process Development Strategies 370


10.3 Regulatory Hurdles for Insect Derived Human Products 374


10.3.1 Case Study: Flublok® Regulatory History 376


10.4 What Comes Next? 377


10.4.1 Improvements in Production Cycle and Yields 377


References 378


11 Avian Suspension Culture Cell Lines for Production of Vaccines and Other Biologicals 391
Manfred Reiter, Daniel Portsmouth, and P. Noel Barrett


11.1 Development of Cell Culture for the Production of Vaccines and Biologicals 391


11.2 Avian Cell Lines 393


11.3 Potential of Avian Cell Lines for the Manufacture of Vaccines and Biologicals 394


11.3.1 Modified Vaccinia Virus Ankara (MVA) Vaccines 394


11.3.2 Yellow Fever Vaccines 394


11.3.3 TBEV Vaccines 395


11.3.4 Influenza Vaccines 395


11.3.5 Monoclonal Antibodies 397


11.4 Development of Avian Cell Lines 397


11.4.1 EB66 (Vivalis) 398


11.4.2 AGE1.CR (Probiogen) 399


11.4.3 QOR2/2E11 (Baxter) 400


11.4.3.1 Establishment of QOR2/2E11 400


11.4.3.2 Characterization and GMP Qualification 401


11.4.3.3 Virus growth in QOR2/2E11 Cells 401


11.4.3.4 MVA Virus Replication on QOR2/2E11 Cells at Different MOIs and Temperature 403


11.4.4 Chicken Embryo Cell Line PBS-12SF (Michigan State University, USA) 405


11.5 Basic Process Design Aspects 405


11.6 Basic Bioreactor Design Aspects 405


11.7 Key Factors Related to Process Economics 405


11.8 Regulatory Aspects 406


11.9 Summary and Outlook 406


References 407


12 Large Scale Suspension Culture of Mammalian Cells 411
Richard M. Alldread, John R. Birch, Hilary K. Metcalfe, Suzanne Farid, Andrew J. Racher, Robert J. Young, and Mohsan Khan


12.1 Introduction to Mammalian Cell Culture 412


12.1.1 Brief History of the Use of Mammalian Cell Culture 412


12.1.2 Why Mammalian Cells for Protein Production? 413


12.1.3 Commercial Importance of Mammalian Cell Culture 414


12.1.4 Mammalian Cell Culture Industry 415


12.2 Cell Lines and Expression Technologies 417


12.2.1 Introduction 417


12.2.2 Host Cell Lines for Manufacturing Therapeutic Proteins 419


12.2.2.1 Regulatory Acceptance 419


12.2.2.2 Productivity of CHO Cell Lines 419


12.2.2.3 Cell Line Development Timeline 420


12.2.2.4 Product Characteristics 420


12.2.2.5 Current Status and Future Developments 420


12.2.3 Selecting Highly Productive Cell Lines 421


12.2.4 Expression Vector Architecture 421


12.2.4.1 Insulator and Chromatin Opening Sequences 423


12.2.5 Selection Markers 424


12.2.6 Targeted Integration 425


12.3 Bioreactor Design 427


12.3.1 Introduction 427


12.3.2 Types of Mammalian Cell Culture Bioreactors 428


12.3.2.1 Stirred-Tank Bioreactor 428


12.3.2.2 Airlift Bioreactor 429


12.3.2.3 Wave-Based Bioreactor 430


12.3.3 Scale Up Considerations 431


12.3.3.1 Mixing 431


12.3.3.2 Mass Transfer 432


12.3.3.3 Shear 432


12.3.3.4 Pressure 432


12.3.3.5 Scale up Strategy 433


12.3.4 Sterilization and Cleaning 433


12.3.5 Single Use Bioreactor Systems 435


12.4 Process Operation 436


12.4.1 Batch and Fed-Batch Culture 436


12.4.2 Perfusion Culture 438


12.4.3 Culture Media and Feeds 439


12.4.4 Non-nutrient Additions 439


12.4.5 Control Parameters 440


12.4.5.1 Temperature 440


12.4.5.2 pH 441


12.4.5.3 Dissolved Oxygen Concentration 442


12.4.5.4 Carbon Dioxide Concentration 442


12.4.5.5 Osmolarity 443


12.5 Process Economics of Mammalian Cell Culture 443


12.5.1 Process Economic Challenges 443


12.5.2 Process Economic Drivers 444


12.5.3 Antibody Process Economics Case Studies 447


12.5.3.1 Stainless Steel versus Single Use Decisions 447


12.5.3.2 Fed-Batch versus Perfusion Decisions 448


12.5.3.3 Robustness of Legacy Purification Facilities to Higher Titer Processes 450


12.6 Regulatory Aspects 450


12.6.1 Source, History, and Generation of the Cell Substrate 451


12.6.2 Cell Banks 452


12.6.3 Cell Substrate Stability 452


12.6.4 Expression Vector 452


12.6.5 Characterization of Cell Banks 452


12.6.6 Quality by Design (QbD) 453


12.7 Summary and Outlook 453


References 455


Part IV Suspension Culture for Special Products 463


13 Pillars of Regenerative Medicine: Therapeutic Human Cells and Their Manufacture 465
Christian van den Bos, Robert Keefe, Carmen Schirmaier, and Michael McCaman


13.1 Introduction 465


13.1.1 Regeneration 465


13.1.2 Therapeutically Valuable Cells 466


13.2 Autologous Therapies 468


13.2.1 T-Cells 470


13.2.2 Dendritic Cells 473


13.2.3 Natural Killer Cells 474


13.2.4 Hematopoietic Stem Cells 475


13.3 Allogeneic Therapies 476


13.3.1 Background 476


13.3.2 Current Definition 477


13.3.3 Activity 477


13.3.4 Animal Models 478


13.3.5 Safety 479


13.3.6 Lack of Rejection 479


13.3.7 Immunity and Manufacturing 480


13.3.8 Manufacturing and Technology Transitions 480


13.3.9 Challenges to Manufacturing 480


13.3.9.1 Dosing 481


13.3.9.2 Biological Limitations to Culture Expansion Yields 482


13.3.9.3 Regulatory Expectations 482


13.3.10 Markers versus Process 483


13.3.11 Current Solutions 483


13.3.12 Forthcoming Solutions, Lessons from Bioproduction versus MSC Biology 484


13.3.13 Adaptation/Directed Evolution of Industrial Cell Lines 486


13.3.14 Therapeutic Cells Should not be Adapted 486


13.3.15 Providing Scalable Adhesion Surfaces in Stirred-Tank Bioreactors: Microcarrier Based Bioreactor Processes 487


13.3.15.1 Expanding Adult Somatic Stem Cells: A Medium Scale Bioreactor Example 488


13.3.16 Critical Quality Attributes (CQAs) for Therapeutic Cells 488


13.3.17 Potency 490


13.3.18 Practical Challenges 493


13.3.19 Future Directions for Cell Testing 493


13.4 Downstream Processing 494


13.5 Key Factors Towards Economic Success 496


13.6 Regulatory Considerations 497


13.7 Summary and Outlook 497


References 498


14 Virus Production Under Suspension Conditions 503
Otto-Wilhelm Merten, Wilfried A.M. Bakker, Jürgen Vorlop, Manfred Reiter, Gabriel Visnovsky, Volker J?ger, Maia Merabishvili, and Udo Reichl


14.1 Introduction 503


14.2 Adherent versus Suspension Culture for Virus Production 504


14.2.1 Viral Vaccines for Human use Produced with Microcarrier Based Manufacturing Processes 506


14.2.2 Towards Single Cell Suspension Processes for Virus Production 506


14.3 Polio Virus/Vaccines 508


14.3.1 Introduction 508


14.3.2 Large-Scale IPV Manufacturing Using Vero Cells Grown on Microcarriers 509


14.3.3 Per.C6 and Other Cell Lines for Future Polio Vaccine Production 509


14.3.4 Future Perspectives in IPV Manufacturing 511


14.4 Influenza Virus/Vaccines 512


14.4.1 Introduction 512


14.4.2 Use of Anchorage Dependent Cell Lines – Development of Microcarrier Based Suspension Processes 512


14.4.3 Use of Cell Lines Adapted to Suspension Growth 514


14.5 Modified Vaccinia Ankara (MVA) Production in Suspension Cell Lines 517


14.6 Production of Viruses for Gene Therapy Purpose 519


14.6.1 Large Scale Adenovirus Production Using Suspension Culture Processes 520


14.6.2 Large Scale AAV Production Using Suspension Culture Processes – Comparison of Different Production Systems 523


14.6.3 LV Vector Production – Towards the Use of Suspension Process for Transient Vector Production 527


14.7 Other Viruses 532


14.7.1 Production of Viruses for Veterinary Vaccines 532


14.7.2 Production of Bio-pesticides using the Insect Cell/Baculovirus System 533


14.7.3 Production of Bacteriophages Using Bacterial Suspension Cultures for Phage-Therapy 537


14.7.3.1 Introduction 537


14.7.3.2 Bacterial Strains – Selection for Bacteriophage Generation 538


14.7.3.3 Bacteriophages – Isolation 539


14.7.3.4 Bacteriophages – Production 540


14.7.3.5 Large Scale Production of Phages 540


14.8 Concluding Remarks 542


References 543


15 Cultivable Marine Organisms as a Source of New Products 555
Jean-Michel Kornprobst


15.1 Introduction 555


15.2 Substances of Interest Isolated from Archaea and Prokaryotes 557


15.2.1 Archaea 557


15.2.2 Non-photosynthetic Bacteria 558


15.2.3 Cyanobacteria 560


15.3 Substances of Interest Isolated from Unicellular Eukaryotes 560


15.3.1 Unicellular Chlorophyta and Rhodophyta 563


15.3.2 Diatoms, Chrysophyceae, Raphidophyceae, and Eustigmatophyceae 564


15.3.3 Haptophyceae (¼ Prymnesiophyceae) 565


15.3.4 Fungi and Thraustochytrids 565


15.3.5 Dinoflagellates 567


15.4 Substances of Interest Isolated from Microorganisms Associated with Pluricellular Organisms 570


15.4.1 Bacteria and Sponges 570


15.4.2 Bacteria and Bryozoans 572


15.4.3 Bacteria, Prochlorophyta, and Didemnidae 572


15.4.4 Dinoflagellates and Cnidaria 573


15.4.5 Dinoflagellates and Platyhelminthes (Flat Worms) 575


15.5 Substances of Interest Produced by Sponge Cell Culture 575


15.6 Substances of Interest Isolated by Culture of Macroorganisms 575


15.6.1 Red Algae and Marine Spermatophyta 578


15.6.2 Green algae and Molluscs 579


15.7 Conclusion and Future Prospects 579


References 584


Index 593

About the Author

The holder of a PhD in microbiology from the University of Fribourg, Switzerland, Hans-Peter Meyer served as VP Strategic Projects Biotechnology at Lonza until his retirement in early 2014. Following three years of postdoctoral studies in Stockholm, at the University of Pennsylvania, Philadelphia, and Lehigh University, Bethlehem, USA, in 1982 he joined Prof. Armin Fiechter's team as group leader at the ETH in Zurich before starting at Lonza in Visp, Switzerland in 1986, where he held a number of positions in R&D, manufacturing, and sales & marketing. He recently joined the faculty of the University of Applied Sciences and Arts of Western Switzerland, and also remains an expert at the Commission for Technology & Innovation (CTI) of the Swiss Federal Confederation. Diego R. Schmidhalter is head of R&T within the pharma and biotechnology custom manufacturing division at Lonza Switzerland. He holds a PhD in microbiology from the University of Fribourg, Switzerland, and carried out two years of postdoctoral studies at Genencor International, California, USA. He has held various management positions at Lonza, including head of microbial anufacturing, head of the Biopharma R&D Services business, and as head of Microbial Manufacturing Science and Technology, as well as being a member of the Biopharmaceuticals business team. Dr. Schmidhalter has over 20 years of experience in the biotechnology industry in biopharmaceuticals and biochemicals process development and manufacturing, technology transfer, scaling-up fermentations right up to the 50,000-liter scale, and within the biopharmaceuticals related regulatory environment.