Engineering Principles in Biotechnology

WEI-SHOU HU is Professor in the Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, USA.

• Preface xviiAbout the CompanionWebsite xix1 An Overview of Bioprocess Technology and Biochemical Engineering 11.1 A Brief History of Biotechnology and Biochemical Engineering 11.1.1 Classical Biotechnology 11.1.2 Recombinant DNA 41.1.3 A Typical Bioprocess 61.1.4 Biochemical Engineering and Bioprocess Technology 81.2 Industrial Organisms 101.2.1 Prokaryotes 121.2.1.1 Eubacteria and Archaea 121.2.2 Eukaryotic Microorganisms 121.2.2.1 Fungi 131.2.2.2 Algae 131.2.3 Multicellular Organisms andTheir Cells 131.2.3.1 Insect Cells 131.2.3.2 Plant Cells, Tissues, and Organs 131.2.3.3 Animal Cells, Tissues, and Organs 141.2.4 Transgenic Plants and Animals 141.3 Biotechnological Products 151.3.1 Metabolic Process 151.3.2 Metabolites 181.3.3 Cells, Tissues, and Their Components 191.3.3.1 Viruses 201.3.4 Secreted Enzymes and Other Biopolymers 201.3.5 Recombinant DNA Products 201.3.5.1 Heterologous rDNA Proteins 201.3.6 Metabolic Engineering and Synthetic Pathways 221.4 Technology Life Cycle, and Genomics- and Stem Cell-Based New Biotechnology 231.4.1 The Story of Penicillin and the Life Cycle of Technology 231.4.2 Genomics, Stem Cells, and Transformative Technologies 25Further Reading 26Problems 262 An Introduction to Industrial Microbiology and Cell Biotechnology 292.1 Universal Features of Cells 292.2 Cell Membranes, Barriers, and Transporters 302.3 Energy Sources for Cells 312.3.1 Classification of Microorganisms According toTheir Energy Source 322.4 Material and Informational Foundation of Living Systems 342.4.1 All Cells Use the Same Molecular Building Blocks 342.4.2 Genes 342.4.3 Genetic Information Processing 362.5 Cells of Industrial Importance 362.5.1 Prokaryotes 382.5.2 Eubacteria 382.5.2.1 CellWall and Cell Membrane 382.5.2.2 Membrane and Energy Transformation 402.5.2.3 Differentiation 412.5.3 Archaea 422.5.4 Eukaryotes 432.5.4.1 The Nucleus 442.5.4.2 Mitochondrion 452.5.4.3 Endoplasmic Reticulum and Golgi Apparatus 462.5.4.4 Other Organelles 472.5.4.5 Cytosol 482.6 Cells Derived from Multicellular Organisms 492.7 Concluding Remarks 50Further Reading 50Problems 503 Stoichiometry of Biochemical Reactions and Cell Growth 533.1 Stoichiometry of Biochemical Reactions 533.1.1 Metabolic Flux at Steady State 583.1.1.1 NAD/NADH Balance in Glycolysis 593.1.1.2 OxidativeMetabolism and NADH 603.1.2 Maximum Conversion of a Metabolic Product 633.2 Stoichiometry for Cell Growth 663.2.1 Cell Composition and Material Flow to Make Cell Mass 663.2.1.1 Composition and Chemical Formula of Cells 663.2.1.2 Material Flow for Biomass Formation 693.2.2 Stoichiometric Equation for Cell Growth 703.2.2.1 Yield Coefficient 713.3 Hypothetical Partition of a Substrate for Biomass and Product Formation 733.4 Metabolic Flux Analysis 743.4.1 Analysis of a Chemical Reaction System 743.4.1.1 Setting Up Material Balance Equations 743.4.1.2 Quasi–Steady State 763.4.1.3 Stoichiometric Matrix, Flux Vectors, and Solution 763.4.2 Analysis of Fluxes in a Bioreaction Network 773.4.3 Metabolic Flux Analysis on a Cellular System 813.4.3.1 Selecting Reactions for Analysis 813.4.3.2 Compartmentalization 833.4.3.3 Biomass 833.4.3.4 Limitations on Accounting of Materials 843.4.3.5 Solution and Analysis 843.5 Concluding Remarks 85Further Reading 85Nomenclature 86Problems 864 Kinetics of Biochemical Reactions 954.1 Enzymes and Biochemical Reactions 954.2 Mechanics of Enzyme Reactions 964.3 Michaelis–Menten Kinetics 984.4 Determining the Value of Kinetic Parameters 1014.5 Other Kinetic Expressions 1044.6 Inhibition of Enzymatic Reactions 1064.7 Biochemical Pathways 1084.7.1 Kinetic Representation of a Reaction Pathway 1084.7.2 Linearity of Fluxes in Biochemical Pathways 1104.8 Reaction Network 1144.9 Regulation of Reaction Rates 1144.9.1 Flux Modulation by Km 1144.9.2 Allosteric Regulation of Enzyme Activities 1154.9.3 Regulation at Transcriptional and Posttranslational Levels 1174.9.4 Modulation of Resource Distribution through Reversible Reactions 1184.10 Transport across Membrane and Transporters 1204.10.1 Transport across the Cell Membrane 1204.10.2 Transport of Electrolytes 1214.10.3 Transport of Charged Molecules across Membrane 1224.10.4 Types of Transporters 1234.10.5 Kinetics of a Facilitated Transporter 1244.11 Kinetics of Binding Reactions 1264.11.1 Binding Reactions in Biological Systems 1264.11.2 Dissociation Constant 1274.11.3 Saturation Kinetics 1284.11.4 Operator Binding and Transcriptional Regulation 1294.11.5 Kinetics of Transcription and Translation 1314.12 Concluding Remarks 135Further Reading 136Nomenclature 136Problems 1385 Kinetics of Cell Growth Processes 1455.1 Cell Growth and Growth Kinetics 1455.2 Population Distribution 1485.3 Description of Growth Rate 1495.4 Growth Stage in a Culture 1505.5 Quantitative Description of Growth Kinetics 1515.5.1 Kinetic Description of Substrate Utilization 1535.5.2 Using the Monod Model to Describe Growth in Culture 1555.6 Optimal Growth 1565.7 Product Formation 1585.8 Anchorage-Dependent Vertebrate Cell Growth 1595.9 Other Types of Growth Kinetics 1615.10 Kinetic Characterization of Biochemical Processes 1625.11 Applications of a Growth Model 1635.12 The Physiological State of Cells 1645.12.1 MultiscaleModel Linking Biotic and Abiotic Phases 1665.13 Kinetics of Cell Death 1685.14 Cell Death and the Sterilization of Medium 1695.15 Concluding Remarks 171Further Reading 172Nomenclature 172Problems 1736 Kinetics of Continuous Culture 1836.1 Introduction 1836.2 Kinetic Description of a Continuous Culture 1856.2.1 Balance Equations for Continuous Culture 1856.2.2 Steady-State Behavior of a Continuous Culture 1876.2.2.1 Monod Kinetics 1876.2.2.2 Steady-State Concentration Profiles 1876.2.2.3 Washout 1896.2.3 Productivity in Continuous Culture 1906.3 Continuous Culture with Cell Recycling 1936.3.1 Increased Productivity with Cell Recycling 1936.3.2 Applications of Continuous Culture with Cell Recycling 1966.3.2.1 Low Substrate Levels in the Feed 1966.3.2.2 Low Residual Substrate Concentration 1976.3.2.3 Labile Product 1976.3.2.4 Selective Enrichment of Cell Subpopulation 1976.3.2.5 High-Intensity Mammalian Cell Culture 1976.4 Specialty Continuous Cultures 1996.4.1 Multiple-Stage Continuous Culture 1996.4.2 Immobilized Cell Culture System 2006.4.3 Continuous Culture with Mixed Populations 2016.5 Transient Response of a Continuous Culture 2026.5.1 Pulse Increase at the Substrate Level 2036.5.2 Step Change in Feed Concentration 2046.6 Concluding Remarks 205Further Reading 205Nomenclature 205Problems 2067 Bioreactor Kinetics 2177.1 Bioreactors 2177.2 Basic Types of Bioreactors 2187.2.1 Flow Characteristics in Idealized Stirred-Tank (Well-Mixed) and Tubular (Plug Flow) Reactors 2197.2.2 Reaction in an Idealized CSTR 2207.2.3 Reaction in an Idealized PFR 2227.2.4 Heterogeneous and Multiphasic Bioreactors – Segregation of Holding Time 2257.3 Comparison of CSTR and PFR 2257.3.1 CSTR versus PFR in Conversion Yield and Reaction Rate 2257.3.2 CSTR versus PFR in Terms of Nutrient Depletion and Scale-Up 2267.3.3 CSTR versus PFR – A Perspective from Residence Time Distribution 2277.4 Operating Mode of Bioreactors 2297.4.1 Batch Cultures 2297.4.2 Fed-Batch Cultures 2297.4.2.1 Intermittent Harvest 2297.4.2.2 Fed-Batch 2307.5 Configuration of Bioreactors 2317.5.1 Simple Stirred-Tank Bioreactor 2317.5.2 Airlift Bioreactor 2337.5.3 Hollow-Fiber Bioreactor 2337.6 Other Bioreactor Applications 2337.7 Cellular Processes through the Prism of Bioreactor Analysis 2357.8 Concluding Remarks 236Further Reading 236Nomenclature 237Problems 2378 Oxygen Transfer in Bioreactors 2418.1 Introduction 2418.2 Oxygen Supply to Biological Systems 2428.3 Oxygen and Carbon Dioxide Concentration in Medium – Henry’s Law 2438.4 Oxygen Transfer through the Gas–Liquid Interface 2448.4.1 A Film Model for Transfer across the Interface 2448.4.2 Concentration Driving Force for Interfacial Transfer 2458.4.3 Mass Transfer Coefficient and Interfacial Area 2468.5 Oxygen Transfer in Bioreactors 2488.5.1 Material Balance on Oxygen in a Bioreactor 2498.5.2 Oxygen Transfer in a Stirred Tank 2518.6 ExperimentalMeasurement of KLa and OUR 2538.6.1 Determination of KLa in a Stirred-Tank Bioreactor 2538.6.2 Measurement of OUR and qO2 2548.7 Oxygen Transfer in Cell Immobilization Reactors 2568.8 Concluding Remarks 256Further Reading 256Nomenclature 256Problems 2589 Scale-Up of Bioreactors and Bioprocesses 2659.1 Introduction 2659.2 General Considerations in Scale Translation 2669.2.1 Process and Equipment Parameters Affected by Scale-Up 2669.2.2 Scale Translation for Product Development and Process Troubleshooting 2669.2.3 How Scale-Up Affects Process Variables, Equipment, and Cellular Physiology 2679.2.4 Scale-Up of Equipment and Geometrical Similarity 2679.3 Mechanical Agitation 2689.4 Power Consumption and Mixing Characteristics 2699.4.1 Power Consumption of Agitated Bioreactors 2699.4.2 Other Dimensionless Numbers 2729.4.3 Correlation of Oxygen Transfer Coefficient 2739.5 Effect of Scale on Physical Behavior of Bioreactors 2739.6 Mixing Time 2769.6.1 Nutrient Enrichment Zone: Mixing Time versus Starvation Time 2769.6.2 Mixing Time 2779.6.3 Mixing Time Distribution 2789.7 Scaling Up and Oxygen Transfer 2799.7.1 Material Balance on Oxygen in Bioreactor 2799.7.1.1 Aeration Rate and the Oxygen Transfer Driving Force 2809.8 Other Process Parameters and Cell Physiology 2819.9 Concluding Remarks 282Further Reading 283Nomenclature 283Problems 28410 Cell Culture Bioprocesses and Biomanufacturing 28910.1 Cells in Culture 28910.2 Cell Culture Products 29010.2.1 Vaccines 29010.2.2 Therapeutic Proteins 29110.2.3 Biosimilars 29210.3 Cellular Properties Critical to Biologics Production 29410.3.1 Protein Secretion 29410.3.1.1 Folding in the Endoplasmic Reticulum 29410.3.1.2 Membrane Vesicle Translocation and Golgi Apparatus 29510.3.2 Glycosylation 29610.3.3 Protein Secretion and Glycan Heterogeneity 29610.4 Nutritional Requirements 29910.4.1 Chemical Environment In Vivo and in Culture 29910.4.2 Types of Media 30010.4.2.1 Basal Medium and Supplements 30010.4.2.2 Complex Medium, Defined Medium 30110.5 Cell Line Development 30110.5.1 Host Cells and Transfection 30110.5.2 Amplification 30210.6 Bioreactors 30410.6.1 Roller Bottles 30410.6.2 Stirred-Tank Bioreactors for Suspension Cells 30510.6.3 Stirred-Tank Bioreactor with Microcarrier Cell Support 30610.6.4 Disposable Systems 30710.7 Cell Retention and Continuous Processes 30710.7.1 Continuous Culture and Steady State 30710.8 Cell Culture Manufacturing – Productivity and Product Quality 30810.8.1 Process and Product Quality 30810.8.2 Product Life Cycle 30910.8.3 Product Manufacturing 31110.8.3.1 Platform Process 31110.8.3.2 Manufacturing 31110.9 Concluding Remarks 312Further Reading 312Problems 31311 Introduction to Stem Cell Bioprocesses 31911.1 Introduction to Stem Cells 31911.2 Types of Stem Cells 32011.2.1 Adult Stem Cells 32011.2.1.1 Hematopoietic Stem Cells 32111.2.1.2 Mesenchymal Stem Cells 32311.2.1.3 Neuronal Stem Cells 32311.2.2 Embryonic Stem Cells 32411.2.3 Induced Pluripotent Stem Cells and Reprogramming 32411.3 Differentiation of Stem Cells 32611.4 Kinetic Description of Stem Cell Differentiation 32811.5 StemCell Technology 33111.6 Engineering in Cultivation of Stem Cells 33211.7 Concluding Remarks 335Further Reading 335Nomenclature 336Problems 33612 Synthetic Biotechnology: FromMetabolic Engineering to Synthetic Microbes 33912.1 Introduction 33912.2 Generalized Pathways for Biochemical Production 34012.3 General Strategy for Engineering an Industrial, Biochemical-Producing Microorganism 34212.3.1 Genomics, Metabolomics, Deducing Pathway, and Unveiling Regulation 34212.3.2 Introducing Genetic Alterations 34312.3.3 Isolating Superior Producers 34512.3.3.1 Screening of Mutants with the Desired Phenotype 34512.3.3.2 Selection of Mutants with the Target Trait 34512.3.4 Mechanisms of Enhancing the Biosynthetic Machinery 34712.3.4.1 Relaxing the Constriction Points in the Pathway 34712.3.4.2 Channeling Precursor Supply 34812.3.4.3 Eliminating Product Diversion 35012.3.4.4 Enhancing Product Transport 35012.3.4.5 Rerouting Pathways 35012.3.5 Engineering Host Cells – Beyond the Pathway 35212.3.5.1 Altering Substrate Utilization 35212.3.5.2 Manipulating the Time Dynamics of Production 35212.3.5.3 Increasing Product Tolerance 35412.4 Pathway Synthesis 35612.4.1 Host Cells: Native Hosts versus Archetypical Hosts 35612.4.2 Expressing Heterologous Enzymes to Produce a Nonnative Product 35712.4.3 Activating a Silent Pathway in a Native Host 35912.5 Stoichiometric and Kinetic Considerations in Pathway Engineering 35912.6 Synthetic Biology 36712.6.1 Synthetic (Cell-Free) Biochemical Reaction System 36712.6.2 Synthetic Circuits 36912.6.2.1 Artificial Genetic Circuits 36912.6.2.2 Synthetic Signaling Pathway 36912.6.3 Synthetic Organisms 37112.6.3.1 Minimum Genome and Reduced Genome 37112.6.3.2 Chemical Synthesis of a Genome 37212.6.3.3 Surrogate Cells from a Synthetic Genome 37412.7 Concluding Remarks 374Further Reading 374Problems 37513 Process Engineering of Bioproduct Recovery 38113.1 Introduction 38113.2 Characteristics of Biochemical Products 38213.3 General Strategy of Bioproduct Recovery 38513.3.1 Properties Used in Bioseparation 38513.3.2 Stages in Bioseparation 38713.3.2.1 Cell and Solid Removal 38713.3.2.2 Product Isolation (Capture) and Volume Reduction 38713.3.2.3 Product Purification 38813.3.2.4 Product Polishing 38813.4 Unit Operations in Bioseparation 38913.4.1 Filtration 38913.4.2 Centrifugation 39013.4.3 Liquid–Liquid Extraction 39313.4.4 Liquid Chromatography 39513.4.5 Membrane Filtration 39613.4.6 Precipitation and Crystallization 39713.5 Examples of Industrial Bioseparation Processes 39813.5.1 Recombinant Antibody IgG 39813.5.2 Penicillin 40113.5.3 Monosodium Glutamate 40413.5.4 Cohn Fractionation 40413.6 Concluding Remarks 404Further Reading 406Nomenclature 407Problems 40814 Chromatographic Operations in Bioseparation 41314.1 Introduction 41314.2 Adsorbent 41514.2.1 Types of Adsorbent 41514.2.2 Ligand and Mechanism of Separation 41814.2.3 Types of Liquid Chromatography 41914.3 Adsorption Isotherm 42014.3.1 Adsorption Equilibrium: Langmuir Isotherm 42014.3.2 Isotherm Dynamics in Adsorption and Desorption 42114.4 Adsorption Chromatography 42514.4.1 Discrete-Stage Analysis 42514.4.2 Breakthrough Curve 42714.4.3 An Empirical Two-Parameter Description of a Breakthrough Curve 42914.4.4 One-Porosity Model for an Adsorption Process 43114.4.5 Elution of Solutes from an Adsorption Column 43314.5 Elution Chromatography 43514.5.1 Discrete-Stage Analysis 43514.5.2 Determination of Stage Number 44114.5.3 Effect of Stage Number and Number of Theoretical Plates 44214.5.4 Two-Porosity Model, Mass Transfer Limitation 44414.6 Scale-Up and Continuous Operation 44714.6.1 Mass Transfer Limitation and the van Deemter Equation 44714.6.2 Scale-Up of Chromatography 44814.6.3 Continuous Adsorption and Continuous Elution Chromatography 45014.7 Concluding Remarks 454Further Reading 454Nomenclature 454Problems 456Index 471