Bioseparations Science and Engineering

Dr. Roger G. Harrison is the David Ross Boyd Professor of Sustainable Chemical, Biological and Materials Engineering at the University of Oklahoma. Dr. Paul W. Todd is retired Research Professor of Chemical Engineering, University of Colorado; and Chief Scientist Emeritus, Techshot, Inc. (now Redwire, Inc.).Dr. Scott R. Rudge is a Founder and Principal Consultant with Syner-G Biopharma Group. Dr. Demetri P. Petrides is the president of Intelligen, Inc., a software company that develops and markets simulation, design, and scheduling tools for the process manufacturing industries.

• Preface xix 1. Introduction to Bioproducts and Bioseparations 1.1 Instructional Objectives 1.2 Broad Classification of Bioproducts 1.3 Small Biomolecules 1.3.1 Primary Metabolites 1.3.2 Secondary Metabolites 1.3.3 Stability of Small Biomolecules1.3.4 Summary of Small Biomolecules 1.4 Macromolecules: Proteins 1.4.1 Primary Structure 1.4.2 Secondary Structure 1.4.3 Tertiary Structure Example 1.1. Effect of a Reducing Agent on Protein Structure and Mobility 1.4.4 Quaternary Structure 1.4.5 Prosthetic Groups and Hybrid Molecules 1.4.6 Functions and Commercial Uses of Proteins 1.4.7 Stability of Proteins 1.4.8 Recombinant Protein Expression 1.5 Macromolecules: Nucleic Acids and Oligonucleotides 1.5.1 Structure of Nucleic Acids 1.5.2 Functions and Commercial Uses 1.5.3 Stability of Nucleic Acids 1.6 Macromolecules: Polysaccharides 1.7 Particulate Products 1.8 Introduction to Bioseparations: Engineering Analysis 1.8.1 Stages of Downstream Processing Example 1.2. Initial Selection of Purification Steps 1.8.2 Basic Principles of Engineering Analysis 1.8.3 Process and Product Quality 1.8.4 Criteria for Process Development 1.9 The Route to Market 1.9.1 The Chemical and Applications Range of the Bioproduct 1.9.2 Documentation of Pharmaceutical Bioproducts 1.9.3 GLP and cGMP 1.9.4 Formulation 1.10 Summary Nomenclature Problems References 2. Analytical Methods and Bench Scale Preparative Bioseparations 2.1 Instructional Objectives 2.2 Specifications 2.3 Assay Attributes 2.3.1 Precision 2.3.2 Accuracy 2.3.3 Specificity 2.3.4 Linearity, Limit of Detection, and Limit of Quantitation 2.3.5 Range 2.3.6 Robustness 2.4 Analysis of Biological Activity 2.4.1 Animal Model Assays 2.4.2 Cell-Line-Derived Bioassays 2.4.3 In vitro Biochemical Assays Example 2.1. Coupled Enzyme Assay for Alcohol Oxidase 2.5 Analysis of Purity 2.5.1 Electrophoretic Analysis Example 2.2. Estimation of the Maximum Temperature in an Electrophoresis Gel 2.5.2 High-Performance Liquid Chromatography (HPLC) 2.5.3 Mass Spectrometry 2.5.4 Coupling of HPLC with Mass Spectrometry 2.5.5 Ultraviolet Absorbance Example 2.3. Determination of Molar Absorptivity 2.5.6 CHNO/Amino Acid Analysis (AAA) Example 2.4. Calculations Based on CHNO Analysis 2.5.7 Protein Assays 2.5.8 Enzyme-Linked Immunosorbent Assay 2.5.9 Gas Chromatography 2.5.10 DNA Hybridization 2.5.11 ICP/MS (AES) 2.5.12 Dry Weight 2.5.13 Flow Cytometry2.6 Microbiology Assays 2.6.1 Sterility 2.6.2 Bioburden 2.6.3 Endotoxin 2.6.4 Virus, Mycoplasma, and Phage 2.7 Bench Scale Preparative Separations 2.7.1 Preparative Electrophoresis 2.7.2 Magnetic Bioseparations2.7.3 Cell Purification by Flow Cytometry 2.8 Summary Nomenclature Problems References 3. Cell Lysis and Flocculation 3.1 Instructional Objectives 3.2 Some Elements of Cell Structure 3.2.1 Prokaryotic Cells 3.2.2 Eukaryotic Cells 3.3 Cell Lysis 3.3.1 Osmotic and Chemical Cell Lysis 3.3.2 Mechanical Methods of Lysis 3.4 Flocculation 3.4.1 The Electric Double Layer Example 3.1. Dependence of the Debye Radius on the Type of Electrolyte 3.4.2 Forces Between Particles and Flocculation by Electrolytes Example 3.2. Sensitivity of Critical Flocculation Concentration to Temperature and Counterion Charge Number 3.4.3 The Schulze-Hardy Rule 3.4.4 Flocculation Rate 3.4.5 Polymeric Flocculants 3.5 Summary Nomenclature Problems References 4. Filtration 4.1 Instructional Objectives 4.2 Filtration Principles 4.2.1 Conventional Filtration Example 4.1. Batch Filtration 4.2.2 Crossflow Filtration Example 4.2. Concentration Polarization in Ultrafiltration Example 4.3. Comparison of Mass Transfer Coefficient Calculated by Boundary Layer Theory Versus by Shear-Induced Diffusion Theory 4.3 Filter Media and Equipment 4.3.1 Conventional Filtration 4.3.2 Crossflow Filtration 4.4 Membrane Fouling 4.5 Scale-up and Design of Filtration Systems 4.5.1 Conventional Filtration Example 4.4. Rotary Vacuum Filtration Example 4.5. Washing of a Rotary Vacuum Filter Cake 4.5.2 Crossflow Filtration Example 4.6. Diafiltration Mode in Crossflow Filtration 4.6 Summary Nomenclature Problems References 5. Sedimentation 5.1 Instructional Objectives 5.2 Sedimentation Principles 5.2.1 Equation of Motion 5.2.2 Sensitivities 5.3 Methods for Analysis of Sedimentation 5.3.1 Equilibrium Sedimentation 5.3.2 Sedimentation Coefficient Example 5.1. Application of the Sedimentation Coefficient 5.3.3 Equivalent Time Example 5.2. Scale-up Based on Equivalent Time 5.3.4 Sigma Analysis 5.4 Production Centrifuges: Comparison and Engineering Analysis 5.4.1 Tubular Bowl Centrifuge Example 5.3. Complete Recovery of Bacterial Cells in a Tubular Bowl Centrifuge 5.4.2 Disk Centrifuge 5.5 Ultracentrifugation 5.5.1 Determination of Molecular Weight 5.6 Flocculation and Sedimentation 5.7 Sedimentation at Low Accelerations 5.7.1 Diffusion, Brownian Motion 5.7.2 Isothermal Settling 5.7.3 Convective Motion and Péclet Analysis 5.7.4 Inclined Sedimentation 5.7.5 Field-Flow Fractionation 5.8 Centrifugal Elutriation 5.9 Summary Nomenclature Problems References 6. Extraction 6.1 Instructional Objectives 6.2 Extraction Principles 6.2.1 Phase Separation and Partitioning Equilibria Example 6.1 Process for Large-Scale Isolation of ?-Galactosidae from E. coli in an Aqueous Two-Phase Sytstem6.2.2 Countercurrent Stage Calculations Example 6.2. Separation of a Bioproduct and an Impurity by Countercurrent Extraction Example 6.3. Effect of Solvent Rate in Countercurrent Staged Extraction of an Antibiotic 6.3 Scale-up and Design of Extractors 6.3.1 Reciprocating-Plate Extraction Columns Example 6.4. Scale-up of a Reciprocating-Plate Extraction Column 6.3.2 Centrifugal Extractors Example 6.5. Increase in Feed Rate to a Podbielniak Centrifugal Extractor 6.4 Summary Nomenclature Problems References 7. Liquid Chromatography and Adsorption 7.1 Instructional Objectives 7.2 Adsorption Equilibrium 7.3 Adsorption Column Dynamics 7.3.1 Fixed-Bed Adsorption Example 7.1. Determination of the Mass Transfer Coefficient from Adsorption Breakthrough Data 7.3.2 Agitated-Bed Adsorption 7.4 Chromatography Column Dynamics 7.4.1 Plate Models 7.4.2 Moment Analysis Example 7.2 Calculation of the HETP Using the Method of Moments7.4.3 Chromatography Column Mass Balance with Negligible Dispersion Example 7.3. Chromatographic Separation of Two Solutes Example 7.4. Calculation of the Shock Wave Velocity for a Nonlinear Isotherm Example 7.5. Calculation of the Elution Profile 7.4.4 Dispersion Effects in Chromatography 7.4.5 Computer Simulation of Chromatography Considering Axial Dispersion, Fluid-Phase Mass Transfer, Intraparticle Diffusion, and Nonlinear Equilibrium Example 7.6 Computer Simulation of a Chromatography Process 7.4.6 Gradients and Modifiers Example 7.7. Equilibrium for a Protein Anion in the Presence of Chloride Ion 7.5 Membrane Chromatography Example 7.8. Comparison of Time for Diffusion Mass Transfer in Conventional Chromatography and Membrane Chromatography 7.6 Simulated Moving Bed Chromatography 7.7 Adsorbent Types 7.7.1 Silica-Based Resins 7.7.2 Polymer-Based Resins 7.7.3 Ion Exchange Chromatography and Adsorption 7.7.4 Reversed-Phase Chromatography 7.7.5 Hydrophobic Interaction Chromatography 7.7.6 Affinity Chromatography 7.7.7 Immobilized Metal Affinity Chromatography (IMAC) 7.7.8 Size Exclusion Chromatography 7.8 Particle Size and Pressure Drop in Fixed Beds 7.9 Equipment 7.9.1 Columns 7.9.2 Chromatography Column Packing Procedures 7.9.3 Detectors 7.9.4 Chromatography System Fluidics 7.10 Scale-up 7.10.1 Adsorption Example 7.9. Scale-up of the Fixed-Bed Adsorption of a Pharmaceutical Product 7.10.2 Chromatography Example 7.10. Scale-up of a Protein Chromatography Example 7.11. Scale-up of Protein Chromatography Using Standard Column Sizes Example 7.12. Scale-up of Elution Buffer Volumes in Protein Chromatography Example 7.13. Consideration of Pressure Drop in Column Scaling 7.11 Summary Nomenclature Problems References 8. Precipitation 8.1 Instructional Objectives 8.2 Protein Solubility 8.2.1 Structure and Size 8.2.2 Charge 8.2.3 Solvent Example 8.1. Salting Out of a Protein with Ammonium Sulfate 8.3 Precipitate Formation Phenomena 8.3.1 Initial Mixing 8.3.2 Nucleation 8.3.3 Growth Governed by Diffusion Example 8.2. Calculation of Concentration of Nuclei in a Protein Precipitation Example 8.3. Diffusion-Limited Growth of Particles 8.3.4 Growth Governed by Fluid Motion Example 8.4. Growth of Particles Limited by Fluid Motion 8.3.5 Precipitate Breakage 8.3.6 Precipitate Aging 8.4 Particle Size Distribution in a Continuous-Flow Stirred Tank Reactor Example 8.5. Dependence of Population Density on Particle Size and Residence Time in a CSTR 8.5 Methods of Precipitation 8.6 Design of Precipitation Systems 8.7 Summary Nomenclature Problems References 9. Crystallization 9.1 Instructional Objectives 9.2 Crystallization Principles 9.2.1 Crystals 9.2.2 Nucleation 9.2.3 Crystal Growth 9.2.4 Crystallization Kinetics from Batch Experiments 9.3 Batch Crystallizers 9.3.1 Analysis of Dilution Batch Crystallization Example 9.1. Batch Crystallization with Constant Rate of Change of Diluent Concentration 9.3.2 Cooling Batch Crystallization Example 9.2 Batch Crystallization by Cooling 9.4 Continuous CrystallizationExample 9.3 Calculation of the Population Density and the Growth and Nucleation Rates for a MSMPR Crystallizer9.5 Process Crystallization of Proteins 9.6 Crystallizer Scale-up and Design 9.6.1 Experimental Crystallization Studies as a Basis for Scale-up 9.6.2 Scale-up and Design Calculations Example 9.4. Scale-up of Crystallization Based on Constant Power per Volume 9.7 Summary Nomenclature Problems References 10. Evaporation 10.1 Instructional Objectives 10.2 Evaporation Principles 10.2.1 Heat Transfer Example 10.1. Evaporation of a Butyl Acetate Stream Containing a Heat-Sensitive Antibiotic in a Falling-Film Evaporator 10.2.2 Vapor-Liquid Separation 10.3 Evaporation Equipment 10.3.1 Climbing-Film Evaporators 10.3.2 Falling-Film Evaporators 10.3.3 Forced-Circulation Evaporators 10.3.4 Agitated-Film Evaporators 10.4 Scale-up and Design of Evaporators 10.5 Summary Nomenclature Problems References 11. Drying 11.1 Instructional Objectives 11.2 Drying Principles 11.2.1 Water in Biological Solids and in Gases Example 11.1. Drying of Antibiotic Crystals 11.2.2 Heat and Mass Transfer Example 11.2. Conductive Drying of Wet Solids in a Tray Example 11.3. Mass Flux During the Constant Rate Drying Period in Convective Drying Example 11.4. Time to Dry Nonporous Biological Solids by Convective Drying 11.3 Dryer Description and Operation 11.3.1 Vacuum-Shelf Dryers 11.3.2 Batch Vacuum Rotary Dryers 11.3.3 Freeze Dryers 11.3.4 Spray Dryers 11.5 Fluidized Bed Dryers11.4 Scale-up and Design of Drying Systems 11.4.1 Vacuum-Shelf Dryers 11.4.2 Batch Vacuum Rotary Dryers 11.4.3 Freeze Dryers 11.4.4 Spray Dryers Example 11.5. Sizing of a Spray Dryer 11.4.5 Fluidized Bed Dryers Example 11.6 Scale-up of a Fluidized Bed Dryer11.5 Summary Nomenclature Problems References 12. Bioprocess Design and Economics 12.1 Instructional Objectives 12.2 Definitions and Background 12.3 Synthesis of Bioseparation Processes 12.3.1 Primary Recovery Stages 12.3.2 Intermediate Recovery Stages 12.3.3 Final Purification Stages 12.3.4 Pairing of Unit Operations in Process Synthesis 12.4 Process Analysis 12.4.1 Spreadsheets 12.4.2 Process Simulators and Their Benefits 12.4.3 Using a Biochemical Process Simulator 12.5 Process Economics 12.5.1 Capital Cost Estimation 12.5.2 Operating Cost Estimation 12.5.3 Profitability Analysis 12.6 Illustrative Examples 12.6.1 Citric Acid Production 12.6.2 Human Insulin Production 12.6.3 Therapeutic Monoclonal Antibody Production 12.6.4 RNA (mRNA) Vaccine Production12.6.5 Hyaluronic Acid Production12.6.6 Monosodium Glutamate (MSG) Production12.7 Summary Problems References 13. Laboratory Exercises in Bioseparations 13.1 Flocculant Screening 13.1.1 Background 13.1.2 Objectives 13.1.3 Procedure 13.1.4 Report 13.1.5 Some Notes and Precautions 13.2 Crossflow Filtration 13.2.1 Background 13.2.2 Objectives 13.2.3 Procedure 13.2.4 Report 13.3 Centrifugation of Flocculated and Unflocculated Particulates 13.3.1 Background 13.3.2 Objectives 13.3.3 Procedure 13.3.4 Report 13.4 Aqueous Two-Phase Extraction 13.4.1 Physical Measurements 13.4.2 Procedure 13.4.3 Calculations and Report 13.4.4 Inverse Lever Rule 13.5 Chromatography Scale-up 13.5.1 Background 13.5.2 Objectives 13.5.3 Procedure 13.5.4 Report References APPENDIX: Table of Units and Constants Index