Biopharmaceutical Production Technology -- Contents -- Preface -- List of Contributors -- Part One: Upstream Technologies -- 1: Strategies for Plasmid DNA Production in Escherichia coli -- 1.1 Introduction -- 1.2 Requirements for a Plasmid DNA Production Process -- 1.3 Structure of a DNA Vaccine Production Process -- 1.4 Choice of Antigen -- 1.5 Vector DNA Construct -- 1.5.1 Popular Amplification Systems -- 1.5.2 Intrinsic Factors -- 1.6 Host Strains -- 1.6.1 endA and recA -- 1.6.2 relA -- 1.6.3 Nucleoside Pathway -- 1.6.4 gyrA -- 1.6.5 Strains for Production Processes -- 1.7 Cultivation Medium and Process Conditions -- 1.8 Lysis/Extraction of Plasmid DNA -- 1.9 Purification -- 1.9.1 Clarification of the Lysate and Intermediate Purification -- 1.9.2 Purification by Chromatography -- 1.9.2.1 Anion-Exchange Chromatography -- 1.9.2.2 Hydrophobic Interaction Chromatography -- 1.9.2.3 Gel Filtration -- 1.9.2.4 Membrane Chromatography -- 1.9.2.5 Chromatography on Porous Monolithic Supports -- 1.10 Formulation -- 1.10.1 Lipoplexes -- 1.10.2 Polyplexes -- 1.10.3 Inorganic Nanoparticles -- 1.11 Conclusions -- References -- 2: Advances in Protein Production Technologies -- 2.1 Introduction -- 2.2 Glycoengineering for Homogenous Human-Like Glycoproteins -- 2.3 Bacteria as Protein Factories -- 2.4 Mammalian Cell Technology -- 2.5 Yeast Protein Production -- 2.6 Baculovirus-Insect Cell Technology -- 2.7 Transgenic Animal Protein Production -- 2.8 Plant Molecular Farming -- 2.9 Cell-Free Protein Production -- 2.10 Future Prospects -- References -- Part Two: Protein Recovery -- 3: Releasing Biopharmaceutical Products from Cells -- 3.1 Introduction -- 3.2 Cell Structure and Strategies for Disruption -- 3.3 Cell Mechanical Strength -- 3.4 Homogenization -- 3.4.1 Mechanisms -- 3.4.2 Modeling -- 3.5 Bead Milling -- 3.5.1 Modeling -- 3.6 Chemical Treatment.

3.7 Cellular Debris -- 3.7.1 Modeling -- 3.8 Conclusions -- References -- 4: Continuous Chromatography (Multicolumn Countercurrent Solvent Gradient Purification) for Protein Purification -- 4.1 Introduction -- 4.1.1 Overview of the Biopharmaceutical Market -- 4.1.2 Overview of Purification of Biopharmaceuticals -- 4.1.3 Introduction to Continuous Chromatographic Processes -- 4.2 Overview of Continuous Chromatographic Processes -- 4.2.1 SMB and Its Derivatives -- 4.2.1.1 Applications of SMB in the Pharmaceutical Industry: Small Molecules -- 4.2.1.2 Limitations of SMB -- 4.2.2 MCSGP Goes Beyond SMB and Makes Continuous Chromatography Possible for Bioseparations -- 4.3 Principles of MCSGP -- 4.3.1 Tasks in Batch Chromatogram -- 4.3.1.1 Generic Purification Problem -- 4.3.2 Six-Column MCSGP Principle -- 4.3.3 Three-Column MCSGP Principle -- 4.3.4 Four-Column MCSGP with Separate CIP Position -- 4.3.5 Four-Column MCSGP with a Separate Position for Continuous Feed -- 4.3.6 MCSGP Process for Separations with More Than Three Fractions -- 4.4 Application Examples of MCSGP -- 4.4.1 Polypeptide Purification with Reversed-Phase Chromatography -- 4.4.2 mAb Charge Variant Separation -- 4.4.3 mAb Capture and Polish from Supernatant -- 4.4.4 Size-Exclusion Chromatographic Purification with MCSGP -- 4.5 Enabling Features and Economic Impact of MCSGP -- 4.6 Annex 1: Chromatographic Process Decision Tree -- References -- 5: Virus-Like Particle Bioprocessing -- 5.1 Introduction -- 5.2 Upstream Processing -- 5.2.1 Intracellular Expression and Assembly -- 5.2.2 Cell-Free Approaches -- 5.3 Downstream Processing -- 5.3.1 Gardasil Downstream Processing -- 5.3.2 VLP Aggregation -- 5.3.3 Purification of Cell-Assembled VLPs -- 5.3.4 Purification for In Vitro Assembly -- 5.4 Analysis -- 5.5 Conclusions -- 5.6 Nomenclature -- Acknowledgments -- References.

6: Therapeutic Protein Stability and Formulation -- 6.1 Introduction -- 6.2 Protein Stability -- 6.2.1 Structural Stability -- 6.2.2 Thermal Stability -- 6.2.3 Chaotropes, Solvents, and pH -- 6.2.4 Shear -- 6.2.5 Freezing -- 6.2.6 Drying -- 6.2.7 Air-Liquid and Solid-Liquid Interfaces -- 6.2.8 Chemical Stability -- 6.2.9 Precipitation, Aggregation, and Fibril Formation -- 6.2.10 Leachables -- 6.3 Formulation and Materials -- 6.3.1 Liquid Formulations -- 6.3.2 pH -- 6.3.3 Amino Acids and Other Organic Buffers -- 6.3.4 Sugars and Polyols -- 6.3.5 Salts -- 6.3.6 Surfactants -- 6.3.7 Specific Binding -- 6.3.8 Chelating Agents -- 6.3.9 Redox Potential -- 6.3.10 Containers and Closures -- 6.3.11 Frozen Formulations -- 6.3.12 Freeze-Dried Formulations -- 6.4 Screening Methods -- 6.4.1 DSC -- 6.4.2 Thermal Scanning with Spectroscopic Detection of Protein Unfolding -- 6.5 Accelerated and Long-Term Stability Testing -- 6.5.1 Regulatory Perspective -- 6.5.2 Accelerated Stability Testing -- 6.6 Analytical Techniques for Stability Testing -- 6.6.1 Cell-Based Bioassays and In Vitro Binding Assays -- 6.6.2 High-Performance Liquid Chromatography and Capillary Zone Electrophoresis -- 6.6.3 Mass Spectrometry-Based Analysis -- 6.6.4 Detection of Protein Aggregates -- 6.6.5 Crude Analytical Assays: PAGE, IEF, Blotting, FTIR, CD, and UV Fluorescence -- 6.7 Conclusions -- References -- 7: Production of PEGylated Proteins -- 7.1 Introduction -- 7.2 General Considerations -- 7.2.1 Efficiency of PEG Conjugation -- 7.2.2 Control of Positional Isomerism -- 7.2.3 Control of the Number of PEG Adducts -- 7.2.4 Purification of Target Products -- 7.3 PEGylation Chemistry -- 7.3.1 Amine Conjugation -- 7.3.2 Thiol Conjugation -- 7.3.3 Oxidized Carbohydrate or N-Terminal Conjugation -- 7.3.4 Transglutaminase-Mediated Enzymatic Conjugation -- 7.3.5 Miscellaneous Conjugation Chemistries.

7.3.6 Reversible PEGylation -- 7.4 PEGylated Protein Purification -- 7.4.1 Removal of Low-Molecular-Weight Contaminants -- 7.4.2 Removal of Free PEG -- 7.4.3 Separation of PEGylated and Native Protein Forms -- 7.4.4 Separation of PEGylated Species -- 7.5 Conclusions -- References -- Part Three: Advances in Process Development -- 8: Affinity Chromatography: Historical and Prospective Overview -- 8.1 History and Role of Affinity Chromatography in the Separation Sciences -- 8.1.1 Introduction -- 8.1.2 Early History -- 8.1.3 Biological Ligands -- 8.1.4 Synthetic and Designed Ligands -- 8.1.5 Alternative Ligands -- 8.1.6 Role of Affinity Chromatography in the Separation Sciences -- 8.2 Overview of Affinity Chromatography: Theory and Methods -- 8.2.1 Basic Chromatographic Theory -- 8.2.2 Matrix Selection and Immobilization of an Affinity Ligand -- 8.2.3 Other Considerations -- 8.3 Affinity Ligands -- 8.3.1 Biological Ligands -- 8.3.1.1 Immunoaffinity Adsorbents -- 8.3.1.2 Bacterial Proteins -- 8.3.1.3 Lectins -- 8.3.1.4 Heparin -- 8.3.1.5 Glutathione -- 8.3.1.6 Avidin and Streptavidin -- 8.3.1.7 Vitamins and Hormones -- 8.3.1.8 Nucleic Acids -- 8.3.1.9 Alternative Affinity Methods -- 8.3.2 Synthetic and Designed Ligands -- 8.3.2.1 Immobilized Metals -- 8.3.2.2 Hydrophobic Ligands -- 8.3.2.3 Thiophilic Ligands -- 8.3.2.4 Histidine -- 8.3.2.5 Mixed-Mode Adsorbents -- 8.3.2.6 Boronate -- 8.3.2.7 Benzhydroxamic Acid -- 8.3.2.8 Dye Ligands -- 8.3.2.9 Biomimetics -- 8.4 Affinity Ligands in Practice: Biopharmaceutical Production -- 8.5 Conclusions and Future Perspectives -- References -- 9: Hydroxyapatite in Bioprocessing -- 9.1 Introduction -- 9.2 Materials and Interaction Mechanisms -- 9.2.1 Apatites for Chromatography -- 9.2.2 Structure-Function Relationship -- 9.2.3 Retention Mechanisms in Apatite Chromatography -- 9.3 Setting up a Separation.

9.3.1 General Considerations -- 9.3.2 Elution Mode -- 9.3.3 Displacement Mode -- 9.4 Separation Examples -- 9.4.1 Proteins in General -- 9.4.2 Antibodies -- 9.4.3 Polynucleotides -- 9.4.4 Others -- 9.5 Conclusions -- References -- 10: Monoliths in Bioprocessing -- 10.1 Introduction -- 10.2 Properties of Chromatographic Monoliths -- 10.3 Monolithic Analytical Columns for Process Analytical Technology Applications -- 10.3.1 Upstream Applications -- 10.3.2 Downstream Applications -- 10.3.2.1 HPLC Analysis of IgG Proteins -- 10.3.2.2 HPLC Analysis of the IgM Samples -- 10.3.2.3 HPLC Anion-Exchange Analysis of the PEGylated Proteins -- 10.3.2.4 Viruses -- 10.4 Monoliths for Preparative Chromatography -- 10.4.1 Protein Purification -- 10.4.2 Purification of Viruses -- 10.4.3 Plasmid DNA Purification -- 10.4.4 Negative Chromatography -- 10.5 Enzyme Reactors -- 10.5.1 Proteome Analysis -- 10.5.2 Biosensors -- 10.5.3 Bioconversion of Target Molecules -- 10.5.4 Study of Enzyme-Intrinsic Properties -- 10.6 Conclusions -- References -- 11: Membrane Chromatography for Biopharmaceutical Manufacturing -- 11.1 Membrane Adsorbers - Introduction and Technical Specifications -- 11.1.1 Introduction -- 11.1.2 Membrane Adsorber Construction -- 11.1.3 Types of Available Ligands -- 11.1.4 Use and Scaling-Up with Membrane Adsorbers -- 11.2 Comparing Resins and Membrane Adsorbers -- 11.2.1 Flow-Through Polishing Applications -- 11.2.2 Bind-and-Elute Applications -- 11.2.3 Economical Modeling and Case Studies -- 11.3 Membrane Chromatography Applications and Case Studies -- 11.3.1 Validation of Membranes into a Purification Process -- 11.3.2 Virus Purification and Vaccine Manufacture -- 11.3.3 Virus Removal -- 11.3.4 Endotoxin Removal -- 11.3.5 HCP Removal -- 11.3.6 DNA Removal -- 11.3.7 Aggregate Reduction -- 11.4 Conclusions -- References.

12: Modeling and Experimental Model Parameter Determination with Quality by Design for Bioprocesses.