Rational Design of Stable Protein Formulations

Recombinant proteins and polypeptides continue to be the most important class of biotechnology-derived agents in today's pharmaceutical industry. Over the past few years, our fundamental understanding of how proteins degrade and how stabilizing agents work has made it possible to approach formulation of protein pharmaceuticals from a much more rational point of view.

This book describes the current level of understanding of protein instability and the strategies for stabilizing proteins under a variety of stressful conditions.

1 Practical Approaches to Protein Formulation Development.- Preparation for Formulation Development.- Resource Requirements for Formulation Development.- Useful Information for Designing Formulations.- Preformulation Development.- Characterization of Protein Pharmaceuticals.- Accelerated Stability Studies.- Development of Analytical Methods.- Evaluation of the Significance of Problems.- Formulation Development.- Formulation Options for Protein Pharmaceuticals.- Typical Protein Stability Problems: Causes and Solutions.- Optimization of Formulation Variables.- Necessary Studies for Formulation Development.- Strategies to Overcome Difficult Formulation Problems.- Formulation in Commercial Product Development.- Critical Formulation Decisions During Pharmaceutical Development.- Formulation for Early Preclinical and Clinical Studies.- Commercial Formulation.- Regulatory Issues in Formulation Development.- Appendix: List of Regulatory Documents.- References.- 2 Recombinant Production of Native Proteins from Escherichia coli.- Distribution of Expressed Proteins.- Cell Washing and Lysis.- Purification of Soluble, Folded Proteins.- Purification and Refolding of Soluble, Misfolded Proteins.- Purification and Refolding of Proteins from Inclusion Bodies.- Washing and Solubilization of Inclusion Bodies.- Purification of Expressed Proteins from Inclusion Bodies.- Refolding Mechanism.- Disulfide Bond Formation.- Removal of Denaturant.- Effects of Tag Sequences.- Effects of Excipients.- Response Surface Methodology.- High Pressure Disaggregation and Refolding.- Methods to Analyze Folded Structures.- Bioactivity.- Binding to Receptors.- Dilsulfide Bond Analysis.- Spectroscopy.- Conformational Stability.- Limited Proteolysis.- References.- 3 Physical Stabilization of Proteins in Aqueous Solution.- Overview of Physical Stability.- Thermodynamic Control of Protein Stability.- Kinetic Control of Protein Stability.- Interactions of Excipients with Proteins.- Preferentially Excluded Cosolvents.- Buffers/Salts.- Specific Binding of Ligands.- Protein Self-Stabilization.- Physical Factors Affecting Protein Stability.- Temperature.- Freeze-Thawing.- Agitation and Exposure to Denaturing Interfaces.- Pressure.- Conclusions.- Appendix: Derivation of the Wyman Linkage Function and Application to the Timasheff Preferential Exclusion Mechanism.- References.- 4 Effects of Conformation on the Chemical Stability of Pharmaceutically Relevant Polypeptides.- Relationship Between Structure and Deamidation Rates.- Primary Structure Effects.- Secondary Structure Effects.- Tertiary Structure Effects.- Summary of Structure Effects on Deamidation.- Role of Structure in Protein Oxidation.- Types of Oxidation Processes.- Effects of Oxidation of Surface and Buried Methionines on Protein Structure.- Limiting Solvent Accessibility of Residues.- Conformational Control of Oxidation in Aqueous Solution.- Structural Control of Oxidation in Lyophilized Products.- Summary of Structural Control of Oxidation.- Summary.- References.- 5 Rational Design of Stable Lyophilized Protein Formulations: Theory and Practice.- Minimal Criteria for a Successful Lyophilized Formulation.- Inhibition of Lyophilization-Induced Protein Unfolding.- Storage at Temperatures Below Formulation Glass Transition Temperature.- The Water Content is Relatively Low.- A Strong, Elegant Cake Structure is Obtained.- Steps Taken to Minimize Specific Routes of Protein Chemical Degradation.- Rational Design of Stable Lyophiilized Formulations.- Choice of Buffer.- Specific Ligands/pH that Optimizes Thermodynamic Stability of Protein.- Trehalose or Sucrose to Inhibit Protein Unfolding and Provide Glassy Matrix.- Bulking Agent (e.g., Mannitol, Glycine or Hydroxyethyl Starch).- Nonionic Surfactant to Inhibit Aggregation.- Acknowledgments.- References.- 6 Spray-Drying of Proteins.- Introduction: Why Spray-Dry a Protein?.- Developments in the Last 10 Years.- The Practice of Spray-Drying Proteins.- Type of Equipment.- Spray-Drying Conditions.- Influence of Formulation.- Pure Proteins.- Formulated Systems.- Use of Added Surface Active Substances.- Concluding Remarks.- References.- Chapter7 Surfactant-Protein Interactions.- Proteins and Surfactants at Surfaces.- Protein-Surfactant Interactions in Solution.- Surfactant Effects on Protein Assembly State.- Surfactant Effects on Proteins During Freezing, Freeze-Drying and Reconstitution.- Enzymatic Degradation of Non-Ionic Surfactants.- Recommendations for Protein Formulation.- References.- 8 High Throughput Formulation: Strategies for Rapid Development of Stahle Protein Products.- Overall Structure of the HTF Approach.- Role of an Established Decision Tree for Formulation Design.- Constraints on a Pharmaceutically Acceptable Protein Formulation.- Proper Choice of Dosage Form.- Preformulation Studies.- Proper Choice of Excipients.- Estimates of Resources Needed for Formulation Development.- Use of Software and Databases to Assist in the HTF Process.- Essential Analytical Methods.- Stability Protocols.- Unified Strategy for HTF.- References.