| Practical Approaches to Protein Formulation Development | |
| Introduction | p. 1 |
| Preparation for Formulation Development | p. 3 |
| Resource Requirements for Formulation Development | p. 3 |
| Useful Information for Designing Formulations | p. 4 |
| Preformulation Development | p. 4 |
| Characterization of Protein Pharmaceuticals | p. 5 |
| Accelerated Stability Studies | p. 5 |
| Developmentof Analytical Methods | p. 6 |
| Evaluation of the Significance of Problems | p. 7 |
| Formulation Development | p. 10 |
| Formulation Options for Protein Pharmaceuticals | p. 10 |
| Typical Protein Stability Problems: Causes and Solutions | p. 13 |
| Optimization of Formulation Variables | p. 13 |
| Necessary Studies for Formulation Development | p. 15 |
| Strategies to Overcome Difficult Formulation Problems | p. 17 |
| Formulation in Commercial Product Development | p. 18 |
| Critical Formulation Decisions During Pharmaceutical Development | p. 18 |
| Formulation for Early Preclinical and Clinical Studies | p. 19 |
| Commercial Formulation | p. 19 |
| Regulatory Issues in Formulation Development | p. 20 |
| List of Regulatory Documents | p. 22 |
| References | p. 23 |
| Recombinant Production of Native Proteins from Escherichia coli | |
| Introduction | p. 27 |
| Distribution of Expressed Proteins | p. 28 |
| Cell Washing and Lysis | p. 32 |
| Purification of Soluble, Folded Proteins | p. 34 |
| Purification and Refolding of Soluble, Misfolded Proteins | p. 35 |
| Purification and Refolding of Proteins from Inclusion Bodies | p. 36 |
| Washing and Solubilization of Inclusion Bodies | p. 36 |
| Purification of Expressed Proteins from Inclusion Bodies | p. 36 |
| Refolding Mechanism | p. 38 |
| Disulfide Bond Formation | p. 41 |
| Removal of Denaturant | p. 41 |
| Effects of Tag Sequences | p. 44 |
| Effects of Excipients | p. 44 |
| Response Surface Methodology | p. 47 |
| High Pressure Disaggregation and Refolding | p. 48 |
| Methods to Analyze Folded Structures | p. 48 |
| Bioactivity | p. 49 |
| Binding to Receptors | p. 49 |
| Dilsulfide Bond Analysis | p. 50 |
| Spectroscopy | p. 50 |
| Conformational Stability | p. 51 |
| Limited Proteolysis | p. 51 |
| References | p. 51 |
| Physical Stabilization of Proteins in Aqueous Solution | |
| Introduction | p. 61 |
| Overview of Physical Stability | p. 62 |
| Thermodynamic Control of Protein Stability | p. 62 |
| Kinetic Control of Protein Stability | p. 63 |
| Interactions of Excipients with Proteins | p. 65 |
| Preferentially Excluded Cosolvents | p. 66 |
| Buffers/Salts | p. 67 |
| Specific Binding of Ligands | p. 68 |
| Protein Self-Stabilization | p. 69 |
| Physical Factors Affecting Protein Stability | p. 70 |
| Temperature | p. 70 |
| Freeze-Thawing | p. 71 |
| Agitation and Exposure to Denaturing Interfaces | p. 71 |
| Pressure | p. 72 |
| Conclusions | p. 73 |
| Derivation of the Wyman Linkage Function and Application to the Timasheff Preferential Exclusion Mechanism | p. 73 |
| References | p. 78 |
| Effects of Conformation on the Chemical Stability of Pharmaceutically Relevant Polypeptides | |
| Introduction | p. 85 |
| Relationship Between Structure and Deamidation Rates | p. 86 |
| Primary Structure Effects | p. 87 |
| Secondary Structure Effects | p. 89 |
| Tertiary Structure Effects | p. 91 |
| Summary of Structure Effects on Deamidation | p. 92 |
| Role of Structure in Protein Oxidation | p. 92 |
| Types of Oxidation Processes | p. 93 |
| Effects of Oxidation of Surface and Buried Methionines on Protein Structure | p. 95 |
| Limiting Solvent Accessibility of Residues | p. 96 |
| Conformational Control of Oxidation in Aqueous Solution | p. 97 |
| Structural Control of Oxidation in Lyophilized Products | p. 99 |
| Summary of Structural Control of Oxidation | p. 100 |
| Summary | p. 101 |
| References | p. 101 |
| Rational Design of Stable Lyophilized Protein Formulations: Theory and Practice | |
| Introduction | p. 109 |
| Minimal Criteria for a Successful Lyophilized Formulation | p. 111 |
| Inhibition of Lyophilization-Induced Protein Unfolding | p. 112 |
| Storage at Temperatures Below Formulation Glass Transition Temperature | p. 113 |
| The Water Content is Relatively Low | p. 114 |
| A Strong, Elegant Cake Structure is Obtained | p. 114 |
| Steps Taken to Minimzie Specific Routes of Protein Chemical Degradation | p. 116 |
| Rational Design of Stable Lyophiilized Formulations | p. 117 |
| Choice of Buffer | p. 118 |
| Specific Ligands/pH that Optimizes Thermodynamic Stability of Protein | p. 119 |
| Trehalose or Sucrose to Inhibit Protein Unfolding and Provide Glassy Matrix | p. 120 |
| Bulking Agent (e.g., Mannitol, Glycine or Hydroxyethyl Starch) | p. 126 |
| Nonionic Surfactant to Inhibit Aggregation | p. 127 |
| Acknowledgments | p. 127 |
| References | p. 127 |
| Spray-Drying of Proteins | |
| Introduction: Why Spray-Dry a Protein? | p. 135 |
| Developments in the Last 10 Years | p. 136 |
| The Practice of Spray-Drying Proteins | p. 139 |
| Type of Equipment | p. 139 |
| Spray-Drying Conditions | p. 140 |
| Influence of Formulation | p. 147 |
| Pure Proteins | p. 147 |
| Formulated Systems | p. 149 |
| Use of Added Surface Active Substances | p. 151 |
| Concluding Remarks | p. 156 |
| References | p. 156 |
| Surfactant-Protein Interactions | |
| Introduction | p. 159 |
| Proteins and Surfactants at Surfaces | p. 161 |
| Protein-Surfactant Interactions in Solution | p. 166 |
| Surfactant Effects on Protein Assembly State | p. 167 |
| Surfactant Effects on Proteins During Freezing, Freeze-Drying and Reconstitution | p. 169 |
| Enzymatic Degradation of Non-Ionic Surfactants | p. 170 |
| Recommendations for Protein Formulation | p. 170 |
| References | p. 171 |
| High Throughout Formulation: Strategies for Rapid Development of Stable Protein Products | |
| Introduction | p. 177 |
| Overall Structure of the HTF Approach | p. 179 |
| Role of an Established Decision Tree for Formulation Design | p. 181 |
| Constraints on a Pharmaceutically Acceptable Protein Formulation | p. 182 |
| Proper Choice of Dosage Form | p. 183 |
| Preformulation Studies | p. 185 |
| Proper Choice of Excipients | p. 186 |
| Estimates of Resources Needed for Formulation Development | p. 188 |
| Use of Software and Databases to Assist in the HTF Process | p. 189 |
| Essential Analytical Methods | p. 191 |
| Stability Protocols | p. 193 |
| Unified Strategy for HTF | p. 194 |
| References | p. 195 |
| Index | p. 199 |
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