Development of High Concentration cetuximab Formulations using Ultrafiltration and Precipitation Techniques

Transcription

1 Dissertation zur Erlangung des Doktorgrades der Fakultat fur Chemie und Pharmazie der Ludwig-Maximilians-Universitat Munchen Development of High Concentration cetuximab Formulations using Ultrafiltration and Precipitation Techniques vorgelegt von Susanne Matheus aus Wittlich Munchen, 2006

2 Table of Content I. INTRODUCTION 1. Current Trends in Antibody Formulations Rationale for High Concentration Formulations Benefits from High Concentration Formulations Common Instabilities of Monoclonal Antibodies Physical Instabilites Chemical Instabilities Analysis of Protein Instabilities Induction of Protein Instabilities Design of Monoclonal Antibody Formulations Stabilisation of Monoclonal Antibody Formulations Primary Packaging Materials Cetuximab What is cetuximab? Pharmacology of cetuximab Mechanism of Action of EGFR Mechanism of Action of cetuximab Manufacturing of High Concentration Antibody Formulations Methods for Concentrating Proteins Ultrafiltration Techniques Ultrafiltration as Approach to High Concentration Formulations Theoretical Aspects of Ultrafiltration Techniques Membrane Characteristics and Different Ultrafiltration Configurations Operation Modes of Ultrafiltration Control of Process Parameters, Protein Stability and Solution Composition during Ultrafiltration Operations Protein Precipitation Techniques Protein Precipitation and Crystallisation as Approach to High Concentration Formulations Theoretical Aspects of Protein Precipitation and Crystallisation Precipitation and Crystallisation Strategy Protein Precipitation and Crystallisation Techniques Control of Protein Precipitation and Crystallisation Drying Techniques as Approach to High Concentration Formulations Other Techniques Dialysis as Approach to High Concentration Formulations Chromatographic Techniques as Approach to High Concentration Formulations Challenges in the Development of High Concentration Antibody Formulations General Considerations for High Concentration Formulations Solubility Challenges Protein Stability Challenges Analytical Considerations Viscosity Challenges Costs of Goods Considerations 54

3 II. OBJECTIVES OF THIS WORK III. MATERIAL AND METHODS Materials 1.1 Cetuximab 1.2 Excipients, Packaging Materials, Reagents and Chemicals 59 CO os Manufacturing of High Concentration cetuximab Formulations Manufacturing of High Concentration cetuximab Formulations by Ultrafiltration Manufacturing of High Concentration cetuximab Formulations by Precipitation Precipitation by Batch Method and Re-dissolution of Precipitates Microscale Crystallisation Methods Compounding and Filling of High Concentration cetuximab Formulations Accelerated Stress Methods 3.1 Agitation Stress Shaking Stress Stirring Stress 3.2 Thermal Stress Elevated Temperature Stress ^ Real Temperature Conditions Freeze-thaw Stress 4. Analytical Methods 4.1 Characterisation and Quantification of Aggregation and Degradation Products Size Exclusion Chromatography (SEC) Dynamic Light Scattering (DLS) Turbidity (Opalescence) by Absorbance Spectroscopy at 350 nm Light Obscuration Visuallnspection Sodium Dodecylsulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE, reducing and non-reducing) Ion Exchange Chromatography (IEC) Protein Content Protein Content by Absorbance Spectroscopy at 280 nm Protein Content by Size Exclusion Chromatography (SEC) 4.3 Conformational Stability and Binding Activity Fourier-transformed Infrared Spectroscopy (FTIR) Fourier-transformed Infrared Spectroscopy (FTIR) Microscope Nano Differential Scanning Calorimetry (ndsc) segfr based cetuximab Enzyme Linked Immunosorbent Assay (ELISA) 4.4 Physicochemical Properties ph-value Critical Micelle Concentration (CMC) Viscosity Syringability Characterisation of cetuximab Precipitates Light Microscopy 7< * Atomic Force Microscopy (AFM) Scanning Electron Microscopy (SEM) 7 * X-ray powder diffraction (XRPD) '* Quantification of Precipitants Design of Experiment (DoE) 75

4 JV. PREPARATION OF HIGH CONCENTRATION CETUXIMAB FORMULATIONS BY ULTRAFILTRATION TECHNIQUES Increase in cetuximab Concentration by Various Ultrafiltration Techniques at Different Scales Study Design Final Concentration, Recovery and Filtrate Flux Cetuximab Stability during Ultrafiltration Maximal Achievable cetuximab Concentration by Ultrafiltration Techniques Conclusions Process Control and Development of TFF Operations for High Concentration cetuximab Formulations Study Design Process Control of Concentration Operations Effects of Different Formulation Conditions on Ultrafiltration Operations Process Development of Combined Concentration and Diafiltration Operations Conclusions 97 V. PREPARATION OF HIGH CONCENTRATION CETUXIMAB FORMULATIONS BY PRECIPITATION TECHNIQUES Precipitation of cetuximab Study Design Screening of Various Precipitating Agents Optimisation and Up-scale of the Precipitation Process Evaluation of Further Crystallisation Techniques and Conditions Evaluation of cetuximab Solubility Conclusions Re-Dissolution of cetuximab Precipitates to High Concentration cetuximab Formulations Study Design Evaluation of Re-Dissolution Conditions Stability of cetuximab during Precipitation and Re-Dissolution Conclusions 125 YL. FORMULATION DEVELOPMENT OF A STABLE LIQUID HIGH CONCENTRATION CETUXIMAB FORMULATION Analytical Tools for the Characterisation of cetuximab in the Formulation Development Studies Characterisation of cetuximab Aggregation and Degradation Products FTIR and ndsc as Analytical Tools for High Concentration Protein Formulations FTIR Spectra and Temperature Induced Structural Changes Determination of the Protein Melting Temperature by FTIR (T m, FTI R ) Determination of the Protein Melting Temperature by ndsc (T m, ndsci) Conclusions 139

5 2. Preformulation of Liquid High Concentration cetuximab Formulations Study Design 2.2 Influence of Protein Concentration and Formulation Buffer on cetuximab Aggregate Formation 2.3 Influence of Mechanical Stress Method on cetuximab Aggregate Formation Influence of Polysorbate 80 on cetuximab Aggregate Formation 2.5 Conclusions 3. Evaluation of the Optimal ph Value for High Concentration cetuximab Formulations ^ Study Design 3.2 Influence of ph Value on cetuximab Mechanical Stress Stability 3.3 Influence of ph Value on cetuximab Thermal Stress Stability Influence of ph value on cetuximab Conformational Stability 1^4 3.5 Conclusions 4. Selection of the Optimal Buffer System for High Concentration cetuximab Formulations 1 5 *> 4.1 Study Design 4.2 Influence of the Buffer System on cetuximab Mechanical Stress Stability Influence of the Buffer System on cetuximab Thermal Stress Stability Influence of the Buffer System on cetuximab Conformational Stability Conclusions 5. Selection of the Optimal Ionic Strength for High Concentration cetuximab Formulations Study Design Influence of the Ionic Strength on cetuximab Mechanical Stress Stability Influence of the Ionic Strength on cetuximab Thermal Stress Stability Influence of the Ionic Strength on cetuximab Conformational Stability Conclusions 1 6. Evaluation of Surfactants and Stabilising Excipients for High Concentration cetuximab Formulations Study Design Surfactants Effect of Surfactants on cetuximab Mechanical Stress Stability Effect of Surfactants on cetuximab Thermal Stress Stability Effect of Surfactants on cetuximab Conformational Stability Sugars, Polyols and Amino Acids I Effect of Sugars, Polyols and Amino Acids on cetuximab Mechanical Stress Stability Effect of Sugars, Polyols and Amino Acids on cetuximab Thermal Stress Stability Effect of Sugars, Polyols and Amino Acids on cetuximab Conformational Stability Conclusions

6 7. Optimisation of Viscosity and Syringability of High Concentration cetuximab Formulations Study Design Evaluation of Parameters Influencing Viscosity Syringability of High Concentration cetuximab Formulations Screening of Viscosity Lowering Excipients Conclusions Formulation Robustness of High Concentration cetuximab Formulations Study Design Formulation Robustness considering cetuximab Mechanical Stress Stability Formulation Robustness considering cetuximab Thermal Stress Stability Conclusions Stability of High Concentration cetuximab Formulations in Prefilled Syringes Study Design Cetuximab Stability at Various Storage Conditions Conclusions Stability of High Concentration cetuximab Formulations obtained by Precipitation Techniques Study Design Cetuximab Stability in High Concentration Formulations obtained by Precipitation Conclusions Chapter Summary 199 VM. GENERAL SUMMARY AND OUTLOOK 201 VIM. ZUSAMMENFASSUNG UND AUSBLICK 205 JX. REFERENCES 209 X. APPENDIX 223