New Ultra Scale-Down Principles for Monoclonal Antibody Recovery And
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2809077300 New Ultra Scale-down Principles for Monoclonal Antibody Recovery and Purifications Nicholas Hutchinson UCL Engineering Doctorate Thesis I, Nicholas Hutchinson, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 1 UMI Number: U592920 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U592920 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract The development of manufacturing processes for biopharmaceuticals is carried out by performing experiments in the laboratory and then the pilot plant. These laboratory - scale experiments are often misleading as they do not allow for the effect of the large-scale processing environment on the properties of biological process material. The research contained within this thesis seeks to develop laboratory techniques capable of eliciting the effects of the processing environment on the material and of predicting the performance of large-scale unit operations used in the purification of monoclonal antibody (MAb) biopharmaceuticals using milliliter volumes of process material. Two key interactive stages were examined; firstly, an early centrifugation clarification stage designed to prepare material for subsequent filtration which precedes the second stage studied, a high affinity antibody capture step. The first focuses on the whole cell broth, containing the contaminants and the antibody product, the latter on the antibody product itself. A scale-down centrifuge technique which employed a rotating-disc, centrifuge feed zone mimic along with a laboratory-scale centrifugation method were developed to predict the clarification performance of two pilot, disc stack centrifuges with differing feed zone configurations and a manufacturing-scale disc stack centrifuge. The material obtained from the scale-down centrifuge protocol was shown to be equivalent to that from one of the pilot centrifuges to which it had been compared and, hence, could be used for the development of subsequent unit operations, such as chromatography processes. A method was developed to predict the elution profile of an 18 L pilot affinity chromatography column using a scale-down column with a column volume of one milliliter. A regime analysis of the laboratory-scale 2 chromatography system was performed using conductivity transitions to determine the extent of additional zone broadening occurring at the small scale so that the prediction of the large-scale elution volume and eluting product concentration could be significantly improved. 3 Acknowledgments The help of so many people at University College London and Lonza Biologies has been required during the course of my doctorate that unfortunately there isn’t room to mention them all. However, if you are reading this and feel you should be acknowledged, I assure you that you are. I would like to thank Professor Mike Hoare for teaching me such a lot about how to conduct and present research. I could never have completed this work without his patience and positive attitude which have been so invaluable over the past five years. I’d like to acknowledge the never ending support of my wonderful family especially Mum and Dad, Vicky, Mike, Spike and Jared. Many thanks to Claire for her support and patience, for her gentle encouragement to get this done and for all the good times we’ve had together. The financial support from the EPSRC and Lonza Biologies is gratefully acknowledged. Finally, thanks to London, an amazing city “full of sordid sinners and splendid sins”, undivided and undefeated by terrorism, home for the past six years and an education in itself. 4 Table of contents Abstract 2 Acknowledgements 4 Table of contents 5 List of figures 9 List of tables 16 Chapter 1- Introduction 19 Literature Review 22 1.1. Antibodies 22 1.1.1. Antibody structure 22 1.1.2. Antibody classes 24 1.1.3. Polyclonal antibodies 25 1.1.4. Monoclonal antibodies 26 1.1.5. Therapeutic monoclonal antibodies 26 1.1.5.1. Murine monoclonal antibodies and the HAMA response 29 1.1.5.2. Chimeric monoclonal antibodies 30 1.1.5.3. Humanized and human antibodies 30 1.1.5.4. Fusion proteins. 31 1.2. Manufacturing therapeutic monoclonal antibodies 31 1.2.1. Cell culture 32 1.2.1.1. Cell lines 34 5 1.2.1.2. Cell culture media 34 1.2.1.3. Recombinant protein expression systems 36 1.2.1.4. Bioreactor formats 37 1.2.1.5 Feeding strategies 38 1.2.1.6. Alternatives to cell culture 39 1.2.2. Product recovery 41 1.2.2.1. Centrifugation 42 1.2.2.2. Normal flow filtration 42 1.2.2.3. Tangential flow filtration 43 1.2.2.4. Expanded bed adsorption 44 1.2.3. Purification 45 1.2.3.1. Chromatography 47 1.2.3.1.1. Affinity chromatography 47 1.2.3.1.2. Ion-exchange chromatography 48 1.2.3.1.3. Hydrophobic interaction chromatography 49 1.2.3.1.4. Size exclusion chromatography 50 1.2.3.2. Viral removal & inactivation 50 1.2.3.3. Ultrafiltration 51 1.2.4. Characterization of therapeutic monoclonal antibodies 52 1.2.4.1. Sources of antibody heterogeneity 53 1.2.4.2. Analytical tests to assess heterogeneity 56 1.2.4.3. Controlling product quality 58 1.3. Challenges of process specification 59 6 1.3.1. Centrifuge specification 59 1.3.2. Normal flow filter specification 64 1.3.3. Tangential flow filter specification 66 1.3.4. Chromatography process specifications 67 Chapter 2 - The use of ultra scale-down centrifugation for the prediction of normal and soft feed, industrial-scale, disc stack centrifuges used for the clarification of mammalian cell culture broth at pilot scale 69 2.1. Introduction 69 2.2. Materials and Methods 71 2.3. Results 82 2.3.1. Ultra scale-down centrifuge technique development and verification of clarification performance predictions. 82 2.3.2. Centrifugation Windows of Operation. 93 2.3.3. Product and process stream comparability study. 99 2.4. Conclusions 107 Chapter 3 - The verification of the ultra scale-down centrifugation technology by a comparison of predictions with a manufacturing-scale, disc-stack centrifuge used to harvest a 20,000 litre cell culture bioreactor. 108 3.1 Introduction 108 7 3.2 Materials and Methods 109 3.3 Results 110 3.4 Conclusions 119 Chapter 4 - The use of an ultra scale-down chromatography column for the prediction of product elution profiles and its verification by comparison with an elution profile from a pilot- scale chromatography column. 116 4.1 Introduction 116 4.2 Materials and Methods 125 4.2.1 Laboratory Studies 125 4.2.2 Pilot Studies 130 4.2.2 Transition Calculation Methodology 131 4.3 Results 132 4.3.1 Transition analysis of ultra scale columns 132 4.3.2 Comparison of ultra scale-down columns with laboratory- scale columns 143 4.3.4 Comparison of ultra scale-down column with pilot-scale column 156 4.4 Discussion 160 Chapter 5 - The construction and use of a semi-empirical model for predicting scale-dependent chromatography bed compression and its use with ultra scale-down chromatography. 161 8 5.1 Introduction 161 5.2 Materials and Methods 165 5.3 Results 171 5.4 Conclusions 183 Chapter 6 - Discussion 184 6.1. Conclusions 184 6.2 Discussion and recommendation for future work 185 6.2.1. Ultra scale-down centrifugation 185 6.2.2. Ultra scale-down chromatography 189 6.2.3. Whole bioprocess scale-down 193 Appendix 1 - Transition analysis sample calculations 196 Appendix 2 - Elution profile correction process sample calculations 198 Nomenclature 202 List of Abbreviations 206 References 207 Publications List 224 9 List of figures Chapter 1 Figure 1.1. Overview of a therapeutic protein production process Figure 1.2. The structure of a human antibody (IgG). Figure 1.3. Schematic of IgG glycoforms. Chapter 2 Figure 2.1 Growth profile of cell culture material used in centrifugation experiments showing harvest point. Figure 2.2. Computational Fluid Dynamic (CFD) analysis of (a) feed zone of a disc-stack centrifuge and (b) the rotating disc device. Figure 2.3. Standard curve used for determining protein concentrations by the Coomassie method. Figure 2.4. Particle size distributions of mammalian cell broth exposed to shear in the rotating-disc device. Figure 2.5 - Light micrographs of NSO cells, (a) pre-shear and (b) 85 after shearing in the rotating disc device at 3.67 x 105 W kg'1. Figure 2.6. Dependence of the percentage of solids remaining in the supernatant following centrifugation on the duration of shear 86 exposure prior to centrifugation. Figure 2.7. Dependence of the percentage of solids remaining in the supernatant following centrifugation on the maximum energy dissipation rate to which the mammalian cell broth sample is exposed 88 prior to centrifugation. Figure 2.8. Relationship between the % solids remaining and the ratio of equivalent flow rate to centrifuge separation area, plotted on 91 probability-logarithmic axes for ultra scale-down method. Figure 2.9. Relationship between the % solids remaining and the ratio of equivalent flow rate to centrifuge separation area, plotted on 92 probability-logarithmic axes for industrial scale centrifugation.