Rapid Optimisation of Preparative Chromatography for Monoclonal Antibody
Total Page：16
File Type：pdf, Size：1020Kb
Rapid Optimisation of Preparative Chromatography for Monoclonal Antibody Purification using High-Throughput Methodology A thesis submitted for the degree of Doctor of Engineering by Vivien Fischer The Advanced Centre for Biochemical Engineering Department of Biochemical Engineering University College London November 2019 Declaration of Authorship 3 Declaration of Authorship I, Vivien Fischer, 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. Signature: Date: Acknowledgements 5 Acknowledgements I would like to thank the many people who helped me along this journey and contributed in various ways. Firstly, I would like to express my gratitude to my academic supervisor, Professor Ajoy Velayudhan, for sharing his deep expertise in the field, his guidance and critical insight throughout this project. Further, I would like to thank my industrial supervisors, Dr. Richard Kucia-Tran and Dr. Will Lewis, for their continuing support and interest in my work, and for giving me the opportunity to pursue this doctorate as part of their team in Downstream Process Research at GSK (Stevenage, UK). I am grateful to my GSK colleagues for providing both a stimulating working environment and an enjoyable social atmosphere. From the downstream team, my gratitude extends in particular to Daniel Gibson for sharing his knowledge of column packing and being a great chromatography mentor across all scales, from micro- to pilot-scale, Dr. Nehal Patel for our enjoyable discussions about automation and modelling, and for all questions around Äkta’s I’m thankful to Dr. Monika Farys, Sophie Russell and Apoorva Alva. For analytical support, special thanks are due to Dr. David Hilton and Richard Caruana who not only allowed me to occupy multiple HPLC systems but also trained me in the development and use of HPLC methods. From the upstream team, I appreciate the help from Yuen-Ting Chim who trained me in the local cell culture protocols, as well as Peter Steward and Alex Livingstone for their bioreactor support. I would like to thank Dr. Haren Arulanantham for his help in setting up the HTP Lunatic method and Dr. Spiros Konstantinidis from UCL for his introduction to the Tecan Evo. The statistical advice and training from Dr. Simon Bate and Sonya Godbert are also greatly acknowledged. In addition, I am thankful to the EPSRC and GSK for financial support of this EngD. Ich danke meinen Freunden, die mir über nah und fern immer treu geblieben sind. Insbesondere danke ich Dan - dafür, dass du mich unermüdlich begleitet, immer an mich geglaubt und die Zeit so viel schöner gemacht hast. Ein besonderer Dank gilt meinen Brüdern und von ganzem Herzen meinen Eltern, die mich ermutigt haben, meinen Interessen zu folgen, mich fortwährend unterstützt und mir stets Rückhalt geboten haben. Abstract 7 Abstract A key challenge for early stage bioprocess development is the need to deliver effective operating conditions against aggressive timelines and stringent material constraints. For antibodies, the largest class of biopharmaceuticals to date, chromatography constitutes a key tool for product purification. The development of these steps typically involves multi-variate and multi-objective optimisation problems with various constraints and often complex trade-offs. This research investigates data-driven experimental optimisation strategies based on the Nelder-Mead simplex algorithm, and other more traditional approaches using DoE and fundamental chromatography understanding. Through three case studies, the polishing chromatography process was developed for a series of industrially- relevant binary and ternary mAb feeds. Bind-and-elute cation-exchange chromatography operations were optimised with regards to the loading and elution conditions, employing step and linear gradients. Experiments were conducted using conventional laboratory-scale chromatography. In the third case study, these were compared head-to-head with micro-scale chromatography methods, on a Tecan Fluent liquid handling station. Process insight was generated, and trends visualised and interpreted for key performance outputs such as product mass, recovery and productivity against pre-defined requirements for product purity. These trends were shown to be reproducible at micro-scale with comparable chromatographic resolution, employing residence time scaling. The proposed experimental optimisation strategies were shown to effectively deal with the high variability typical of antibodies, revealing interesting trade-offs for high loading conditions and in the vicinity of product breakthrough, leading to high- performing counter-intuitive operating regions. In these practical scenarios, the simplex was demonstrated to be an easy-to-use and dynamic tool that guides development efforts towards feasible and promising operating regions whilst also being interactive by accepting information from different sources to maximise the value gained from the available experimental data. Hence, this work has demonstrated how state-of-the-art experimental optimisation methods can be 8 Abstract deployed for rapid and rational process development, enabling the identification of high-performing operating conditions that may not necessarily align with conventional assumptions and traditional expectations. Impact Statement 9 Impact Statement The modern biopharmaceutical drug development landscape is highly competitive in nature. Key challenges the biopharmaceutical industry is facing include cost pressure and the need to accelerate development timelines on one hand, and a trend towards increasingly complex and diverse biomolecules on the other. During the production of medicines, polishing chromatography is a critical component that contributes not only to overall process productivity and cost but most importantly plays a role in assuring product safety. The development of chromatography steps can be complex with often conflicting objectives. The optimisation of these types of problems is an important field in process research and was investigated in this work. Monoclonal antibodies constitute a major proportion of biological therapies. In this work, three industrially-relevant and challenging purification scenarios were selected which showcase the variety of this class of molecules and demonstrate how state-of-the-art tools for experimental optimisation can be applied to deliver high- performing operating conditions for polishing chromatography. One such tool is the simplex algorithm which was employed to systematically search for optimal parameter settings. By demonstrating how the method operates in different scenarios and in combination with other tools (local fitting, high-throughput technologies), this work promotes the uptake of the technique for efficient optimisation of bioprocesses at the early stage of development. Key outcomes of the work have been published in Biotechnology Progress journal (Fischer et al. 2019) and communicated within the academic and industrial community at two major conferences ‒ American Chemical Society (ACS) spring conference, New Orleans, USA 2018; Poster Presentation, ‘Optimisation of preparative polishing chromatography for a ternary antibody mixture’ and at the 17 th International Symposium on Preparative and Industrial Chromatography and Allied Techniques (SPICA), Darmstadt, Germany, 2018; Oral Presentation, ‘Hybrid optimisation of preparative chromatography for a difficult and realistic ternary protein mixture’. 10 Impact Statement High-throughput technologies have been recognised in academia and industry for their capacity to reduce development time and material consumption. This research describes the implementation of a high-throughput method to operate miniature columns (RoboColumns) as a scale-down strategy for chromatography process development. The outputs from the scale-down system and traditional laboratory- scale chromatography experiments are compared and data presented to support method qualification. The developed high-throughput chromatography workflow adds direct value to the process development group at GSK as the industrial collaborator of this doctorate and will have an immediate impact in facilitating the scale-down of optimisation studies. Outside this group, the output of the studies contributes to the body of literature highlighting the potential and limitations of a scale-down strategy in direct comparison to conventional chromatography methods. Therefore the impact of the Engineering Doctorate (EngD) is considerable, both on the fundamental development of state-of-the-art experimental optimisation approaches and on the practical deployment of an industrially important experimental mode. Table of Contents 11 Table of Contents 1 Introduction .................................................................................................. 23 1.1 Nature of the bioprocess industry ............................................................ 23 1.2 Monoclonal antibodies and production processes ................................... 24 1.3 Chromatography development for mAb purification .............................. 29 1.3.1 Elution chromatography modes .......................................................... 30 1.3.2 Separation mechanisms ....................................................................... 31 1.3.3 Operational parameters ....................................................................... 33 1.4 Bioprocess development .........................................................................