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Protein Precipitation for the Purification of Therapeutic Proteins

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Protein Precipitation for the Purification of Therapeutic Proteins Protein Precipitation for the Purification of Therapeutic Proteins by Christopher W Morris A thesis submitted for the degree of Doctor of Engineering in The Department of Biochemical Engineering, UCL 1 Abstract This thesis documents the application of precipitant scouting and analytical tools for the development of a precipitation process for the purification of therapeutic proteins in biopharmaceuticals. Precipitation has the potential to bypass the bottlenecks in productivity experienced with packed bed chromatographic separations, offering a fast, robust first purification step with volume reduction at low cost. In order to evaluate a large number of precipitation candidates, microscale investigations aided by automated liquid handling robotics were chosen, which conferred precision, speed, and complex experimental design with microliter material requirements and in-line analytics. A precipitation screening methodology was successfully built onto a Tecan liquid handling platform including product recovery and techniques for measuring soluble protein, liquid volumes, and recovered protein precipitate. The protocol was supplemented with a custom- build Excel VBA driven Tecan control tool. This accelerated progress by freeing up the potential of the liquid handling arm, which was curtailed by system software. These techniques were then used to characterise effective precipitants for the purification of monoclonal antibodies. Optimal conditions were identified on pure protein models, which were the bridged to process relevant cell culture fluid. Process performance, notably recovery yield and product quality were the investigated on the precipitant conditions brought forward. Process integration aspects were explored, by linking precipitation with an anion exchange chromatography step. This led to discussion on future work and process scale up considerations. 2 Impact Statement This impact statement serves as a suitable place to reflect on the work presented in this thesis and how it relates to academic and industrial applications. The core subject of precipitation is an established technology, and as stated in this thesis the work was focused on applying precipitation to modern downstream processing challenges. As part of an industrial sponsored doctorate, the investigation of precipitation and development of techniques helped the internal research and development at UCB, and a further doctorate was subsequently carried out, building on the foundations of this work. A near full-time placement of this engineering doctorate on-site at UCB working as part of a larger team saw opportunities to set up automated high-throughput techniques on the liquid handling platforms for other teams in the company. 96-well plate assays were created for the team in upstream, and over the course of the final 2 years these techniques were employed weekly to a high level of precision and reproducibility by different people, saving time and increasing analytical throughput significantly. These techniques were written up as internal SOPs and it was rewarding to see tangible benefits of the skills developed throughout work detailed in this thesis. I had the honour of achieving a finalist place in Cogent’s UK life sciences student placement of the year award, very kindly nominated by Edith Lecomte-Norrant, due not only to my work at UCB, but also as a result of voluntarily running student open days, tours, and presentations. Regular opportunities to present my work both internally, at regional conferences, and at Recovery 2014 in Rostok were great experiences and I benefitted greatly from the feedback and sharing of ideas. A silver lining of finalising this engineering doctorate later than planned, is that I have directly seen how industrial experience at UCB combined with academic grounding at UCL have helped me develop a specialist focus in the downstream processing of therapeutic proteins which have concretely led to the successful development of new purification technologies in my current role. The global launch of our new technology of fibre-based chromatography 3 (planned in the next few months at the time of writing), will have a high impact and I attribute a lot of the contributions I have made to this project to the experience and skills garnered during this doctoral thesis. I, Christopher Morris 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. ______________________________________________________Date________________ 4 Acknowledgements “I sometimes try to be miserable so that I may do more work, but find it is a foolish experiment.” William Blake 1757-1827 My first acknowledgement is to my supervisor Nigel, whose steering hand and wisdom guided me through all the various distractions and tangents I would have elsewise wilfully travelled down. Secondly, a huge thank-you to Stefanos Grammatikos, Edith Lecomte-Norrant, Mari Spitali, Mark Pearce-Higgins and everyone else at UCB who guided me throughout the project. Their help, encouragement, knowledge, and experience have been invaluable. Finally, I am extremely grateful to my family and friends who have shown inexhaustible encouragement and support throughout. 5 Table of Contents Abstract .................................................................................................................................... 2 Impact Statement ..................................................................................................................... 3 Acknowledgements .................................................................................................................. 5 Table of Contents ..................................................................................................................... 6 List of Figures ........................................................................................................................ 10 List of Tables ......................................................................................................................... 12 List of Abbreviations ............................................................................................................. 13 Chapter One: Introduction & Literature Review ................................................................... 14 1.1 Project Relevance ................................................................................................... 14 1.2 Project Aims ........................................................................................................... 18 1.3 The Production and Purification of Monoclonal Antibodies ................................. 19 1.3.1 Upstream Processing ...................................................................................... 21 1.3.2 Downstream Processing ................................................................................. 23 1.4 Review of Protein Purification by Precipitation .................................................... 24 1.4.1 Overview ........................................................................................................ 24 1.4.2 Salting-In ........................................................................................................ 27 1.4.3 Precipitation by Salting-Out ........................................................................... 28 1.4.4 Metal Ion Precipitation ................................................................................... 39 1.4.4 Non-ionic Polymer Precipitation.................................................................... 40 1.4.4 Isoelectric Precipitation .................................................................................. 41 1.4.5 Polyelectrolyte Precipitation .......................................................................... 42 1.4.6 Affinity Precipitation ..................................................................................... 47 1.4.7 Organic Solvent Precipitation ........................................................................ 51 1.4.8 Protein Stabilising Agents .............................................................................. 52 1.4.9 Conclusions & Direction of Research ............................................................ 55 1.5 Precipitate Formation & Properties ........................................................................ 55 1.5.1 DLVO theory and precipitation ..................................................................... 55 1.5.2 Precipitate formation – nucleation and growth by diffusion .......................... 56 1.5.3 Precipitate formation - growth by fluid motion ............................................. 57 1.5.4 Precipitate conditioning / Camp Number ....................................................... 57 1.5.5 Precipitate breakage ....................................................................................... 58 1.5.6 Precipitate properties ...................................................................................... 59 1.6 The effect of mixing on precipitation .................................................................... 59 6 1.7 Centrifugation ........................................................................................................ 60 1.8 Filtration ................................................................................................................. 60 1.9 Bioprocess Development through Scale Down techniques.................................... 61
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