Development of aqueous two-phase separations by combining high-throughput screening and process modelling A thesis submitted to University College London for the degree of DOCTOR OF ENGINEERING by Nehal Patel Friday 21st July 2017 The Advanced Centre for Biochemical Engineering Department of Biochemical Engineering University College London Gower Street, London, WC1E 6BT, UK 1 I, Nehal Patel, 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. 2 Abstract Separation based on aqueous two-phase extraction (ATPE) is a promising downstream separation technology for the production of biological products. The advantages of using aqueous two-phase systems include but are not limited to easy scalability, ease of continuous operation and a favourable environment for biological compounds. One of the main challenges associated with aqueous two-phase systems is process development. This is in part due to the many factors which influence the separation of biological materials in such systems such as polymer and salt type, pH and charge. The large number of factors to consider makes the development of aqueous two-phase systems challenging due to the need to find a robust and efficient separation in a large experimental space. This work addresses this issue by considering the use of dynamic process models and high-throughput experimentation for the development of aqueous two-phase extraction processes for biological products. The use of a dynamic equilibrium stage process model to simulate aqueous two-phase extraction is considered in Chapter 3. The process model is capable of simulating various modes of operation; and both multi-cycle batch and continuous counter-current modes of operation are considered. The capabilities of the model are demonstrated using a case study separation of enzyme α-amylase from impurities in a PEG 4,000- phosphate aqueous two-phase system containing NaCl. The dynamic model allowed investigation into the impact of upstream process variability on a continuous counter- current extraction process. The development of aqueous two-phase systems requires detailed knowledge of the phase diagram. In Chapter 4, PEG 4,000-citrate aqueous two-phase system phase diagrams are determined using a combination of high-throughput screening and lab scale experiments. This involved the development of a systematic two-stage screening approach to determine the binodial curve location to a high accuracy using ~50% of the experimental resources that a single high-resolution screen would use. In addition, a novel method was developed to quantify uncertainty in the phase diagram due to the binodial curve location and tie-line fitting. The characterised phase diagrams were then used to estimate thermodynamic interaction parameters which are used in process models to describe phase equilibria. In Chapter 5, the simulation and high-throughput screening methods of Chapter 3 and 4 are combined to develop an aqueous two-phase extraction separation process. The 3 approach is demonstrated by separating enzyme α-amylase from myoglobin in a PEG 2,000-phosphate aqueous two-phase system containing 6wt% NaCl. High-throughput experimentation is used to determine partitioning behaviour of α-amylase and myoglobin at different tie-line lengths and phase ratios. The experimental partitioning and phase diagram data was then used to simulate a counter-current extraction process. The insights gained using the process model allowed for better decisions to be made regarding selection, control and operation of aqueous two-phase separation equipment. Therefore, the combined approach of using process modelling and high-throughput experimentation allowed for greater amounts of process understanding to be gained for aqueous two-phase systems using limited resources where there is a large experimental space to be navigated. 4 Impact Statement Currently the major expense in the production of new biopharmaceuticals is the cost of downstream separation processes (Azevedo et al., 2009). The work in this thesis is centred on the use of aqueous two-phase extraction, a potentially cheaper alternative to expensive chromatographic techniques currently used in biopharmaceutical manufacturing. Specifically, the work presented in this thesis looks at the potential of using a combined high-throughput experimental and modelling approach for the development of aqueous two-phase extraction separations for the purification of biopharmaceuticals. The motivation for using high-throughput experimentation is that greater amounts of information can be extracted from limited experimental resources. This is particularly important in early stage development of new drugs where the availability of material to purify is limited. The use of appropriate process models allows for exploration of separation systems beyond the scope of the high-throughput experimentation without the need to perform additional experiments. Such an approach provides greater process insights to researchers so that robust separation processes can be developed more efficiently. The methods presented in this work will help researchers develop commercially relevant aqueous two-phase extraction processes. This would benefit patients and the general public as the cost of a new biopharmaceutical drug could be cheaper for two reasons: 1) The combined approach using experiments and modelling allows for research and development efforts to be conducted more efficiently and 2) The use of aqueous two-phase extraction could save money as the inherent process may be cheaper than using conventional separation techniques. 5 Acknowledgement I would like to thank my UCL supervisors, Dan Bracewell and Eva Sorensen for their continuous guidance and support throughout this project. Eva, I have never met someone who is as passionate as you are in the personal development of their students, it is truly inspiring. I feel very fortunate to have had your support during this project. Dan, throughout this project you have given me the freedom and flexibility to try new things. At the same time, you have always been very approachable when help was needed which is something I will always appreciate. I would like to thank BioMarin for making this project possible. The input of Eric, Dan and John in project meetings was invaluable. In addition I would like to thank all the BioMarin employees I met for their hospitality during a visit to their facilities. I would like to thank the many staff members at UCL for all their help in various forms. Dhushy and Elaine, thank you for your continuous support in all lab related matters. I would like to thank the many contributors to online computing websites such as Stack Overflow and Stack Exchange for taking the time to share their invaluable knowledge. I would like to thank the many friends I have made at UCL for their endless support and motivation. The EngD would not have been as fun without all of you. I would like to thank Amira. You made the last few years of the EngD the best they could possibly be. Finally, I would like to thank my family for their never ending support throughout all of my education and encouraging me to pursue what I enjoy. 6 Table of contents Abstract ............................................................................................................................. 3 Impact Statement ............................................................................................................... 5 Acknowledgement............................................................................................................. 6 Table of contents ............................................................................................................... 7 Table of figures ............................................................................................................... 10 List of tables .................................................................................................................... 17 Nomenclature .................................................................................................................. 20 Chapter 1: Introduction ................................................................................................... 22 1.1 Biopharmaceutical market ................................................................................ 22 1.2 Challenges in biopharmaceutical production ................................................... 23 1.3 Motivations and objectives ............................................................................... 25 1.4 Outline of thesis ................................................................................................ 25 1.5 Contributions of this thesis ............................................................................... 27 Chapter 2: Literature Review .......................................................................................... 28 2.1 Overview of aqueous two-phase systems ......................................................... 28 2.2 Aqueous two-phase system experimentation ................................................... 45 2.3 Modelling of liquid-liquid extraction ............................................................... 49 2.4 Concluding remarks ......................................................................................... 54 2.5 Aim of this thesis by chapter ...........................................................................