Development of aqueous two-phase separations by combining high-throughput screening and process modelling

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 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.