Towards Intensified Protein Refolding and Purification on Size Exlusion Chromatography: Fundamental Studies and Mathematical Modelling

For industrial recombinant-protein processes, protein refolding and purification are crucial steps towards the recovery of considerable numbers of active and safe therapeutic products. In this thesis, intensification strategies for protein refolding and purification processes are explored. Development of an intensified process aims at simultaneous optimization of process performance indicators, namely: refolding yield, product purity, volumetric productivity and solvent consumption which in turn decrease the cost and time constraints to market. The first strategy investigated related to multivariable experimental work using size exclusion chromatography (SEC) as a refolding method and a denatured/reduced model protein (lysozyme). SEC was selected due to its potential for refolding of higher concentrations of protein compared to conventional refolding methods used currently in industry. The investigated variables were protein loading concentration, refolding buffer composition including pH, sodium chloride salt and ʟ-arginine, aggregation prevention additive, concentrations. The interplay of these process variables was studied and it was shown when ʟ-arginine is used, over the experimental space, the effects of pH and protein loading concentration on refolding yield are insignificant. This observation introduced the possibility of manipulating pH in a wider range without concerns for protein aggregation; for instance, to adjust the redox potential of the buffer without the need for costly redox couple chemicals to assist reformation of disulfide bridges in oxidative refolding of the protein. The results also provide more experimental evidence on the mechanism of aggregation prevention by ʟ-arginine. Secondly an experimentally-verified model of oxidative protein refolding on SEC was developed, with the goal of high-throughput process screening and optimization using the aforementioned model. Model development involved exploration of methods to find characteristic information on short-lived refolding kinetic species and lysozyme oxidative refolding kinetic schemes and constants under the two studied refolding environments, namely with and without ʟ-arginine additive. It was shown that ʟ-arginine prevents aggregation without considerable impact on the kinetics of lysozyme oxidative refolding. Finally, SEC in a multi-column continuous simulated moving bed configuration (SMB-SEC) was evaluated to fully exploit the potential of SEC for intensified protein refolding and purification. This configuration offers several advantages compared to single-column operation, including increased productivity per unit mass of solid phase, lower solvent consumption, and less diluted products, provided that operation parameters are screened and tuned for simultaneous optimization of process performance indicators. In this phase of the project, the effect of scale-up was predicted and considered for modifying and utilizing single-column model towards design/operation of a SMB-SEC. This thesis presents a framework for protein refolding and purification process development and optimization, including reduced cost of chemicals, improving the refolding yield, high-throughput measurements of parameters and finding a suitable reaction scheme of refolding and aggregation for mathematical model development applicable to both single-column and multi-column continuous operations, and defining appropriate process performance indicators for optimized operation of SMB-SEC