Abstract

Aqueous two-phase systems (ATPS) have begun to be used industrially for the isolation and separation of recombinant enzyme proteins. However, until now most studies have concentrated on investigating factors affecting the partition coefficients of molecules and even the pilot plant and scale-up developments have lacked engineering, optimization and design considerations. This paper investigates such considerations in the process of protein separation and purification in ATPS. A cost function for the process of protein extraction that takes into account the effect of variables in ATPS has been developed to study the influence of this extraction stage over the rest of the stages in the process (production, concentration and purification). The effect of the flow rate ratios, the relative sizes of the PEG and Phosphate phases and the NaCl concentration have been analysed. An extended mathematical model was used to describe the continuous, steady state operation of an aqueous two-phase system for protein extraction. The basic model is based on steady state mass balances of the main components and phase equilibrium data. Experimental data on the separation of ?-amylase from Bacillus subtilis supernatant in a PEG/salt system was used. Simulations were carried out to study the effect of flow rate ratio as a function of NaCl concentration in the feed. At a flow rate ratio above 2.4 the extraction process was very sensitive to NaCl concentration. A rigorous optimization was carried out using the Sequential Quadratic Programming Method (SQP) with a fixed initial NaCl concentration of 8.8%. An optimal Flow Rate Ratio of 2.27 was obtained. Thus, the optimization strategy developed in this paper was mathematically implementable and successful. The optimal steady state process conditions corresponded to conditions of a large and continuous bottom phase in both the forward and back extractions. This is very favourable for phase separation as it results in fast settling rates which are also virtually independent of phase volume ratio, which are extremely favourable conditions for the design of appropriate settlers. A cost function for the isolation and separation of recombinant enzyme proteins process has been developed to study the influence of this process over the rest of the stages in the process. The effect of the flow rate ratios, the relative sizes of the polyethylene glycols (PEG) and phosphate phases and the NaCl concentration have been analyzed. An extended mathematical model was used to describe the continuous, steady state operation of an aqueous two-phase system for protein extraction. Simulations were carried out to study the effect of flow rate ratio as a function of NaCl concentration. A rigorous optimization was carried out using the sequential quadratic programming (SQP) method with a fixed initial NaCl concentration of 8.8%.