Evaluation of Driving Forces for Protein Partition in PEG-Salt Aqueous Two-Phase Systems and Optimization by Design of Experiments

Abstract

In the present study, the partitioning of four model proteins (bovine serum albumin, ovalbumin, α-chymotrypsin and lysozyme) was investigated in polyethylene glycol (PEG)-salt aqueous two-phase systems (ATPS) addressing the effects of PEG molecular weight (MW) and concentration, phase-forming salt type (potassium phosphate/sodium citrate (KPP/NaCit)) and concentration, sodium chloride (NaCl) concentration and pH, as well as MW, isoelectric point, charge and hydrophobicity of the proteins. It was found that protein partitioning was influenced by different coexisting effects, such as hydrophobic and electrostatic interactions, related to the system parameters and physicochemical/surface properties of proteins. Moreover, a rigorous analysis and optimization of the significant forces for protein partitioning in PEG-salt ATPS was performed by design of experiments (DoE). Initially, KPP/NaCit, NaCl and PEG concentrations were considered as statistically significant factors by a fractional factorial design. Subsequently, the optimal values of the significant factors were determined by a central composite face-centered design coupled with response surface methodology. Furthermore, linear and quadratic models were obtained in the experimental designs, respectively, and evaluated by statistical regression analysis and analysis of variance. Finally, a validation of the quadratic model confirmed a good correlation between predicted and experimental results, thus verifying the validity of the model.