Tailoring the partitioning of proteins using ionic liquids as adjuvants in polymer-polymer aqueous biphasic systems

Aqueous biphasic systems (ABS) are promising for proteins purification; however, when dealing with samples comprising several proteins, the selectivity towards a target protein is difficult to achieve. In this work, the addition of ionic liquids (ILs) as adjuvants (5 and 10 wt%) in ABS composed of polyethyleneglycol (PEG) and Dextran was investigated to tailor proteins’ partitioning between the coexisting phases. The liquid-liquid phase diagrams were determined at 298 K, followed by partition studies of three proteins (bovine serum albumin (BSA), immunoglobulin G (IgG), and cytochrome C (Cyt C)). Partition coefficients of IgG and BSA indicate the preference of both proteins to the dextran-rich phase, whereas Cyt C has no preferential partitioning between the phases. The addition of chloride-based ILs as adjuvants allows to increase or decrease the partition coefficients, thus tailoring the proteins partitioning between the phases. BSA partitioning essentially depends on the IL content in each phase, whereas Cyt C and IgG partitioning is ruled by the ILs chemical structure and established interactions. Molecular docking was carried out to address the ILs effect on the proteins partitioning, supporting experimental observations, while identifying the specific interactions occurring. The partition of each protein in polymer-salt ABS with ILs as adjuvants was determined, demonstrating the higher tailoring ability of polymer-polymer ABS when adding ILs. Finally, the partition of each protein in presence of the remaining ones was determined, as well as the selectivity of the studied systems to separate each pair of proteins, paving the way for their use in liquid-liquid chromatography.publishe