Towards Enhanced Tunability of Aqueous Biphasic Systems: Furthering the Grasp of Fluorinated Ionic Liquids in the Purification of Proteins.

This work unfolds functionalized ABSs composed of FILs ([C(2)C(1)Im][C(4)F(9)SO(3)] and [N1112(OH)][C(4)F(9)SO(3)]), mere fluoro-containing ILs ([C(2)C(1)Im][CF(3)SO(3)] and [C(4)C(1)Im][CF(3)SO(3)]), known globular protein stabilizers (sucrose and [N1112(OH)][C(4)F(9)SO(3)]), low-molecular-weight carbohydrate (glucose), and even high-charge density salt (K(3)PO(4)). The ternary phase diagrams were determined, stressing that FILs highly increased the ability for ABS formation. The functionalized ABSs (FILs vs. mere fluoro-containing ILs) were used to extract lysozyme (Lys). The ABSs' biphasic regions were screened in terms of protein biocompatibility, analyzing the impact of ABS phase-forming components in Lys by UV-VIS spectrophotometry, CD spectroscopy, fluorescence spectroscopy, DSC, and enzyme assay. Lys partition behavior was characterized in terms of extraction efficiency (% EE). The structure, stability, and function of Lys were maintained or improved throughout the extraction step, as evaluated by CD spectroscopy, DSC, enzyme assay, and SDS-PAGE. Overall, FIL-based ABSs are more versatile and amenable to being tuned by the adequate choice of the phase-forming components and selecting the enriched phase. Binding studies between Lys and ABS phase-forming components were attained by MST, demonstrating the strong interaction between Lys and FILs aggregates. Two of the FIL-based ABSs (30 %wt [C(2)C(1)Im][C(4)F(9)SO(3)] + 2 %wt K(3)PO(4) and 30 %wt [C(2)C(1)Im][C(4)F(9)SO(3)] + 25 %wt sucrose) allowed the simultaneous purification of Lys and BSA in a single ABS extraction step with high yield (extraction efficiency up to 100%) for both proteins. The purity of both recovered proteins was validated by SDS-PAGE analysis. Even with a high-charge density salt, the FIL-based ABSs developed in this work seem more amenable to be tuned. Lys and BSA were purified through selective partition to opposite phases in a single FIL-based ABS extraction step. FIL-based ABSs are proposed as an improved extraction step for proteins, based on their biocompatibility, customizable properties, and selectivity.