We demonstrate that fluorinated ionic liquids reduce the impact of the addition of water upon the ionic liquid's H-bond acceptance ability. This is a key factor to obtain functionalized materials to be used e.g. in the dissolution of biomolecules, extraction processes or material engineering.

Interferon alpha-2b (IFN-$\alpha$2b) is an essential cytokine widely used in the treatment of chronic hepatitis C and hairy cell leukemia, and serum albumin is the most abundant plasma protein with numerous physiological functions. Effective single-step aqueous biphasic system (ABS) extraction for the simultaneous purification of IFN-$\alpha$2b and BSA (serum albumin protein) was developed in this work. Effects of the ionic liquid (IL)-based ABS functionalization, fluorinated ILs (FILs; [C${}_2$C${}_1$Im][C${}_4$F${}_9$SO${}_3$] and [N${}_{1112\left(\mathrm{OH}\right)}$][C${}_4$F${}_9$SO${}_3$]) vs. mere fluoro-containing IL ([C${}_4$C${}_1$Im][CF${}_3$SO${}_3$]), in combination with sucrose or [N${}_{1112\left(\mathrm{OH}\right)}$][H${}_2$PO${}_4$] (well-known globular protein stabilizers), or high-charge-density salt K${}_3$PO${}_4$ were investigated. The effects of phase pH, phase water content (%wt), phase composition (%wt), and phase volume ratio were investigated. The phase pH was found to have a significant effect on IFN-$\alpha$2b and BSA partition. Experimental results show that simultaneous single-step purification was achieved with a high yield (extraction efficiency up to 100%) for both proteins and a purification factor of IFN-$\alpha$2b high in the enriched IFN-$\alpha$2b phase (up to 23.22) and low in the BSA-enriched phase (down to 0.00). SDS-PAGE analysis confirmed the purity of both recovered proteins. The stability and structure of IFN-$\alpha$2b and BSA were preserved or even improved (FIL-rich phase) during the purification step, as evaluated by CD spectroscopy and DSC. Binding studies of IFN-$\alpha$2b and BSA with the ABS phase-forming components were assessed by MST, showing the strong interaction between FILs aggregates and both proteins. In view of their biocompatibility, customizable properties, and selectivity, FIL-based ABSs are suggested as an improved purification step that could facilitate the development of biologics.

This work unfolds functionalized ABSs composed of FILs ([C₂C₁Im][C₄F₉SO₃] and [${\mathrm{N}}_{1112\left(\mathrm{OH}\right)}$][C₄F₉SO₃]), mere fluoro-containing ILs ([C₂C₁Im][CF₃SO₃] and [C₄C₁Im][CF₃SO₃]), known globular protein stabilizers (sucrose and [${\mathrm{N}}_{1112\left(\mathrm{OH}\right)}$][C₄F₉SO₃]), low-molecular-weight carbohydrate (glucose), and even high-charge density salt (K₃PO₄). 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 [C₂C₁Im][C₄F₉SO₃] + 2 %wt K₃PO₄ and 30 %wt [C₂C₁Im][C₄F₉SO₃] + 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.