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Projeto de investigação
Bridging Nature-Inspired Ionic Fluids and Microfluidic Aqueous Biphasic Systems for the Purification of Therapeutic Biomolecules
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Towards Enhanced Tunability of Aqueous Biphasic Systems
Publication . Carvalho, Sara F.; Custódio, Margarida H.; Pereiro, Ana B.; Araújo, João M. M.; LAQV@REQUIMTE; DQ - Departamento de Química; MDPI - Multidisciplinary Digital Publishing Institute
This work unfolds functionalized ABSs composed of FILs ([C2C1Im][C4F9SO3] and [(Formula presented.)][C4F9SO3]), mere fluoro-containing ILs ([C2C1Im][CF3SO3] and [C4C1Im][CF3SO3]), known globular protein stabilizers (sucrose and [(Formula presented.)][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.
Simultaneous Purification of Human Interferon Alpha-2b and Serum Albumin Using Bioprivileged Fluorinated Ionic Liquid-Based Aqueous Biphasic Systems
Publication . Carvalho, Sara F.; Pereiro, Ana B.; Araújo, João M. M.; LAQV@REQUIMTE; DQ - Departamento de Química; MDPI - Multidisciplinary Digital Publishing Institute
Interferon alpha-2b (IFN- (Formula presented.) 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- (Formula presented.) 2b and BSA (serum albumin protein) was developed in this work. Effects of the ionic liquid (IL)-based ABS functionalization, fluorinated ILs (FILs; [C (Formula presented.) C (Formula presented.) Im][C (Formula presented.) F (Formula presented.) SO (Formula presented.)] and [N (Formula presented.)][C (Formula presented.) F (Formula presented.) SO (Formula presented.)]) vs. mere fluoro-containing IL ([C (Formula presented.) C (Formula presented.) Im][CF (Formula presented.) SO (Formula presented.)]), in combination with sucrose or [N (Formula presented.)][H (Formula presented.) PO (Formula presented.)] (well-known globular protein stabilizers), or high-charge-density salt K (Formula presented.) PO (Formula presented.) 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- (Formula presented.) 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- (Formula presented.) 2b high in the enriched IFN- (Formula presented.) 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- (Formula presented.) 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- (Formula presented.) 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.
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Fundação para a Ciência e a Tecnologia
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SFRH/BD/139677/2018