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- Title
Simultaneous Purification of Human Interferon Alpha-2b and Serum Albumin Using Bioprivileged Fluorinated Ionic Liquid-Based Aqueous Biphasic Systems.
- Authors
Carvalho, Sara F.; Pereiro, Ana B.; Araújo, João M. M.
- Abstract
Interferon alpha-2b (IFN- α 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- α 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 (OH) ][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 (OH) ][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- α 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- α 2b high in the enriched IFN- α 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- α 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- α 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.
- Subjects
ACRYLONITRILE butadiene styrene resins; HAIRY cell leukemia; BLOOD proteins; CHRONIC hepatitis C; INTERFERONS; SERUM albumin; IONIC liquids; GLOBULAR proteins
- Publication
International Journal of Molecular Sciences, 2024, Vol 25, Issue 5, p2751
- ISSN
1661-6596
- Publication type
Article
- DOI
10.3390/ijms25052751