Simultaneous Purification of Human Interferon Alpha-2b and Serum Albumin Using Bioprivileged Fluorinated Ionic Liquid-Based Aqueous Biphasic Systems

Research output: Contribution to journalArticlepeer-review

9 Downloads (Pure)


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.
Original languageEnglish
Article number2751
Number of pages31
JournalInternational Journal of Molecular Sciences
Issue number5
Publication statusPublished - 27 Feb 2024


  • aqueous biphasic systems
  • bioactive proteins
  • bioprivileged fluorinated ionic liquids
  • BSA
  • human IFN-α2b
  • purification
  • serum albumin


Dive into the research topics of 'Simultaneous Purification of Human Interferon Alpha-2b and Serum Albumin Using Bioprivileged Fluorinated Ionic Liquid-Based Aqueous Biphasic Systems'. Together they form a unique fingerprint.

Cite this