Enhanced performance of polymer-polymer aqueous two-phase systems using ionic liquids as adjuvants towards the purification of recombinant proteins

authors	Castro, LS; Pereira, P; Passarinha, LA; Freire, MG; Pedro, AQ
nationality	International
journal	SEPARATION AND PURIFICATION TECHNOLOGY
author keywords	Adjuvants; Aqueous two-phase systems; Ionic liquids; Purification; Recombinant protein
keywords	HUMAN INTERFERON ALPHA2B; BIPHASIC SYSTEMS; POLYETHYLENE-GLYCOL; ESCHERICHIA-COLI; HUMAN-ANTIBODIES; PRIMARY RECOVERY; SEPARATIONS; EXTRACTION; BEHAVIOR; BOOST
abstract	Protein biopharmaceuticals, among which interferon alpha-2b (IFN alpha-2b) that can be used in the treatment of chronic hepatitis C and hairy cell leukemia, have become an indispensable product of current medicine. However, their current high costs derived from the lack of cost-effective downstream strategies still limits their widespread use. Polymer-based aqueous two-phase systems (ATPS) or aqueous biphasic systems (ABS) can be used in biopharmaceuticals purification. This work investigates the application of ionic liquids (ILs) as adjuvants (at 5 wt%) in ATPS constituted by polyethylene glycol with a molecular weight of 600 g mol(-1) (PEG 600) and polypropylene glycol with a molecular weight of 400 g mol(-1) (PPG 400) at constant pH (8) to purify the recombinant protein IFN alpha-2b from Escherichia coli lysates. IFN alpha-2b was produced from isopropyl beta-d-1-thiogalactopyranoside (1 mM)-induced Escherichia coli BL21 (DE3) cultures and recovered from inclusion bodies after mechanical lysis, involving glass beads and a solubilization step with urea (8 M) in alkaline pH (12.5). PEGPPG-based ATPS involving ILs as adjuvants were subsequently applied for IFN alpha-2b purification, in which the target protein tends to migrate to the PEG-rich phase (being the phase also enriched in IL) and the remaining proteins tend to precipitate at the interface (fitting within the three-phase partitioning approach). In comparison with the ATPS without adjuvant, most systems comprising ILs as adjuvants lead to enhancements in the purification factors of IFN alpha-2b, namely from 2.28 +/- 0.06 up to 6.77 +/- 0.49. The purity of IFN alpha-2b is maximized using ILs composed of aromatic cations and anions with high hydrogen-bond basicity. The secondary structure of IFN alpha-2b is preserved during the purification step, as appraised by circular dichroism and western-blot studies. Overall, the obtained results demonstrate the ILs ability to tune the characteristics of the ATPS coexisting phases towards improved purification processes, paving the way for their investigation in the purification of other highvalue biological products.
publisher	ELSEVIER
issn	1383-5866
isbn	1873-3794
year published	2020
volume	248
digital object identifier (doi)	10.1016/j.seppur.2020.117051
web of science category	Engineering, Chemical
subject category	Engineering
unique article identifier	WOS:000538827600054