Abstract
Protein biopharmaceuticals, among which interferon alpha-2b (IFNα-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 IFNα-2b from Escherichia coli lysates. IFNα-2b was produced from isopropyl β-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). PEG-PPG-based ATPS involving ILs as adjuvants were subsequently applied for IFNα-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 IFNα-2b, namely from 2.28 ± 0.06 up to 6.77 ± 0.49. The purity of IFNα-2b is maximized using ILs composed of aromatic cations and anions with high hydrogen-bond basicity. The secondary structure of IFNα-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 high-value biological products.
Highlights
The advent of biopharmaceuticals in modern medicine for the treatment of numerous diseases brought a significant impact on the improvement of global health
The time growth profile of interferon alpha-2b (IFNα-2b) in the SOB medium was evaluated by SDS-PAGE (Fig. S2 in the Supplementary Information), whereas the densitometric analysis of the band corresponding to the target protein revealed that higher IFNα-2b levels are achieved after 3 h of induction
The purification of IFNα-2b as a major protein biopharmaceutical using polymerpolymer aqueous two-phase systems (ATPS) and ionic liquids (ILs) as adjuvants was attempted in this work
Summary
The advent of biopharmaceuticals in modern medicine for the treatment of numerous diseases brought a significant impact on the improvement of global health. A common drawback associated to polymer-polymer-based ATPS is that they exhibit a small range of polarities between the two phases [20], hampering their effective application in the purification of target proteins from complex samples To overcome such limitation, it has been proposed the functionalization of PEG with glutaric acid to improve the recovery yields of immunoglobulins [29, 30]. IL-based TPP have been proposed by Alvarez et al [40, 41] to recover model food proteins at the ATPS interface It is described the production and purification of IFNα-2b from E. coli BL21 (DE3) inclusion bodies using PEG -600-PPG-400-based ATPS/TPP with ILs as adjuvants (at 5 wt%). The ATPS process was combined with TPP, in which the purification of the target protein was aimed at the PEG-rich phase with the simultaneous precipitation of the remaining proteins at the ATPS interface
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