Improving the purification of recombinant proteins by the use of ionic liquids as adjuvants in aqueous two-phase systems

Abstract

The development of new drugs has a high impact on the global health, and since the approval of humulin (recombinant insulin) by FDA, several protein-based biopharmaceuticals have been introduced in the market. These products are usually attained through biotechnological processes using engineered biological sources and have led to remarkable advances in the treatment of distinct human diseases, as for example the case of interferon alfa 2b (IFNα2b) which has been used in in the treatment of oncological diseases for more than 30 years [1]. Currently, the (bio)pharmaceutical industry is committed to improve the quality of their products while endeavouring a reduction of its production costs, which is intimately related to the necessity of improving their downstream processes, since this field is mainly dominated by chromatographic techniques [2] and are responsible for their current high-cost. Therefore, envisaging to develop new, cost-effective, efficient and sustainable techniques for proteins purification, polymer-polymer aqueous two-phase systems (ATPS) with ionic liquids (ILs) as adjuvants are herein proposed for the extraction and purification of IFNα2b from Escherichia coli BL21 cultures. The recombinant protein was produced in the form of inclusion bodies, followed by a dialysis step in order to solubilize the target biomolecule in a specific buffer, with the goal of maintaining the biomolecule stability. Afterwards, several polymers where studied as phase-forming components of ATPS, namely polyethylene glycol (PEG), polypropylene glycol (PPG), as well as Dextran. Furthermore, several ILs belonging to the cholinium, imidazolium, tetralkylammonium and tetralkylphosphonium families, were tested as co-adjuvants for ATPS formation in order to extract and purify of IFNα2b.