Bioprocessing of recombinant proteins using alternative purification platforms

Abstract

The advent of biopharmaceuticals in modern medicine brought enormous benefits to diverse human diseases and improved the well-being of many people worldwide. Since the FDA approval of humulin (recombinant insulin) in 19821, remarkable advances in the treatment of chronic diseases have been achieved with biopharmaceutical-based therapies2. This sector represents 40% of ca. 6000 products currently in clinical development2, and is dominated by therapeutic proteins with over 200 protein drugs in the market1. Along the years, many improvements in the biopharmaceuticals upstream stage resulted in high titers of the desired product, and shifted the bioprocess bottleneck to the downstream processing, which is currently dominated by chromatography, accounting with more than 70% of total downstream costs3. Aiming at finding new cost-effective, efficient and sustainable technologies for proteins purification, novel polymer-polymer aqueous biphasic systems (ABS) with ionic liquids (ILs) as adjuvants are investigated as alternative purification platforms for the downstream of interferon alfa 2b (IFNα2b) from Escherichia coli BL21 cultures. Initial experiments showed that the production of IFNα2b is higher using the SOB culture medium and the western-blot analysis revealed that it is present in the inclusion body fraction. This fraction was washed, solubilized using a specific buffer and, finally dialyzed. After analyzing the stability of the target protein in several phase-forming components, the ternary phase diagrams of ABS composed by polyethylene glycol (PEG), polypropylene glycol (PPG) with ILs as adjuvants were determined at 25 °C, as well as the corresponding tie-lines, tie-line lengths and critical points. Chloride-based ILs combined with cholinium, imidazolium, pyrrolidinium, piperidinium, tetralkylammonium and tetralkylphosphonium cations were investigated. In summary, this study reports effective IFNα2b purification platforms from E. coli based on polymer-polymer-ABS, being highlighted the beneficial role of ILs in the downstream processing of proteins, either as adjuvants in ABS or by exploring new ILs features in protein stabilization.