Ionic-liquid-based aqueous biphasic systems, three phase partitioning methods and hybrid processes combined with ultrafiltration are sustainable strategies for the downstream processing of monoclonal antibodies.

Given the biotechnology advances observed in recent years in terms of upstream, the development of effective downstream processes becomes mandatory to decrease the associated costs of biotechnological-based products. Although a large interest has been devoted to ionic-liquid-based aqueous biphasic systems (IL-based ABS) as tailored separation platforms, imidazolium-based ILs have been the preferred choice as phase-forming agents. To overcome some toxicity and biodegradability issues associated to imidazolium-based ILs, novel ABS composed of ILs analogues of glycine-betaine (AGB-ILs) are here proposed and investigated. Five AGB-ILs were synthesized, characterized in terms of ecotoxicity, and applied toward the development of novel ABS formed with Na SO . Three commercial ILs were also investigated for comparison purposes. The respective ABS ternary phase diagrams, as well as the tie-lines and tie-line lengths, were determined at 25°C. Finally, their performance as extraction strategies was evaluated with five amino acids (L-tryptophan, L-phenylalanine, D-phenylalanine, L-tyrosine and L-3,4-dihydroxyphenylalanine/L-dopa). In all studied systems amino acids preferentially migrate to the IL-rich phase, and with AGB-ILs, the amino acid extraction efficiencies to the IL-rich phase range between 65% and 100%, obtained in a single-step. Furthermore, the studied AGB-ILs display a higher ability to form ABS and to extract amino acids than ABS composed of more traditional and commercial ILs. In summary, novel ABS composed of AGB-ILs can be formed and used as separation routes of value-added compounds of biotechnological interest. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1205-1212, 2018.

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In this work, the extraction and separation of bovine serum albumin (BSA) from its original matrix, i.e., bovine serum, was performed using a novel ionic-liquid-based aqueous biphasic system (IL-based ABS). To this end, imidazolium-, phosphonium-, and ammonium-based ILs, combined with the anions’ acetate, arginate and derived from Good Buffers, were synthesized, characterized, and applied in the development of ABS with K2HPO4/KH2PO4 buffer aqueous solutions at pH 7. Initial studies with commercial BSA revealed a preferential migration of the protein to the IL-rich phase, with extraction efficiencies of 100% obtained in a single-step. BSA recovery yields ranging between 64.0% and 84.9% were achieved, with the system comprising the IL tetrabutylammonium acetate leading to the maximum recovery yield. With this IL, BSA was directly extracted and separated from bovine serum using the respective ABS. Different serum dilutions were further investigated to improve the separation performance. Under the best identified conditions, BSA can be extracted from bovine serum with a recovery yield of 85.6% and a purity of 61.2%. Moreover, it is shown that the BSA secondary structure is maintained in the extraction process, i.e., after being extracted to the IL-rich phase. Overall, the new ABS herein proposed may be used as an alternative platform for the purification of BSA from serum samples and can be applied to other added-value proteins.

Aqueous biphasic systems (ABS) can integrate multiple unit operations and operate under continuous mode, contributing to the development of sustainable separation processes. Encouraged by the designer solvent features of ionic liquids (ILs), we herein propose their use as components of double-stimuli-responsive (temperature- and pH-driven) ABS. Resorting to choline-alkanoate-based ILs as the pH-responsive components and poly­(propylene glycol) (PPG 400) as the thermo-responsive component, the ABS ternary phase diagrams are determined at various temperature (25–45 °C) and pH (3–7) conditions. While the liquid–liquid phase diagram response to temperature obeys a lower critical solution temperature-like behavior, the response to pH correlates with the pK a of the IL anion parent acid. The simultaneous responsiveness to temperature and pH is then shown, whose results inspire the development of customizable separation techniques as proved with the simultaneous (one-step) separation of two dyes. By a proper customization of the IL chemical structure and stimuli applied, ABS may be designed to improve the performance and sustainability of separation processes.