Production and purification of antibodies using aqueous biphasic systems composed of ionic liquids as adjuvants


Currently, monoclonal antibodies (mAbs) are the most prevalent class of recombinant protein therapeutics for the treatment of several diseases, namely cancer (e.g.: breast cancer, leukemia and prostate cancer), auto-immune disorders (rheumatoid arthritis and Crohn’s disease), asthma, and cardiovascular and infectious diseases. Although the upstream processing of mAbs, especially immunoglobulin G (IgG), has improved considerably in the last years, the downstream processing has not evolved at the same pace and is currently considered the bottleneck in the manufacturing of therapeutic IgG. The established platform for IgG purification comprises a capture primary stage of the antibody using protein A affinity chromatography, followed by two subsequent chromatographic purification steps. Although this is the industry golden standard purification platform, there are some problems arising from the use of chromatography, such as the high cost associated to the process and also some intrinsic limitations of the ligand itself. In this context, aqueous biphasic systems (ABS) can be highlighted as a promising non-chromatographic alternative, taking into account their advantages resulting from a reduction of the costs and number of steps involved in the process when compared with conventional methods. However, traditional ABS mainly composed of salts and/or polymers are not selective, resulting in low yields and purification factors. On the other hand, ionic liquids (ILs) are a new class of solvents which allow the manipulation of the polarity and affinity of the coexistent phases by an appropriate choice of the chemical structure of the IL. However, and despite this possibility, their application in the formation of ABS for the extraction and purification of IgG has not yet been reported in the literature. In this context, herein in this work, several ILs were studied as adjuvants in different concentrations, in conventional PEG/Phosphate and PEG/Dextran ABS, for the selective extraction and further purification of IgG directly from Chinese Hamster Ovary (CHO) cell culture supernatants. According to the obtained results, it was demonstrated that the presence of ILs as adjuvants allowed the manipulation and the tailoring of phases’ polarities/affinities, increasing ABS extraction potential towards IgG. Moreover, the system composed of PEG 3350 + dextran 500 kDa + H2O + 20% [C4mim]Br has lead to extremely promising results concerning the selective extraction and further purification of IgG, achieving extraction yields of 82% for the PEG-rich phase with a purity level of 64% in a single-step. In this context, novel ABS composed of IL as adjuvant is proposed as an effective platform for the extraction/purification of value-added IgG directly from CHO cell cultures supernatants, and their application at a large scale is thus envisaged.


Emanuel V. Capela

our authors


Dr. Mara G. Freire, Dr. Ana M. Azevedo, Prof. M. Raquel Aires-Barros

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