Suitability of bio-based ionic liquids for the extraction and purification of IgG antibodies

Abstract

In the past decade, remarkable advances in the production and use of antibodies as therapeutic drugs and in research/diagnostic fields have led to their recognition as value-added proteins. These biopharmaceuticals have become increasingly important, reinforcing the current demand for the development of more benign, scalable and cost-effective techniques for their purification. Typical polymer-polymer and polymer-salt aqueous biphasic systems (ABS) have been studied for such a goal; yet, the limited polarity range of the coexisting phases and their low selective nature still are their major drawbacks. To overcome this limitation, in this work, ABS formed by bio-based ionic liquids (ILs) and biocompatible polymers were investigated. Bio-based ILs composed of ions derived from natural sources, namely composed of the cholinium cation and anions derived from plants natural acids, have been designed, synthesized, characterized and used for the creation of ABS with polypropyleneglycol (PPG 400). The respective ternary phase diagrams were initially determined at 25 °C to infer on mixture compositions required to form aqueous systems of two phases, further applied in the extraction of pure immunoglobulin G (IgG) to identify the most promising bio-based ILs, and finally employed in the purification of IgG from complex and real matrices of rabbit serum. Remarkably, the complete extraction of IgG to the IL-rich phase was achieved in a single-step. With pure IgG a recovery yield of 100% was obtained, while with rabbit serum this value slightly decreased to ca. 85%. Nevertheless, a 58% enhancement in the IgG purity was achieved when compared with its purity in serum samples. The stability of IgG before and after extraction was also evaluated by size exclusion high-performance liquid chromatography (SE-HPLC), sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared spectroscopy (FTIR). In most ABS formed by bio-based ILs, IgG maragfreire@ua.pt; Fax: +351 234370084; Tel: +351 234370200; kamlesh@csmcri.org, drkamaleshp@gmail.com; Fax: +91-278-2567562; Tel: +91-278 2567760. † Electronic supplementary information (ESI) available: Source of natural acids, NMR spectra of biobased ILs, experimental data on binodal curves, and SE-HPLC data of rabbit serum samples. See

Separation of immunoglobulin G using aqueous biphasic systems composed of cholinium‐based ionic liquids and poly(propylene glycol)

“…previously applied PPG–IL ABS for the separation of IgG from serum samples. Albeit high recovery yields were reported, the purification percentages were considerably lower than those obtained in this work (< 30%) . Further optimization studies aiming at both the complete partitioning of IgG to the IL‐rich phase and the precipitation of albumin at the interface may be thus envisioned with IL‐based ABS to confirm their potential as alternative purification systems for antibodies.…”

“…Although the ILTPP approach does not induce the recovery of IgG at the interface, it however allows the precipitation of the major contaminant protein from serum (albumin), leading to an increase in the IgG purity at the IL‐rich phase. Mondal et al . previously applied PPG–IL ABS for the separation of IgG from serum samples.…”

Controlling the l‐asparaginase extraction and purification by the appropriate selection of polymer/salt‐based aqueous biphasic systems

“…Recently, polymer/salt‐based ABS using cholinium ([Ch] + )‐based salts (or ionic liquids, ILs) were proposed as platforms for solutes partition, particularly, due to the relative hydrophobicity/hydrophilicity balance between the coexisting phases . Furthermore, it was demonstrated that [Ch] + ‐based salts can promote specific protein‐ions interactions, acting directly on the protein partitioning capacity, as shown for beta‐lactoglobulin, A and B, lysozyme, and ribonuclease A and B, as well as on protein stabilization, as previously observed for monoclonal antibodies, cytochrome c, rubisco, bovine serum albumin (BSA) and α‐chymotrypsin …”

“…Recently, polymer/salt-based ABS using cholinium ([Ch] + )-based salts (or ionic liquids, ILs) were proposed as platforms for solutes partition, [22][23][24][25] particularly, due to the relative hydrophobicity/hydrophilicity balance between the coexisting phases. 26 Furthermore, it was demonstrated that [Ch] + -based salts can promote specific protein-ions interactions, acting directly on the protein partitioning capacity, as shown for beta-lactoglobulin, A and B, lysozyme, and ribonuclease A and B, 27 as well as on protein stabilization, 11 as previously observed for monoclonal antibodies, 28 cytochrome c, 24 rubisco, 19 bovine serum albumin (BSA) 29 and -chymotrypsin. 30 These previous studies demonstrated that polymer/salt ABS can provide biocompatible environments to maintain the protein structural conformation 11,19,24,[28][29][30] and to enlarge the range of protein partition driving mechanisms, 27 but a careful selection of the phase forming agents is crucial.…”

“…These previous studies demonstrated that polymer/salt ABS can provide biocompatible environments to maintain the protein structural conformation and to enlarge the range of protein partition driving mechanisms, but a careful selection of the phase forming agents is crucial. Nevertheless, only a few works have investigated the mechanisms behind the purification of ASNase using ABS .…”