Purification of monoclonal antibodies, IgG1, from cell culture supernatant by use of metal chelate convective interaction media monolithic columns

Abstract

Monoclonal antibodies (MAbs) have diverse applications in diagnostics and therapeutics. The recent advancement in hybridoma technology for large-scale production of MAbs in bioreactors demands rapid and efficient purification methods. Conventional affinity purification systems have drawbacks of low flow rates and denaturation of antibodies owing to harsh elution conditions. Here, we attempted purification of MAbs by use of a high-throughput metal-chelate methacrylate monolithic system. Monolithic macroporous convective interaction media-iminodiacetate (CIM-IDA) disks immobilized with four different metal ions (Cu²⁺, Ni²⁺, Zn²⁺ and Co²⁺) were used and evaluated for purification of anti-human serum albumin IgG1 mouse MAbs from cell culture supernatant after precipitation with 50% ammonium sulfate. Elution with 10 mM imidazole in the equilibration buffer (25 mM MMA = MOPS (Morpholino propane sulfonic acid) + MES (Morpholino ethane sulfonic acid) + Acetate + 0.5 M NaCl, pH 7.4) resulted in a purification of 25.7 ± 2.9-fold and 32.5 ± 2.6-fold in experiments done using Zn²⁺ and Co²⁺ metal ions, respectively. The highest recovery of 85.4 ± 1.0% was obtained with a CIM-IDA-Zn(II) column. SDS-PAGE, ELISA and immuno-blot showed that the antibodies recovered were pure, with high antigen-binding efficiency. Thus, metal chelate CIM monoliths could be a potential alternative to conventional systems for fast and efficient purification of MAbs from the complex cell culture supernatant.

Functional monolithic platforms: Chromatographic tools for antibody purification

“…In addition to affinity‐based monoliths, anion‐exchange methacrylate monolithic systems constituted by a monolithic macroporous convective interaction media (CIM) were tested and proved to be effective in the isolation of anti‐glycophorin‐A IgG1 mouse mAbs from cell culture supernatant. Also, CIM‐ iminodiacetate (IDA) disks with four different metal ions (Zn 2+ , Cu 2+ , Co 2+ and Ni 2+ ) immobilized were employed for mAb isolation from cell culture supernatant, achieving a maximum recovery of 85.4% of purified antibody [39].…”

Prediction of the isotherms of human IgG adsorption on Ni(II)-IDA-PEVA membrane using artificial neural networks

“…It is based on specific and reversible binding ability of molecules in solution to complementary molecules, the ligands, usually immobilized on a solid chromatographic media, such as agarose gels, membranes, monoliths, cryogels or alternative novel support materials (Labrou and Clonis 1994;Serpa et al 2005;Tscheliessnig and Jungbauer 2009;Prasanna and Vijayalakshmi 2010;Gondim et al 2012;Dogan et al 2012;Rajak et al 2012;BorsoiRibeiro et al 2013;Pavan et al 2014).…”

Purification of Anti-Interleukin-6 Monoclonal Antibody Using Precipitation and Immobilized Metal-Ion Affinity Chromatography

“…Several studies have applied this method for improving the concentration of MAbs (Bresolin et al 2010;El-kak and Vijayalakshmi 1991;Gagnon 2012;Rajak et al 2012;Serpa et al 2005). However, for highresolution purification of MAbs, selective adsorption techniques based on hydrophobic interaction, ion exchange or affinity principles are often used.…”

“…With regard to purification of antibodies, previous studies have used immobilized ligands (e.g. thiophilic and biomimetic ligands), peptides, amino acids, dyes and metal ions or multimodal agents (Boschetti 2001;Bresolin et al 2010;Ghose et al 2013;Gondim et al 2012;Maltezos et al 2014;Rajak et al 2012;Roque et al 2007;Sadavarte et al 2014;Sugita et al 2013;Wolfe et al 2014).…”

“…To improve the formation of coordination bonds, thus minimizing ionic interactions, NaCl was added to the buffer (Porath et al 1975). The nature of the buffer also affects the adsorption of MAbs, irrespective of the support material and chelating ligands used (Bresolin et al 2010;Rajak et al 2012;Serpa et al 2005).…”