title: 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.
authors.affiliation: Centre for Bioseparation Technology, VIT University, Vellore-632014, Tamil Nadu, India.
authors.forename: Poonam
authors.initials: P
authors.lastname: Rajak
authors.forename: M A
authors.initials: MA
authors.lastname: Vijayalakshmi
authors.forename: N S
authors.initials: NS
authors.lastname: Jayaprakash
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002478:Cells, Cultured; D002614:Chelating Agents / Q000737:chemistry* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000295:instrumentation* / Q000379:methods*; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006825:Hybridomas; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008689:Methacrylates / Q000737:chemistry; D051379:Mice; D011485:Protein Binding; D012709:Serum Albumin / Q000378:metabolism; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D007074:Immunoglobulin G; D008689:Methacrylates; D012709:Serum Albumin; D003300:Copper; D015032:Zinc