Purification of Monoclonal Antibodies from Cell-Culture Supernatant by Use of Anion-Exchange Convective Interaction Media (CIM) Monolithic Columns

• Published in: Chromatographia 2010-12, Vol.72 (11-12), p.1183-1188
• Main Authors: Dhivya, Ambur P, Kumar, Bellie Prem, Prasanna, Rajasekar R, Jayaprakash, Naatamai S, Vijayalakshmi, Mookambeswaran A
• Format: Article
• Language: English
• Subjects: Analytical Chemistry; Analytical, structural and metabolic biochemistry; Anion exchange and buffers; Biological and medical sciences; Chemistry; Chemistry and Materials Science; Chromatography; CIM disk; Column liquid chromatography; Full Short Communication; Fundamental and applied biological sciences. Psychology; Glycoproteins; Laboratory Medicine; Monoclonal antibody; Pharmacy; Proteins; Proteomics; purification
• ISSN: 0009-5893; 1612-1112
• DOI: 10.1365/s10337-010-1787-3

Purified monoclonal antibodies (mAb) have been used in therapeutics and some analytical procedures. Purification of mAb by use of high-throughput anion-exchange methacrylate monolithic systems has been attempted in this work. Monolithic macroporous convective interaction media (CIM) with diethylaminoethyl (DEAE) and ethylene diamine (EDA) as anion-exchange ligands were used and evaluated for purification of anti-glycophorin-A IgG1 mouse mAbs from cell culture supernatant (CCS) after precipitation with 50% ammonium sulfate. The adsorption and elution of mAb from the CCS on CIM-DEAE and CIM-EDA disks were studied with three different buffer systems, acetate, MOPS (3-(N-morpholino)propanesulfonic acid), and Tris, to study the effect of the nature of buffer ions and to find the optimum buffer conditions for purification of mAb. The optimum buffers for purification of mAb using CIM-DEAE and CIM-EDA were 50 mM acetate buffer, pH 5.1 and 20 mM Tris buffer, pH 8.0, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunosorbent assay (ELISA) showed the antibody fractions obtained were highly pure, with high antigen-binding efficiency. High specific activity with purification factors of 130 ± 34 (unretained fraction with acetate buffer) and 74 ± 13 (fraction eluted with Tris buffer containing 0.6 M NaCl) was obtained for IgG1 using the CIM-DEAE and CIM-EDA disks, respectively. The results indicate that rapid separation and efficient recovery of high-purity anti-glycophorin-A mAbs could be achieved by use of anion-exchange CIM disks.