Characterization of metal chelate methacrylate monolithic disk for purification of polyclonal and monoclonal immunoglobulin G

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• 作者：Rajasekar R. Prasanna ; Mookambeswaran A. Vijayalakshmi
• 关键词：Immobilized metal affinity chromatography (IMAC) ; Convective interaction media (CIM) monolithic disk ; Dynamic binding capacity ; Buffers ; IgG purification
• 刊名：Journal of Chromatography A
• 出版年：2010
• 出版时间：4 June 2010
• 年：2010
• 卷：1217
• 期：23
• 页码：3660-3667
• 全文大小：595 K

文摘

Dynamic binding capacity (DBC) of commercial metal-chelate methacrylate monolith-convective interaction media (CIM) was performed with commercial human immunoglobulin G (IgG) (Cohn fraction II, III). Monoliths are an attractive stationary phase for purification of large biomolecules because they exhibit very low back pressure even at high flow rates and flow-unaffected binding properties. Adsorption of IgG onto CIM-IDA disk immobilized with Cu²⁺, Ni²⁺ and Zn²⁺ were studied with Tris-acetate (TA), phosphate-acetate (PA) and MMA (MES, MOPS and acetate) buffer systems at different flow rates. Adsorption and elution of IgG varied with different buffers and adsorption of IgG was maximum with MMA buffer. Adsorption of human IgG from Cohn fractions (II, III) was high when Cu²⁺ was used as ligand. CIM-IDA disk showed dynamic binding capacity in the range of 14–16 mg/ml with Cu²⁺ and 7–9 mg/ml with Ni²⁺ for human IgG with MMA buffer. In the case of CIM-IDA-Zn²⁺ column, the binding capacity was only about 0.5 mg/ml of support. Different desorption strategies like lowering of pH and increasing of competitive agent were also studied to achieve maximum recovery. Chromatographic runs with human serum and mouse ascites fluid were also carried out with metal chelate methacrylate monolithic disk and the results indicate the potential of this technique for polyclonal human IgG and monoclonal IgG purification from complex biological samples.