Characterization of metal chelate methacrylate monolithic disk for purification of polyclonal and monoclonal immunoglobulin G.
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 Cu(2+), Ni(2+) and Zn(2+) 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 Cu(2+) was used as ligand. CIM-IDA disk showed dynamic binding capacity in the range of 14-16 mg/ml with Cu(2+) and 7-9 mg/ml with Ni(2+) for human IgG with MMA buffer. In the case of CIM-IDA-Zn(2+) column, the binding capacity was only about 0.5mg/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.
Prasanna
Rajasekar R
RR
Centre for Bioseparation Technology, VIT University, Vellore 632014, Tamil Nadu, India.
Vijayalakshmi
Mookambeswaran A
MA
D000327:Adsorption; D000818:Animals; D000911:Antibodies, Monoclonal / Q000097:blood / Q000302:isolation & purification*; D001201:Ascites; D002021:Buffers; D002614:Chelating Agents / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000097:blood / Q000302:isolation & purification*; D019216:Metals, Heavy / Q000737:chemistry*; D008689:Methacrylates / Q000737:chemistry*; D051379:Mice; D011485:Protein Binding
D000911:Antibodies, Monoclonal; D002021:Buffers; D002614:Chelating Agents; D007074:Immunoglobulin G; D019216:Metals, Heavy; D008689:Methacrylates