Purification of human IgG by negative chromatography on omega-aminohexyl-agarose.
The omega-aminohexyl diamine immobilized as ligand on CNBr- and bisoxirane-activated agarose gel was evaluated for the purification of human immunoglobulin G (IgG) from serum and plasma by negative affinity chromatography. The effects of matrix activation, buffer system, and feedstream on recovery and purity of IgG were studied. A one-step purification process using Hepes buffer at pH 6.8 allowed a similar recovery (69-76%) of the loaded IgG in the nonretained fractions for both matrices, but the purity was higher for epoxy-activated gel (electrophoretically homogeneous protein with a 6.5-fold purification). The IgG and human serum albumin (HSA) adsorption equilibrium studies showed that the adsorption isotherms of IgG and HSA obeyed the Langmuir-Freundlich and Langmuir models, respectively. The binding capacity of HSA was high (210.4 mg mL(-1) of gel) and a positive cooperativity was observed for IgG binding. These results indicate that immobilizing omega-aminohexyl using bisoxirane as coupling agent is a useful strategy for rapid purification of IgG from human serum and plasma.
de Souza
Maria Cristiane Martins
MC
Department of Biotechnological Processes, School of Chemical Engineering, University of Campinas, UNICAMP, P.O. Box 6066, CEP 13083-970, Campinas, SP, Brazil.
Bresolin
Igor Tadeu Lazzarotto
IT
Bueno
Sonia Maria Alves
SM
D000327:Adsorption; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000302:isolation & purification* / Q000378:metabolism; D008024:Ligands; D011485:Protein Binding; D012685:Sepharose / Q000031:analogs & derivatives* / Q000737:chemistry; D012709:Serum Albumin / Q000302:isolation & purification / Q000378:metabolism
D007074:Immunoglobulin G; D008024:Ligands; D012709:Serum Albumin; C024903:aminohexyl-sepharose; D012685:Sepharose