Purification of human IgG by negative chromatography on ω-aminohexyl-agarose

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2010-02, Vol.878 (5), p.557-566
• Main Authors: de Souza, Maria Cristiane Martins, Bresolin, Igor Tadeu Lazzarotto, Bueno, Sonia Maria Alves
• Format: Article
• Language: English
• Subjects: Adsorption; Aminohexyl; Analysis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography, Affinity - methods; Fundamental and applied biological sciences. Psychology; General pharmacology; Human IgG; Human plasma; Human serum; Humans; Hydrogen-Ion Concentration; Immunoglobulin G - isolation & purification; Immunoglobulin G - metabolism; Ligands; Medical sciences; Negative chromatography; Pharmacology. Drug treatments; Protein Binding; Purification; Sepharose - analogs & derivatives; Sepharose - chemistry; Serum Albumin - isolation & purification; Serum Albumin - metabolism
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2009.12.034

The ω-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 ω-aminohexyl using bisoxirane as coupling agent is a useful strategy for rapid purification of IgG from human serum and plasma.