4-(1H-imidazol-1-yl) aniline: A new ligand of mixed-mode chromatography for antibody purification

4-(1H-imidazol-1-yl) aniline (AN) was immobilized on Sepharose CL-6B (AN-Sepharose) for use as a new ligand of mixed-mode chromatography. Adsorption equilibria of immunoglobulin G (IgG) and bovine serum albumin (BSA) to AN-Sepharose were studied at extensive pH values (4.0–8.8) and salt concentrations (0–1.0 mol/L). Static binding studies indicated that AN-Sepharose had a good salt-tolerance property for IgG adsorption up to 1.0 mol/L NaCl. This was attributed to the combined ligand–protein interactions (hydrophobic interaction, hydrogen bonding and charge transfer interaction). By contrast with BSA, AN-Sepharose showed a high binding selectivity for IgG at NaCl > 0.2 mol/L. Dynamic binding capacities (DBC) of IgG and BSA at 10% breakthrough were measured at pH 4.0–8.8 by frontal analysis chromatography. IgG had DBC values over 40 mg/mL at pH 7.0–8.8, and the maximum reached 59 mg/mL at pH 8.0. At pH 5.0, a distinct drop in DBC to 8.5 mg/mL was observed, but that for BSA kept over 22 mg/mL. The result suggested that IgG could be selectively desorbed from AN-Sepharose by decreasing pH to about 5. Therefore, compared to BSA, AN-Sepharose exhibited a dual-selectivity for IgG in both adsorption and elution. Purification of IgG from bovine serum also confirmed the dual-selectivity. IgG purity of the pooled fractions by elution at pH 4.0, 4.5 and 5.0 reached 55% and the highest purity, 80%, was obtained at pH 4.5. The average purification factor of IgG was over 25. The results indicate that AN is a promising ligand of mixed-mode chromatography for antibody purification from a complex feedstock.