Mechanism of protein desorption from 4-mercaptoethylpyridine resins by arginine solutions

Abstract

A multimodal chromatography column containing 4-mercaptoethylpyridine (4-MEP) as a resin ligand is capable of binding antibodies. Arginine has been proven effective for eluting bound antibodies from the 4-MEP resin column. In this study, the effect of arginine on the binding of antibodies to 4-MEP resin was examined through equilibrium adsorption experiments using bovine γ-globulin (BGG). Arginine was found to efficiently reduce antibody adsorption onto 4-MEP resin more than solvents containing guanidine, urea and NaCl. Organic solvents, including ethylene glycol and glycerol, were also found to be effective for antibody desorption. These results suggest that the attraction between BGG and 4-MEP is based on hydrophobic interactions that can be weakened by arginine or the organic solvents. The use of arginine yielded similar results with globular proteins. Molecular dynamics simulations used to illuminate the mechanism of binding of an arginine molecule to a 4-MEP resin ligand molecule showed that the alkyl chain of the arginine side chain primarily interacts with the pyridine ring of the 4-MEP resin ligand through hydrophobic interactions. Interaction of the backbone or guanidinium group of arginine with the pyridine ring of the 4-MEP resin ligand played an insignificant role in their bindings. The hydrophobic interaction of the arginine side chain with the pyridine ring reduces the attraction between the antibodies and 4-MEP, leading to the efficient dissociation of the antibodies from the 4-MEP resin in the presence of arginine-containing solutions. Thus, utilization of arginine as an eluent can improve antibody purification by multimodal column chromatography.