Reversed-phase liquid chromatography of immunoglobulin G molecules and their fragments with the diphenyl column

Abstract

A diphenyl column was able to resolve two closely related monoclonal IgG2 molecules, while a C8 column failed to separate these IgGs under identical chromatographic conditions. The diphenyl column also showed a better separation of a mixture of two light and two heavy chains than the C8 column. The influence of amino acid side chains from protein sequences in binding to the diphenyl and C8 stationary phases was studied by using a set of synthetic peptides with the sequence GXXLLLKK, where X represents substitution with all of the 20 amino acids. Peptides containing aromatic amino acids showed a greater binding on the diphenyl column than on the C8 column. This increase in retention was attributed to π–π interactions between the aromatic amino acid side chains and the diphenyl ligand. Based on the retention of peptides on the diphenyl column, new retention coefficients were assigned for the separation of proteins. A good correlation was observed between the sum of retention coefficients (ΣRc) for IgGs and their retention time on the diphenyl column. On-column hydrogen–deuterium exchange showed that the diphenyl column had a larger surface of interaction with protein than the C8 column. π–π interactions and the large contact surface resulted in improved resolution of IgGs and their fragments on the diphenyl column.