Abstract
Quality control of monoclonal antibodies is challenging due in part to the diversity of post-translational modifications present. The regulation of the N-glycans of IgG-Fc domain is one of the key factors to maintain the safety and efficacy of antibody drugs. The FcγRIIIa affinity column is an attractive tool for the precise analysis of the N-glycans in IgG-Fc domain. We used the mutant FcγRIIIa, which is produced in Escherichia coli and is therefore not glycosylated, as an affinity reagent to analyze the N-glycans of monoclonal antibodies expressed in Expi293 and ExpiCHO cells. The monoclonal antibodies expressed in these cells showed very different chromatograms, because of differences in terminal galactose residues on the IgG-Fc domains. We also carried out kinetic and thermodynamic analyses to understand the interaction between monoclonal antibodies and the mutant FcγRIIIa. Expi293 cell-derived monoclonal antibodies had higher affinity for the mutant FcγRIIIa than those derived from ExpiCHO cells, due to slower off rates and lower binding entropy loss. Collectively, our results suggest that the FcγRIIIa column can be used to analyze the glycosylation of antibodies rapidly and specifically.