Chromatographic Analysis of the N-Glycan Profile on Therapeutic Antibodies Using Fc gamma RIIIa Affinity Column Chromatography

N-Linked glycosylation on IgG has a profound impact on antibody functions. The relationship between the N-glycan structure and the binding affinity of Fc gamma RIIIa, relating to antibody-dependent cell-mediated cytotoxicity (ADCC) activity, is important for the efficient development of a therapeutic antibody. Here, we report an influence of the N-glycan structure of IgGs, Fc fragments, and antibody-drug conjugates (ADCs) on Fc gamma RIIIa affinity column chromatography. We compared the retention time of several IgGs with heterogeneous and homogeneous N-glycans. IgGs with a heterogeneous N-glycan structure provided several peaks in column chromatography. On the other hand, homogeneous IgGs and ADCs gave a single peak in column chromatography. The length of glycan on IgG also affected the retention time of the Fc gamma RIIIa column, suggesting that the length of glycan is also impacted by binding affinity to Fc gamma RIIIa, resulting in ADCC activity. This analytic methodology provides evaluation of the binding affinity of Fc gamma RIIIa and ADCC activity, not only full-length IgG but also Fc fragments, which are difficult to measure in a cell-based assay. Furthermore, we showed that the glycan-remodeling strategy controls the ADCC activity of IgGs, Fc fragment, and ADCs.