Systematic Preparation of a 66-IgG Library with Symmetric and Asymmetric Homogeneous Glycans and Their Functional Evaluation.

Abstract

Immunoglobulin G (IgG) antibodies possess a conserved N-glycosylation site in the Fc domain. In FcγRIIIa affinity column chromatography, unglycosylated, hemiglycosylated, and fully glycosylated IgG retention times differ considerably. Using retention-time differences, 66 different trastuzumab antibodies with symmetric and asymmetric homogeneous glycans were prepared systematically, substantially expanding the scope of IgGs with homogeneous glycans. Using the prepared trastuzumab with homogeneous glycans, thermal stability and antibody-dependent cellular cytotoxicity were investigated. In some glycan series, a directly proportional relationship was observed between the thermal unfolding temperature (Tm) and the calorimetric unfolding heat (ΔHcal). Antibody function could be deduced from the combination of a pair of glycans in an intact form. Controlling glycan structure through the combination of a pair of glycans permits the precise tuning of stability and effector functions of IgG. Overall, our technology can be used to investigate the effects of glycans on antibody functions.