Glycoengineering of Monoclonal Antibodies with Homogeneous Glycan Isomers with Asymmetric Antennae

Glycosylation is an important post-translational modification that can alter the biological activities of antibodies. In monoclonal antibody biotherapeutic products, glycosylation of the Fc-domain, which is often sensitive to manufacturing conditions, can alter product potency by affecting its interactions with host immune cells. Improving control of antibody glycosylation to produce more chemically defined products and to better understand glycan function is gaining interest, with chemoenzymatic glycan engineering emerging as a powerful technique. This chapter describes methods to obtain purified glycans, including isomers with asymmetric antennae, their enzymatic incorporation into monoclonal antibodies, and a natural killer T cell-based binding assay to determine FcγRIIIA (CD16A) binding affinity.