Spotlight on Glycan Pairing: The Generation and Impact of Monoclonal Antibody Asymmetrical Fc N‑Glycan Pairs on Fc Receptor Interaction.

Monoclonal antibodies' Fc N-glycans play a crucial role in their therapeutic efficacy, as they influence effector functions through Fc receptor binding. However, the impact of asymmetrical Fc glyco-pairs is often overlooked in assessing Fc receptor binding and effector functions. This study addresses this gap by generating pure asymmetrical Fc glyco-pairs and evaluating their Fc receptor binding properties, thereby providing a comprehensive understanding of the impact of Fc N-glycans. Utilizing redox pairing and affinity chromatography, homogeneously asymmetrical Fc glyco-pairs were generated, and their interaction properties toward Fcγ receptors IIIa, IIa, IIb, and I were determined by surface plasmon resonance. The results underscore the importance of considering the apparent glycan distribution of Fc N-glycans as glycan pairing was found to individually influence Fc receptor binding. Notably, single afucosylation significantly increased the affinity for FcγRIIIa, while the effect of galactosylation was detectable but less pronounced. Galactosylation, however, played a crucial role in FcγRIIa binding, with asymmetrical galactosylation being sufficient for the whole effect. In contrast, for FcγRIIb, afucosylation was more important, while galactosylation played a minor role. Furthermore, glycosylation-dependent Fc-FcγRI complex stability differences could be resolved, challenging the commonly held belief that this interaction is glycosylation independent.