Influence Of Galactosylation On Fc-Mediated Binding And Functional Properties Of Adalimumab

By Agata Burzawa, Yu-Ting Hsu, Stuart Kain, Craig Devine, Pamela Hamill, Marian McKee, Global Operational Development Services, MilliporeSigma; BioReliance® Biotesting Services, Stirling, UK

The N-glycan profile of an antibody plays a critical role in product stability, immunogenicity or pharmacokinetics and can also significantly influence the Fc-region mediated effector functions of antibodies, such as antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC).

β-galactosylation is known to be crucial for CDC activity, since terminal galactose enhances binding of antibody Fc regions to the C1q complement protein, which is required for CDC. β-galactosylated glycans can also increase antibody Fc-region binding to the FcγIIIa receptor, which can give rise to enhanced ADCC activity.

Product characterization is key to successful biological drug development. Comprehensive characterization of new therapeutic monoclonal antibodies requires a deep understanding of their structural and functional critical quality attributes (CQAs), which greatly impact product potency, stability and safety.

We evaluated the criticality of glycan structure on antibody Fc-region mediated effector functions through the application of a highly resolving N-glycan assay, in combination with binding and cell-based assays. In order to demonstrate the influence of terminal galactose on the effector functions of adalimumab, we removed βgalactose from N-glycans using β-1,4 galactosidase and evaluated the impact of the changes in glycosylation profile on CDC and ADCC activity.