The Impact of Immunoglobulin G1 Fc Sialylation on Backbone Amide H/D Exchange

Kuhne F, Bonnington L, Malik S, Thomann M, Avenal C, Cymer F, Wegele H, Reusch D, Mormann M, Bulau P

Abstract

The usefulness of higher-order structural information provided by hydrogen/deuterium exchange-mass spectrometry (H/DX-MS) for the structural impact analyses of chemical and post-translational antibody modifications has been demonstrated in various studies. However, the structure-function assessment for protein drugs in biopharmaceutical research and development is often impeded by the relatively low-abundance (below 5\%) of critical quality attributes or by overlapping effects of modifications, such as glycosylation, with chemical amino acid modifications; e.g., oxidation or deamidation. We present results demonstrating the applicability of the H/DX-MS technique to monitor conformational changes of specific Fc glycosylation variants produced by in vitro glyco-engineering technology. A trend towards less H/DX in Fc C gamma 2 domain segments correlating with larger glycan structures could be confirmed. Furthermore, significant deuterium uptake differences and corresponding binding properties to Fc receptors (as monitored by SPR) between alpha-2,3- and alpha-2,6-sialylated Fc glycosylation variants were verified at sensitive levels.