Discovery and Characterization of Unusual O-Linked Glycosylation of IgG4 Antibody Using LC-MS.

BACKGROUND: Consensus is that immunoglobulin IgG4 contains only N-linked glycosylation. The analysis of several batches of commercial biopharmaceutical product Dupixent using top-down intact mass spectrometry revealed that this IgG4 features a small amount of O-linked glycosylation in the Fab region. This is the first report of an O-linked glycosylation in an IgG4 antibody. METHODS: Monoclonal antibody solutions were subjected to cation exchange (CEX) and reverse phase (RP) chromatography and/or additional preconcentration/fractionation methods to prepare samples for subsequent analysis. Advanced MS analysis and fragmentation techniques (HCD, ETD, and EThcD) were employed to localize the O-linked glycosylation as well as elucidate the structure of the glycan(s). RESULTS: O-linked glycosylation in the IgG4 dupilumab was discovered by intact-MS. The probable location was narrowed down to four sites in the CH1 domain, and the structure of the O-linked glycan was determined to be of Core 1 type. The relative quantities of the modifications were low, but the glycosylation was consistently detected in several batches of Dupixent. CONCLUSIONS: We discovered a rare glycosylation modification on dupilumab, an IgG4 antibody. The O-linked glycosylation was characterized and localized in the Fab region.