Discovery and Characterization of Novel G0 Glycan Isomers in an Afucosylated Therapeutic Antibody Using Liquid Chromatography–Mass Spectrometry

Therapeutic antibodies are a class of glycoproteins that commonly carry conserved N-glycans at the Fc domain, and the attached N-glycans play a pivotal role in their biological function and efficacy. In this study, we conducted a detailed N-glycan profiling of an afucosylated monoclonal antibody using the Waters GlycoWorks RapiFluor-MS kit and hydrophilic interaction liquid chromatography coupled with fluorescence detection and mass spectrometry (HILIC-FLD-MS). We discovered and reported for the first time novel G0 glycan isomers on a monoclonal antibody. The G0 glycan has a composition of two additional N-acetylglucosamine (GlcNAc) units in addition to the core pentasaccharide structure of N-glycans. The MS/MS profile revealed few fragmentation differences for RapiFluor-MS-labeled glycan isomers. To enhance structural elucidation, a reduction and permethylation assay was performed. Reversed-phase liquid chromatography (RP-LC) separated permethylated glycans due to their altered hydrophobic properties and revealed the presence of additional isomers. The fragmentation of sodium adducts of the permethylated glycans provided distinct patterns among isomers, indicating a bisecting structure for the novel G0 glycan isomers previously identified. Since the bisecting glycans possess one GlcNAc on mannose with 1-4 linkage (bisecting GlcNAc), the other GlcNAc could occupy either branching antenna, resulting in the additional subtle positional isomers, which agrees with our observation. This study underscores the utility of permethylation coupled with advanced chromatography and mass spectrometry techniques to resolve glycan isomers and contributes to a deeper understanding of glycan structural diversity in biologic therapeutic development.