Glycosylation is an important attribute of monoclonal antibodies (mAbs) for assessing manufacturing quality. Analysis of non-human glycans containing terminal galactose-α1,3-galactose and N-glycolylneuraminic acid is essential due to the potential immunogenicity and insufficient efficacy caused by mAb expression in non-human mammalian cells. Using parallel sequencing of isobaric glycopeptides and isomeric glycans that were separated by reversed-phase and porous graphitic carbon LC, we report a highly sensitive LC MS/MS method for the comprehensive characterization of low-abundance non-human glycans and their closely related structural isomers. We demonstrate that the straightforward use of high-abundance diagnostic ions and complementary fragments under the positive ionization low-energy collision-induced dissociation is a universal approach to rapidly discriminate branch-linkage structures of biantennary glycans. Our findings reveal the structural diversity of non-human glycans and sulfation of α-galactosylated glycans, providing both an analytical method and candidate structures that could potentially be used in the crucial quality control of therapeutic mAb products.
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