Comprehensive N-Glycan Mapping using Parallel Reaction Monitoring LC-MS/MS

Glycan composition can impact a biotherapeutic's safety and efficacy. For example, changes in the relative abundance of different glycan attributes like afucosylation, galactosylation or high-mannose content can change the properties or functions of a monoclonal antibody (mAb). While established methods can effectively characterize major glycan species in biotherapeutic drug products, there is still a need for more sensitive and specific methods that can effectively monitor low abundance species which may impact mAb function.

Glycans released from two mAbs, adalimumab and trastuzumab, were derivatized with Rapifluor-MS™. Glycans were separated using HILIC and detected using either fluorescence (FLD) or mass spectrometry (MS). A parallel reaction monitoring (PRM) workflow was used for the MS analysis.

FLD analysis identified 18 and 19 glycan peaks in adalimumab and trastuzumab, respectively. Glycan identities were determined using MS-analysis and a high number of FLD peaks containing co-eluting glycan species were observed. PRM analysis quantified 38 and 39 glycan species in adalimumab and trastuzumab, respectively, and the increase in glycans that could be identified was due to superior sensitivity and selectivity compared to FLD. Notably, many low abundance glycans identified by PRM included species that were not reported in other studies. PRM also offered several additional advantages; unique structural features could be identified using the collected MS/MS spectra and de-coupling MS acquisition and data processing simplified the transfer of methods between instruments. The results established PRM as a precise, informative tool for glycan analysis and quantitation.