Glycine additive facilitates site-specific glycosylation profiling of biopharmaceuticals by ion-pairing hydrophilic interaction chromatography mass spectrometry.

Many biotherapeutics such as monoclonal antibodies (mAb) and Fc-domain fusion proteins contain heterogeneous glycan contents at one or multiple glycosylation site(s). Site-specific glycan profile characterization is critical for monitoring the quality of these molecules during different stages of drug development. Hydrophilic interaction chromatography (HILIC) as an orthogonal separation method to reversed-phase liquid chromatography (RPLC) can achieve better glycopeptide identification due to the effective separation between individual glycoforms as well as the separation of glycopeptides from high-abundance non-glycosylated peptides, which can be further improved by modifying the mobile phases with ion-pairing agents (IP-HILIC). However, an online IP-HILIC coupled to mass spectrometry (MS) detection may suffer from the suppression of mass spectrometry signal during electrospray ionization due to the trifluoroacetic acid (TFA), commonly used as an ion-pairing agent. Here, we reported an optimized experimental condition for IP-HILIC-MS where glycine is added in the TFA-containing mobile phases to enhance the MS detection sensitivity for glycopeptides up to ~ 50-fold by eliminating the ion-suppression effect of an ion-pairing agent while still retaining excellent separation capacity. We demonstrated that with enhanced detection sensitivity, IP-HILIC-MS can confidently identify an increased number of site-specific N-linked glycans for IgG1, and IgG4 mAbs as well as an Fc-domain fusion protein (containing five N-glycosylation sites) through MS/MS-based search in the data-dependent acquisition mode, meanwhile, achieve comparable quantitative results compared with the traditional methods. We also demonstrated that IP-HILIC-MS can be used to identify low-level O-glycosylation and non-consensus N-glycosylation on mAbs without any enrichment prior to LC-MS analysis. Graphical abstract.