ArticleAnalytical and bioanalytical chemistry2021
Glycine additive facilitates site-specific glycosylation profiling of biopharmaceuticals by ion-pairing hydrophilic interaction chromatography mass spectrometry.
Article in Analytical and bioanalytical chemistry, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 2 papers.
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2 citing papers in PubMed, 6 citations in OpenAlex.
- Biopharmaceutical analysis - current analytical challenges, limitations, and perspectives.Analytical and bioanalytical chemistry · 2026Review
- A novel hydrophilic polymer-coated magnetic nanomaterial based on the HILIC strategy for fast separation of glycopeptides and glycosylated exosomes.Analytical and bioanalytical chemistry · 2023Article
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4 authors at 1 institution in 1 country.
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Abstract
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.
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