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

Glycine additive facilitates site-specific glycosylation profiling of biopharmaceuticals by ion-pairing hydrophilic interaction chromatography mass spectrometry.
Anal Bioanal Chem. 2020 Nov 26;:
Authors: Zhao Y, Raidas S, Mao Y, Li N
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-supp...

Source: Analytical and Bioanalytical Chemistry - November 26, 2020 Category: Chemistry Authors: Zhao Y, Raidas S, Mao Y, Li N Tags: Anal Bioanal Chem Source Type: research