Glycine additive facilitates site-specific glycosylation profiling of biopharmaceuticals by ion-pairing hydrophilic inte
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RESEARCH PAPER
Glycine additive facilitates site-specific glycosylation profiling of biopharmaceuticals by ion-pairing hydrophilic interaction chromatography mass spectrometry Yunlong Zhao 1 & Shivkumar Raidas 1 & Yuan Mao 1 & Ning Li 1 Received: 14 September 2020 / Revised: 19 November 2020 / Accepted: 21 November 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
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 lowlevel O-glycosylation and non-consensus N-glycosylation on mAbs without any enrichment prior to LC-MS analysis. Keywords Ion-pairing hydrophilic interaction chromatography . Mass spectrometry . Glycopeptide analysis . Glycine-assisted electrospray . Biopharmaceuticals
Introduction Glycosylation is a critical quality attribute of biotherapeutics including monoclonal antibodies (mAb) and Fc-domain (fragment crystallization domain) fusion proteins.1 The Fc-domain glycosylation profile at the conserved asparagine-297 site is strongly associated with effector functions such as antibodydependent cellular cytotoxicity (ADCC) and complementdependent cytotoxicity (CDC), which can impact the drug Yunlong Zhao and Shivkumar Raidas contributed equally to this work. * Yuan Mao [email protected] 1
Analytical Chemistry, Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, N
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