Mass Spectrometric-Based Stable Isotopic 2-Aminobenzoic Acid Glycan Mapping for Rapid Glycan Screening of Biotherapeutics

Abstract

Fast, sensitive, robust methods for "high-level" glycan screening are necessary during various stages of a biotherapeutic product's lifecycle, including clone selection, process changes, and quality control for lot release testing. Traditional glycan screening involves chromatographic or electrophoretic separation-based methods, and, although reproducible, these methods can be time-consuming. Even ultrahigh-performance chromatographic and microfluidic integrated LC/MS systems, which work on the tens of minute time scale, become lengthy when hundreds of samples are to be analyzed. Comparatively, a direct infusion mass spectrometry (MS)-based glycan screening method acquires data on a millisecond time scale, exhibits exquisite sensitivity and reproducibility, and is amenable to automated peak annotation. In addition, characterization of glycan species via sequential mass spectrometry can be performed simultaneously. Here, we demonstrate a quantitative high-throughput MS-based mapping approach using stable isotope 2-aminobenzoic acid (2-AA) for rapid "high-level" glycan screening.

LC–MS analysis of glycopeptides of recombinant monoclonal antibodies by a rapid digestion procedure

“…Methods for comparative quantitation have been reported in the literature by analyzing either the released stable isotope labeled oligosaccharides [27][28][29][30][31][32][33][34][35][36] or peptide moieties with stable isotope labels at the peptide termini and the glycosylation sites [37][38][39]. Here, the procedure of dimethyl labeling was employed to specifically label the peptide's N-terminal primary amine and the side chain of lysine (Lys) residue using reagents with stable isotopes and thus allowing detection and quantification of glycopeptides by mass spectrometry [40].…”

Mass spectrometry for glycosylation analysis of biopharmaceuticals

“…Permethylation is therefore commonly performed prior to MALDI-MS of released N-or O-glycans, as it improves sensitivity, stabilizes the labile sialic-acid bonds, and enables the detection of non-sialylated and sialylated glycan species in positive-ion mode [8,20,21,29,30]. This approach was recently applied to characterize N-glycans from influenza antigen [31].…”

Quantitative isomer-specific N-glycan fingerprinting using isotope coded labeling and high performance liquid chromatography–electrospray ionization-mass spectrometry with graphitic carbon stationary phase

“…Xia et al introduced glycan labeling by reductive amination with differentially stable-isotope-coded tags [ 12 C 6 ]-and [ 13 C 6 ]-aniline for quantification of N-glycans by MALDI-TOF-MS [27]. Differentially labeled 2-aminobenzoic acid has been used for glycan screening of biopharmaceuticals as well [28]. Here, we follow the concept of stable isotope-coded labeling using aniline, as [ 13 C 6 ]-aniline is a readily available and inexpensive reagent and the presence of 6 13 C atoms is not expected to cause any shift in retention with most HPLC stationary phases.…”