A LC-MS All-in-One Workflow for Site-Specific Location, Identification and Quantification of N-/O- Glycosylation in Human Chorionic Gonadotropin Drug Products

Abstract

Site-specific characterization of the N- and O-linked glycosylation on a set of different human chorionic gonadotropin (hCG) drug products was performed by a LC-MS method combining high resolution (120K at m/z 200) mass spectrometry, multiple dissociation methods, tandem mass tag (TMT 10plex) labeling, and partial least squares-discriminant analysis (PLS-DA). In total, the data provided identification, relative quantification, and comparison of site-specific glycosylation of protein therapeutics with a single experiment. Ten different lots and/or brands of commercial therapeutic hCG were labeled with TMT 10plex reagents after tryptic digestion. The labeled intact glycopeptides were then analyzed by high resolution LC-MS with online alternating HCD/ETD/CID dissociation methods. For digested hCG drugs, 1000 intact N- and O-linked glycopeptides were identified. The relative amount of each glycopeptide from hCG products was determined based on the reporter signal intensities of the TMT labeling reagents. Moreover, with the help of TMT 10plex, through just one LC-MS run, PLS-DA was performed to ascertain the differences in glycosylation among different sources of hCG drug products. The results of PLS-DA showed that 167 glycopeptides were found to be significantly different between the naturally derived and recombinant hCG products. The results demonstrate the suitability of this method for similarity assessments and counterfeit identification of hCG as well as other glycoproteins.

Keywords: LC-MS; PLS-DA; glycopeptide; hCG; protein therapeutic.