Tian Wang, Lily Chu, Wenzhou Li, Ken Lawson, Izydor Apostol and Tamer Eris
One Amgen Center Dr, MS 30E-1-B, Thousand Oaks, CA 91320, USA
In traditional methods for N-linked oligosaccharide characterization, all the glycans are released enzymatically from proteins prior to labeling and analysis. A significant drawback of these methods is that they do not provide site specific glycosylation information. In this paper, we describe the development, qualification, and application of a LC-MS-based peptide mapping method for site specific quantitation of N-linked glycans present on an IgG1 molecule containing two distinct Nlinked glycosylation sites; one on HC Fab region and the other on HC Fc region. LC and MS conditions were optimized to achieve optimal separation and quantitation of glycoforms using a high-resolution, accurate-mass Orbitrap MS instrument, Q ExactiveTM, with automated quantitation software, Pinpoint (Thermo). The LC/MS method was shown to have acceptable accuracy, reproducibility, precision, and linearity for routine glycan profiling. The results from the quantitation of individual glycan species obtained from the LC-MS method were consistent with those obtained from traditional HILIC analysis of enzymatically released and fluorescently labelled glycans. Our work has demonstrated the quantitative LC/MS method using automated software is suitable for providing site-specific glycan information for routine profiling of N-glycans on immunoglobulins. This high resolution analysis is important for determining product comparability and biosimilarity.