Site-specific N- and O-glycosylation analysis of atacicept

The Fc-fusion protein atacicept is currently under clinical investigation for its biotherapeutic application in autoimmune diseases owing to its ability to bind the two cytokines B-Lymphocyte Stimulator (BLyS) and A PRoliferation-Inducing Ligand (APRIL). Like typical recombinant IgG-based therapeutics, atacicept is a glycoprotein whose glycosylation-related heterogeneity arises from the glycosylation-site localization, site-specific occupation and structural diversity of the attached glycans. Here, we present a first comprehensive site-specific N- and O-glycosylation characterization of atacicept using mass spectrometry-based workflows. First, N- and O-glycosylation sites and their corresponding glycoforms were identified. Second, a relative quantitation of the N-glycosylation site microheterogeneity was achieved by glycopeptide analysis, which was further supported by analysis of the released N-glycans. We confirmed the presence of one N-glycosylation site, carrying 47 glycoforms covering 34 different compositions, next to two hinge region O-glycosylation sites with core 1-type glycans. The relative O-glycan distribution was analyzed based on the de-N-glycosylated intact protein species. Overall, N- and O-glycosylation were consistent between two individual production batches.