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

Kathrin Stavenhagen*, Rabah Gahoual, Elena Dominguez Vega, Angelo Palmese, Agnes L.Hipgrave Ederveen, Francesca Cutillo, Wolf Palinsky, Horst Bierau, Manfred Wuhrer

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review


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.

Original languageEnglish
Pages (from-to)1053-1063
Number of pages11
Issue number6
Early online date26 Jul 2019
Publication statusPublished - 18 Aug 2019


  • atacicept
  • Fc-fusion protein
  • glycosylation
  • N-glycans
  • N-glycopeptides
  • O-glycopeptides


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