An Integrated Strategy Reveals Complex Glycosylation of Erythropoietin Using Mass Spectrometry

The characterization of therapeutic glycoproteins is challenging
due to the structural heterogeneity of the therapeutic protein glycosylation.
This study presents an in-depth analytical strategy for glycosylation
of first-generation erythropoietin (epoetin beta), including a developed
mass spectrometric workflow for N-glycan analysis, bottom-up mass
spectrometric methods for site-specific N-glycosylation, and a LC-MS
approach for O-glycan identification. Permethylated N-glycans, peptides,
and enriched glycopeptides of erythropoietin were analyzed by nanoLC-MS/MS,
and de-N-glycosylated erythropoietin was measured by LC-MS, enabling
the qualitative and quantitative analysis of glycosylation and different
glycan modifications (e.g., phosphorylation and O-acetylation). The
newly developed Python scripts enabled the identification of 140 N-glycan
compositions (237 N-glycan structures) from erythropoietin, especially
including 8 phosphorylated N-glycan species. The site-specificity
of N-glycans was revealed at the glycopeptide level by pGlyco software
using different proteases. In total, 114 N-glycan compositions were
identified from glycopeptide analysis. Moreover, LC-MS analysis of
de-N-glycosylated erythropoietin species identified two O-glycan compositions
based on the mass shifts between non-O-glycosylated and O-glycosylated
species. Finally, this integrated strategy was proved to realize the
in-depth glycosylation analysis of a therapeutic glycoprotein to understand
its pharmacological properties and improving the manufacturing processes.