EThcD Mass Spectrometry Workflow Characterizes Aflibercept N-Glycosylation, O-Glycosylation, and Aspartic Acid Isomerization

Characterizing post-translational modifications (PTMs), particularly glycosylation, is paramount for developing safe and effective biotherapeutics. These modifications profoundly impact a protein's structure, stability, pharmacokinetics, biological activity, and potential immunogenicity. Current analytical methods often struggle with the integrated, site-specific characterization of complex PTMs like glycosylation heterogeneity and aspartic acid (Asp) isomerization within a single workflow. Aflibercept, a recombinant fusion protein, possesses five N-glycosylation sites and multiple potential O-glycosylation sites, making its comprehensive PTM analysis critical for quality control and comparability assessments.

Researchers employed an advanced EThcD data-dependent MS2 (ddMS2) peptide mapping workflow to thoroughly characterize Aflibercept. The analysis was performed on a Thermo Scientific Orbitrap Excedion Pro mass spectrometer equipped with the EASY-ETD option, specifically designed for detailed PTM analysis. This approach aimed to provide an integrated characterization of glycosylation heterogeneity and aspartic acid isomerization. The study focused on identifying and relatively quantifying site-specific N-glycosylation profiles, detecting O-glycopeptides, and differentiating isoAsp residues, alongside other PTMs like deamidation and oxidation, all within a unified analytical framework.

The EThcD workflow successfully identified and relatively quantified site-specific N-glycosylation profiles across all five known glycosylation sites of Aflibercept. This comprehensive mapping provides unprecedented detail on the N-glycan heterogeneity. Furthermore, the study provided direct experimental evidence for site-specific O-glycosylation, identifying an O-glycopeptide localized at S12. This finding addresses a previously less characterized aspect of Aflibercept's PTM landscape. The EThcD fragmentation approach proved highly effective in differentiating and precisely localizing isoAsp residues, even within low-abundance peptides containing multiple Asp residues, through the detection of characteristic diagnostic fragment ions. This capability is crucial for understanding protein stability and potential degradation pathways. The integrated analytical workflow also simultaneously characterized several other important PTMs, including deamidation and oxidation, demonstrating its versatility.

The results highlight that EThcD peptide mapping supports the integrated multi-attribute method (MAM) concept for complex glycoproteins, combining glycosylation analysis, Asp/isoAsp differentiation, and PTM profiling in a single experiment.