LC-MS/MS-Based Site-Specific N-Glycosylation Analysis of VEGFR-IgG Fusion Protein for Sialylation Assessment Across IEF Fractions

Abstract

The glycosylation profile of therapeutic proteins significantly influences their efficacy, stability, and immunogenicity. Sialylation is crucial for the biological activity and pharmacokinetics of fusion proteins used in treating angiogenic disorders, making sialic acid levels a critical quality attribute in the development and production of biologics. In this study, we employed a mass spectrometry-based approach to assess sialylation levels through site-specific N-glycosylation analysis. To validate the method’s effectiveness, IEF fractions (acidic, main, and basic) obtained from the production media of the VEGFR-IgG fusion protein and anticipated to exhibit varying sialylation levels were analyzed. Our analytical method successfully evaluated the sialylation levels of each domain—IgG, VEGFR-1, and VEGFR-2—within the Fc-fusion protein. The results confirm that the overall sialylation level of the Fc-fusion protein correlated with the levels observed across the IEF fractions. This finding highlights the value of LC-MS/MS-based sialylation monitoring as a crucial tool for biosimilar development and quality control, particularly in optimizing target protein production. Additionally, glycopeptide-based LC-MS analysis enables site-specific sialylation evaluation, ensuring consistent profiles for robust quality assurance.

Keywords

• Eylea fusion protein
• sialylated N-glycopeptide
• mass spectrometry