백주희 (충남대학교)

안현주 (충남대학교)

백주희 (충남대학교)

정혜경 (충남대학교)

오명진 (충남대학교)

안현주 (충남대학교)

Glycosylation is a critical quality attribute (CQA) of biotherapeutics, influencing efficacy, stability, and immunogenicity. Since immunogenicity risks are heavily modulated by the precise glycan localization, comprehensive site-specific analysis is essential. However, hyperglycosylated darbepoetin alfa (NESP) presents analytical challenges due to five densely adjacent N-glycosylation sites. As conventional tryptic digestion fails to resolve these closely spaced regions, we conducted a sequential digestion using trypsin, followed by non-specific Pronase E to cleave adjacent regions into single-site short glycopeptides. These resulting glycopeptides were analyzed using an integrated CID-EAD LC-MS/MS strategy, which we previously established for monoclonal antibody characterization. Our results demonstrated that combining the complementary fragmentation strategy of CID and EAD enabled significantly enhanced glycopeptide identification and high-confidence assignment. Specifically, CID provided critical glycan composition information via abundant oxonium ions, although it lacked sufficient peptide backbone details. Conversely, EAD successfully generated an extensive series of complementary  backbone ions while keeping the labile multi-sialylated glycans fully intact. This robust performance enabled unambiguous assignment of the highly heterogeneous peptide backbones and precise glycan localization. Using this approach, we successfully mapped the detailed site-specific N-glycosylation profiles across all five adjacent sites of darbepoetin alfa. Consequently, our findings demonstrate that the integrated CID-EAD approach is an essential tool for the advanced characterization of glycopeptides in complex biotherapeutic systems.​