YINDONGTAN (충남대학교)

YINDONGTAN (충남대학교)

Capillary electrophoresis (CE) is widely employed for high-resolution analysis of biotherapeutics, providing key data on glycosylation and charge variants. While CE is often paired with laser-induced fluorescence (LIF) detection for glycan analysis, recent advancements have enabled direct hyphenation of CE to MS, enhancing the analysis of native glycans even from limited sample quantities. In this study, we developed and optimized a label-free CE-MS method for characterizing the N-glycan critical quality attributes(CQAs) of erythropoietin(EPO) products. N-glycans released from three commercial EPOs were separated based on their sialic acid content and O-acetylation using a polyethylene oxide-coated capillary and an acidic, volatile buffer (10 mM ammonium formate, pH 2.5). This setup effectively minimized electroosmotic flow(EOF), providing sharp peak shapes and reproducible migration times. The total analysis was under 15 minutes, with most glycan species showing relative standard deviations (RSDs) below 20% in quantitative measurements. Comparative profiling revealed distinct sialylation and O-acetylation pattern, further confirmed by PCA. Notably, Aranesp contained a higher degree of O-acetylation, while NESP and Recormon displayed differing distributions of tri- and tetra-sialylated glycans. These results demonstrate the suitability of CE-MS for glycan analysis in terms of speed, sensitivity, and environmentally friendliness. This approach is well-suited for evaluating glycan CQAs during drug development and ensuring glycoprotein efficacy and safety of glycoprotein therapeutics