earticle

영어

Protein glycosylation with structural diversity is one of the most abundant post translational modifications. A wide range of functions for glycans has been described, from structural to regulatory roles in molecular and cellular level. Especially, sialic acid and its modifications are known to affect stability, efficacy, antigenicity, and half-life of biopharmaceuticals. Current glycosylation profiling is mainly focused on N-glycans due to availability of enzymes for their release. In contrast, there is no corresponding enzymes for O-glycan liberation. In general, O-glycans are liberated using alkaline β-elimination which conditions are highly destructive toward sialic acid and its modifications. For this reason, comprehensive O-glycosylation profiling method which preserves the sialic acid and its modifications is required to demonstrate in respect of quality, safety and efficacy of therapeutic glycoproteins. In this study, we developed a new analytical strategy for non-destructive O-glycosylation profiling of biopharmaceuticals using O-glycopeptides produced by non-specific protease. Erythropoietins (EPOs) were digested with pronase E for six hours, and then resulting glycopeptides were enriched using a solid phase extraction with porous graphitized carbon. The biopharmaceutical O-glycosylation profiling and structural characterization were conducted on nanoLC-chip/Q-TOF MS system. We successfully applied this method to analysis of batch-to-batch variation study of darbepoetin alfa, and O-glycosylation profiling of epoetin alfa and beta.