earticle

영어

Improved methods are required for glyco-characterization of biopharmaceuticals and comparison between their corresponding biologics and biosimilar, where they have multiple glycosylation sites such as Etanercept of a fusion protein. In order to enhance sensitivity of discovery and minimize the sample loss, the combination of a TMT labeling method and fractionation using high flow HPLC has contributed to detect low amount of peptide in data dependent mode of LC-MS/MS. Here, we report a new method combined with TMT labeling, fractionation using ZIC-HILIC HPLC, LC-MS/MS using HCD triggered CID/EThcD, and computational software for identification of site-specific N- and O-glycopeptide and comparison of their corresponding biologics and biosimilar. Of three different Etanercept, such as Enbrel (biologics), Etoloce and Eucept biosimilars), a total of 115 N- and O- glycopeptides were identified and quantified using TMT-11plex labeling including triplicates and 22 ZIC-HILIC fractionation in three and 15 of N- and O- glycosylation sites, respectively. Two O-glycopeptides in different O-glycosylation site and seven core 2 type of O-glycopeptides were first reported. In particular, three kinds of O-glycopeptides with NeuGC which could be a foreign agent of immune response. In depth of quantification, our strategy shows site-specific similarity is quite not similar between biologics and biosimilar.