Structure-based comprehensive identification of erythropoiesis-stimulating agents and their biosimilars

Erythropoietin (EPO) is a glycoprotein that stimulates production of red blood cells. After the patents for recombinant EPO (epoetin) expired in 2007, biosimilar erythropoiesis-stimulating agents (ESAs) have become widely circulated in the black market. However, the heterogeneity of ESAs in the post-translational glycosylation hinders the identification of these compounds. In the present study, after cleaving N-linked glycans from tryptic peptides of ESAs with N-glycosidase F, we analyzed the resulting eight glycan-free peptides by liquid chromatography–electrospray ionization-tandem mass spectrometry in the multiple reaction monitoring mode. It has been confirmed that all ESAs studied gave rise to common signature peptides SLTTLLR, VYSNFLR, and YLLEAK. In darbepoetin alfa (DPO), glycan-free tryptic peptide GQALLVNSSQVNETLQLHVDK was detected instead of GQALLVNSSQPWEPLQLHVDK in the epoetin. One of the signature peptides VNFYAWK was found to be significantly suppressed in continuous erythropoietin receptor activator (CERA), indicating pegylation at Lys52. Our method allowed simultaneous identification of ESAs including epoetin, DPO, CERA, and the biosimilars.