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.
Erythropoietin Biosimilars CERA Darbepoetin alfa Performance-enhancing drug LC–MS–MS
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Part of this research was funded by a grant from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) for 2010 and by the Japan Sports Council for 2011.
Conflict of interest
There are no financial or other relations that could lead to a conflict of interest.
Lai PH, Everett R, Wang FF, Arakawa T, Goldwasser E (1986) Structural characterization of human erythropoietin. J Biol Chem 261:3116–3121PubMedGoogle Scholar
Recny MA, Scoble HA, Kim Y (1987) Structural characterization of natural human urinary and recombinant DNA-derived erythropoietin. Identification of des-arginine 166 erythropoietin. J Biol Chem 262:17156–17163PubMedGoogle Scholar
Kang MJ, Shin SM, Yoo HH, Kwon OS, Jin C (2010) Characteristics of IEF patterns and SDS-PAGE results of Korean EPO biosimilars. Bull Korean Chem Soc 31:2493–2496CrossRefGoogle Scholar
Lasne F, Martin L, Crepin N, de Ceaurriz J (2002) Detection of isoelectric profiles of erythropoietin in urine: differentiation of natural and administered recombinant hormones. Anal Biochem 311:119–126PubMedCrossRefGoogle Scholar
Reichel C, Kulovics R, Jordan V, Watzinger M, Geisendorfer T (2009) SDS-PAGE of recombinant and endogenous erythropoietins: benefits and limitations of the method for application in doping control. Drug Test Anal 1:43–50PubMedCrossRefGoogle Scholar
Kohler M, Ayotte C, Desharnais P, Flenker U, Lüdke S, Thevis M, Völker-Schänzer E, Schänzer W (2008) Discrimination of recombinant and endogenous urinary erythropoietin by calculating relative mobility values from SDS gels. Int J Sports Med 29:1–6PubMedGoogle Scholar
Reichel C, Abzieher F, Geisendorfer T (2009) SARCOSYL-PAGE: a new method for the detection of MIRCERA- and EPO-doping in blood. Drug Test Anal 1:494–504PubMedCrossRefGoogle Scholar
Reichel C (2012) SARCOSYL-PAGE: a new electrophoretic method for the separation and immunological detection of PEGylated proteins. Methods Mol Biol 869:65–79PubMedCrossRefGoogle Scholar
Schellekens H (2004) Biosimilar epoetins: how similar are they? Eur J Hosp Pharm 3:43–47Google Scholar
Guan F, Uboh CE, Soma LR, Maylin G, Jiang Z, Chen J (2010) Confirmatory analysis of continuous erythropoietin receptor activator and erythropoietin analogues in equine plasma by LC–MS for doping control. Anal Chem 82:9074–9081PubMedCrossRefGoogle Scholar
Chang Y, Maylin GM, Matsumoto G, Neades SM, Catlin DH (2011) Screen and confirmation of PEG-epoetin β in equine plasma. Drug Test Anal 3:68–73PubMedCrossRefGoogle Scholar
Yu NH, Ho EN, Wan TS, Wong AS (2010) Doping control analysis of recombinant human erythropoietin, darbepoetin alfa and methoxy polyethylene glycol-epoetin beta in equine plasma by nano-liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem 396:2513–2521PubMedCrossRefGoogle Scholar
Okano M, Sato M, Kageyama S (2014) Identification of the long-acting erythropoiesis-stimulating agent darbepoetin alfa in human urine by liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem 406:1317–1329Google Scholar