Anti-Doping Laboratory, Mitsubishi Chemical Medience Corporation, Tokyo, Japan.
Drug Test Anal. 2011 Nov-Dec;3(11-12):798-805. doi: 10.1002/dta.369. Epub 2011 Oct 19.
Since the expiration of patent protection, a number of new recombinant erythropoietin (rEPO) biosimilars have appeared on the worldwide market. In 2010, epoetin kappa, which is biosimilar to epoetin alfa, was clinically approved in Japan. Currently, both isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) are approved by the World Anti-Doping Agency (WADA) for detection of rEPO doping. Because it was unclear whether epoetin kappa could be detected by WADA-accredited detection methods, intravenous administration studies of epoetin kappa, epoetin alfa, and epoetin beta were performed to test the applicability of these methods. The isoform bands of epoetin kappa expanded more widely towards the basic area and the profile appeared to be composed of at least eight bands, which were clearly different from those of other epoetins. The results showed that epoetin kappa also contains isoforms of higher molecular masses than those of originator epoetins on SDS-PAGE; the mass distribution was confirmed by electrospray ionization time-of-flight mass spectrometry. We clearly detected epoetin kappa after its administration up to 10 h by IEF-PAGE and 24 h by SDS-PAGE; the detection window of the SDS-PAGE is longer than that of the IEF-PAGE. SDS-PAGE compensates for the disadvantages of IEF-PAGE in detecting urinary epoetin kappa. We also concluded that athletes abusing rEPO might move to intravenous injections for shorter clearance times instead of subcutaneous injections. In conclusion, out-of-competition tests need to be applied more frequently to improve the effectiveness of the rEPO detection.
自专利保护期届满以来，许多新的重组促红细胞生成素（rEPO）生物类似物已在全球市场上出现。2010 年，与促红细胞生成素阿尔法具有生物相似性的促红素 kappa 在日本获得临床批准。目前，等电聚焦聚丙烯酰胺凝胶电泳（IEF-PAGE）和十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）均被世界反兴奋剂机构（WADA）批准用于检测 rEPO 兴奋剂。由于不清楚促红素 kappa 是否可以被 WADA 认可的检测方法检测到，因此进行了促红素 kappa、促红素阿尔法和促红素 beta 的静脉给药研究，以测试这些方法的适用性。促红素 kappa 的同工型条带向碱性区域更广泛地扩展，其形态似乎由至少 8 条带组成，这与其他促红素明显不同。结果表明，SDS-PAGE 上促红素 kappa 还含有比原研促红素更高分子量的同工型；通过电喷雾电离飞行时间质谱法证实了质量分布。我们通过 IEF-PAGE 清楚地检测到给药后 10 小时的促红素 kappa，通过 SDS-PAGE 检测到 24 小时的促红素 kappa；SDS-PAGE 的检测窗口比 IEF-PAGE 长。SDS-PAGE 弥补了 IEF-PAGE 检测尿液促红素 kappa 的缺点。我们还得出结论，滥用 rEPO 的运动员可能会转向静脉注射以缩短清除时间，而不是皮下注射。总之，需要更频繁地进行场外测试，以提高 rEPO 检测的有效性。
