Clarification on the detection of epoetin delta and epoetin omega using isoelectric focusing.

To the Editor: In a recent article in your journal, Delanghe et al. [1]. reviewed both the indirect and direct methods for the detection of recombinant human erythropoietin (rhEPO) in doping analysis. However, some of the statements made are incorrect and some others deserve clarification. The direct detection of the misuse of rhEPO and analogues in sports is currently done in World Antidoping Agency (WADA) accredited laboratories following the method developed by Lasne [2], which is based on the differences in the isoelectric focusing (IEF) behavior between endogenously produced urinary EPO (uEPO) and the recombinant products (epoetin alpha, beta, etc.) and analogues (i.e., darbepoetin alpha). Those differences are claimed to reside in the glycans of those glycoproteins, resulting from the enzymatic endowment of the particular cell in which the protein is expressed as well as from the selective purification applied to obtain each pharmaceutical product. The article, however, is wrong in stating that epoetin omega is produced in human cells. Epoetin omega is produced in Baby Hamster Kidney (BHK) cells [3]. Its glycosylation profile is monitored using the same IEF method and it is perfectly distinguishable from endogenous EPO (Fig. 1). Epoetin delta (Dynepo, Shire Pharmaceuticals) is expressed in human cells, although it is not produced in the kidney cells where endogenous EPO is produced, but rather in fibrosarcoma cells (HT-1080). The statement that ‘‘dynepo/erythropoietin-delta, which is produced in human cells, has no distinct glycosylation pattern that enables its detection by the direct Epo test’’ is wrong. As can be seen in Fig. 1, epoetin delta can be detected—as can the other recombinant products—by using the IEF method, and its IEF profile is distinguishable from the endogenous EPO. The profile shows six isoforms in the basic area and two extra isoforms (one very faint) in the endogenous area, as described also in a recent article [4]. Last but not least, CERA (Mircera, Roche), a pegylated derivative of epoetin beta, can also be detected using the same method. It shows five intense bands plus a weaker one also in the basic area [5]. However, because of its higher molar mass (ca. 60,000 Da), it is not expected to be significantly excreted in urine so its detection will probably have to be done in serum. The application of the same IEF method to serum can also be performed, although it requires a different purification approach as recently described [6].