CEPO: Synthesis, purification and doping-relevant electrophoretic characterization of carbamylated

Code: 18A22CR

Chapter S2 of WADA’s Prohibited List 2017 (“Peptide hormones, growth factors, related substances, and mimetics”) lists carbamylated EPO under sub-chapter 1.2 (“Non-erythropoietic EPO-Receptor agonists”). Carbamylated EPO (also called “CEPO”) is not an erythropoiesis stimulating agent (ESA) but acts tissue protective and can be used for therapeutic or prophylactic treatment of human diseases. In this function, it may also be misused by cheating athletes. However, CEPO is still under clinical investigation and not available as a pharmaceutical or analytical standard. Aim of the project is the synthesis of CEPO by controlled reaction of rEPO with cyanate, followed by purification, mass spectrometric characterization and electrophoresis (IEF-, SAR-, SDS-PAGE according to WADA TD2014EPO). Carbamylation will lead to a loss of positive charges and an increase in molecular mass (43 Da for each carbamylated amino group). Consequently, it is expected that the isoform cluster of CEPO is shifted towards the anode on IEF-PAGE, i.e. towards the “endogenous area” – thus making CEPO undetectable. Contrary to that, the relatively small increase in molecular mass (in total 387 Da, if the N-terminus and all 8 lysines are carbamylated) will lead to hardly any changes on SDS- or SAR-PAGE, i.e. CEPO will be detected within the same mass range as recombinant EPO and hence misusers of CEPO will also be tested positive. Additionally, the project will answer the question if CEPO remains detectable by the highly sensitive anti EPO antibody used for Western blotting in doping control (clone AE7A5). The antibody is directed against the first 26 amino acids of the N-terminus of EPO, which contains two modified (carbamylated) amino acids in CEPO. The synthesized compound will be made available to all WADA-accredited labs for use as standard in routine ESA-testing.

Main findings

CEPO was successfully synthesized and characterized by mass spectrometry. It is detectable by SAR-, SDS-, and IEF-PAGE and can be differentiated from other ESAs by these methods. Differentiation between CEPO and rEPO is possible by digestion with endopeptidase Lys-C. Since Lys-C cannot cleave at carbamylated lysines only rEPO is degraded and CEPO remains intact. Conclusions: CEPO is detectable by the currently applied electrophoretic methods for ESA-doping control. Although there is a band-overlap with rEPO and endogenous EPO on SAR- and SDS-PAGE, it can be relatively easy differentiated from them by Lys-C digestion. However, since usage of CEPO is prohibited, this differentiation may be not necessary.