Combined immuno‐purification and detection of recombinant erythropoietins and activin receptor type II‐Fc fusion proteins by isoelectric focusing for application in doping control

Abstract

Iso-electric focusing (IEF) was the first method established to discriminate endogenous and recombinant erythropoietins (rEPOs). It is still approved by the World Anti-Doping Agency (WADA) as an initial testing procedure to detect erythropoiesis stimulating agents (ESAs) in doping control samples. However EPO-Fc, one of the prohibited rEPOs designated by WADA, is not detectable with the actual IEF conditions. Other newly developed ESAs - luspatercept and sotatercept, both activin receptor type II-Fc fusion proteins (ActRII-Fc) - are also now prohibited and could be used in combination with rEPOs. Methods of identification of ActRII-Fc in blood by SAR/SDS-PAGE have been described, but not by IEF. Here we detail improvements in blood sample preparation and IEF analysis: A combined immuno-purification of EPOs and ActRII-Fc proteins in a single procedure, an appropriate isoforms separation for all proteins using new pre-loading and gel conditions, and a single detection of all rEPOs and ActRII-Fc proteins after successive incubation with anti-EPO and anti-ActRII antibodies. With these changes, distinctive profiles for all the ESAs were obtained by IEF. Therefore, IEF could be used as a screening method to detect a wide spectrum of prohibited ESAs in blood samples prior to specific confirmation for the identified rEPO or ActRII-Fc.

Annual banned‐substance review – Analytical approaches in human sports drug testing

“…The successful implementation of additional target analytes such as the transforming growth factor‐beta (TGF‐β) inhibitors sotatercept and luspatercept into different conventional ESA‐specific ITPs was reported by various research units. For instance, Martin et al described a strategy employing magnetic nanoparticles coated with monoclonal anti‐EPO‐, anti‐ActRIIA‐, and anti‐ActRIIB antibodies used to immunoextract 0.3–1.0 mL of serum or plasma . By means of a pH gradient ranging from pH 2–10, isoelectric focusing (IEF) and double blotting, sotatercept, luspatercept, various ActRII‐Fc fusion proteins and the commonly tested erythropoietins were jointly analyzed, enabling LODs of approximately 5 ng/mL for sotatercept and luspatercept and 15–100 pg/mL for erythropoietin and its derivatives, meeting the requirements of corresponding WADA technical documents .…”

Detection of a nonerythropoietic erythropoietin, Neuro‐EPO, in blood after intranasal administration in rat

“…The IEF‐PAGE needs extended pH gradient to 2–8 or 2–10 instead of the conventional pH gradient at 2–6. The use of these extended gradients would still allow identification of all classic prohibited EPOs: rEPOs, NESP, and CERA 32 . Interestingly, it can also be useful to identify EPO‐Fc (which cannot be seen with the pH 2–6 gradient) and using additional antibodies to detect other erythropoiesis stimulating agents like activin‐receptor‐Fc constructs 32 .…”

“…The use of these extended gradients would still allow identification of all classic prohibited EPOs: rEPOs, NESP, and CERA 32 . Interestingly, it can also be useful to identify EPO‐Fc (which cannot be seen with the pH 2–6 gradient) and using additional antibodies to detect other erythropoiesis stimulating agents like activin‐receptor‐Fc constructs 32 . This increased pH gradient could therefore be used by anti‐doping laboratories to simplify the detection of all these molecules in a doping control sample.…”

Transcriptomic Markers of Recombinant Human Erythropoietin Micro-Dosing in Thoroughbred Horses

“…In human sports, various methods of detecting rHuEPO administration have been employed. These include setting upper limits for hemoglobin and hematocrit levels [ 8 ], using isoelectric focusing (IEF) to detect differences in the charge profiling of endogenously versus exogenously produced EPO in urine and blood [ 9 , 10 ], and using sarcosyl polyacrylamide gel electrophoresis (SAR-PAGE) [ 11 ], to differentiate between exogenous and endogenous EPO by differences in migration, which are indicative of the small changes in molecular weight. For the latter two methods, the detection window used to be quite short, ranging from 24–85 h from the time of administration [ 9 , 11 ], but recent advances in detection methods have increased the detection window.…”

“…An added challenge is the advent of micro-dosing, where small amounts of rHuEPO are administered over a long period of time to avoid detection [ 12 ]. However, with the improvements in IEF, SAR-PAGE, and sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) detection methodologies, micro-doses can be detected up to 72–104 h after administration [ 10 , 13 ]. Currently, the Athlete Biological Passport (ABP) regulates rHuEPO abuse and other blood doping agents in human athletes indirectly by monitoring for changes in hematological values such as hemoglobin, reticulocytes, and red blood cell count in an individual over time [ 12 ].…”