Simultaneous detection of erythropoietins, TGF-ß signalling inhibitors (Sotatercept, Luspatercept) and their analogs by SAR- and SDS-PAGE

Code: 23B02CR

Chapter S2 of WADA’s Prohibited List 2023 (“Peptide hormones, growth factors, related substances, and mimetics”) lists Erythropoietin Receptor Agonists (ERAs) and Transforming growth factor beta (TGF-ß) signalling inhibitors (luspatercept, sotatercept) under chapter 1 (“Erythropoietins (EPO) and agents affecting erythropoiesis”) as prohibited substances. Currently, SAR- and SDS-PAGE are the most frequently applied techniques for the initial testing and confirmation procedures (ITP, CP) for ERAs in WADA accredited laboratories worldwide (TD2022EPO). While electrophoretic detection methods for TGF-ß signalling inhibitors were also developed, they are not yet part of the technical document. In 2019 and 2020, the first luspatercept-based pharmaceutical (Reblozyl®, “luspatercept-aamt”) was approved by FDA (USA) and EMA (Europe).

Hence, routine testing for TGF-ß signalling inhibitors will become necessary in the near future. For the detection of erythropoietins and TGF-ß signalling inhibitors in blood and urine immunoaffinity purification is required before electrophoretic separation. Due to significant structural differences between these compounds, three different capture antibodies have to be employed, i.e. anti-EPO, anti-activin receptor type IIA (ACVR2A), and type IIB (ACVR2B) antibodies for epoetins, sotatercept and luspatercept, respectively. A protocol for the combined immunopurification of ERAs, sotatercept and luspatercept followed by isoelectric focusing (IEF-PAGE) was published in 2019, another one for ERAs and luspatercept in combination with SDS- and SAR-PAGE in 2021. Both protocols were based on covalent immobilization of relatively large amounts of the capture antibodies on magnetic beads.

Consequently, the beads had to be re-used several times for cost-reduction. Additionally, sotatercept was not included in the protocol for SDS- and SAR-PAGE. We already developed individual protocols for capturing sotatercept and luspatercept in serum samples, which use non-covalent immobilization of very small antibody amounts on anti-antibody coated magnetic beads. Additionally, we presented a similar cost-minimized protocol for the purification of EPOs from blood and urine at the Cologne workshop in March 2023. The plan is to combine these protocols in order to simultaneously purify all three compounds from the three sample matrices (serum/plasma/urine). Another disadvantage of the 2021 protocol was, that EPOs and luspatercept could not be detected in a truly multiplexed way after electrophoretic separation and Western blotting.

The reason were interferences caused by non-specific binding of the two detection antibodies with co-eluted proteins (the protocol used the same polyclonal antibody for capture and detection of luspatercept). Hence, the membrane had to be first incubated with an anti-EPO antibody followed by re-incubation with the anti-ACVR2B antibody. Contrary to that, the proposal of this project is that (1) the target proteins will be simultaneously detected by incubating the blot membrane with a mixture of all three detection antibodies, (2) it will also include sotatercept, and (3) the protocol will also be applicable to urine (it was shown in 2022 that luspatercept is also partly excreted in urine).