Mutant Human Erythropoietin from Insect Cells as Novel Low Molecular‐Weight Internal Standards for ERAs Analysis

Doping analysis of erythropoietin receptor agonists (ERAs) is frequently hindered by the "no band" phenomenon, in which endogenous erythropoietin (EPO) becomes undetectable owing to a variety of pre-analytical factors. The absence of an internal standard (ISTD) poses the risk of an undetected sample preparation failure. Furthermore, the EPO gene variant c.577del presents a substantial analytical challenge. Specifically, the failure to identify variant-encoded EPO (VAR-EPO) during reverse immunopurification (RI) may cause false-positive results for recombinant EPO (rEPO), highlighting the necessity of an ISTD for RI process monitoring. Nevertheless, the degradation of high molecular weight PEG-modified ISTDs could interfere with ERAs detection. To address these technical limitations, low molecular weight ISTDs represent a superior alternative for methodological development. This study reports the strategic development of two novel low molecular weight ISTDs: a wild-type EPO-based designed mutant (INS1) and a mutant EPO encoded by the c.557del variant (INS2). Both mutant proteins were expressed in the H5 insect cell line via the baculovirus expression vector system. The two engineered EPO mutants possess simplified paucimannose-type N-glycans, conferring a distinctly lower molecular weight profile than endogenous EPO and mammalian cell-expressed rEPO. Functionally, INS1 is designed to monitor the integrity of the initial testing procedure (ITP), whereas INS2 is engineered for process monitoring of RI to enable accurate rEPO confirmation. The results demonstrate that the developed low molecular weight ISTDs can effectively monitor procedural integrity and improve the detection performance of ERAs analytical assays.