Introduction of a PEGylated EPO conjugate as internal standard for EPO analysis in doping controls

Immunopurification of doping control samples is a mandatory necessity in erythropoietin (EPO) analysis during a confirmation procedure; moreover, it has become common practice to also immunopurify samples for the initial testing procedure. Typically used materials (e.g., Stemcell purification plate and MAIIA purification kit) rely on anti‐EPO antibodies for purification. Also, the detection of EPO after electrophoretic separation and western blotting is based on a monoclonal anti‐EPO antibody, clone AE7A5, directed against a 26 amino acid sequence of the N‐terminal region of human EPO. While the electrophoretic separation and blot transfer efficiency can be monitored with reference standards and quality control samples, it is presently not possible to monitor the functionality of the entire sample preparation procedure. The reliance on antibodies for both purification and detection has complicated the implementation of an internal standard (ISTD). In this study, customized EPO‐polyethylene glycol (PEG) conjugates were synthesized as potential ISTDs and assessed as to their compatibility with existing sample preparation procedures for urine and blood sample analysis using the most common immunopurification techniques. Moreover, probing for the impact of the ISTD on sodium N‐lauroylsarcosinate (“sarcosyl”) polyacrylamide gel electrophoresis (SAR‐PAGE)‐based EPO analysis concerning potential interference with target analytes was conducted. The presented data demonstrate that a 12‐kDa PEG residue attached to human EPO represents a particularly useful construct to serve as ISTD for erythropoietin‐receptor agonist (ERA) analysis. The conjugate is applicable to both urine and blood testing using the commonly employed purification techniques, supporting and improving result interpretations especially concerning specimens where the natural abundance of human EPO is low. Recombinant erythropoietin (EPO) was conjugated with a selection of polyethylene glycol (PEGs) in order to asses a candidate to be utilized as internal standard, monitoring preparational procedures during antidoping analysis of erythropoietin‐receptor agonists (ERAs) prior to sodium N‐lauroylsarcosinate (“sarcosyl”) polyacrylamide gel electrophoresis (SAR‐PAGE). The conjugate C115 did not influence ERA analysis or identification in urine or serum when utilized and was found fit‐for‐purpose as internal standard (ISTD).