Detection of the GH Analogue Redalsomatropin Alfa in Sports Drug Testing: Immunological Approaches and LC-HRMS/MS

Human growth hormone (hGH) is a peptide hormone exerting different growth-promoting and metabolic functions through binding to the GH receptor (GHR). Due to the presumed lipolytic and anabolic effects, the misuse of recombinant 22-kDa hGH in sports is prohibited both in- and out-of-competition, and also synthetic long-acting GH analogues were added to the WADA Prohibited List in 2022. Within this research project, the detectability of redalsomatropin alfa (JR-142) with the immunoassays routinely employed by anti-doping laboratories for rhGH testing was evaluated. The drug candidate represents a recombinant fusion protein of 22-kDa hGH (191 amino acids) and human serum albumin (HSA, 585 amino acids) and is currently undergoing Phase III of its clinical development. Due to the attachment of HSA to the C-terminus of 22-kDa hGH, only "Kit 2" of the routinely employed immunoassay was found to recognize the drug, and the cross-reactivity was significantly reduced when compared to native hGH. As the misuse of redalsomatropin alfa in sports can therefore remain undetected when using this assay alone, the drug was implemented into the existing qualitative initial testing procedure (ITP) for the hGH analogue somatrogon employing affinity purification with GHR-Fc-conjugated magnetic beads for sample extraction and tryptic digestion in combination with LC-HRMS/MS for protein identification. Method validation demonstrated that the assay allows for a sensitive and specific detection of the fusion protein down to concentrations of 50 ng/mL. Moreover, these findings show that this LC-HRMS/MS assay can be further expanded to simultaneously test for multiple long-acting growth hormones (LAGHs).