A Generic Detection Method for the Doping Control Analysis of Fc-Fusion Proteins and Monoclonal Antibodies in Equine...

An increasing number of novel Fc-fusion proteins and monoclonal antibodies (mAbs) are being developed as therapeutic agents for treating various diseases. Among these, there are inhibitors of the activin Type II receptor (ActRIIA and ActRIIB) signaling pathways and mAbs against nerve growth factor (NGF), which may be misused for performance enhancement in horseracing and equestrian sports. This study is aimed at developing a generic detection method for doping control analysis of nine targeted proteins, each containing the Fc domain of human IgG or IgG from other species in equine plasma, namely, three recombinant Fc-fusion proteins (sotatercept, follistatin-Fc (FST-Fc), and erythropoietin-Fc (EPO-Fc)) and six mAbs (bimagrumab, domagrozumab, garetosmab, landogrozumab, bedinvetmab (Librela), and frunevetmab (Solensia)). A generic workflow has been developed, involving affinity purification with commercially available Protein A magnetic beads followed by tryptic digestion and detection of 20 targeted peptides (with 2-3 diagnostic peptides for each targeted protein) using capillary flow high-performance liquid chromatography-high-resolution tandem mass spectrometry (HPLC-HRMS). The method identified all nine targeted proteins in spiked equine plasma with adequate sensitivity and precision, and for the first time, bedinvetmab (Librela) was detected and identified in plasma for at least 34 days after a single subcutaneous administration (0.5 mg/kg) to a Thoroughbred horse. The results have demonstrated the method's applicability to equine doping control. This generic method involving affinity purification by Protein A has provided a pragmatic and effective approach to cope with the doping control of novel Fc domain-containing proteins.