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Drug testing and analysis2025; doi: 10.1002/dta.3878

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

Abstract: 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.
© 2025 John Wiley & Sons Ltd.
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Publication Date: 2025-03-03 PubMed ID: 40033065DOI: 10.1002/dta.3878Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research article presents a new method for detecting certain proteins that are currently being developed for medicinal purposes but have potential for being misused in horse racing to enhance performance. This new detection method was developed specifically to identify nine different proteins, and its successful application in equine doping control is detailed in the study.

Research Purpose

  • The primary goal of the research was to develop a generic detection method for doping control analysis of nine targeted proteins that are presently being developed as therapeutic agents.
  • These proteins, which include Fc-fusion proteins and monoclonal antibodies (mAbs), could possibly be misused to improve performance in horseracing and equestrian sports.

Methodology

  • The researchers developed a generic workflow that involved affinity purification with commercially available Protein A magnetic beads.
  • The process continued with tryptic digestion and identification of 20 targeted peptides using capillary flow high-performance liquid chromatography-high-resolution tandem mass spectrometry (HPLC-HRMS).
  • The targeted proteins included three recombinant Fc-fusion proteins and six mAbs, all of which have the Fc domain of human IgG or IgG from other species.

Findings

  • The proposed detection method was successful in identifying all nine targeted proteins in spiked equine plasma with adequate sensitivity and precision.
  • For the first time, the protein bedinvetmab (Librela) was detected and identified in plasma for at least 34 days after a single subcutaneous administration to a Thoroughbred horse.

Implications

  • The research findings indicate that the method developed offers a practical and effective approach to handle the doping control of novel Fc domain-containing proteins.
  • The detection method offers potential value in equine sports to maintain fair play and animal welfare by cracking down on misuse of such proteins.

Cite This Article

APA
Cheung HW, Wong KS, Choi YC, Kwok WH, So YM, Farrington AF, Bond AJ, Wan TSM, Ho ENM. (2025). A Generic Detection Method for the Doping Control Analysis of Fc-Fusion Proteins and Monoclonal Antibodies in Equine Plasma. Drug Test Anal. https://doi.org/10.1002/dta.3878

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English

Researcher Affiliations

Cheung, Hiu Wing
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, New Territories, Hong Kong, China.
Wong, Kin-Sing
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, New Territories, Hong Kong, China.
Choi, Yung-Ching
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, New Territories, Hong Kong, China.
Kwok, Wai Him
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, New Territories, Hong Kong, China.
So, Yat-Ming
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, New Territories, Hong Kong, China.
Farrington, Adrian F
  • Veterinary Clinical Services, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, New Territories, Hong Kong, China.
Bond, Amanda J
  • Equestrian Affairs, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, New Territories, Hong Kong, China.
Wan, Terence S M
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, New Territories, Hong Kong, China.
Ho, Emmie N M
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, New Territories, Hong Kong, China.

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