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Drug testing and analysis2023; doi: 10.1002/dta.3525

High-throughput untargeted screening of biotherapeutic macromolecules in equine plasma by UHPLC-HRMS/MS: Application to monoclonal antibodies and Fc-fusion proteins for doping control.

Abstract: Many innovative biotherapeutics have been marketed in the last decade. Monoclonal antibodies (mAbs) and Fc-fusion proteins (Fc-proteins) have been developed for the treatment of diverse diseases (cancer, autoimmune diseases, and inflammatory disorders) and now represent an important part of targeted therapies. However, the ready availability of such biomolecules, sometimes characterized by their anabolic, anti-inflammatory, or erythropoiesis-stimulating properties, raises concerns about their potential misuse as performance enhancers for human and animal athletes. In equine doping control laboratories, a method has been reported to detect the administration of a specific human biotherapeutic in equine plasma; but no high-throughput method has been described for the screening without any a priori knowledge of human or murine biotherapeutic. In this context, a new broad-spectrum screening method involving UHPLC-HRMS/MS has been developed for the untargeted analysis of murine or human mAbs and related macromolecules in equine plasma. This approach, consisting of a "pellet digestion" strategy performed in a 96-well plate, demonstrates reliable performances at low concentrations (pmol/mL range) with high-throughput capability (≈100 samples/day). Targeting species-specific proteotypic peptides located within the constant parts of mAbs enables the "universal" detection of human biotherapeutics only by monitoring 10 peptides. As proof of principle, this strategy successfully detected different biotherapeutics in spiked plasma samples, and allowed, for the first time, the detection of a human mAb up to 10 days after a 0.12 mg/kg administration to a horse. This development will expand the analytical capabilities of horse doping control laboratories towards protein-based biotherapeutics with adequate sensitivity, throughput, and cost-effectiveness.
© 2023 John Wiley & Sons Ltd.
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Publication Date: 2023-06-20 PubMed ID: 37337992DOI: 10.1002/dta.3525Google Scholar: Lookup
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  • Journal Article

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 study presents a screening method for detecting the misuse of certain biotherapeutic proteins in horse racing. The method is based on ultra-high performance liquid chromatography and high-resolution mass spectrometry, and can be effective in controlling doping by providing broad-spectrum screening of human or murine proteins in equine plasma.

Research Objectives and Motivation

  • The main objective of this research was to develop a high-throughput, untargeted screening method to detect the presence of biotherapeutic macromolecules – specifically, monoclonal antibodies (mAbs) and Fc-fusion proteins – in equine plasma.
  • This development was motivated by the concern that such biotherapeutic proteins, which have been developed for human medical treatment purposes, might be misused as performance enhancers in horse racing.

Methodology

  • The proposed screening method uses Ultra High Performance Liquid Chromatography coupled with High-Resolution Mass Spectrometry/Mass Spectrometry (UHPLC-HRMS/MS).
  • A “pellet digestion” strategy is employed, enabling high-throughput screening capability of approximately 100 samples per day.
  • The unique feature of this method is that it can detect mAbs and related macromolecules without any prior knowledge of their presence.

Findings

  • This screening method demonstrated reliable performance at low concentrations, specifically in the picomolar per milliliter range.
  • It was found that it could universally detect human biotherapeutics by monitoring just 10 peptides located within the constant parts of mAbs.
  • In practical tests, this strategy successfully detected various biotherapeutics in spiked plasma samples, and even managed to detect a human mAb up to 10 days after administration to a horse, marking a first in this field of research.

Impact and Utility

  • This development enhances the analytical capabilities of horse doping control laboratories, enabling them to detect and monitor protein-based biotherapeutics with adequate sensitivity and cost-effectiveness.
  • As such, it can contribute significantly to the fight against doping in horse racing and other equestrian sports, helping to ensure fair competition and animal welfare.

Cite This Article

APA
Pinetre J, Delcourt V, Becher F, Garcia P, Barnabé A, Loup B, Popot MA, Fenaille F, Bailly-Chouriberry L. (2023). High-throughput untargeted screening of biotherapeutic macromolecules in equine plasma by UHPLC-HRMS/MS: Application to monoclonal antibodies and Fc-fusion proteins for doping control. Drug Test Anal. https://doi.org/10.1002/dta.3525

Publication

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

Researcher Affiliations

Pinetre, Justine
  • GIE LCH, Laboratoire des Courses Hippiques, Verrières-le-Buisson, Essonne, France.
  • Université Paris-Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), MetaboHUB, Gif sur Yvette, Ile de France, France.
Delcourt, Vivian
  • GIE LCH, Laboratoire des Courses Hippiques, Verrières-le-Buisson, Essonne, France.
Becher, François
  • Université Paris-Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), MetaboHUB, Gif sur Yvette, Ile de France, France.
Garcia, Patrice
  • GIE LCH, Laboratoire des Courses Hippiques, Verrières-le-Buisson, Essonne, France.
Barnabé, Agnès
  • GIE LCH, Laboratoire des Courses Hippiques, Verrières-le-Buisson, Essonne, France.
Loup, Benoit
  • GIE LCH, Laboratoire des Courses Hippiques, Verrières-le-Buisson, Essonne, France.
Popot, Marie-Agnès
  • GIE LCH, Laboratoire des Courses Hippiques, Verrières-le-Buisson, Essonne, France.
Fenaille, François
  • Université Paris-Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), MetaboHUB, Gif sur Yvette, Ile de France, France.
Bailly-Chouriberry, Ludovic
  • GIE LCH, Laboratoire des Courses Hippiques, Verrières-le-Buisson, Essonne, France.

Grant Funding

  • Association Nationale de la Recherche et de la Technologie
  • Institut Franu00e7ais du Cheval et de l'Equitation

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