A new method for high-throughput screening for the analysis of therapeutic monoclonal antibodies and fusion proteins for the control of equine anti-doping

Many innovative biotherapeutic products have been commercialized during the last decade. Monoclonal antibodies (AbM) and Fusion proteins Fc (Fc proteins) have been developed to treat different diseases (cancer, auto-immune diseases and inflammatory disorders), now representing an important part of targeted therapies. However, the immediate availability of these biomolecules, sometimes characterized by their anabolizing, anti-inflammatory or erthropoiesis stimmulating properties, raise questions concerning their potential abusive use as performance enhancers for human and animal athletes. Today, only one method has been reported to detect the administration of a human-specific fusion protein in equine plasma. However, in principle no high-throughput method has been described for the screening of biotherapeutic macromolecules in equine samples.

Within this context, a new large spectrum screening method implicating UHPLC-HRMS/MS has been developed for the analysis of therapeutic AcM and fusion proteins in equine plasma. This targeted proteomics approach has been performed using a 96-well plate and shown reliable performances at low concentrations (around pmol/mL) at high-throughput (≈100 échantillons/jour). The targeting of specific proteotypic peptides, situated in the constant parts of AcM, allow the detection of "universal" biotherapeutic products made up of human and murine IgG protein sequences, by testing only 10 peptides. As proof of principle, this strategy allowed the successful detection of several AcM and fusion proteins in supplemented plasma samples and enables for the first time, to detect a human AcM up to 10 days after a unique administration of 1,2 mg/kg to a horse. This development will provide the extension of the analytical capacities of laboratories in the fight against doping of horses with biotherapeutic macromolecules including immunoglobulins, using a high-throughput, sensitive, specific and robust method with a high efficiency/cost ration.

Contact :

• Patrice Garcia