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.

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.

miRNAs detection in equine plasma by quantitative polymerase chain reaction for doping control: Assessment of blood sampling and study of eca-miR-144 as potential erythropoiesis stimulating agent biomarker.

Short half-life doping substances are, quickly eliminated and therefore difficult to control with traditional analytical chemistry methods. Indirect methods targeting biomarkers constitute an alternative to extend detection time frames in doping control analyses. Gene expression analysis (i.e., transcriptomics) has already shown interesting results in both humans and equines for erythropoietin (EPO), growth hormone (GH), and anabolic androgenic steroid (AAS) misuses. In humans, circulating cell-free microRNAs in plasma were described as new potential biomarkers for control of major doping agent (MDA) abuses. The development of a quantitative polymerase chain reaction (qPCR) method allowing the detection of circulating miRNAs was carried out on equine plasma collected on different type of tubes (EDTA, lithium-heparin [LiHep]). Although analyzing plasma collected in EDTA tubes is a standard method in molecular biology, analyzing plasma collected in LiHep tubes is challenging, as heparin is a reverse transcription (RT) and a PCR inhibitor. Different strategies were considered, and attention was paid on both miRNAs extraction quality and detection sensitivity. The detection of endogenous circulating miRNAs was performed and compared between the different types of tubes. In parallel, homologs of human miRNAs characterized as potential biomarkers of doping were sought in equine databases. The miRNA eca-miR-144, described as potential erythropoiesis stimulating agents (ESAs) administration candidate biomarker was retained and assessed in equine post-administration samples. The results about the qPCR method development and optimization are exposed as well as the equine miRNAs detection. To our knowledge, this work is the first study and the proof of concept of circulating miRNAs detection in plasma dedicated to equine doping control.