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.
Drug Test Anal. 2024 Feb;16(2):199-209. doi: 10.1002/dta.3525. Epub 2023 Jun 20.
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.
过去十年中，已有许多创新型生物疗法上市。单克隆抗体（mAbs）和 Fc 融合蛋白（Fc-蛋白）已被开发用于治疗多种疾病（癌症、自身免疫性疾病和炎症性疾病），现在已成为靶向治疗的重要组成部分。然而，此类生物分子的易得性，有时具有合成代谢、抗炎或促红细胞生成作用，引起了人们对其作为人类和动物运动员的潜在滥用的关注，作为性能增强剂。在赛马兴奋剂检测实验室，已经报道了一种检测马血浆中特定人用生物疗法的方法；但是，在没有关于人用或鼠用生物疗法的先验知识的情况下，还没有描述高通量筛选方法。在这种情况下，已经开发了一种涉及 UHPLC-HRMS/MS 的新的广谱筛选方法，用于在马血浆中对鼠用或人用 mAbs 和相关大分子进行非靶向分析。这种方法由 96 孔板中的“沉淀消化”策略组成，在低浓度（pmol/mL 范围内）具有可靠的性能，高通量能力（≈100 个样品/天）。针对 mAbs 恒定部分内的种特异性肽型肽，可通过监测 10 个肽来实现“通用”检测人用生物疗法。作为原理验证，该策略成功地在加标血浆样品中检测到了不同的生物疗法，并首次允许在马中以 0.12mg/kg 剂量给药 10 天后检测到人用 mAb。这一发展将提高马兴奋剂检测实验室对蛋白质生物疗法的分析能力，具有足够的灵敏度、通量和成本效益。
