Pérez Malena M, Veronez Luciana C, Cordeiro Francielle A, de Castro Figueiredo Bordon Karla, Arantes Eliane C, Munari Carla, Marquele-Oliveira Franciane, Muller Vinícius, Guimarães Danielle A
Research and Development Department, Ourofino Animal Health Company, SP-330 Km298 Rodovia Anhanguera, Cravinhos, SP 14140-000, Brazil.
BioMolecular Sciences Department, School of Pharmaceutical Science of Ribeirao Preto, University of São Paulo, 3900 Bandeirantes Avenue, Ribeirao Preto, SP 14049-900, Brazil.
ACS Omega. 2025 Feb 13;10(7):7211-7219. doi: 10.1021/acsomega.4c10780. eCollection 2025 Feb 25.
Equine-chorionic gonadotropin (eCG) is widely used in fixed-time artificial ovulation and superovulation protocols to improve reproductive performance. Commercial products available in the market for veterinary use consist of partially purified preparations of pregnant mare serum gonadotropin (PMSG). eCG is a heterodimeric glycoprotein and is thus extremely challenging to quantify through chromatography. Pharmaceutical companies usually carry out methods to measure the potency of finished products containing eCG. Considering the three Rs principle (refinement, replacement, and reduction), the aim of this study was to develop assays using high-performance liquid chromatography (HPLC) and cell culture techniques to predict the biological activity of products containing eCG. The experimental conditions established for the chromatographic method allowed an efficient separation between the eCG peaks and the excipients present in the formulation, with a consistent chromatographic profile between batches of eCG-products and the analytical standard (PMSG). In cell culture, stimulation of MA-10 cells with both PMSG and products containing eCG resulted in significant progesterone production in all tested concentrations and a similar profile between the products and control, indicating substantial biological activity. The data presented corroborate the potential use of the combination of the chromatographic profile and cell culture method for the successful quality control of commercial preparations containing eCG. Beyond the practical benefits of reduced time and cost, these approaches align with a growing recognition of the need to reduce the reliance on animal models for the quality control of products containing eCG.
马绒毛膜促性腺激素（eCG）广泛用于定时人工排卵和超排卵方案，以提高繁殖性能。市场上可买到的兽用商业产品由孕马血清促性腺激素（PMSG）的部分纯化制剂组成。eCG是一种异源二聚体糖蛋白，因此通过色谱法进行定量极具挑战性。制药公司通常采用方法来测量含eCG成品的效价。考虑到3R原则（优化、替代和减少），本研究的目的是开发利用高效液相色谱（HPLC）和细胞培养技术来预测含eCG产品生物活性的检测方法。为色谱法建立的实验条件能够有效分离eCG峰与制剂中存在的赋形剂，不同批次的eCG产品与分析标准品（PMSG）之间具有一致的色谱图。在细胞培养中，用PMSG和含eCG的产品刺激MA - 10细胞，在所有测试浓度下均导致显著的孕酮产生，且产品与对照之间具有相似的图谱，表明具有显著的生物活性。所呈现的数据证实了色谱图和细胞培养方法相结合在含eCG商业制剂成功质量控制中的潜在用途。除了减少时间和成本的实际益处外，这些方法符合人们日益认识到的减少对含eCG产品质量控制中动物模型依赖的需求。
