An Alternative <i>In Vitro</i> Methodology for the Quality Control of Equine-Chorionic Gonadotropin in Commercial Products

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 <i>in vivo</i> 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 <i>in vitro</i> 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.