Biological Activity of Different Batches of Equine Chorionic Gonadotropin as Determined by Reversed-phase High-performance Liquid Chromatography and in vivo Assay

Abstract

Aims: To evaluate the physicochemical profile of commercial batches of eCG, in order to find if differences can be related to their biological activity. Study Design: Commercial eCG was analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) and in vivo bioassay. Place and Duration of Study: Department of Biotechnology (IPEN-CNEN) and Animal antibody production Laboratory (Animal Science Institute), between June 2013 and April 2014. Methodology: Two commercial eCG batches for veterinary use (I and II) and an eCG official International Standard from the World Health Organization (WHO) were analyzed by RP-HPLC. Additionally, two experiments were designed to validate the biological activity. In experiment 1, groups of prepubertal 21–25 day old Wistar female rats  (n = 6/group) received the equivalent to 0 UI (saline) and 10 IU of eCG of each one of these preparations. Autopsy was performed 48 h later and ovaries were removed and weighed. The experiment 2 was designed to determine whether increasing the dose of less active eCG batches could increase the ovarian response. Therefore, groups of prepubertal rats (n = 6/group) were treated with 10 and 30 IU eCG from batch II, while eCG from WHO (10 IU) and saline were control. The evaluation of ovarian response was done similar to Experiment 1. Differences among treatments were analyzed by one-way ANOVA. Results: Results of RP-HPLC showed differences in the main tR peak profile (tR 26.7) of the standard WHO compared with eCG batches I and II. In experiment 1, the average ovarian weight of rats treated with eCG from WHO (60.0 ± 12.1 mg) was higher (P < .01) than saline (23.1 ± 1.6 mg) and batches I (37.6 ± 1.4 mg) and II (31.0 ± 4.3 mg). In experiment 2, the ovarian weight of rats treated with 30 IU of eCG of batch II (45.7 ± 4.1 mg) was higher (P < .01) than saline (32.6 ± 1.4 mg) and significantly lower (P = .05) than 10 UI of the standard WHO (63.3 ± 8.1 mg). Conclusion: The low ovarian response to eCG treatments can be related to differences in the physicochemical profile of eCG batches and RP-HPLC is a fast and reliable tool for detecting these differences.