基于人卵巢颗粒细胞KGN建立的卵泡刺激素体外生物活性效价测定法

Establishment of a bioassay for follicle stimulating hormone titer in vitro based on human ovarian granulosa cells KGN

目的:基于哺乳动物体内激素具有刺激卵巢颗粒细胞产生黄体酮(孕酮)的机理,建立尿促性素(human menopausal gonadotropin, HMG)刺激人卵巢颗粒细胞KGN分泌孕酮(progesterone, PG)法,用于评价HMG中活性成分卵泡刺激素(follicle stimulating hormone, FSH)的效价。方法:用不同浓度的HMG溶液刺激KGN细胞,检测分泌上清中的PG含量,建立FSH的体外生物学活性效价测定方法;参照《中华人民共和国药典》2020年版四部通则9101分析方法验证和9401生物制品生物活性/效价测定方法,验证指导原则对所建立的方法进行专属性、精密度、耐用性等方法学研究;以HMG国家标准品作为基准,检测HMG国际标准品和制剂(合格和不合格)的效价。结果:不同浓度的HMG作用KGN细胞后具有时间和剂量依赖性。结论:本方法的专属性、精密度和耐用性符合《中华人民共和国药典》四部通则的要求;以HMG国家标准品为基准,能有效检测HMG国际标准品和注射用HMG的效价,与药典方法一致。

Objective: To establish a method of stimulating human ovarian granulosa cells KGN to secrete progesterone(PG) by human menopausal gonadotropin(HMG), based on the mechanism of estrogen stimulating the production of progesterone in ovarian granulosa cells in mammalian, to evaluate the titer of follicle-stimulating hormone(FSH), the active component of HMG. Methods: KGN cells were stimulated with different concentrations of HMG solution, the content of PG in the secreted supernatant was detected, and the biological activity assay of FSH in vitro was established. According to the 9101 analysis method verification and 9401 biological products bioactivity/potency determination method verification guiding principles in the General Rules of the fourth part of Chinese Pharmacopoeia, the established method was validated in terms of specificity, precision and durability. The national standard of HMG was used as the benchmark to detect the potency of HMG international standard and preparation(qualified and unqualified). Results: Different concentrations of HMG displayed effect on KGN cells in a time-and dose-dependent manner. Conclusion: The specificity, precision and durability of the method meet the requirements of the General Principles of the fourth part of Chinese Pharmacopoeia. Based on HMG national standard, the titers of HMG international standard and HMG for injection can be effectively determined, with consistent results with the pharmacopoeia method.