第1批重组人绒促性素(生物测定用)国家标准品的制备与协作标定

Preparation and collaborative calibration of the 1st national standard of recombinant human chorionic gonadotrophin for bioassay

目的:制备并标定第1批重组人绒促性素(recombinant human chorionic gonadotrophin,rHCG)国家标准品(生物测定用)。方法:以WHO第5批绒促性素国际标准品07/364效价为162 IU·瓶^(-1)作为标准,采用2020年版《中华人民共和国药典》四部1209绒促性素生物测定法,联合5个实验室对第1批rHCG待标品进行生物活性协作标定。对待标品原液进行了等电点、肽图、相对分子质量测定等结构确认。对待标品进行了紫外分光光度法蛋白含量测定、SEC-HPLC法纯度和聚体分析、RP-HPLC法氧化亚基分析等关键理化性质分析,并进行了稳定性考察。结果:,rHCG素待标品经协作标定,最终定值其生物效价为6400 IU·瓶^(-1);,rHCG素含量约为290μg·瓶^(-1),SEC-HPLC纯度约为100%。结构确认结果符合要求;聚体、解离亚基、氧化亚基等各项理化指标符合规定;稳定性考察结果满足要求。结论:本批待标品可作为第1批rHCG国家标准品,以供rHCG及相关制剂的生物效价测定用,定值为6400 IU·瓶^(-1)。

Objective:To prepare and calibrate the first batch national standard(NS)of recombinant human chorionic gonadotrophin(rHCG)for bioassay.Methods:The bioactivity of the first batch of the candidate preparationof rHCG was collaboratively calibratedby five laboratories taking the fifth batch of rHCG of WHO international standard 07/364 with a titer of 162 IU per vial as the standard,the 1209 bioassay method of rHCG in VolumeⅣof the Chinese Pharmacopoeia was adopted.The structure of candidate preparation concentrate was confirmed by imaging capillary isoelectric focusing(icIEF),peptide mapping and determination of relative molecular mass.The key physicochemical properties were measured,such as the protein content by ultraviolet spectrophotometry,purity and polymers by SEC-HPLC,oxidation of subunits by RP-HPLC.The stability tests for the candidate preparation were carried out.Results:The biopotency of the candidate preparation of rHCG was finally determined to be 6400 IU per vial after collaborative calibration.The content of rHCG was about 290μg per vial,and the purity by SEC-HPLC was about 100%.Structure confirmation results meet the requirements;Physical and chemical indicators such as polymer,dissociated subunit,and oxidized subunits were conformed to the standard;the stability test results meet the requirements.Conclusion:The candidate preparation is recognized as rHCG to sever as the 1 st NS rHCG for bioassay of rHCG and related preparations,and its biopotency is 6400 IU per vial.