冻干重组结核杆菌融合蛋白(EC)原液效力评价用国家参考品的初步建立

Preliminary establishment of national reference of the freeze-dried recombinant Mycobacterium tuberculosis allergen for the bulk potency evaluation

目的以冻干重组结核杆菌融合蛋白(EC)[该制品名称是国家药典委员会确定的药品中文通用名称,"EC"为重组融合蛋白"结核分枝杆菌早期分泌性抗原靶6(ESAT-6)和培养滤液蛋白10(CFP-10)"](以下简称"EC")原液为原材料,研制EC变态反应原原液效力评价用国家参考品。方法以电泳法、高效液相色谱法分别测定备选EC原液纯度,纯度合格后经精密称量、分装、冻干制成候选国家参考品,再测定其蛋白质和水分含量。将候选国家参考品稀释成不同稀释度后,皮内注射结核分枝杆菌活菌致敏的豚鼠,根据皮肤试验硬结或红晕反应平均直径大小(简称"反应平均直径大小")结果探索原液效价测定的适宜稀释度。在此基础上,将候选国家参考品用于EC原液的效价测定并进行方法验证,初步评价其适用性能。研究还初步观察了候选品在-20℃放置24个月的稳定性。结果 EC原液电泳法纯度为100.00%,高效液相色谱法纯度为95.33%,候选国家参考品蛋白质含量为508μg/瓶,水分含量为1.57%,分装精度控制在±1%以内。稀释度探索结果显示:候选品稀释度为2.5μg/ml(12.5U/ml)、5μg/ml(25U/ml)和10μg/ml(50U/ml)时剂量对数反应平均直径大小曲线有良好的线性关系(R^2=0.9944),可作为后续研究的适宜稀释度。将候选国家参考品用于EC原液效价测定,两者剂量对数反应平均直径大小曲线趋势基本一致(R^2=0.9878,R^2=0.9643),且每个稀释度EC原液与相应稀释度候选国家参考品反应平均直径大小的比值均满足1.0±0.2。方法验证结果显示:候选国家参考品和EC原液的剂量对数反应平均直径大小曲线趋势基本一致(R^2=0.9999,R^2=0.9815),且两者相应稀释度反应平均直径大小的比值均满足1.0±0.2,方法可行。初步稳定性研究结果显示:-20℃放置24个月的候选国家参考品效价测定性能稳定。结论候选国家参考品的纯度、蛋白质含量、水分含量及均匀性检测均符合国家参考品质量要求,将其稀释到适宜稀释度后可用于EC原液的效价测定;经方法验证与稳定性观察,显示其性能良好,为国家参考品申报提供了基础。

Objective To develop a national reference of the freeze-dried recombinant Mycobacterium tuberculosis allergen for the bulk potency evaluation,in which the recombinant Mycobacterium tuberculosis fusion protein(EC)(Which is the Chinese generic name of the drug determined by the National Pharmacopoeia Committee,EC stands for recombinant fusion protein 6 kDa early secretory antigenic target(ESAT-6)and 10 kDa culture filtrate protein(CFP-10))bulk was used as the material. Methods The EC bulk with qualified purity determined by electrophoresis and HPLC was weighed,packaged and freeze-dried accurately to make as national reference candidate,and the content of protein and moisture was further verified.The candidate of different dilution was injected into guinea pig infected with live Mycobacterium tuberculosis,aproper dilution was designated according to the reaction of skin test.And then the applicability of national reference candidate was evaluated preliminarily through the potency testing of the recombinant EC bulk.Stability of candidate storing in-20℃for 24 months was also evaluated. Results The purity of EC stock was 100.00% by electrophoresis,and 95.33% by HPLC.The protein content of national reference candidate was 508μg/bottle,moisture content was 1.57%,and sub-packaging precision was controlled within ±1%.There was agood linear relationship in the dose response curve(R^2=0.9944)when the dilution of reference candidate was 2.5μg/ml(12.5 U/ml),5μg/ml(25 U/ml),and 10μg/ml(50 U/ml).Assaying the national reference candidate by EC stock,the trend of both logarithmic reaction curve was corresponding(R^2=0.9878,R^2=0.9643),and ratio met 1.0±0.2.Method validation showed that the trend of logarithmic reaction curve of national reference candidate and EC bulk was corresponding(R^2=0.9999,R^2=0.9815),and ratio met 1.0±0.2.The result of stability study showed that national reference candidates were stable when they were kept at-20℃ for 24 months. Conclusion The candidate meets the demand of national reference in terms of purity,protein content,moisture content and uniformity.It will be possible to be used in the standization of potency assay of the freeze-dried recombinant Mycobacterium tuberculosis allergen.in a proper dilution.After method validation and stability evaluation,the candidate shows good performance,which will provide a good basis for the application of national reference.