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目的建立人性腺激素释放激素类似物—绿脓杆菌外毒素A功能片段重组蛋白GP38的质控方法和质量标准。方法采用HeLa细胞/结晶紫比色法测定GP38蛋白的生物学活性,并进行精密性和回收率验证;反相高效液相色潽法(RP-HPLC)测定其纯度,用自编CPM软件进行肽图比较分析;其他检测项目按《中国药典》三部(2005版)规定进行。结果GP38蛋白生物学活性测定方法经验证,原液试验内变异系数(CV)为10%,试验间CV值为20%;成品试验内CV值为12%,试验间CV值为21%。用该法对GP38蛋白原液和成品进行检测,其生物学活性分别为(3736±483)和(1777±260)U/ml。原液经RP-HPLC分析,纯度为(97.1±0.2)%,符合其质量标准大于95.0%的规定。用自编CPM软件分析肽图结果显示,相同酶切条件重复检测的相似度大于0.99,不相同次酶切、相同条件重复检测的相似度大于0.90。其他各项检测结果均符合《中国药典》三部(2005版)的相关要求。结论所建立的质控方法和质量标准可用于GP38蛋白产品的常规检定。
Objective To establish a method and quality standard for quality control of human gonadotropin - releasing hormone analogue - Pseudomonas aeruginosa exotoxin A functional fragment recombinant protein GP38. Methods The biological activity of GP38 protein was determined by HeLa cell / crystal violet colorimetric method, and its precision and recovery were verified. The purity of GP38 protein was determined by reverse-phase high performance liquid chromatography (RP-HPLC) Peptide map comparative analysis; other test items according to “Chinese Pharmacopoeia” three (2005 version) requirements. Results The method of determination of GP38 protein biological activity was validated. The coefficient of variation (CV) in the solution was 10% and the CV value was 20%. The CV value in the finished product was 12% and the CV value was 21%. The method of GP38 protein solution and finished products were tested, the biological activity were (3736 ± 483) and (1777 ± 260) U / ml. The stock solution was analyzed by RP-HPLC with a purity of (97.1 ± 0.2)%, meeting the requirement of more than 95.0% of its quality standard. The results of peptide analysis using self-compiled CPM software showed that the similarity of repeated detection of the same conditions was greater than 0.99, different digestion, the similarity of repeated detection under the same conditions was greater than 0.90. Other test results are in line with the “Chinese Pharmacopoeia” three (2005 version) of the relevant requirements. Conclusion The established quality control methods and quality standards can be used for routine testing of GP38 protein products.