绿脓杆菌外毒素A功能片段重组蛋白GP38质控方法和质量标准的建立  

Development of Method and Standard for Quality Control of Freeze-dried Functional Fragment GP38 of Pseudomonas Exotoxin A

作　　者：史新昌[1] 韩春梅[1] 李响[1] 刘兰[1] 丁有学[1] 饶春明[1] 

机构地区：[1]中国药品生物制品检定所重组技术产品室,北京100050

出　　处：《中国生物制品学杂志》2010年第4期399-402,共4页Chinese Journal of Biologicals

摘　　要：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 develop a method and a standard for quality control of freeze-dried functional fragment GP38 of Pseudomonas exotoxin A（PEA）, an analog of human gonad hormone releasing hormone（GHRH）. Methods HeLa cells / crystal violet colorimetric method was used for determination of biological activity of GP38, and verified for precision and recovery rate. The purity of GP38 was determined by RP-HPLC, and the peptide maps of test sample and reference were compared by the CPM software developed by the authors. Other control tests were carried out according to the requirements in Chinese Pharmacopoeia （Volume Ⅲ, 2005）. Results The intra-and inter-coefficients of variations （CVs）of determination results of bulk of GP38 by HeLa cells / crystal violet colorimetric method were 10% and 20%, while those of final product were 12% and 21%, respectively. The biological activities of bulk and final product of GP38 determined by the developed method were （3 736 ± 483）and （1 777 ± 260）U / ml respectively. The RP-HPLC purity of bulk was（97. 1 ± 0. 2）%, which met the quality standard（more than 95%）. The similarity of peptide maps of test sample and reference repeatedly tested under the same digestion conditions was more than 0.99, while that digested for different times and repeatedly tested under the same conditions was more than 0. 90. All the other quality indexes met the requirements in Chinese Pharmacopoeia （Volume Ⅲ, 2005）. Conclusion The developed method and standard may be used for the routine quality control of GP38.

关 键 词：绿脓杆菌外毒素A 功能片段 质量控制 质量标准 

分 类 号：R378[医药卫生—病原生物学] R392.33[医药卫生—基础医学]