重组结核杆菌融合蛋白(EC)产品质量标准的建立  

Establishment of quality standards for recombinant Mycobacterium tuberculosis fusion protein

作　　者：张凯 沈小兵[2] 陶立峰 韦芬 陈保文[2] 仇晶晶 陈伟 卢锦标[2] 朱银猛 程兴 钟再新 赵爱华[2] 蒲江 ZHANG Kai;SHEN Xiao-bing;TAO Li-feng;WEI Fen;CHEN Bao-wen;QIU Jing-jing;CHEN Wei;LU Jin-biao;ZHU Yin-meng;CHENG Xing;ZHONG Zai-xin;ZHAO Ai-Hua;PU Jiang(Anhui Zhifei Longcom Biopharmaceutical Co.,Ltd.,Hefei 230000,China)

机构地区：[1]安徽智飞龙科马生物制药有限公司,合肥230000 [2]中国食品药品检定研究院结核病疫苗和过敏原产品室中国医学科学院生物技术产品检定方法及其标准化重点实验室

出　　处：《中国防痨杂志》2020年第8期814-820,共7页Chinese Journal of Antituberculosis

基　　金：“十二五”国家科技重大专项(2014ZX10003002-004,2015ZX09108-004);“十三五”国家科技重大专项(2017ZX10201301-001-003,2018ZX10103001-001-008)。

摘　　要：目的建立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)";简称"EC"]原液及成品的质量标准。方法选取6批次EC原液及11批次EC成品、体质量300~400g无特定病原体(SPF)级白色豚鼠21只、卡介苗(BCG)活菌菌液(1mg/ml)、结核分枝杆菌减毒株H37Ra活菌菌液(2mg/ml)作为实验材料,建立EC原液质量标准,包括残余抗生素活性检测、致敏效应实验、效价测定及动物法鉴别实验;建立EC成品质量标准,包括鉴别实验及效价测定;应用建立的质量检测标准对连续6批次EC原液和11批次EC成品进行检测,验证其适用性和可行性。结果(1)EC原液残余抗生素活性检测。①专属性实验:磷酸盐缓冲液的吸光度值(A值)为1.736,卡那霉素标准品(0.0ng/ml)的A值为2.178;②线性与范围实验:卡那霉素标准品在0.5~40.5ng/ml范围内,连续3次实验回归方程拟合优度的判定系数R^2均大于0.99;③准确性实验:连续3批次EC原液的卡那霉素残留量回收率分别为112.163%、117.285%、103.527%;④重复性实验:连续3批EC原液重复性实验A值相对标准偏差(RSD)分别为5.59%、9.18%、8.07%;⑤中间精密度实验:同一实验员不同时间及不同实验员检测连续3批次EC原液卡那霉素残留量检测均小于定量限0.5ng/ml,为限量测定;⑥耐用性实验:改变读板时间(0、5、10min)对EC原液卡那霉素残留量检测无影响,卡那霉素残留量均小于定量限0.5ng/ml。(2)EC原液效价测定:稀释度为1μg/ml(5U/ml)、2.5μg/ml(12.5U/ml)、5μg/ml(25U/ml)及10μg/ml(50U/ml)时,每个稀释度对应的2批次EC原液动物皮肤试验的硬结或红晕平均直径及硬结或红晕平均直径总和比值分别为(15.17±0.88)mm、(13.67±1.25)mm、1.11(91.00/82.00);(17.00±1.76)mm、(16.08±1.32)mm、1.06(102.00/96.50);(19.58±1.69)mm、(17.67±1.37)mm、1.11(117.Objective To establish the quality standard for 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))stock solution and recombinant EC.Methods Six batches of recombinant EC stock solution,11 batches of recombinant EC,21 no specific pathogen(SPF)white guinea pigs with the weight of 300-400 g,viable BCG bacterial solution(1 mg/ml),and viable bacteria solution of Mycobacteriumtuberculosis attenuated strain H37 Ra(2 mg/ml)were selected to establish the quality standard of the recombinant EC stock solution,including residual antibiotic activity detection,sensitization effect test,potency determination and animal identification experiment.Establishment of quality standards for recombinant EC included identification experiment and potency determination.Six consecutive batches of the recombinant EC stock solution and 11 batches of the recombinant EC were tested using to the established quality standard,to verify the applicability and feasibility of the standard.Results 1.Data of the residual antibiotic activity detection in the recombinant EC stock solution:(1)Specific experiment:the absorbance(A)value of PBS buffer was 1.736,and that of kanamycin standard(0.0 ng/ml)was 2.178;(2)Linear and range experiments:the determination coefficient R^2 of goodness of fit of regression equation for three consecutive experiments was greater than 0.99 when kanamycin ranged between 0.5 and 40.5 ng/ml;(3)Accuracy test:the recovery rates of residual kanamycin in three consecutive batches of recombinant EC were 112.163%,117.285%and 103.527%,respectively;(4)Repeatability test:the relative standard deviation(RSD)of A values of repeatability test in three consecutive batches of recombinant EC were5.59%,9.18%and 8.07%,respectively;(5)Intermediate precision test:kanamycin residues of three consecutive batches of the

关 键 词：重组结核杆菌融合蛋白(EC) 质量控制 参考值 药物评价 体外研究 

分 类 号：R927.1[医药卫生—药学]