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中国防痨杂志 ›› 2020, Vol. 42 ›› Issue (8): 814-820.doi: 10.3969/j.issn.1000-6621.2020.08.007

• 论著 • 上一篇    下一篇

重组结核杆菌融合蛋白(EC)产品质量标准的建立

张凯*, 沈小兵, 陶立峰, 韦芬, 陈保文, 仇晶晶, 陈伟, 卢锦标, 朱银猛, 程兴, 钟再新, 赵爱华(), 蒲江()   

  1. 230000 合肥,安徽智飞龙科马生物制药有限公司(张凯、陶立峰、韦芬、仇晶晶、陈伟、朱银猛、程兴、钟再新、蒲江);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(沈小兵、陈保文、卢锦标、赵爱华)
  • 收稿日期:2020-06-19 出版日期:2020-08-10 发布日期:2020-08-10
  • 通信作者: 赵爱华,蒲江 E-mail:zhaoaihua2016@126.com;pujiang@zhifeishengwu.com
  • 基金资助:
    “十二五”国家科技重大专项(2014ZX10003002-004);“十二五”国家科技重大专项(2015ZX09108-004);“十三五”国家科技重大专项(2017ZX10201301-001-003);“十三五”国家科技重大专项(2018ZX10103001-001-008)

Establishment of quality standards for recombinant Mycobacterium tuberculosis fusion protein

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()   

  1. *Anhui Zhifei Longcom Biopharmaceutical Co., Ltd., Hefei 230000, China
  • Received:2020-06-19 Online:2020-08-10 Published:2020-08-10
  • Contact: ZHAO Ai-Hua,PU Jiang E-mail:zhaoaihua2016@126.com;pujiang@zhifeishengwu.com

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R2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(117.50/106.00);(20.75±1.57)mm、(20.25±1.17)mm、1.02(124.50/121.50)。(3)EC原液动物法鉴别实验:连续3批次EC原液动物皮肤试验的硬结或红晕平均直径均为0,TB-PPD动物皮肤试验的硬结平均直径为(15.13±5.06)mm。(4)EC成品鉴别试验:连续3个批次EC成品动物皮肤试验的硬结或红晕平均直径均为0,TB-PPD动物皮肤试验的硬结平均直径为(16.50±2.65)mm。(5)质量标准验证:①EC原液:残余抗生素活性检测结果均低于定量限0.5ng/ml;致敏效应实验结果均为皮肤试验后豚鼠注射部位局部反应无明显区别,也无全身反应;每个稀释度[2.5μg/ml(12.5U/ml)、5μg/ml(25U/ml)及10μg/ml (50U/ml)]在皮内注射后24h所产生的局部硬结或红晕反应平均直径均≥8mm,与相应稀释度参考品的局部硬结或红晕平均直径总和的比值为0.9±0.1;动物法鉴别实验结果为EC原液对BCG活菌致敏组豚鼠的皮肤试验结果均呈阴性反应(硬结或红晕平均直径<5mm),而TB-PPD呈阳性反应(硬结平均直径≥5mm)。②EC成品检测:各批次成品对BCG活菌致敏组豚鼠的皮肤试验均呈阴性反应(硬结或红晕平均直径<5mm),而TB-PPD呈阳性反应(硬结平均直径≥5mm)。结论 所建立的质量标准可用于EC原液及成品的检定。

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

Abstract:

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 (1mg/ml), and viable bacteria solution of Mycobacterium tuberculosis attenuated strain H37Ra (2mg/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 R2 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 were 5.59%, 9.18% and 8.07%, respectively; (5) Intermediate precision test: kanamycin residues of three consecutive batches of the recombinant EC stock solution were detected by the same experimenter at different times and by different experimenters at the same time, and the results were all less than the quantitative limit of 0.5 ng/ml; (6) Durability test: the change of reading time (0, 5, 10min) had no effect on the detection of kanamycin residues in recombinant EC stock solution, and kanamycin residues were all less than the quantitative limit of 0.5 ng/ml. 2. Potency determination of recombinant EC stock solution: when the dilutions were 1μg/ml (5 U/ml), 2.5μg/ml (12.5 U/ml), 5μg/ml (25 U/ml) and 10μg/ml (50 U/ml), the average diameters and sum ratios of the average diameter of induration or redness of 2 batches of recombinant EC stock in animal experiments were (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 (117.50/106.00); (20.75±1.57) mm, (20.25±1.17) mm, 1.02 (124.50/121.50), respectively. 3. Animal identification experiment for recombinant EC stock solution: the average diameters of induration or redness in 3 consecutive batches of recombinant EC were 0, while the average diameter of induration was (15.13±5.06) mm in TB-PPD. 4. Identification test of the recombinant EC: the average diameters of induration or redness in 3 consecutive batches of recombinant EC were 0, while the average diameter of induration was (16.50±2.65) mm in TB-PPD. 5. Quality standard verification: (1) Recombinant EC stock solution: the results of residual antibiotic activity were all lower than the quantitative limit of 0.5 ng/ml; the sensitization effect test results showed that there was no significant difference in the local reactions of the injection site and no systemic reaction were found; the results of potency determination showed that in different dilutions (2.5μg/ml (12.5 U/ml), 5μg/ml (25 U/ml), 10μg/ml (50 U/ml)), the average diameter of local induration or redness reaction of each dilution were all ≥8 mm 24 h after injection. The ratio of the sum of local induration or redness reaction of the corresponding controls was 0.9±0.1. Results of animal identification experiment showed that the skin tests of BCG viable bacteria sensitized guinea pigs were all negative (mean diameter of induration or redness <5 mm), while the skin tests of guinea pigs with TB-PPD were positive (mean induration diameter ≥5 mm); (2)Tests for the recombinant EC: the results showed that the skin test were all negative (mean induration or redness diameter <5 mm), while TB-PPD were positive (mean induration diameter ≥5 mm). Conclusion The established quality standards could be used for verification in recombinant EC stock solution and recombinant EC.

Key words: Recombinant Mycobacterium tuberculosis fusion protein (EC), Quality control, Reference values, Drug evaluation, In vitro