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磷脂化重组人铜锌超氧化物歧化酶的质量研究
磷脂化重组人铜锌超氧化物歧化酶的质量研究
付志浩,韩春梅,李永红,王 兰,陶 磊,李 响,毕 华,饶春明,王军志

中国食品药品检定研究院重组药物室,卫生部生物技术产品检定方法及标准化重点实验室,北京 100050
Quality study of phosphatidyl choline Cu/Zn superoxide dismutase
(Division of Recombinant Products, National Institutes for Food and Drug Control, Key Laboratory of the Ministry  of Health for Research on Quality and Standardization of Biotech Products, Beijing 100050, China)

摘要参考文献相关文章

起始页:1855

摘要:目的:磷脂化重组人铜锌超氧化物歧化酶(phosphatidyl choline Cu/Zn superoxide dismutase,简称PC-SOD)是由重组人铜锌超氧化物歧化酶(rhCu/Zn-SOD,简称SOD)经磷脂化修饰而成,对该创新药物的质量研究,需要针对SOD原液、PC-SOD原液和PC-SOD成品的关键质量属性建立质控方法,从而为其质量标准的建立和申报临床研究奠定基础。方法:采用凯氏定氮法测定制品的蛋白含量;采用经典的氮蓝四唑(NBT)还原法测定其生物学活性;采用原子吸收光谱法检测其铜、锌含量;应用磷脂检测试剂盒,采用碱水解显色法检测PC-SOD的胆碱含量;采用SDS-PAGE法和RP-HPLC两种方法测定SOD原液的纯度,采用RP-HPLC和SEC-HPLC两种方法测定PC-SOD原液的纯度;采用Pico-Green荧光法测定外源性DNA残留量。结果:SOD原液、PC-SOD原液和PC-SOD成品的蛋白含量分别为96.46 mg·mL-1,50.53 mg·mL-1和每剂40.07 mg;SOD原液、PC-SOD原液和PC-SOD成品的生物学活性分别为5.11×105 U·mL-1,1.4×105 U·mL-1和每剂1.04×105 U,比活性分别为5 298,2 771和2 595 U·mg-1;SOD原液的铜含量为0.37%,锌含量为0.39%,PC-SOD原液的铜含量为0.35%,锌含量为0.36%;SOD原液的SDS-PAGE测定纯度为>99%,RP-HPLC法测定纯度为99.22%,PC-SOD原液的RP-HPLC法测定纯度为98.75%,SEC-HPLC法测定纯度为99.71%;外源性DNA残留量为每剂6.498 ng;其他各项指标均应符合《人用重组DNA制品质量控制技术指导原则》和2010版《中国药典》(三部)的要求。结论:初步建立了PC-SOD的质控方法,并已用于该制品的质量控制,同时为其他类型磷脂化修饰生物技术药物的质量控制提供参考。

关键词:超氧化物歧化酶;磷脂化超氧化物歧化酶;质量控制;蛋白含量;生物学活性;铜锌含量;纯度

通讯作者:饶春明,男,研究员,主要从事生物技术药物质量标准研究。

基金项目:国家“重大新药创制”科技重大专项资助项目(2012ZX09304010)

作者简介:付志浩,男,助理研究员,主要从事生物技术药物质量控制研究。

Abstract:Objective: To establish quality control methods for SOD bulk, phosphatidyl choline Cu/Zn superoxide dismutase (PC-SOD) bulk and PC-SOD product, making a basis for the requirement establishment and clinical trial application. Methods: The protein content was determined by Kjeldahl nitrogen determination method. The biological activity was determined by the classic NBT disoxidation method. The copper and zinc contents were determined by atomic absorption method. The choline content was determined by base hydrolysis color display method. The purity of SOD bulk was determined by SDS-PAGE method and RP-HPLC method, and the purity of PC-SOD bulk was determined by RP-HPLC method and SEC-HPLC method. The DNA residual was determined by Pico-Green method. Results: The protein contents of SOD bulk, PC-SOD bulk and PC-SOD product were 96.46 mg·mL-1, 50.53 mg·mL-1 and 40.07 mg·dose-1. The biological activity of SOD bulk, PC-SOD bulk and PC-SOD product was 5.11×105 U·mL-1, 1.4×105 U·mL-1 and 1.04×105 U·dose-1. The ratio of biological activity to protein content was 5 298, 2 771 and 2 595 U·mg-1. The copper content of SOD bulk was 0.37%, and the zinc content of SOD bulk was 0.39%. The copper content of PC-SOD bulk was 0.35%, and the zinc content of PC-SOD bulk was 0.36%. The purity of SOD bulk was >99% with SDS-PAGE method and 99.22% with RP-HPLC method. The purity of PC-SOD bulk was 98.75% with RP-HPLC method and 99.71% with SEC-HPLC method. The DNA residual was 6.498 ng·dose-1. Other quality control items were complied with corresponding requirements in the guidance for quality control of human recombinant DNA products and Chinese Pharmacopeia volume Ⅲ 2010 edition. Conclusion: The methods and requirements have been established for the quality control of PC-SOD, and they also are a reference for quality control of other PC modified products.

Key words:superoxide dismutase; phosphatidyl choline Cu/Zn superoxide dismutase(PC-SOD); quality control; protein content; biological activity; copper and zinc content; purity

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