血管内皮生长因子抑制剂质控方法和质量标准研究

Study on methods and requirements for quality control of vascular endothelial growth factor inhibitor

目的:建立血管内皮生长因子抑制剂的质控方法和质量标准。方法:利用HEK293/D9/Flt-18R(alpha)/Flt-IL18R(beta)细胞系,通过荧光素酶检测系统测定血管内皮生长因子抑制剂的生物学活性;SDS-PAGE和SEC-HPLC测定纯度;实时定量PCR检测CHO宿主DNA残留量;采用ELISA法分别测定VEGF结合力和残留蛋白A含量;用反相HPLC进行唾液酸含量分析;阴离子交换HPLC对寡糖性质进行测定以检查该制品的唾液酸化程度;水平等电聚焦电泳法测定等电点;其余检测项目按中国药典2005年版三部规定进行。结果:用建立的方法对血管内皮生长因子抑制剂原液和成品进行了检定,各项指标均符合《人用重组DNA制品质量控制技术指导原则》和中国药典2005年版三部的要求。结论:建立的质控方法和质量标准具有保证产品安全、有效、质量可控的特点,可用于该类产品的常规检定。

Objective：To establish methods and requirements for quality control of vascular endothelial growth factor inhibitor.Methods：Using HEK293/D9/Flt-18R（alpha）/Flt-IL18R（beta）,biological potency of VEGF inhibitor was determined by luciferase reporter system.Purity of VEGF inhibitor was analyzed by SDS-PAGE and SEC-HPLC.CHO residual DNA was detected by real-time PCR.The binding activity of VEGF inhibitor to human VEGF,and residual protein A content were measured using sensitive ELISA.In addition,sialic acid content was analyzed by RP-HPLC.Oligosaccharide profiling by anion exchange HPLC was used to examine sugar chain quality and degree of sialylation.Isoelectric focusing was used for the qualitative analysis of VEGF inhibitor isoforms with their pI.Routine tests were all carried out according to Pharmacopoeia of People＇s Republic of China（volume Ⅲ,2005 edition）.Results：The requirements and methods for quality control of VEGF inhibitor were established and used for quality control of bulk and final product.The results of all tests complied with the requirements of guideline for quality control of recombinant DNA products for human use and Pharmacopoeia of People＇s Republic of China（volume Ⅲ,2005 edition）.Conclusion：The methods and requirements for quality control of VEGF inhibitor showed characteristics of assuring the products safety and efficacy,which can be used for routine quality control of VEGF inhibitor.