重组人肿瘤坏死因子相关凋亡诱导配体突变体质控方法与质量标准的建立  

Establishment of method and requirement for quality control of recombinant human tumor necrosis factor-related apoptosis-inducing ligand mutant

作　　者：毕华[1] 李永红[1] 杨靖清[1] 丁有学[1] 范文红[1] 韩春梅[1] 李响[1] 史新昌[1] 裴德宁[1] 饶春明[1] 

机构地区：[1]中国食品药品检定研究院重组药物室,北京100050

出　　处：《中国生物制品学杂志》2016年第3期270-274,278,共6页Chinese Journal of Biologicals

基　　金：863计划项目(2012AA020805);国家科技重大专项项目(2012ZX09304010)

摘　　要：目的建立重组人肿瘤坏死因子相关凋亡诱导配体突变体的质控方法和质量标准。方法利用乳腺癌细胞MDA-MB-231增殖抑制试验测定重组人肿瘤坏死因子相关凋亡诱导配体突变体的生物学活性;RP-HPLC和SECHPLC测定其纯度;胰酶酶切,HPLC测定其肽图;荧光定量PCR检测E.coli宿主DNA残留量;ELISA法测定宿主蛋白残留量;Edman降解法进行N-末端序列测定;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》三部(2010版)规定进行。同时对通常在原液中进行检定的宿主DNA残留、菌体蛋白残留、N-末端序列测定进行初步的方法验证,考察其在成品中进行检测的可行性。结果经初步验证,在成品中进行宿主DNA残留、菌体蛋白残留、N-末端序列测定具有可行性,用建立的方法对重组人肿瘤坏死因子相关凋亡诱导配体突变体成品进行检定,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》三部(2010版)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。同时只对成品进行质控的方法和质量标准对同类产品的质量控制具有借鉴意义。Objective To establish the method and requirement for quality control of recombinant human tumor necrosis factor-related apoptosis-inducing ligand（TRAIL） mutant. Methods Recombinant human TRAIL mutant was determined for bioactivity by breast cancer MDA-MB-231 cell proliferation inhibition test, and for purity by RP-HPLC and SEC-HPLC,for peptide map by trypsin digestion, for residual E. coli DNA by fluorescent quantitative PCR, for residual host protein content by ELISA, for N-terminal sequence by Edman degradation method, and for isoelectric point by isoelectric focusing electrophoresis. Other control tests were performed according to the requirements in Chinese Pharmacopoeia（VolumeⅢ,2010 edition）. Meanwhile, residual DNA test, residual host protein test and N-terminal amino acid sequencing, which were usually performed on bulk, were validated to evaluate their feasibility in final product. Results It was feasible to perform residual DNA test, residual host protein test and N-terminal amino acid sequencing in final product. All the indexes of final product of recombinant human TRAIL mutant determined by the developed method met the requirements in the Guideline for Quality Control of Recombinant DNA Products for Human Use and in Chinese Pharmacopoeia（Volume Ⅲ,2010 edition）. Conclusion The established method and requirement were safe and effective, which can be used for routine quality control of recombinant human TRAIL mutant. The method and requirement only used for final product provided a reference for the quality control of products of the same kind.

关 键 词：重组人肿瘤坏死因子相关凋亡诱导配体突变体 质量控制 生物学活性 

分 类 号：Q255[生物学—细胞生物学] R951[医药卫生—药学]