摘要
目的建立酵母表达的重组人胰高血糖素样肽-1类似物融合蛋白的质控方法和质量标准。方法采用荧光素酶报告基因法进行效价测定;分别用非还原聚丙烯酰胺凝胶电泳(SDS-PAGE)和分子筛液相色谱(SE-HPLC)测定纯度;反相高效液相色谱(RP-HPLC)进行原液肽图分析;荧光定量PCR方法测定外源DNA残留量;液质联用技术分析重组人胰高血糖素样肽-1类似物融合蛋白理化对照品的相对分子质量和氨基酸序列;其余检测项目按《中国药典》2010年版三部规定进行。结果用建立的方法对重组人胰高血糖素样肽-1类似物融合蛋白3批原液、3批成品及1批理化对照品进行了质量控制研究,建立了针对该制品原液和成品的质量标准,原液和成品的检验结果均在质量标准控制范围内,符合《人用重组DNA制品质量控制技术指导原则》和《中国药典》2010年版三部的要求;理化对照品质谱相对分子质量测定结果为71 361.0,与理论相对分子质量一致,氨基酸序列覆盖率达到97%。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 the quality control system for the recombinant human GLP-1 analogue fusion protein. METHODS The potency of the fusion protein was determined by luciferase reporter gene assay. The purity was analyzed by non-reduced SDS-PAGE and SEC-HPLC respectively. RP-HPLC was used for the peptide mapping. ELISA was used to analyze the identifica- tion of the final products. The molecular mass and peptide mass spectra were analyzed by LC-ESI-MS technique. Other control tests were performed according to the requirements in the Chinese Pharmacopoeia （Volume m, 2010 edition）. RESULTS Control tests were performed on three different lots of bulks and final products of recombinant GLP-1 analog fusion protein by the developed methods. The results showed that all the indexes met the requirements in the Guideline for Quality Control of Recombinant DNA Products for Hu- man Use and Chinese Pharmacopoeia （ Volume HI, 2010 edition）. The molecular weight of the recombinant human GLP-1 analogue fu- sion protein was 71 361.0, which was in conformity with the theoretical value. CONCLUSION The developed methods and standard may assure the safety, effectiveness, and controllability of the recombinant human GLP-1 analogue fusion protein, which might be used for the routine quality control of products of the same kind.
出处
《中国药学杂志》
CAS
CSCD
北大核心
2016年第13期1085-1090,共6页
Chinese Pharmaceutical Journal
基金
国家科技重大专项课题资助项目(2015ZX09501008)
