摘要
细菌内毒素是革兰氏阴性菌细胞壁的脂多糖成分,是受污染的注射剂、制剂辅料和医疗器械等引起热原反应的主要原因,故应进行严格控制。本研究评价了阴离子交换层析、超滤和纳滤三种方法对一种哺乳动物细胞表达的重组蛋白中内毒素的去除效果。该蛋白的等电点为5.4~6.2,相对分子质量约为150000。采用鲎试剂法检测内毒素残留,并采用SEC-HPLC和紫外-可见分光光度法检测蛋白纯度及浓度。结果表明,阴离子交换层析对内毒素的去除效果不显著,30000的超滤膜将目的蛋白及内毒素同时截留。而纳滤膜将该重组蛋白中的内毒素含量从初始的大于160 EU/mg降低至10 EU/mg以下,该膜对目的蛋白无截留作用,且不影响蛋白纯度。推测蛋白溶液中钙离子等的存在使内毒素聚合成了更大的分子,故可利用纳滤膜进行去除。纳滤法操作简单,无需进行样品调节,故无需进行后续纯化、换液等工作,是精制内毒素超标样品的优选方法。
Bacterial endotoxins are lipopolysaccharide components liberated by the cell wall of gram-negative bacteria and they are the most important cause of pyrogen reaction caused by contaminated injections,excipients of preparations and medical devices.Therefore,the content of endotoxin in the final products of biological products must be strictly controlled.In this study,the authors evaluated the removal effect of different purification processes on endotoxin from a recombinant protein expressed in mammalian cells.The pI of the recombinant protein was 5.4~6.2 and the relative molecular weight was 150000.The removal ability of endotoxin contaminated with recombinant protein by anion exchange chromatography,ultrafiltration and nanofiltration was investigated.The residual endotoxin was detected by Limulus Amebocyte Lysate(LAL)method,the protein purity was detected by SEC-HPLC,and the concentration of the target protein was determined by Ultraviolet-visible spectrophotometry.The results showed that the removal effect of endotoxin was not significant by anion exchange chromatography flow-through mode,and the 30000 ultrafiltration membrane retained the target protein and endotoxin at the same time.However,the virus removal nanofiltration membrane effectively retained endotoxin while having no retention effect on the target protein,and there was no significant change in the purity of the target protein before and after nanofiltration.It is speculated that endotoxin molecules polymerize in the recombinant protein solution due to the action of calcium ions or other components,forming large aggregates,so they can be removed by virus removal nanofiltration membrane.Using the nanofiltration process,the endotoxin content in the recombinant protein was reduced from more than 160 EU/mg to less than 10 EU/mg,and the yield of the recombinant proprotein was 100%.The nanofiltration method is simple and does not require the adjustment of pH and composition of the sample,so it does not require subsequent purification,liquid replacement or other work,so nanofiltration method is the optimal method for refining the sample with excessive endotoxin.
作者
陈梅梅
秦宇
刘世萍
胡宏飞
张甜甜
刘翔
李盈淳
CHEN Mei-mei;QIN Yu;LIU Shi-ping;HU Hong-fei;ZHANG Tian-tian;LIU Xiang;Li Ying-chun(Research Institute of Chia Tai Tianqing Pharmaceutical Group Co.,Ltd.,Nanjing 211100,China)
出处
《药物生物技术》
CAS
2024年第1期15-19,共5页
Pharmaceutical Biotechnology
关键词
内毒素
阴离子交换层析
超滤
纳滤
重组蛋白
去除
回收率
Endotoxin
Anion exchange chromatography
Ultrafiltration
Nanofiltration
Recombinant protein
Removal
Yield
