Study of the Removal Method of Endotoxin from A Recombinant Protein
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 pl of the recombinant protein was 5.4～6.2 and the relative molecular weight was 150 000.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 30 000 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.
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