Analysis of Selective Metal-Salt-Induced Endotoxin Precipitation in Plasmid DNA Purification Using Improved Limulus Amoebocyte Lysate Assay and Central Composite Design

摘要

Recent advancements in plasmid DNA (pDNA) produc-ntion involve the development of innovative and cost-neffective methods as well as reduced number of unitnoperations. This study investigates the feasibilities of usingna metal salt to selectively remove endotoxins from clarifiedncell lysates containing plasmid DNA. Screening of endot-noxin precipitation in various metal salt solutions andnoptimization of process conditions (pH, ion concentration,ntemperature, and incubation time) using central compos-nite design experiments have been carried out successfully.nResults show that selective endotoxin precipitation ( 0.05nEU/µg) can economically be carried out during thenalkaline cell lysis procedure (neutralization step) at a pHncondition similar to that of alkaline-lysed cell lysate, a lownZnSO4 concentration (0.5 M), a minimum incubationntime (30 min), and a temperature of 15 °C. Innsummary, this method provides ease of subsequentnplasmid DNA purification due to reduced bulk impuri-nties and cost-effective and most importantly high en-ndotoxin removal ( 80%) and plasmid recovery ( 90%).