Anion exchange purification of plasmid DNA using expanded bed adsorption.

摘要: Recent developments in gene therapy with non-viral vectors and DNA vaccination have increased the demand for large amounts of pharmaceutical-grade plasmid DNA. The high viscosity process streams is major concern purification plasmids, since it can cause back pressures column operations, thus limiting throughput. In order to avoid these pressures, expanded bed anion exchange chromatography was evaluated as an alternative fixed chromatography. A Streamline 25 filled 100 ml QXL media, equilibrated 0.5 M NaCl TE (10 mM Tris, 1 EDTA, pH=8.0) buffer at upward flow 300 cmh-1, E. coli lysates (obtained from up 3 liters fermentation broth) were injected column. After washing out unbound material, media allowed sediment eluted a downward 120 cmh-1. Purification factors 36±1 fold, 26±0.4 purity, close 100% yields obtained when less than one settled volume feed injected. However, both recovery yield purity abruptly decreased larger processed–values 35±2 5±0.7 respectively, 250 feedstock processed. cases, gel clogging expansion collapse observed. processing volumes, quantities, only possible by performing isopropanol precipitation step prior chromatographic step. This led enhancement