Development of Process Flow Sheets for the Purification of Supercoiled Plasmids for Gene Therapy Applications

A cationic cetylpyridinium chloride (CPC) surfactant-based micellar-enhanced ultrafiltration (MEUF) process was used to recover thuringiensin from the supernatant of Bacillus thuringeinsis fermentation broth. Several different strategies of manipulating process variables, such as ionic strength, pH, CPC concentration, micelle formation temperature, and membrane pore size, were investigated. It was found that CPC concentration and membrane pore size were two major factors to the increase of thuringiensin recovery, while the ionic strength and pH adjustments were not necessary and micelle formation temperature was not important within the temperature range studied. Finally, a novel two-step MEUF processing scheme was developed which includes microfiltration and two-step ultrafiltration (UF) processes. The thuringiensin recovery up to 94.6% was attained when an initial CPC concentration of 4% (w/v) and UF membrane of molecular weight cutoff (MWCO) of 3000 was used in conjunction with the previous UF process using a MWCO 30 000 membrane. The bioassay results showed that the spray-dried thuringiensin with CPC was more toxic to fly larvae than without CPC. This research demonstrated that CPC not only facilitated thuringiensin recovery but also improved the insecticidal effect.