Flocculate removal after alkaline lysis in plasmid DNA production.
Alkaline lysis is the most commonly used method following harvest of bacterial cells for production of plasmid DNA. The method was originally developed for laboratory scale experiments and has shown to be challenging at larger scales. A major problem prior to further downstream processing is the risk of filter clogging without efficient removal of the flocculate that occurs after neutralization. For this purpose we here present a scalable method where the clarification of alkaline lysate is greatly simplified. Through a rapid procedure, involving the addition of ammonium hydrogen carbonate to the neutralized alkaline lysate, the flocculate is lifted to the surface of the solution by the released carbon dioxide and ammonium. After this step a clarified solution can be drained from the bottom of the vessel. The procedure does not impact pH, plasmid DNA concentration or the ratio of open circular to supercoiled plasmid DNA in the solution.
Processes and Applications, BioProcessing R&D, GE Healthcare Bio-Sciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden. hans.blom@ge.com
Hans
H
Blom
Mia
M
Bennemo
Mikael
M
Berg
Raf
R
Lemmens
D001431:Bacteriological Techniques / Q000379:methods*; D005591:Chemical Fractionation / Q000379:methods*; D004269:DNA, Bacterial / Q000302:isolation & purification*; D005427:Flocculation; D008967:Molecular Biology / Q000379:methods*; D010957:Plasmids / Q000302:isolation & purification*
D004269:DNA, Bacterial