Scalable recovery of plasmid DNA based on aqueous two-phase separation.
Future developments in gene therapy and DNA vaccination depend on cost-effective large-scale production of pharmaceutical-grade pDNA (plasmid DNA). Given the large amount of impurities present in the feedstock, purification processes that have high specificity and capacity at a moderate cost are required. In the present study, we describe a non-chromatographic procedure based on aqueous two-phase extraction allowing a fast and simply scalable capture step. PEG [poly(ethylene glycol)] in combination with potassium citrate or potassium phosphate was tested as phase component for extraction. By increasing either PEG or salt concentration, the partitioning of nucleic acids changed from bottom to top phase. Phase systems with a composition of 15% PEG 800 and 20% potassium phosphate at pH 7.0 showed a strong partitioning of pDNA to the bottom phase, linked to a clear decrease in open circular pDNA, while proteins, genomic DNA and RNA remain at the top or at the interphase. A great advantage of the current process is that the complete procedure of lysis, precipitation, clarification and extraction can be performed in a single vessel. The number of denatured and sheared genomic DNAs in a spiking experiment was found to be depleted by more than 99%.
Frerix
Andreas
A
Institut für Biotechnologie 2, Forschungszentrum Jülich, 52426 Jülich, Germany.
Müller
Markus
M
Kula
Maria-Regina
MR
Hubbuch
Jürgen
J
D005591:Chemical Fractionation / Q000379:methods*; D044367:Phase Transition; D010710:Phosphates / Q000737:chemistry*; D010865:Pilot Projects; D010957:Plasmids / Q000737:chemistry* / Q000302:isolation & purification*; D011092:Polyethylene Glycols / Q000737:chemistry*; D019357:Potassium Citrate / Q000737:chemistry*; D017680:Potassium Compounds / Q000737:chemistry*; D014867:Water / Q000737:chemistry*
D010710:Phosphates; D017680:Potassium Compounds; D014867:Water; D011092:Polyethylene Glycols; C013216:potassium phosphate; D019357:Potassium Citrate