Using a single hydrophobic-interaction chromatography to purify pharmaceutical-grade supercoiled plasmid DNA from...

The recent developments in non-viral gene therapy and DNA vaccine have fostered the development of efficient plasmid DNA (pDNA) purification processes.

This work aimed to establish a cost-effective purification process for the large-scale production of plasmid DNA for gene therapy and DNA vaccine.

E. coli DH5α harboring pCDNA3.1-GFP (7200 base pairs) was used as a model plasmid. Hydrophobic-interaction chromatography (HIC) was employed to purify supercoiled plasmid DNA (sc pDNA).

With this method, not only host contaminants, but also open circular plasmid DNA (oc pDNA) could be removed from sc pDNA. Anion-exchange HPLC analysis proved that the recovery of HIC could reach 75%. The plasmid DNA exhibited high purity with supercoiled percentage of 98 ± 1.2% and undetectable residual endotoxins, genomic DNA, RNA and protein. The purity of pDNA had nothing to do with the flow rate in the range at least up to 400 cm/h. Liposomes transfection experiment prove that the purified pDNA in this article had higher transfection efficiency than the control pDNA.

In the present work, we confirmed the possibility of separation of sc pDNA from oc pDNA and other host contaminants using a single HIC chromatography.