Purification Process Design And Quality Control Of Therapeutic Supercoiled Plasmid DNA

There are increasing applications of various plasmid DNA constructs in many areas of medical research,agricultural,medical diagnosis and therapy.Specifically,for the postential uses in DNA vaccines and other genetic therapies,it is very important to ensure the highest qualities of production and purification of supercoiled plasmid DNA as injectable.In this study,we designed a purification process for obtain high quality plasmid DNA by chromatography in three steps.The process employeed different chromatographic mechanisms incuding molecular sieve chromatography,affinity chromatography,and anion exchange chromatography.The resins used were Sepharose^TM6 Fast Flow,Plasmid Select Xtra,and SOURCE^TM30Q.Various flow conditions for separation and purification were explored and optimized.The optimized process enabled successful separation of supercoiled plasmid DNA from impurities including genomic DNA,protein,RNA,endotoxin and other forms of plasmid DNA.Compared to other commonly used methods(plasmid DNA kits,CTAB,method,etc.),our methods had higher capacity,higher yields,and easy scale up.The stability and compatibility of this process were verified by testing different plasmid constructs.We finally focused on the purification products of a CRISPR/Cas9 plasmid DNA targeted to the ccc DNA of the hepatitis B virus.We tested the plasmid quality based on the“Technical Guidelines for Preclinical Research in DNA Vaccines for Prevention”and the quality attributes of other similar products(recombinant human hepatocyte growth factor plasmid DNA,HIV DNA vaccines,etc.).Based on these studies,we proposed a set of quality standards for plasmid DNA that may be used in gene therapy clinical studies...