Purification of Plasmid DNA Vectors Produced in Escherichia coli for Gene Therapy and DNA Vaccination Applications

A bench-scale protocol for the purification of plasmid DNA (pDNA) produced in Escherichia coli is described. The method is specifically designed to prepare pDNA vectors for gene therapy and DNA vaccination applications. The method comprises alkaline lysis, concentration with iso-propanol, prepurification by (NH4)2SO4 precipitation and purification by hydrophobic interaction chromatography (HIC), and desalting in gravity-operated 10-mL plastic columns. Analytical techniques used to control the performance of the method and to assess the quality of the pDNA vaccine are also described. Anion-exchange HPLC is used to determine pDNA concentration, whereas the presence of impurities such as RNA, proteins, E. coli genomic DNA, and endotoxins is determined by agarose gel electrophoresis, micro-bicinchoninic acid (BCA), real-time polymerase chain reaction and kinetic-quantitative chromogenic lymulus amoebacyte lysate (QCL LAL) assays, respectively. The method performs very well in terms of yield, purity and biological activity of the final pDNA. Furthermore, it is rapid, very easy to perform, and cost-efficient.