An Investigation on E. coli Host Strain Influences and Strategies to Improve Supercoiled Plasmid DNA Production for Gene Therapy and Vaccination

The growing demand for quick and effective methods of producing large amounts of plasmid DNA for human therapy and vaccination has increased the practical challenges associated with process optimisation to improve supercoiled plasmid DNA yields obtained through current methods. The supercoiled isoform of DNA is the preferred form for use in gene therapy and vaccination as this isoform is known to produce higher levels of in vitro and in vivo transgene expression than other forms of plasmid DNA. This study was designed to investigate whether different strategies can be implemented early on in a process to improve supercoiled plasmid DNA yields obtained upstream, with the view to aid and/or ease further downstream stages. The main theme investigated is the influence of the host strain on supercoiled plasmid DNA production. Seventeen strains of Escherichia coli and three different plasmids were investigated at shake flask scale, before two strains were selected for scale up to 7L fermentation scale. The results obtained indicated that the host strain plasmid combination can heavily influence both the quantity and quality of plasmid DNA obtained and this behaviour cannot simply be determined by looking at the host strain genotype. Fermentation runs on the two strains selected for scale up (BL21 DE3 gWiz and HB101 gWiz) demonstrated that these two strains scale up very well, maintaining high specific pDNA yields (1.5mg/L/OD for BL21 DE3 gWiz) and high SC-DNA yields (98% for HB101 gWiz). Temperature amplification studies using strains harbouring pUC18 have shown that although most strain-plasmid combinations yielded more plasmid at a higher temperature of 40°C, the extent of this increase is highly influenced by the host strain. Indeed in some cases, such as for the