An investigation on E. coli host strain influences and strategies to improve supercoiled plasmid DNA production for
gene therapy and vaccination.
Doctoral thesis, UCL (University College London).
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 strains ABLE K, W3110, W1485, a higher plasmid yield was obtained at 37°C. However, as the growth rates of these cultures were not measured, the extent of the accumulation of plasmid DNA due to the effects of the growth rate and/or temperature during the exponential phase of growth is unknown at this time. Similarities to what has been reported as temperature induced runaway plasmid replication have been observed in this study, although no experiments were conducted to confirm whether these observations were indeed as result of runaway replication as defined in the literature. Potential alternative strategies investigated included implementing anaerobiosis to test if these conditions can improve supercoiled plasmid DNA production at fermentation scale, and whether a ‘Quiescent cell expression system’ (a state where chromosomal replication and expression is temporarily shut down but residual proteins remain metabolically active) can be implemented to improve plasmid DNA yields by redirecting resources away from biomass production. The results suggest that under the conditions set in this study, these strategies do not increase plasmid DNA production or the percentage of supercoiled plasmid obtained. In conclusion, the results from this investigation have demonstrated that a highly effective and influential strategy for improving the quality and quantity of plasmid DNA obtained is the initial choice of the host strainplasmid combination. Further improvements can then be obtained through the application of other reported fermentation strategies.
|Title:||An investigation on E. coli host strain influences and strategies to improve supercoiled plasmid DNA production for gene therapy and vaccination|
|Open access status:||An open access version is available from UCL Discovery|
|UCL classification:||UCL > School of BEAMS > Faculty of Engineering Science > Biochemical Engineering > Advanced Centre for Biochemical Engineering|
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