Plasmid-based gene therapy and vaccination require the production of purified plasmid DNA. There is a current understanding that supercoiled plasmid DNA is the best form for administration and FDA guidelines state that a specification for the minimum level of supercoiled plasmid DNA, in the final product, should be made. Currently, plasmids of 10 kb or smaller are being used in gene therapy trials, but if plasmid-based vaccination is to become a reality then much larger plasmids, with the ability to carry multi-variant genes, will be required, this raises questions of feasibility of production. This study examined two main issues. Firstly a fed-batch strategy to maximise the level of supercoiling of a 6.9 kb plasmid resulting from fermentation of a recombinant Escherichia coli strain was developed. Secondly issues relating to the production of larger plasmids, for gene therapy and vaccination, were examined. A fed-batch fermentation strategy for the production of a 6.9 kb plasmid, pSV, in E. coli DH5, on a semi-defined medium, was developed. Starvation of amino acids was shown to induce plasmid amplification and in batch fermentation a maximum biomass of 3.5 g/L dry cell weight (DCW) and maximum plasmid yields of 40 mg/L and 11.3 mg/g DCW were achieved. However, amplified plasmid levels were not maintained. After 31 h 90% of plasmid was in the supercoiled form but only remained so for 1 h. Maximum plasmid yield and maximum plasmid supercoiling did not occur simultaneously. Hence a strategy to delay amplification was investigated. A dual feeding strategy consisting of a linear amino acids feed and an exponential D-glucose feed was employed. In fed-batch culture a mean maximum biomass of 4.9 g/L dry cell weight and mean maximum plasmid yields of 44 mg/L and 9.1 mg/g DCW were achieved. 90% of plasmid was in the supercoiled form after 25 h and remained at this level until harvest at 35 h. An important consideration in the production of large plasmids is the ability to supply sufficient oxygen to the fermentation. E. coli DH5 harbouring either the plasmid pBGS18, a 4.4 kb pUC-based plasmid, or pQR150, a 20 kb derivative of pBGS18, was grown in D-glucose-limited chemostat culture to investigate the effects of plasmid size and recombinant protein production on oxygen demand. Under conditions where no recombinant protein was expressed the cellular oxygen demand of the two strains was not significantly different. When recombinant protein was expressed cells harbouring pBGS18 demonstrated a statistically insignificant increase in mean specific oxygen uptake rate while those harbouring pQR150 demonstrated a statistically significant increase. It was concluded that plasmid size does not significantly affect oxygen demand. The increase in oxygen demand reported with an increase in plasmid size by other researchers is due to production of recombinant protein. The production of a 116 kb plasmid, p5176, in E. coli DH10 on complex and semi-defined media was investigated. Fermentations on complex medium were poorly reproducible while those on semi-defined medium were highly reproducible. Maximum plasmid yields were comparable between the two media, while higher biomass yields and lower oxygen requirements were observed with semi-defined medium. Lysis and primary recovery were investigated and indicated that, using current methods, the manufacture of a large plasmid product may be difficult. A fed-batch fermentation strategy which allows an increased yield of supercoiled plasmid DNA has been developed. It has been established that oxygen supply is not an issue in the production of large plasmids and a base for the production of large plasmids has been established for future work.

Not all DNA has the familiar Watson-Crick, right-handed double helical structure. Left-handed helixes, triplexes, quadruplexes, bent DNA and regions of increased stiffness have all been encountered in nature. Some of these DNA structures are formed as a result of protein binding, while others are intrinsic. Such intrinsic structures may be incorporated into future plasmid constructs for gene therapy and DNA vaccine products. Intrinsic DNA structures were included at a defined point in a 2.9 kb plasmid, and their effects on cell growth rate, total plasmid yield, and topology (i.e. the relative proportions of supercoiled plasmid, open circular and linear forms), were determined. The stability of the inserted sequences was assessed using gel electrophoresis. Results suggest that Z-DNA is unstable in a batch Escherichia coli DH1 production system grown in complex medium. Encouragingly other sequences studied (triplex, bend and quadruplex) did not cause spontaneous deletions, and no detrimental effect was found on growth rate or on total plasmid yield indicating that such sequences could be included in future DNA products without any detrimental effect on plasmid yields. Although the intramolecular triplex studied significantly decreased the proportion of supercoiled species. The effect of different topological forms on transcription of a DNA vaccine in a cell free system was investigated, in order to determine the implications of this on specifications of plasmid products. Open circular plasmid was found to be expressed around 3.5 fold more than the supercoiled form. Current guidelines suggest that plasmid products should contain a minimum of 90% supercoiled species. The supercoiled form is not a single isoform, but contains plasmids with different numbers of supercoils (linking number). Measurement of the levels of supercoiled species in comparison to the other plasmid forms allows monitoring of the production process, however it is recommended that the guidelines be set on individual product basis i.e. drug efficacy will depend on delivery method, longevity and transcription levels.

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