The synthesis of supercoiled plasmid DNA (SC‐pDNA) for therapeutic use will involve large‐scale production in bioreactors. The success of these fermentations will be dependent on the interactions between the host organism, the recombinant plasmid vector and the growth environment. In the present study, the recombinant host, Escherichia coli DH5α bearing the recombinant plasmid pSVβ, was grown in shake flasks, batch and exponentially fed‐batch bioreactors. Specific and volumetric pDNA yields were increased 8‐ and 25‐fold respectively using exponentially fed‐batch cultures in comparison with shake‐flask cultures. The percentage of SC‐pDNA as a proportion of total plasmid DNA decreased over time in batch cultures, but remained relatively constant during fed‐batch cultures. The relative merits of different modes of fermentation and their effects on the quality of alkaline lysate extracts of pDNA with respect to genomic contamination and the percentage of SC‐pDNA are discussed.