AbstractEscherichia coli producing a plasmid DNA (pDNA) vaccine was cultivated in fed‐batch mode at small scale (1 L) using oxygen‐enriched air, and at pilot scale (50 L) using a pressurized bioreactor, to maintain aerobic conditions. In the small scale, the attained oxygen transfer rate (OTRMAX) using an oxygen concentration in the inlet gas of 68.2%, reached 0.42 mol L−1 h−1. The OTRMAX in the pressurized reactor with an overpressure of 8 bar was 0.5 mol L−1 h−1. In the small‐ and pilot‐scale cultivations, the final biomass concentrations (74 and 79 g/L, respectively), pDNA concentrations (236 and 215 mg/L), overall productivity and pDNA topology were very similar. Therefore, the pressurized cultivation is a viable option to scale up pDNA production processes.