Plasmid DNA is used as a cloning vector to deliver recombinant genetic information into microorganisms. Since the 1990s, this principle has also been applied for the delivery of therapeutic genes in gene therapy and genetic vaccination. This non-viral gene delivery is afflicted with fewer safety concerns in comparison to viral systems. Processes for the production of high-quality plasmid DNA at multi-and kilogram scale are necessary to meet the needs of clinical trials as well as future therapeutics. Cell disruption, the separation of structurally-related impurities and analytical techniques for process and quality control are the main challenges for bioengineering. This review summarizes the development in these fields over the past recent years.