Processes yielding >500 mg plasmid DNA/L have previously been reported by Merck, Boehringer Ingelheim, and Nature Technology (NTC). Common features among these high-yield processes include the use of fed-batch fermentation with high copy number plasmids.
Due to the controlled addition of nutrients, much higher cell densities can be achieved with a fed-batch process (as opposed to a batch process), and specific growth rates can be reduced, which generally results in increased plasmid copy number.
Commonly used therapeutic and DNA vaccine vectors also contain a high copy pUC-type origin of replication that results in increased copy numbers at temperatures above 30°C. Selection of host strain also has a strong influence on the quality and quantity of plasmid produced.
Host strains are generally K-12 derived and should be recA- to avoid homologous recombination, and endA- to avoid plasmid nicking by the nonspecific endonuclease during purification.
Popular plasmid host strains that meet these requirements include DH5α, DH10B, and XL1-Blue. Stabilizing host strains are commercially available for production of unstable plasmids. However, in our experience such strains often result in lower plasmid yields.
Nature Technology’s HyperGRO™ process is an inducible fed-batch fermentation process consisting of a cell bank and fermentation process that reduce plasmid-mediated metabolic burden, enabling high yield production of optimized plasmids, as well as successful production of previously known unstable or toxic vectors.