Production of Unstable Plasmids
Based on our data, alternative high-yield plasmid production processes, which use 37ºC continuously, are unlikely to perform well with many unstable or suboptimal plasmids due to increased metabolic burden. For example, pVLTrap, a 6.7 kb retroviral plasmid vector containing two long terminal repeats (LTRs, known to cause stability problems) was produced successfully in DH5α at 785 mg/L using the aforementioned low metabolic burden fermentation process.
When grown under identical conditions, but at 37ºC, the region between the LTRs had been entirely deleted by the end of the fermentation (Figure 3), and the plasmid yield only reached 214 mg/L. Thus, the use of the low metabolic burden process facilitates production of otherwise unstable plasmids in high-yielding strains, such as DH5α, eliminating the need to use specialized stabilizing host strains.
Importantly, maintaining a low metabolic burden should begin as early as the transformation process. This is especially true with toxic and unstable plasmids. Numerous inserts can confer low-yield plasmid production on an otherwise high-yielding plasmid backbone. Propagation of cultures at 30°C, rather than 37°C, for manufacturing glycerol stocks can dramatically improve yields when producing plasmids containing toxic inserts.
This has been demonstrated by the following experiment: Three DNA Vaccine plasmids containing influenza hemagglutinin (HA) genes from H1, H3, or H5 serotypes were evaluated. Two of these genes, from H1, and H3 serotypes, are inserts known to lower plasmid yield. Fermentation yields from DH5α glycerol stocks of these DNA vaccine plasmids, manufactured at either 30ºC or at 37ºC, are summarized in the table.
Glycerol stock viability was determined after >1 week at -80ºC storage. Application of reduced-temperature glycerol stock production, coupled with the inducible fermentation process, solved the problem of manufacturing difficult plasmids, and resulted in successful production of 3/3 plasmids compared to only 1/3 using standard transformation at 37ºC.
Nature Technology’s temperature-inducible fermentation process has been successfully scaled up to 300 L and has been used for GMP production of DNA vaccine plasmids. Because of the ability to produce previously unstable and toxic plasmid DNA, as well as optimized plasmids at high yields, this low metabolic burden process is ideal as a generic plasmid DNA production platform.