DNA vaccines, wherein a new antigen is inserted into a validated vector, have tremendous potential for deployment in pandemic applications as they can be rapidly produced in a validated, fermentation-purification process.
For this application, it is essential that the vector and fermentation process function with a variety of different antigen genes. Antigen genes may be unpredictably toxic or otherwise low yielding in standard fermentation processes, however. Careful consideration of host strain, plasmid, and production process factors have led to innovations that allow Nature Technology to routinely achieve high plasmid yields, even with plasmids known to be toxic or unstable when using standard production methods.
Several studies on various plasmid host strains indicate that plasmid yield and quality are significantly affected by the choice of host strain. Together, these studies also demonstrate that plasmid production in shake flasks is poorly predictive of plasmid production in fermentation, and that a strain’s performance can be affected by the fermentation process. In general, recA, endA, and relA mutations in a host strain are beneficial for plasmid production. We have found that DH5a is a good host strain to use as it consistently produces high-quality plasmid DNA, even with difficult plasmids.
Plasmid copy number within a strain is largely set by vector-intrinsic factors. Gene therapy or DNA vaccine plasmids typically contain either the pUC or pMM1 temperature-sensitive origin of replication. This temperature sensitivity is especially useful for inducing high-yield plasmid production in fermentation. Standard ROP-minus replication origins derived from pBR322 have also been used, but have much lower copy number than pUC vectors, and consequently lower fermentation yields.