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March 01, 2009 (Vol. 29, No. 5)

Low Metabolic Burden Plasmid Production

Nature Technology’s DNA Manufacturing Platform Tackles Instability and Toxicity Issues

  • 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.

  • Antibiotic-Free Selection

    Click Image To Enlarge +
    Figure 1. NTC antibiotic-free selection

    High-copy plasmids can impose a metabolic burden on their hosts, giving plasmid-free cells an advantage in culture. Consequently, even a small number of plasmid-free cells can quickly overtake an entire fermentation in the absence of selection. Plasmid-free cells can be controlled by using a selectable marker in the plasmid. Antibiotic-resistance markers, typically kanamycin resistance (KanR), allow selective retention of plasmid DNA during bacterial fermentation and are the most commonly utilized selectable markers. 

    To ensure safety, however, regulatory agencies recommend elimination of antibiotic-resistance markers from therapeutic and vaccine plasmid DNA vectors. The presence of an antibiotic resistance gene in the plasmid backbone is considered undesirable by regulatory agencies, due to the potential transfer of antibiotic resistance to endogenous microbial flora and the potential activation and transcription of the genes from mammalian promoters after cellular incorporation into the genome.

    Elimination of antibiotic resistance from the plasmid offers additional advantages, including elimination of potential metabolic stress caused by expression of the antibiotic marker gene product, and increased therapeutic potency if removal of the antibiotic marker results in a reduced plasmid size (e.g., the KanR gene is ~1 kb). Further, the use of antibiotics in fermentation culture requires expensive process validation of antibiotic removal during plasmid purification, to prevent contamination of the final product with residual antibiotics.

    Nature Technology has designed an antibiotic-free selection system (Figure 1). Vectors with this selection system incorporate and express a 150 bp RNA-OUT antisense RNA. RNA-OUT represses expression of a counter-selectable marker (SacB) from the host chromosome. SacB encodes a levansucrase, which is toxic in the presence of sucrose. Plasmid selection is achieved in the presence of 0.5% sucrose.

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