Effects of medium composition on the production of plasmid DNA vector potentially for human gene therapy

Abstract

Plasmid vector is increasingly applied to gene therapy or gene vaccine. The production of plasmid pCMV-AP3 for cancer gene therapy was conducted in a modified MBL medium using a recombinant E. coli BL21 system. The effects of different MMBL components on plasmid yield, cell mass and specific plasmid DNA productivity were evaluated on shake-flask scale. The results showed that glucose was the optimal carbon source. High plasmid yield (58.3 mg/L) was obtained when 5.0 g/L glucose was added to MMBL. Glycerol could be chosen as a complementary carbon source because of the highest specific plasmid productivity (37.9 mg DNA/g DCW). After tests of different levels of nitrogen source and inorganic phosphate, a modified MMBL medium was formulated for optimal plasmid production. Further study showed that the initial acetate addition (less than 4.0 g/L) in MMBL improved plasmid production significantly, although it inhibited cell growth. The results will be useful for large-scale plasmid production using recombinant E. coli system.

Scalable Technology to Produce Pharmaceutical Grade Plasmid DNA for Gene Therapy

“…In addition, other authors suggest that the plasmid yields obtained from culture were highest into stationary phase. High specific growth rates are associated with acetate production, plasmid instability and lower percentages of supercoiled plasmid overall (Xu et al, 2005). The FDA recognizes that open-circle, linear and nicked forms may be fewer effectives therapeutically than supercoiled DNA.…”

“…It was reported that the media composition affected cell specific growth rate, and thus influenced in plasmid copy number. Therefore, the effects of medium components on cell growth and plasmid productivity should be evaluated (Xu et al, 2005). Media composition can dramatically affect yield and consequently the overall cost of production.…”

“…The trend toward testing DNA vaccines as part of combination trials can be seen in both the large vaccine producers like Merck, Aventis Pasteur and Wyeth as well as in smaller research companies (Powell, 2004). Although the processes of production for many genetherapy vectors have been developed in pharmaceutical companies, the information of large-scale pDNA production is scarce and usually not available to the scientific community (Xu et al, 2005).…”

Biomedical application of plasmid DNA in gene therapy: A new challenge for chromatography

“…Normally, the effects of the components in a medium on cell growth and plasmid production should be evaluated to solve the trade-off between higher copy number and reduced growth rate. Acetate accumulation is also a major concern for medium design and high density fermentation of recombinant E.coli because it inhibits cell growth and protein expression (Xu et al 2005). Nevertheless, sometimes acetate addition improves pDNA yield in the experimental system.…”

“…Nevertheless, sometimes acetate addition improves pDNA yield in the experimental system. This suggests that low rates lead to high specific pDNA yield provided the cell growth is not seriously inhibited by acetate (Xu et al 2005). The use of reduced growth rate is the unifying principle in high quality, high yield plasmid fermentations.…”