DNA-Vaccine Production With E.coli In High Cell Density Culture

Abstract:
Plasmid DNA is commonly produced using Escherichia coli strains as host. The use of fedbatch mode for plasmid production will then involve the common challenges observed for aerobic high-cell density cultures at large and pilot-scales: presence of environmental gradients and high oxygen demand among others. An approach for transferring enough oxygen to the culture broth is to enhance the mass transfer capacity of the bioreactor by increasing the head-space pressure. For this reason this study wants to show a pDNAdevelopment above the limit of biomass which is reached in conventional (industrial) bioreactors.

Therefore E. coli DH5α has been transformed with the plasmid pCMV-S. It contains a sequence encoding the Hepatitis B surface antigen under control of the cytomegalovirus promoter. Consequently, the antigen is not expressed in E. coli. Hence this plasmid can be used as an appropriate model of a DNA vaccine.

In this case the development of pDNA-concentration should be investigated using fed-batch processes with different carbon sources in feed solution to reach high cell density. To avoid irreproducible effects on plasmid production caused by anaerobic metabolism and the formation of byproducts an exponential feeding strategy with a constant growth rate (μ = 0.15 1/h) was chosen (see FIG. 1).

In this study maximum dry cell weight concentration of 79 g/L and 85 g/L for feeding glucose solution and glycerol solution could be detected. Thereby maximum oxygen transfer capacities of 0.506 mol/(L*h) for using glucose feed and of 0.784 mol/(L*h) for using glycerol feed were reached. Final values for pDNA-concentration were 80 mg/L (glycerol feed) and 215 mg/L (glucose feed) as shown in FIG. 1.

The pDNA-concentration reaches different levels after starting the feed (9 hours) depending on the carbon source in the feed solution (glucose or glycerol) though a comparable trend in growth could be observed. In comparison to the glycerol feed the pDNA-yield at the end is approx. three times higher using a glucose feed. FIG. 1: Development of dry cell weight and pDNA-concentration with different feed carbonsources