Scale up of Fed-Batch Culture to Produce Plasmid DNA in Escherichia coli (Peer Reviewed)

The authors present scale-up from a 5-L fermentor to a 50-L pilot-scale using the criterion of constant power consumption per unit liquid volume.


Scale-up from a 5-L fermentor to a 50-L pilot-scale fermentor was carried out successfully using the criterion of constant power consumption per unit liquid volume (P/V). Fed-batch mode of cultivation using constant feeding of glucose and yeast extract mixture was employed for the production of plasmid DNA in Escherichia coli. Results showed that final biomass concentration and specific plasmid DNA yield were similar between small and large bioreactors.

Gene therapy and DNA immunization are promising possibilities for the prevention, treatment, and cure of various diseases (1, 2). In general, DNA-based vaccines are considered safe due, in part, to the lack of genetic integration, and to the absence of a specific immune response to the plasmid itself (3). This process requires considerable amounts of plasmid DNA (pDNA) that should be homogeneous with respect to structural form and DNA sequence (4). In spite of considerable scientific effort over the past few years, no gene-therapy product has yet reached the market.

For the production of large quantities of pDNA, an efficient fermentation process needs to be established. Optimization of the fermentation conditions of Escherichia coli (E. coli) for pDNA production could be fundamental, however, experimental data are limited compared with the extensive recombinant protein production literature. However, some general rules and methodologies pertaining to the production of pDNA by cultivation of E. coli are beginning to emerge (1).

A number of recent reports discuss fermentation strategies used for production of pDNA, but have not addressed the effect of fermentation conditions on the quality of the resulting pDNA. Because the location of pDNA is intracellular, productivity is proportional to the final cell density and the specific productivity (i.e., the amount of pDNA per unit cell mass) (5).

A feature of fermentation technology for large-scale plasmid production is the performance of high-density culture to obtain large amounts of biomass. Fed-batch fermentation provides higher biomass yields than batch fermentation because substrate is supplied at a rate such that it is nearly completely consumed, so nutrients are delivered over an extended period of time (6).

When a molecular biologist thinks of large-scale pDNA production, the range of 10–100 mg of DNA usually comes to mind. However, at pharmaceutical production-scale, pDNA requirements may exceed 50 g per batch. In extreme cases, many kilograms of pDNA per year may be needed to fill the demand for DNA vaccines currently in clinical trials (1).

The transfer from research-scale technology to manufacturing-scale requires management of the scale-up process. Scale-up is not just a simple multiplication of relevant factors, but instead requires skill, time investment, and incurs cost (7).

Many widely used fermentation processes were successfully scaled up on the basis of a constant volumetric oxygen transfer coefficient (KLa) and power consumption per unit volume (P/V). The use of traditional empirical methods, such as P/V leads to an increase in mixing and circulation times at large scale. In addition, high oxygen demands and high viscosity can cause concentration gradients in oxygen, shear, and pH which can have a significant impact on fermentation yield (8). Therefore, the choice of scale-up criteria in not an easy task, given the potentially sensitive and diverse responses of cells to each of the transport phenomena influenced by impeller design, system geometry, scale, fluid properties, and operating parameters (9).

In this investigation, scale-up of plasmid DNA (pIDKE2) production from a 5-L fermentor to a 50-L fermentor was carried out using power consumption per unit liquid volume (P/V) constant in a fed-batch process design as demonstrated by Ruiz and collaborators in 2009 (10).

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