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


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.

BioPharm International
Volume 25, Issue 7


High cell-density fermentation requires a balanced medium that supplies adequate amounts of nutrients needed for energy, biomass, and cell maintenance and that commonly contains carbon and nitrogen sources, various salts, and trace metals. By employing this approach, Ruiz and collaborators in 2009 designed a culture medium according to bacterial element composition (10). Using that methodolgy, fed-batch fermentation was scaled up to 50 L in this study.

The scale of the fermenter, together with the expected biomass yield and product content, are key parameters in designing a manufacturing process, if the amount of product required is already known. The primary scale-up criterion of the process should be selected based upon the transport property most critical to the performance of the process. If oxygen transfer is the limiting factor, then scale up by equal P/V will be essential. This method is adopted for many authors using larger-scale fermentors, such as these below 1000 L capacity (8).

Table I: Reactor geometries for 5-L and 50-L fermentation process.
Current scale-up methods assume that, as in a small-scale fermentor, the environmental conditions are homogeneously distributed within the large-scale fermentation,. However, there are many factors, such as hydrodynamics, height, and geometric configuration of the reactor (see Table I) that can affect the environment of the fluid in large-scale reactors (8). Moreover, the suitability of scale-up methods is usually confirmed by experimental results.

Physical and chemical parameters remain constant over the scale and in some cases are optimized on a laboratory scale. The physical properties of the culture medium are:

  • Temperature = 37 °C
  • Density (ρ) = 1050 kg m-3
  • Acidity pH) = 7
  • Viscosity (μ) = 10-3 Pa·s

Scale-up of plasmid DNA (pIDKE2) production in E. coli from laboratory scale to pilot scale was carried out keeping the scaling criterion (P/V) constant. Large-scale manufacturing was completed using a 50-L Marubishi fermentor; relevant reactor geometric data are shown in Table I. The height of the liquid was calculated ifor the Marubishi 5-L fermenter assuming a flat bottom using equation (1) and for a 50-L fermenter with an ellipsoidal bottom using equation (2).

The fermenters generally met the geometric relationships in equation 3:

The correction factor for each scale was calculated using the following expression:

When taking into account the geometrical parameters, leading to one base of a fermentor with standard dimensions, and including the amount and type of propellant and the correction to be agitated and aerated fermenter, whereas the Re ≥104 and the Re power number (Np)=6, the expression for the calculation of agitation on the upper scale would be (14):

When the values from Table I are substituted into equation 5, the following expression is obtained:

Assuming that the number of aeration is constant for both scales, the air flow to each fermenter can be calculated employing equations 7 and 8:

Table II: Calculated parameters for 5-L and 50-L fermentation process.
The values of the operating parameters resulting from the calculation for 50 L are shown in Table II. Pilot plant scale-up is dominated by empirical criteria requiring geometric similarity, which is rarely achievable in practice, but which is necessary for adequate correlation of the biological responses of cells to the effects of changing scale.

Table III: Results of the final fermentations in 5-L and 50-L fed-batch cultures.
The results of final biomass concentration, pDNA concentration, specific pDNA yield, volumetric pDNA yield, and percent of plasmid supercoil show that there is no significant difference in fermentation between small and large bioreactors carried out in fed-batch condition (see Table III). Because several studies have revealed that supercoiling linking number would be an important factor to consider when processing pDNA for therapeutic use, we analyzed the percentage of super coils at 24 h of culture, and it was maintained around 90% of total plasmid DNA (see table III) in both scales (4).

Figure 1: Cell growth kinetics of recombinant Escherichia coli DH10B transformed with pIDKE2 in the design medium using fed-batch fermentation process at 5-L and 50-L scale. DCW is dry cell weight. (ALL FIGURES ARE COURTESY OF THE AUTHORS)
The process characterization at 5-L and 50-L scale (see Figure 1), have shown that cell density increased to the logarithmic phase between 1–21 h and to the stationary phase at around 22–24 h. Figure 1 shows that carbon source exhaustion in both scales occurred after 5 h of batch growth. This depletion was indicated by an increase in pH, which was caused by the consumption of alternative carbon sources, and was confirmed by a reducing-sugar assay. Precisely at this time, a mixture of glucose and yeast extract was fed at constant flow and glucose accumulation was observed only when feeding started.

During the initial batch portion, a specific growth rate (μ) of 0.60/h was achieved while a lower μ= 0.062/h was maintained during the fed batch portion. It has been reported that the low growth rate leads to a high specific plasmid DNA yield then improved plasmid DNA yield if the cell growth was not inhibited (10).

Figure 2: Effects of culture strategy and scale on pDNA production and cell growth.
Final biomass concentration and specific pDNA yield were increased in comparison with cultures grown on a standard laboratory medium (TB) in batch mode, as has been reported by some authors (15, 16) (see Figure 2).

Maintaining a robust scale-up with a consistent impurity profile was important for implementation of this process. The fermentation provides the primary control for minimizing unwanted impurities, which greatly enhances the efficiency of the downstream separation of plasmid DNA. This fermentation process is very easy to scale up and has been used to provide plasmid yields that are becoming acceptable from a manufacturing viewpoint.

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