Growth Rate Options
High growth rates, close to the maximum theoretical growth rate of E. coli in glucose-based media, were used in some early experiments. High growth rates often result in overfeeding, which may in
turn result in detrimental acetate accumulation in the culture.3 Several exponential growth rates were evaluated with the best results observed at lower growth rates. This is to be expected
for a number of reasons. There is little risk of substrate overfeeding with lower growth rates because the feed is added at
a controlled rate that has been optimized such that glucose is consumed quickly upon addition. This minimizes the risk, allowing
waste products (such as acetate) to accumulate to levels that may become detrimental to growth. Lower growth rates also have
been shown to allow plasmid replication to synchronize with cell division resulting in higher percentages of supercoiled plasmid
and better plasmid stability. Both defined and feedback-controlled growth rates were evaluated. For typical plasmids, less
than 10 kB in size, production levels often exceed 1 g/L. In the production of larger plasmids, fed-batch cultures are grown
to lower cell densities (A600 = 40–80) because slower growth rates are often required for optimal production. Yields can be
much lower (75–100 mg/L), but are 50–80 times higher than what is achieved in batch fermentation. Moreover, there are several
commercial advantages in using a lower growth rate. Our HCD strategy uses a lower growth rate that is timed for a single shift
operation.
Specifications
Table 1. The high cell density process met specifications in all assays
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In addition to achieving goals such as increasing cell density and plasmid yield per gram of cell paste, the HCD process that
we have developed has successfully demonstrated that it can also maintain plasmid quality. Specifically, our process has shown
that it can meet standard release specifications for clinical and commercial grade products (Table 1).
Conclusion
A number of parameters have been evaluated in adaptation of a high cell density process for plasmid DNA production. In the
evaluation, special consideration was made to ensure that the quality of the plasmid DNA was not compromised. It was established
that industry and regulatory specifications may be achieved using the HCD process. This objective was balanced with the need
to develop a cost-effective process that can be used to produce larger quantities of these products that will be required
at commercial launch. A fed-batch fermentation strategy using minimal media fed at a predefined rate produced the highest
yields without compromising quality.
Marvin Peterson, PhD, is the director of biologics manufacturing and Bill Brune is a senior process engineer at Althea, San Diego, CA, 858.882.0123, info@althea.com
References
1. US Food and Drug Administration. Guidance for industry. Guidance for human somatic cell therapy and gene therapy. Rockville,
MD; 1998 Mar.
2. O'Kennedy RD, Baldwin C, Keshavarz-Moore E. Effects of growth medium selection on plasmid DNA production and initial processing
steps. J Biotechnol. 2000 Jan 21;76(2–3):175–83.
3. Thatcher DR, et al. Method of Plasmid DNA Production and Purification. US Patent 6,503,738 (Cobra Therapeutics, Ltd.,
Keele, UK) 2003 Jan 7.
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