Fermentation Options

Once the vector has been designed and the host cell line selected, one of the first decisions that must be made is whether to initiate production with batch, fed-batch, or continuous fermentation processes. With the demands for plasmid DNA in late clinical trials approaching the 100-gram scale or greater , a batch fermentation, with typical yields of 10–20 mg/L, becomes unfeasible. Limitations are caused by uncontrolled growth rates and waste product accumulation in this mode. A better choice for avoiding these issues and thereby increasing productivity is the use of fed-batch or continuous high cell density fermentation. Continuous fermentation processes are conducive to the production of large amounts of a single product but maintaining sterility is a major challenge. Fed-batch fermentation, which starts with a short batch fermentation followed by the addition of media at a defined rate, offers many advantages for DNA production, particularly in circumstances where many vector types are produced. Fed-batch offers more flexibility and consistency than batch production. Specifically, it allows for relatively simple optimization of fermentation profiles for each plasmid DNA product. Even with modifications to fermentation profiles from plasmid to plasmid, this feed strategy produces controlled cell growth results that are inherently more consistent and predictable than batch fermentation. Scale-up from process development activities to production of commercial quantities is a fairly streamlined process.

Fermentation Feed Strategy Options

Media Options

Media composition can dramatically affect yields and consequently the overall cost of production. Options vary greatly, including minimal (defined) media to complex (semi-defined). Complex media formulations often contain ingredients like yeast extract, peptones, and other growth factors that may allow for a higher cell density, but may present challenges with reproducibility and with contaminant removal in downstream processing. Minimal media contains known quantities of essential nutritional components including a carbon source, a nitrogen source, and salts, and excludes components known to be inhibitory to bacterial growth. Fermentation processes using minimal media are highly reproducible and plasmid copy number may even be higher when using minimal media.² Our process uses a minimal media for its high cell density manufacturing processes. Minimal media also allows for adjustment and optimization through the addition of components that may increase yields. Glucose is routinely used in E. coli fermentations and was chosen as the carbon source. It is easily obtained in a purified form and easy for the E. coli to metabolize. A nitrogen source and trace elements are also required for bacterial growth, metabolism, and enzymatic reactions. Particular consideration has been made in eliminating the use of any animal-derived components or any genetically modified organisms to alleviate potential regulatory concerns and to comply with current US and European Union regulatory recommendations. Source documentation for all media components including certificates of analysis and certificates of compliance should be available for review and regulatory submission.