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
Fermentation Feed Strategy Options
The most straightforward feed strategy is a defined growth rate strategy. When using a defined growth rate, feed media is
added at rates as determined by a pre-established growth profile. In our process, the feed is triggered by a DO2 spike, which is caused by the exhaustion of the initial bolus of glucose in the media. Following this initial spike, the
program feeds media based on a predefined feed profile.
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.