(378g) Process Design for Plasmid DNA Production Using Microbioreactors

When designing a biopharmaceutical production process, a trade-off exists between the use of shake flask cultures versus bench-top bioreactors for process development. Shake flasks are relatively high throughput, but lack the monitoring and control provided by bioreactors, which are in turn more resource and time intensive. Micro-scale bioreactors (?microbioreactors?) have the potential to combine the respective advantages of shake flasks and bioreactors in a single system. Many designs of microbioreactors have been reported in the literature for the cultivation of E. coli cells, but there have been very few applications of this technology to industrially-relevant processes. We have been using microbioreactors to study plasmid DNA production in E. coli for gene therapy and DNA vaccine applications. High-yield plasmid production generally occurs via a fed-batch process and is often induced by a temperature shift, providing complexities that can be used to demonstrate the utility of microbioreactors as a process development tool.

We have developed microbioreactors with a 1-mL working volume that contain sensors for online measurement of optical density, pH, and dissolved oxygen concentration. Temperature can also be controlled and modulated by the reactors. We have shown that the growth profile of a plasmid DNA production strain of E. coli is very similar in the microbioreactor and a shake flask (50-100 mL working volume) in rich culture medium. We have also demonstrated that temperature-inducible amplification of a pUC-based plasmid can be replicated across these scales. Plasmid DNA yield was measured from microliter-scale culture samples using a quantitative PCR assay we developed. We are currently working to run the microbioreactors in a fed-batch configuration to determine how well they mimic a bench-scale fed-batch plasmid production process.