Fed-Batch Fermentation for eGFP Production in E. coli

Shannadora Hollis, Andria Armstrong, Jake Davis, Brittany Lanier

North Carolina State University, Department of Chemical and Biomolecular Engineering

North Carolina State University’s Biomanufacturing Training and Education Center (BTEC) will begin to offer courses in large-scale biomanufacturing. As part of the course, students will produce enhanced Green Fluorescent Protein (eGFP) by fermentation of recombinant E. coli. Batch operations are currently in place. However, such processes are limited by the oxygen transfer and accumulation of acetate; an undesirable by-product that subsequently results in lower product yields.

Experiments have shown that controlling the growth rate below the maximum specific growth rate by limiting glucose will prevent oxygen limitation and organic acid production. Controlling the feedrate of glucose throughout fermentation allows E. coli to grow to higher cell densities and thus facilitates an increased eGFP yield. Therefore, we propose to design a fed-batch fermentation process that limits glucose.

We have investigated this alternative by designing a process model created from experimental process development data and specifications provided by BTEC. The model is based on a feedback control algorithm for the glucose feeding strategy. A supplemental control scheme for the glucose feed using measurements of carbon dioxide in the fermenter off-gas has also been considered. The resulting fed-batch design not only reduces undesirable acetate byproduct formation, but also leads to substantial eGFP yield and cell growth. The process control scheme also achieves the target harvest goal in the required time.

Fed-batch processes are very common and widely used in industry due to their success in obtaining high cell density cultures during the fermentation of E. coli. Processes such as the one designed are critical in the development of biotechnology-derived products, which have had an increasing market demand over the last two decades. As the demand for such products continues to rise, a need for scale-up of therapeutic proteins such as eGFP will likely ensue, and a process such as the one proposed in this work will prove to be an excellent training tool for those aiming to gain experience in this very relevant biomanufacturing process.