Avoiding overfeeding in high cell density fed-batch cultures of E. coli during the production of heterologous proteins

Heterologous protein production often causes a significant metabolic burden in Escherichia coli cells which manifests itself in a substantial decrease in their physiological characteristics such as the maximal specific growth rate on a given substrate, the maximal substrate uptake rate as well as the maximal specific oxygen uptake rate. In high-cell-density cultures, the substrate feed rate must be adapted to this changing capabilities of the cells in order to avoid overfeeding and thus the formation of by-products that inhibit the cell performance further. This requires the precise knowledge about the changes in these specific rates, particularly during the product formation phase. In order to precisely investigate the time profile of the critical specific substrate uptake rate σcrit of microorganisms, i.e. the maximal rate at which the cells can fully oxidize their substrate, a new online tracking technique is presented. The feed rate F is modulated in such a way that the specific substrate uptake rate σ is linearly raised towards its critical value σcrit. When this is reached the feed rate is automatically reduced and the procedure is repeated. In this way the method automatically follows the changing time profile of σcrit during the entire cultivation and avoids significant acetate formation rates. This procedure considerably increases the identifiability of σcrit. The high precision of the technique also results from replacing the pO2 measurements that seem to suggest themselves for monitoring maximal oxygen uptake rate, by measuring the total oxygen consumption rate tOUR, which is available at a much higher signal-to-noise ratio and is not as prone to distortions. An important advantage of measuring tOUR is that it allows keeping pO2 controlled at its optimal value. The applicability of the new tracking technology is demonstrated at E. coli cultures. The resulting σcrit(t) profile allows determining the substrate feed rate profile and other key variables that can be used for advanced feedback control in protein production processes. The technique can be considered a PAT tool and is well suited to industrial fermentations.