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.

kLa of stirred tank bioreactors revisited.

By means of improved feedback control kLa measurements become possible at a precision and reproducibility that now allow a closer look at the influences of power input and aeration rate on the oxygen mass transfer. These measurements are performed online during running fermentations without a notable impact on the biochemical conversion processes. A closer inspection of the mass transfer during cultivations showed that at least the number of impellers influences mass transfer and mixing: On the laboratory scale, two hollow blade impellers clearly showed a larger kLa than the usually employed three impeller versions when operated at the same agitation power and aeration rate. Hollow blade impellers are preferable under most operational conditions because of their perfect gas handling capacity. Mixing time studies showed that these two impeller systems are also preferable with respect to mixing. Furthermore the widths of the baffle bars depict a significant influence on the kLa. All this clearly supports the fact that it is not only the integral power density that finally determines kLa.

Improving cultivation processes for recombinant protein production.

An new cascade control system is presented that reproducibly keeps the cultivation part of recombinant protein production processes on its predetermined track. While the system directly controls carbon dioxide production mass and carbon dioxide production rates along their setpoint profiles in fed-batch cultivation, it simultaneously keeps the specific biomass growth rates and the biomass profiles on their desired paths. The control scheme was designed and tuned using a virtual plant environment based on the industrial process control system SIMATIC PCS 7 (Siemens AG). It is shown by means of validation experiments that the simulations in this straightforward approach directly reflect the experimentally observed controller behaviour. Within the virtual plant environment, it was shown that the cascade control is considerably better than previously used control approaches. The controller significantly improved the batch-to-batch reproducibility of the fermentations. Experimental tests confirmed that it is particularly suited for cultivation processes suffering from long response times and delays. The performance of the new controller is demonstrated during its application in Escherichia coli fed-batch cultivations as well as in animal cell cultures with CHO cells. The technique is a simple and reliable alternative to more sophisticate model-supported controllers.

Simple and efficient control of CHO cell cultures.

Cell cultures must tightly be kept under control in order to guarantee a sufficiently small variability in the protein product quality. A simple and efficient technique for CHO-cell cultures is presented that allows keeping the viable cell count X(v) and the specific growth rate µ of the cells on predefined trajectories. As X(v) and µ cannot directly be measured online, they are controlled indirectly via the total mass of oxygen consumed. Online values of the latter can precisely be estimated from off gas analysis, i.e. from the O2 volume ratio measured in the vent line and air flow rate measurements. In glutamine-limited fed-batch cultivations, the glutamine feed rate can be manipulated in such a way that the viable cell density and the specific growth rate are kept on predefined profiles for nearly the entire cultivation time. The viability of the cells is not affected by the closed loop control actions. The technique was validated with CHO-cells cultured in a 2.5-L fully instrumented stirred tank bioreactor. It is shown that the controller is able to run the process exactly on predefined tracks with a high batch-to-batch reproducibility. By means of six fed-batch cultivations of CHO cells it was shown that a remarkable reproducibility of viable cell concentration could be achieved throughout 140 h cultivation time.

Increasing batch-to-batch reproducibility of CHO cultures by robust open-loop control.

In order to guarantee the quality of recombinant therapeutic proteins produced in mammalian cell systems, the straightforward approach in industry is to run the processes as reproducible as possible. It is first shown that considerable distortions in the currently operated processes appear when the initial cell density deviates from its nominal value. Small deviations in the initial cell mass may lead to severe deviations from the desired biomass trajectory. Next, it is shown how to design a fed-batch production process in such a way that it is robust with respect to variations in the viable cell density. A simple open loop strategy is proposed for that purpose. Here we show for the first time at animal cell cultures (CHO cells) that by means of an appropriate glutamine feed rate profile F(t), which keeps the specific growth rate of the cells on a predefined value below its maximal value while maintaining the viabilities on a high level, the diverging viable cell count profiles change over into a robust converging set of profiles. The CHO cells used to validate the procedure could be focused to any specific growth rates below µ(max).