Selective expression of the soluble product fraction in Escherichia coli cultures employed in recombinant protein production processes

Abstract

Recombinant proteins produced in Escherichia coli hosts may appear within the cells' cytoplasm in form of insoluble inclusion bodies (IB's) and/or as dissolved functional protein molecules. If no efficient refolding procedure is available, one is interested in obtaining as much product as possible in its soluble form. Here, we present a process engineering approach to maximizing the soluble target protein fraction. For that purpose, a dynamic process model was developed. Its essential kinetic component, the specific soluble product formation rate, if represented as a function of the specific growth rate and the culture temperature, depicts a clear maximum. Based on the dynamic model, optimal specific growth rate and temperature profiles for the fed-batch fermentation were determined. In the course of the study reported, the mass of desired soluble protein was increased by about 25%. At the same time, the formation of inclusion bodies was essentially avoided. As the optimal cultivation procedure is rather susceptible to distortions, control measures are necessary to guarantee that the real process can be kept on its desired path. This was possible with robust closed loop control. Experimental process validation revealed that, in this way, high dissolved product fractions could be obtained at an excellent batch-to-batch reproducibility.

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

“…Hence, the process becomes highly reproducible as demanded from the product quality point of view. As these processes are deterministic in nature, a high reproducibility in the biomass concentration profiles will lead to a high reproducibility in other state variables, e.g., the product profiles as well, as was already found in microbial systems (Gnoth et al 2010). …”

Improving cultivation processes for recombinant protein production

“…The complexity of the model depends on the active knowledge of the user. Here we took the quite general model described in detail by Gnoth et al [7]. The essential point with respect to a virtual plant, however, is Fig.…”

“…Initial medium and feed solution are described in detail by Gnoth et al [7]. Respiration was monitored via exhaust gas analysis in the vent line, i.e., O 2 was measured on-line with a paramagnetic oxygen sensor (Maihak Oxor 610) while CO 2 was detected with an infrared detector (Maihak Unor 610).…”

“…The process under consideration was modeled with a set of ordinary differential equations based on the mass balances of biomass, substrate, acetate and product [7].…”

“…the fermentation run can tightly be kept on its predefined tracks for various recombinant protein production processes [2,7,9]. However, in some situations stability problems might appear when such a controller does not react fast enough on deviations from the set point profile.…”