Physiological capacities decline during induced bioprocesses leading to substrate accumulation

During the cultivation of E. coli for recombinant protein production, substrate accumulation is often observed in induction phase. Uncontrolled substrate accumulation leads to difficulties in transferring or scaling processes and even to failed batches. The phenomenon of metabolite/substrate accumulation occurs as a result of exceeding the physiological capacity to metabolize substrate (q_Scrit ). In contrast to the common understanding of q_Scrit as "static" value, we hypothesize that q_Scrit essentially has a dynamic nature. Following the state of the art approach of physio logical strain characterization, substrate pulse experiments were used to quantify q_Scrit in induction phase. The q_Scrit was found to be temperature and time dependent. Subsequently, q_Scrit was expressed through a linear equation, to serve as boundary for physiologically controlled experiments. Nevertheless, accumulation was observed within a physiologically controlled verification experiment, although the q_Scrit boundary was not exceeded. A second set of experiments was conducted, by oscillating the q_S set point between discrete plateaus during physiologically controlled experiments. From the results, we deduced a significant interrelation between the metabolic activity and the timely decline of qScrit. This finding highlights the necessity of a comprehensive but laborious physiological characterization for each strain or alternatively, to use physio logical feedback control to facilitate real time monitoring of q_Scrit , in order to effectively avoid substrate accumulation.