The strain that was used in the study exhibited excellent protein expression in the first four to six hours after induction.
Afterwards, no notable target protein formation was observed. The researchers speculated that factors affecting continued
expression were inadequate for growth rate caused by the linear feeding profile, genetic instability, or missing medium additives.
Genetic instability was eliminated as a possible cause with the help of subsequent tests. Product yields were slightly improved
to ~1.5 g/L by adding peptone to the feeding medium and by means of exponential feeding at a growth rate of µw = 0.2 h-1.
However, significant protein expression occurred only during the first few hours after induction.
In the third step, various expression temperatures and higher cell densities were examined with a parallel approach to reduce
cell stress caused by the high induction temperature and high expression yield. The experiments were planned applying the
principles of Design of Experiments (DOE), which is supported by DASGIP Control 4.0. The input parameters were defined as
induction temperature and optical density at induction start. The specified target parameters were product yield and the ratio
of insoluble to soluble protein expression (IB/sol-ratio).
The results of the parallel approach are illustrated in Figure 1. All four of the parallel fermenters used identical medium
and feeding profiles. Compared to an induction temperature of 42 °C, these results showed that the product expression phase
could be slightly prolonged at 40 °C. By further reducing the induction temperature to 38 °C, target protein formation was
sustained with the same high expression yield during the entire process, thus significantly improving the product titer to
A subsequent induction of the cultures at five hours after initial feeding, combined with an induction temperature of 38 °C,
further raised the end concentration of the target protein to 7.5 g/L. Figure 2 shows the influence of induction temperature
and cell density using statistical test planning. These results show that a low induction temperature in conjunction with
a high cell density yields the optimum product titer.
The researchers were able to increase the product concentration to 11 g/L by varying the peptone concentration and prolonging
the expression phase. The process was then successfully scaled to 10 L to yield a product concentration of >20 g/L through
improved oxygen input and the prolonged expression phase.
"By implementing parallel processing and using identical start conditions for the same precultures, we were able to carry
out fast and efficient parameter screening. Simple automation of the fermentation processes ensured the comparability and
reproducibility of the cultivations," explains Christian Kaiser from Hamburg-based Richter-Helm Biologics. "The DASGIP system
combines a wide range of centrally monitored and controlled parameters with numerous parallel reactors. As a result, we experienced
a near 10-fold increase in the product yield, from 1.2 to 11 g/L," Kaiser adds.