Analytical tools for monitoring and control of fermentation processes

The overall objective of this work has been to adopt new developments and techniques in the area of measurement, modelling and control of fermentation processes. Flow cytometry and software sensors are techniques which were considered ready for application and the focus was set on developing tools for research aiming at understanding the relationship between measured variables and process quality parameters. In this study fed-batch cultivations have been performed with two different strains of Escherichia coli (E.coli) K12 W3110 with and without a gene for the recombinant protein promegapoietin. Inclusion body formation was followed during the process with flow cytometric detection by labelling the inclusion bodies with first an antibody against the protein promegapoietin and then a second fluorescent anti-antibody. The approach to label inclusion bodies directly in disintegrated and diluted cell slurry could be adopted as a method to follow protein production during the process, although the labelling procedure with incubation times and washings was somewhat time-consuming (1.5 h). The labelling of inclusion bodies inside the cells to follow protein production was feasible to perform, although an unexplained decrease in the relative fluorescence intensity occurred late in process. However, it is difficult to translate this qualitative measurement into a quantitative one, since a quantitative protein analysis should give data proportional to the volume, while the labelling of the spheric inclusion bodies gives a signal corresponding to the area of the body, and calibration is not possible. The methods were shown to be useful for monitoring inclusion body formation, but it seems difficult to get quantitative information from the analysis. Population heterogeneity analysis was performed, by using flow cytometry, on a cell population, which lost 80-90% viability according to viable count analysis. It was possible to show that the apparent cell death was due to cells incapable of dividing on agar plates after induction. These cells continued to produce the induced recombinant protein. It was shown that almost all cells in the population (≈97%) contained PMP, and furthermore total protein analysis of the medium indicated that only about 1% of the population had lysed. This confirms that the "non-viable" cells according to viable count by cfu analysis produced product. The software sensors XNH3 and μNH3, which utilises base titration data to estimate biomass and specific growth rate was shown to correlate well with the off-line analyses during cultivation of E. coli W3110 using minimal medium. In rich medium the μNH3 sensor was shown to give a signal that may be used as a fingerprint of the process, at least from the time of induction. The software sensor KLaC* was shown to respond to foaming in culture that probably was caused by increased air bubble dispersion. The RO/S coefficient, which describes the oxygen to substrate consumption, was shown to give a distinct response to stress caused by lowered pH and addition of the inducing agent IPTG. The software sensor for biomass was applied to a highly automated 6-unit multibioreactor system intended for fast process development. In this way also specific rates of substrate and oxygen consumption became available without manual sampling.