On-line monitoring and control of animal-cell cultures.

On-line analysis and control of biotechnological processes is still the stepchild in industry. In general, only parameters as dissolved-oxygen concentration, pH and temperature are controlled on-line. Important parameters as substrate and inhibitor concentrations are only measured offline. Controlling these parameters could not only result in more efficient production and higher product quality, but could also save manpower. The attitude in industry could change if analytical systems were available which are selective, reliable and robust, have low maintenance requirements and a large detection range (chapter 6 of this thesis). However, such systems are commercially seldom available. Therefore, an analytical system based on the flow-injection analysis (FIA) method was developed, which could fulfil the requirements mentioned above. The possibilities of this new analytical system are described in chapter 2 of this thesis. A software program (FIACCO) was developed which could control a high amount of hardware components and was able to carry out peak evaluation, automatic (re)calibration and outlier tests. This program in combination with precisely working pumps enabled the automatic and reproducible application of almost every known FIA method for sample pretreatment as dilution, pH adjustment and the elimination of interfering substances. Up to five analytes could be determined with the analytical system using enzymatic and/or chemical assays and only one detector.The developed analytical system was applied for the on-line monitoring of the glucose, glutamine, lactate and ammonium concentration during the culture of animal cells, which is described in chapter 3 of this thesis. Although some small disturbances occurred during a long term culture of 1000 h, the system proved to be reliable and robust and had very low maintenance requirements. The high analysis frequency could be used to estimate the growth rate during start-up periods of the culture. Moreover, it showed that in contradiction with general opinion, animal cells can react quickly to changes in the environment.The on-line control of animal-cell cultures by using the analytical system is described in chapter 4 of this thesis. The closed-loop control was realized by the application of an Extended Kalman filter and a minimum variance controller. Cultures could be started up at either a constant glutamine or glucose concentration, whereby the perfusion rate was adapted automatically to the increasing consumption rate of the controlled substrate. Glutamine was the better control substrate for the increasing perfusion rate with respect to avoiding substrate limitations. During a culture period of more than 800 h, the glucose and glutamine concentrations were controlled independently at different perfusion rates. The concentrations deviated in general maximally 10 % of the setpoint.Since the product concentration and/or productivity are also important process parameters, an analytical setup was developed for the online monitoring of the product concentration. A commercially available immunoassay (ABICAP) was coupled to an animal-cell culture by a newly developed sample system, which is described in chapter 5 of this thesis. The concentration of product, a monoclonal antibody, was monitored successfully for nearly 900 h. During the culture, the specific monoclonal antibody production rate increased with increasing temperature of the culture