ONLINE GAS-ANALYSIS IN ANIMAL-CELL CULTIVATION .2. METHODS FOR OXYGEN-UPTAKE RATE ESTIMATION AND ITS APPLICATION TO CONTROLLED FEEDING OF GLUTAMINE

Different methods for oxygen uptake rate (OUR) determinations in anima l cell cultivation were investigated using a high quality mass spectro meter. Dynamic measurements have considerable disadvantages because of disturbances of the growing cells by the necessary variations of diss olved oxygen concentration. Only infrequent discrete measurements are possible using this method. Stationary liquid phase balance yielded be tter results with much higher frequency. Gas phase balancing has the a dvantage of not requiring dissolved oxygen measurement and knowledge o f K(L)a, both of them are easily biased. It was found that simple gas phase balancing is either very inaccurate (error larger than expected signal) or very slow, with gas phase residence times of several hours. Therefore, a new method of aeration was designed. Oxygen and CO2 tran sfer are mainly achieved via sparging. The gas released to the headspa ce is diluted with a roughly 100-fold stream of an inert gas (helium). Through this dilution, gas ratios are not changed for O-2, CO2, Ar, a nd N-2. The measurement of lower concentrations (parts per million and below) is easy using mass spectrometry with a secondary electron mult iplier. With this new method an excellent accuracy and sufficient spee d of analysis were obtained. All these on-line methods for OUR measure ment were tested during the cultivation of animal cells. The new metho d allowed better study of the kinetics of animal cell cultures as was shown with a hybridoma cell line (HFN 7.1, ATCC CRL 1606) producing mo noclonal antibodies against human fibronectin. With the aid of these m ethods it was possible to find a correlation between a rapid decrease in oxygen uptake rate (OUR) and glutamine concentration. The sudden de crease in OUR can be attributed to glutamine depletion. This provided a basis for the controlled addition of glutamine to reduce the formati on of ammonia produced by hydrolysis. This control method based on OUR measurement resulted in increased cell concentration and threefold hi gher product concentration. (C) 1995 John Wiley and Sons, Inc.