On‐line gas analysis in animal cell cultivation: II. Methods for oxygen uptake rate estimation and its application to controlled feeding of glutamine

Abstract

Different methods for oxygen uptake rate (OUR) determinations in animal cell cultivation were investigated using a high quality mass spectrometer. Dynamic measurements have considerable disadvantages because of disturbances of the growing cells by the necessary variations of dissolved oxygen concentration. Only infrequent discrete measurements are possible using this method. Stationary liquid phase balance yielded better results with much higher frequency. Gas phase balancing has the advantage of not requiring dissolved oxygen measurement and knowledge of 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 CO(2) transfer are mainly achieved via sparging. The gas released to the headspace is diluted with a roughly 100-fold stream of an inert gas (helium). Through this dilution, gas ratios are not changed for O(2), CO(2), Ar, and N(2). The measurement of lower concentrations (parts per million and below) is easy using mass spectrometry with a secondary electron multiplier. With this new method an excellent accuracy and sufficient speed of analysis were obtained. All these on-line methods for OUR measurement were tested during the cultivation of animal cells. The new method 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 monoclonal antibodies against human fibronectin. With the aid of these methods it was possible to find a correlation between a rapid decrease in oxygen uptake rate (OUR) and glutamine concentration. The sudden decrease in OUR can be attributed to glutamine depletion. This provided a basis for the controlled addition of glutamine to reduce the formation of ammonia produced by hydrolysis. This control method based on OUR measurement resulted in increased cell concentration and threefold higher product concentration. (c) 1995 John Wiley & Sons, Inc.

Responses of CHO-DHFR cells to ratio of asparagine to glutamine in feed media: cell growth, antibody production, metabolic waste, glutamate, and energy metabolism

“…Amino acid concentrations were assayed by reversedphase HPLC (1525 Binary HPLC pump, 717plus Autosampler, 2487 Dual λ absorbance detector, Nova-Pak C18 4 μm 3.9 × 150 mm column, Waters) after derivatization with ACCQ-Tag kit purchased from Waters. The oxygen uptake rate (OUR) was determined through the online dynamic method (Eyer et al 1995).…”

Effects of cell density and temperature on oxygen consumption rate for different mammalian cell lines

“…In the continuous methods, the aeration system is not disturbed and the OCR can be calculated from mass balances using gas inlet and outlet composition. Together with the gas flow rate, the difference in gas composition allows on-line determination of the OCR (Bonarius et al, 1995;Eyer et al, 1995). The OCR can also be measured using gas phase oxygen composition (measured by a gas analyzer) and dissolved oxygen (measured by DO probe) using a mass transfer method (Ramirez and Mutharasan, 1990;Eyer et al, 1995).…”

On-line measurement of oxygen uptake in cell culture using the dynamic method

“…Such small uptake rates result in a very small decrease in oxygen concentration in the bioreactor exhaust gas stream. Attempts have been made to improve conventional offgas analysis techniques using inert carrier gases and special mass spectrometry equipment (Eyer et al, 1995) to achieve the required accuracy. Other researchers have used external recycle loops to estimate OUR (Yoon and Kon-* Telephone: 908-820-6869; fax: 908-820-6995; e-mail vijaysingh @ spcorp.com stantinov, 1994).…”