Glucose and lactate profiles in Chinese hamster ovary cell cultures were accurately monitored in real time and in situ during three bioreactor batch cultures lasting 11,15, and 15 days performed within a 60-day period. Monitoring was accomplished using in situ-collected mid-infrared spectra analyzed with a priori one-time established partial least-squares regression models. The robustness of the technique was demonstrated by application of these models without modification after 2.3 years. Neither recalibration nor instrument maintenance was required during the 2.3-year period, except for the daily filling of liquid nitrogen for detector cooling during operation. The lactate calibration model yielded accurate absolute concentration estimations during each of the batch cultures with standard errors of estimate from 1 to 3 mM. The a priori-established glucose calibration model yielded concentration estimations with an off-set, which was constant throughout a culture. Adjustment of the off-set before inoculation resulted in accurate concentration estimations with Standard errors of estimate of approximately 1 mM for each of the bioreactor cultures. Sensitivity in detecting differences of 0.5 mM and selectivity against variation of one metabolite while the other was kept constant was demonstrated during standard additions of either glucose or lactate. The sensor system proved to be reliable, simple, accurate, sterile, and capable of long-term automatic operation and is considered to be mature enough to be routinely applied for in situ (on-line) cell culture monitoring.

A new approach for the indirect determination of cell concentration in the case of nonconstant metabolic rates has been developed. The specific glucose‐uptake rate was shown to be nonconstant in batch cultures of free suspended and immobilized CHO SSF3 cells. Time‐independent models correlating the specific rate to the limiting substrate concentration were established, thus providing a continuous determination of the specific rate through on‐line measurement of the limiting substrate. The method could be applied to determine on‐line cell concentration in both free suspended and immobilized cell cultures. Results were verified off‐line by crystal violet nuclei counting. The predicted cell concentration was in very good agreement with the off‐line reference during the whole exponential‐growth phase, until the specific glucose‐uptake rate tended to zero. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 72: 515–522, 2001.

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