Physiological state multiplicity was observed in continuous cultures of the hybridoma cell line ATCC CRL‐1606 cultivated in glutamine‐limited steady state chemostats. At the same dilution rate (0.04 h−1), two physiologically different cultures were obtained which exhibited similar growth rates and viabilities but drastically different cell concentrations (7.36 × 105 and 1.36 × 106 cells/mL). Metabolic flux analysis conducted using metabolite and gas exchange rate measurements revealed a more efficient culture for the steady state with the higher cell concentration, as measured by the fraction of pyruvate carbon flux shuttled into the TCA cycle for energy generation. The low‐efficiency steady state was achieved after innoculation by growing the cells in a nutrient rich environment, first in batch mode followed by a stepwise increase of the dilution rate to its set point at 0.04 h−1. The high‐efficiency steady state was achieved by reducing the dilution rate to progressively lower values to 0.01 h−1 resulting in conditions of stricter nutrient limitation. The high energetic efficiency attained under such conditions was preserved upon increasing the chemostat dilution rate back to 0.04 h−1 with a higher nutrient consumption, resulting in approximate doubling of the steady state cell concentration. This metabolic adaptation is unlikely due to favorable genetic mutations and could be implemented for improving cell culture performance by inducing cellular metabolic shifts to more efficient flux distribution patterns. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 675–683, 1999.

Metabolic flux analysis is a useful tool for unraveling relationships between metabolism and cell function. Material balancing can be used to provide estimates of major metabolic pathway fluxes, provided all significant metabolite uptake and production rates are measured. Potential sources of metabolizable material in many serum‐free media formulations are low molecular weight digests of biological material such as yeast extracts and plant or animal tissue hydrolysates. These digests typically contain large amounts of peptides, which may be utilized as amino acids. This article demonstrates the need for accounting for amino acids liberated from peptides in order to accurately estimate pathway fluxes in Chinese hamster ovary cells grown in a complex (hydrolysate containing) medium. A simplified model of central carbon metabolism provides the framework for analyzing external metabolite measurements. Redundant measurements are included to ensure the consistency of data and assumed biochemistry by comparing redundant measurements with their predicted values from a minimum data set, and by expressing the degree of agreement using a statistical “consistency index.” The consistency index tests whether redundancies are satisfied within expected experimental error. For chemostat steady states of CHO cultures grown in a hydrolysate‐supplemented medium, consistent data were obtained only when amino acids liberated from peptides were taken into account. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 324–335, 1999.

Hydrodynamic phenomena in microcarrier cultures are investigated with regard to the development of improved reactor designs for large‐scale operations. New concepts and theoretical models that describe new data as well as previously published data are presented.

Animal cells are exposed to turbulent fluid flow in many cell culture processes. If the turbulence in the flow is sufficiently strong, the cells will be damaged or killed by fluid-mechanical forces. Through an increase in viscosity, the turbulence can be damped and the hydro-dynamic damage can be reduced. In this article, new experimental results are presented which illustrate the protective effect of thickening agents. The results follow the prediction of a model based on Kolmogorov's theory of universal equilibrium in turbulent flow fields.

Asparagine linked (N‐linked) glycosylation is an important modification of recombinant proteins, because the attached oligosaccharide chains can significantly alter protein properties. Potential glycosylation sites are not always occupied with oligosaccharide, and site occupancy can change with the culture environment. To investigate the relationship between metabolism and glycosylation site occupancy, we studied the glycosylation of recombinant human interferon‐γ (IFN‐γ) produced in continuous culture of Chinese hamster ovary cells. Intracellular nucleotide sugar levels and IFN‐γ glycosylation were measured at different steady states which were characterized by central carbon metabolic fluxes estimated by material balances and extracellular metabolite rate measurements. Although site occupancy varied over a rather narrow range, we found that differences correlated with the intracellular pool of UDP‐N‐acetylglu‐ cosamine + UDP‐N‐acetylgalactosamine (UDP‐GNAc). Measured nucleotide levels and estimates of central carbon metabolic fluxes point to UTP depletion as the cause of decreased UDP‐GNAc during glucose limitation. Glucose limited cells preferentially utilized available carbon for energy production, causing reduced nucleotide biosynthesis. Lower nucleoside triphosphate pools in turn led to lower nucleotide sugar pools and reduced glycosylation site occupancy. Subsequent experiments in batch and fed‐batch culture have confirmed that UDP‐sugar concentrations are correlated with UTP levels in the absence of glutamine limitation. Glutamine limitation appears to influence glycosylation by reducing amino sugar formation and hence UDP‐GNAc concentration. The influence of nucleotide sugars on site occupancy may only be important during periods of extreme starvation, since relatively large changes in nucleotide sugar pools led to only minor changes in glycosylation. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 336–347, 1999.