The use of glutamate and dipeptides as substitutes for glutamine in animal cell cultures

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Christie, Andrew D.
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The chemical decomposition and cellular metabolism of glutamine in animal cell cultures results in ammonium accumulation in the growth medium. Less ammoniagenic substrates including glutamate and dipeptides (ala-gln and gly-gln) were investigated as substitutes for glutamine in cell culture. The CC9C10 murine hybridoma and BHK-21 cells were sensitive to ammonium. Consequently, these two cell lines were used as models to evaluate the effects of substitutes on growth and metabolism and the factors influencing the ability to utilize glutama e and dipeptides. Glutamate was found to inhibit the growth of CC9C10, cells although ala-gln and gly-gln supported the growth of the hybridoma. High cell densities were obtained in the presence of 6 mM ala-gln and 20 mM gly-gln. The final cell density in gly-gln was 14% higher than in glutamine medium although monoclonal antibody production was not improved. Substrate utilization and metabolism were affected by the dipeptides, particularly with gly-gln. The accumulation ofammonium and lactate was significantly lower. The higher concentration of gly-gln was required because a cellular peptidase had a lower affinity for gly-gln. An investigation of the mechanism of dipeptide utilization implicated extracellular hydrolysis following release of peptidase into the culture medium. The dipeptides were also effective as substitutes for BHK cells although high gly-gln concentrations (up to 40 mM) were required. In addition, glutamate, asparagine and a low level of glutamine or dipeptides supported high growth rates and cell yields of BHK cells. Significant changes in metabolism were characterized in glutamate-based medium including markedly reduced ammonium production although growth was not improved with respect to glutamine-based medium. Cell yields were limited by surface area or nutrient depletion in batch cultures. In fed-batch microcarrier cultures, glutamate substitution resulted in the accumulation of inhibitory lactate levels. Growth in glutamate-based medium was not limited by glutamate uptake. Changes in intracellular enzymes such as glutamine synthetase were probably more important for the adaptation to utilize glutamate. Substitutes for glutamine are effective for controlling ammonium levels in culture. However, achieving substantial increases in growth and productivity may depend on the regulation of other substrates in the medium, particularly glucose.