As mentioned before, ammonia is generated mainly by the spontaneous degradation and cellular metabolism of glu-tamine. Ammonia generation can be minimized by controlling these processes.
Minimization of Glutamine Degradation. As previously mentioned, the glutamine degradation rate is dependent on several factors, and it can be minimized by several strategies. Temperature is the most obvious factor, and the media should be kept cold when stored. In the reactor, however, the temperature cannot be lowered much. Other factors, such as media pH, can be used to minimize the degradation. The pH optimum for cells varies between pH 7.0 and 7.4 (22,23). Cultivation at lower pH values should decrease the degradation rate and ammonia generation. Serum, if used, should be inactivated to minimize any glutaminase activity. Finally, glutamine degradation is dependent on several ions, such as phosphate. Various media should be evaluated for a given cell line, and glutamine degradation rates for each should be compared.
Attempts have been made to replace glutamine by glutamine-containing peptides (133,134). Peptides such as alanyl-glutamine or glycyl-glutamine are more stable, and the media prepared with these peptides can be autoclaved. These peptides were also shown to generate less ammonia. Although the cells exhibited a considerable lag phase, the cell yields from peptide-substituted media were comparable to those obtained from standard media. Holmlund et al. (135) used glycyl-glutamine and A-acetyl-glutamine for CHO cultures. Although cells did not perform well in the presence of A-acetyl-glutamine (low growth and t-PA production), glycyl-glutamine provided good culture perfor-
Control of Cellular Metabolism. The specific ammonia production rate is affected by the cellular environment, and variables such as pH, dissolved oxygen (DO), temperature, and the level of metabolites can be manipulated to minimize the production rate.
There seems to be an optimal pH for reducing the ammonia production rate. For instance, Ozturk and Palsson (93) observed the ammonia production rates to be minimal at pH 7.2. The cell growth rates were optimal, and glutamine consumption was also observed to be minimal at this pH. The ammonia yield from glutamine, on the other hand, was reduced at low pH values (92,136). Cells seem to produce less ammonia when they are growing under optimal conditions. At very low (1% air saturation) and very high (100% air saturation) DO levels, the ammonia generation rates increased (93,136,137).
In most cases, the ammonia production follows closely with the glutamine consumption with a relatively constant yield coefficient. Reducing the glutamine concentration ([Gln]) decreases the glutamine consumption (qGln), and Monod-type saturation kinetics can be used to describe the data:
where qGln and Km are constants. The value of Km is in the order of 0.2 to 1 mM (10,87).
Reducing glutamine not only decreases the glutamine consumption and ammonia production rates, but also decreases the yield of ammonia from glutamine. When glu-tamine was fed in a controlled fashion below 1 mM levels, Glacken et al. (50) observed a substantial decrease in ammonia production in Madin-Darby bovine kidney (MDCK) and human fibroblast cells. A similar strategy was successfully used to reduce ammonia production by 50% in hybridoma cells (36,38).
Although a glucose limitation alone did not cause a change in ammonia secretion, Ljunggren and Haggstrom (38) observed an enhancement in ammonia reduction when glucose and glutamine were limited. The replacement of glucose by fructose, mannose, and galactose virtually eliminated the ammonia-induced generation of UDP-GNAc.
Altering the amino acid composition of the media seems to affect ammonia generation. Hiller at al. (138) increased the concentrations of leucine, isoleucine, valine, and lysine and observed a decrease in ammonia secretion in a hybridoma line. In these experiments, alanine production rates increased, indicating an elevation in the activity of alanine transaminase.
Replacement of Glutamine. The replacement of gluta-mine in cell culture media can eliminate most of the ammonia generation in culture. Although the idea of replacement is a good one, it is difficult to implement in most cases because the cells seem to be strongly dependent on gluta-mine for energy and biomass production. Cells need to adapted or genetically altered to grow in the absence of supplemented glutamine.
Mammalian cells can use other amino acids as a substitute for glutamine. Studies were conducted to investigate the replacement of glutamine by glutamate, a-ketoglutarate, and asparagine. The efficiency ofglutamate for supporting cell growth is very low, and high concentrations of glutamate (up to 20 mM) are required (139,140). The replacement of glutamine by glutamate was possible for mouse LS cells (141) and for the McCoy cells (142). On the other hand, MDCK cells could not be adapted to glutamine-free media (143). Cells can convert glutamate to glutamine via the glutamine synthetase reaction, and the success of growing the cells on glutamate can depend on the concentration and activity of this enzyme. However, McDermott and Butler (143) observed that the key factor in cell adaptation to glutamine-free media is not the glu-tamine synthetase, but the uptake rate of glutamate by the cells.
Asparagine is another amino acid used to replace glu-tamine. Although asparagine is also unstable in media (25), the degradation rate is much lower (half-life, 87 days). Kurano et al. (85) observed the growth of CHO cells on asparagine after an initial lag phase. The asparagine-containing cultures generated 40% less ammonia than cultures in standard media. The use of a-ketoglutarate instead of glutamine was also effective in reducing ammonia generation (142).
The success of growing cells in the absence of glutamine can be increased by genetic engineering. Scientists at Celltech Ltd. developed a vector containing glutamine synthe-tase and infected NS0 myeloma and CHO cells with plas-mid containing this vector (144-146). The construction of this vector is presented in Figure 8. The cells use glutamate as a substrate in glutamine-free media. The genes for protein expression were also integrated into the vector; thus, the glutamine synthetase gene was used as an am-plifiable, selectable marker; in the glutamine-free media only the cells containing plasmid could grow. This resulted in the selection of high-producing clones. The system works best for NS0 myeloma cells because these cells lack any endogenous glutamine synthetase activity. CHO cells, on the other hand, contain endogenous glutamine synthe-tase genes, and the selective pressure induced by the absence of glutamine does not work effectively. The use of a specific inhibitor, methionine sulphoximine (MSX), increases the efficiency of selection and amplification.
Adaptation of Cells to High Ammonia. The adaptation of cells to grow at high ammonia concentrations can result in
more ammonia-tolerant cultures. Adaptation is a complex process, and it is not clear whether the cells alter themselves or a particular clone is selected as a result of this process (147,148). Regardless of the mechanism, the cells can tolerate higher ammonia levels after the adaptation, and this method of ammonia adaptation can be used to minimize the ammonia inhibition. The adaptation of hybridoma cells to ammonia has been demonstrated by several investigators (52,54,149).
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