In mammalian cell culture, glucose and glutamine are required for biomass synthesis. They are also a major energy source. Main products of glucose and glutamine metabolism are lactate and ammonia. It is believed that, as waste products, their accumulation in the medium will limit cell growth and product formation (Schneider et al., 1996; Genzel et al., 2005).
Generally, lactate production in mammalian cell culture is owing to the metabolism of glucose. When glucose is metabolized inefficiently or overflow
37 metabolism occurs (i.e. the rate of glycolysis exceeds some critical value), a higher concentration of lactate is produced (Zhou et al., 1997; Gambhir et al., 1999). Lactate is also one of the products of glutaminolysis. Production of lactic acid during mammalian cells growth generally lowers the culture pH because of limited buffering capacity of the medium. This inhibits cell growth. An increase in lactate concentration causes a remarkable increase in specific death rate, mainly because of the increase of osmotic pressure due to a build up of sodium lactate in the medium (Omasa et al., 1992; Ozturk
et al., 1992). A similar observation was reported by Glacken et al. (1986), where
sodium lactate formed by the reaction between sodium bicarbonate in the medium and the lactic acid produced by the cells, significantly inhibited antibody production by hybridoma cells in an isotonic (325 mOsm) solution. Inhibitory effect of lactate on cell growth has been reported at a concentration range of 22 – 55 mM (Legazpi et al., 2005; Macmillan et al., 1987; Ozturk et al., 1992).
Ammonia formation in mammalian cell culture is generally attributed to glutamine catabolism. A second important source is through chemical decomposition of glutamine. Glutamine is not chemically stable in culture media. It tends to decompose chemically to produce ammonia and pyrrolidonecarboxylic acid (Ozturk and Palsson, 1990b; Schneider et al., 1996). Thus, the disappearance of glutamine in the culture cannot be ascribed exclusively to uptake by cells and the measured rate of disappearance must be corrected for chemical degradation to accurately establish cellular uptake.
Chemical degradation of glutamine follows first order kinetics: - d[Gln]/dt = k[Gln]
where [Gln] is the glutamine concentration, t is time and k is the first order rate constant (Ozturk and Palsson, 1990b). The rate constant k is influenced by the medium composition, pH and temperature (Ozturk and Palsson, 1990b; Schneider et al., 1996). Thus, the k value must be determined at the temperature and pH of use and in the specific medium of interest (Ozturk and Palsson, 1990b; Schneider et al., 1996).
As quoted by Genzel et al. (2005), depending on both the cell line and the medium, cell growth will be severely inhibited at ammonia concentration as low as 1 - 5 mM in the culture medium. Martinelle and Häggström (1993) suggested that one of the
38 important toxic mechanisms of ammonia/ammonium ion exposure is an increased demand for maintenance energy, caused by the need to keep up ion gradients over the cytoplasmic membrane. They demonstrated that ammonia crosses the cell membrane through simple diffusion, while ammonium ion needs a transport protein to do so. Doyle and Butler (1990) examined the effect of pH on the toxicity of ammonia to a murine hybridoma. They showed that at high pH, a lower concentration of ammonium chloride was able to inhibit cell growth compared to at low pH. Hence, Doyle and Butler (1990) suggested that a decreasing of pH during cultivation may counteract the ammonia accumulation effect, which could be advantageous for maximizing cell growth. Nonetheless, it has to be very clear that the effect of adding ammonia to the medium could be very different from that of ammonia produced within the cells.
The common approach in avoiding accumulation of the waste products in mammalian cell culture is to replace the spent medium with fresh medium. Without a doubt, this strategy increases the production costs, especially if using a serum- containing medium. A few strategies have been proposed, which include substitution of glutamine by its stable derivatives, controlled addition of glutamine and glucose, replacement of glucose by other sugars, and removing of ammonium using ion- exchange resins, non-porous ion-exchange membranes, gas-permeable hydrophobic porous membranes and electrodialysis (Butler and Jenkins, 1989; Schneider et al., 1996).
Dalili et al. (1990) found that minimum initial glutamine concentration required for a hybridoma cell line that they studied was 2 mM, below it the specific growth rate and specific antibody production rate were reduced. Yet, Genzel et al. (2005) successfully replaced 2 mM glutamine with 10 mM pyruvate in the medium without growth rate reduction for several different adherent commercial cell lines and this greatly reduced the production of ammonia and lactate.
Ljunggren and Häggström (1990, 1994) effectively reduced ammonium ion production in the culture medium of myeloma and hybridoma cells by controlled feeding of glutamine in fed-batch culture. A similar finding was reported by other researchers working on 293 HEK cells (Lee et al., 2003), recombinant murine myeloma cells (Gambhir et al., 1999), PER.C6TM cells (Maranga and Goochee, 2006) and
39 hybridoma cells (Kurokawa et al., 1994; Li et al., 2005). Different cell lines and culture media were used in these studies and their methodology of feeding the components that were being controlled were not identical. When only glucose was controlled at low concentration, lactate production was reduced, but ammonia accumulation increased (Ljunggren and Häggström, 1994; Kurokawa et al., 1994). On the other hand, if only glutamine was controlled at a low concentration, lactate, alanine and ammonia accumulation were all reduced (Ljunggren and Häggström, 1994; Kurokawa et al., 1994;
Lee et al., 2003). Conversely, when both glucose and glutamine were controlled at low
levels, accumulation of both lactate and ammonia were almost totally eliminated (Maranga and Goochee, 2006; Kurakawa et al., 1994; Li et al., 2005; Ljunggren and Häggström, 1994; Gambhir et al., 1999). The feeding concentrations for glucose and glutamine of these researchers were 0.28 – 1.10 mM and 0.085 – 0.700 mM, respectively.
Glacken et al. (1986) has explored the possibility of substituting glucose with other sugars. Galactose was found to be able to substitute for glucose and reduce the specific lactic acid productivity dramatically. Altamirano et al. (2004) developed a fed- batch strategy to cultivate t-PA (tissue type plasminogen activator) producing CHO cells by substituting glutamine with glutamate and alternating glucose with galactose. Lactate consumption in glucose/galactose-fed culture was significantly higher than in glucose-fed culture, which meant that lactate was co-metabolized with galactose. They concluded that the higher cell concentration and viability obtained were mainly the consequence of glucose substitution by galactose, rather than lactate detoxification.
Brose and van Eikeren (1990) developed a membrane-based method to remove toxic ammonia from mammalian cell culture. Their gas-permeable hydrophobic porous membrane was able to selectively remove ammonia to well below 1 mM. BHK cells were grown in the culture medium that was spiked with 14 mM ammonia, Brose and van Eikeren (1990) successfully used the membrane they developed to strip off the ammonia. The growth rate for BHK cells was found to be similar to those of cells grown in the fresh medium.
40 Control of initial glucose and glutamine concentrations in the culture medium seems to be critical in ensuring the availability of these nutrients for growth and production, and also reducing the formation of waste products that affect growth.