Media pH and Ammonium Concentration

It is well known that the ammonium ion content in the media alters intracellular pH and thereby affects cell growth and protein glycosylation (87). Miller et al. (88) conducted a comprehensive study of hybridomas in chemostats. The pH was varied and cell densities, viability, and antibody concentration were measured. Miller et al. (88) determined that pH was a very important parameter for successful cell culture. Borys et al. (89,90) investigated the effects of pH on mouse placental lac-togen expressed in CHO cells. Their initial studies used tissue culture flasks (T-flasks), yet clearly demonstrated that the culture pH shifted the glycosylation pattern. Later experiments used cells grown on microcarriers; again, their results demonstrated that pH effected glycosylation. Next, the extracellular pH was held constant and the ammonium concentration was varied. Increased ammonium ion concentrations decreased intracellular pH, and resulted in less glycosylated proteins (90).

Valley et al. (91) noted that ammonia inhibited cell growth and influenced glycosylation, but were unsure of the mechanism. Valley et al. (91) used labeled NH4Cl to detect N-labeled species. They observed that 60% of the UDP-GlcNAc pool was labeled, and 60-80% of the N-acetylated sugars in N-glycan structures were labeled. Therefore, they concluded that ammonium was used as a building block.

Gawlitzek et al. (81) observed that ammonium altered N-glycosylation in CHO expressing TNFR-IgG. Ammonium levels were increased from 1.0 to 15mM, which resulted in a 40% decrease in terminal galactosylation and sialylation. Extracellular b-galactosidase and sialidase activity increased throughout the culture. The b-galactosidase activity was proportional to the initial ammonium level; however, the sialidase activity was independent of the initial ammonium level. Also, the incubation of TNFR-IgG with b-galactosidase and sialidase in vitro did not alter glycosylation. These results suggested that the effect of ammonium on gly-cosylation was biosynthetic and not degradative. mRNA levels and the enzyme activity of b1,4-Gal-T and a2,3-ST enzymes were observed in response to ammonium (13 mM). Both the mRNA and the enzyme activities were not observed to change. The b1,4-Gal-T and a2,3-ST enzymes were observed in this study to be very sensitive to pH in vitro. Therefore, Gawlitzek et al. (81) concluded that ammonium altered the carbohydrate biosynthesis of TNGR-IgG by a pH-mediated effect on glycosyltrans-ferase activity. Other studies have shown that increased nucleotide sugar pools, do not increase glycosylation, as would be expected (61-63,79).

Yang and Butler (61,62) investigated the effects of ammonia on the glycosylation of EPO expressed in CHO cells. It was observed that increased ammonia concentration resulted in a reduction of the tetra-antennary structures and increased the tri- and biantennary structures. Also, the sialic acid content decreased with increased ammonia; however, the oligosaccharide sequence did not change. A later study by Yang and Butler (60) investigated ammonia and glucosamine levels on glycosylation. Both ammonia and glucosamine reduced the terminal sialylation, increased heterogeneity, and reduced the level of O-glycosylation. It was also observed that the UDP-N-acetylhexsamine pools increased (60).

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