Effect of Ammonia on Product Quality

Ammonia was reported to affect the conditions in intra-cellular compartments and enzymatic reactions for protein processing. High ammonia concentrations were reported to alter protein after translations, glycosylation, and secretion. Ammonia can alter the intracellular or intracom-partmental pH, alter membrane potentials, and directly interact with enzymes. Thorens and Vassalli (98) reported inhibition of sialic acid transferase for Immunoglobulin (Ig) M at 10 mM ammonia. The results indicate a pH increase in Golgi because of ammonia load. Similarly, Andersen and Goochee (99) reported significant reduction in the terminal sialylation of O-linked glycosylation of recombinant granulocyte colony-stimulating factor. Borys et al. (100) studied the N-linked glycosylation of recombinant mouse placental lactogen-I from CHO cells and observed inhibition of glycosylation by ammonia. The effect of ammonia was also dependent on extracellular pH. Gawlitzek et al. (101,102) studied the glycosylation pattern of recombinant proteins expressed by BHK-21 cells. For the production of HuIL-2 variant in BHK cells, ammonia was observed to increase the intracellular uridine diphosphate-N-actylglucosamine (UDP-GlcNAc) pool. UDP-GlcNAc is a precursor substrate for the glycosylation process in the cy-tosol and Golgi. High ammonia levels led to a decrease in terminal sialyation and to an increase in branching. To test the hypothesis that UDP-GlcNAc is involved, the authors used glucosamine, which is the precursor for UDP-GlcNAc. Kopp et al. (103) studied product consistency and glycosylation patterns in recombinant CHO-expressed glycoproteins. High ammonia levels influenced glycosylation patterns significantly for INF-ffl and t-PA. The alteration of product glycosylation by ammonia was described in monoclonal antibody production (104). To minimize the effects of ammonia, a multilevel pH control was proposed for commercial production. Genetic and process engineering strategies for control of ammonia in cell cultures were recently described to yield enhanced glycosylation (104).

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