Effects of elevated ammonium on glycosylation gene expression in CHO cells

The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the N-glycosylation profile of a monoclonal antibody

“…Culture conditions such as accumulation of ammonia, dissolved oxygen level, nutrient contents, pH level and temperature, may have great effects on the distribution of glycan structures (microheterogeneity and macroheterogeneity) found on the final recombinant protein products. Those factors may cause one or more mechanisms in the following: (1) the depletion of the cellular energy state (Rearick et al, 1981;Valley et al, 1999); (2) disruption of the local ER and Golgi environment, such as pH (Andersen and Goochee, 1995) or redox potential (Restelli et al, 2006); (3) alteration of glycosyltransferase activities (Chen and Harcum, 2006;Chotigeat et al, 1994); and (4) changes in the Nglycosylation precursor pool, such as nucleotides, nucleotide sugars (Kochanowski et al, 2008;Nyberg et al, 1999) and lipid precursors (Stoll and Krag, 1988). …”

Tailoring recombinant protein quality by rational media design

“…Increased ammonium levels in processes B, C and D were correlated with supplementary Asn additions either combined with the CD-feed or as a separate feed solution. Processes B and D were characterized by high levels of ammonium (≥ 18 mM) that were well beyond the reported galactosylation inhibiting levels 126,237 . The lower cell densities as well as reduced productivity suggest that the high ammonium levels entailed toxic effects for the cells.…”

“…It was observed that elevated ammonium levels (≥ 10 mM) during the cell culture reduced both GalT gene expression and activity. Furthermore, these conditions significantly impacted the post-glycosylation process in the Golgi apparatus, while it had less impact on ER and cytosol compartments 126 . Structural analogs and mimics of the natural sugar-nucleotide UDP-galactose (UDP-Gal) have a great potential to specifically inhibit GalT.…”

“…Ammonium levels not only inhibit galactosylation, but also have an impact on the abundance of sialylated species, due to the sparsity of galactosylated glycoforms and limited expression levels of α-2,3-sialyltransferase (SiaT) 126 . In contrast to human IgG, sialic acids are attached to the terminal galactose residues of antibodies expressed in CHO cells via an α-2,3 linkage exclusively 68 .…”

“…Ammonium supplementation increased the transGolgi pH, thus reducing galactosyltransferase activities, which yielded lower galactosylation levels 237 . It was found that high ammonium concentration in the supernatant (10 mM NH 4 Cl) diminished galactosyltransferase gene expression levels 126 . While supplementation of uridine, manganese and galactose offers a wide range of galactosylation enhancement, which proves to be sufficient in the frame of the quality modulation exercise of new biological entities and biosimilars, a limited number of effective compounds to decrease galactosylation during cell culture is available.…”