The effect of ammonium on the glycosylation pattern of the recombinant immunoadhesin tumor necrosis factor–IgG (TNFR‐IgG) produced by Chinese hamster ovary cells is elucidated in this study. TNFR‐IgG is a chimeric IgG fusion protein bearing one N‐linked glycosylation site in the Fc region and three complex‐type N‐glycans in the TNF‐receptor portion of each monomer. The ammonium concentration of batch suspension cultures was adjusted with glutamine and/or NH4Cl. The amount of galactose (Gal) and N‐acetylneuraminic acid (NANA) residues on TNFR‐IgG correlated in a dose‐dependent manner with the ammonium concentration under which the N‐linked oligosaccharides were synthesized. As ammonium increased from 1 to 15 mM, a concomitant decrease of up to 40% was observed in terminal galactosylation and sialylation of the molecule. Cell culture supernatants contained measurable β‐galactosidase and sialidase activity, which increased throughout the culture. The β‐galactosidase, but not the sialidase, level was proportional to the ammonium concentration. No loss of N‐glycans was observed in incubation studies using β‐galactosidase and sialidase containing cell culture supernatants, suggesting that the ammonium effect was biosynthetic and not degradative. Several biosynthetic mechanisms were investigated. Ammonium (a weak base) is known to affect the pH of acidic intracellular compartments (e.g., trans‐Golgi) as well as intracellular nucleotide sugar pools (increases UDP‐N‐acetylglucosamine and UDP‐N‐acetylgalactosamine). Ammonium might also affect the expression rates of β1,4‐galactosyltransferase (β1,4‐GT) and α2,3‐sialyltransferase (α2,3‐ST). To separate these mechanisms, experiments were designed using chloroquine (changes intracellular pH) and glucosamine (increases UDP‐GNAc pool [sum of UDP‐GlcNAc and UDP‐GalNAc]). The ammonium effect on TNFR‐IgG oligosaccharide structures could be mimicked only by chloroquine, another weak base. No differences in N‐glycosylation were found in the product synthesized in the presence of glucosamine. No differences in β1,4‐galactosyltransferase (β1,4‐GT) and α2,3‐sialyltransferase (α2,3‐ST) messenger RNA (mRNA) and enzyme levels were observed in cells cultivated in the presence or absence of 13 mM NH4Cl. pH titration of endogenous CHO α2,3‐ST and β‐1,4‐GT revealed a sharp optimum at pH 6.5, the reported trans‐Golgi pH. Thus, at pH 7.0 to 7.2, a likely trans‐Golgi pH range in the presence of 10 to 15 mM ammonium, activities for both enzymes are reduced to 50% to 60%. Consequently, ammonium seems to alter the carbohydrate biosynthesis of TNFR‐IgG by a pH‐mediated effect on glycosyltransferase activity. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 637–646, 2000.

We have engineered two Chinese hamster ovary cell lines secreting different recombinant glycoproteins to express high levels of human beta1,4-galactosyltransferase (GT, E.C. 2.4.1.38) and/or alpha2, 3-sialyltransferase (ST, E.C. 2.4.99.6). N-linked oligosaccharide structures synthesized by cells overexpressing the glycosyltransferases showed greater homogeneity compared with control cell lines. When GT was overexpressed, oligosaccharides terminating with GlcNAc were significantly reduced compared with controls, whereas overexpression of ST resulted in sialylation of >/=90% of available branches. As expected, GT overexpression resulted in reduction of oligosaccharides terminating with GlcNAc, whereas overexpression of ST resulted in sialylation of >/=90% of available branches. The more highly sialylated glycoproteins had a significantly longer mean residence time in a rabbit model of pharmacokinetics. These experiments demonstrate the feasibility of genetically engineering cell lines to produce therapeutics with desired glycosylation patterns.