Mary B. Sliwkowski

Ammonium alters N-glycan structures of recombinant TNFR-IgG: degradative versus biosynthetic mechanisms.

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 NH(4)Cl. 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 beta-galactosidase and sialidase activity, which increased throughout the culture. The beta-galactosidase, but not the sialidase, level was proportional to the ammonium concentration. No loss of N-glycans was observed in incubation studies using beta-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 beta1, 4-galactosyltransferase (beta1,4-GT) and alpha2,3-sialyltransferase (alpha2,3-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 beta1, 4-galactosyltransferase (beta1,4-GT) and alpha2,3-sialyltransferase (alpha2,3-ST) messenger RNA (mRNA) and enzyme levels were observed in cells cultivated in the presence or absence of 13 mM NH(4)Cl. pH titration of endogenous CHO alpha2,3-ST and beta-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.

Biochemical Characterization of Recombinant Human Nerve Growth Factor

Abstract: Recombinant human nerve growth factor (rhNGF) was expressed and secreted by Chinese hamster ovary cells and purified to homogeneity using ion‐exchange and reversed‐phase (RP) chromatography. The isolated product was shown to be consistent with a 120‐amino‐acid residue polypeptide chain by amino acid composition, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), RP‐HPLC, and mass spectrometry and with an N‐terminal sequence consistent with that expected from the cDNA for human nerve growth factor. By size‐exclusion chromatography, rhNGF behaves like a noncovalent dimer. Limited enzymatic digests of the 120‐residue monomer produced additional species of 118 (trypsin, removal of the C‐terminal Arg119‐Ala120 sequence) and 117 (trypsin plus carboxypeptidase B, removal of the C‐terminal Arg118‐Arg119‐Ala120 sequence) residues. Each of these species was isolated by high‐performance ion‐exchange chromatography and characterized by amino acid and N‐terminal sequence analyses, SDS‐PAGE, RP‐HPLC, and mass spectrometry. All three species were present in the digests as both homodimeric and heterodimeric combinations and found to be equipotent in both the chick dorsal root ganglion cell survival and rat pheochromocytoma neurite extension assays.

Alteration in Glycosylation: Controlling the Diversity of Recombinant Glycoproteins

N-linked Oligosaccharides on a recombinant protein play a critical role in the metabolic behavior of the molecule. The extent of glycosylation on the protein can determine clearance rates and the ultimate pharmacokinctic profile. The amount of terminal sialylation is particularly important in this respect. Changes in the glyeosylation pattern due to changes in culture conditions will be shown for a model glyeoprotein produced in CHO cells. The parameter determining the sialic acid content will be discussed together with a method to control for this type of heterogeneity.