title: Glycosylation-related genes in NS0 cells are insensitive to moderately elevated ammonium concentrations.
abstract: NS0 and Chinese hamster ovary (CHO) cell lines are used to produce recombinant proteins for human therapeutics; however, ammonium accumulation can negatively impact cell growth, recombinant protein production, and protein glycosylation. To improve product quality and decrease costs, the relationship between ammonium and protein glycosylation needs to be elucidated. While ammonium has been shown to adversely affect glycosylation-related gene expression in CHO cells, NS0 studies have not been performed. Therefore, this study sought to determine if glycosylation in NS0 cells were ammonium-sensitive at the gene expression level. Using a DNA microarray that contained mouse glycosylation-related and housekeeping genes, these genes were analyzed in response to various culture conditions - elevated ammonium, elevated salt, and elevated ammonium with proline. Surprisingly, no significant differences in gene expression levels were observed between the control and these conditions. Further, the elevated ammonium cultures were analyzed using real-time quantitative reverse transcriptase PCR (qRT-PCR) for key glycosylation genes, and the qRT-PCR results corroborated the DNA microarray results, demonstrating that NS0 cells are ammonium-insensitive at the gene expression level. Since NS0 are known to have elevated nucleotide sugar pools under ammonium stress, and none of the genes directly responsible for these metabolic pools were changed, consequently cellular control at the translational or substrate-level must be responsible for the universally observed decreased glycosylation quality under elevated ammonium.
authors: Department of Bioengineering, 301 Rhodes Research Center, Clemson University, Clemson, SC 29634-0905, USA. Electronic address: abrodsk@clemson.edu.
authors: Arthur Nathan
authors: AN
authors: Brodsky
authors: Department of Bioengineering, 301 Rhodes Research Center, Clemson University, Clemson, SC 29634-0905, USA. Electronic address: caldwe5@clemson.edu.
authors: Mary
authors: M
authors: Caldwell
authors: Department of Bioengineering, 301 Rhodes Research Center, Clemson University, Clemson, SC 29634-0905, USA. Electronic address: sbae@clemson.edu.
authors: Sooneon
authors: S
authors: Bae
authors: Department of Bioengineering, 301 Rhodes Research Center, Clemson University, Clemson, SC 29634-0905, USA. Electronic address: harcum@clemson.edu.
authors: Sarah W
authors: SW
authors: Harcum
mesh_terms: D064751:Ammonium Compounds / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D019149:Bioreactors; D016466:CHO Cells; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media; D015870:Gene Expression / Q000187:drug effects*; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis
chemical_list: D064751:Ammonium Compounds; D003470:Culture Media; D016695:Glycosyltransferases
keywords: Biopharmaceuticals; CHO cells; DNA microarrays; Glycosylation; Mammalian cells