Ammonium toxicity and amino acid protection in Chinese hamster ovary cells

Mammalian cell cultures are widely used to produce recombinant proteins for medical treatment. These recombinant proteins are usually glycosylated for better efficacy. Bioprocess parameters significantly affect the protein productivity and glycosylation profiles. Ammonium is one of the most important inhibitory factors in cell culutre. Ammonium negatively inhibits glycosylation and productivity of glycoproteins. Chinese hamster ovary (CHO) cells are the most commonly used mammalian cell line for the production of recombinant glycoproteins; however, the impact of ammonium toxicity and prevention of ammonium toxicity have not been fully investigated. In this work, the prevention of ammonium toxicity by various amino acid supplements was examined. The effects of ammonium toxicity on gene expression levels were examined by two methods, differential display and quantitative real time reverse transcription polymerase chain reaction (QRT-PCR) analysis. Differential display analysis was used to identify unknown genes with varied expression, while QRT-PCR was used to quantify the expression of known genes.; Amino acid supplementations were investigated as a means to mitigate the negative effects of ammonium on CHO cell cultures. Threonine, proline and glycine were found to partially improve CHO cell growth rates, recombinant protein tissue plasminogen activator (t-PA) levels, and glycosylation profiles. Additionally, threonine, proline, and glycine additions positively impacted other key metabolic parameters. Thus, it was determined that threonine, proline, and glycine can protect CHO cells stressed by elevated ammonium levels.; Elevated ammonium has been reported to inhibit glycosylation profiles; however, the interaction of ammonium and glycosylation gene expression has not been completely elucidated. In this work, twelve glycosylation related C. griseus genes were evaluated by QRT-PCR. It was determined that protein glycosylation, and specifically sialylation, were sensitive to elevated ammonium due to altered gene expression levels of Gal (beta1-3/4)-GlcNAc-alpha (2,3)-sialyltransferase and CMP-sialic acid transporter. Additionally, the enzyme activity and gene expression levels of sialidase, a glycosylation degradation enzyme, was insensitive to ammonium and culture time, which confirms that reduced synthesis rates, not increased degradation rates, were responsible for decreased glycosylation under elevated ammonium. The gene expression levels of selected precursor genes were insensitive to ammonium and culture time, which indicates these precursor pathways were not responsible for the observed glycosylation variations due to elevated ammonium. (Abstract shortened by UMI.)...