Reduction of CMP-N-acetylneuraminic acid hydroxylase activity in engineered Chinese hamster ovary cells using an antisense-RNA strategy

Chenu, S.; Grégoire, Anne; Malykh, Yanina N.; Visvikis, Athanase; Monaco, Lucía; Shaw, Lee; Schauer, Roland; Marc, Annie; Goergen, Jean‐Louis

doi:10.1016/s0304-4165(03)00137-5

Cited by 43 publications

(26 citation statements)

References 44 publications

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“…In contrast, there are very few literature reports describing the impact of culture conditions on glycoprotein Neu5Gc levels. Neu5Gc levels have been modulated using antisense-RNA techniques (Chenu et al, 2003), and there are reports of Neu5Gc being affected by environmental conditions such as high pCO 2 (Kimura and Miller, 1997), manipulation of nucleotide-sugar levels by feeding N-acetylmannosamine (Baker et al 2001), and by HEPES buffer and high pH (Muthing et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Effects of culture conditions on N‐glycolylneuraminic acid (Neu5Gc) content of a recombinant fusion protein produced in CHO cells

Borys

Dalal

Abu‐Absi

et al. 2010

Biotech & Bioengineering

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CHO cells express glycoproteins containing both the N-acetylneuraminic acid (Neu5Ac) and minor amounts of the N-glycolylneuraminic acid (Neu5Gc) forms of sialic acid. As Neu5Gc is not expressed in humans and can be recognized as a foreign epitope, there is the potential for immunogenicity issues for glycoprotein therapeutics. During process development of a glycosylated fusion protein expressed by CHO cells, a number of culture conditions were identified that affected the Neu5Gc content of the recombinant glycoprotein. Sodium butyrate (SB), a well-known additive reported to enhance recombinant protein productivity in specific cases, minimally affected product titers here, but did decrease Neu5Gc levels by 50-62%. A shift in culture temperature to a lower value after the exponential growth phase was used to extend the culture period. It was found that the Neu5Gc levels were 59% lower when the temperature shift occurred later near the stationary phase of the culture compared to an early-temperature shift, near the end of the exponential growth phase. Studies on the effects of pCO(2) with this product showed that the Neu5Gc levels were 46% lower at high pCO(2) conditions (140 mmHg) compared to moderate pCO(2) levels (20-80 mmHg). Finally, a comparison of sodium carbonate versus sodium hydroxide as the base used for pH control resulted in a reproducible 33% decrease in Neu5Gc in bioreactors using sodium hydroxide. These results are of practical importance as SB is a commonly tested additive, and the other factors affecting Neu5Gc can conveniently be used to reduce or control Neu5Gc in processes for the manufacture of glycoprotein therapeutics.

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Section: Introductionmentioning

confidence: 99%

Effects of culture conditions on N‐glycolylneuraminic acid (Neu5Gc) content of a recombinant fusion protein produced in CHO cells

Borys

Dalal

Abu‐Absi

et al. 2010

Biotech & Bioengineering

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“…The glycoform distribution and the shifting pattern of microheterogeneity on glycoproteins has been reported to be affected by cell culture conditions (Andersen et al, 2000;Chee Furng Wong et al, 2005;Cruz et al, 2000;Gawlitzek et al, 1995;Nyberg et al, 1999;Yang and Butler, 2000). In the efforts to control glycoform diversity, attempts have been made to engineer enzymes involved in the protein N-glycosylation pathway (Chenu et al, 2003;Davies et al, 2001;Lee et al, 1989;Mori et al, 2004;Potvin et al, 1990;Sburlati et al, 1998;Weikert et al, 1999), and modify the media composition (Baker et al, 2001;Gu and Wang, 1998).…”

Section: Introductionmentioning

confidence: 99%

GlycoVis: Visualizing glycan distribution in the protein N‐glycosylation pathway in mammalian cells

Hossler

Goh

2006

Biotech & Bioengineering

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Glycosylation has profound effects on the quality of recombinant proteins produced in mammalian cells. The biosynthetic pathways of N-linked glycans on glycoproteins involves a relatively small number of enzymes and nucleotide sugars. Many of these glycoconjugate enzymes can utilize multiple N-glycans as substrates, thus generating a large number of glycan intermediates, and making the biosynthetic pathway resemble a network with diverging and converging paths. The N-glycans on secreted glycoprotein molecules include not only terminal glycans, but also pathway intermediates. To better assess the glycan distribution and the potential route of their synthesis, we created GlycoVis, a visualization program that displays the distribution and the potential reaction paths leading to each N-glycan on the reaction network. The substrate specificities of the enzymes involved were organized into a relationship matrix. With the input of glycan distribution data, the program outputs a reaction pathway map which labels the relative abundance levels of different glycans with different colors. The program also traces all possible reaction paths leading to each glycan and identifies each pathway on the map. Glycoform distribution of Chinese Hamster Ovary cell-derived tissue plasminogen activator (TPA), and human and mouse IgG were used as illustrations for the application of GlycoVis. In addition, the intracellular and secreted IgG from an NS0 producer cell line were isolated, and their glycoform profiles were displayed using GlycoVis for comparison. This visualization tool facilitates the analysis of potential reaction paths utilized under different physiological or culture conditions, and may provide insight on the potential targets for metabolic engineering.

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“…In most animal cells, and in particular rodent cells including CHO, the synthesis of sialic acid sugar nucleotides proceeds to N-glycolyl-neuraminic acid (Neu5Gc) because these cells typically possess CMP-N-acetylneuraminic acid hydroxylase (CMP-Neu5Ac hydroxylase) activity [121]. However, human cells lack this enzyme and therefore glycostructures containing terminal Neu5Gc-sialic acid can be considered a foreign epitope to the human immune system.…”

Section: Glycoengineering Methods Targeting the Sugar Nucleotide Metamentioning

confidence: 99%

“…Therefore, the absence of Neu5Gc-sialic acid is an important quality attribute of biotherapeutics. In order to mitigate this potential problem Chenu et al have engineered CHO lines to achieve a downregulated CMP-Neu5Ac hydroxylase activity [121].…”

Section: Glycoengineering Methods Targeting the Sugar Nucleotide Metamentioning

confidence: 99%

3.5 Manipulation of Cell Growth, Metabolism and Product Quality Attributes

Horsten¹,

Winkler²,

Sandig³

2014

Animal Cell Biotechnology

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Reduction of CMP-N-acetylneuraminic acid hydroxylase activity in engineered Chinese hamster ovary cells using an antisense-RNA strategy

Cited by 43 publications

References 44 publications

Effects of culture conditions on N‐glycolylneuraminic acid (Neu5Gc) content of a recombinant fusion protein produced in CHO cells

Effects of culture conditions on N‐glycolylneuraminic acid (Neu5Gc) content of a recombinant fusion protein produced in CHO cells

GlycoVis: Visualizing glycan distribution in the protein N‐glycosylation pathway in mammalian cells

3.5 Manipulation of Cell Growth, Metabolism and Product Quality Attributes

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