Sindbis Virus Glycoproteins Are Abnormally Glycosylated in Chinese Hamster Ovary Cells Deprived of Glucose

Davidson, Sandra K.; Hunt, Lawrence A.

doi:10.1099/0022-1317-66-7-1457

Cited by 26 publications

(11 citation statements)

References 35 publications

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“…Reduced site-occupancy of N-glycans was also shown in immunoglobulin synthesis from mouse myeloma cells at low glucose concentration (<0.5 mM; Stark and Heath 1979). Davidson and Hunt (1985) showed under-glycosylation and the presence of abnormal truncated glycans in viral proteins derived from CHO cells deprived of glucose.…”

Section: Supplementsmentioning

confidence: 80%

Optimisation of the Cellular Metabolism of Glycosylation for Recombinant Proteins Produced by Mammalian Cell Systems

2005

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Many biopharmaceuticals are now produced as secreted glycoproteins from mammalian cell culture. The glycosylation profile of these proteins is essential to ensure structural stability and biological and clinical activity. However, the ability to control the glycosylation is limited by our understanding of the parameters that affect the heterogeneity of added glycan structures. It is clear that the glycosylation process is affected by a number of factors including the 3-dimensional structure of the protein, the enzyme repertoire of the host cell, the transit time in the Golgi and the availability of intracellular sugar-nucleotide donors. From a process development perspective there are many culture parameters that can be controlled to enable a consistent glycosylation profile to emerge from each batch culture. A further, but more difficult goal is to control the culture conditions to enable the enrichment of specific glycoforms identified with desirable biological activities. The purpose of this paper is to discuss the cellular metabolism associated with protein glycosylation and review the attempts to manipulate, control or engineer this metabolism to allow the expression of human glycosylation profiles in producer lines such as genetically engineered Chinese hamster ovary (CHO) cells.

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Section: Supplementsmentioning

confidence: 80%

Optimisation of the Cellular Metabolism of Glycosylation for Recombinant Proteins Produced by Mammalian Cell Systems

2005

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“…It has been reported that a shortage of oligosaccharide precursors caused the addition of abnormally small oligosaccharides at the initial step of glycosylation from dolichol precursors (Davidson and Hunt, 1985;Elbein, 1987;Rearick et al, 1981). Several studies have shown that glucose limitation results in incomplete protein glycosylation.…”

Section: Discussionmentioning

confidence: 95%

Effect of Ammonia on the Glycosylation of Human Recombinant Erythropoietin in Culture

Yang

Butler

2000

Biotechnol. Prog.

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Recombinant human erythropoietin (EPO) was produced by a stable transfected CHO-K1 cell clone (EPO-81) grown in serum-free medium. Our previous work showed that there was a significant increase in the heterogeneity of the glycoforms of EPO and a reduction of the sialylation at 20 mM NH(4)Cl. In the work presented here, the effects of ammonia on EPO N-linked oligosaccharides were analyzed. EPO was purified from culture supernatants by immunoaffinity chromatography. The N-linked oligosaccharides were released enzymatically and analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) and HPLC. The FACE N-linked oligosaccharide profile showed that the sialylated glycans contain one prominent band at a position corresponding to eight glucose units. The density of the major band was greatly diminished and the width was significantly increased in cultures containing added ammonia. The proportion of tetraantennary structures was reduced by 60%, while the tri- and biantennary structures were increased proportionally in the presence of ammonia. Glycan analysis by HPLC using a weak anion exchange column showed that the most significant characteristic effect of ammonia was a reduction of the proportion of glycans with four sialic acids from 46% in control cultures to 29% in ammonia-treated cultures. Analysis of the desialylated glycans by normal phase chromatography indicated a distribution of tetra-, tri-, and biantennary structures similar to that shown by FACE. The N-linked glycan sequence was determined by sequential exoglycosidase digestion followed by FACE. The results indicated a typical N-linked complex oligosaccharide structure. Glycans from ammonia-containing cultures showed the same sequence pattern. In conclusion, we showed that ammonia in the culture medium affected EPO glycosylation, which was observed as a reduction of the tetraantennary and tetrasialylated oligosaccharide structures. However, the presence of ammonia in the cultures did not change the oligosaccharide sequence.

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“…Reduced N-glycan occupancy was also shown in immunoglobulin synthesis from mouse myeloma cells at low glucose concentration (<0.5 mM) (Stark and Heath, 1979). In addition, abnormal glycosylation was found on viral proteins derived from CHO cells grown in a glucose-limited culture (Davidson and Hunt, 1985). …”

Section: Glucosementioning

confidence: 96%

The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the N-glycosylation profile of a monoclonal antibody

Liu

Spearman

Doering

et al. 2014

Journal of Biotechnology

102

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Sindbis Virus Glycoproteins Are Abnormally Glycosylated in Chinese Hamster Ovary Cells Deprived of Glucose

Cited by 26 publications

References 35 publications

Optimisation of the Cellular Metabolism of Glycosylation for Recombinant Proteins Produced by Mammalian Cell Systems

Optimisation of the Cellular Metabolism of Glycosylation for Recombinant Proteins Produced by Mammalian Cell Systems

Effect of Ammonia on the Glycosylation of Human Recombinant Erythropoietin in Culture

The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the N-glycosylation profile of a monoclonal antibody

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