In vitro glycosylation of proteins: An enzymatic approach

Meynial-Salles, Isabelle; Combes, Didier

doi:10.1016/0168-1656(95)00174-3

Cited by 29 publications

(21 citation statements)

References 68 publications

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“…However, in lower eukaryotes such as fungi and yeast the high-mannose structures are released from the cell as end-products of glycosylation with multiple mannose residues and also in some cases, hybrid structures have been found (Figure 4). These consist of branches from the mannose core of both complex and high-mannose type structures (Meynial-Salles and Combes 1996).…”

Section: N-glycansmentioning

confidence: 99%

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: N-glycansmentioning

confidence: 99%

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

2005

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“…These effects depend on several cellular factors: (1) enzyme repertoire and localization (Ferrara et al, 2006;Meynial-Salles and Combes, 1996); (2) competition between different enzymes for one substrate (Umaña and Bailey, 1997); (3) transit time of the glycoproteins through the Golgi apparatus (Hooker et al, 1999;Nabi and Dennis, 1998); (4) levels of nucleotide sugar donors (Nyberg et al, 1999;Valley et al, 1999); and last but not the least (5) competition between different glycosylation sites on the protein for the same pool of enzymes (Schachter et al, 1983).…”

Section: Heterogeneity In N-glycosylation Pathwaymentioning

confidence: 99%

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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“…Development of a yeast secretion system which glycosylates heterologous proteins properly and applies carbohydrates of minimal immunogenicity in humans could be achieved as we understand more about yeast protein secretion and glycosylation. The in vitro reconstitution of a yeast-generated recombinant protein into the glycoform identical to the authentic glycoprotein could also be expected as more advances are made in the chemical production of oligosaccharides (Meynial-Salles and Combes, 1996). The heterogeneity in the glycosylation of proteins is known to be affected not only by the different types of host cells but also by the culture conditions that have a profound influence on the physiological status of the host cell.…”

Section: Discussionmentioning

confidence: 99%

Glycosylation of human α1‐antitrypsin in Saccharomyces cerevisiae and methylotrophic yeasts

Kang¹,

Sohn²,

Choi³

et al. 1998

Yeast

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Human alpha 1-antitrypsin (alpha 1-AT) is a major serine protease inhibitor in plasma, secreted as a glycoprotein with a complex type of carbohydrate at three asparagine residues. To study glycosylation of heterologous proteins in yeast, we investigated the glycosylation pattern of the human alpha 1-AT secreted in the baker's yeast Saccharomyces cerevisiae and in the methylotrophic yeasts, Hansenula polymorpha and Pichia pastoris. The partial digestion of the recombinant alpha 1-AT with endoglycosidase H and the expression in the mnn9 deletion mutant of S. cerevisiae showed that the recombinant alpha 1-AT secreted in S. cerevisiae was heterogeneous, consisting of molecules containing core carbohydrates on either two or all three asparagine residues. Besides the core carbohydrates, variable numbers of mannose outer chains were also added to some of the secreted alpha 1-AT. The human alpha 1-AT secreted in both methylotrophic yeasts was also heterogeneous and hypermannosylated as observed in S. cerevisiae, although the overall length of mannose outer chains of alpha 1-AT in the methylotrophic yeasts appeared to be relatively shorter than those of alpha 1-AT in S. cerevisiae. The alpha 1-AT secreted from both methylotrophic yeasts retained its biological activity as an elastase inhibitor comparable to that of alpha 1-AT from S. cerevisiae, suggesting that the different glycosylation profile does not affect the in vitro activity of the protein.

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In vitro glycosylation of proteins: An enzymatic approach

Cited by 29 publications

References 68 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

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

Glycosylation of human α1‐antitrypsin in Saccharomyces cerevisiae and methylotrophic yeasts

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