Structural Analysis of the Sialylated N- and O-Linked Carbohydrate Chains of Recombinant Human Erythropoietin Expressed in Chinese Hamster Ovary Cells. Sialylation Patterns and Branch Location of Dimeric N-acetyllactosamine Units

Hokke, Cornelis H.; Bergwerff, Aldert A.; Dedem, G.W.K. van; Kamerling, J.P.; Vliegenthart, Johannes F.G.

doi:10.1111/j.1432-1033.1995.tb20350.x

Cited by 137 publications

(70 citation statements)

References 42 publications

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“…For NS0-derived glycoproteins, the NGNA can be >50% of the total sialylation, whereas in CHO cells the proportion is lower but not insignificant; 1-5% for one study (Hokke et al 1995) and 15% in another (Baker et al 2001). …”

Section: Immunogenicity Of Non-human Structuresmentioning

confidence: 95%

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: Immunogenicity Of Non-human Structuresmentioning

confidence: 95%

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

2005

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“…For example, Asn-38 and Asn-83 of the human EPO play an essential role in EPO secretion, since site-directed mutagenesis removing one of these glycosylation sites prevents EPO secretion (39,40) and capping of external galactose residues by sialic acids prevents the recognition of EPO by the asialo glycoprotein binding protein of the liver (41,42). Characterization of the glycoforms of the commercial form of human EPO obtained from recombinant CHO cells have been highly documented (37,38,(43)(44)(45)(46)(47). However, data on the influence of the culture conditions on the glycosylation of this protein remain very sparse.…”

Section: Introductionmentioning

confidence: 98%

HPCE Monitoring of the N-Glycosylation Pattern and Sialylation of Murine Erythropoietin Produced by CHO Cells in Batch Processes

et al. 2004

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Using capillary electrophoresis coupled to laser-induced fluorescence (HPCE-LIF), it was possible to profile N-linked oligosaccharides from EPO, including species containing sialic acid, during the course of batch cultures performed either in serum-free or serum-containing medium. Although an unusual high heterogeneity of the N-linked oligosaccharides was observed by both SDS-PAGE and HPCE analysis, the patterns of mEPO glycans after desialylation by mild acid hydrolysis were found to be quite constant over the course of the cultures either with or without serum supplementation. In contrast, when the protein was analyzed by HPCE without acidic desialylation, fingerprints of N-linked oligosaccharides changed with time in serum-containing conditions. This phenomenon appeared to be mainly due to the desialylation of mEPO as a result of a sialidase activity released upon cell lysis. These results demonstrate that though a higher EPO titer was obtained in serum supplemented conditions, sialylation of EPO was severely affected by the presence of serum in the culture medium.

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“…As expected from the similarity of its N-linked oligosaccharide profiles to those of r-shIFNAR2, the types of N-linked oligosaccharides in EPO consist of the core-fucosylated structures with di-, tri-, tetraantennae, and N-acetyllactosamine-containing higher-order structures. These structures in EPO were first determined using NMR and/or GC-MS methods [26,29,53], then later by FACE [16,51]. Differences in the Nlinked oligosaccharides between the two glycoproteins include the tetra-antennary structure with three N-acetyllactosamines in EPO that was not observed in r-shIF-NAR2 and the penta-antennary structure in r-shIFNAR2 that was not observed in EPO.…”

Section: Discussionmentioning

confidence: 99%

Structural characterization of oligosaccharides in recombinant soluble human interferon receptor 2 using fluorophore-assisted carbohydrate electrophoresis

Frado¹,

Strickler²

2000

Electrophoresis

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The N-linked oligosaccharide profiles (banding patterns in gels) and structures of recombinant soluble human interferon receptor 2 (r-shIFNAR2) were determined using fluorophore-assisted carbohydrate electrophoresis (FACE, Glyko, Novato, CA). The method involves releasing N-linked oligosaccharide moieties from a glycoprotein by digestion with peptide-N glycanase (PNGase F), labeling the released oligosaccharides with the fluorescent dye 8-aminonaphthalene-1,3,6-trisulfonate (ANTS), and separating the labeled oligosaccharides by gel electrophoresis. The isolated oligosaccharides in the bands from the profiling gels can then be sequenced using exoglycosidases to reveal the oligosaccharide structures. The oligosaccharide profile of r-shIFNAR2 consists of at least nine oligosaccharide bands. The relative amount of oligosaccharide in each band can vary, depending on the culture conditions of the source cells. FACE structural analysis shows that r-shIFNAR2 contains only core-fucosylated N-linked oligosaccharides, most of which are fully sialylated (approximately 92%). The major types and relative amounts of the oligosaccharides from a representative sample are: disialylated, galactosylated, biantennary (15%); trisialylated, galactosylated, triantennary (19%), tetrasialylated, galactosylated, tetraantennary (30%), and N-acetyllactosamine-containing higher-order oligosaccharides including tri-, tetra-, and pentaantennary (28%). The remaining oligosaccharides are not fully sialylated and/or not fully galactosylated di-, tri-, and tetraantennary structures (approximately 5%) and unidentified structures (approximately 3%). A method for determining the types and structures of the N-acetyllactosamine containing oligosaccharides is also reported in this study.

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Structural Analysis of the Sialylated N- and O-Linked Carbohydrate Chains of Recombinant Human Erythropoietin Expressed in Chinese Hamster Ovary Cells. Sialylation Patterns and Branch Location of Dimeric N-acetyllactosamine Units

Cited by 137 publications

References 42 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

HPCE Monitoring of the N-Glycosylation Pattern and Sialylation of Murine Erythropoietin Produced by CHO Cells in Batch Processes

Structural characterization of oligosaccharides in recombinant soluble human interferon receptor 2 using fluorophore-assisted carbohydrate electrophoresis

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