1Heat-Induced Aggregation of Recombinant Erythropoietin in the Intact and Deglycosylated States as Monitored by Gel Permeation Chromatography Combined with a Low-Angle Laser Light Scattering Technique1

Endo, Yoshiyuki; Nagai, Hiroshi; Watanabe, Yoshihiko; Ochi, Kiyoshige; Takagi, Toshio

doi:10.1093/oxfordjournals.jbchem.a123961

Cited by 51 publications

(27 citation statements)

References 19 publications

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“…Similar results were found for erythropoietin (EPOGEN ® , PROCIT ® ; Amgen, Ortho) by Endo et al . 222. Hoiberg-Nielsen et al also reported increased colloidal stability for the glycosylated form of phytase 218.…”

Section: Physical Instabilities Prevented By Glycosylationmentioning

confidence: 98%

Effects of glycosylation on the stability of protein pharmaceuticals

Solá

Griebenow

2009

Journal of Pharmaceutical Sciences

555

393

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In recent decades, protein-based therapeutics have substantially expanded the field of molecular pharmacology due to their outstanding potential for the treatment of disease. Unfortunately, protein pharmaceuticals display a series of intrinsic physical and chemical instability problems during their production, purification, storage, and delivery that can adversely impact their final therapeutic efficacies. This has prompted an intense search for generalized strategies to engineer the long-term stability of proteins during their pharmaceutical employment. Due to the well known effect that glycans have in increasing the overall stability of glycoproteins, rational manipulation of the glycosylation parameters through glycoengineering could become a promising approach to improve both the in vitro and in vivo stability of protein pharmaceuticals. The intent of this review is therefore to further the field of protein glycoengineering by increasing the general understanding of the mechanisms by which glycosylation improves the molecular stability of protein pharmaceuticals. This is achieved by presenting a survey of the different instabilities displayed by protein pharmaceuticals, by addressing which of these instabilities can be improved by glycosylation, and by discussing the possible mechanisms by which glycans induce these stabilization effects.

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Section: Physical Instabilities Prevented By Glycosylationmentioning

confidence: 98%

Effects of glycosylation on the stability of protein pharmaceuticals

Solá

Griebenow

2009

Journal of Pharmaceutical Sciences

555

393

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“…The EPO molecule has been well-characterized [5][6][7][8][9] and is a stable molecule that remains predominantly in monomeric form when stored at 2-8 • C. However, when the product is exposed to higher temperatures or to certain stress conditions, dimer and higher order aggregates of r-HuEPO can be formed [10,11]. As with many other marketed biopharmaceuticals, to protect the active protein against denaturation or aggregate formation, nonionic surfactants such as polysorbate 80 are included as stabilizers [12][13][14][15].…”

Section: Introductionmentioning

confidence: 99%

Development of a sensitive size exclusion HPLC method with fluorescence detection for the quantitation of recombinant human erythropoietin (r-HuEPO) aggregates

Gunturi¹,

Ghobrial²,

Sharma³

2007

Journal of Pharmaceutical and Biomedical Analysis

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“…These carbohydrates can be attached to the hydroxyl group on a serine or threonine (O-linked glycosylation) or the amine of an asparagine via an N-glycosidic bond (N-linked glycosylation). The addition of carbohydrate chains to the polypeptide backbone of a protein may have an impact on the structure, solubility, antigenicity, folding, secretion, and stability of the protein (1)(2)(3)(4)(5)(6)(7)(8). The carbohydrate may also affect the clearance rate and in vivo activity of the protein (9 -12).…”

mentioning

confidence: 99%

Structural Requirements for Additional N-Linked Carbohydrate on Recombinant Human Erythropoietin

Elliott

Chang

Delorme

et al. 2004

Journal of Biological Chemistry

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N-Linked glycosylation is a post-translational event whereby carbohydrates are added to secreted proteins at the consensus sequence Asn-Xaa-Ser/Thr, where Xaa is any amino acid except proline. Some consensus sequences in secreted proteins are not glycosylated, indicating that consensus sequences are necessary but not sufficient for glycosylation. In order to understand the structural rules for N-linked glycosylation, we introduced N-linked consensus sequences by site-directed mutagenesis into the polypeptide chain of the recombinant human erythropoietin molecule. Some regions of the polypeptide chain supported N-linked glycosylation more effectively than others. N-Linked glycosylation was inhibited by an adjacent proline suggesting that sequence context of a consensus sequence could affect glycosylation. One N-linked consensus sequence (Asn 123 -Thr 125 ) introduced into a position close to the existing O-glycosylation site (Ser 126 ) had an additional O-linked carbohydrate chain and not an additional Nlinked carbohydrate chain suggesting that structural requirements in this region favored O-glycosylation over N-glycosylation. The presence of a consensus sequence on the protein surface of the folded molecule did not appear to be a prerequisite for oligosaccharide addition. However, it was noted that recombinant human erythropoietin analogs that were hyperglycosylated at sites that were normally buried had altered protein structures. This suggests that carbohydrate addition precedes polypeptide folding.Secreted proteins are often glycosylated during transit through the secretory apparatus in eukaryotic cells. These carbohydrates can be attached to the hydroxyl group on a serine or threonine (O-linked glycosylation) or the amine of an asparagine via an N-glycosidic bond (N-linked glycosylation). The addition of carbohydrate chains to the polypeptide backbone of a protein may have an impact on the structure, solubility, antigenicity, folding, secretion, and stability of the protein (1-8). The carbohydrate may also affect the clearance rate and in vivo activity of the protein (9 -12).The nature of the signal for carbohydrate addition is partially understood. N-Linked carbohydrate addition is mediated by oligosaccharide transferase and occurs at asparagine residues that are part of the consensus sequence Asn-Xaa-(Ser/ Thr), where Xaa can be any amino acid, except proline (13-16). The observation that not all consensus sequences are glycosylated suggests that there are additional sequence or conformational requirements essential for efficient carbohydrate attachment (17,18). Although at least 12-14 amino acids must be synthesized and have entered the luminal surface of the endoplasmic reticulum for carbohydrate addition, the synthesis of the protein need not be completed for glycosylation to take place (19,20). This suggests that the structures for carbohydrate addition are recognized in partially folded molecules. The sequence context of the glycosylation site has also been shown to influence the efficiency of glycos...

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1Heat-Induced Aggregation of Recombinant Erythropoietin in the Intact and Deglycosylated States as Monitored by Gel Permeation Chromatography Combined with a Low-Angle Laser Light Scattering Technique1

Cited by 51 publications

References 19 publications

Effects of glycosylation on the stability of protein pharmaceuticals

Effects of glycosylation on the stability of protein pharmaceuticals

Development of a sensitive size exclusion HPLC method with fluorescence detection for the quantitation of recombinant human erythropoietin (r-HuEPO) aggregates

Structural Requirements for Additional N-Linked Carbohydrate on Recombinant Human Erythropoietin

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