Inactivation of Erythropoietin by Neuraminidase and by Mild Substitution Reactions

Lowy, Peter H.; Keighley, Geoffrey; Borsook, Henry

doi:10.1038/185102a0

Cited by 103 publications

(18 citation statements)

References 16 publications

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“…For rHuEPO, in particular, it has been shown that the addition of carbohydrate is required for secretion from the cell, and for increasing the solubility of the molecule (Dube et al, 1988;Narhi et al, 1991;Delorme et al, 1992). Early research on EPO from natural sources indicated that the sialic acid residues were necessary for biological activity in vivo (Lowry et al, 1960;Lukowsky and Painter, 1972;Goldwasser et al, 1974). Removal of the sialic acid from either native EPO or rHuEPO resulted in molecules having an increased activity in vitro, but very low activity in vivo, presumably due to removal from circulation by the asialoglycoprotein receptor in the liver (Fukuda et al, 1989;Spivak and Hogans, 1989).…”

Section: Role Of Carbohydrate In Biological Activitymentioning

confidence: 99%

Development and characterization of novel erythropoiesis stimulating protein (NESP)

2001

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Studies on human erythropoietin (EPO) demonstrated that there is a direct relationship between the sialic acid-containing carbohydrate content of the molecule and its serum half-life and in vivo biological activity, but an inverse relationship with its receptor-binding affinity. These observations led to the hypothesis that increasing the carbohydrate content, beyond that found naturally, would lead to a molecule with enhanced biological activity. Hyperglycosylated recombinant human EPO (rHuEPO) analogues were developed to test this hypothesis. Darbepoetin alfa (novel erythropoiesis stimulating protein, NESP, ARANESP TM , Amgen Inc, Thousand Oaks, CA), which was engineered to contain 5 N-linked carbohydrate chains (two more than rHuEPO), has been evaluated in preclinical animal studies. Due to its increased sialic acid-containing carbohydrate content, NESP is biochemically distinct from rHuEPO, having an increased molecular weight and greater negative charge. Compared with rHuEPO, it has an approximate 3-fold longer serum half-life, greater in vivo potency, and can be administered less frequently to obtain the same biological response. NESP is currently being evaluated in human clinical trials for treatment of anaemia and reduction in its incidence.© 2001 Cance Cance Cancer Research Campaign

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Section: Role Of Carbohydrate In Biological Activitymentioning

confidence: 99%

Development and characterization of novel erythropoiesis stimulating protein (NESP)

2001

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“…by guest www.bloodjournal.org From bioactivity of hEPO. 11,[77][78][79][80] Asialo-hEPO is rapidly cleared from the circulation by the galactose-receptor in the liver and has, therefore, no in vivo bioactivity. Several reports on endogenous hEPO have shown that circulating hEPO contains fewer acidic glycoforms than urinary hEPO.…”

Section: Discussionmentioning

confidence: 99%

Sugar profiling proves that human serum erythropoietin differs from recombinant human erythropoietin

Skibeli

Nissen-Lie

Torjesen

2001

Blood

201

160

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Erythropoietin (EPO) from sera obtained from anemic patients was successfully isolated using magnetic beads coated with a human EPO (hEPO)-specific antibody. Human serum EPO emerged as a broad band after sodium dodecyl sulfatepolyacrylamide gel electrophoresis, with an apparent molecular weight slightly smaller than that of recombinant hEPO (rhEPO). The bandwidth corresponded with microheterogeneity because of extensive glycosylation. Two-dimensional gel electrophoresis revealing several different glycoforms confirmed the heterogeneity of circulating hEPO. The immobilized anti-hEPO antibody was capable of binding a representative selection of rhEPO glycoforms. This was shown by comparing normal-phase high-performance liquid chromatography profiles of oligosaccharides released from rhEPO with oligosaccharides released from rhEPO after isolation with hEPO-specific magnetic beads. Charge analysis demonstrated that human serum EPO contained only mono-, di-, and tri-acidic oligosaccharides and lacked the tetra-acidic structures present in the glycans from rhEPO. Determination of charge state after treatment of human serum EPO with Arthrobacter ureafaciens sialidase showed that the acidity of the oligosaccharide structures was caused by sialic acids. The sugar profiles of human serum EPO, describing both neutral and charged sugar, appeared significantly different from the profiles of rhEPO. The detection of glycan structural discrepancies between human serum EPO and rhEPO by sugar profiling may be significant for diagnosing pathologic conditions, maintaining pharmaceutical quality control, and establishing a direct method to detect the misuse of rhEPO in sports. IntroductionHuman erythropoietin (EPO) is a glycoprotein that is synthesized mainly in the kidney and that stimulates erythropoiesis through actions on erythroid progenitor cells. [1][2][3] Human EPO (hEPO) was the first hematopoietic growth factor to be cloned. 4,5 Recombinant hEPO (rhEPO) has been available as a drug since 1988 and is used in the clinical treatment of anemia, especially anemia caused by renal failure. In sports the misuse of rhEPO has been suspected among athletes for several years. 6 Active hEPO consists of a single 165-amino acid polypeptide chain with three N-glycosylation sites at Asn24, Asn38, and Asn83, respectively, and one O-glycosylation site at Ser126. The average carbohydrate content is approximately 40%. 7-9 Glycosylation is important for the biologic activity of EPO. Removal or modification of the glycan chains results in altered in vivo and in vitro activity. [9][10][11][12][13][14] Furthermore, the number of sialic acid residues and the branching pattern of the N-linked oligosaccharides modify the pharmacodynamics, speed of catabolism, and biologic activity of hEPO. 11,15,16 Oligosaccharide units, or glycans, of glycoproteins serve a variety of functions, including folding of nascent polypeptide chains in the endoplasmic reticulum, protection of the protein moieties from the action of proteases, and modulation of biologic act...

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“…Reaction with p-chloromercuriben zoate, on the other hand, did not·inacti vate, nor did esterifica tion of primary alcohol groups with anhydrous formic acid (19).…”

Section: Purity Of Erythropoietinmentioning

confidence: 99%

Purification and Properties of Erythropoietin.

Goldwasser¹,

Kung²

1972

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Inactivation of Erythropoietin by Neuraminidase and by Mild Substitution Reactions

Cited by 103 publications

References 16 publications

Development and characterization of novel erythropoiesis stimulating protein (NESP)

Development and characterization of novel erythropoiesis stimulating protein (NESP)

Sugar profiling proves that human serum erythropoietin differs from recombinant human erythropoietin

Purification and Properties of Erythropoietin.

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