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

Abstract

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...

Anti-Erythropoietin Antibodies and Pure Red Cell Aplasia

“…Regarding the association with the formulation of epoetin alfa marketed outside the United States, there is no reason to believe that the amino acid sequence of the molecule is different in this particular brand. Subtle differences exist between the carbohydrate moieties of epoetin alfa and beta (31,32), but it seems unlikely that they have significant impact on immunogenicity. Moreover, there is no indication that the glycosylation pattern of epoetin alfa has changed over time and could therefore explain the uspsurge of PRCA cases.…”

Relationships between the N‐glycan structures and biological activities of recombinant human erythropoietins produced using different culture conditions and purification procedures

“…However, only limited data have been reported relating to the detailed N-glycan structures of the isoforms of EPO with their biological activities (Takeuchi et al, 1989). More detailed structural information about differences in glycosylation between EPOs can be obtained by N-glycan profiling techniques (Rice et al, 1992;Nimtz et al, 1993;Kanazawa et al, 1999;Skibeli et al, 2001), based on the release of N-glycans and their subsequent mapping. We have recently also described such a method for profiling the N-glycans of glycoproteins (Yuen et al, 2002), which can be applied to small (2-5 lg) amounts of EPO.…”

Biosimilar epoetins and other “follow‐on” biologics: Update on the European experiences

“…In contrast to the other epoetins, epoetin omega (Repotin 1 , Bioclones, Cape Town, South Africa) is produced in EPO cDNA-transfected baby hamster kidney (BHK) cells. Epoetin omega has an N-glycan with phosphorylated oligomannoside chains, and it possesses less O-glycans than the CHO-cell derived rhEPOs [81,84]. The clinical consequences of these glycosylation differences have not been studied, probably because epoetin omega is not widely used.…”

“…The amino acid sequence of all epoetins is identical with that of endogenous EPO, but the glycans of the products exhibit structural differences [11]. Epoetin alfa is more homogenous and possesses less basic isoforms than epoetin beta [80,81]. Reportedly, structural differences even exist between the established epoetin alfa formulations, Epogen 1 (Amgen) and Eprex 1 (J&J) [82].…”