1998-British_Journal_of_Haematology.pdf - British Journal of Haematology 1998 100 79\u201389 Epoetin alfa and beta differ in their erythropoietin isoform

1998-British_Journal_of_Haematology.pdf - British Journal...

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Epoetin alfa and beta differ in their erythropoietin isoform compositions and biological properties P. L. S TORRING , R. J. T IPLADY , R. E. G AINES D AS , B. E. S TENNING , A. L AMIKANRA , B. R AFFERTY AND J. L EE The National Institute for Biological Standards and Control (W.H.O. International Laboratory for Biological Standards), Blanche Lane, South Mimms, Potters Bar, Herts. Received 8 July 1997; accepted for publication 14 October 1997 Summary. Epoetin alfa and beta are the two forms of recombinant DNA-derived erythropoietin (rEPO), both synthesized in Chinese hamster ovary cells, which are used for the treatment of erythropoietin (EPO)-responsive anae- mias. Several batches of each of these rEPOs were compared for differences in their EPO isoform compositions by iso- electric focusing (IEF) and in a range of lectin-binding assays, and for differences in their EPO activities by in-vivo and in-vitro mouse bioassays and by immunoassay. Epoetin beta was found to differ from epoetin alfa in containing: (a) a greater proportion of more basic isoforms, (b) a greater proportion of EPO binding to Erythrina cristagalli agglutinin (which binds N-glycans with non- sialylated outer Gal b 1-4GlcNAc moieties), and (c) isoforms with higher in-vivo : in-vitro bioactivity ratios. Epoetin beta also contained slightly more than epoetin alfa of EPO binding to Lycopersicon esculentum agglutinin (which binds N-glycans containing repeating Gal b 1-4GlcNAc sequences), to the leucoagglutinin of Phaseolus vulgaris (which binds tetra- antennary and 2,6-branched triantennary N-glycans) and to Agaricus bisporus agglutinin (which binds Gal b 1-3GalNAc containing O-glycans). No differences were found between the two rEPOs in their binding to a further five lectins. The differences between the isoform composition of epoetin alfa and beta, and the smaller inter-batch differences appear to be due to differences in glycosylation. The higher murine in-vivo : in-vitro bioactivity ratio of epoetin beta compared to epoetin alfa could not be explained in terms of differences in their degrees of sialylation, but was consistent with differences in their pharmacokinetics and pharmaco- dynamics observed in human subjects. There have been no reports that epoetin alfa differs from epoetin beta in its clinical efficacy, but the differences between epoetin alfa and beta in some analytical systems suggest that there might be a need for separate international standards for these two types of rEPO. Keywords: epoetin alfa, epoetin beta, erythropoietin isoforms, lectin-binding, biological properties. Erythropoietin (EPO) is the principal regulator of erythropoi- esis (Krantz, 1991; Jelkmann, 1992). Like other glycoprotein hormones, EPO exists as mixtures of a multitude of isoforms, differing mainly in their glycosylation (Storring, 1992). The isoform composition of EPO preparations varies with their source, differing between samples from serum and urine (Tam et al , 1991; Wide et al , 1995), between samples obtained from subjects under different pathophysiological conditions (Wide & Bengtsson, 1990), and between human urinary EPOs
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