title: An alternative to animal testing in the quality control of erythropoietin.
abstract: A physico-chemical method has been developed as an alternative to the current bioassay in normocythaemic mice for estimating the biological activity of erythropoietin batches. Capillary zone electrophoresis was used for quantification of the isoforms and their substructures were further elucidated by N-glycan mapping techniques. The analytical study was carried out on a total of 40 batches of epoetin beta which were selected to cover an adequate range of precisely established potency values. The relationship between the biological and chemical parameters was evaluated statistically in order to identify suitable covariates for the prediction of the biological activity. Out of several alternatives, a prediction model which is based on the percentages of isoforms per batch and the degree of sialidation was selected and tested. This model is comparable in terms of accuracy to the established in vivo bioassay, but is far superior in terms of precision. Further advantages of the method are improved animal welfare and savings in time and effort. The question whether the prediction model already meets the requirements for replacing the bioassay according to the ICH guideline Q6B is discussed.
authors.affiliation: Roche Diagnostics GmbH, Bioassays, Sandhoferstrasse 116, 68305 Mannheim. hanne.zimmermann@roche.com
authors.forename: H
authors.initials: H
authors.lastname: Zimmermann
authors.forename: D
authors.initials: D
authors.lastname: Gerhard
authors.forename: L A
authors.initials: LA
authors.lastname: Hothorn
authors.forename: T
authors.initials: T
authors.lastname: Dingermann
mesh_terms: D000595:Amino Acid Sequence; D000826:Animal Testing Alternatives / Q000379:methods / Q000592:standards; D000818:Animals; D000837:Anion Exchange Resins; D001681:Biological Assay; D002852:Chromatography, Ion Exchange; D019075:Electrophoresis, Capillary; D004921:Erythropoietin / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology / Q000592:standards; D006031:Glycosylation; D006397:Hematinics / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology / Q000592:standards; D006410:Hematopoiesis / Q000187:drug effects; D016014:Linear Models; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D025341:Principal Component Analysis; D011487:Protein Conformation; D020033:Protein Isoforms; D011786:Quality Control; D011994:Recombinant Proteins; D015203:Reproducibility of Results; D012794:Sialic Acids / Q000032:analysis; D020349:Surface Plasmon Resonance
chemical_list: D000837:Anion Exchange Resins; D006397:Hematinics; D020033:Protein Isoforms; D011994:Recombinant Proteins; D012794:Sialic Acids; C103998:epoetin beta; D004921:Erythropoietin