Collaborative study for the establishment of erythropoietin BRP batch 2.

Abstract

The preparation and establishment of the 2nd European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for erythropoietin was the goal of a project run within the framework of the European Biological Standardisation Programme. The project, coded BSP062, was carried out between October 2002 and July 2003. The candidate preparation (cBRP2) was prepared in a similar manner to the first BRP batch (BRP1), as follows: -50:50 (weight/weight) blending of the two erythropoietin preparations currently available on the European market (epoietin-alpha and epoietin-beta), -lyophilisation using a protein-free carrier formulation to allow use of the standard for both biological and physico-chemical assay methods, -each vial contains approximately 250 microg erythropoietin. The cBRP2 was analysed in a collaborative study, carried out with the following aims: -to calibrate cBRP2 by in vivo bioassay in terms of the International Standard for erythropoietin, and assign a unitage, -to demonstrate continuity of unitage with BRP1, -to evaluate the suitability of cBRP2 to serve as a reference material for physico-chemical tests of erythropoietin. The collaborative study involved 14 laboratories both from Europe, and from Australia, Canada, South-Korea and the United States of America. Participants carried out biological and physicochemical assays on the candidate BRP batch 2, using BRP 1 and the 2nd World Health Organization (WHO) International Standard (IS) for recombinant erythropoietin as the reference standards. It was demonstrated that: -an assigned potency of 32,500 U per vial would maintain continuity between BRP1 and BRP2 in terms of the IS for erythropoietin, -the replacement batch was appropriate for use as erythropoietin BRP in the context of the control of erythropoietin concentrated solutions according to the Ph. Eur. monograph 1316. In July 2003, the Ph. Eur. Commission established the proposed standard as 'Erythropoeitin BRP batch 2' for use as a reference preparation for the polycythaemic and normocythaemic mouse bioassay, with an assigned potency of 32,500 U/vial, the identification by capillary zone electrophoresis (CZE), by polyacrylamide gel electrophoresis, immunoblotting and peptide mapping and as a reference for checking the system suitability of size exclusion chromatographic procedures used in the test for 'Dimers and related substances of higher molecular mass'.