Multicenter collaborative study to calibrate salmon calcitonin by bioassayand high-performance liquid chromatography: Establishment of the Third International Standard

Salmon calcitonin (sCT) is widely used therapeutically in the treatment of patients with postmenopausal osteoporosis, Paget's disease, and some forms of hypercalcemia. Preparations of synthetic calcitonin peptides of high pur ity and reproducibility are now routinely produced and physicochemical meth ods, particularly reverse-phase high-performance liquid chromatography (RP- HPLC), are replacing the in vivo biological assay for monitoring and calibr ation. Although the bioassay is no longer required for routine batch contro l in Europe, calcitonin bioassays are still required in some countries and in the development of new products. Stocks of the Second International Stan dard (IS) for salmon calcitonin are now depleted and, to replace it with a new calibrant, an international collaborative study was organized in which the aims were to: determine the activity of the candidate sCT by in vivo bi oassay in terms of the second IS; assess the stability of the preparation a fter accelerated thermal degradation; estimate the purity of the ampouled c andidate preparation; and determine the sCT content in gravimetric units by HPLC. The HPLC data in terms of ampoule content were in good agreement giv ing an estimate of 23.1 (coefficient of variation [CV] 3.8%) mug per ampoul e. The HPLC chromatograms revealed a small, but detectable, degree of heter ogeneity, which possibly occurred during the formulating or ampouling proce dures, resulting in a reduction in monocomponent content (purity) from 96% to 92%. The biological activity of the ampoule contents in international un its (IU) was calculated from the mass value and the internationally agreed- upon figure of 6000 IU/per mg for the specific activity of salmon calcitoni n. This gave a value of 138 IU per ampoule, which was in good agreement wit h the biological assay estimate (140 IU per ampoule). The preparation of sC T was subsequently adopted as the Third International Standard by the World Health Organization with an assigned content of 138 IU per ampoule. (C) 20 01 by Elsevier Science Inc. All rights reserved.