The International Reference Preparation of Tetracosactide for Bioassay: characterization and estimation of its (1-24)corticotrophin-tetracosapeptide content by physicochemical and biological methods.

The preparation and nature of the International Reference Preparation of Tetracosactide for Bioassay (IRP; in ampoules coded 80/590) are described. The IRP was studied by six laboratories in five countries using in-vivo and in-vitro bioassays and various physicochemical methods. The bulk (1-24)corticotrophin-tetracosapeptide (batch 000179) from which the IRP was prepared contained 10.4% (w/w) acetic acid and 8.3% (w/w) water; its (1-24)corticotrophin-tetracosapeptide content was estimated to be 71.7% (w/w) by amino acid analysis, 74.2% (w/w) by high performance liquid chromatography (HPLC) and 77.5% (w/w) by spectrophotometry. (1-24)Corticotrophin-tetracosapeptide accounted for more than 90% (w/w) of the total peptide in the IRP as judged by HPLC, thin-layer chromatography, carboxymethyl-cellulose chromatography, isoelectric focusing (IEF) and electrophoresis. The homogeneity of the peptide in the IRP was similar by all methods to that in batch 000179 from which it was prepared. The (1-24)corticotrophin-tetracosapeptide content of the IRP (with 95% confidence limits), in terms of batch 000179, was found to be 491 micrograms/ampoule by HPLC and spectrophotometry, 473 (433-513) micrograms/ampoule by IEF and 505 (473-539) micrograms/ampoule by the in-vitro rat adrenocortical cell assay. A comparison in the same bioassay system of the IRP with a laboratory house standard of (1-24)corticotrophin-tetracosapeptide, which originated from a different manufacturer, gave similar results. Accelerated thermal degradation studies of the IRP by adrenocortical cell assay, HPLC and IEF suggested that more than 99.9% of its original content of (1-24)corticotrophin-tetracosapeptide would remain after 10 years under normal storage conditions of -20 degrees C in the dark. Bioassay estimates of samples of the IRP which had undergone significant degradation were higher than estimates by HPLC, indicating that molecular species other than (1-24)corticotrophin-tetracosapeptide contributed to their corticotrophic activity. The corticotrophic activity of the IRP was demonstrated by cytochemical bioassay and by in-vivo bioassays as well as by the adrenocortical cell assay. After consideration of these data, the Expert Committee on Biological Standardization of the World Health Organization established the ampoule d preparation, coded 80/590, as the International Reference Preparation of Tetracosactide for Bioassay and assigned to it a potency of 490 i.u./ampoule; thus the i.u. is represented by 1 microgram (1-24)corticotrophin-tetracosapeptide.