Purification of human recombinant anti-mullerian hormone and its derivatives

Abstract

Anti-mullerian hormone (AMH) is a cytokine of transforming growth factor β (TGF-β) superfamily able to induce apoptosis in cells bearing specific AMH type II receptors (AMHRII). AMHRII is overexpressed in some malignant cells, so at present recombinant AMH (rAMH) is considered as a new candidate antineoplastic drug. The use of rAMH may be especially effective in case of such severe diseases as ovarian, prostate and breast cancer. However, the development of a new drug is hampered by the laboriousness of obtaining highly purified rAMH and by the lack of data about the pharmacological characteristics of rAMH derivatives. In this work, we obtained preparations of prohormone, half-cleaved rAMH and a C-terminal fragment of rAMH, which was confirmed by qualitative and quantitative analyses. To obtain rAMH and its derivatives we used a previously developed highly effective producer strain containing the optimized human AMH gene. The production process has been divided into several stages: (a) rAMH biosynthesis in the bioreactor; (b) culture media preparation; (c) purification of rAMH and its derivatives using immunoaffinity chromatography and reversed-phase HPLC; (d) identification of the purified proteins by immunoblotting and analytical reversed-phase HPLC; and (e) evaluation of the hormone forms activity. The obtained proteins may be used in preclinical trials and in vitro study of rAMH derivatives properties.