Laboratory production and first characterization of purified recombinant human thyrotropin - IPEN

Abstract

Authentic recombinant human thyrotropin (rec-hTSH), an heterodimeric glycoprotein hormone: containing two non-covalently finked subunits alpha(s) and beta (B).secreted by transformed Chinose Hamster Ovary (CHO) colls, was obtained in our laboratory at IPEN-CNEN/SP. Tho production of rec-HTSH was carriod out in hollow fiber bioreactor of in 162 em* culture flasks, tho concentration of the protein of interest in the hollow fibor bioreactor being ~ 20pg/mL. in progence of dialysed fetal bovine sorum (dFBS) or ~7ug/mL when dFBS is absent and - Sug/mL in culture flasks, with or without dFBS. The medium total protein content in these two production systoms, evaiuatod by micro BCA assay, was - 15 tmes highor in bloreactor-derived medium. Consequently, the starling material for puriication, gonerated in this system, presenied a3 timos lowor spocific actvity. A three-step puritication procedure involving cation-exchange, dye affinity and sizo exclusion chromatography was ulized for obtaining purified, biologically active, heterodimeric rec-HTSH. A high punty level (-95%) and an overall yiald of - 20% were obtained thanks o this purification process. Identty and purity of rec-HTSH were evaluated through physico-chemical methods (size excluson HPLG, reverzed phase HPLC and SDS-PAGE), immunological methods (mmunoradiometric assay, radioimmunoassay and Wostem blotiing), N-terminal aminoacid sequencing and Maldi-Tol mass spectrometry. The physico-chemical and immunological mothods were always caried out comparing our product with two well known roference proparations (rec-hTSH-Thyrogen" and pit-HTSH-NIDDK). An "in vivo* bioassay confirmed that the relative blological activity of the purified homone was 2.13 with reſation 10 the intomational reforonce preparation of pituitary hTSH(NIDDK, USA). This homone, produced for the first time in our country, thanks to an original process, is extromaly important for thyrold cancer diagnosls and therapy and for hypenhyroidism treatment.”