Development Of New Long-acting Mutants Of Human Follicle Stimulating Hormone And Comparation Of In Vivo And In Vitro Bioactivity

Human follicle stimulating hormone (FSH) is a heterodimeric glycoprotein comprising of non-covalently attachedαandβsubunits. Theαsubunit is common to all four members of a glycoprotein hormone family,which includes pituitary luteinizing hormone (LH),pituitary thyroid stimulating hormone and placental human chorionic gonadotrophin (HCG). Reproductive function in both female and male is mainly regulated by FSH. In females,FSH stimulates the development of ovarian follicles carrying the oocyte,and in males it plays a role in spermatogenesis. As a result, FSH is widely used clinically to the treatment of infertility as a proten drug wihch is at the top list of bioenginerring drug sale in these years. One issue regarding the clinical use of FSH is its short half-life in the circulation. To address this point, in this paper,we construct and evaluate three new type of long-acting FSH mutants including the long-acting FSH-CTP mutants which developed by Organon company in Netherland. New mutant of FSH-CTP was proved to be 2~3 times of half time in human circulation and owned higher level of in vitro and in vivo biological activity compared with wild type of FSH. One new mutant named FSH-CTPM was contructed by addition of three N-linked glycosylated sites through site mutagenesis in based on mutant of FSH-CTP. One another new mutant named FSH-CTPN was contructed by addition of two N-linked glycosylated sites on C-terminal ofβsubunit through gene coding ANITVNITV amino sequence based on mutant of FSH-CTP.These constructs of wild type of FSH and new long-acting mutants were transfected into Chinese hamster ovary (CHO) cells and H293 cells. We eastablished double antibody sandwich ELISA system to detect and quantify the expression of wild type FSH and new FSH mutants, the sensitivity of ELISA is no less than 2.5 mIU/ml. The ELISA data show that wild type FSH and two new type of three FSH mutants including FSH-CTP and FSH-CTPN transient expressed in level about 100ng/106 cell·24hour. After acquiring small amout of protein of wild type FSH and FSH-CTP,FSH-CTPN mutants , we evaluate two new FSH mutants of FSH-CTP, FSH-CTPN in vitro and in vivo bioactivity through in vitro FSH receptor bioassay and in vivo Steelman and Pohley bioassay compared with wild type of FSH. The results show that new muntants of FSH-CTP and FSH-CTPN own high level of in vitro and in vivo biological activity compared with wild type FSH. New long –acting FSH mutants by addition of N-linked or O-linked glycosylated site can apparently increase in vivo and in vitro biological potency. New mutant of FSH-CTPN is better than FSH-CTP compared wtih in vitro and in vivo biological activity. New mutant of FSH-CTPN may form the basis for an improved treatment of intertility in the future and this research may form the basis of futher developing new protein drug for treatment of infertility.In the end, we try to screen FSH-CTPN mutant in high expression level of stable cell strains. When the MTX concentration is 5×10-8mol/L, the expression level of one cell clone reached 550mIU/106cell·24hour.