Preparation And Properties Study Of Long-acting Follicle Stimulating Hormone Analog

Follicle Stimulating Hormone is a glycoprotein synthesized and secreted from the anterior lope of the gland. Like the other members of the pituitary glycoprotein hormone family, FSH is produced through the heterodimeric assembly of α and βsubunits, which are encoded by separate genes. FSH is essential for reproduction in both female and male for it’s physiological function. FSH could stimulate the development of follicle and promote ovulation for female animals and in male animals it plays an important role in spermatogenesis. The preparations of FSH are widely used for curing anovulatory infertile patients in human and developing multiple follicles in mammalian embryo implantation in animals. Currently, all available FSH preparations for animals are made from pituitary extracts in China.However, the FSH preparations made from animals are unsafe because of the virus and the pituitary from animals is insufficient. Otherwise, the half-life of natural FSH is short in animals, thus the superstimulatory treatment protocols consist of twice daily intramuscular injections over 4 to 5 days. This requires frequent attention by farm personnel and increases the possibility of failures because of noncompliance.Therefore our team converted the α and β subunit of bovine FSH with a linker sequence which contains two additional N-linked glycosylation sites. The recombinant FSH analog was demonstrated it’s effectiveness and long acting for that the ovarian weight increase is no significant difference between one injection of the long-acting FSH analog and eight consecutive injections of the commercial FSH preparation.In this research, we selected monoclonal cell by limiting dilution assay and the supernatant collected was detected by Western Blotting and ELISA to obtain a high expression cell strain(CHO-FSH). The major improvements in cellular productivity were achieved by using continuous suspension cultures with cell recycling instead of an adherent culture system or batch-mode suspension cultures wherefore we acclimate the CHO-FSH to serum free medium and suspension culture by a gradual reduction of serum domesticated. Amplify the cells to manufacture a working cell bank(WCB)stored in liquid nitrogen to ensure that cells from the same cell batch could be used in all experiment. Bioreactors play a key role in the field of biologics, where they are used for the production of recombinant therapeutic proteins by large scale cultivation of animal cells. We research the large scale cultivation process of CHO-FSH in a Labors 5 cell bioreactor and obtain FSH analog successfully. The continuous suspension cultures of CHO-FSH demonstrate the cell we stored in WCB is stable for the stable expression and in vivo bioactivity, the expressions of 4 batches are 2.75μg/ml, 2.98 μg/ml, 2.89 μg/ml, 2.64 μg/ml and the in vivo activity are 12066 IU/mg,11294 IU/mg, 10811 IU/mg, 11101 IU/mg. The FSH analog is purified by isoelectric precipitation first to remove most of the impurities, then use dye affinity chromatography and the last the protein we need is harvested and concentrated by a3000 D cut-off membrane. The purified FSH analog was identified by Western Blotting.The current study is performed to obtain a novel long-acting FSH analog preparation to simply the protocol of developing multiple follicles in mammalian embryo implantation. For this purpose, we selected high expression cell strain and acclimated the cell to serum free medium and suspension culture, researched the large scale cultivation process and the purified protocol.