Purification And Characterization Of The Recombinant Human Follicle Stimulating Hormone

Human follicle stimulating hormone (hFSH) is a glycoprotein hormone of synthesized and secreted from the anterior lope of the pituitary gland, comprising non-covalently attachedαandβsubunits. Theαsubunit contains 92 amino acid residues, with five disulphide bonds contributing to its tertiary structure. Theβsubunit contains 111 amino acid residues, with six disulphide bonds. Of the four Asn-linked glycosylation sites in FSH , two are located on theαsubunit(Asn52 and 78 ) and two on theβsubunit(Asn 7 and 24 ).Reproductive function in both female and male mammals, including humans, is regulated by FSH. In females, FSH stimulates the development of ovarian follicles carrying the oocyte, and in males it plays a role in spermatogenesis. The synthesis of FSH by gonadotroph cells takes place within the anterior pituitary gland, before secretion into the general circulation. The synthesis and secretion of FSH are regulated by gonadotrophin releasing hormone (GnRH), secreted by specialized neurones within the hypothalamus, and steroidal and nonsteroidal products secreted from the gonads. Through high-affinity binding to its respective receptor, FSH affects the function of specific target cells in the ovaries and testes and triggers intracellular mechanisms that regulate steroidogenesis, cell replication, and the expression of specific proteins and growth factors that control gametogenesis.Inadequate concentrations of FSH, caused either by deficient FSH synthesis or secretion, are a common cause of infertility in men and women. In women, this state is characterized by abnormal or the absence of ovulation. In men, it may lead to infertility because of the production of inadequate numbers of viable spermatozoa. Administration of FSH, either alone or in combination with LH, has been used successfully to treat these fertility disorders. Previously, commercial preparations of FSH for therapeutic use were derived from a urinary source (u-hFSH from post-menopausal women). However, the development of recombinant DNA technology to produce recombinant human FSH (r-hFSH) has resulted in significant improvements in the quality and availability of this product for clinical use.In this reach, the complete process, from the purification of rFSH and its results about it, is described.rhFSH purificationrhFSH was developed for clinical use, so a high degree of purity is needed. The downstream purification process was designed to remove cell culture medium-related proteins as well as degraded FSH molecules and to recover the greatest amount of rhFSH at a high level of purity. This was accomplished by a series of five manufacturing steps. The separation techniques were designed to take advantage of both the differences in molecularsize, charge and hydrophobicity between FSH and contaminants, and the high selectivity of the immunoaffinity chromatographic step performed.Purification methodsThe production of highly purified r-hFSH was accomplished by a sequence of five chromatographic steps. The first three steps (ultrafiltration ion-exchange and immunoaffinity chromatography) were designed to remove most of the impurities, while the remaining two steps (in order: ion exchange and size-exclusion chromatography) further refined the product by eliminating contaminants present in trace amounts.Cell culture supernatant was harvested and concentrated/ diafiltered using a 10 000 D cut-off membrane. The concentrated solution was adsorbed onto a diethylaminoethyl sepharose CL 6B chromatographic column, previously equilibrated in ammonium acetate buffer (0.2 M, pH 8.0). Semipurified FSH was recovered in the unbound fraction and, after concentration (10 000 D cut-off membrane), was loaded onto an immunoaffinity resin, equilibrated in 0.01 M Tris-HCl buffer (pH 8.0) containing 0.5 M NaCl. After washing steps with Citric Acid (0.02M,pH4.0), and neutralizationand with phosphate buffer(0.5M,pH 8.0). The eluate was concentrated and diafiltered in 0.02 M ammonium acetate (pH 8.5) for adsorption on a Q Sepharose HP resin. Elution was performed with 0.2M ammonium acetate buffer (pH 8.5). The eluate was concentrated and loaded onto a size-exclusion chromatographic column packed with Sephacryl S 200, previously equilibrated in phosphate buffer (pH 7.0). The highly purified rhFSH was then collected and concentrated/ dialysed against water by ultrafiltration (10 000D cut-off membrane). After microfiltration on 0.2μm membrane, the rhFSH bulk was stored frozen at–20°C.rhFSH characterizationThe electrophoretic mobility in SDS-PAGE showed an apparent molecular weitht of the dissociated subunits (estimated using molecular weight markers in the range of (14.4-97.4kDa), of 23kDa.Isoelectric focusingThe isoelectric profile of rhFSH was established by carrier amholine isoelectric focusing, pH rang 3.5-10. The typical isoeletric focusing pattern showed 6 or 7 bands in the pH interval 4.0-5.2. The charge heterogeneity of the molecule reflects the differences in the glycoshlated moiety.PurityThe purification strategy has been shown to produce a stock solution of rhFSH that is≥95% pure.Dissociated and Aggregated of rhFSHThe consistency and integrity of the rhFSH manufacturing process was assessed by the quantification of oxidized forms of FSH using HPLC. It was demonstrated that rhFSH contained only trace quantities of the degraded forms(data not shown).Analysis by high-performance size-exclusion chromatography and SDS-PAGE-under non-denaturing conditions showed only very low concentrations(?1%)of aggregated or dissociated FSH in the final purified drug solution.In-vivo bioactivityThe potency of rh FSH was assessed by determining wister rat ovarian weight gain in response to FSH administration. The result was 12000~13000IU/mg protein,which was higher than that of literature.Pharmacodynamic trialsrhFSH contributes to the development of female rat's follicle, the evocation of ovulation, and multiplication the number of embryo of conception rat.Production of FSH by recombinant technology using new purification and process technology results in a high batch to batch consistency, high purity, absence of contaminating human proteins including LH and HCG, the likelihood of reducing the risk of infectious particles, and the elimination of drugs co-extracted from urine, which is collected from numerous donors. To date, rhFSH has been used in thousands of patients worldwide. rhFSH has been shown to be more effective at stimulating ovarian follicle growth than uhFSH, and therefore it is likely to replace the urinar preparations for the treatment of human infertility future.