Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Glycoprotein – e.g. – mucins – proteoglycans – etc.
Reexamination Certificate
1999-11-16
2002-07-02
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Glycoprotein, e.g., mucins, proteoglycans, etc.
C530S397000, C530S344000, C530S350000, C530S412000, C530S413000, C530S415000
Reexamination Certificate
active
06414123
ABSTRACT:
TECHNICAL FIELD
The present invention relates to the protein purification arts and more specifically to methods for producing highly purified follicle stimulating hormone (FSH) to high purity FSH compositions.
BACKGROUND OF THE INVENTION
Follicle stimulating hormone (FSH, or follitropin) is a pituitary heterodimeric glycoprotein hormone synthesized and released from gonadotrope cells of the anterior pituitary gland. As a circulating hormone, FSH interacts with high affinity with receptor molecules on the surface of granulosa cells in the ovary. This interaction evokes a series of intracellular events, including increasing intracellular levels of the second messenger, cyclic AMP, and elicitation of a steroidogenic response by the granulosa cells resulting in estrogen production. Local estrogen and FSH stimulation promote the growth and maturation of ovarian follicles.
The amount of circulating FSH is dependent upon several other endocrine and neural factors. Gonadotropin releasing hormone (GnRH) is a peptide elaborated by hypothalamic neurons. Released GnRH interacts with receptors on pituitary gonadotrope cells to control the synthesis and release of FSH and leutenizing hormone (LH) from the anterior pituitary gland. FSH secretion also is affected by circulating levels of steroid hormones. The steroidogenic response of granulosa cells to FSH results in gradually increasing estradiol levels. When serum estradiol reaches a critical level, it triggers a large increase in the rate of LH and FSH release from the anterior pituitary. The resultant LH surge induces ovulation and luteinization of granulosa cells. Progesterone is released from the corpus luteum following ovulation and this steroid prepares the uterus for implantation of the fertilized ovum. Elevated levels of estrogens and progesterone exert a negative feedback inhibition at hypothalamic sites to lower FSH and LH synthesis and release. Hence, the effects of steroids on gonadotropin release depend on the circulating levels; at low levels of estrogen, FSH and LH are positively regulated while higher levels result in negative feedback inhibition.
In males, FSH induces spermatogenesis through a proliferative effect on spermatocytes. Sperm production also requires testosterone, which is under positive regulatory control by LH.
An apparent paradox of the above-described hormonal control process is the production of both LH and FSH by the gonadotrope cell while GnRH serves as a positive regulatory agent for both hormones. Work within the last 10 years suggests that the peptides activin, follistatin, art and inhibin selectively regulate FSH secretion from the anterior pituitary gland. FSH synthesis and release are activated by activin, while inhibin and follistatin have negative feedback effects. The inhibitory effect of follistatin is thought to be mediated by its high-affinity binding to activin and blockage of its biological activity. There is evidence for both autocrine and paracrine local regulatory effects of these peptides and for feedback effects of inhibin released from gonadal tissue. Both inhibin and activin are structurally related and are members of the diverse transforming growth factor beta family of peptides. Study of the physiological roles of activin, follistatin, and inhibin is a current area of active research (reviewed by Knight, 1996).
FSH has been used extensively as a drug to treat human infertility by induction of follicular development in females. Earlier products were crude preparations of LH and FSH, i.e., Pergonal®. More recently developed products contain purer FSH preparations. Metrodin® has low levels of LH and the FSH specific activity is about 100 IU of FSH per mg total protein. This drug requires intramuscular injections every day for 5 to 7 days, followed by a single injection of human chorionic gonadotropin (hCG) to induce ovulation. A recent advance is Fertinex® which is affinity-purified FSH from human menopausal gonadotrophin (hMG). This product exhibits a FSH potency of 8500 to 13,500 IU of FSH per mg total protein at 95% purity as reported by the manufacturer. The high purity of Fertinex® allows delivery by subcutaneous injection, which can be administered at home. Following administration of Fertinex®, hCG is used for induction of ovulation. Depending on the dosage of FSH administered, it may be used to promote in vivo fertility or, at higher dosages, it may be used to induce multiple oocyte formation for in vitro fertilization procedures. Recombinant forms of FSH (Puregon® and Gonal®) also are used as fertility drugs; these versions of FSH have potencies and-purities similar to that of Fertinex®.
FSH has been purified from pituitary glands, human postmenopausal urine, and from culture media collected from genetically engineered cells. FSH purification has been an active area of research over the past 30 years. Older methods rely on procedures such as ion exchange chromatography, size exclusion chromatography, polyacrylamide gel electrophoresis, and chromatography on hydroxylapatite. In one method (Roos, et al., 1968) a FSH preparation of 14,000 IU of FSH per mg total protein was obtained from fresh frozen human pituitary glands with an overall recovery of activity of 5.0%. A similar procedure applied to urinary FSH resulted in a preparation of 780 IU of FSH per mg total protein at a 7.7% overall yield.
Because of the similar physicochemical properties of FSH and LH, i.e., similar molecular weight and overlapping isoelectric profiles, affinity chromatography methods have been employed to improve the separation of LH from FSH. Affinity methods also afford the possibility of high purification in a single step (up to 100-fold) thereby reducing the number of steps in a purification method and improving overall yield. The latter is a critical factor in the commercial production of FSH as overall yield is a major determinant of cost. Group-specific affinity adsorbents such as the lectin Concanavalin A or chitosan (Japanese patent number 8,027,181) bind glycoproteins via specific carbohydrate groups. Concanavalin A has been used to characterize microheterogeneity of purified FSH preparations (Chapped, et al., 1983). However, these ligands are ineffective in the separation of two glycoproteins such as FSH and LH.
Immunoaffinity chromatography (IAC) relies upon the specificity of mono- or polyclonal antibodies for capture of specific protein antigens from crude mixtures. Antibodies may first be screened for use in IAC (Bonde, et al., 1991). The selected antibodies are coupled to a chromatographic solid phase, e.g., cross-linked agarose, through covalent bonds, e.g., cyanogen bromide (CNBr) or other coupling chemistries targeting surface amino, hydroxyl, carboxyl or sulfhydryl groups of immunoglobulins to form a solid matrix. Recent coupling methods attempt site-directed immobilization of antibodies in an effort to optimize antigen-binding efficiencies, which are typically low, using classical coupling chemistries. One method of site-directed coupling is through carbohydrate groups of the F
c
immunoglobulin region to hydrazide-activated solid supports (Hoffman and O'Shannessy, 1988). The solid phase coupled to antibody then is packed in a chromatography column and equilibrated with buffer for binding to antigen. Mixtures of target protein and contaminants are equilibrated with binding buffer and then applied to the column. Non-adsorbed contaminants are removed by washout with various buffers. Elution occurs by use of chaotropic agents, extremes of pH, changes in ionic strength, etc.. Elution is a critical aspect of IAC since the elution conditions may alter the biological activity of the immobilized antibody or the eluted antigen or both (reviewed by Jack, 1994).
IAC may be used to remove specific contaminants from a crude mixture. This mode first was applied to FSH purification using antibodies to hCG, which through cross reactivity to LH, effectively reduced LH contamination levels (Donini, et al., 1966). Other methods (Jack, et al., 1987 and Great Britain patent number 8,510,177) utili
Musick James R.
Van Horn Erik
Hubl Susan T.
Low Christopher S. F.
Mohamed Abdel A.
Quine Intellectual Property Law Group P.C.
Vitro Diagnostics, Inc.
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