TWI322692B - Fsh and lh pharmaceutical formulations - Google Patents

Description

1322692 发明, the invention description: [Technical Field of the Invention] The present invention relates to the field of pharmaceutical formulations for promoting follicle stimulating hormone (FSH), luteinizing hormone (lh), and a mixture comprising FSH and luteinizing hormone (LH), And methods of making such formulations. [Prior Art] Follicle stimulating hormone (FSH), luteinizing hormone (LH), and chorionic gonadotropin (CG) are classified as injectable proteins of gonadotropins. FSH, and hCG are used alone or in combination to treat infertility and fertility disorders in women and men. Under natural conditions, FSH and LH are made from the pituitary gland. For medical use, FSH and LH and their variants can be prepared recombinantly (rFSH and rLH), or they can be prepared from the urine of menopausal women (uFSH and uLh) 〇FSH for inducing ovulation in female patients ( 〇 1) and control ovarian hyperstimulation (COH) for assisted reproductive technology (ART). In a representative course of induction of ovulation, the patient is administered FSH or H/day for about 6 to about 12 days. In a representative course of control printing, the patient is administered daily FSH or variant (about 1 50-600 IU FSH/day) for about 6 to about 12 days. FSH is also used in male patients with oligospermia to induce sperm production. A treatment with 15 〇 W FSH 3 times a week and 2, 5 〇〇仞 coffee twice a week has successfully achieved sperm metering in male patients with low gonadotropin-induced sexual dysfunction! . 1322692 LH and FSH are used in combination with 〇i and c〇H in female patients, especially for patients with very low endogenous LH concentrations or resistance, such as hypogonadotropic sexual dysfunction Women (HH, WH〇 Group I) or older patients (ie, patients 35 years of age or older) and patients with problems with embryo implantation or early abortion. LH combined with FSH has traditionally been obtained in the preparation of so-called human menopausal mitogenic hormone (hMG) extracted from post-menopausal women. hMG has a 1:1 ratio of FSH: LH activity 〇 CG acts on the same receptor as LH and initiates the same reaction. CG has a longer circulating half-life than LH and is therefore generally used as a long-term source of LH activity. CG was used to simulate the peak of natural LH and induce ovulation in the 〇1 and C0H treatments. Ovulation is induced by injection of human chorionic gonadotropin (hCG) when stimulated with FSH or a mixture containing FSH and LH. CG can also be used in conjunction with FSH for stimulation of 01 and COH to provide LH-activity in a patient who is expected to have LH-activity (e.g., as mentioned above) during stimulation. FSH, LH and CG are members of the heterodimeric, glycoprotein hormone family, which also includes thyroid stimulating hormone (TSH). Members of this family are heterodimeric, containing alpha- and beta-subunits. The sub-units are combined by non-covalent non-covalent interactions. The human FSH (hFSH) heterodimer is composed of (i) mature 92 amino acid glycoprotein Aval (the subunit, which is also commonly found in other human family members (ie, chorionic gonadotropin ("CG, ,) Luteinizing hormone ("LH") and thyroid stimulating hormone ("τ,)); and (u) FSH-specific mature ill amino acid beta (beta) subunits. 2. Human LH heterogeneous The dimer consists of 1322692 (i) mature 92 amino acid glycoprotein Aval subunit; and (丨丨) unique mature 112 beta subunit consists of 3. The alpha and beta units of these glycoproteins may Due to its interaction with preservatives, surfactants and other excipients, it is easy to decompose in the formulation. Dissociation of these subunits will result in loss of biological efficacy. FSH is formulated for intramuscular (iM) or subcutaneous ( Sc) injection. FSH is supplied in a vial or lyophilized (solid) form in a vial or ampoule at a dose of 75 IU per vial and 150 IU per vial. The storage period is one and a half to two when stored at 2_25 °c. Injectable solution was formed by reconstituting the lyophilized product with water for injection (hepatic sputum). For induction of ovulation and control of ovarian hyperstimulation, daily injections of up to 75 IU to 600 IU are recommended for up to approximately 10 days. Depending on the patient's response, up to three treatment cycles can be performed with increasing doses of FSH. To lyophilize the formulation, the patient needs to reconstitute a small vial of fresh lyophilized material daily and reconstitute immediately after reconstitution [package insert description N1 7001 01A, published in February 1996, for subcutaneous injection FertinexTM (Urine Follicles for Injection), by Ser〇n〇 Laboratories, Inc., Randolph, MA]. FSH has also been formulated into single-dose and multi-dose formulations in small vials or ampoules. Single-dose formulations must be stable and effective during storage prior to use. Multi-dose formulations must not only remain stable and effective during storage prior to use, but must also be administered in multiple doses after the sealed ampoule has been opened. It is also stable, effective and relatively sterile during this period. For this purpose, multiple dose formulations often contain bacteriostatic agents. LH is formulated for intramuscular (IM) Subcutaneous (sc) injection. 11{Series 1322692 Z is 75 HI / vial in vial or ampoules; Donggan (solid) form & for 'the storage period of one of the preservation powers when hungry is - year Half. Reconstruct the product with water for injection (WFI) (4) Solution for the injection. For the induction of typography and control of the over-stimulation of the nest (with the coffee combination, the daily dose of 75 IU to 600 IU is injected up to about Ten days. EP 〇 618 8G8 (Applied Research System ARS Holding Share nv) discloses a pharmaceutical composition comprising a solid intimate mixture of gonadotropin and a stabilizing amount of vegetable sugar alone or in combination with glycerin. EP 0 814 841 (Applied Research System ARS Holding Share N v ) discloses a stable, liquid pharmaceutical composition comprising recombinant human chorionic gonadotropin (hCG) and a stabilized amount of mannitol. EP 0 448 146 (AKZO NV) discloses a lyophilization comprising a dicarboxylate stabilizer containing one (heavy 1) gonadotropin, 200 to 1 〇, 〇〇〇 by weight and gonadotropin-associated with gonadotropin Stabilized gonadotropin of the product. EP 0 853 945 (Azozo Nobel N_V.) discloses a liquid formulation containing gonadotropin' characterized in that the formulation comprises gonadotropin and a stabilizing amount of polytoxaic acid or a salt thereof and a sulfur bond compound. W0 00/04913 (Erly and the company) reveals a variant containing FSH or FSH (containing Alva and beta units) and is selected from the group consisting of phenol, m-cresol, p-to-be, and o-- Gas cresol, benzoquinone, alkyl hydroxybenzoate (methyl, ethyl, propyl, butyl, etc.), benzalkonium, benzethonium chloride, sodium dehydroacetate, and thimerosal, Or a formulation of a preservative dissolved in a mixture of aqueous diluents. There is still a need for a 丄322692 stable liquid formulation containing a mixture of FSH or FSH variants and a mixture of FSH and LH for single or multiple dose administration. SUMMARY OF THE INVENTION [Summary of the Invention] One item of the present invention provides novel lyophilized and liquid formulations of FSH or FSH variant, LH or LH variants, providing methods for their preparation and their use in the treatment of fertility Medical or veterinary use of sexual disorders. Another item of the present invention provides novel lyophilized and liquid formulations of a mixture of FSH and LH, providing methods for their preparation and their pharmaceutical or veterinary use for the treatment of fertility disorders. In a first aspect, the present invention provides a block copolymer comprising FSH or a variant thereof, and selected from the group consisting of ethylene oxide and propylene oxide, preferably

A lyophilized and liquid pharmaceutical composition of a surfactant of Pluronic® F77, Pluronic F87, Pluronic F88 and Pluronic F68. In a second aspect, the present invention provides a method for producing a liquid pharmaceutical composition comprising forming a block copolymer comprising FSH or a variant thereof and selected from the group consisting of ethylene bromide and propylene propylene oxide. A solution of a surfactant of piur〇nic8F 77 , Pluronic F87, Pluronic F88 and Pluronic F68. In a third aspect, the invention provides a method for the manufacture of a packaged pharmaceutical composition comprising a block copolymer comprising FSH and selected from the group consisting of ethylene oxide and propylene oxide, preferably piur〇nic A solution of the surfactant of @F77, piur〇nic F87, Pluronic F88 and Pluronic F68 was dispensed into a container. In a fourth aspect, the present invention provides a product of 10 1322692 for human medical use, which comprises a copolymer comprising FSH or FSH variants and a copolymer selected from the group consisting of ethylene oxide and epoxy propylene. a vial of a solution of Pluronic® F77, Pluronic F87, a surfactant of Pluronic F88 and Pluronic F68, and indicating that the solution can be maintained for a period of about twenty-four hours or more after the first use. Text description. In a fifth aspect, the present invention provides a block copolymer comprising FSH and LH, and a block copolymer selected from the group consisting of ethylene oxide and propylene oxide, preferably Pluronic® F77, Pluronic F87, Pluronic F88 and Pluronic F68. A dry and liquid pharmaceutical composition of the agent. In a sixth aspect, the present invention provides a method for the manufacture of a lyophilized and liquid pharmaceutical composition comprising forming a copolymer containing FSH and LH and selected from the group consisting of Ethylene Ethylene and Epoxy Acetate A solution of the surfactants of Pluronic® F77, Pluronic F87, Pluronic F88 and Pluronic F68. According to a seventh aspect, the present invention provides a method for producing a packaged pharmaceutical composition comprising a copolymer comprising FSH and LH, and a copolymer selected from the group consisting of ethylene oxide and epoxy propylene. A solution of a surfactant such as Pluronic® F77, Pluronic F87, Pluronic F88 and Pluronic F68 is dispensed into a container. In an eighth aspect, the present invention provides a product for human medical use comprising a block copolymer comprising FSH and LH, and selected from the group consisting of ethylene oxide and propylene propylene, preferably Pluronic® F77, A small vial of a solution of Pluronic F87, a surfactant of Pluronic F88 and Pluronic F68, and a textual description indicating that the solution can be maintained for about twenty-four hours, 1322692 or longer after the first use. In a ninth aspect, the present invention provides a product for human medical use comprising a copolymer comprising a freeze-dried FSH or FSH variant, and a segment selected from the group consisting of ethylene bromide and propylene bromide, preferably Pi ur〇n丨F77,

Pluronic F87, a first barn of the surfactant of Pluronic F88 and Pluronic F68 and a second container containing a solvent for reconstitution, preferably an aqueous solution containing a bacteriostatic agent (preferably inter-system). In a tenth aspect, the present invention provides a product for human medical use, comprising a block copolymer comprising a lyophilized LH or LH variant, and a block copolymer selected from the group consisting of epoxy bromide and propylene bromide, preferably Pluur〇nic@ F77,

A first container of Pluronic F87, a surfactant of Pluronic F88 and Pluronic F68, and a second container containing a solvent for reconstitution, preferably an aqueous solution containing a bacteriostatic agent, preferably interstitial-nonylphenol. In an eleventh aspect, the present invention provides a product for human medical use comprising a block copolymer comprising lyophilized FSH and LH or FSH or LH variants and selected from the group consisting of ethylene oxide and propylene oxide Better system

a first container of Pluronic® F77, Pluronic F87, a surfactant of Pluronic F88 and Pluronic F68, and a second container containing a solvent for reconstitution, preferably an aqueous solution containing a bacteriostatic agent (preferably inter-p-phenol) . DETAILED DESCRIPTION OF THE INVENTION The liquid and lyophilized FSH or FSH and LH formulations of the present invention have improved or more suitable properties or stability and are useful for the medicinal treatment of women and/or men. These formulations and articles are also suitable for use in injectable and other delivery systems selected for 12 1322692, such as (but not limited to) nasal, pulmonary, transmucosal, transdermal, oral, subcutaneous, intramuscular or non- Continuous release through the intestine. In a particularly preferred embodiment, the formulation of the invention is for subcutaneous and/or intramuscular injection. The FSH or FSH and LH variant solutions and formulations provided may also be prevented or reduced in activity or loss of stability, or by any aspect of the effectiveness or desirability of the drug, such as at least a dosage form, Frequency, dose, comfort, ease of use, in vitro or in vivo

Biological activity and the like, and after a period of time, it has enhanced in vivo efficacy compared to known commercial products. Follicle stimulating hormone (or FSH) as used herein means FSH produced as a full-length mature protein, including but not limited to, FSH or, hFSH, regardless of whether it is produced recombinantly or from human origin ( For example, the urine of menopausal women) is a single leaver. The protein sequence series of the human glycoprotein Avalanche unit is not in SEQ ID N (M, and the protein sequence series of human FSH beta subunits is shown in SEQ ID NO: 2.

The expression "FSH variant" is intended to encompass a molecule that is equal to the amino acid sequence glycosylation pattern or sub-unit linkage and human-FSH + but still FSH-active. Examples include ap FsH, which is a species The long-acting repaired recombinant FSH consists of the wild-created „+Kun; B + the β-subunit of the C-terminal of the carboxy terminal i FSH of the hCG, UP〇lt et al., endocrinology, stain, and cut , 2514_252〇; or gram

The development and characteristics of Li human = type recombination _ activator, described in Human Health Chain VIII-56. Also included is the single-stranded CTP-FSH, a chain knife consisting of the following sequence "from the N-terminus to the C-terminus": 13 Ί322692

PFSH phCG-CTP (113-145) aFSH where PFSH represents the β-subunit of FSH, phCG-CTP (113-145) represents the carboxy terminal peptide of hCG and aFSH represents the a-subunit of FSH, Klein et al. Other examples of 5 ° FSH variants include FSH molecules having additional glycosidation sites incorporated into a- and/or β-subunits, as disclosed in WO 01/58493 (Maxygen) 'especially in the scope of patent applications ! And the FSH molecules having sub-unit S-S linkages as disclosed in WO 98/58957. The FSH variants cited herein also include the deletion of the carboxy terminus of the beta cell and the shorter than the full length mature protein of SEQ ID NO: 2. The carboxy terminal deletion of the human beta subunit is shown in SEQ ID NO: 3, 4 and 5. It is understood that the carboxy terminal variant of the beta chain forms a dimer with the known avalanche unit and becomes a FSH variant heterodimer. The FSH heterodimeric or FSH variant heterodimer can be prepared by any suitable method, such as by recombinant methods, by isolation or purification from a possible natural source, or by chemical synthesis, or any combination thereof. obtain. The use of sf recombination means that FSH, LH or FSH and LH variants are produced by using recombinant]) NA technology (see for example w〇85/01958). For Avalanche from several species The sequence of the fsh genomic and cDNA pure strains is known for the beta-subunit. An example of a method for expressing FSH or LH using recombinant techniques is by encoding eukaryotic cells with FSH or LH Avala The DNA sequence of the subunit (whether or not it is placed on U22b92; on the first plant, or on two carriers in which each unit has a separate promoter) is transfected, as in European Patent EP 0 21 1 894 pp Λ . u 487 512. Another example of the use of recombinant techniques for the manufacture of FSH or LH is by using an intervening link to insert a heterologous regulatory fragment into an FSH or LH subunit. Homologous recombination of sequences, as described in European Patent EP 〇 505 500 (Applied Research System ARS Holding Shares NV). The FSH or FSH variants used in accordance with the present invention are not only recombined (including from mammals) Made of cells, or from other organisms Sources are, for example, purified from urine sources. Acceptable methods include those described in Hakkola, K. Molecular and Endocrinology, 127: 59-69, 997; Keen et al., J. Biol. Chem., 264: 4769-4775, 1989; Serpa-Poyak et al., Endocrinology, 132: 351-356, 1993; Dias et al., J. Biol. Chem., 269: 25289-25294 , 1994; Farak et al. 'J. Biol. Chem., 269: 14015-14020, 1994; and Warov et al., Endocrinology, 135: 2657-2661, 1994, US Patent 3, 1 19, 740 and U.S. Patent No. 5,767,067. Luteinizing hormone (or LH) as used herein means LH produced as a full-length mature protein, including but not limited to LH or 'hLH', regardless of its Individually obtained by reconstitution or isolated from a human source, such as the urine of a postmenopausal woman. The protein sequence series of the human glycoprotein Avalanche unit is shown in SEQ ID NO: 1, and the protein sequence of human LH beta subunit 7 is shown in SEQ ID NO: 6. In a preferred embodiment, LH is recombinant. The expression "LH variant" is intended to encompass molecules which are equal to the amino acid sequence, the sugar 15 or the sub-unit linkages which are different from the human LU but which still exhibit LH_activity. LH isoindole or LH variant heterodimers can be obtained by any suitable method, for example, by recombinant methods, by isolation or purification from a possible natural source, or by chemical synthesis, or any combination thereof. . ° °. A peony or "administration" means to introduce a formulation of the present invention into a patient in need thereof to treat a disease or condition. A patient is a mammal who is intended to receive treatment for a disease or condition. Limited to: a person selected from the group consisting of human sheep, pigs, horses, cows, rabbits, etc. The term "efficacy" associated with FSH activity means that a fsh formulation or a mixed formulation can initiate a biological reaction linked to FSH. For example, the increase in the weight of the nest in the Stilman-Poly analysis 8 or the growth of the female patient's blister growth can be achieved by, for example, ultrasonic measurement on the 8th day of the stimulation. The amount of follicles having an average diameter of or about 16 mm is estimated to be measurable. The bioactivity is evaluated in relation to the accepted FSH standard. The term "efficacy" in relation to LH activity means lh formulation or blend. Tune: the substance can initiate a biological reaction linked to LH, such as the increase in seminal vesicle weight. Method 9. The biological activity is dissolved in a liquid containing water with the term "aqueous diluent" as accepted; the solvent system can be water Into, or it may be water plus one or more mixed solvents, and may contain dissolved solutes such as sugars, buffers, salts thereof, or elsewhere. The more commonly used non-aqueous solvents are short-chain organic alcohols (eg, Yin 16 1322692 alcohol, ethanol, propanol) such as glycerin). Short chain ketones (eg, C) and polyols (Isotonizing Agent I is physiologically tolerant and gives the formulation a suitable sheet to prevent clean water from passing through the cell membrane in contact with the formulation. Known concentrations are used in this project. Other suitable isotonic pressures include, but are not limited to, amino acids or proteins f (eg glycine or white), salts (eg sodium chloride) and sugars (eg For example, glucose and lactose).

(d) (d) agent or "bacteriostat," means a compound or composition that acts as an anti-bacterial agent added to the formulation. The preservative-containing formulation of the invention comprising a cut or FSH variant or FSH Xiao LH, preferably It is in compliance with the laws or regulations governing the preservative efficacy of the mouthpiece (preferably for humans) that is commercially available. Examples of the bacteriostatic agent include phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzofural, alkyl p-hydroxybenzoic acid (mercapto, ethyl, propyl, butyl Base and other classes), stupid ammonium, benzodiazepine, sodium argonate and sulphur.

The term "buffer" or "physiologically acceptable buffer" means containing a formulation known to be safe for pharmaceutical or veterinary use, and having the pH of the formulation maintained or controlled to the desired pH of the formulation. A solution of a compound of efficacy within the range of values. Acceptable buffers for controlling the pH to a moderately acidic pH to a moderately alkaline pH include, but are not limited to, phosphates, acetates, citrates. , arginine, TRIS and histidine, etc. "TRIS" means 2-amino-2-hydroxymethyl-1,3-propanediol, and any pharmaceutically acceptable salt thereof. Preferably, the buffer is a phosphate buffer having saline 13 1322692 or an acceptable salt. The term "phosphate buffer" means a solution containing phosphoric acid or a salt thereof, adjusted to a desired pH. The liquid system is prepared from phosphoric acid, or a salt of phosphoric acid including, but not limited to, sodium and potassium salts. There are several phosphates known to the art, such as sodium and potassium of the acid, valence, divalent and Trivalent salts. Phosphates are also known to form waters of the salts formed. Thereof.

4 acid delay buffer may cover a pH range ', for example, from about pH 4 to about pH 10, and preferably ranges from about ρίΙ 5 to about pH 9, and the optimum range

0。 From about pH 6.0 to about pH 8. 0, optimally or about 7. 0. The term "small vial," broadly refers to a storage vial suitable for maintaining a FSH in a solid or liquid form in a sealed sterilized state. Examples of vials for use herein include ampoules, cartridges, blister packs, or This applies to storage bottles that can be delivered to patients via syringes, sputum (including osmosis), catheters, skin patches, lungs or transmucosal sprays. Suitable for packaging for parenteral, pulmonary Small drug forms of products that are administered by mucosal or transdermal administration are well known and recognized by the art.

The term "stability," means rationality, chemistry, and conformational stability (including maintenance of biological potency). Proteins are due to chemical degradation or protein molecules _ = 乍, sugar correction, oxidation (special unit $ any other Decrease by at least a structural change. The solution or formulation of the biologically active beta is a protein in which degradation, modification 18 1322692, aggregation, loss of biological activity is acceptablely controlled and does not follow Time and unacceptably increased. Preferably, the formulation remains at or about 2-8 ° C (more preferably at or about 2-8, 〇, more preferably at or after about 4 months) Maintaining at least or at least about 8% of the labeled activity and at least or at least about 80% of the labeled LH activity. The FSH activity can be measured using the Stiemann-Poly Nest Weighted Bioassay 5. The LH activity can be used with the seminal vesicle Weight gain bioassay 1Q is measured. ' Terminology' treatment, means a patient who is expected to have follicular or testicular stimulating purposes or = two other FSH and / or LH-regulated reactions for FSH and / or lh administration Give medication, follow Treatment and care. Thus treatment may include, but is not limited to, administration of FSH and/or LH to induce or improve semen quality, stimulate male release of steroids, or female follicular development or ovulation. - Dosage is intended to encompass a single small vial, ampule or cartridge FSH formulation or FSH to LH formulation for more than one injection, such as 2, 3, 4, 5, 6 or more injections. Preferably, the injections are for at least about 12 hours, 24 hours, 48 hours, etc., preferably up to or about 12 days. The injections can be separated by, for example, 6, 12, 24, 48 or 72 hours. A "salt" of a protein is an acid or base addition salt. These salts are preferably any one or more charged groups in the protein and any one or more physiologically acceptable, non-toxic cations. Or formed between anions. Organic and inorganic salts include, for example, such as hydrochloric acid, sulfuric acid, acid reflux, tartaric acid, trans-butyric acid, hydrobromic acid, glycolic acid, citric acid, maleic acid, phosphoric acid, amber Acid, acetic acid, nitric acid, benzoic acid Acid acid, p-benzoate 1322692 acid, benzoic acid, Caixian acid, propionic acid, acid and other acids, or such as Syrian, sodium, potassium or magnesium. The inventor has found that by FSH and a mixture of FSH and LH and a block copolymer selected from the group consisting of ethylene oxide and propylene oxide, preferably Pluronic® F77, Pluronic F87, Pluronic F88 and Pluronic F68, particularly preferably Pluronic F68 (BASF, Pluronic F68 is also known as a surfactant formulation called Poloxa mer 188), which is available for active adsorption on the surface of vials and/or delivery devices (eg syringes, pumps, catheters, etc.). A stable formulation in which the loss of ingredients (FSH or FSH and LH) is minimized. The inventors of the present invention have found that by combining FSH and a mixture of FSH and LH with a block copolymer selected from the group consisting of ethylene oxide and propylene oxide, preferably Pluronic® F77, Pluronic F87, Pluronic F88 and Pluronic F68, Preferably, Pluronic F68 (BASF, Pluronic F68 is also known as Pol oxa mer 188) is formulated with surfactants, and they are obtained to avoid precipitation problems caused by the presence of bacteriostatic agents such as m-nonphenol and phenol. Stabilization formulation. When Tween TWEEN 20 is used in combination with m-nonylphenol or phenol, precipitation can occur which results in turbid or emulsion solution formation. These Pluronic surfactants are block copolymers of ethylene oxide (E0) and propylene oxide (P0). Propylene oxide (P0) is interposed between two ethylene oxide (E0) block interlayers. CH. ! 3

H〇-(CH2CH2〇)x-(CH2CHO)y-(CH2CH2〇)x-H

EO P〇 EO 20

The Piuronic surfactant is synthesized in a two-step process: 2. A hydrophobe having a desired molecular weight is produced by controlled addition of propylene oxide to propylene glycol to produce a hydrophobe having a desired molecular weight; Oxygen is added to sandwich the hydrophobe between the hydrophilic double layer in Pluronic® F77, and the percentage of polyethylene oxide (hydrophilic) is 70% 'and the hydrophobe (polypropylene oxide) Molecular weight is about 2, 3〇6

Da. In Pluronic F87, the percentage of polyethylene oxide (hydrophilic) is 70% for the case and the molecular weight of the hydrophobe (polypropylene oxide) is about 2, 644.

Da. In Pluronic F88, the percentage of polyethylene oxide (hydrophilic) is 80%' and the molecular weight of the hydrophobe (polypropylene oxide) is about 2, 644.

Da. In Pluronic F68, the percentage of polyethylene oxide (hydrophilic) is 80%, and the molecular weight of the hydrophobe (polypropylene oxide) is about 1967.

Da. _

The representative properties of Pluronic F77 are listed below: Average molecular weight: 6600; Melting point/casting temperature: 48 ° C; Physical form @20 ° C: Solid state; Viscosity (Brookfield) cps: 480 [Liquid at 25 ° C, paste Body at 60 ° C and solid at 77 ° C]; surface tension, dyne / cm @25 ° C; 21 1322692 0. 1% concentration: 47. Ο 0. 01% concentration: 49. 3 0. 001% concentration The concentration of the interfacial tension, dyne/cm @25°C vs. Nujol; 0. 1% concentration: 17. 7 0. 01% concentration: 20. 8

0. 001% concentration: 25. 5 Draves fish, second 25 ° C 1. 0% concentration: > 360 0. 1% concentration: > 360 foam height

Ross Miles » 0. 1% > mm@50°C : 100 Ross Miles, 0. 1%, mm@26°C : 47 Kinetics, 0.1%, mm@ 400 ml/min : > 600 Cloud point in aqueous solution, °C 1% concentration: > 100 10% concentration: > 100 HLB (hydrophile-lipophile balance value): 25

The representative properties of Pluronic F87 are listed below: Average molecular weight: 7700; Melting point/casting temperature: 49 ° C; Physical form @20 ° C: Solid state; Viscosity (Brookfield) cps: 700 [Liquid at 25 ° C, paste Temperature at 60 ° C and solid at 77 ° C ]; 22 1322692 Surface tension, dyne / cm @25 ° C; 0. 1% concentration: 44. 0 0. 01% concentration: 47. 0 0. 001% concentration : 50. 2 Interfacial tension, dyne / cm @25 ° C to Nujol; 0. 1% concentration: 17. 4 0. 01% concentration: 20. 3

0. 01% concentration: 23. 3 Draves fish, second 25 ° C 1. 0% concentration: > 360 0. Γ /. Concentration: > 360 Foam Height

Ross Miles > 0. 1% » mm@50°C : 80 Ross Miles » 0. 1% » mm@26〇C : 37 Kinetics, 0.1%, mm@ 400 ml/min : > 600 Cloud point in aqueous solution, °C 1% concentration: > 100 10% concentration: > 100 HLB (hydrophile-lipophile balance value): 24

The representative properties of Pluronic F88 are listed below: Average molecular weight: 1 1400; Melting point/casting temperature: 54 ° C; Physical form @20 ° C: Solid state; Viscosity (Brookfield) cps: 2300 [Liquid at 25 ° C, paste The content is 23 1322692 60 ° C and the solid is at 77 ° C]; surface tension, dyne / cm ® 25 ° C; 0. 1% concentration: 48. 5 0. 01% concentration: 52. 6 0. 001% Concentration: 55. 7 Interfacial tension, dyne/cm @25°C vs. Nujol; 0. 1% concentration: 20. 5 0. 01% concentration: 23.3

0. 001% concentration: 27. 0 Draves fish, second 25 ° C 1. 0% concentration: > 360 0. Γ /. Concentration: > 360 Bubble height

Ross Miles > 0. 1% * mm@50°C : 80 Ross Miles, 0.1%, mm@26°C : 37 Kinetics, 0.1%, mm@ 400 ml/min : > 600 Cloud point in aqueous solution, °C 1% concentration: > 100 10% concentration: > 100 HLB (hydrophile-lipophile balance value): 28

The representative properties of Pluronic F68 are listed below: Average molecular weight: 8400; Melting point/casting temperature: 52 ° C; Physical form @20 ° C: Solid state; 1322692 Viscosity (Brookfield) cps: 1000 [Liquid at 25 ° C, paste The concentration is 60 ° C and the solid is at 77 ° C.; Surface tension, Dyne / cm @25 ° C; 0. 1% concentration: 50. 3 0. 01% concentration: 51. 2 0. 001 ° /. Concentration: 53.6 Interfacial tension 'Dyne/cm @25. (: For Nujol; 0.1% concentration: 19.8 0. 01% concentration: 24. 0 0.001% concentration: 26.0 Draves Wet, second 25 °C 1.0% concentration: > 360 0.1% concentration: > 360 bubble Zhu Nandu

Ross Miles, 0. 1%, mm@50〇C : 35

Ross Miles > 0. 1% , mm@26〇C : 40 Kinetics, 0.1% ’ mm@4〇〇 ml/min : > 600 Cloud point in aqueous solution. c 1% concentration: > 100 10% concentration: > 100 HLB (hydrophile-lipophile balance): 29 Other polymers having properties similar to those listed above can also be used in the formulations of the present invention. Preferred surfactants are piur〇nic f68, and surfactants having similar properties. 25 "^ Resistence to maintain the desired storage period (up to 24 months), B + , D main 12 Bu & and also enough to prevent the egg self-quality mouth and attached to such as small bottles, The concentration of the ampoule or the canister 戎^ is lost on the surface of the gemstone. (d) The concentration of P1Ur〇niC (especially P1Ur〇nic F68) in liquid preparation is or is about 〇.01 mg/ml to or is about! Mg/ml, more preferably or as about G.G5 mg/ml to or about q. 5 mg/ml, ^ which is preferably or about Q2 mg/ml to or about U# mg /ml, optimally or for about 1 mg/ml. The pro-foaming hormone (FSH) in the Linggan formulation is preferably present or is present at a concentration (w/w) of the overall formulation of from about 1 to ίομg/μl. In one embodiment, the follicle stimulating hormone (FSH) is present or is present at a concentration of about 3 to 5 micrograms per house liter of the overall formulation. In another embodiment, the follicle stimulating hormone (FSH) is present at a concentration of or as a steroidal formulation of about 37 to 2 micrograms per milliliter. The luteinizing hormone (LH) in the lyophilized formulation is preferably present at a concentration of or from about 0.1 to 3 micrograms per milliliter of the total formulation. In one embodiment, luteinizing hormone (LH) is present or is present at a concentration of about 1 to 1 microgram per milliliter of the total formulation. In another embodiment, the luteinizing hormone (LH) is present or is present at a concentration of from about 0.1 to about 6 micrograms per milliliter of the total formulation. Preferably, in a liquid formulation comprising FSH, including a reconstituted formulation, the concentration of the FSH in the formulation is from about 150 IU/ml to about 26 liters, preferably 2, IU/ml. Or is about _ ΠΙ / ml to f 丨 '5.0 IU / ml 'particularly more preferably or about 450 ΠΙ / ml or about IU / ml, optimally or about _ W ML. In a liquid formulation comprising LH - including a reconstituted formulation, preferably "H is in the formulation at a concentration of about 2,000 iu/ml, more preferably from Mago to .1, nn. Or is about (5) IU/ml to or as:: liter, especially preferably or, about 3. The liter is up to or as '.spoon 750 IU/ml, optimally or about 625! U / ml. (fs:i!iVsh, LH in the formulation, the ratio of fsh to lh is increased by the weight of the big sac semipod, about W or about 1:6, better range is better Between or is, i- j: * V.,,. is between or about 4:1 to or about 1:2, especially preferably between or about 3. j is better than 1:1 and 2 Between 卜 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约The ground is or is about _ 〇 1 to gram (four) gram of concentration is present. To or is about 0.075 mg / mM preferably Plur 〇nic (concentration in the special compound is or about = UrGniC F68) in the reconstruction ML, more preferably & mg/ml to or about 1 mg / pen open better The ground is or is about gram/ml, especially preferably ^mg/ml to or about 0.50 milligrams/mg; milligrams, the best ^ is about 〇.2 gram / ml to Or is about or about 1 赤 赤/荟# preferably FSH and lh are made with tongue, .mg/ml. ', heavy, and made, especially preferably they are 27 1322692 Produced by transfected Chinese hamster-printed cells containing DNA encoding human glycoprotein Avalatin units and FSH or LH beta-units. The DNA encoding the Aval and Beta-Subunits may be identical. Or different carriers. Recombinant FSH and LH have several advantages over their urine counterparts. The use of recombinant cell culture and isolation techniques can maintain values between batches. Conversely, urine FSH and LH In terms of purity, glycosylation pattern, sialylation and oxidation of subunits, there is a large difference between batches. Because of the large batch-to-batch consistency and purity of recombinant FSH and LH Therefore, the hormones can be easily identified and quantified using techniques such as isoelectric focusing (IEF). LH readily identified and quantified, it is enabled for the filling of vials (fill mass) rather than a mass to be filled via a hormone bioassay Mi residence invention π <. R on possess to ligand having

---, n %, from j 〇 · u to J is about U' more preferably or about 6 8 to or about 7 8 of the heart circumference 'including about pH 70, nH 7 9 u „ , Acid sight i and 1 p .4. Preferred buffer (4) 酉 long-term salt, which has better anti-charge

氽拉佐4 reviews the carvings. Dish salts, and liquids are well known in the art, such as Dulber, # brine. The concentration of the buffer in the bulk solution can be about 5 m, .mM, mM, 50 mM, 1 mM, 15 mM and 500 mM. Change between. Preferably, m 250 is preferably A»sharp/morning or about 10 mM. Buffer. ''' has a plateau value of 7.°, and preferably has a Fsh. 〇 to or is about 9. 〇, a formulation with a mixture of LH is preferably or is about The value of the pH value of 28 8.5, including about pH 7. Ο, pH 8.0 and pH 8.2, optimally or about 8. 0. This invention relates to liquid formulations and reconstitutable cold; East Dry (lyophilized) formulations wherein the solvent (also for reconstitution) is water for injection. Liquid formulations can be in single or multiple doses. The liquid and freeze-dried FSH and/or LH formulations of the present invention to be used in multiple applications preferably comprise a bacteriostatic agent such as phenol, m-cresol, p-cresol, o-cresol, gas.代甲, 笨甲8|, burnt-based benzoic acid vinegar (methyl, ethyl, propyl, butyl, etc.), benzoza gas recorded, benzodiazepine, degassed sodium acetate and sulfur fruit. Particularly preferred are age, benzyl alcohol and m-methyl 19, and more preferably, the same is true. The amount of the bacteriostatic agent is effective to produce a formulation for a period of multiple doses (which may be or is about 12 or 24 hours to or about 12 or "days, preferably or about 6 to Or for about 12 days) to maintain a substantially sterile concentration (for injection). The agent is preferably or is about 〇%% (in the bacteriostatic quality, in the solvent) to or about 2%.乂 乂 “ “ “ “ “ “ “ “ “ 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Or as a spoon, 0·5%. Taking m-nonylphenol as an example, a particularly preferred concentration is about 丨8丨14 八

). (for example, a liquid pharmaceutical composition for use in the present invention, which is or is about 3 mg/ml dissolved in WFI, in a preferred embodiment, for: a preferred multi-dose D1. It contains FSH or its variant Type, $6

Pluronic® F77. Piur〇ni U from

VnQ ^ „ F87, Pluronic F88 盥p] llrn . The surfactant of F68 ^ ^luronic is selected from the bacteriostatic agent of 'A singularity (preferably inter-age) 29 1322692. 1 In a specific aspect, the present invention provides a liquid pharmaceutical composition preferably for use in a multi-dose comprising LH, a surfactant selected from the group consisting of piur〇nic8 F77, Pluronic F87, Pluronic F88 and piur〇nic ρ68, and selected from the group consisting of A bacteriostatic agent of m-cresol and phenol (preferably m-nonylphenol). In another preferred embodiment, the present invention provides a liquid pharmaceutical composition preferably for use in multiple doses, comprising FSH and , selected from

Pluronic® F77, Pluronic F87, Pluronic F88 and Pluronic call F68 surfactants and bacteriostatic agents selected from the group consisting of m-nonylphenol and phenol (preferably m-cresol). Preferably, the FSH and LH systems are present in a ratio of (FSH:LH) of from about 2:1 to about or about 1:1. In another preferred embodiment, the present invention provides a method for making a liquid pharmaceutical composition preferably for use in multiple doses comprising forming FSH-containing or variants thereof, selected from the group consisting of piur〇nic@F77, piur界面nic F87, a surfactant of Pluronic F88 and Pluronic F68, and an antibacterial agent selected from the group consisting of m-cresol and phenol (preferably m-cresol) and an aqueous solution of WFI. In another preferred embodiment, the present invention provides a method for making a liquid pharmaceutical composition preferably for multiple doses comprising forming 3 LH selected from the group consisting of piur〇nic8 ρ77, Pluronic F87, Pluronic F88 A surfactant with Piur〇nic F68 and an antibacterial agent selected from the group consisting of decylphenol and phenol (preferably meta-phenol) and an aqueous solution of WFI. In another preferred embodiment, the present invention provides a method for providing A method of making a liquid pharmaceutical composition for use in multiple doses comprising forming 30 1322692 comprising FSH or a variant thereof selected from the group consisting of Plur〇nic8F77, piu(7)μ. F87, a surfactant of Pluronic F88 and Pluronic F68, and an antibacterial agent selected from the group consisting of m-cresol and phenol (preferably m-cresol) and an aqueous solution of WFI. In another preferred embodiment, the present invention provides a method for making a liquid pharmaceutical composition for use in multiple doses comprising forming FSH and LH, selected from Piuronic® F77, piur〇nic F87,

A surfactant of Pluronic F88 and piuronic F68 and a bacteriostatic agent selected from the group consisting of m-cresol and phenol (preferably m-cresol) and an aqueous solution of WF丨. In still another preferred embodiment, the present invention provides a method for making a packaged pharmaceutical composition comprising fsh-containing, selected from the group consisting of P1Ur〇niC® F77, Pluronic F87, Pluronic F88 and piur〇nic F68 The surfactant and a solution of a bacteriostatic agent selected from the group consisting of m-nonylphenol and phenol (preferably m-cresol) are dispensed. In yet another preferred embodiment, the present invention provides a method for making a packaged pharmaceutical composition comprising FSH and lh, selected from the group consisting of P1Ur〇nic8 F7®, Pluronic F87, Pluronic F88 and Piuronic F68 The surfactant and the solution selected from the group consisting of meta- phenol and phenol (preferably m-phenol) bacteriostatic agents are dispensed. In yet another preferred embodiment, the invention provides an article for human medical use comprising a variant comprising FSH or FSH, selected from the group consisting of

a pill of Pluronic® F77, Piuronic F87, Pluronic F88 and Piuronic F68 and a solution of a bacteriostatic agent selected from the group consisting of m-nonphenol and phenol (preferably m-nonphenol), and labeled as a solution After the first use, keep a paragraph for a period of about twenty-four hours or more, 31 1322692. Preferably, the written instructions indicate that the solution can be maintained for up to or about 12 or 14 days after the first use. In yet another preferred embodiment, the present invention provides a product for human medical use comprising a FSH and LH, selected from P-® m, Ρ-F87, Pluronic F88 and Plur〇nic F68. a small vial of a surfactant and a solution of a bacteriostatic agent selected from the group consisting of m-cresol and phenol (preferably m-cresol), and indicating that the solution can be maintained for a period of about twenty after the first use What is the text of the four-hour or longer period? The instructions indicate that the solution can be maintained at φ 逹 or about 12 or Μ days after owing to use. In a particularly preferred embodiment, the formulation comprises m-cresol and

PlUr〇niG F68. The inventors of the present invention have surprisingly found that formulations containing Pluronic do not precipitate in the presence of m-methyl, which is a problem observed with other surfactants. The preparation of the present invention may be stored for at least or at least about 6 months, 12 months, or 24 months before the first use (i.e., after the vials, ampoules, or cans have been removed). Under preferred storage conditions, the formulation is placed away from glare (preferably in the dark) at the first use of the sputum, and is maintained at or about 2-8. (:, more preferably, or about 4_5 〇 Ct. In 7 particularly preferred embodiments, the present invention provides a reconstituted lyophilized formulation for use in a preferred sputum, comprising FSH or Variant pseudo and & from P1Ur〇nic® F77, Pluronic F87, Pluronic F88, 111 (2 F68 (preferably Pluronic F68) surfactant. In another special car Jiaojia specific state temple installation, 纟 invention S A reconstituted bed dry formulation of preferably 32 1322692 for multiple doses comprising LH and interfacial activity selected from the group consisting of Pluronic® F77, Pluronic F87, Pluronic F88 and Pluronic F 68 (preferably P1 uron ic F68) In another particularly preferred embodiment, the present invention provides a reconstituted lyophilized formulation for use in multiple doses comprising FSH and LH, and selected from the group consisting of Pluronic® F77, Pluronic F87, and Pluronic F88. A surfactant with Pluronic F68 (preferably Pluronic F68). Preferably, the FSH and LH are present in a ratio of (FSH:LH) of from about 2:1 to about or about 1:1. In a particular embodiment, the invention provides a method for manufacturing A method for reconstituting a lyophilized formulation for multiple doses after reconstitution, comprising forming a mixture comprising FSH or a variant thereof and a surfactant selected from the group consisting of Pluronic® F77, Pluronic F87, Pluronic F88, and Pluronic F68, and The mixture is lyophilized. In another particular embodiment, the present invention provides a method for making a reconstituted lyophilized formulation for use in multiple doses after reconstitution, comprising forming LH-containing and selective a mixture of surfactants from Pluronic® F 77, Pluronic F8 7 , Pluronic F88 and Pluronic F68, and lyophilizing the mixture. In another particular embodiment, the invention provides a preferred embodiment for use in manufacturing A method of reconstituting a reconstituted lyophilized formulation for use in multiple doses comprising forming a mixture comprising FSH and LH and a surfactant selected from the group consisting of Pluronic® F77, Pluronic F87, Pluronic F88 and Pluronic F68, and subjecting the mixture to a mixture Freeze-drying. The lyophilized mixture of F68 surfactant is dispensed into a container. In yet another preferred embodiment, it is packaged. Pharmaceutical compositions thereof methods of,

Pluronic® F77, Pluronic F87, in yet another preferred embodiment, a method of packaging a pharmaceutical composition,

Pluronic® F77, Pluronic F87 'The present invention provides a process for the manufacture comprising the inclusion of FSH and a selected one, Pluronic F88 and Pluronic'. The invention provides a method for making it comprising LH and a selected one, Pluronic F88 and Pluronic F68 The lyophilized mixture of surfactant is dispensed into the container

In yet another preferred embodiment, the present invention provides a method for making a packaged pharmaceutical composition comprising: comprising FSH and lh and selected from Piuronic® F77, Pluronic F87, piur〇nic F88 and piur〇 Ni (F68's lyophilized mixture of surfactants is dispensed into a container. In yet another preferred embodiment, the invention provides an article for human medical use comprising a lyophilized FSH or FSH variation Type, and selected from Pluronic® F77, Plur〇nic F87, piur〇nic F88

The first container or vial of Pluronic F68 surfactant. The second container or vial contains a diluent (preferably water) for reconstitution and a bacteriostatic agent selected from the group consisting of a mixture and a discriminating (preferably a medium-to-A). In still another preferred embodiment, the present invention provides an article for human medical use comprising a lyophilized LH or LH variant and selected from Pluronic® F77, Piuronic F87, piur〇nic F88 and The first container or vial of Pluronic F68 surfactant. The second benefit or vial contains a diluent (preferably water) for reconstitution and a bacteriostatic agent selected from the group consisting of 34. In yet another preferred embodiment, the present invention provides an article for human medical use comprising a lyophilized fsh & variant, and an LH or LH variant selected from the group consisting of p-F77, Piuronic F87, Pl: The first container of the surfactant of 〇niC F88 and Pluronic F68 or the vial of the i-vial or the vial containing the diluent for reconstitution (preferably water) and selected from the In a particularly preferred embodiment of the bacteriostatic agent, preferably, the solvent for reconstitution comprises m-cresol. The inventors of the present invention have found that a lyophilized formulation comprising Pluronic F68 does not swell when it has been reconstituted with a diluent, which is a problem observed with other surfactants (e.g., Tween). The lyophilized formulations of the present invention can be stored for at least or at least about 6 months, months, or 24 months. Under preferred storage conditions, prior to the first use, the formulation is placed away from glare (preferably in the dark), at or about 2-8 ° C, more preferably Or for about 4_5^. After the first use of the liquid or reconstituted multiple dose formulation, it can be maintained and used for at least or at least about 24 hours, preferably at least or at least about 4, 5 or 6 days, more preferably Up to 12 or 14 days. Preferably, after the first use, the formulation is stored at a temperature below room temperature (ie, below or below about 25 ° C), preferably more preferably or about 2 _ c, optimally Preferably, the formulation of the present invention contains an antioxidant such as a salt of methionine, sodium hydrogen sulfite, ethylene diamine tetraacetic acid (10) TA, a tert-butyl pair, and a tert-butyl group. Methoxy hope _. The best is methionine. Anti-35 1322692 Oxidants prevent oxidation of FSH and LH (especially alpha-subunits). Preferably, the liquid and/or the reconstituted formulation comprises methionine of from about 0.01 to about or about U mg/ml, more preferably or from about: to or about 0.5 mg/ml, more preferably Or exist for a concentration of about 〇. Preferably, the formulations of the present invention comprise mono- or disaccharides or sugar alcohols as an anchoring agent and a tonicity adjusting agent, such as a sucrose, (tetra) sugar, lactose, mannitol, or glycerin. The best one is a recommended sugar, preferably at a concentration of or

ML. Brother/As mentioned above, the present invention provides a liquid formulation for single use and multiple dose use, which contains a bacteriostatic agent, or a bacteriostatic agent is added thereto during the formulation. As described above, in a preferred embodiment, the article of the present invention comprises a packaging material and a containing, ', 3, ΜΗ or FSH variant, LH, or FSH and LH, Plur〇nic F68 and Since the confession

a bacteriostatic agent, optionally with a buffer and/or other excipients, a vial of a solution formed in an aqueous diluent, wherein the inclusion material comprises a label indicating that the solution can be retained after the first use - A textual description of the period of twenty-four hours or more. The present invention further comprises a kit comprising a packaging material, a vial containing a FSH<clear variant formulation according to the present invention, wherein the (four) material comprises a patient indicating that the patient is reconstructed to form a segment A textual description of the solution for a period of twenty-four hours or more. As described above, in a preferred embodiment of the present invention, the present invention provides a 36 1322692 article comprising a packaging material and a small inclusion of Kanggan Fsh or fsh type, LH, or FSH and LH, and Pluronic F68. Medicine bottle. The bacteriostatic agent in the container containing the diluent is selected from the group consisting of arbitrarily and inter alia, and the other materials are required, wherein the packaging material comprises the labeling solution and can be retained after the first use. A period of twenty-four hours or more = in the text of the manual. The range of protein hormones in the formulation of the present invention comprises a concentration which can be produced at a concentration of from about 1.0 microgram/ml to about 50 mg/ml upon reconstitution, however lower and more south concentrations can be administered and depending on the desired delivery. Loaded, for example, solution formulations may differ from percutaneous patches, lungs, transmucosal or methods such as perforation or micro-pull. The protein hormone concentration is preferably from about 5.0 micrograms per milliliter to about 2 milligrams per milliliter, more preferably from about 0.25 micrograms per milliliter to about or about! The mg/ml is optimally or about 5 〇 micrograms/ml to or about 200 micrograms/ml.

Preferably, the formulations of the invention retain at least or at least about 80% of FSH activity and/or Lh activity after 24 months (before the first use) packaging period. FSH activity can be measured using a Stiemann-Poly ovarian weight gain bioanalyzer 5 . LH activity can be measured using seminal vesicle weight bioassay. The liquid formulation of the present invention may be prepared by mixing a mixture of FSH or FSH variant, LH, or a combination of FSH and LH with Pluronic F68 and a bacteriostatic agent selected from the group consisting of P- or FSH or FSH. Variant, lh, or a mixture of FSH and LH ("protein") and piur〇nic F68 and a bacteriostatic agent selected from the group consisting of bismuth and cresol dissolved in an aqueous diluent to prepare the mixture and They are dissolved in aqueous thinners and are used by Xi 37 1322692

Know that the dissolution and mixing process is complete. For the preparation of suitable formulations, for example, FSH or FSH variants, LH, or a mixture of FSH and LH in a buffer solution and piur〇nic F68 dissolved in a buffer solution and selected from phenol and The m-cresol bacteriostatic agent is quantitative enough to bring the protein, piur〇nic F68 and bacteriostatic agent to the desired concentration. The resulting solution is then dispensed into vials, ampoules or cans. Those skilled in the art should understand that this method has various variations. For example, the order in which the ingredients are added, whether additional additives are used, the temperature, and the pH of the prepared formulation are all factors that are optimal for the concentration and method of administration.

In a preferred embodiment, the liquid formulation of the present invention is prepared by making all of the ingredients in the formulation (eg, buffered sodium sulphate 'sucrose, tween, sulphur sulphate, FSH, and/or LH). The individual stock solutions of known concentrations, M are equally divided into equal volumes to form "mother tears" having the same composition as the final formulation. Preferably, the "mother liquor" is passed through DurDp〇re@(MiUip〇re) 〇2, =PDF membrane to remove microorganisms, and then the aliquot is dispensed into (iv) containers, such as vials, ampoules or In the medicine can.

The dry formulation of the present invention may comprise a fsh "fsh variant, LH, or FSH with LH, LH or LH variants, or a mixture of _ and heart with Hu and ic F68 and such as antioxidants and/or buffers. The other excipients of the agent are mixed and cooled; the method of East drying is prepared. Mix the names and mix them with cold-drying %m % *.. Drying is done using a known process. Prepare a suitable formulation, such as a system, combine the measured amount of ΜΗ ^ ^ L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L It is packaged in 38 1322692 vials, ampoules or cans. Those skilled in the art should be aware of this method and have various variations. For example, the order in which the components are added, whether or not the ingredients are added, the additional additives, the temperature, and the value of the prepared formulation are all factors that can be optimized for the concentration and the mode of administration used. Formulations of the invention can be administered using approved devices. Examples including such single vial systems include for delivery such as 仏叮 to 8, Gonabu F® pen, Humaject®, NovoPen®, B_D(g)Pen,

Pen type injector device for solutions such as AutoPen® and 〇ptipen®. The products claimed in this case include packaging materials. In addition to providing the information required by the regulatory agency, the packaging material also provides the conditions under which the product can be used. The packaging material of the present invention provides an indication to the patient to reconstitute the FSH or FSH variant in an aqueous diluent to form a solution, and to use the solution for twenty-four hours or more (for two bottles (wet/dry) product) Description. For single vials, solution products, the label indicates that the solution can be stored for a period of up to twenty-four hours or more, preferably 12 or 14 days, after the first use. The products claimed in this case can be used for human medical products. A suitable antiseptic formulation can be provided to the patient as a clear solution. The solution can be used singly or it can be reused multiple times and can satisfy single or multiple patient treatment cycles, thus providing a more convenient treatment than currently available to the sputum present in the stable or antiseptic formulations described herein. Or FSH or FSH variants, LH, or a mixture of FSH and U in solution, according to the invention via various delivery methods including SC or IM injection, transdermal, pulmonary, 39 1322692 transmucosal, implant, infiltration pump The patient is administered in a manner known to the person skilled in the art (and the skill). [Embodiment] The following examples are provided only to further illustrate the preparation of the formulations and compositions of the present invention. The scope of the invention should not be construed as being limited to the following examples. Example 1 Comparative formulation materials

Project manufacturer bulk r-hFSH for candidate formulation Serono laboratory SA D-mannitol (DBA, Ph Eur, BP, FU, USP, FCC, E421) Merk sucrose (DBA, Ph Eur, BP , NF) Merk NaCl (ACS * ISO) Merk Na? HP04 2H20 (analytical grade) Merk NaH9P04 H20 (analytical grade) Merk benzofuran (analysis) Grade) Merk-indophenol (for synthesis) Merk TWEEN 20 (polysorbate 20) (for synthesis) Merk Pluronic F68 (Poloxaraer 188) Sigma /-曱 thiaminic acid (for biochemistry) Merk Orthocalyptus 85% (Ph Eur, BP, NF) Merk 1. 5 ml glass cartridge SFAM (in Aguettant dream) Type A rubber western Company Crim Cover Aguettant Millex-GV Syringe Drive Filter Unit - Durapore Mi Mi ipore Durapore Membrane Filter 0. 22 micron GV Mi Mi ipore 20 ml plastic syringe package Beckon Dickinson Filtration stainless steel holder Sartorius 40

1022WZ Equipment HPLC System Detector Model 486 or 490 Controller Model 600S Pump Model 626 1 Dynamic Sampler Model __ 717 Waters 2 pH Meter _sJ! No. 654 Metrohm 1 Osmometer - 0 3 0 - D_ Osmomat 1 The following studies evaluated the following parameters for large quantities of formulation: Interface/tongue and bacteriostatic compatibility • Avatar to subunit oxidative formulations are multi-dose formulations and contain TWEEN 20 Or Pluronic F68 and a bacteriostatic agent. The following three bacteriostatic agents were evaluated: • Benzyl alcohol 0.9% • m-cresol 0. 3% • Phenol 0. 5% TWEEN 20 and Pluronic F68 were used in the following concentrations: • TWEEN 20: range from 10 To 1 μg/g • Pluronic F68: Solutions prepared from 10 to 100 μg/g are listed in Table 1. 1322692 Table 1: Comparative Formulation ID# Na2HP04 NaH2P04 r-hFSH* Pluroni TWEEN Antibacterial Excipient 2H20 H20 c F68 20 (mg/g) (rag/g) (rag/g) (pg/g) (Mg /g) IP 1.11 0. 45 600 10 - 0. 5% sucrose IU / g phenol 70. 6 2P 1.11 0.45 600 1 0 - 0.5% mannitol IU / g phenol 38.7 3P 1.11 0.45 600 100 - 0. 5% Sucrose IU/g Phenol 70.6 4P 1.11 0. 45 600 100 - 0. 5% Mannitol IU/g Phenol 38.7 5P 1.11 0. 45 600 - 10 0.5% Sucrose IU/g Phenol 70. 6 6P 1.11 0.45 600 10 0.5 % mannitol IU/g phenol 38. 7 7 1.11 0. 45 600 - 100 0. 9% NaCl IU/g phenylhydrazine 6.0 alcohol 8 1.11 0. 45 600 - 100 0.9% sucrose IU/g phenylhydrazine 62. 3 Alcohol 9 1.11 0. 45 600 - 100 0.9% mannitol IU/g Benzene 34.1 Alcohol

42 1322692 Table 1: Comparative Formulation ID# Na2HP04 NaH2P04 r-hFSH* Pluroni TWEEN Bacteriostatic Excipient 2H20 H20 c F68 20 (rag/g) (mg/g) (rag/g) (μδ/g) ( μΕ/g) 10 1.11 0. 45 600 - 100 0.3% NaCl IU/g m-phenol 7.6 11 1.11 0.45 600 - 100 0. 3% sucrose IU/g m-cresol 78.0 12 1.11 0. 45 600 - 100 0. 3% mannose IU/g m-cresol 42. 7 13 1.11 0.45 600 - 10 0. 9% NaCl IU/g phenyl sterol 6.0 14 1.11 0. 45 600 - 10 0.9% sucrose IU/g phenyl sterol 62 3 15 1.11 0. 45 600 - 10 0.9% mannose IU/g phenylhydranol 34. 1 16 1.11 0.45 600 - 10 0. 3% NaCl IU/g m-phenol 7.6 17 1.11 0. 45 600 - 10 0.3% sucrose IU/g m-cresol 78.0 18 1.11 0. 45 600 - 10 0. 3% mannose IU/g m-phenolol 42. 7

43 1322692 Table 1: Comparative Formulation ID# Na2HP04 NaH2P04 r-hFSH* Pluroni TWEEN Bacteriostatic Excipient 2H20 h2o c F68 20 (mg/g) (rag/g) (rag/g) (Mg/g) ( Mg/g) 19 1.11 0. 45 600 100 - 0.9% NaCl IU/g phenyl sterol 6.0 20 1.11 0.45 600 100 - 0. 9% sucrose IU/g benzyl alcohol 62. 3 21 1.11 0.45 600 100 - 0.9% nectar Sugar IU/g phenylhydranol 34. 1 22 1.11 0.45 600 100 - 0. 3% NaCl IU/g m-phenol 7.6 23 1.11 0.45 600 100 - 0. 3% sucrose IU/g m-phenol 78.0 24 1.11 0 45 600 100 - 0.3% mannose IU/g m-phenolic alcohol 42. 7 25 1.11 0. 45 600 10 - 0.9% NaCl IU/g phenyl sterol 6.0 26 1.11 0.45 600 10 - 0. 9% sucrose IU/ g 曱 曱 6 62.3 27 1.11 0. 45 600 10 - 0. 9% mannose IU / g benzyl alcohol 34.1

44 1322692 Table 1: Comparative Formulation ID# Na2HP04 2H20 (rag/g) NaH2P04 H20 (rag/g) r-hFSH* Pluroni c F68 (μΕ/g) TWEEN 20 (Mg/g) Bacteriostatic Excipient ( Mg/g) 28 1.11 0.45 600 IU/g 10 - 0. 3% m-cresol NaCl 7.6 29 1.11 0. 45 600 IU/g 10 - 0. 3% m-cresol sucrose 78. 0 30 1.11 0.45 600 IU/ g 10 - 0.3% m-phenol mannitol 42. 7

* FSH is added to the formulation based on its bioavailability rather than protein content.

45 1322692 From the results of visual inspection of the formulations, TWEEN 2〇 was not compatible with m-nonylphenol and phenol because of the presence of a white creamy suspension of TWEEN 20 and m-prepared FSH formulations. Conversely, FSH formulations containing Pluronic F68 did not present the problems associated with this and cresol. With the Pluronic F68, you can also use the room. Combination of FSH and Pluronic F68 with Antioxidants The following antioxidants are evaluated for their ability to inhibit alpha-subunit oxidation in the presence of p 1 ur〇n ic F68: • Methionine: ranging from 10 to 100 μg / gram. Ascorbic acid: ranging from 1 〇 to 1 〇〇 microgram / gram of sucrose and mannose pure as a tonicity agent, and TWEEN 2 〇 P1Ur 〇 niC is added at a concentration of 100 μg / gram. 5 The formulations prepared are listed in Table 2.

46 1322692 Table 2. Comparative formulations containing and R containing methionine ID# Na2HP04 NaH2P04 RhFSH Pluroni TWEEN Anti-deteriomethyl bacteriostatic agent 2H20 h2o c F68 (Mg/g) Acid acid reagent (mg /g) (mg/g) (μβ/g) (μβ/g (pg/g)) 31 1.11 0. 45 600 100 - - - 0.33⁄4 sucrose IU/g cresol 32 1.11 0. 45 600 100 - — — 0.3% nectar IU/g cresol alcohol 33 1.11 0.45 600 - 100 - - 0. 9% phenyl sucrose IU/g methanol 34 1. 11 0.45 600 - 100 - - 0· 9% Benzanol IU/g Methanol sugar alcohol 35 1.11 0. 45 600 100 - - - 0.93⁄4 phenyl sucrose IU / g sterol 36 1.11 0.45 600 100 - - - 0_ 93⁄4 Benzanol IU / g sterol sugar alcohol 37 1.11 0.45 600 100 - - 10 0.3 3⁄4 sucrose IU/g cresol 38 1. 11 0. 45 600 100 - - 10 0.33⁄4 mannose IU/g cresol 39 1. 11 0.45 600 100 - - 100 0.3% sucrose IU/g cresol 47 1322692 Table ί. Comparative formulation with and without methionine ID# Na2HP04 NaH2P04 RhFSH Pluroni TWEEN Anti-bad sulphur bacteriostatic Shape 2Η20 H20 c F68 (μβ/g) Serum acid (mg/g) (mg/g) (μβ/g) (pg/g) (pg/g) 40 1. 11 0. 45 600 100 - - 100 0.3% mannose IU/g cresol 41 1. 11 0. 45 600 100 - 10 - 0. 3% sucrose IU/g phenol 42 1. 11 0. 45 600 100 - 10 - 0.3 Between mannose IU/g cresol 49. 11 0. 45 600 100 - 100 - 0.3% sucrose IU/g cresol 44 1. 11 0. 45 600 100 - 100 - 0.3% nectar IU/g Indophenolol 45 1.11 0. 45 600 - 100 - 10 0· 9% phenyl sucrose IU / g methanol 46 1. 11 0. 45 600 - 100 - 10 0.93⁄4 Benzanol IU / g methanol sugar alcohol 47 1. 11 0. 45 600 - 100 - 100 0. 9% phenyl sucrose IU / g methanol 48 1.11 0. 45 600 - 100 - 100 0.9% phenylmannan IU / g methanol sugar alcohol 48 1322692 Table 2. with and without methyl thiamin Acid ratio 4 biting formulation ID# Na2HP04 NaH2P04 RhFSH Pluroni TWEEN Ascorbic acid thiamine bacteriostatic agent Excipient 2H20 h20 c F68 (pg/g) Acid acid (mg/g) (mg/g) (Mg/ g) (pg/g) (μβ/g) 49 1. 11 0. 45 600 - 100 10 - 0.93⁄4 sucrose IU/g methanol 50 1.11 0. 45 600 - 100 10 - 0.93⁄4 phenylmannose IU/g methanol alcohol 51 1. 11 0. 45 600 - 100 100 - 0.93⁄4 sucrose IU/g methanol 52 1. 11 0. 45 600 - 100 100 - 0.9% phenylmannose IU/g sterol 53 1. 11 0. 45 600 100 - - 10 0.9% phenyl sucrose IU/g sterol 54 1.11 0.45 _ 600 100 - - 10 0.9% phenylmannose IU/g methanolol 55 1. 11 0. 45 600 100 - - 100 0.93⁄4 phenyl sucrose IU/g methanol 56 1.11 0. 45 600 100 - - 100 0.93⁄4 phenylmannose IU/ g Methanol Yeast 57 1.11 0.45 600 100 - 10 - 0.9% phenyl sucrose IU/g Methanol 49 1322692 Table 2. Containing and not containing acetamino acid ratio L Formulation ID# Na2HP04 NaH2P04 RhFSH Pluroni TWEEN Anti-Methane Bacteriostatic phlegm 2H20 H20 c F68 (pg/g) arginine (mg/g) (mg/g) (pg/g) (Mg/g) (pg/g) 58 1.11 0.45 600 IU/ g 100 - 10 0. 9% phenylmannan methanolitol 59 1.11 0.45 600 IU/g 100 - 100 - 0.9% phenyl sucrose methanol 60 1. 11 0 .45 600 IU/g 100 - 100 - 0. 93⁄4 Benzenol methanolitol 61 1. 11 0.45 600 IU/g 100 - - - Phenol sucrose 62 1.11 0.45 600 IU/g 100 - - - Phenol mannitol 63 1 11 0.45 600 IU/g phenol sucrose 64 1. 11 0.45 600 IU/g 100 - - 10 phenol mannitol 65 1. 11 0. 45 600 IU/g 100 - - 100 phenol sucrose 66 1.11 0.45 600 IU/g 100 - - 100 phenol mannitol 67 1. 11 0.45 600 IU/g 100 - 10 - phenol sucrose 68 1. 11 0.45 600 IU/g 100 - 10 - phenol mannitol 69 1.11 0.45 600 IU/g 100 — 100 - Phenol sucrose 70 1. 11 0.45 600 IU/g 100 - 100 - Phenolmannitol FSH is added to the formulation based on its bioavailability rather than protein content. 20 grams of each formulation is prepared into Falc〇 n The polypropylene tube is driven by a 3 - 0.22 micron Millex-GV syringe to filter the unit, and (4) (4) > The solution is then stored under the armpit and tested according to the following table:

Analytical test T = 0 1 week 2 weeks 3 weeks 4 adjusted reverse phase-HPLC analysis of oxidized avalanche units (%) XXXXX size exclusion-HPLC analysis protein quantification (pg/g) XXXXX size exclusion-HPLC analysis quantification Free subunit XXXXX (X): Tested by reverse phase HPLC, shown in a formulation containing FSH, Pluronic F68, m-cresol and methionine (concentration 1 〇 and 1 〇〇 microgram/ml) When the system is stored at 4 (TC), the oxidation of the FSH α-subunit has been greatly reduced (relative to the formulation without methionine), as shown in Figure 1. Based on the average of the two experiments, The percentage of oxidized α-subunits in the methionine-containing formulation is 2.3 at T = 0, 4.0 at T = 1 week, and 7.1 at T = 2 weeks. The percentage of oxidized α_' subunits in the microgram/ml methionine formulation is 2.0. Τ =0, 3.2 in T = 1 week, and 3.8 in T = 2 weeks. In the formulation of 100 μg/ml guanidine thioglycol, the percentage of oxidized cardiac subunits was 1.8 at Τ = 0, 1.7 at T = 1 week, and 1 · 3 at τ = 2 weeks. 51 1322692 Example 2 Under the skin Or a single dose formulation of recombinant Fsh liquid injected intramuscularly. Based on the results of Example 1, the following formulations were prepared. Compositions 1 to 7 listed in Table 3 were prepared as a volume solution dissolved in WFI. Each solution is added to the mixing vessel to form a "mother liquor." The mother liquor is dispensed into a vial to contain 1 〇 9 μg (15 〇 11}) or 5 45 μg (75 IU) of FSH » Using recombinant FSH The biological activity and specific activity can be maintained, and the FSH can be filled with mass instead of bioanalyzed. Table 3. FS composition number I single dose liquid helium preparation also describes the composition of f 150 IU FSH 75 IU FSH 1 rhFSH (pg/pill bottle) 10.9 (150 IU) 5 45 (75 IU) 2 sucrose (mg/pill bottle) 15. 00 7 50 3 NaH2P04 H20 (mg/pill bottle) 0. Ill 0.0555 4 Na2HP04 2H20 (mg /Pill bottle) 0. 273 0.1365 5 Pluronic F68 (mg/pill bottle) 0.025 0.0125 6 Methionine (rag/pill bottle) 0.025 nm 25 7 m-cresol (mg/pill bottle) 0.75 0.375 8 PH 7. 0 7 0 9 WFI qs to 1 ml n_ s. to 0. 5 ml Fill and seal the vial in a sterile state. The formulation has a shelf life of up to two years at the enclosed temperature. Example 3 Recombinant FSH Liquid Multi-Dose Formulation for Subcutaneous or Intramuscular Injection 52 1322692 Based on the results of Example 1, the following multiple dose formulations were prepared. Compositions 1 to 7 listed in Table 4 were prepared as a volume solution dissolved in WFI. An aliquot of each solution is added to the mixing vessel to form a "mother liquor." The mother liquor was dispensed into a vial containing 22.2 micrograms (305 IU), 33.3 micrograms (458 IU) or 66.7 micrograms (916 IU) of FSH. The resulting formulation delivered a total of 300, 450 and 900 IU of FSH. The canister is filled and sealed under aseptic conditions. The multiple dose formulation can be stored at or about 2-8 ° C, more preferably at about 4-5 ° C until the first use. After the first use, the canister should be stored at or about 2-8 ° C, more preferably at or about 4-5 ° C for a period of multiple doses, which can be 24 hours, 2 days Or up to 12 or 14 days. Table 4. FSH Multiplexers] I: Composition of Liquid Formulations Number Description 300 IU FSH 450 IU FSH 900 IU FSH 1 rhFSH (μ8/drug) 22.2 (305 IU) 33.3 (458 IU) 66.7 (916 IU) 2 Sucrose (mg/drug) 30. 0 ' 45. 0 90. 0 —* 3 NaH2P04 H20 (mg/drug 5 0.225 0.337 0. 675 4 Na2HP04 2H20 (rag/drug) ϋ. 555 0.832 1.665 5 Pluronic F68 (mg/drug) 0. 0 5 0 0.075 0.150 6 Methionine (mg/drug) U. ϊ) 50 ~ 0.075 0.150 — 7-indolol (mg/drug) TTTO '~~ " 2. 25 4. 5 ~ 8 PH Y. U 7.0 7.0 9 WFI qs to U. 5 ml qs to 0. 75 ml q. s· to 1. 5 ml

Example 4 Recombinant LH Liquid Single Dose Formulation for Subcutaneous or Intramuscular Injection 53 1322692 The following formulations were prepared. Compositions 1 to 7 listed in Table 5 were prepared as a body essence solution dissolved in WFI. The aliquots of each solution were added to the mixing vessel to form a "mother wave". The mother liquor was dispensed into a vial containing 3 micrograms (75 liters of LH. The resulting formulation delivered a single dose of 75 IU). LH. Using recombinant LH, biological activity and specific activity can be maintained constant, while LH can be filled in mass rather than bioanalyzed. Table 5. L" - Composition of a dose of liquid formulation number description LH 75 IU 1 rhLH (pg/pill bottle) 3. 0 2 curtain sugar (mg/pill bottle) 52. 5 3 NaH2P04 HW (rag/pill bottle) 0.052 4 Na2HP04 2H4 (mg/pill bottle) 0. 825 5 Pluronic F68 (mg /Medication, ------- 0.0125 6 Methionine (mg / vial) 0. 125 ' 7 - indophenol (mg / vial) 0.375 — 9 WFI Q· s. to 0 5 ml The vial is filled and sealed under aseptic conditions. The formulation has a storage and storage life of two years.

In the package

Example 5 Recombinant Dosage Formulation for Subcutaneous or Intramuscular Injection The following FHS to LH FSH:LH ratio of 2:1 was prepared. A multi-dose formulation of FSH and LH (2:1) solution, which is shown in Table 6, is prepared as a solution of Zhao Zhao 54 1^22692. An aliquot of each solution is added to the mixing vessel to form a "mother liquor." If necessary, adjust the pH of the mother liquor to 8 藉 by adding NaOH or Hci. The mother liquor was dispensed into a vial containing 18 3 μg LH (457 1{]) and 66.7 μg FSH (916 IU) for 6 doses of 150 IU FSH; 9.2 μg LH (230 IU) For 33.3 μg FSH (458 IU), 3 doses of 150 IU FSH; and 6.1 μg LH (152.5 IU) and 22.23 μg FSH (305 IU), for 2 doses of 150 IU FSH each〇 The canister is filled and sealed under aseptic conditions. The multiple dose formulation can be stored at or about 2-8 ° C, more preferably at or about 4-5X: until the first use. After the first use, the canister should be stored at or about 2-8 ° C, more preferably at or about 4-5 ° C for a period of multiple doses '24 hours, 2 days Or up to 12 or 14 days. Table 6. FSH and LH (2:1) multiple dose liquid formulation composition number description 6 doses 3 doses i 2 doses 1 rhLH (pg/drug) 18.3 (457 IU) 9.2 (230 IU) 6. 1 (152. 5 IU) 2 rhFSH Ug/drug) 66.7 (916 IU) 33.3 (458 IU) 22.23 (305 IU) 3 Sucrose (mg/drug) 115.5 57. 75 38.5 0.49 4 Η3Ρ04 (rag/canister) 1. 35 0.735 5 NaOH (mg/drug) qs to pH 8.0 q_s. to pH 8· 〇qs to pH 8.0 6 Pluronic F68 (rag/drug) 375. 0 187.5 125.0 7 Methionine 225 112.5 75.0 55 (rag / canister) -------1 8 - cresol (mg / canister) 4. 5 2.25 1.5 9 PH 8. 0 8.0 8. 0 10 WFI — g. s· to 1. 5 ml qs to 0·75 ml as to 0. 5 ml Example 6 Recombinant FSH and LH (1:1) liquid multi-dose formulations for subcutaneous or intramuscular injection The following FHS and LH multiple dose formulations were prepared. The FSH:LH ratio is 1:1. Compositions 1 to 8 listed in Table 7 were prepared in a volume > trough solution dissolved in WFI. An aliquot of each solution is added to the mixing vessel to form a "mother liquor." If necessary, the pH of the mother liquor is adjusted to 8. 0 ^ by adding NaOil or HCl. The mother liquor is divided into small vials to contain 36 6 μg LH (914 III) and 66. 7 μg FSH (916 IU) For 6 doses of 150 IU FSH; 18. 4 μg LH (460 IU) and 33·3 μg FSH (458 IU), for 3 doses of 150 IU FSH each; and 12.2 μg LH (305 IU) and 22. 23 μg FSH (305 IU) 'For a dose of 150 IU FSH twice, 充 Fill and seal the canister aseptically. The multi-dose formulation can be stored at or about 2-8 ° C. 'More preferably at or about 4-5 ° C until the first use. After the first use, the canister should be stored at or about 2-8 ° C, more preferably at or about 4-5 ° C for a period of multiple doses, which can be 24 hours, 2 days Or up to 12 or 14 days. 56

I3226W Table composition number [· fsh^TITT] Description ' 1 rhLH (μβ/^^Λ 2 rhFSH - (με/drug) sucrose (mg/ 4 W ~^ (mg/drug) 5 NaOH ~ (mg/ Medicine tank) V Pluronic (mg/drug) methionine (mg/drug) 8 rooms-A ('mg/drug can) PH Γο ΨΓι --- | 3 doses--ΡΖ|Ι(9Τ4ΤϋΥΐ8 .4 (46Τ1ΙΙΐ〇^(3〇Γ^· 66· 7 (916 IU) 33.3 (458 IU) 22?23^~~^ '75. 'O'

Example 7 Experimental literature on FSH mixing and lh liquid multi-dose formulation 7.1 Inverse phase HpLc analysis of protein content Evaluation of protein content of FHS and LH of Example 5 (6 doses) , using reverse phase HPLC method. The protein content (fsh and lH) was measured at 0:00 and after storage of the formulation at 4〇c for 2, 3 and 6 months. The results are shown in Table 8, expressed as grams of FSH or LH per gram of solvent. 7. Analysis of oxidized Alfa-subunits The oxidized Alfa-subunit percentage values present in the formulation of Example 5 were measured by reverse phase HPLC (RP-HPLC). Store at 4 o'clock and store the formulation at 4. (: The next 1, 2, 3, and 6 57 1322692 months after the measurement of the percentage value of the oxidized Alfa-subunit. The results are shown in Table 8. 〇7.3 3 FSH in vivo analysis at zero hour and The formulation was stored at 4 ° C for 1, 2, 3, and 6 months, and the formulation of Example 5 (6 doses) was tested using the ovarian weight gain bioassay in the Stilman-Poly Analysis 8. Its FSH activity. The results are shown in Table 8 'in International Units (ιυ) per gram of solvent. 7. 4 In vivo analysis of LH at 0 o'clock and storage of the formulation at 4 ° C 1, 2 After 3 and 6 months, using the rat seminal vesicle weight gain bioassay, the FSH activity of the formulation of Example 5 (6 doses) was measured. The results are shown in Table 8, in International Units (IU). Expressed per gram of solvent. 7.5 Evaluation of free subunits (rFSH + rLH) The free subunit percentage values were evaluated for the formulation of Example 5 by SDS-PAGE. The formulation was stored at zero time and stored. 4. (: Measurements were taken after 1, 2, 3 and 6 months. The results are expressed as a percentage of the total protein (rFSH + rLH) and the results are shown in Table 8. 7.5 Evaluation of Aggregates The formulation of Example 5 was evaluated for the percentage of aggregates by SDS-PAGE as described above for the evaluation of free subunits in 7.5, except that the higher molecular weight aggregate system was determined to be the total protein (rFSH+ The percentage value of rLH) was measured at 0:00 and after storage of the formulation at 1, 2, 3 and 6 months at 4 ° C. The results are shown in Table 8. 1322692 7. 6 Visually detectable The particles were visually evaluated for the formulation of Example 5 at zero time and after the formulation was stored at 4 ° C for 1, 2, 3 and 6 months. The results are shown in Table 8. ' 7_ 3 The pH of the formulation of Example 5 was measured at pH 0 and after the formulation was stored at 1, 1, 2, and 3 months. The results are shown in Table 8 i I AVvc%1 !^ 0:4 6.50 Analysis _ rFSH content measured by RP-HPLC (μg/g) rLH content (μg/g) as measured by RP-HPLC % oxidized Avalanche unit

EXAMPLE 8 FSH and LH freeze-dried multi-dose formulations Two formulations with the following composition, A, were prepared to dry formulations A and B:

FSH microgram (45〇 U.) 59 1322692

LH sucrose NaH2P04 H20 Na2HP04 2H20 Pluronic F68 L-methionine formulation B microgram 9. 0 (255 I. u.) mg 15. 0 mg 0. 052 mg 0. 825 mg 0. 05 mg 0. 05 FSH micro LH microsucrose milli NaH2P04 H20 milli Na2HP04 2H20 milliP1uron ic F68 milliL-methionine gram 65· 5 (900 I. u.) gram 18. 0 (450 I. u.) gram 30. 0 gram 0_ 104 g 1. 65 g 0·10 mg 0. 10 The method of manufacture comprises mixing the drug directly with the ingredients, filtering the resulting solution and lyophilizing the filtrate. The description of each step of the preparation is as follows: - Add WFI, hydrogen dihydrogen dihydrate dihydrate, sodium dihydrogen monohydrate monohydrate, sucrose, 5% Pluronic F68 to a container of 5 weights And L-methionine' and stir for 1 〇 minutes until completely dissolved.

- Measure the pH and correct it to pH 7.00 soil 0. 2 with NaOH 10% or dilute IPO4. 1322692 - FSH and LH were added to the mixture prepared above and the resulting solution was gently stirred for 10 minutes. - again measure the pH and correct it to pH 7.0 with i〇% Na0H or dilute Ο/Ο4. 1 〇- will be the 0.22 micron Durapore membrane at a filtration rate of not less than i5g/cm2, Filtration was carried out under a nitrogen stream with a pressure not higher than 15 atmospheres. - The solution was collected in a previously sterilized flask. - Fill the glass container with the passed solution, install the stopper and place the filled vial into the stainless steel tray. - Load the tray in a freeze dryer and freeze the product using the following freeze-drying cycle: • at +4. (: Lower the balance for about 20 minutes. Bring the rack temperature to -25 ° C for 2 hours. Bring the rack temperature to -15. 〇: and keep it for 1 hour. Bring the rack temperature to -45 °C for 3 hours • Bring the condensed Is temperature to – 65 ° C. • Apply a vacuum to the chamber • Increase the rack temperature to _ 10 ° C when the vacuum reaches 7 X 1 (Γ2 mBar) And hold for 14 hours. Increase the rack temperature to + 35X in 8 hours: and keep it until the end of the cycle (14 hours). Break the vacuum to allow dry nitrogen to enter the chamber. With the freeze dryer The automatic system completes the plugging. 1322692 • The filled vials will be sealed with a suitable split lid. The formulations A and B have been stored at 25 ± 2. (: and tested as follows) Stability and biological activity. Before the analysis of the composition, they were first reconstituted with water for injection containing 3% decyl phenol as a bacteriostatic agent. The stability and biological activity values were determined as follows: . Internal analysis: FSH activity was tested using a Stiemann-Poly ovarian weight gain bioassay. • In vivo analysis of LH: using rat sperm Weight gain bioassay to test LH activity • Analysis of oxidized Alfa-subunits: Percentage of oxidized Alfa-subunits by reverse phase HPLC (Rp_HPLC) method • Free state subunit (rFSH + rLH) Evaluation: The percentage value of the free subunits was evaluated by SDS_PAGE. • Assessment of aggregates: The percentage value of aggregates was evaluated by SDS-PAGE as described above for the evaluation of free subunits. Completed in accordance with the European Pharmacopoeia Biological tests. In particular, these tests are reported in “Protrophotropin, Monograph. Eight Table 9 provides a comprehensive analysis of the results of the knife test related to the stability and biological activity of Formulation A.” These values are The test was carried out at 4 test points: at 0:00, after the preparation was stored at 25±2 for 1 month, 3 months and 6 months. 丄:>zz〇y;z ΓΓΤ-- -—----- Table 9 Jade Activity I _ υ FSH ' Biological Activity IU L Η % Oxidized Product Zero Time ~416 276 1 QC; 1 ^ ~~42〇~^~ ~~25Τ~^~ i~ 3 months 4l5 259 6 months~ ^4Π 27〇^~ % dimer % free state single 亓. <2 Γ <5 1 · ο 1 <~Γ^~ &l t;τ^~ 1.95 <2 <5 ΓΤ57 ^ <1 < 5^ ------ , Table 10 comprehensively lists the analytical test results related to the stability and biological activity of the formulation B. The test was carried out at 4 test points of the numerical value: at 0:00, the formulation was stored at 25±2 t for 3 months, 6 months and after . 1 solid

It can be concluded from Tables 9 and 10 that the biological activities of Formulations A and B remain good after 9 months. The formulations have high stability. This high stability is not affected by a large number of recombinant FSH and recombinant LH sequences: SEQ ID NO. 1: human glycoprotein alpha_subunit; SEQ ID NO. 2: hFSH beta-subunit SEQ ID NO. : hFSH β-subunit variant i SEQ ID NO. 4 : hFSH β-subunit variant 2 63 ^^692 SEQ id n〇. 5 : hFSH β-subunit variant 3 SEQ ID NO. 6 : hLH β -Secondary unit [Simple description of the schema] Figure 1 shows the ρ 1 uron i C F68 at a time point of 0, 1 week and 2 weeks, and the concentration of bismuth thiocyanate is ι〇μg / 3⁄4 liter ("Meth i〇 The FSH formulation of mCg/mi") and 100 μg/ml ("Meth 100 mcg/ml") has been oxidized in the formulation of α-- in the formulation containing no thiosulphate ("non-hydrazine thioglycolate") Percentage of subunits. 1322692 References 1 Perkins et al; Highly purified follicle stimulating hormone for the treatment of male gonadotropin deficiency and subcutaneous self-administration of human chorionic gonadotropin. Spanish Gonadotropin Deficiency Hypofunction Cooperative Group; Hum. Repord.; 1997, 12, 980-6; 2 Schum et al., j. Clin. Endocrinol. Metab. 39:1 87-205 (1 974); Xiaom et al, J. Prot. Chem., 7:325-339, 1988; 3 Kutman et al; Structure of human luteinizing hormone beta-beta unit: for related carboxy-terminal sequences in specific peptide hormones evidence;

Biochem. Biophys. Res. Commun.; 1979, 90, 842-848; Talmitch et al; Gene evolution of human chorionic gonadotropin and beta-producing units of luteinizing hormone; Nature; 1984, 307, 37 -40 'Fedis and Talmeqi; Gene Structure, Expression and Evolution of Human Glycoprotein Hormone; Recent Prog Horm. Res. ; 1984, 40, 43-78 4 Reichert LE, Ramsey RB; Human Dissociation of follicle stimulating hormone; J. Biol. Chem.; 1975, 250, 3034-3040 5 Klein et al; single-stranded recombinant human follicle stimulating hormone containing human chorionic gonadotropin carboxy terminal peptide in macaque Pharmacokinetics and Pharmacodynamics; Fertility &Sterility; 2()()2,77, 1248-1255 6a) Fadis, jc et al.,] of Mol. and AppHed Genetics, 1:3_18 (1981) b) Ai Shi Fs et al., dna 65 1322692 5:363-369 (1986); e) Watkins PC et al., DNA 6:2〇5_ 21 2 (1 987); d) Hirai T. et al. Man, J. Mol. Endocrinol. 5:1 47-1 58 (1 990) ; e) Moller, RA et al., Mol

Endocrinol. 1:717-723 (1987); f) Kuzman K. et al., DNA Cell Biol. 10:593-601 (1991); g) Kumar tr et al., Gene. 1995 Dec 12, 166 ( 2): 333-4 7 Biochem. Biophys. Res. Commun.; 1979, 9〇, 842-848 8 Stilman et al; Follicle stimulating hormone assay based on the increase of human chorionic gonadotropin; endocrinology ; 1953, 53, 604-616 Van Hor et al; human fertility gonadotropin preparations are not

The efficacy of the same bioanalytical method 1964, 47, 409-418 Van Hor et al. 'Human fertility gonadotropin preparations are not

Efficacy in the same bioanalytical method; 1964, 47, 409-418 66 1322692 Sequence Listing

<110> ARES TRADING SA <12〇> FSH and FSH variant formulation <130> US 847 Y <160> 6 <170> Patentln version 3.1 <210> 1 <211>

<212> PRT <213> Human <400> 1

Ala Pro Asp Val Gin Asp Cys Pro Glu Cys Thr Leu Gin Glu Asn Pro 1 5 10 15

Phe Phe Ser Gin Pro Gly Ala Pro lie Leu Gin Cys Met Gly Cys Cys 20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu 35 " 40 45

Val Gin Lys Asn Val Thr Ser Glu Ser Thr Cvs Cys Val Ala Lys Ser 50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Val Glu Asn His Thr Ala 65 70 75 80

Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser 85 90 21322692 <210> 2 <211> 129 <212> PRT <2i3> Human <400> 2 Met Lys Thr Leu Gin Phe Phe Phe 1 5 Cys Cys Asn Ser Cys Glu Leu Thr 20 Glu Glu Cys Arg Phe Cys lie Ser 35 40 Tyr Cys Tyr Thr Arg Asp Leu Val 50 55 lie Gin Lys Thr Cys Thr Phe Lys 65 70

Leu Phe Cys Cys Trp Lys Ala He 10 15

Asn lie Thr lie Ala lie Glu Lys 25 30 lie Asn Thr Thr Trp Cys Ala Gly 45

Tyr Lys Asp Pro Ala Arg Pro Lys 60

Glu Leu Val Tyr Glu Thr Val Arg 75 80

Val Pro Gly Cys Ala His His Ala 85 Ala Thr Gin Cys His Cys Gly Lys 100 Thr Val Arg Gly Leu Gly Pro Ser 115 120 Glu <210> 3 <211> 103 <212> PRT <213> Human <;400> 3

Asp Ser Leu Tyr Thr Tyr Pro Val 90 95

Cys Asp Ser Asp Ser Thr Asp Cys 105 110

Tyr Cys Ser Phe Gly Glu Me t Lys " 125

Asn Ser Cys Glu Leu Thr Asn lie Thr lie Ala lie Glu Lys Glu Glu 1322692 X 5 10 15

Cys Arg Phe Cys lie Ser lie Asn Thr Thr Trp Cys Ala Gly Tyr Cys 20 25 30

Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Lys lie Gin 35 40 45

Lys Thr Cys Thr Phe Lys Giu Leu Val Tyr Glu Thr Val Arg Val Pro 50 55 60

Gly Cys Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro Val Ala Thr 65 70 75 80

Gin Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys Thr Val 85 90 95

Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe Gly Glu 100 105 <210> 4

<211> 106 <212> PRT Human <400> 4

Asn Ser Cys Glu Leu Thr Asn He Ala He Glu Lys Glu Glu Cys Arg 1 5 10 15

Phe Cys lie Ser lie Asn Thr Trp Cys Ala Gly Tyr Cys Tyr Thr Arg 20 25 30

Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Lys lie Gin Lys Thr Cys 35 ή〇 45

Thr Phe Lys Glu Leu Vel Tyr Glu Thr Val Arg Val Pro Gly Cys Ala 50 55 60

His His Ala Asp Ser Leu Tyr Thr Val Pro Val Ala Thr Gin Cys His 65 70 75 80

Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys Thr Val Arg Gly Leu 85 90 95 4 41322692

Gly Pro Ser Tyr Cys Ser Phe Gly Glu Met 100 105 <210> 5 <211> 110 <212> PRT <213> Human <_> 5

Asn Ser Cys Glu Leu Thr Asn lie Thr lie Ala lie Glu Lys Glu Glu 15 10 15

Cys Arg Phe Cys lie Ser lie Asn Thr Thr Trp Cys Ala Gly Tyr Cys 20 25 30

Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Ara Pro Lys lie Gin 35 40 45

Lys Thr Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val Arg Val Pro 50 55 60

Gly Cys Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro Val Ala Thr 65 70 75 80

Gin Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys Thr Val 85 90 95

Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe Gly Glu Met Lys 100 105 110

<210> 6 <211> 112 <212> PRT <213> Human <400> 6

Ser Arg Glu Pro Leu Arg Pro Trp Cys His Pr o He Asn Ala He Leu 1 5 · 10 15 1322692

Ala Val GIu Lys Glu Gly Cys Pro Val Cys lie Thr Val Asn Thr Thr 20 25 30

He Cys Ala Gly Tyr Cys Pro Thr Me t Arg Val Leu Gin Ala Val Leu 35 40 45

Pro Pro Leu Pro Gin Val Cys Thr Tyr Arg Asp Val Arg Phe Glu Ser 50 55 60

He Arg Levi Pro Gly Cys Pr o Arg Gly Val Asp Pro Val Val Ser Phe 65 70 75 80

Pro Val Ala Leu Ser Cys Arg Cys Gly Pro Cys Arg Arg Ser Thr Ser 85 90 95

Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp His Pro Gin 100 105 110

Claims (1)

1322692 r?年丨月爻Fi repair (more) 正本^本本,本专利 igT 一~ 1. A liquid pharmaceutical composition containing follicle stimulating hormone (FSH) or a surfactant for Pluronic F68 And further comprising methionine and a bacteriostatic agent selected from the group consisting of phenol or m-nonylphenol. a liquid pharmaceutical composition comprising a follicle stimulating hormone (FSH) or a variant thereof and luteinizing hormone (LH) or a variant thereof, and a surfactant of pi_nic and further comprising methionine and selected from the group consisting of Hope or between - A bacteriostatic agent. a liquid pharmaceutical composition comprising luteinizing hormone (lh) or a variant thereof, and a surfactant of piur〇nic F68 and further comprising methionine and an antibacterial agent selected from the group consisting of phenol or m-phenol Agent. The liquid pharmaceutical composition according to any one of claims 3 to 3, wherein the follicle stimulating hormone (FSH) is or is about 15 〇 iu / ir1 to or about 1200 υ ml / ml The concentration is present. The liquid pharmaceutical composition according to claim 4, wherein the follicle stimulating hormone (FSH) is present at a concentration of from about 300 IU/ml to or about 9 IU IU/ml. 6. The liquid pharmaceutical composition according to claim 5, wherein the follicle stimulating hormone (FSH) is present at or at a concentration of about 600 IU/ml. 7. The liquid according to claim 2 or 3 A pharmaceutical composition wherein the luteinizing hormone (LH) is present at a concentration of from about 15 〇iU/ml to or at about 1 200 Ιϋ/ml. 8. The liquid pharmaceutical composition according to claim 7 wherein the luteinizing hormone (LH) is present at a concentration of from about 300 IU/ml to about 750 IU/ml. 9. According to the application The pharmaceutical composition of any one of claims 1 to 3 wherein the follicle stimulating hormone is a human follicle stimulating hormone and/or the luteinizing hormone (LH) is human luteinizing hormone (LH). 10. The pharmaceutical composition according to claim 9, wherein the pro-buffering hormone is urine human pro-follicle stimulating hormone and/or the luteinizing hormone (LH) is urine human luteinizing hormone (LH). 11. The pharmaceutical composition according to claim 9, wherein the follicle stimulating hormone is a recombinant human stimulating hormone and/or the luteinizing hormone (LH) is recombinant human luteinizing hormone (LH). The pharmaceutical group of any of items 1 to 3 is in the range of from about 6:1 to about 12. The range of FSH to LH is 1:6 according to the scope of the patent application. 13. According to the scope of the patent application, the ratio of FSH to LH is between or within the range. A pharmaceutical composition of 12, wherein about 4. 丨 is or is a pharmaceutical composition of about 1:2, wherein • 1 to or is about 1 · 丨 14 14. According to the scope of application patent u FSH The ratio to LH is between or within the range. The FSH is called the 14th item of the wood. The ratio of the LH is the composition of the music. Μ is about 2:1 to or is about 1: 16. According to the scope of the patent application of the first to third of the 13226 compounds, wherein the bacteriostatic agent is m-cresol. According to the pharmaceutical composition of claim 16 of the patent application, the dragon wave is or is about 3% (ft/f amount of solvent). And the medical compound according to any one of the claims 1 to 3, which further comprises sucrose. Le New 19_ according to the scope of the patent application! Contains a pH of about or about 6 〇 to the oleate buffer. 4 8. 〇 2〇. The pharmaceutical composition according to claim 19 of the patent application - the step comprises a pH value of about 7 。. Phosphate buffer. —— 21. The pharmaceutical composition according to the scope of claim 2, which comprises the following components: rFSH, Pluronic F68, cane mites, methionine, inter- 3 _ Η value or about 7 · 〇 water Acid buffer. 22_ The pharmaceutical composition according to claim 21, wherein the rFSH is present or at a concentration of about IU/inl, and the Pluronic F68 is present or at a concentration of about mgmg/ml, and the sucrose is or is about The concentration of 60 mg/ml is present, the methionine is present at a concentration of about 0.1 mg/ml, the m-cresol is present at a concentration of about 3 mg/ml, and the citrate buffer is Or it is present at a concentration of about 10 mM phosphate. 23' An article comprising a lyophilized formulation comprising a follicle stimulating hormone (FSH) or a variant thereof, a surfactant of Pluronic F68, and methionine, the article further comprising a phenol or a mixture selected from a solvent for reconstitution of a cresol bacteriostatic agent. 24. An article comprising a lyophilized formulation comprising corpus luteum 1322692 (LH) or a variant thereof, a surfactant of piuronic F68, and methionine, the article further comprising a phenol selected from the group consisting of Or a solvent for reconstitution of a cresol bacteriostatic agent. 25. An article comprising a lyophilized formulation comprising a follicle stimulating hormone (FSH) or a variant thereof and luteinizing hormone (LH) or a variant thereof, a surfactant of Pluronic F68, and guanidine thiamine The acid further comprises a solvent for reconstitution comprising a bacteriostatic agent selected from the group consisting of phenol or m-cresol. 26. The article according to claim 23, wherein the follicle stimulating hormone (FSH) is present or is present in a concentration (w/w) of about 〇" to 1 〇/蜎 overall formulation. 27. The article of claim 26, wherein the follicle stimulating hormone (FSH) is present at or as a concentration of from about 0.3 to 5 Bg/mg of the total formulation of A' x horses. The product of item 27, wherein the follicle 〇·37 to 2 pg/mg of the total formulation is concentrated 28_ according to the patent application of the hormone (F S Η) is or is present. 29. Luteinizing hormone (LH) is present at or about a concentration according to the scope of the patent application. A product of 24 or 25, wherein the 0.1 to 3 pg/mg of the total formulation is 3 0. According to the article of the application of the true sinister ‧ 'Liwei, item 29, wherein the luteinizing hormone (LH) is or Aw is present at a concentration of about 0 · 1 to 1 pg / mg of the total formulation. 31. According to the application for the search for the product of the 30th item, wherein the luteinizing hormone (LH) is considered to be a bite rabbit, about 0.11 to 0.6 pg/mg of the total formulation concentration of 1322692 degrees. 32. An article according to claim 25, comprising 32.75 Kg of recombinant FSH, 9. 0 pg of recombinant lh, 15.0 mg of sucrose, 〇. 052 mg of NaHzP〇4 H2O '0.825 rag Na2HP〇4 2 Hz O '0.05 mg Pluronic F68 and 0.05 mg L-indole thioglycolic acid. 33. According to Article 25 of the patent application, which comprises 65. 5 Mg recombinant FSH, 10.0 pg recombinant LH, 30.0 mg cane toad, 〇. 1〇4 mg NaH2P〇4 H2O ' 1.65 mg Na2HP〇4 2H2〇' 0.10 mg Pluronic F68 and 0.10 mg L-guanidine thioglycolic acid. The preparation according to any one of claims 23 to 25 wherein the follicle stimulating hormone is a human follicle stimulating hormone and/or the luteinizing hormone (LH) is human luteinizing hormone (lh). 35. The article of claim 34, wherein the follicle stimulating hormone is urine human follicle stimulating hormone and/or the luteinizing hormone (LH) is urine human luteinizing hormone (LH). 36. The article according to claim 34, wherein the follicle is a recombinant human follicle stimulating hormone and/or the luteinizing hormone (10) is recombinant human luteinizing hormone (LH). The article of any one of the items is about 6:1 to or about 37. According to the scope of claim 23, wherein the ratio of FSH to LH is in the range of or 1 : 6. _ 38. According to the scope of the patent application, the product is in the range of about 4: 1 39. According to the scope of the patent application, the product F is in the range of about 1:2. The product of item 38, wherein the ratio of FSH to Lh 1322692 is between or about 3: ι to 40. According to the application of sputum; 丨^ ‘, ', s is about 1:1. The ratio of 0 „ τη of the 39th patent range of τ ° month is in the range of 9 s, which is the range of FSH to 41 in the range of about 1:1. According to claim 23, wherein the bacteriostatic agent is m-cresol. Any one of the items may be used. 42. According to the scope of the patent application No. 4 or about 3% (mass/f solvent) Between the two and the second, which contain the concentration 43_ according to the scope of the patent application, the further step includes any one of the 25 items... According to the 23rd article of the patent application, the step further comprises a PH value of about 6 - Acid salt buffer. 々飞为,, 々8. 45. According to the article of the scope of the patent application, the dream contains a pH value or a phosphate buffer of about 7. 46. The product according to claim 45, and comprising the following ingredients: _, Pluronic F68, sucrose, methionine and an aqueous phosphate buffer having a pH of about 7.5. 47. The article of claim 46, wherein the rFSH is present at or about a concentration of about _IU/ml, pi_icF68 is present at or at a concentration of about 0.1 mg/mi, and the sugar is mg/m 1 The concentration is present, the methionine is or is present at a concentration of about Ά1 Άβ〇, the m-cresol is or is a concentration of about 3 mg/nU of the phosphate buffer or is about 1 mM phosphate. The concentration t is in the form of a pharmaceutical composition comprising 48. ^ fSH> 1322692 is a solution of a surfactant of Pluronic F68, and a liquid diluent, and a step of further adding methionine and a bacteriostatic agent selected from the group consisting of bismuth and m-cresol. 49. A method of making a packaged pharmaceutical composition comprising filling a solution containing FSH, a surfactant of Piuronic F68, into a vial, ampoule or canister and further reacting the thiol amide with a phenol and The bacteriostatic agent of m-cresol is placed in a vial, ampoule or canister. A method of producing an article according to any one of claims 23 to 25, which comprises forming a mixture of a surfactant comprising FSH and LH or no lh, or only LH alone and piur〇nic F68 The hydrazine thio acid is added and the mixture is lyophilized, and a step of providing a solvent for reconstitution containing a bacteriostatic agent selected from the group consisting of bismuth and methicillin is provided. Pick up, pattern: like the next page 7