CN1795012A

CN1795012A - Liquid pharmaceutical formulations of fsh and lh together with a non-ionic surfactant

Info

Publication number: CN1795012A
Application number: CN 200480014504
Authority: CN
Inventors: F·萨马利塔尼; P·多纳蒂
Original assignee: Ares Trading SA
Current assignee: Ares Trading SA
Priority date: 2003-04-02
Filing date: 2004-04-02
Publication date: 2006-06-28
Anticipated expiration: 2024-04-02
Also published as: ES2358330T5; UA91960C2; ES2358330T3; CN100488566C; ZA200507287B

Abstract

The invention relates to the field of pharmaceutical formulations of follicle -stimulating hormone (FSH), luteinising hormone (LH), and mixtures of FSH and luteinising hormone (LH), and to methods of producing such formulations. The invention provides a liquid or freeze-dried formulation of FSH, or LH, or FSH and LH comprising a surfactant selected from Pluronic (R) F77, Pluronic F87, Pluronic F88 and Pluronic F68.

Description

The liquid pharmaceutical formulation of FSH and LH and non-ionic surface active agent

Technical field

The present invention relates to the field of follicule-stimulating hormone (FSH) (FSH) pharmaceutical preparation, luteotropic hormone (LH) preparation and FSH and LH mixture, the invention still further relates to the production method of these preparations.

Background of invention

Follicule-stimulating hormone (FSH) (FSH), luteotropic hormone (LH) and chorionic-gonadotropin hormone (CG) all are injectable protein, belong to gonadotrophins.FSH, LH and hCG can use separately or coupling, are used for the treatment of male and female patient infertility and dysgenesia.

Natural FSH and LH are produced by pituitary gland.Can be at the FSH that pharmaceutically uses and LH and variant thereof by recombinant technique production (rFSH and rLH), perhaps they can produce (uFSH and uLH) from postmenopausal women's urine.

The aeg uses FSH to be used for ovulation induction (OI) and controlled super ovulation (COH) in the auxiliary procreation technology (ART).The typical treatment regimen of ovulation induction is to inject FSH or variant (about 75 to 300 IU FSH/ days) every day to patient, continues about 6 to 12 days.Controlled superovulated typical treatment regimen is to inject FSH or variant (about 150 to 600 IU FSH/ days) every day to patient, continues about 6 to 12 days.

FSH also is used for the Aeg of spermacrasia, induces spermatogenesis.To give 3 times 150 IUFSH weekly, to give birth to the hypofunction Aeg in conjunction with the scheme that gives 2 times 2,500 IU hCG weekly at low gonadotropin and achieve success, its sperm quantity is significantly improved 1.

Use LH and be used for aeg's OI and COH in conjunction with FSH, particularly those endogenous LH levels are extremely low or LH is had a patient of resistance, for example low gonadotropin is given birth to hypofunction women (HH, and the patient who is difficult for embryo nidation or early abortion the I of World Health Organization (WHO) group) or auld patient (promptly 35 years old or more than).LH and the bonded preparation of FSH have been arranged now, and promoting sexual gland hormone (hMG) after the title people menopause is extracted from the postmenopausal women.The FSH of hMG: the LH activity is 1: 1.

CG and LH act on same receptor, cause same replying.The circulating half-life of CG is longer than LH, therefore usually as long-acting LH activated source.CG is used for OI and COH scheme, simulates natural LH peak and causes ovulation.During with FSH or FSH and LH mixture stimulation end, injection human chorionic gonadotropin (hCG) can cause ovulation.Between OI and COH stimulation period, also can use CG and FSH together, between stimulation period, provide the LH activity for needing the active patient of LH such as those above-mentioned patients.

FSH, LH and CG are the members of heterodimer glycoprotein hormones family, and this family also comprises thyrotropin (TSH).The member of this family is a heterodimer, comprises α-and β-subunit.These subunits connect together by noncovalent interaction.People FSH (hFSH) dimer is made up of following subunit: (i) sophisticated 92 aminoacid glycoprotein α subunits, this also with other people family member's (being that chorionic-gonadotropin hormone (" CG "), luteotropic hormone (" LH ") and thyrotropin (" TSH ") are identical); And (ii) sophisticated 111 amino acid beta subunits, this subunit be FSH exclusive 2.People LH dimer is made up of following subunit: (i) sophisticated 92 aminoacid glycoprotein α subunits; And (ii) sophisticated 112 amino acid beta subunits, this subunit be LH exclusive 3.The α of glycoprotein and β subunit are owing to tend to disassociation with antiseptic, surfactant or the interaction of other excipient in preparation.The disassociation of subunit causes biopotency to disappear 4.

FSH is mixed with suitable intramuscular (IM) or subcutaneous (SC) injection.FSH is placed in bottle or the ampoule (75IU/ bottle and 150IU/ bottle) with lyophilizing (solid) form and supplies with, if can preserve 1.5 years to 2 years in 2-25 ℃ of storage.With lyophilized products and injection water (WFI) reconstruct, can be made into injection solution.Want ovulation induction or controlled super ovulation, suggestion injection every day, initial dose is 75IU to 600IU, continues about 10 days at most.According to patient's reaction, can increase FSH dosage and handle, be no more than 3 circulations.When using lyophilized formulations, patient need use one bottle of new freeze-dried material of diluent reconstruct, and wants reconstruct every day, uses [the Fertinex that subcutaneous injection is used then immediately ^TMIndividual N1700101A of description, Serono Laboratories, Inc. delivered in February, 1996, Randolph, MA].

FSH also can be made into single agent or the multi-agent liquid form in bottle or ampoule.It is stable and effective that list agent form must keep between the storage life before use.It is stable and effectively that the multi-agent form not only requires must keep between before use storage life, and when the ampoule unseal after, also must protection during multi-agent uses dosage regimen stable, effective, relatively aseptic.For this reason, the multi-agent form contains antibacterial usually.

LH can be mixed with intramuscular (IM) or subcutaneous (SC) injection.LH is placed in bottle or the ampoule (75IU/ bottle) with lyophilizing (solid) form and supplies with, if can preserve 1.5 years to 2 years in 2-25 ℃ of storage.With lyophilized products and injection water (WFI) reconstruct, can be made into injection solution, be used for.Want ovulation induction or controlled super ovulation, suggestion combines with FSH, every day LH injection, initial dose is 75IU to 600IU, continues maximum about 10 days.

EP 0 618 808 (Applied Research Systems ARS Holding N.V.) discloses a kind of pharmaceutical composition, and it only contains the solid uniform homogeneous blend that the sucrose by promoting sexual gland hormone and stable quantity forms, and perhaps mixes glycine again.

EP 0 814 841 (Applied Research Systems ARS Holding N.V.) discloses a kind of stable composition of liquid medicine, and it contains the mannitol of R-HCG (hCG) and stable quantity.

EP 0 448 146 (AKZO N.V.) discloses a kind of stable freeze-dried material that contains promoting sexual gland hormone, and it contains portion (weight) the promoting sexual gland hormone dicarboxylate stabilizing agent relevant with promoting sexual gland hormone with 200 to 10,000 parts (weight).

EP 0 853 945 (Akzo Nobel N.V.) discloses a kind of liquid preparation that contains promoting sexual gland hormone, and the characteristics of said preparation are that it contains polycarboxylic acid or its salt and the sulfide compound of promoting sexual gland hormone and stable quantity.

WO 00/04913 (Eli Lilly and Co.) discloses a kind of preparation, its FSH or FSH variant contain α-and β-subunit, and its antiseptic is selected from the water diluent of following material: phenol, metacresol, paracresol, orthoresol, chlorocresol, benzyl alcohol, alkyl paraben class (as methyl ester, ethyl ester, propyl ester, butyl ester or the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal or its mixture.

People still need the liquid preparation of stable FSH or FSH variant and FSH and LH mixture at present, are used for single agent or multi-agent administration.

Summary of the invention

One object of the present invention is the FSH of novelty or the lyophilizing and the liquid preparation of FSH variant, LH or LH variant are provided, and their preparation method and the medicine of their treatment infertility or the using method on the veterinary also is provided.

Another object of the present invention is the FSH of novelty and the lyophilizing and the liquid preparation of LH mixture are provided, and their preparation method and the medicine of their treatment infertility or the using method on the veterinary also is provided.

A first aspect of the present invention provides a kind of lyophilizing and composition of liquid medicine, and said composition contains FSH or its variant and surfactant, and described surfactant is selected from the epoxy ethane-epoxy propane block polymer, preferred Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68.

A second aspect of the present invention provides a kind of method for preparing composition of liquid medicine, and this method comprises the solution that forms FSH or its variant and surfactant, and described surfactant is selected from the epoxy ethane-epoxy propane block polymer, preferred Pluronic ^F77, Pluronic F87, Pluronic F88, Pluronic F68 and WFI.

A third aspect of the present invention provides a kind of preparation packaged pharmaceuticals method for compositions, this method comprises that the solution that will contain FSH and surfactant is dispensed in the container, described surfactant is selected from the epoxy ethane-epoxy propane block polymer, preferred Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68.

A fourth aspect of the present invention provides a kind of goods that pharmaceutically use for the people, and these goods comprise a bottle that contains FSH or its variant and surface-active solution, and described surfactant is selected from the epoxy ethane-epoxy propane block polymer, preferred Pluronic ^F77, Pluronic F87, Pluronic F88 and PluronicF68; And illustrate that this solution can use the back to preserve about 24 hours or above printed instructions first.

A fifth aspect of the present invention provides a kind of lyophilizing and composition of liquid medicine, and said composition contains FSH and LH and surfactant, and described surfactant is selected from the epoxy ethane-epoxy propane block polymer, preferred Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68.

A sixth aspect of the present invention provides a kind of method for preparing lyophilizing and composition of liquid medicine, and this method comprises the solution that forms FSH, LH and surfactant, and described surfactant is selected from the epoxy ethane-epoxy propane block polymer, preferred Pluronic ^F77, Pluronic F87, Pluronic F88 and PluronicF68.

A seventh aspect of the present invention provides a kind of preparation packaged pharmaceuticals method for compositions, this method comprises that the solution that will contain FSH, LH and surfactant is dispensed in the container, described surfactant is selected from the epoxy ethane-epoxy propane block polymer, preferred Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68.

A eighth aspect of the present invention provides a kind of goods that pharmaceutically use for the people, and these goods comprise a bottle that contains the solution of FSH, LH and surfactant, and described surfactant is selected from the epoxy ethane-epoxy propane block polymer, preferred Pluronic ^F77, Pluronic F87, Pluronic F88 and PluronicF68; And illustrate that this solution can use the back to preserve about 24 hours or above printed instructions first.

A ninth aspect of the present invention provides a kind of goods that pharmaceutically use for the people, and these goods comprise first container that contains lyophilizing FSH or FSH variant and surfactant, and described surfactant is selected from the epoxy ethane-epoxy propane block polymer, preferred Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68; And second container, contain the solvent that is useful on reconstruct, the aqueous solution of preferred bacteriostatic agent, antibacterial is metacresol preferably.

A tenth aspect of the present invention provides a kind of goods that pharmaceutically use for the people, and these goods comprise first container that contains lyophilizing LH or LH variant and surfactant, and described surfactant is selected from the epoxy ethane-epoxy propane block polymer, preferred Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68; And second container, contain the solvent that is useful on reconstruct, the aqueous solution of preferred bacteriostatic agent, antibacterial is metacresol preferably.

A eleventh aspect of the present invention provides a kind of goods that pharmaceutically use for the people, these goods comprise first container that contains lyophilizing FSH and LH or FSH or LH variant and surfactant, described surfactant is selected from the epoxy ethane-epoxy propane block polymer, preferred Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68; And second container, contain the solvent that is useful on reconstruct, preferably contain the aqueous solution of metacresol.

Description of drawings

Figure 1 shows that FSH preparation that contains Pluronic F68,10 μ g/ml methionines (" methionine 10mcg/ml ") and 100 μ g/ml methionines (" methionine 100mcg/ml ") and the preparation that does not contain methionine (" no the methionine ") oxidation α-subunit percentage rate when 0 time, 1 week and 2 weeks.

The specific embodiment

Liquid of the present invention and lyophilizing FSH or FSH and LH preparation have performance or stability improved or that be more suitable for, are used for the male and female infertility treatment.These preparations and goods also are applicable to injection and interchangeable transfer system, such as but not limited to nose, lung, stride mucosa, percutaneous, oral, subcutaneous, intramuscular or parenteral slow release transfer system.In a particularly preferred embodiment, preparation of the present invention is used for subcutaneous and/or intramuscular injection.Owing to stop or reduced active or stable forfeiture, perhaps owing to improved operational efficiency or desirability (for example having improved wherein one of use pattern, frequency, dosage, comfort level, convenience, inside and outside biological activity or the like at least), so FSH provided by the present invention or FSH compare with known commercially available prod with LH variant solution and preparation, the time of doing the trick has obtained prolongation in vivo.

Follicule-stimulating hormone (FSH) used herein or FSH refer to the FSH that obtains as the total length mature protein, include but not limited to recombinant production or the people FSH or " hFSH " that obtain from people source (for example menopausal women urine) separation.The protein sequence of human glucoprotein α subunit is shown in SEQ ID NO:1, and the protein sequence of people FSH β subunit is shown in SEQ ID NO:2.

Term " FSH variant " refers to and comprises between aminoacid sequence, glycosylation pattern or subunit and to connect different with people FSH but show active those molecules of FSH.CTP-FSH for example, it is the Recombinant FSH of a long-acting modification, is made up of wild type α-subunit and hybridization type β-subunit, wherein the β of the carboxyl terminal peptide of hCG and FSH-subunit C-terminal merges, and sees (Endocrinology such as LaPolt; 1992,131,2514-2520) or (Development and characterization of a long-actingrecombinant hFSH agonist such as Klein; Human Reprod.2003,18, description 50-56).Also comprise strand CTP-FSH, it is a single chain molecule, is made up of following sequence (from N-terminal to C-terminal):

βFSH

βhCG-CTP(113-145)

αFSH

Wherein, β FSH represents β-subunit of FSH, and β hCG CTP (113-145) represents the carboxyl terminal peptide of hCG, and α FSH represents α-subunit of FSH, describes and to see Klein etc. 5.Other FSH variants comprise α-and/or β-subunit on contain the FSH molecule of additional glycosylation site, referring to the claim 10 of WO 01/58493 (Maxygen), particularly WO 01/58493 and 11 described those; And the FSH molecule with S-S key between subunit, referring to WO 98/58957.

FSH variant used herein also comprises the carboxyl-terminal deletion of β-subunit, and they are shorter than the total length mature protein shown in the SEQ ID NO:2.The carboxyl-terminal deletion of people's β-subunit is shown in SEQ ID NO:3,4 and 5.The carboxyl terminal variant and the known α-subunit that should be appreciated that the β chain form dimer, thereby form FSH variant heterodimer.

FSH heterodimer or FSH variant heterodimer all can be by suitable method productions, for example recombinant technique, obtain from the natural origin isolated or purified, or by chemosynthesis, or its combination in any.

The use of term " reorganization " is meant FSH, LH or FSH and the LH variant that adopts recombinant DNA technology (for example referring to WO 85/01958) to produce.The FSH genome of some species α and β subunit or cDNA clone's sequence 6 have been known.For example, a kind of method of utilizing recombinant technique to express FSH or LH is by coding FSH or the α of LH and the DNA sequence transfection eukaryotic cells of β subunit, and no matter it still is on two carriers that each subunit with single promoter is contained in a carrier, describes and see European Patent Application No. EP 0 211 894 and EP 0 487 512.Another example that utilizes recombinant technique to produce FSH or LH is to insert allos with homologous recombination to regulate fragment, this fragment links to each other with the endogenous sequence operability of the subunit of coding FSH or LH, describes and sees European Patent Application No. EP 0 505 500 (Applied ResearchSystems ARS Holding NV).

FSH that the present invention uses or FSH variant not only can be by comprising the recombination method production that mammalian cell is implemented, and can for example originate that the urine purification obtains by other biological.Acceptable method comprises those of following document description: Hakola, K.Molecular and Cellular Endocrinology, 127:59-69,1997; Keene etc., J.Biol.Chem., 264:4769-4775,1989; Cerpa-Poljak etc., Endocrinology, 132:351-356,1993; Dias etc., J.Biol.Chem., 269:25289-25294,1994; Flack etc., J.Biol.Chem., 269:14015-14020,1994; Valove etc., Endocrinology, 135:2657-2661,1994, and United States Patent (USP) 3,119,740 and 5,767,067.

Luteotropic hormone used herein or LH refer to the LH that obtains as the total length mature protein, include but not limited to recombinant production or the people LH or " hLH " that obtain from people source (for example menopausal women urine) separation.The protein sequence of human glucoprotein α subunit is shown in SEQ ID NO:1, and the protein sequence of people LH β subunit 7 is shown in SEQ ID NO:6.LH recombinates in a preferred embodiment.

Term " LH variant " refers to and comprises between aminoacid sequence, glycosylation pattern or subunit and to connect different with people LH but show active those molecules of LH.

LH heterodimer or LH variant heterodimer all can be by suitable method productions, for example recombinant technique, obtain from the natural origin isolated or purified, or by chemosynthesis, or its combination in any.

Term " administration " or " giving " refer to be sent into preparation of the present invention in the patient body who needs treatment disease or disease.

Term " patient " refers to the mammal of accepting disease or treatment for diseases.Without any restriction, patient can be people, sheep, pig, horse, cattle, rabbit or the like.

" render a service or effect " ability that refers to FSH preparation or mix preparation initiation FSH dependency biological respinse with the active relevant term of FSH, for example record the weight of ovary increase or aeg's ovarian follicular growth with Steelman-Pohley analytic process 8.Aeg's ovarian follicular growth situation can be estimated by ultrasound wave, for example stimulates the follicle number of the about 16mm of the 8th per day diameter.Estimate the biological activity of FSH with respect to acceptable FSH standard.

" render a service or effect " ability that refers to LH preparation or mix preparation initiation LH dependency biological respinse, for example seminal vesicle weightening finish method 9 with the active relevant term of LH.Estimate the biological activity of LH with respect to acceptable LH standard.

Term " aqueous diluent " refers to the liquid flux that contains water.The aqueous solvent system can only be made up of water, also can be by water and one or more miscible solvent composition, and can contain soluble solute as sugar, buffer, salt or other excipient.Nonaqueous solvent commonly used is such as organic alcohol of short chain such as methanol, ethanol, propanol, such as chain ketones such as acetone, such as polyhydric alcohol such as glycerol.

That term " isotonic agent " refers to physiological tolerance and bring the suitable Zhang Du of preparation and cross the chemical compound of the cell membrane that contacts with preparation to stop the water net flow.Usually be used as isotonic agent (concentration is known) with chemical compounds such as glycerol.Other suitable isotonic agents include but not limited to aminoacid or protein (for example glycine or albumin), salt (for example sodium chloride) and sugar (for example glucose, sucrose and lactose).

Term " bacteriostatic " or " antibacterial " refer to chemical compound or the compositions that adds as antibacterial in preparation.The storage preparation that the present invention contains FSH or FSH variant or FSH and LH preferably meets the particularly legal or regulation guide of implementing about commerce of the anticorrosion effectiveness of the product of confession human of multipurpose product.For example, antibacterial comprises phenol, metacresol, paracresol, orthoresol, chlorocresol, benzyl alcohol, alkyl paraben class (as methyl ester, ethyl ester, propyl ester, butyl ester or the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal.

It is safe and compound solution with the effect in the desirable pH value scope of pH value at said preparation of keeping or control preparation to preparation in the application on pharmacy or the veterinary that term " buffer " or " physiologically acceptable buffer " refer to known.The control pH value includes but not limited to following chemical compound at moderate acid pH value to the buffer accepted of medium basic pH value: phosphate, acetate, citrate, arginine, TRIS and histidine." TRIS " refers to 2-amino-2-methylol-1, ammediol and pharmaceutically acceptable salt thereof.Preferred buffer is phosphate buffer normal saline and acceptable salt thereof.

Term " phosphate buffer " refers to the solution that contains phosphoric acid or phosphate and transferred to desirable pH value.Usually, phosphate buffer is from phosphoric acid or phosphate (including but not limited to sodium phosphate and potassium phosphate) preparation.Some phosphate have been known in this area, for example sodium dihydrogen phosphate and potassium dihydrogen phosphate, disodium-hydrogen and potassium phosphate,monobasic and sodium phosphate and potassium phosphate.Known that phosphate is that hydrate with salt exists.The pH value scope of phosphate buffer is very wide, and for example about pH4 better is the scope of about pH5 to about pH9 to the scope of about pH10, is more preferably the scope of about pH6 to about pH8, preferably about pH7.0.

Term " bottle " general reference is fit to preserve with the sealed, sterile state reservoir of solid or liquid form FSH.For example, the bottle that herein uses comprises that ampoule, injection tube, bubble wrap or other are fit to by syringe, pumping (comprise etc. ooze), conduit, percutaneous plaster, lung or stride mode such as mucosa injection FSH is delivered to the intravital reservoir of patient.Be fit to parenteral, lung, to stride bottle that mucosa and transdermal administration packaging product use known to those skilled in the art and confirm.

Term " stability " refers to physics, chemistry and the conformational stability (comprising keeping of biopotency) of FSH and LH in the preparation of the present invention.The chemical degradation of protein molecule or the polymer, the heterodimer that assemble to form the higher order of magnitude are dissociated at least a bioactive other structural modifications of the included FSH polypeptide of single aggressiveness, de-glycosylation, glycosylation modified, oxidation (particularly α-subunit oxidation) or any minimizing the present invention, can both cause the protein formulation instability.

Term " stable " solution or preparation are meant such solution or preparation: the degree of aspects such as protein degradation wherein, modification, gathering, loss of bioactivity is to control acceptably, and the unacceptable increase that can not become along with the time.Preferably, preparation kept active and at least about the LH activity of 80% labelling at least about the FSH of 80% labelling in 6 months under about 2-8 ℃ temperature under better about 4-5 ℃ of temperature.The FSH activity can measure 5 by Steelman-Pohley ovary weightening finish bioanalysis.The LH activity can measure 10 by the seminal vesicle bioanalysis that increases weight.

Term " treatment " refers to patient's administration, tracking, processing and/or nursing, wherein this patient to be given FSH and/or LH purpose be the physiological reaction that stimulates follicle or testis or any FSH and/or LH to regulate.Therefore, treatment can include but not limited to give FSH and/or LH to induce or to improve sperm quality, stimulate the man to discharge testosterone or stimulate women's follicular development or induced ovulation.

Term " multiple dose use " comprises that the use of the FSH preparation of single bottle, ampoule or injection tube or FSH and LH preparation surpasses once, for example inject 2,3,4,5,6 times or more than.Preferably, at least about in 12 hours, 24 hours, 48 hours equal times, in about 12 days time, inject at most.Also can inject, for example every injection in 6,12,24,48 or 72 hours by interval.

Proteinic " salt " is acid or base addition salts.These salt are preferably between proteinic any or a plurality of charged group and physiologically acceptable avirulence cation or the anion and form.For example, organic salt and inorganic salt comprise by acid such as hydrochloric acid, sulphuric acid, sulfonic acid, tartaric acid, fumaric acid, hydrobromic acid, glycolic, citric acid, maleic acid, phosphoric acid, succinic acid, acetic acid, nitric acid, benzoic acid, ascorbic acid, p-methyl benzenesulfonic acid, benzenesulfonic acid, LOMAR PWA EINECS 246-676-2, propanoic acid, carbonic acid, perhaps by the salt of preparations such as ammonium, sodium, potassium, calcium or magnesium.

The inventor has been found that described surfactant is selected from the epoxy ethane-epoxy propane block polymer the mixture of FSH and FSH and LH and surfactant mixed preparing, preferred Pluronic ^F77, Pluronic F87, Pluronic F88, preferred especially Pluronic F68 (BASF, Pluronic F68 also claims Poloxamer 188), they can obtain stable formulation, and effective ingredient (FSH or FSH and the LH) forfeiture that the absorption of bottle and/or transporter (for example syringe, pump, conduit etc.) surface is caused reduces to minimum.

The present invention finds that also the mixture of FSH and FSH and LH and surfactant mixed preparing, described surfactant is selected from the epoxy ethane-epoxy propane block polymer, preferred Pluronic ^F77, Pluronic F87, Pluronic F88, preferred especially Pluronic F68 (BASF, Pluronic F68 also claims Poloxamer 188), they can obtain stable formulation, owing to antibacterial such as metacresol and phenol have been arranged have been avoided sedimentation problem.When TWEEN 20 uses with metacresol or phenol, precipitate, form muddiness or milky solution.

General stream Buddhist nun gram (Pluronic) surfactant is oxirane (EO)-expoxy propane (PO) block polymer.Expoxy propane (PO) is embedded between two oxirane (EO) section, is sandwich shape.

EO

PO

EO

The Pluronic surfactant can be synthetic by two-step method:

1. add expoxy propane with control mode in two hydroxyls in propylene glycol, produce hydrophobe with desired molecular weight; And

2. adding oxirane is clipped between two hydrophilic groups described hydrophobe.

Pluronic ^The polyoxyethylene of F77 (hydrophilic) percentage rate is 70%, and hydrophobe (polyoxypropylene) molecular weight is about 2,306Da.

Pluronic ^The polyoxyethylene of F87 (hydrophilic) percentage rate is 70%, and hydrophobe (polyoxypropylene) molecular weight is about 2,644Da.

Pluronic ^The polyoxyethylene of F88 (hydrophilic) percentage rate is 80%, and hydrophobe (polyoxypropylene) molecular weight is about 2,644Da.

Pluronic ^The polyoxyethylene of F68 (hydrophilic) percentage rate is 80%, and hydrophobe (polyoxypropylene) molecular weight is about 1,967Da.

The typical performance of Pluronic F77 is as follows:

Mean molecule quantity: 6600;

Molten/pour point: 48 ℃;

20 ℃ of physical form: solid;

25 ℃ of viscosity (Brookfield) cps:480[liquid, 60 ℃ of pastes, 77 ℃ of solids];

25 ℃ of surface tension, dynes/cm;

0.1% concentration: 47.0

0.01% concentration: 49.3

0.001% concentration: 52.8

25 ℃ of interfacial tensions, dynes/cm (with respect to Nujol);

0.1% concentration: 17.7

0.01% concentration: 20.8

0.01% concentration: 25.5

25 ℃ of Draves wetting times (second)

1.0% concentration:＞360

0.1% concentration:＞360

Foam height

Ross Miles method, 0.1%, mm50 ℃: 100

Ross Miles method, 0.1%, mm26 ℃: 47

Dynamically, 0.1%, mm, 400ml/min:＞600

Cloud point in the aqueous solution, ℃

1% concentration:＞100

10% concentration:＞100

HLB (hydrophile-lipophile balance): 25

The typical performance of Pluronic F87 is as follows:

Mean molecule quantity: 7700;

Molten/pour point: 49 ℃;

20 ℃ of physical form: solid;

25 ℃ of viscosity (Brookfield) cps:700[liquid, 60 ℃ of pastes, 77 ℃ of solids];

25 ℃ of surface tension, dynes/cm;

0.1% concentration: 44.0

0.01% concentration: 47.0

0.001% concentration: 50.2

25 ℃ of interfacial tensions, dynes/cm (with respect to Nujol);

0.1% concentration: 17.4

0.01% concentration: 20.3

0.01% concentration: 23.3

25 ℃ of Draves wetting times (second)

1.0% concentration:＞360

0.1% concentration:＞360

Foam height

Ross Miles method, 0.1%, mm50 ℃: 80

Ross Miles method, 0.1%, mm26 ℃: 37

Dynamically, 0.1%, mm, 400ml/min:＞600

Cloud point in the aqueous solution, ℃

1% concentration:＞100

10% concentration:＞100

HLB (hydrophile-lipophile balance): 24

The typical performance of Pluronic F88 is as follows:

Mean molecule quantity: 11400;

Molten/pour point: 54 ℃;

20 ℃ of physical form: solid;

25 ℃ of viscosity (Brookfield) cps:23.0[liquid, 60 ℃ of pastes, 77 ℃ of solids];

25 ℃ of surface tension, dynes/cm;

0.1% concentration: 48.5

0.01% concentration: 52.6

0.001% concentration: 55.7

25 ℃ of interfacial tensions, dynes/cm (with respect to Nujol);

0.1% concentration: 20.5

0.01% concentration: 23.3

0.01% concentration: 27.0

25 ℃ of Draves wetting times (second)

1.0% concentration:＞360

0.1% concentration:＞360

Foam height

Ross Miles method, 0.1%, mm50 ℃: 80

Ross Miles method, 0.1%, mm26 ℃: 37

Dynamically, 0.1%, mm, 400ml/min:＞600

Cloud point in the aqueous solution, ℃

1% concentration:＞100

10% concentration:＞100

HLB (hydrophile-lipophile balance): 28

The typical performance of Pluronic F68 is as follows:

Mean molecule quantity: 8400;

Molten/pour point: 52 ℃;

20 ℃ of physical form: solid;

25 ℃ of viscosity (Brookfield) cps:1000[liquid, 60 ℃ of pastes, 77 ℃ of solids];

25 ℃ of surface tension, dynes/cm;

0.1% concentration: 50.3

0.01% concentration: 51.2

0.001% concentration: 53.6

25 ℃ of interfacial tensions, dynes/cm (with respect to Nujol);

0.1% concentration: 19.8

0.01% concentration: 24.0

0.01% concentration: 26.0

25 ℃ of Draves wetting times (second)

1.0% concentration:＞360

0.1% concentration:＞360

Foam height

Ross Miles method, 0.1%, mm50 ℃: 35

Ross Miles method, 0.1%, mm26 ℃: 40

Dynamically, 0.1%, mm, 400ml/min:＞600

Cloud point in the aqueous solution, ℃

1% concentration:＞100

10% concentration:＞100

HLB (hydrophile-lipophile balance): 29

Preparation of the present invention also can use has other polymer similar to those performances of listing above.Preferred surfactants is Pluronic F68 and surfactant with similar performance.

The contained Pluronic surfactant of preparation, Pluronic F68 particularly, concentration preferably to be enough in the desirable storage life (for example 6 to 12 or to 24 months) and keep FSH and/or LH stability, this concentration also will be enough to prevent absorb such as bottle, syringe or injection tube surface and causes the protein forfeiture.

Preferably, Pluronic surfactant in the liquid preparation, particularly Pluronic F68, concentration at about 0.01mg/ml to about 1mg/ml scope, more about 0.05mg/ml to about 0.5mg/ml scope, better at about 0.2mg/ml extremely in about 0.4mg/ml scope, preferably about 0.1mg/ml.

The concentration (w/w) of follicule-stimulating hormone (FSH) (FSH) is preferably every milligram of about 0.1 to 10 microgram of preparation total amount in the lyophilized formulations.In one embodiment, the concentration of follicule-stimulating hormone (FSH) (FSH) is every milligram of about 0.3 to 5 microgram of preparation total amount.In another embodiment, the concentration of follicule-stimulating hormone (FSH) (FSH) is every milligram of about 0.37 to 2 microgram of preparation total amount.

The concentration (w/w) of luteotropic hormone (LH) is preferably every milligram of about 0.1 to 3 microgram of preparation total amount in the lyophilized formulations.In one embodiment, the concentration of luteotropic hormone (LH) is every milligram of about 0.1 to 1 microgram of preparation total amount.In another embodiment, the concentration of luteotropic hormone (LH) is every milligram of about 0.1 to 0.6 microgram of preparation total amount.

In containing the liquid preparation of FSH, comprise the reconstruct preparation, the FSH concentration of described preparation preferably at about 150IU/ml to about 2, in the 000IU/ml scope, better at about 300IU/ml to about 1, in the 500IU/ml scope, good especially be at about 450IU/ml to about 750IU/ml scope, preferably about 600IU/ml.

In containing the liquid preparation of LH, comprise the reconstruct preparation, the LH concentration of described preparation preferably at about 50IU/ml to about 2, in the 000IU/ml scope, better at about 150IU/ml to about 1, in the 500IU/ml scope, good especially be at about 300IU/ml to about 750IU/ml scope, preferably about 625IU/ml.

In the preparation that contains FSH and LH, the ratio of FSH and LH (FSH: LH, IU: IU, FSH records with rat ovary weightening finish analytic process, LH records with rat seminal vesicle weightening finish analytic process) preferably in about 6: 1 to about 1: 6 scopes, better in about 4: 1 to about 1: 2 scopes, good especially is in about 3: 1 to about 1: 1 scopes, best 1: 1 and 2: 1.

The concentration of lyophilized formulations internal surfactants (as Pluronic F68) is about 0.001 to 0.1 milligram of every milligram of preparation total amount preferably, is more preferably about 0.01 to 0.075 milligram of every milligram of preparation total amount.

Preferably, the Pluronic surfactant of reconstruct preparation, particularly the concentration of Pluronic F68 at about 0.01mg/ml to about 1mg/ml scope, better at about 0.05mg/ml to about 0.5mg/ml scope, good especially be at about 0.2mg/ml to about 0.4mg/ml scope, preferably about 0.1mg/ml.

FSH and LH be preferably by recombinant technique production, particularly with one or more carrier transfection Chinese hamster ovary cell of the DNA of the human glucoprotein α-subunit that contains coding FSH or LH and β-subunit, and reuse this cells produce FSH and LH.Coding for alpha-and the DNA of β-subunit can be present on the identical or different carrier.

Recombinant FSH and LH have some advantages with respect to their urine homologue.Use the cultivation of reconstitution cell and isolation technics can keep the concordance of producing in batches.On the contrary, the characteristic (as purity, glycosylation pattern, sialylated and subunit oxidation) of urine FSH and LH differs greatly between different batch process the in batches.Because Recombinant FSH and LH can keep higher concordance and purity between in batches, so (isoelectric focussing, IEF) etc. technology is easy to these hormones are identified and quantitative analysis employing such as isotope electron focusing.Evaluation and the quantitative analysis of Recombinant FSH and LH are easy, also can realize carrying out filling toward a large amount of hormones of bottle filling (fill-by-mass) rather than by biologic test.

Preferably, the pH of FSH preparation of the present invention is about 6.0 to about 8.0 scopes, better about 6.8 to about 7.8 scopes, comprises about pH7.0, pH7.2 and 7.4.Preferred buffer is a phosphate buffer, and preferred equilibrium ion is sodium or potassium ion.The phosphoric acid brine buffer solution has been well known in the art, for example Dulbecco ' s phosphoric acid brine buffer solution.The concentration of buffer can be about 5mM, 9.5mM, 10mM, 50mM, 100mM, 150mM, 200mM, 250mM and 500mM in total solution.Preferably, the about 10mM of buffer concentration.Particularly preferred situation is a 10mM pH7.0 phosphate buffer.

Preferably, the pH of FSH of the present invention and LH mixture preparation is about 6.0 to about 9.0 scopes, better about 6.8 to about 8.5 scopes, comprises about pH7.0, pH8.0 and 8.2, preferably about pH8.0.

The present invention relates to liquid preparation and lyophilized formulations that can reconstruct, wherein (reconstruct) solvent is an injection water.Liquid preparation can be single agent or multi-agent.The present invention is used for the liquid of multi-agent purpose and lyophilizing FSH and/or LH preparation and preferably contains a kind of antibacterial, for example phenol, metacresol, paracresol, orthoresol, chlorocresol, benzyl alcohol, alkyl paraben class (as methyl ester, ethyl ester, propyl ester, butyl ester or the like), thymol, benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal.Particularly preferably be phenol, benzyl alcohol and metacresol, be more preferably benzyl alcohol and metacresol, preferably metacresol.The consumption of antibacterial is that the concentration that obtains can be kept preparation effectively do not have antibacterial (being fit to injection) basically during the multi-agent injection, and the described multi-agent injection phase can about 12 or 24 hours to about 12 or 14 days, better about 6 days to 12 days.Preferably, the concentration of antibacterial in about 0.1% (antibacterial/solvent, mass/mass) to about 2.0% scope, better about 0.2% to about 1.0% scope.For benzyl alcohol, its preferred concentration is 0.9%.For phenol, its preferred concentration about 0.5%.For metacresol, about 0.3% (for example about 3mg/ml in WFI) of its preferred concentration.

In a preferred embodiment, the invention provides a kind of composition of liquid medicine, it preferably uses as multi-agent, contains FSH or its variant, surfactant and antibacterial, and wherein surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68, antibacterial is selected from metacresol and phenol, preferred metacresol.

In another preferred embodiment, the invention provides a kind of composition of liquid medicine, it preferably uses as multi-agent, contains LH, surfactant and antibacterial, and wherein surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68, antibacterial is selected from metacresol and phenol, preferred metacresol.

In another preferred embodiment, the invention provides a kind of composition of liquid medicine, it preferably uses as multi-agent, contains FSH and LH, surfactant and antibacterial, and wherein surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68, antibacterial is selected from metacresol and phenol, preferred metacresol.Preferably, the ratio of FSH and LH (FSH: LH) about 2: 1 to about 1: 1.

In another preferred embodiment, the invention provides a kind of method for preparing composition of liquid medicine, described compositions is preferably used as multi-agent, and described method comprises that formation contains the aqueous solution of FSH or its variant, surfactant and antibacterial, and wherein surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68, antibacterial is selected from metacresol and phenol, preferred metacresol, and WFI.

In another preferred embodiment, the invention provides a kind of method for preparing composition of liquid medicine, described compositions is preferably used as multi-agent, and described method comprises that formation contains the aqueous solution of LH, surfactant and antibacterial, and wherein surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68, antibacterial is selected from metacresol and phenol, preferred metacresol and WFI.

In another preferred embodiment, the invention provides a kind of method for preparing composition of liquid medicine, described compositions is preferably used as multi-agent, and described method comprises that formation contains the aqueous solution of FSH and LH, surfactant and antibacterial, and wherein surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68, antibacterial is selected from metacresol and phenol, preferred metacresol and WFI.

In a preferred embodiment again, the invention provides a kind of preparation packaged pharmaceuticals method for compositions, described method comprises distributes the solution that contains FSH, surfactant and antibacterial, and wherein surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68, antibacterial is selected from metacresol and phenol, preferred metacresol.

In a preferred embodiment again, the invention provides a kind of preparation packaged pharmaceuticals method for compositions, described method comprises distributes the solution that contains FSH and LH, surfactant and antibacterial, and wherein surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68, antibacterial is selected from metacresol and phenol, preferred metacresol.

In a preferred embodiment again, the invention provides a kind of goods that pharmaceutically use for the people, these goods comprise a bottle that contains the solution of FSH or FSH variant, surfactant and antibacterial, wherein surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68, antibacterial is selected from metacresol and phenol, preferred metacresol; And illustrate that this solution can use the back to preserve about 24 hours or above printed instructions first.Preferably, printed instructions illustrates that this solution can use the back to preserve maximum approximately 12 or 14 days first.

In a preferred embodiment again, the invention provides a kind of goods that pharmaceutically use for the people, these goods comprise a bottle that contains the solution of FSH and LH and surfactant and antibacterial, wherein surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68, antibacterial is selected from metacresol and phenol, preferred metacresol; And illustrate that this solution can use the back to preserve about 24 hours or above printed instructions first.Preferably, printed instructions illustrates that this solution can use the back to preserve maximum approximately 12 or 14 days first.

In a special preferred embodiment, preparation contains metacresol and Pluronic F68.The inventor is surprised to find that the preparation that contains Pluronic F68 can not produce precipitation in the presence of metacresol, uses other surfactants then can see deposited phenomenon.

Before using first, promptly open before bottle, ampoule or the injection tube sealing, preparation of the present invention can be preserved at least about 6 months, 12 months or 24 months.The storage of preparation will be away from high light (be better than most and be stored in the dark) before using first, and the about 2-8 of storage temperature ℃, better about 4-5 ℃.

In one embodiment, the invention provides a kind of lyophilized formulations of reconstruct, it preferably uses as multi-agent, contains FSH or its variant and surfactant, and wherein surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68, preferred Pluronic F68.

In another specific embodiment, the invention provides a kind of lyophilized formulations of reconstruct, it preferably uses as multi-agent, contains LH and surfactant, and wherein surfactant is selected from Pluronic ^F77, PluronicF87, Pluronic F88 and Pluronic F68, preferred Pluronic F68.

In another specific embodiment, the invention provides a kind of lyophilized formulations of reconstruct, it preferably uses as multi-agent, contains FSH and LH and surfactant, and wherein surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68, preferred Pluronic F68.Preferably, the ratio of FSH and LH (FSH: LH) about 2: 1 to about 1: 1.

In another specific embodiment, the invention provides a kind of method for preparing lyophilized formulations, described lyophilized formulations preferably uses as multi-agent after reconstruct, and described method comprises that formation contains FSH or its variant and surfactant mixtures, and wherein surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68; And make described mixture lyophilizing.

In another specific embodiment, the invention provides a kind of method for preparing lyophilized formulations, described lyophilized formulations preferably uses as multi-agent after reconstruct, and described method comprises that formation contains LH and surfactant mixtures, and wherein surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68; And make described mixture lyophilizing.

In another specific embodiment, the invention provides a kind of method for preparing lyophilized formulations, described lyophilized formulations preferably uses as multi-agent after reconstruct, and described method comprises that formation contains FSH and LH and surfactant mixtures, and wherein surfactant is selected from Pluronic ^F77, Pluronic F87, PluronicF88 and Pluronic F68; And make described mixture lyophilizing.

In a preferred embodiment again, the invention provides a kind of preparation packaged pharmaceuticals method for compositions, described method comprises a freeze-dried mixture that contains FSH and surfactant is dispensed in the container that wherein surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68.

In the preferred embodiment, the invention provides a kind of preparation packaged pharmaceuticals method for compositions again, described method comprises that a freeze-dried mixture that contains LH and surfactant is dispensed in the container, and wherein surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68.

In a preferred embodiment again, the invention provides a kind of preparation packaged pharmaceuticals method for compositions, described method comprises a freeze-dried mixture that contains FSH and LH and surfactant is dispensed in the container that wherein surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and PluronicF68.

In a preferred embodiment again, the invention provides a kind of goods that pharmaceutically use for the people, these goods comprise first container or the bottle that contain lyophilizing FSH or FSH variant and surfactant, wherein surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68.Second container or bottle contain the diluent that reconstruct is used, preferred water and antibacterial, and described antibacterial is selected from metacresol and phenol, is preferably metacresol.

In a preferred embodiment again, the invention provides a kind of goods that pharmaceutically use for the people, these goods comprise first container or the bottle that contain lyophilizing LH or LH variant and surfactant, wherein surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68.Second container or bottle contain the diluent that reconstruct is used, preferred water and antibacterial, and described antibacterial is selected from metacresol and phenol, is preferably metacresol.

In a preferred embodiment again, the invention provides a kind of goods that pharmaceutically use for the people, these goods comprise first container or the bottle that contain lyophilizing FSH or FSH variant and LH or LH variant and surfactant, wherein surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68.Second container or bottle contain the diluent that reconstruct is used, preferred water and antibacterial, and described antibacterial is selected from metacresol and phenol, is preferably metacresol.

In a special preferred embodiment, the solvent that is used for reconstruct contains metacresol.The inventor is surprised to find that the lyophilized formulations that contains Pluronic F68 produces precipitation when be reconstructed Shi Buhui with the diluent that contains metacresol, uses other surfactants (as Tween) then can see deposited phenomenon.

Lyophilized formulations of the present invention can be preserved at least about 6 months, 12 months or 24 months.The storage of preparation will be away from high light (be better than most and be stored in the dark) before using first, about 25 ℃ of storage temperature, preferably about 2-8 ℃, better about 4-5 ℃.

After liquid or reconstruct multi-agent preparation used first, it can preserve use at least about 24 hours, preferably at least about 4,5 or 6 days, and better maximum 12 or 14 days.Preparation better is stored in below 10 ℃ using the back to suit to be stored in (for example about below 25 ℃) below the room temperature first, better is stored in 2-8 ℃, preferably is stored in 5-0 ℃.

Preferably, preparation of the present invention contains antioxidant, for example methionine, sodium sulfite, ethylenediaminetetraacetic acid (EDTA) salt, Yoshinox BHT (BHT) and Butylated hydroxyanisole (BHA).Methionine preferably.Antioxidant prevents that FSH and LH (particularly α-subunit) from oxidation taking place.

The methionine concentration of liquid and/or reconstruct preparation is about 0.01 to about 1.0mg/ml scope, better about 0.05 to about 0.5mg/ml scope, and preferably about 0.1mg/ml.

Preferably, preparation of the present invention with sugar alcohol as stabilizing agent and tonicity contributor, for example sucrose, glucose, lactose, mannitol and/or glycerol.Sucrose preferably, the about 60mg/ml of its preferred concentration.

As mentioned above, the invention provides the liquid preparation that uses as single agent and multi-agent, it or contain antibacterial or add antibacterial during the reconstruct preparation.Preparation of the present invention is fit to pharmacy or the veterinary uses.

As mentioned above, a preferred embodiment of the present invention provides a kind of goods, it contains packaging material and bottle, described bottle contains FSH or FSH variant, LH or FSH and LH, Pluronic F68 and the antibacterial in aqueous diluent, described antibacterial is selected from phenol and metacresol, randomly can add buffer and/or other excipient, wherein said packaging material comprise and show that this solution can use the back to preserve about 24 hours or above printed instructions first.The present invention also comprises a kind of goods, it contains packaging material and bottle, described bottle contains FSH of the present invention or FSH variant formulations, and wherein said packaging material comprise printed instructions, illustrates that to patient use solvent reconstruct FSH or FSH variant can preserve 24 hours or above solution to form.

As mentioned above, a preferred embodiment of the present invention provides a kind of goods, and it contains packaging material and bottle, and described bottle contains lyophilizing FSH or FSH variant, LH or LH variant or FSH and LH, Pluronic F68.Antibacterial in second container comprises the diluent that is selected from phenol and metacresol, randomly can add other excipient, and wherein said packaging material comprise and show that this solution can use the back to preserve 24 hours or above printed instructions first.

The scope of preparation internal protein hormone of the present invention produces the quantity of about 1.0 μ g/ml to about 50mg/ml concentration after comprising reconstruct, but low and higher concentration all is feasible, depend on and plan the medium send into, for example the proteohormone content of pharmaceutical solutions according to percutaneous plaster, lung, to stride mucosa or infiltration or micro pump delivery method be different.Proteohormone concentration suits at about 5.0 μ g/ml to about 2mg/ml scope, and better about 10.0 μ g/ml are preferably in about 50 μ g/ml to about 200 μ g/ml scopes to about 1mg/ml scope.

Preferably, preparation of the present invention 24 months packings in the phase (before using first) keep at least about 80%FSH activity and/or LH activity.The FSH activity can be measured by Steelman-Pohley ovary weightening finish bioanalysis ⁵The LH activity can be measured by rat seminal vesicle weightening finish bioanalysis.

Liquid preparation of the present invention can be by such method preparation, described method comprises to be made solid FSH or FSH variant, LH or FSH and LH compound, Pluronic F68 and is selected from phenol and the mixing of the antibacterial of metacresol, perhaps dissolves FSH or FSH variant, LH or FSH and LH mixture (" protein "), Pluronic F68 and be selected from phenol and the antibacterial of metacresol in aqueous diluent.Utilize conventional dissolving and combination process can carry out mixing and their dissolvings in aqueous diluent of each constituent.In order to prepare suitable preparation, for example, the FSH of measured quantity that will be in buffer or FSH variant, LH or FSH and LH mixture are with Pluronic F68 in buffer and be selected from phenol and the combination of the antibacterial of metacresol, and the quantity of Pluronic F68 and antibacterial enough provides protein, Pluronic F68 and the antibacterial of desired concn.Then, the solution that obtains is dispensed in bottle, ampoule, the injection tube.Those of ordinary skills can do various variations to the method.For example, the addition sequence of each component, whether need to use the temperature and the pH of additional additives, preparation preparation, all of these factors taken together can be according to the administration concentration that adopts and mode and optimization.

In a preferred embodiment of the invention, liquid preparation of the present invention is made like this: each storing solution of the preparation whole components of said preparation (for example sodium phosphate buffer, sucrose, TWEEN, methionine, FSH and/or LH) concentration known, and the cubical content five equilibrium contained " mother solution " with final preparation same composition with formation." mother solution " the most handy Duropore ^(Millipore) 0.22 micron PDF membrane filtration to remove microorganism, distributes each aliquot to enter each container, for example bottle, ampoule or injection tube then.

Lyophilized formulations of the present invention is made by following method: this method comprises FSH or FSH variant, LH or LH variant or FSH and LH mixture, Pluronic F68 and other excipient such as antioxidant and/or buffer mixing, and makes described mixture carry out lyophilizing.Use routine techniques to mix each component and lyophilizing.For example, prepare appropriate formulation, the FSH of measured quantity or FSH variant, LH or LH variant or FSH and LH mixture are mixed with Pluronic F68, resulting mixture is by lyophilizing, and bottle, ampoule or injection tube are gone in reallocation.Those of ordinary skills can make various variations to the method.For example, the addition sequence of each component, whether need to use the temperature and the pH of additional additives, preparation preparation, all of these factors taken together can be according to the administration concentration that adopts and mode and optimization.

Also available generally acknowledged device gives preparation of the present invention.For example comprise single bottle system, described system comprises that transmission is such as EasyJect ^, Gonal-F ^Pen, Humaject ^, NovoPen ^, B-D ^Pen, AutoPen ^And OptiPen ^Pen injector device Deng solution.

The claimed product of the present invention comprises packaging material.This comprises material except the desired information of statutory body is provided, and the service condition of product also is provided.Packaging material of the present invention furnish an explanation for patient: get two bottle products (wet/do), then FSH or FSH variant are joined in the aqueous diluent wiring solution-forming and will use this solution in 24 hours or above time.For single bottle of solution product, its label show this solution can use first the back preserved about 24 hours or more than, preferably be no more than 12 or 14 days.The claimed product of the present invention can be for using on people's pharmaceutical applications.

The preparation that offers invalid stable preservation is a settled solution.But this solution single uses also repeatedly reuse, and is enough one or more cycles of patient, therefore more convenient than existing therapeutic scheme.

Described herein stable or preserve FSH in preparation or the solution or FSH variant, LH or FSH and LH mixture and can pass through various carrying methods, comprise subcutaneous or intramuscular injection, percutaneous, lung, stride mucosa, implantation, osmotic pumps, injection tube, micro pump, oral or other well known to a person skilled in the art that mode gives patient of the present invention.

The embodiment that below provides only illustrates the preparation of preparation of the present invention and component.Scope of the present invention does not should be understood to and is limited to these embodiment.

Embodiment 1

Comparative formulations

Material

Title	Manufacturer
Title	Manufacturer	The r-hFSH bulk solution that candidate's preparation is used	Laboratoires Serono SA
D-mannitol (DAB, Ph Eur, BP, FU, USP, FCC, E421)	Merck		Laboratoires Serono SA
D-mannitol (DAB, Ph Eur, BP, FU, USP, FCC, E421)	Merck	Sucrose (DAB, PhEur, BP, NF)	Merck
NaCl(ACS，ISO)	Merck	Sucrose (DAB, PhEur, BP, NF)	Merck
NaCl(ACS，ISO)	Merck	Na ₂HPO ₄2H ₂O (analytical pure)	Merck
NaH ₂PO ₄H ₂O (analytical pure)	Merck	Na ₂HPO ₄2H ₂O (analytical pure)	Merck

Title	Manufacturer
Title	Manufacturer	Benzyl alcohol (analytical pure)	Merck
Metacresol (synthetic using)	Merck	Benzyl alcohol (analytical pure)	Merck
Metacresol (synthetic using)	Merck	TWEEN 20 (polysorbate20) (synthetic using)	Merck
Pluronic F68(Poloxamer 188)	Sigma	TWEEN 20 (polysorbate20) (synthetic using)	Merck
Pluronic F68(Poloxamer 188)	Sigma	L-methionine (biochemistry is used)	Merck
Orthophosphoric acid 85% (Ph Eur, BP, NF)	Merck	L-methionine (biochemistry is used)	Merck
Orthophosphoric acid 85% (Ph Eur, BP, NF)	Merck	1.5mL glass injection tube	SFAM (siliconed at Aguettant)
A type rubber	West Company	1.5mL glass injection tube	SFAM (siliconed at Aguettant)
A type rubber	West Company	The Crim lid	Aguettant
Filter-the Durapore of Millex-GV injector drive	Millipore	The Crim lid	Aguettant
Filter-the Durapore of Millex-GV injector drive	Millipore	Durapore filter membrane 0.22 μ m GV	Millipore
20mL plastic injector Plastipak	Becton Dickinson	Durapore filter membrane 0.22 μ m GV	Millipore
20mL plastic injector Plastipak	Becton Dickinson	The filtration steelframe	Sartorius

Equipment

The HPLC system	Detector models: 486 or 490 controller models: 600S pump type: 626 Autosampler models: 717	Waters	2
The HPLC system		Waters	2	Acidometer	Model: 654	Metrohm	1
Osmometer	030-D	Osmomat		Acidometer	Model: 654	Metrohm	1	1

Following experimental evaluation the following parameters of big volume preparation:

The compatibility of surfactant and antibacterial

The oxidation of α-subunit

Preparation is the multiple dose preparation, contains TWEEN 20 or Pluronic F68 and antibacterial.Estimate following antibacterial:

Benzyl alcohol 0.9%

Metacresol 0.3%

Phenol 0.5%

The concentration range of TWEEN 20 and Pluronic F68 is as follows:

TWEEN 20:10 to 100 μ g/g

Pluronic F68:10 to 100 μ g/g

Following table 1 is listed the solution of preparation.

Table 1: comparative formulations
Table 1: comparative formulations								ID#	Na ₂HPO ₄ 2H ₂O (mg/g)	NaH ₂PO ₄ H ₂O (mg/g)	r-hFSH ^*	Pluronic F68 (μg/g)	TWEEN 20 (μg/g)	Antibacterial	Excipient (mg/g)
1P	1.11	0.45	600IU/g	10	-	0.5% phenol	Sucrose 70.6	ID#	Na ₂HPO ₄ 2H ₂O (mg/g)	NaH ₂PO ₄ H ₂O (mg/g)	r-hFSH ^*	Pluronic F68 (μg/g)	TWEEN 20 (μg/g)	Antibacterial	Excipient (mg/g)
1P	1.11	0.45	600IU/g	10	-	0.5% phenol	Sucrose 70.6	2P	1.11	0.45	600IU/g	10	-	0.5% phenol	Mannitol 38.7
3P	1.11	0.45	600IU/g	100	-	0.5% phenol	Sucrose 70.6	2P	1.11	0.45	600IU/g	10	-	0.5% phenol	Mannitol 38.7
3P	1.11	0.45	600IU/g	100	-	0.5% phenol	Sucrose 70.6	4P	1.11	0.45	600IU/g	100	-	0.5% phenol	Mannitol 38.7
5P	1.11	0.45	600IU/g	-	10	0.5% phenol	Sucrose 70.6	4P	1.11	0.45	600IU/g	100	-	0.5% phenol	Mannitol 38.7
5P	1.11	0.45	600IU/g	-	10	0.5% phenol	Sucrose 70.6	6P	1.11	0.45	600IU/g	-	10	0.5% phenol	Mannitol 38.7
7	1.11	0.45	600IU/g	-	100	0.9% benzyl alcohol	NaCl6.0	6P	1.11	0.45	600IU/g	-	10	0.5% phenol	Mannitol 38.7
7	1.11	0.45	600IU/g	-	100	0.9% benzyl alcohol	NaCl6.0	8	1.11	0.45	600IU/g	-	100	0.9% benzyl alcohol	Sucrose 62.3
9	1.11	0.45	600IU/g	-	100	0.9% benzyl alcohol	Mannitol 34.1	8	1.11	0.45	600IU/g	-	100	0.9% benzyl alcohol	Sucrose 62.3
9	1.11	0.45	600IU/g	-	100	0.9% benzyl alcohol	Mannitol 34.1	10	1.11	0.45	600IU/g	-	100	0.3% metacresol	NaCl7.6
11	1.11	0.45	600IU/g	-	100	0.3% metacresol	Sucrose 78.0	10	1.11	0.45	600IU/g	-	100	0.3% metacresol	NaCl7.6

Table 1: comparative formulations
Table 1: comparative formulations								ID#	Na ₂HPO ₄ 2H ₂O (mg/g)	NaH ₂PO ₄ H ₂O (mg/g)	r-hFSH ^*	Pluronic F68 (μg/g)	TWEEN20 (μg/g)	Antibacterial	Excipient (mg/g)
12	1.11	0.45	600IU/g	-	100	0.3% metacresol	Mannitol 42.7	ID#	Na ₂HPO ₄ 2H ₂O (mg/g)	NaH ₂PO ₄ H ₂O (mg/g)	r-hFSH ^*	Pluronic F68 (μg/g)	TWEEN20 (μg/g)	Antibacterial	Excipient (mg/g)
12	1.11	0.45	600IU/g	-	100	0.3% metacresol	Mannitol 42.7	13	1.11	0.45	600IU/g	-	10	0.9% benzyl alcohol	NaCl6.0
14	1.11	0.45	600IU/g	-	10	0.9% benzyl alcohol	Sucrose 62.3	13	1.11	0.45	600IU/g	-	10	0.9% benzyl alcohol	NaCl6.0
14	1.11	0.45	600IU/g	-	10	0.9% benzyl alcohol	Sucrose 62.3	15	1.11	0.45	600IU/g	-	10	0.9% benzyl alcohol	Mannitol 34.1
16	1.11	0.45	600IU/g	-	10	0.3% metacresol	NaCl7.6	15	1.11	0.45	600IU/g	-	10	0.9% benzyl alcohol	Mannitol 34.1
16	1.11	0.45	600IU/g	-	10	0.3% metacresol	NaCl7.6	17	1.11	0.45	600IU/g	-	10	0.3% metacresol	Sucrose 78.0
18	1.11	0.45	600IU/g	-	10	0.3% metacresol	Mannitol 42.7	17	1.11	0.45	600IU/g	-	10	0.3% metacresol	Sucrose 78.0
18	1.11	0.45	600IU/g	-	10	0.3% metacresol	Mannitol 42.7	19	1.11	0.45	600IU/g	100	-	0.9% benzyl alcohol	NaCl6.0
20	1.11	0.45	600IU/g	100	-	0.9% benzyl alcohol	Sucrose 62.3	19	1.11	0.45	600IU/g	100	-	0.9% benzyl alcohol	NaCl6.0
20	1.11	0.45	600IU/g	100	-	0.9% benzyl alcohol	Sucrose 62.3	21	1.11	0.45	600IU/g	100	-	0.9% benzyl alcohol	Mannitol 34.1
22	1.11	0.45	600IU/g	100	-	0.3% metacresol	NaCl7.6	21	1.11	0.45	600IU/g	100	-	0.9% benzyl alcohol	Mannitol 34.1
22	1.11	0.45	600IU/g	100	-	0.3% metacresol	NaCl7.6	23	-	0.45	600IU/g	100	-	0.3% metacresol	Sucrose 78.0
24	1.11	0.45	600IU/g	100	-	0.3% metacresol	Mannitol 42.7	23	-	0.45	600IU/g	100	-	0.3% metacresol	Sucrose 78.0
24	1.11	0.45	600IU/g	100	-	0.3% metacresol	Mannitol 42.7	25	1.11	0.45	600IU/g	10	-	0.9% benzyl alcohol	NaCl6.0
26	1.11	0.45	600IU/g	10	-	0.9% benzyl alcohol	Sucrose 62.3	25	1.11	0.45	600IU/g	10	-	0.9% benzyl alcohol	NaCl6.0
26	1.11	0.45	600IU/g	10	-	0.9% benzyl alcohol	Sucrose 62.3	27	1.11	0.45	600IU/g	10	-	0.9% benzyl alcohol	Mannitol 34.1
28	1.11	0.45	600IU/g	10	-	0.3% metacresol	NaCl7.6	27	1.11	0.45	600IU/g	10	-	0.9% benzyl alcohol	Mannitol 34.1
28	1.11	0.45	600IU/g	10	-	0.3% metacresol	NaCl7.6	29	1.11	0.45	600IU/g	10	-	0.3% metacresol	Sucrose 78.0
30	1.11	0.45	600IU/g	10	-	0.3% metacresol	Mannitol 42.7	29	1.11	0.45	600IU/g	10	-	0.3% metacresol	Sucrose 78.0

^*Add FSH in the preparation and be based on the biopotency of FSH but not protein content.

Preparation is carried out visual inspection, find that TWEEN20 can not reinstate with metacresol and phenol one, this is because contain TWEEN20 and metacresol or contain TWEEN20 and the FSH preparation of phenol presents milky suspension.On the contrary, the FSH preparation that contains Pluronic F68 can not produce such problem when using with metacresol and phenol, and promptly Pluronic F68 can use with phenol and metacresol.The combination of FSH and Pluronic F68 and antioxidant

Estimate following antioxidant suppresses α-subunit oxidation in the presence of Pluronic F68 ability:

Methionine: 10 to 100 μ g/g

Ascorbic acid: 10 to 100 μ g/g

Be used as tonicity agent with sucrose and mannitol, add 100 μ g/g TWEEN 20 or Pluronic surfactants.

Table 2 is listed the preparation of preparation.

Table 2. contains or does not contain the comparative formulations of methionine
										ID#	Na ₂HPO ₄ 2H ₂O (mg/g)	NaH ₂PO ₄ H ₂O (mg/g)	RhFSH	Pluroni c F68 (μg/g)	TWEEN (μg/g)	Ascorbic acid (μ g/g)	Methionine (μ g/g)	Antibacterial	Excipient
31	1.11	0.45	600IU/g	100	-	-	-	0.3% metacresol	Sucrose	ID#	Na ₂HPO ₄ 2H ₂O (mg/g)	NaH ₂PO ₄ H ₂O (mg/g)	RhFSH	Pluroni c F68 (μg/g)	TWEEN (μg/g)	Ascorbic acid (μ g/g)	Methionine (μ g/g)	Antibacterial	Excipient
31	1.11	0.45	600IU/g	100	-	-	-	0.3% metacresol	Sucrose	32	1.11	0.45	600IU/g	100	-	-	-	0.3% metacresol	Mannitol
33	1.11	0.45	600IU/g	-	100	-	-	0.9% benzyl alcohol	Sucrose	32	1.11	0.45	600IU/g	100	-	-	-	0.3% metacresol	Mannitol
33	1.11	0.45	600IU/g	-	100	-	-	0.9% benzyl alcohol	Sucrose	34	1.11	0.45	600IU/g	-	100	-	-	0.9% benzyl alcohol	Mannitol
35	1.11	0.45	600IU/g	100	-	-	-	0.9% benzyl alcohol	Sucrose	34	1.11	0.45	600IU/g	-	100	-	-	0.9% benzyl alcohol	Mannitol
35	1.11	0.45	600IU/g	100	-	-	-	0.9% benzyl alcohol	Sucrose	36	1.11	0.45	600IU/g	100	-	-	-	0.9% benzyl alcohol	Mannitol
37	1.11	0.45	600IU/g	100	-	-	10	0.3% metacresol	Sucrose	36	1.11	0.45	600IU/g	100	-	-	-	0.9% benzyl alcohol	Mannitol
37	1.11	0.45	600IU/g	100	-	-	10	0.3% metacresol	Sucrose	38	1.11	0.45	600IU/g	100	-	-	10	0.3% metacresol	Mannitol
39	1.11	0.45	600IU/g	100	-	-	100	0.3% metacresol	Sucrose	38	1.11	0.45	600IU/g	100	-	-	10	0.3% metacresol	Mannitol
39	1.11	0.45	600IU/g	100	-	-	100	0.3% metacresol	Sucrose	40	1.11	0.45	600IU/g	100	-	-	100	0.3% metacresol	Mannitol
41	1.11	0.45	600IU/g	100	-	10	-	0.3% metacresol	Sucrose	40	1.11	0.45	600IU/g	100	-	-	100	0.3% metacresol	Mannitol
41	1.11	0.45	600IU/g	100	-	10	-	0.3% metacresol	Sucrose	42	1.11	0.45	600IU/g	100	-	10	-	0.3% metacresol	Mannitol
43	1.11	0.45	600IU/g	100	-	100	-	0.3% metacresol	Sucrose	42	1.11	0.45	600IU/g	100	-	10	-	0.3% metacresol	Mannitol
43	1.11	0.45	600IU/g	100	-	100	-	0.3% metacresol	Sucrose	44	1.11	0.45	600IU/g	100	-	100	-	0.3% metacresol	Mannitol
45	1.11	0.45	600IU/g	-	100	-	10	0.9% benzyl alcohol	Sucrose	44	1.11	0.45	600IU/g	100	-	100	-	0.3% metacresol	Mannitol

Table 2. contains or does not contain the comparative formulations of methionine
										ID#	Na ₂HPO ₄ 2H ₂O (mg/g)	NaH ₂PO ₄ H ₂O (mg/g)	RhFSH	Pluroni c F68 (μg/g)	TWEEN (μg/g)	Ascorbic acid (μ g/g)	Methionine (μ g/g)	Antibacterial	Excipient
46	1.11	0.45	600IU/g	-	100	-	10	0.9% benzyl alcohol	Mannitol	ID#	Na ₂HPO ₄ 2H ₂O (mg/g)	NaH ₂PO ₄ H ₂O (mg/g)	RhFSH	Pluroni c F68 (μg/g)	TWEEN (μg/g)	Ascorbic acid (μ g/g)	Methionine (μ g/g)	Antibacterial	Excipient
46	1.11	0.45	600IU/g	-	100	-	10	0.9% benzyl alcohol	Mannitol	47	1.11	0.45	600IU/g	-	100	-	100	0.9% benzyl alcohol	Sucrose
48	1.11	0.45	600IU/g	-	100	-	100	0.9% benzyl alcohol	Mannitol	47	1.11	0.45	600IU/g	-	100	-	100	0.9% benzyl alcohol	Sucrose
48	1.11	0.45	600IU/g	-	100	-	100	0.9% benzyl alcohol	Mannitol	49	1.11	0.45	600IU/g	-	100	10	-	0.9% benzyl alcohol	Sucrose
50	1.11	0.45	600IU/g	-	100	10	-	0.9% benzyl alcohol	Mannitol	49	1.11	0.45	600IU/g	-	100	10	-	0.9% benzyl alcohol	Sucrose
50	1.11	0.45	600IU/g	-	100	10	-	0.9% benzyl alcohol	Mannitol	51	1.11	0.45	600IU/g	-	100	100	-	0.9% benzyl alcohol	Sucrose
52	1.11	0.45	600IU/g	-	100	100	-	0.9% benzyl alcohol	Mannitol	51	1.11	0.45	600IU/g	-	100	100	-	0.9% benzyl alcohol	Sucrose
52	1.11	0.45	600IU/g	-	100	100	-	0.9% benzyl alcohol	Mannitol	53	1.11	0.45	600IU/g	100	-	-	10	0.9% benzyl alcohol	Sucrose
54	1.11	0.45	600IU/g	100	-	-	10	0.9% benzyl alcohol	Mannitol	53	1.11	0.45	600IU/g	100	-	-	10	0.9% benzyl alcohol	Sucrose
54	1.11	0.45	600IU/g	100	-	-	10	0.9% benzyl alcohol	Mannitol	55	1.11	0.45	600IU/g	100	-	-	100	0.9% benzyl alcohol	Sucrose
56	1.11	0.45	600IU/g	100	-	-	100	0.9% benzyl alcohol	Mannitol	55	1.11	0.45	600IU/g	100	-	-	100	0.9% benzyl alcohol	Sucrose
56	1.11	0.45	600IU/g	100	-	-	100	0.9% benzyl alcohol	Mannitol	57	1.11	0.45	600IU/g	100	-	10	-	0.9% benzyl alcohol	Sucrose
58	1.11	0.45	600IU/g	100	-	10	-	0.9% benzyl alcohol	Mannitol	57	1.11	0.45	600IU/g	100	-	10	-	0.9% benzyl alcohol	Sucrose
58	1.11	0.45	600IU/g	100	-	10	-	0.9% benzyl alcohol	Mannitol	59	1.11	0.45	600IU/g	100	-	100	-	0.9% benzyl alcohol	Sucrose
60	1.11	0.45	600IU/g	100	-	100	-	0.9% benzyl alcohol	Mannitol	59	1.11	0.45	600IU/g	100	-	100	-	0.9% benzyl alcohol	Sucrose
60	1.11	0.45	600IU/g	100	-	100	-	0.9% benzyl alcohol	Mannitol	61	1.11	0.45	600IU/g	100	-	-	-	Phenol	Sucrose
62	1.11	0.45	600IU/g	100	-	-	-	Phenol	Mannitol	61	1.11	0.45	600IU/g	100	-	-	-	Phenol	Sucrose
62	1.11	0.45	600IU/g	100	-	-	-	Phenol	Mannitol	63	1.11	0.45	600IU/g	-	-	-	-	Phenol	Sucrose
64	1.11	0.45	600IU/g	100	-	-	10	Phenol	Mannitol	63	1.11	0.45	600IU/g	-	-	-	-	Phenol	Sucrose
64	1.11	0.45	600IU/g	100	-	-	10	Phenol	Mannitol	65	1.11	0.45	600IU/g	100	-	-	100	Phenol	Sucrose
66	1.11	0.45	600IU/g	100	-	-	100	Phenol	Mannitol	65	1.11	0.45	600IU/g	100	-	-	100	Phenol	Sucrose
66	1.11	0.45	600IU/g	100	-	-	100	Phenol	Mannitol	67	1.11	0.45	600IU/g	100	-	10	-	Phenol	Sucrose
68	1.11	0.45	600IU/g	100	-	10	-	Phenol	Mannitol	67	1.11	0.45	600IU/g	100	-	10	-	Phenol	Sucrose
68	1.11	0.45	600IU/g	100	-	10	-	Phenol	Mannitol	69	1.11	0.45	600IU/g	100	-	100	-	Phenol	Sucrose
70	1.11	0.45	600IU/g	100	-	100	-	Phenol	Mannitol	69	1.11	0.45	600IU/g	100	-	100	-	Phenol	Sucrose

In preparation, add FSH and be based on the biopotency of FSH but not protein content.

Every kind of preparation is got 20g, makes Falcon polypropylene test tube, and filters through the filter Durapore of 3cm20.22 μ m Millex-GV injector drive, analyzes the numerical value of t=0 then.Solution is stored in 40 ℃,

Test according to following scheme:

Analytical test	T＝0	1 week	2 weeks	3 weeks	4 weeks
Analytical test	T＝0	1 week	2 weeks	3 weeks	4 weeks	Analyze the reversed-phase HPLC (%) of oxidation α subunit	X	X	X	X	X
Exclusion-HPLC (μ g/g) that quantification of protein is analyzed	X	X	X	X	X	Analyze the reversed-phase HPLC (%) of oxidation α subunit	X	X	X	X	X
	X	X	X	X	X	Exclusion-the HPLC of free subunit qualitative analysis	X	X	X	X	X

(X): expression is tested

Reversed-phase HPLC discloses, contain in the preparation of FSH, Pluronic F68, metacresol and methionine (concentration is 10 and 100 μ g/ml), when said preparation when 40 ℃ store, the α of its FSH-subunit oxidation is compared with the preparation that does not contain methionine greatly and is reduced, and sees Fig. 1.According to the meansigma methods of two experiments, do not contain that oxidation α-subunit percentage rate is to be that 4.0, the 2 weeks were 7.1 in 2.3, the 1 weeks in the preparation of methylthio-aminoacids when T=0.Contain that oxidation α-subunit percentage rate is to be that 3.2, the 2 weeks were 3.8 in 2.0, the 1 weeks in the preparation of 10 μ g/ml methylthio-aminoacids when T=0.Contain that oxidation α-subunit percentage rate is to be that 1.7, the 2 weeks were 1.3 in 1.8, the 1 weeks in the preparation of 100 μ g/ml methylthio-aminoacids when T=0.

Embodiment 2

The Recombinant FSH liquid list agent formulation that is used for subcutaneous or intramuscular injection

According to the result of embodiment 1, prepare following preparation.

The component 1 to 7 that table 3 is listed is to make as the equivalent standard solution in WFI.In mixer, add the aliquot of every kind of solution, form in " mother solution ".This mother solution is dispensed in the bottle, contains 10.9 micrograms (150IU) or 5.45 micrograms (75IU) FSH.

Because used Recombinant FSH,, can realize a large amount of filling FSH (fill-by-mass) like this rather than carry out filling by biologic test so biological activity is consistent with specificity.

The component of the single agent liquid preparation of table 3.FSH
				Component #	Title	150IU FSH	75IU FSH
1	RhFSH (μ g/ bottle)	10.9(150IU)	5.45(75IU)	Component #	Title	150IU FSH	75IU FSH
1	RhFSH (μ g/ bottle)	10.9(150IU)	5.45(75IU)	2	Sucrose (mg/ bottle)	15.00	7.50
3	NaH ₂PO ₄·H ₂O (mg/ bottle)	0.111	0.0555	2	Sucrose (mg/ bottle)	15.00	7.50
3	NaH ₂PO ₄·H ₂O (mg/ bottle)	0.111	0.0555	4	Na ₂HPO ₄·2H ₂O (mg/ bottle)	0.273	0.1365
5	Pluronic F68 (mg/ bottle)	0.025	0.0125	4	Na ₂HPO ₄·2H ₂O (mg/ bottle)	0.273	0.1365
5	Pluronic F68 (mg/ bottle)	0.025	0.0125	6	Methionine (mg/ bottle)	0.025	0.0125
7	Metacresol (mg/ bottle)	0.75	0.375	6	Methionine (mg/ bottle)	0.025	0.0125
7	Metacresol (mg/ bottle)	0.75	0.375	8	pH	7.0	7.0
9	WFI	Be assigned to 1ml in right amount	Be assigned to 0.5ml in right amount	8	pH	7.0	7.0

Filling and sealed vial under aseptic condition.These preparations at room temperature can be preserved maximum 2 years.

Embodiment 3

The Recombinant FSH liquid multi-agent preparation that is used for subcutaneous or intramuscular injection

According to the result of embodiment 1, prepare following multi-agent preparation.

The component 1 to 7 that table 4 is listed is to make as the equivalent standard solution in WFI.In mixer, add the aliquot of every kind of solution, form in " mother solution ".This mother solution is dispensed in the bottle, contains 22.2 micrograms (305IU), 33.3 micrograms (458IU) and 66.7 micrograms (916IU) FSH.The preparation that obtains carries totally 300,450 and 900IU FSH.

Filling and sealing injection tube under aseptic condition.These multi-agent preparations can better be preserved down at about 4-5 ℃ in about 2-8 ℃, until using the back to preserve maximum 2 years first.After using first, injection tube should better be preserved down at about 4-5 ℃ at about 2-8 ℃, and storage life can be 24 hours, 2 days, perhaps maximum 12 or 14 days.

The component of table 4.FSH multi-agent liquid preparation
The component of table 4.FSH multi-agent liquid preparation					Component #	Title	300IU FSH	450IU FSH	900IU FSH
1	RhFSH (μ g/ injection tube)	22.2(305IU)	33.3(458IU)	66.7(916IU)	Component #	Title	300IU FSH	450IU FSH	900IU FSH
1	RhFSH (μ g/ injection tube)	22.2(305IU)	33.3(458IU)	66.7(916IU)	2	Sucrose (mg/ injection tube)	30.0	45.0	90.0
3	NaH ₂PO ₄·H ₂O (mg/ injection tube)	0.225	0.337	0.675	2	Sucrose (mg/ injection tube)	30.0	45.0	90.0
3	NaH ₂PO ₄·H ₂O (mg/ injection tube)	0.225	0.337	0.675	4	Na ₂HPO ₄·2H ₂O (mg/ injection tube)	0.555	0.832	1.665
5	Pluronic F68 (mg/ bottle)	0.050	0.075	0.150	4	Na ₂HPO ₄·2H ₂O (mg/ injection tube)	0.555	0.832	1.665
5	Pluronic F68 (mg/ bottle)	0.050	0.075	0.150	6	Methionine (mg/ bottle)	0.050	0.075	0.150
7	Metacresol (mg/ bottle)	1.50	2.25	4.50	6	Methionine (mg/ bottle)	0.050	0.075	0.150
7	Metacresol (mg/ bottle)	1.50	2.25	4.50	8	pH	7.0	7.0	7.0
9	WFI	Be assigned to 0.5ml in right amount	Be assigned to 0.75ml in right amount	Be assigned to 1.5ml in right amount	8	pH	7.0	7.0	7.0

Embodiment 4

The reorganization LH liquid list agent formulation that is used for subcutaneous or intramuscular injection

Prepare following preparation.

The component 1 to 7 that table 5 is listed is to make as the equivalent standard solution in WFI.In mixer, add the aliquot of every kind of solution, form in " mother solution ".This mother solution is dispensed in the bottle, contains 3 micrograms (75IU) LH.The preparation that obtains is carried single agent 75IU LH.

Because used reorganization LH,, can realize a large amount of filling LH (fill-by-mass) like this rather than carry out filling by biologic test so biological activity is consistent with specificity.

The component of the single agent liquid preparation of table 5.LH
			Component #	Title	LH 75IU
1	RhLH (μ g/ bottle)	3.0	Component #	Title	LH 75IU
1	RhLH (μ g/ bottle)	3.0	2	Sucrose (mg/ bottle)	52.5

3	NaH ₂PO ₄·H ₂O (mg/ bottle)	0.052
3	NaH ₂PO ₄·H ₂O (mg/ bottle)	0.052	4	Na ₂HPO ₄·2H ₂O (mg/ bottle)	0.825
5	Pluronic F68 (mg/ bottle)	0.0125	4	Na ₂HPO ₄·2H ₂O (mg/ bottle)	0.825
5	Pluronic F68 (mg/ bottle)	0.0125	6	Methionine (mg/ bottle)	0.125
7	Metacresol (mg/ bottle)	0.375	6	Methionine (mg/ bottle)	0.125
7	Metacresol (mg/ bottle)	0.375	9	WFI	Be assigned to 0.5ml in right amount

Embodiment 5

The Recombinant FSH and LH (2: 1) the liquid multi-agent preparation that are used for subcutaneous or intramuscular injection

Prepare following FSH and LH multi-agent preparation, wherein FSH: LH is 2: 1.

The component 1 to 8 that table 6 is listed is to make as the equivalent standard solution in WFI.The aliquot that adds every kind of solution in mixer is mixed them and is formed " mother solution ".Having needs, and adds the pH to 8.0 that sodium hydroxide or hydrochloric acid are transferred mother solution.This mother solution is dispensed in the injection tube, contains 18.3 micrograms (457IU) LH and 66.7 micrograms (916IU) FSH, divide 6 doses with, every dose contains 150IU FSH; 9.2 microgram (230IU) LH and 33.3 micrograms (458IU) FSH, divide 3 doses with, every dose contains 150IU FSH; 6.1 microgram (152.5IU) LH and 22.23 micrograms (305IU) FSH, divide 2 doses with, every dose contains 150IUFSH.

The component of table 6.FSH and LH (2: 1) multi-agent liquid preparation
					Component #	Title		6 doses	3 doses	2 doses
1	RhLH (μ g/ injection tube)	18.3(457IU)	9.2(230IU)	6.1(152.5IU)	Component #	Title		6 doses	3 doses	2 doses
1	RhLH (μ g/ injection tube)	18.3(457IU)	9.2(230IU)	6.1(152.5IU)	2	RhFSH (μ g/ injection tube)	66.7(916IU)	33.3(458IU)	22.23(305IU)
3	Sucrose (mg/ injection tube)	115.5	57.75	38.5	2	RhFSH (μ g/ injection tube)	66.7(916IU)	33.3(458IU)	22.23(305IU)
3	Sucrose (mg/ injection tube)	115.5	57.75	38.5	4	H ₃PO ₄(mg/ injection tube)	1.35	0.735	0.49

5	NaOH (mg/ injection tube)	Be assigned to pH 8.0 in right amount	Be assigned to pH 8.0 in right amount	Be assigned to pH 8.0 in right amount
5	NaOH (mg/ injection tube)	Be assigned to pH 8.0 in right amount	Be assigned to pH 8.0 in right amount	Be assigned to pH 8.0 in right amount	6	Pluronic F68 (mg/ injection tube)	375.0	187.5	125.0
7	Methionine (μ g/ injection tube)	225	112.5	75.0	6	Pluronic F68 (mg/ injection tube)	375.0	187.5	125.0
7	Methionine (μ g/ injection tube)	225	112.5	75.0	8	Metacresol (mg/ injection tube)	4.5	2.25	1.5
9	pH	8.0	8.0	8.0	8	Metacresol (mg/ injection tube)	4.5	2.25	1.5
9	pH	8.0	8.0	8.0	10	WFI	Be assigned to 1.5 ml in right amount	Be assigned to 0.75ml in right amount	Be assigned to 0.5 ml in right amount

Embodiment 6

The Recombinant FSH and LH (1: 1) the liquid multi-agent preparation that are used for subcutaneous or intramuscular injection

Prepare following FSH and LH multi-agent preparation, wherein FSH: LH is 1: 1.

The component 1 to 8 that table 7 is listed is to make as the equivalent standard solution in WFI.The aliquot that adds every kind of solution in mixer is mixed them and is formed " mother solution ".Having needs, and adds the pH to 8.0 that sodium hydroxide or hydrochloric acid are transferred mother solution.This mother solution is dispensed in the injection tube, contains 36.6 micrograms (914IU) LH and 66.7 micrograms (916IU) FSH, divide 6 doses with, every dose contains 150IU FSH; 18.4 microgram (460IU) LH and 33.3 micrograms (458IU) FSH, divide 3 doses with, every dose contains 150IU FSH; 12.2 microgram (152.5IU) LH and 22.23 micrograms (305IU) FSH, divide 2 doses with, every dose contains 150IUFSH.

The component of table 7.FSH and LH (1: 1) multi-agent liquid preparation
					Component #	Title	6 doses	3 doses	2 doses
1	RhLH (μ g/ injection tube)	36.6(914IU)	18.4(460IU)	12.2(305IU)	Component #	Title	6 doses	3 doses	2 doses

2	RhFSH (μ g/ injection tube)	66.7(916IU)	33.3(458IU)	22.23(305IU)
2	RhFSH (μ g/ injection tube)	66.7(916IU)	33.3(458IU)	22.23(305IU)	3	Sucrose (mg/ injection tube)	115.5	57.75	38.5
4	H ₃PO ₄(mg/ injection tube)	1.35	0.735	0.49	3	Sucrose (mg/ injection tube)	115.5	57.75	38.5
4	H ₃PO ₄(mg/ injection tube)	1.35	0.735	0.49	5	NaOH (mg/ injection tube)	Be assigned to pH 8.0 in right amount	Be assigned to pH 8.0 in right amount	Be assigned to pH 8.0 in right amount
6	Pluronic F68 (mg/ bottle)	375.0	187.5	125.0	5	NaOH (mg/ injection tube)	Be assigned to pH 8.0 in right amount	Be assigned to pH 8.0 in right amount	Be assigned to pH 8.0 in right amount
6	Pluronic F68 (mg/ bottle)	375.0	187.5	125.0	7	Methionine (μ g/ injection tube)	225	112.5	75.0
8	Metacresol (mg/ injection tube)	4.5	2.25	1.5	7	Methionine (μ g/ injection tube)	225	112.5	75.0
8	Metacresol (mg/ injection tube)	4.5	2.25	1.5	9	pH	8.0	8.0	8.0
10	WFI	Be assigned to 1.5 ml in right amount	Be assigned to 0.75ml in right amount	Be assigned to 0.5 ml in right amount	9	pH	8.0	8.0	8.0

Embodiment 7

The liquid multi-agent stability of formulation experiment of FSH and LH mixture

7.1. the reversed-phase HPLC of analysing protein content

Utilize protein content in the preparation of reversed-phase HPLC method evaluation embodiment 5 (6 doses).

Said preparation is stored in 4 ℃, at time 0 point,

preservation

1,2,3 and its protein content (FSH and LH) of mensuration after 6 months.The results are shown in Table 8, represents with every gram solvent microgram FSH or LH.

7.2. oxidation α-subunit analysis

Utilize reversed-phase HPLC (RP-HPLC) method to measure oxidation α-subunit percentage rate in the preparation of embodiment 5.

Said preparation is stored in 4 ℃, at time 0 point,

preservation

1,2,3 and its oxidation α-subunit percentage rate of mensuration after 6 months.The results are shown in Table 8.

7.3.FSH the body inner analysis

Utilize Steelman-Pohley ovary weightening finish bioanalysis, time 0 point, 4 ℃ preserve 1,2,3 and 6 months after FSH activity in the preparation of test implementation example 5 (6 doses).The results are shown in Table 8, represents with every gram solvent iu (IU).

7.4.LH the body inner analysis

Utilize rat seminal vesicle weightening finish bioanalysis, time 0 point, 4 ℃ preserve 1,2,3 and 6 months after LH activity in the preparation of test implementation example 5 (6 doses).The results are shown in Table 8, represents with every gram solvent iu (IU).

7.5. the evaluation of free subunit (rFSH+rLH)

By free subunit percentage rate in the preparation of SDS-PAGE evaluation embodiment 5.

Said preparation is stored in 4 ℃, time 0 point, preserve 1,2,3 and 6 months after measure.The result represents with the percentage rate that accounts for total protein (rFSH+rLH), lists in the table 8.

7.6. the evaluation of aggregation

With reference to the evaluation methodology of the described free subunit of above-mentioned 7.5 parts,, measure the percentage rate that high molecular gathering thing accounts for total protein (rFSH+rLH) but change into by aggregation percentage rate in the preparation of SDS-PAGE evaluation embodiment 5.Mensuration is in time 0 point,

preservation

1,2,3 and carries out after 6 months.The results are shown in Table 8.

7.7. visible particle

Time 0 point, 4 ℃ preserve 3 and 6 months after the granule of preparation of visual assessment embodiment 5.The results are shown in Table 8.

7.8.pH value

Time 0 point, 4 ℃ preserve 1,2,3 and 6 months after measure the pH value of the preparation of embodiment 5.The results are shown in table 8.

Table 8. time 0 point, 4 ℃ preserve 1,2,3 and 6 months after the analytical parameters of FSH and LH (2: 1) liquid preparation
						Analytical test	Time	0 point	1 month	2 months	3 months	6 months
RP-HPLC measures	46.50	46.98	46.71	46.31	44.98	Analytical test	Time	0 point	1 month	2 months	3 months	6 months

RFSH content (microgram/g)
RFSH content (microgram/g)						The rLH content that RP-HPLC measures (microgram/g)	11.74	11.81	12.68	12.67	13.21
Oxidation α-subunit %	2.29	2.17	2.08	2.48	2.95	The rLH content that RP-HPLC measures (microgram/g)	11.74	11.81	12.68	12.67	13.21
Oxidation α-subunit %	2.29	2.17	2.08	2.48	2.95	The body inner analysis 553 (IU/g) of FSH	566	Do not do test	Do not do test	Do not do test	578 (23 weeks)
The body inner analysis (IU/g) of LH	331	Do not do test	Do not do test	311	286	The body inner analysis 553 (IU/g) of FSH	566	Do not do test	Do not do test	Do not do test	578 (23 weeks)
The body inner analysis (IU/g) of LH	331	Do not do test	Do not do test	311	286	Free subunit (the rFSH+rLH of SDS-PAGE; %)	≤5	Do not do test	Do not do test	≤5	≤ 5 (23 weeks)
SDS-PAGE aggregation (rFSH+rLH; %)	≤2	Do not do test	Do not do test	＞3	4	Free subunit (the rFSH+rLH of SDS-PAGE; %)	≤5	Do not do test	Do not do test	≤5	≤ 5 (23 weeks)
SDS-PAGE aggregation (rFSH+rLH; %)	≤2	Do not do test	Do not do test	＞3	4	Visible particle	No	Do not do test	Do not do test	No	No
pH	8.262	8.215	8.216	8.188	8.283	Visible particle	No	Do not do test	Do not do test	No	No

Embodiment 8

FSH and LH lyophilizing multi-agent preparation

Preparation contains two the lyophilized formulations A and the B of following component:

Preparation A

FSH μg32.75(450I.U.)

LH μg9.0(225I.U.)

Sucrose mg15.0

NaH ₂PO ₄H ₂O mg0.052

Na ₂HPO ₄2H ₂O mg0.825

Pluronic F-68 mg0.05

L-methionine mg0.05

Preparation B

FSH μg65.5(900I.U.)

LH μg18.0(450I.U.)

Sucrose mg30.0

NaH ₂PO ₄H ₂O mg0.104

Na ₂HPO ₄2H ₂O mg1.65

Pluronic F68 mg0.10

L-methionine mg0.10

Preparation method comprises directly mixes drug substance with each composition, filter the solution and the lyophilizing filtrate that obtain.

Each step of preparation method below is described:

-in gas-mixing balance container, add WFI, disodium phosphate dihydrate, biphosphate sodium-hydrate, sucrose, 5%Pluronic F68 and L-methionine, stirred 10 minutes, until dissolving fully.

-check pH value, with NaOH 10% or dilution H ₃PO ₄Transfer solution to last pH7.00 ± 0.2.

-in the mixture of above-mentioned preparation, add FSH and LH, stirred the solution that obtains gently 10 minutes.

-check pH value once more, with NaOH10% or dilution H ₃PO ₄Transfer solution to last pH7.0 ± 0.1.

-with 0.22 μ m Durapore membrane filtration solution, specific filtration resistance is no more than 15g/cm ², feeding nitrogen, nitrogen gas pressure is below 1.5atm.

-solution is collected in the sterilized in advance flask.

-toward glass container filling filtrate, stopper and the bottle after the filling placed on the rustless steel plate beyond the Great Wall.

-plate is placed on the freeze dryer, freeze-drying prods, the lyophilizing circulation is as follows:

About 20 minutes of+4 ℃ of balances.

Transferring backing temp is-25 ℃, keeps 2 hours.

Transferring backing temp is-15 ℃, keeps 1 hour.

Transferring backing temp is-45 ℃, keeps 3 hours.

Transferring condenser temperature is-65 ℃.

The vessel evacuation.

When vacuum arrives 7 * 10 ^-2During mBar, backing temp is increased to-10 ℃, kept 4 hours.

In 8 hours, backing temp is risen to+35 ℃, be retained to this loop ends (14 hours).

Suspend evacuation, charge into nitrogen to vessel.

Automated system by freeze dryer adds stopper.

The bottle that adds stopper with suitable gland device sealing.

Preparation A and B are stored in 25 ± 2 ℃, test their stability and biological activity as follows.Before the analysis, with containing 0.3% metacresol each component of injection water reconstruct as antibacterial.

Stable and bioactive method of testing is as follows:

The body inner analysis of FSH: the FSH activity of utilizing Steelman-Pohley ovary weightening finish bioanalysis test formulation.

The body inner analysis of LH: the LH activity of utilizing rat seminal vesicle weightening finish bioanalysis test formulation.

Oxidation α-subunit analysis: utilize reversed-phase HPLC (RP-HPLC) method to measure oxidation α-subunit percentage rate.

The evaluation of free subunit (rFSH+rLH): estimate free subunit percentage rate by SDS-PAGE.

The evaluation of aggregation:, estimate the aggregation percentage rate by SDS-PAGE with reference to the evaluation methodology of above-mentioned free subunit.

Regulation according to European Pharmacopoeia is carried out biologic test." menotrophin " monograph is seen in concrete test.

Table 9 briefly provides the preparation A stability analytical test result relevant with biological activity.These numerical value are measured at following 4 test points: after time 0 point, 25 ± 2 ℃ of

preservations

1,3 and 6 months.

Table 9

Test	Time	0 point	1 month	3 months	6 months
Test	Time	0 point	1 month	3 months	6 months	Biological activity I.U.FSH	416	420	415	417
Biological activity I.U.LH	276	250	259	270		Biological activity I.U.FSH	416	420	415	417
Biological activity I.U.LH	276	250	259	270	The % oxidation product	1.95	1.81	1.95	1.57
% dimer/aggregation	＜2	＜2	＜2	＜2	The % oxidation product	1.95	1.81	1.95	1.57
% dimer/aggregation	＜2	＜2	＜2	＜2	The free subunit of %	＜5	＜5	＜5	＜5

Table 10 briefly provides the preparation B stability analytical test result relevant with biological activity.These numerical value are measured at following 4 test points: after time 0 point, 25 ± 2 ℃ of

preservations

3,6 and 9 months.

Table 10

Test	Time	0 point	3 months	6 months	9 months
Test	Time	0 point	3 months	6 months	9 months	Biological activity I.U.FSH	821	850	830	838
Biological activity I.U.LH	570	564	580	622		Biological activity I.U.FSH	821	850	830	838
Biological activity I.U.LH	570	564	580	622	The % oxidation product	1.0	0.9	1.0	1.0
% dimer/aggregation	＜2	＜2	＜2	＜2	The % oxidation product	1.0	0.9	1.0	1.0
% dimer/aggregation	＜2	＜2	＜2	＜2	The free subunit of %	＜5	＜5	＜5	＜5

Can draw from table 9 and 10, the biological activity of preparation A and B still keeps finely after storing 9 months.These preparations have advantages of higher stability.

How Recombinant FSH and reorganization LH quantity can't influence high stability.

Sequence:

SEQ ID NO.1: human glucoprotein α-subunit;

SEQ ID NO.2:hFSH β-subunit

SEQ ID NO.3:hFSH β-subunit variant 1

SEQ ID NO.4:hFSH β-subunit variant 2

SEQ ID NO.5:hFSH β-subunit variant 3

SEQ ID NO.6:hLH β-subunit

List of references:

¹Burgues et al.；Subcutaneous self-administration of highly purified folliclestimulating hormone and human chorionic gonadotrophin for the treatment of malehypogonadotrophic hypogonadism.Spanish Collaborative Group on MaleHypogonadotrophic Hypogonadism；Hum.Reprod.；1997，12，980-6；

²Shome et al.，J.Clin.Endocrinol.Metab.39：187-205(1974)；Shome，et al.，J.Prot.Chem，7：325-339，1988；

²Keutmann et al.；Structure of human luteinizing hormone beta subunit：evidencefor related carboxyl-terminal sequence among certain peptide hormones；Biochem.Biophys.Res.Commun.；1979，90，842-848；Talmadge et al.；Evolution of the genes forthe beta subunits of human chorionic gonadotropin and luteinizing hormone；Nature；1984，307，37-40；Fiddes & Talmadge；Structure，expression，and evolution of the genesfor the human glycoprotein hormones；Recent Prog.Horm.Res.；1984，40，43-78

⁴Reichert LE，Ramsey RB；Dissociation of human follicle-stimulating hormone；J.Biol.Chem.；1975，250，3034-3040

⁵Klein et al.；Pharmacokinetics and pharmacodynamics of single-chainrecombinant human follicle-stimulating hormone containing the human chorionicgonadotrophin carboxyterminal peptide in the rhesus monkey；Fertility & Sterility；2002，77，1248-1255

⁶a)Fiddes，J.C.，et al.，J of Mol.and Applied Genetics，1：3-18(1981)；b)Esch F.S.，et al.DNA 5：363-369(1986)；c)Watkins P.C.，et al.，DNA 6：205-212(1987)；d)Hirai T.，et al.，J.Mol.Endrocrinol.5：147-158(1990)；e)Maurer，R.A.，et al.，Mol.Endocrinol.1：717-723(1987)；f)Guzman K.，et al.，DNA Cell Biol.10：593-601(1991)；g)Kumar TR，et al.，Gene.1995Dec 12；166(2)：335-6；h)Kumar TR，et al.，Gene.1995Dec 12；166(2)：333-4

⁷Biochem.Biophys.Res.Commun.；1979，90，842-848

⁸Steelman et al.；Assay of the follicle stimulating hormone based on theaugmentation with human chorionic gonadotrophin；Endocrinology；1953，53，604-616

⁹Van Hell et al.；Effects of human menopausal gonadotrophin preparations indifferent bioassay methods；Acta Endocrinologica；1964，47，409-418

¹⁰Van Hell et al.；Effects of human menopausal gonadotrophin preparations indifferent bioassay methods；Acta Endocrinologica；1964，47，409-418

Sequence table

＜110〉Ares Trading S.A.

＜120〉liquid pharmaceutical formulation of FSH and LH and non-ionic surface active agent

<130>US 847 Y

<160>6

＜170〉PatentIn version 3 .1

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＜213〉homo sapiens (Homo sapiens)

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Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

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50 55 60

Ile Gln Lys Thr Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val Arg

65 70 75 80

Val Pro Gly Cys Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro Val

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20 25 30

Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Lys Ile Gln Lys Thr Cys

35 40 45

Thr Phe Lys Glu Leu Val 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 Gln Cys His

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＜213〉homo sapiens (Homo sapiens)

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35 40 45

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50 55 60

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Claims

1. a composition of liquid medicine is characterized in that, described compositions contains follicule-stimulating hormone (FSH) (FSH) or its variant and surfactant, and described surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68.

2. a composition of liquid medicine is characterized in that, described compositions contains follicule-stimulating hormone (FSH) (FSH) or its variant and luteotropic hormone (LH) or its variant and surfactant, and described surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68.

3. a composition of liquid medicine is characterized in that, described compositions contains luteotropic hormone (LH) or its variant and surfactant, and described surfactant is selected from Pluronic ^F77, PluronicF87, Pluronic F88 and Pluronic F68.

4. each described composition of liquid medicine as in the above-mentioned claim is characterized in that, the concentration of follicule-stimulating hormone (FSH) (FSH) is extremely about 1 at about 150IU/ml, in the scope of 200IU/ml.

5. composition of liquid medicine as claimed in claim 4 is characterized in that, the concentration of follicule-stimulating hormone (FSH) (FSH) at about 300IU/ml to the scope of about 900IU/ml.

6. composition of liquid medicine as claimed in claim 5 is characterized in that, the about 600IU/ml of concentration of follicule-stimulating hormone (FSH) (FSH).

7. as each described composition of liquid medicine in claim 2 or 3, it is characterized in that, the concentration of luteotropic hormone (LH) at about 150IU/ml to about 1, in the scope of 200IU/ml.

8. composition of liquid medicine as claimed in claim 7 is characterized in that, the concentration of luteotropic hormone (LH) at about 300IU/ml to the scope of about 750IU/ml.

9. a lyophilized formulations is characterized in that, described preparation contains follicule-stimulating hormone (FSH) (FSH) or its variant and surfactant, and described surfactant is selected from Pluronic ^F77, Pluronic F87, PluronicF88 and Pluronic F68.

10. a lyophilized formulations is characterized in that, described preparation contains luteotropic hormone (LH) or its variant and surfactant, and described surfactant is selected from Pluronic ^F77, Pluronic F87, PluronicF88 and Pluronic F68.

11. a lyophilized formulations is characterized in that, described preparation contains follicule-stimulating hormone (FSH) (FSH) or its variant and luteotropic hormone (LH) or its variant and surfactant, and described surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68.

12., it is characterized in that the concentration (w/w) of follicule-stimulating hormone (FSH) (FSH) is every milligram of preparation total amount 0.1 to 10 microgram approximately as each described lyophilized formulations among the claim 9-11.

13. lyophilized formulations as claimed in claim 12 is characterized in that, the concentration (w/w) of follicule-stimulating hormone (FSH) (FSH) is every milligram of preparation total amount 0.3 to 5 microgram approximately.

14. lyophilized formulations as claimed in claim 13 is characterized in that, the concentration (w/w) of follicule-stimulating hormone (FSH) (FSH) is every milligram of preparation total amount 0.37 to 2 microgram approximately.

15., it is characterized in that the concentration (w/w) of luteotropic hormone (LH) is every milligram of preparation total amount 0.1 to 3 microgram approximately as each described lyophilized formulations among the claim 9-11.

16. lyophilized formulations as claimed in claim 15 is characterized in that, the concentration (w/w) of luteotropic hormone (LH) is every milligram of preparation total amount 0.1 to 1 microgram approximately.

17. lyophilized formulations as claimed in claim 16 is characterized in that, the concentration (w/w) of luteotropic hormone (LH) is every milligram of preparation total amount 0.1 to 0.6 microgram approximately.

18., it is characterized in that described surfactant is Pluronic F68 as each described pharmaceutical composition in the claim 1 to 17.

19., it is characterized in that follicule-stimulating hormone (FSH) is that HFSH and/or luteotropic hormone are the human luteinizing hormones as each described pharmaceutical composition in the above-mentioned claim.

20. pharmaceutical composition as claimed in claim 19 is characterized in that, follicule-stimulating hormone (FSH) is that people's urinary fsh and/or luteotropic hormone are the Urina Hominis luteotropic hormones.

21. pharmaceutical composition as claimed in claim 19 is characterized in that, follicule-stimulating hormone (FSH) is that recombined human follicule-stimulating hormone (FSH) and/or luteotropic hormone are the recombined human luteotropic hormones.

22. as each described pharmaceutical composition in the above-mentioned claim, it is characterized in that FSH: LH is in about 6: 1 to about 1: 6 scopes.

23. pharmaceutical composition as claimed in claim 22 is characterized in that, FSH: LH is in about 4: 1 to about 1: 2 scopes.

24. pharmaceutical composition as claimed in claim 23 is characterized in that, FSH: LH is in about 3: 1 to about 1: 1 scopes.

25. pharmaceutical composition as claimed in claim 24 is characterized in that, FSH: LH is in about 2: 1 to about 1: 1 scopes.

26. as each described pharmaceutical composition in the above-mentioned claim, it is characterized in that described compositions also comprises antibacterial, this antibacterial is selected from phenol and metacresol.

27. pharmaceutical composition as claimed in claim 26 is characterized in that, described antibacterial is a metacresol.

28. pharmaceutical composition as claimed in claim 27 is characterized in that, about 0.3% (the mass/mass solvent) of the metacresol concentration of described compositions.

29., it is characterized in that described compositions also comprises sucrose as each described pharmaceutical composition in the above-mentioned claim.

30., it is characterized in that described compositions also comprises methionine as each described pharmaceutical composition in the above-mentioned claim.

31. as above-mentioned claim in each described pharmaceutical composition, it is characterized in that described compositions also comprises phosphate buffer, the pH value of this buffer about 6.0 to about 8.0 scopes.

32. pharmaceutical composition as claimed in claim 31 is characterized in that, described compositions also comprises phosphate buffer, the pH value of this buffer about 7.0.

33. pharmaceutical composition as claimed in claim 32 is characterized in that, described compositions comprises the phosphoric acid buffer aqueous solution of following composition: rFSH, Pluronic F68, sucrose, methionine, metacresol and pH value about 7.0.

34. pharmaceutical composition as claimed in claim 33, it is characterized in that, the about 3mg/ml of concentration of the about 0.1mg/ml of concentration of the about 600IU/ml of the concentration of rFSH, Pluronic F68, the about 60mg/ml of concentration of sucrose, the about 0.1mg/ml of concentration of methionine, metacresol, and the about 10mM of the phosphate concn of phosphate buffer.

35. lyophilized formulations as claimed in claim 11 is characterized in that, the Recombinant FSH that described preparation comprises is that 32.75 μ g, reorganization LH are that 9.0 μ g, sucrose are 15.0mg, NaH ₂PO ₄H ₂O is 0.052mg, Na ₂HPO ₄2H ₂O is that 0.825mg, Pluronic F68 are that 0.05mg and L-methionine are 0.05mg.

36. lyophilized formulations as claimed in claim 11 is characterized in that, the Recombinant FSH that described preparation comprises is that 65.5 μ g, reorganization LH are that 18.0 μ g, sucrose are 30.0mg, NaH ₂PO ₄H ₂O is 0.104mg, Na ₂HPO ₄2H ₂O is that 1.65mg, Pluronic F68 are that 0.10mg and L-methionine are 0.10mg.

37. the method for a pharmaceutical compositions is characterized in that, described method comprises the step that forms the solution that contains FSH, surfactant and liquid diluent, and described surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68.

38. method as claimed in claim 37 is characterized in that, described surfactant is PluronicF68.

39. as claim 37 or 38 described methods, it is characterized in that described method also comprises the step that adds antibacterial, described antibacterial is selected from phenol and metacresol.

40. one kind prepares the packaged pharmaceuticals method for compositions, it is characterized in that, this method comprises packs in bottle, ampoule or the injection tube containing FSH and surface-active solution branch, and described surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68.

41. method as claimed in claim 40 is characterized in that, described surfactant is PluronicF68.

42. goods is characterized in that described goods comprise first container, this container filling as each described lyophilized formulations in the claim 9 to 11; And second container, this container contains the reconstruct solvent.

43. goods as claimed in claim 42 is characterized in that, described second container contains the aqueous diluent that comprises metacresol.

44. one kind prepares as the method for lyophilized formulations as described in each in the claim 9 to 11, it is characterized in that, described method comprises the independent FSH of formation or FSH and LH or independent LH and surfactant mixtures and makes the freeze dried step of described mixture that described surfactant is selected from Pluronic ^F77, Pluronic F87, Pluronic F88 and Pluronic F68.

45. method as claimed in claim 44 is characterized in that, described surfactant is PluronicF68.