CN116549403A - Follicle-stimulating hormone freeze-dried preparation, and preparation method and application thereof - Google Patents
Follicle-stimulating hormone freeze-dried preparation, and preparation method and application thereof Download PDFInfo
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- CN116549403A CN116549403A CN202310223677.0A CN202310223677A CN116549403A CN 116549403 A CN116549403 A CN 116549403A CN 202310223677 A CN202310223677 A CN 202310223677A CN 116549403 A CN116549403 A CN 116549403A
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/24—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Reproductive Health (AREA)
- Endocrinology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Gynecology & Obstetrics (AREA)
- Pregnancy & Childbirth (AREA)
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention relates to a follicle stimulating hormone freeze-dried preparation, a preparation method and application thereof. Specifically, the invention provides a follicle stimulating hormone freeze-dried preparation, which is characterized by comprising follicle stimulating hormone and a pharmaceutically acceptable carrier; the pharmaceutically acceptable carrier comprises 100-500 parts by weight of sugar alcohol, 0.1-10 parts by weight of amino acid, 0.05-5 parts by weight of surfactant and buffer. The follicle-stimulating hormone freeze-dried preparation has excellent acceleration and long-term stability, and can improve the stability of the follicle-stimulating hormone.
Description
Technical Field
The invention relates to the field of medicine. In particular, the invention relates to a follicle stimulating hormone freeze-dried preparation, a preparation method and application thereof.
Background
Follicle stimulating hormone (Follicle Stimulating Hormone, FSH), which is a glycoprotein gonadotrophin synthesized and secreted by the anterior pituitary She Shi basic cells and controlled by hypothalamic secreted follicle stimulating hormone releasing hormone, is a heterodimeric glycoprotein composed of non-covalently bound alpha and beta subunits. At present, recombinant human follicle stimulating hormone (rhFSH) produced by using a DNA recombination technology is known, and is mainly used for treating reproductive disorder diseases in clinical treatment, including ovulation induction, superovulation control, sperm production induction and the like.
Glycoprotein hormone is used as a biological macromolecule with complex structure and function, and is affected by various factors in the process of storage and transportation to cause aggregation, degradation, even oxidation and other phenomena of protein molecules, so that the quality of products and the safety of medication are affected. Therefore, it is important to select a formulation recipe that maintains its stability over a long period of time. Biomolecules are more difficult to stabilize by formulation than small molecules due to the number of biomacromolecule groups and the dependence of maintaining natural folding on stability. Thus, many biomolecules remain stable in a lyophilized state.
Because follicle stimulating hormone (such as recombinant follicle stimulating hormone) is a bioactive heterodimer glycoprotein, the stability is poor, even if the follicle stimulating hormone preparation is prepared by adopting a strict production process in the prior art, the follicle stimulating hormone can not be well maintained, so that the drug effect is reduced or the effect is invalid, and the application of the follicle stimulating hormone is limited.
Therefore, there is a need in the art to develop a formulation that can improve the stability of follicle stimulating hormone, increase the shelf life of the follicle stimulating hormone formulation, and improve the quality of the follicle stimulating hormone formulation.
Disclosure of Invention
The invention aims to provide a freeze-dried preparation capable of improving stability of follicle stimulating hormone.
In a first aspect of the invention, there is provided a lyophilized formulation of follicle stimulating hormone, said lyophilized formulation comprising follicle stimulating hormone and a pharmaceutically acceptable carrier;
the pharmaceutically acceptable carrier comprises 100-500 parts by weight of sugar alcohol, 0.1-10 parts by weight of amino acid, 0.05-5 parts by weight of surfactant and buffer.
In another preferred embodiment, the follicle stimulating hormone is a human follicle stimulating hormone.
In another preferred embodiment, the follicle stimulating hormone is selected from the group consisting of: natural follicle stimulating hormone, recombinant follicle stimulating hormone, or a combination thereof.
In another preferred example, the amino acid sequence of the alpha subunit gene of follicle stimulating hormone is shown as SEQ ID NO. 1, and the amino acid sequence of the beta subunit gene of FSH is shown as SEQ ID NO. 2.
In another preferred embodiment, the sum of the contents of all components of the lyophilized formulation is 100wt%.
In another preferred embodiment, the lyophilized preparation is a lyophilized preparation for injection.
In another preferred example, the lyophilized preparation is a lyophilized powder for injection.
In another preferred embodiment, the lyophilized formulation comprises 10-800IU of follicle stimulating hormone and a pharmaceutically acceptable carrier;
the pharmaceutically acceptable carrier comprises 100-500 parts by weight of sugar alcohol, 0.1-10 parts by weight of amino acid, 0.05-5 parts by weight of surfactant and buffer.
In another preferred embodiment, the lyophilized formulation has a pH of 6.5-7.5, preferably 6.8-7.2, as measured after reconstitution with water.
In another preferred embodiment, the water is selected from the group consisting of: water for injection, ultrapure water, or a combination thereof.
In another preferred embodiment, the follicle stimulating hormone is 10-800IU, preferably 20-600IU, more preferably 20-500IU, more preferably 20-400IU, more preferably 20-300IU, more preferably 20-200IU, more preferably 20-160IU, more preferably 30-150IU, more preferably 40-120IU, more preferably 60-100IU, more preferably 70-90IU, more preferably 75-85IU, and most preferably 77-83IU.
In another preferred embodiment, the lyophilized formulation comprises 20-500IU of follicle stimulating hormone and a pharmaceutically acceptable carrier;
the pharmaceutically acceptable carrier comprises 100-500 parts by weight of sugar alcohol, 0.1-10 parts by weight of amino acid, 0.05-5 parts by weight of surfactant and 0.5-35 parts by weight of Na 2 HPO 4 And 0.5 to 30 parts by weight of NaH 2 PO 4 ·H 2 O。
In another preferred embodiment, the ratio of said follicle stimulating hormone to said sugar alcohol (IU: g) is 10-800:100-500, preferably 20-500:150-450, more preferably 20-400:150-400, more preferably 60-100:200-400, more preferably 75-85:250-350, optimally 77-83:280-320.
In another preferred embodiment, the lyophilized formulation has a moisture content of less than 3.0%, preferably less than 2.0%.
In another preferred embodiment, the lyophilized formulation has less than 0.5% polymer.
In another preferred embodiment, the sugar alcohol is selected from the group consisting of: saccharides, sugar alcohols, or combinations thereof.
In another preferred embodiment, the saccharide is selected from the group consisting of: monosaccharides, disaccharides, polysaccharides, or combinations thereof.
In another preferred example, the monosaccharide is a monosaccharide having 3 to 6 carbon atoms in the molecular structure.
In another preferred example, the monosaccharide is a monosaccharide having a molecular structure containing 5 to 6 carbon atoms.
In another preferred embodiment, the monosaccharide is selected from the group consisting of: five-carbon sugars, six-carbon sugars, or combinations thereof.
In another preferred embodiment, the monosaccharide is selected from the group consisting of: arabinose, ribose, xylose, lyxose, glucose, mannose, fructose, galactose, or a combination thereof.
In another preferred embodiment, the disaccharide is selected from the group consisting of: maltose, lactose, sucrose, trehalose, or a combination thereof.
In another preferred embodiment, the sugar alcohol is selected from the group consisting of: mannitol, xylitol, erythritol, sorbitol, isomalt, maltitol, lactitol, or a combination thereof.
In another preferred embodiment, the sugar alcohol is selected from the group consisting of: maltose, lactose, sucrose, trehalose, mannitol, xylitol, erythritol, sorbitol, isomalt, maltitol, lactitol, or a combination thereof.
In another preferred embodiment, the sugar alcohol comprises sucrose.
In another preferred embodiment, the sugar alcohols are 150 to 450 parts by weight, preferably 150 to 400 parts by weight, more preferably 200 to 400 parts by weight, still more preferably 250 to 350 parts by weight, most preferably 280 to 320 parts by weight.
In another preferred embodiment, the amino acid is a natural amino acid.
In another preferred embodiment, the amino acid is an L-amino acid.
In another preferred embodiment, the amino acid (e.g., L-amino acid) is selected from the group consisting of: glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, aspartic acid, glutamic acid, asparagine, glutamine, lysine, arginine, phenylalanine, tyrosine, tryptophan, histidine, proline, or a combination thereof.
In another preferred embodiment, the amino acid comprises methionine.
In another preferred embodiment, the amino acid is present in an amount of 0.2 to 8 parts by weight, preferably 0.4 to 6 parts by weight, more preferably 0.5 to 5 parts by weight, still more preferably 0.5 to 2 parts by weight, still more preferably 0.5 to 1.5 parts by weight, and most preferably 0.8 to 1.2 parts by weight.
In another preferred embodiment, the surfactant is selected from the group consisting of: nonionic surfactants, or combinations thereof.
In another preferred embodiment, the nonionic surfactant is selected from the group consisting of: cationic surfactants, anionic surfactants, or combinations thereof.
In another preferred embodiment, the nonionic surfactant is selected from the group consisting of: polysorbate (tween), poloxamer, sorbitan fatty acid esters (span), or combinations thereof.
In another preferred embodiment, the polysorbate (tween) is selected from the group consisting of: polysorbate 20 (tween-20), polysorbate 40 (tween-40), polysorbate 60 (tween-60), polysorbate 80 (tween-80), or a combination thereof.
In another preferred embodiment, the sorbitan fatty acid ester (span) is selected from the group consisting of: sorbitan fatty acid ester-20 (span-20), sorbitan fatty acid ester 80 (span-80), or a combination thereof.
In another preferred embodiment, the surfactant is selected from the group consisting of: polysorbate (tween), poloxamer, sorbitan fatty acid esters (span), or combinations thereof.
In another preferred embodiment, the poloxamer is selected from the group consisting of: poloxamer-188, poloxamer-407, or combinations thereof.
In another preferred embodiment, the surfactant is polysorbate (tween).
In another preferred embodiment, the surfactant is 0.08 to 4 parts by weight, preferably 0.1 to 3 parts by weight, more preferably 0.1 to 2 parts by weight, still more preferably 0.1 to 1.5 parts by weight, still more preferably 0.1 to 1 part by weight, and most preferably 0.3 to 0.8 part by weight.
In another preferred embodiment, the buffering agent is selected from the group consisting of: acetic acid-acetate buffer, phosphate buffer, HEPES buffer, MES buffer, MOPS buffer, or a combination thereof.
In another preferred embodiment, the buffer comprises a phosphate buffer.
In another preferred embodiment, the phosphate buffer comprises Na 2 HPO 4 And NaH 2 PO 4 ·H 2 O。
In another preferred example, the Na 2 HPO 4 0.5 to 35 parts by weight, preferably 1 to 30 parts by weight, more preferably 2 to 25 parts by weight, still more preferably 2 to 20 parts by weight, still more preferably 2 to 15 parts by weight, still more preferably 3 to 12 parts by weight, still more preferably 6 to 12 parts by weight, and most preferably 7 to 12 parts by weight.
In another preferred embodiment, the NaH 2 PO 4 ·H 2 O is 0.5 to 30 parts by weight, preferably 0.8 to 25 parts by weight, more preferably 1 to 20 parts by weight, still more preferably 1 to 15 parts by weight, more preferably 1 to 10 parts by weight, still more preferably 3 to 8 parts by weight, most preferably 4 to 8 parts by weight.
In another preferred embodiment, the phosphate buffer comprises 0.5 to 35 parts by weight of Na 2 HPO 4 And 0.5 to 30 parts by weight of NaH 2 PO 4 ·H 2 O。
In another preferred embodiment, the buffering agent has a pH of 6.5 to 7.5, preferably 6.8 to 7.2, as measured after dissolution with water.
In another preferred embodiment, the weight ratio of said buffer to said amino acid is 10-40:1, preferably 10-30:1, more preferably 10-20:1, most preferably 12-16:1.
In another preferred embodiment, the lyophilization preparation comprises one or more characteristics selected from the group consisting of:
(i) The sugar alcohols are selected from the following groups: maltose, lactose, sucrose, trehalose, mannitol, xylitol, erythritol, sorbitol, isomalt, maltitol, lactitol, or a combination thereof;
(ii) The amino acid is selected from the group consisting of: glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, aspartic acid, glutamic acid, asparagine, glutamine, lysine, arginine, phenylalanine, tyrosine, tryptophan, histidine, proline, or a combination thereof;
(iii) The surfactant is selected from the group consisting of: polysorbate (tween), poloxamer, sorbitan fatty acid esters (span), or combinations thereof; and/or
(iv) The buffering agent is selected from the group consisting of: acetic acid-acetate buffer, phosphate buffer, HEPES buffer, MES buffer, MOPS buffer, or a combination thereof.
In another preferred embodiment, the polysorbate is selected from the group consisting of: polysorbate-20, polysorbate-80, or a combination thereof.
In another preferred embodiment, the lyophilized formulation comprises 20-500IU of follicle stimulating hormone and a pharmaceutically acceptable carrier;
the pharmaceutically acceptable carrier comprises 200-400 parts by weight of sugar alcohol, 0.5-2 parts by weight of amino acid, 0.1-1.5 parts by weight of surfactant and buffer.
In another preferred embodiment, the buffer comprises 2-15 parts by weight Na 2 HPO 4 And 1 to 15 parts by weight of NaH 2 PO 4 ·H 2 O。
In another preferred embodiment, the lyophilized formulation comprises 20-500IU of follicle stimulating hormone and a pharmaceutically acceptable carrier;
the pharmaceutically acceptable carrier comprises 200-400 parts by weight of sugar alcohol, 0.5-2 parts by weight of amino acid, 0.1-1.5 parts by weight of surfactant and 2-15 parts by weight of Na 2 HPO 4 And 1 to 15 parts by weight of NaH 2 PO 4 ·H 2 O。
In another preferred embodiment, the lyophilized formulation comprises 20-500IU of follicle stimulating hormone and a pharmaceutically acceptable carrier;
the pharmaceutically acceptable carrier comprises 200-400 parts by weight of sucrose, 0.5-2 parts by weight of methionine, 0.1-1.5 parts by weight of polysorbate and buffer.
In another preferred embodiment, the buffer comprises 2-15 parts by weight Na 2 HPO 4 And 1 to 15 parts by weight of NaH 2 PO 4 ·H 2 O。
In another preferred embodiment, the lyophilized formulation comprises 20-500IU of follicle stimulating hormone and a pharmaceutically acceptable carrier;
the pharmaceutically acceptable carrier comprises 200-400 parts by weight of sucrose, 0.5-2 parts by weight of methionine, 0.1-1.5 parts by weight of polysorbate and 2-15 parts by weight of Na 2 HPO 4 And 1 to 15 parts by weight of NaH 2 PO 4 ·H 2 O。
In another preferred embodiment, the parts by weight are grams (g).
In another preferred embodiment, the lyophilized formulation comprises 20-500IU of follicle stimulating hormone and a pharmaceutically acceptable carrier;
the pharmaceutically acceptable carrier comprises 100-500g sugar alcohol, 0.1-10g amino acid, 0.05-5g surfactant and buffer.
In another preferred embodiment, the phosphate buffer comprises 0.5-35g Na 2 HPO 4 And 0.5-30g NaH 2 PO 4 ·H 2 O。
In another preferred embodiment, the lyophilized formulation comprises:
(i) 10-800IU, preferably 20-600IU, more preferably 20-500IU, more preferably 20-400IU, more preferably 20-300IU, more preferably 20-200IU, more preferably 20-160IU, more preferably 30-150IU, more preferably 40-120IU, more preferably 60-100IU, more preferably 70-90IU, more preferably 75-85IU, and most preferably 77-83IU follicle stimulating hormone;
(ii) 100-500g, preferably 150-450g, preferably 150-400g, more preferably 200-400g, more preferably 250-350g, most preferably 280-320g sugar alcohols (preferably sucrose);
(iii) 0.1-10g, preferably 0.2-8g, preferably 0.4-6g, more preferably 0.5-5g, more preferably 0.5-2g, more preferably 0.5-1.5g, most preferably 0.8-1.2g of an amino acid (preferably methionine);
(iv) 0.05-5g, preferably 0.08-4g, preferably 0.1-3g, more preferably 0.1-2g, more preferably 0.1-1.5g, more preferably 0.1-1g, most preferably 0.3-0.8g of surfactant (preferably polysorbate); and
(v) A buffer.
In another preferred embodiment, the buffering agent is selected from the group consisting of: acetic acid-acetate buffer, phosphate buffer, HEPES buffer, MES buffer, MOPS buffer, or a combination thereof.
In another preferred embodiment, the buffer comprises a phosphate buffer.
In another preferred embodiment, the phosphate buffer comprises Na 2 HPO 4 And NaH 2 PO 4 ·H 2 O。
In another preferred example, the Na 2 HPO 4 0.5-35g, preferably 1-30g, more preferably 2-25g, more preferably 2-20g, more preferably 2-15g, more preferably 3-12g, most preferably 6-12g.
In another preferred embodiment, the NaH 2 PO 4 ·H 2 O is 0.5-30g, preferably 0.8-25g, more preferably 1-20g, more preferably 1-15g, more preferably 1-10g, most preferably 3-8g.
In another preferred embodiment, the phosphate buffer comprises 0.5-35g Na 2 HPO 4 And 0.5-30g NaH 2 PO 4 ·H 2 O。
In another preferred embodiment, the lyophilized formulation comprises:
(i) 10-800IU, preferably 20-600IU, more preferably 20-500IU, more preferably 20-400IU, more preferably 20-300IU, more preferably 20-200IU, more preferably 20-160IU, more preferably 30-150IU, more preferably 40-120IU, more preferably 60-100IU, more preferably 70-90IU, more preferably 75-85IU, and most preferably 77-83IU follicle stimulating hormone;
(ii) 100-500g, preferably 150-450g, preferably 150-400g, more preferably 200-400g, more preferably 250-350g, most preferably 280-320g sugar alcohols (preferably sucrose);
(iii) 0.1-10g, preferably 0.2-8g, preferably 0.4-6g, more preferably 0.5-5g, more preferably 0.5-2g, more preferably 0.5-1.5g, most preferably 0.8-1.2g of an amino acid (preferably methionine);
(iv) 0.05-5g, preferably 0.08-4g, preferably 0.1-3g, more preferably 0.1-2g, more preferably 0.1-1.5g, more preferably 0.1-1g, most preferably 0.3-0.8g of surfactant (preferably polysorbate);
(v) 0.5-35g, preferably 1-30g, more preferably 2-25g, more preferably 2-20g, more preferably 2-15g, more preferably 3-12g, most preferably 6-12g Na 2 HPO 4 The method comprises the steps of carrying out a first treatment on the surface of the And
(vi) 0.5-30g, preferably 0.8-25g, more preferably 1-20g, more preferably 1-15g, more preferably 1-10g, most preferably 3-8g NaH 2 PO 4 ·H 2 O。
In a second aspect of the invention there is provided a method of preparing a lyophilized formulation according to the first aspect of the invention, the method comprising the steps of:
(1) Dissolving follicle stimulating hormone and a pharmaceutically acceptable carrier with water to obtain a solution to be freeze-dried;
(2) And (3) freeze-drying the solution to be freeze-dried obtained in the step (1), thereby obtaining the freeze-dried preparation according to the first aspect of the invention.
In another preferred embodiment, in the step (1), the water is selected from the group consisting of: water for injection, ultrapure water, or a combination thereof.
In another preferred embodiment, in the step (1), the pH of the solution to be lyophilized is 6.5-7.5, preferably 6.8-7.2.
In another preferred embodiment, the step (1) includes the steps of:
(1-1) dissolving a pharmaceutically acceptable carrier in water to form a diluent;
(1-2) adding follicle stimulating hormone to said dilution, optionally with water, to obtain a solution to be lyophilized.
In another preferred embodiment, in step (1-1), the pH of the diluent is from 6.5 to 7.5, preferably from 6.8 to 7.2.
In another preferred embodiment, in the step (1-2), the pH of the solution to be lyophilized is from 6.5 to 7.5, preferably from 6.8 to 7.2.
In another preferred embodiment, in the step (1-1), the temperature is maintained at 2-10 ℃ during the preparation of the diluent.
In another preferred embodiment, in the step (1-2), the temperature is maintained at 2-10 ℃ during the preparation of the solution to be lyophilized.
In another preferred embodiment, the volume of the solution to be lyophilized is 1-10L, preferably 2-8L, preferably 3-7L, more preferably 4-6L, and most preferably 5L.
In another preferred embodiment, the volume ratio of the dilution liquid in the step (1-1) to the solution to be freeze-dried in the step (1-2) is 2-6:3-7, preferably 3-5:4-6.
In another preferred embodiment, in the step (1), the solution to be freeze-dried is sterilized by filtration.
In another preferred embodiment, the filter sterilization is microporous filter membrane filter sterilization.
In another preferred embodiment, the pore size of the microporous filter membrane is 0.1-0.4. Mu.m, preferably 0.22. Mu.m.
In another preferred embodiment, the microporous filter membrane is made of cellulose acetate membrane.
In another preferred embodiment, the microporous filter membrane is a cellulose acetate membrane (Sartobran 150 from Sartor IUs Co.).
In another preferred embodiment, in the step (1-2), the solution to be lyophilized is obtained by stirring.
In another preferred embodiment, the stirring time is 10-20min.
In another preferred embodiment, in the step (1-2), the solution to be lyophilized is placed in a penicillin bottle for lyophilization.
In another preferred example, the penicillin bottle is a penicillin bottle made of neutral borosilicate glass.
In another preferred embodiment, the bottle stopper of the penicillin bottle is a chlorinated butyl rubber stopper.
In another preferred embodiment, the surface area of the solution to be lyophilized is pi r 2 Where pi is the circumference (preferably 3.14) and r is 2-15mm, preferably 3-10mm, more preferably 5-9mm, most preferably 6-8mm.
In another preferred embodiment, in the step (2), the freeze-drying includes the steps of:
step 1, prefreezing: the temperature is-30 ℃ to-60 ℃;
step 2, heating 1: the temperature is between 20 ℃ below zero and 40 ℃ below zero, the time is between 10 and 25 hours, and the vacuum degree is between 4 and 40pa;
step 3, heating 2: the temperature is between-5 ℃ and-25 ℃, the time is between 1 and 4 hours, and the vacuum degree is between 4 and 40pa;
step 4, heating up 3: the temperature is between-10 ℃ and 10 ℃, the time is between 1 and 4 hours, and the vacuum degree is between 4 and 40pa;
step 5, heating 4: the temperature is 15-35 ℃, the time is 1-5h, and the vacuum degree is 4-40pa;
step 6, heating up 5: the temperature is 20-40 ℃, the time is 0.4-3h, and the vacuum degree is 4-40pa;
and (5) finishing freeze-drying.
In another preferred embodiment, in the step 1 prefreezing, the vacuum is at a standard atmospheric pressure (e.g., 1 atm).
In another preferred embodiment, in said step 1 prefreezing, the temperature is from-35 ℃ to-55 ℃, preferably from-40 ℃ to-50 ℃, preferably from-42 ℃ to-47 ℃, most preferably-45 ℃.
In another preferred embodiment, in said step 1 pre-freezing, the time is 4-16h, preferably 4-12h, more preferably 4-8h, more preferably 5-8h, most preferably 5h.
In another preferred embodiment, in said step 2, the temperature is increased by 1, the temperature is from-20 ℃ to-35 ℃, preferably from-25 ℃ to-35 ℃, more preferably from-27 ℃ to-33 ℃, most preferably from-30 ℃.
In another preferred embodiment, in said step 2, temperature increase 1, the time is 10-20h, preferably 12-18h, more preferably 14-16h, most preferably 15h.
In another preferred embodiment, in said step 2 elevated temperature 1, the vacuum is 4-40pa, preferably 3-30pa, more preferably 5-25pa, more preferably 8-20pa, more preferably 8-16pa, most preferably 9-13pa.
In another preferred embodiment, in said 3 rd step of increasing the temperature 2, the temperature is from-10 ℃ to-20 ℃, preferably from-13 ℃ to-17 ℃, most preferably-15 ℃.
In another preferred embodiment, in said step 3, temperature increase 2, the time is 1-3 hours, preferably 1.5-2.5 hours, more preferably 1.7-2.3 hours, most preferably 2 hours.
In another preferred embodiment, in said 3 rd step of heating 2, the vacuum is 4-40pa, preferably 3-30pa, more preferably 5-25pa, more preferably 8-20pa, more preferably 8-16pa, most preferably 9-13pa.
In another preferred embodiment, in said 4 th step of increasing the temperature 3, the temperature is from-5 ℃ to 5 ℃, preferably from-3 ℃ to 3 ℃, more preferably from-1 ℃ to 1 ℃, most preferably 0 ℃.
In another preferred embodiment, in said 4 th step of heating 3, the time is 1 to 3 hours, preferably 1.5 to 2.5 hours, more preferably 1.7 to 2.3 hours, most preferably 2 hours.
In another preferred embodiment, in said 4 th step of heating up 3, the vacuum is 4 to 40pa, preferably 3 to 30pa, more preferably 5 to 25pa, more preferably 8 to 20pa, more preferably 8 to 16pa, most preferably 9 to 13pa.
In another preferred embodiment, in said 5 th step of increasing the temperature 4, the temperature is 20 ℃ to 30 ℃, preferably 23 ℃ to 27 ℃, more preferably 25 ℃.
In another preferred embodiment, in said step 5 warming 4, the time is 1-4 hours, preferably 2-3 hours, more preferably 2.3-2.7 hours, most preferably 2.5 hours.
In another preferred embodiment, in said step 5 warming 4, the vacuum is 4-40pa, preferably 3-30pa, more preferably 5-25pa, more preferably 8-20pa, more preferably 8-16pa, most preferably 9-13pa.
In another preferred embodiment, in said step 6 elevated temperature 5, the temperature is 20 ℃ to 35 ℃, preferably 25 ℃ to 35 ℃, more preferably 26 ℃ to 30 ℃, most preferably 28 ℃.
In another preferred embodiment, in said step 6, the temperature is raised 5 for a period of time ranging from 0.5 to 2 hours, preferably from 0.5 to 1.5 hours, more preferably from 0.8 to 1.2 hours, most preferably 1 hour.
In another preferred embodiment, in said step 6 heating 5, the vacuum is 4-40pa, preferably 3-30pa, more preferably 5-25pa, more preferably 8-20pa, more preferably 8-16pa, most preferably 9-13pa.
In another preferred embodiment, in the step (2), the freeze-drying includes the steps of:
step 1, prefreezing: the temperature is-40 ℃ to-50 ℃;
step 2, heating 1: the temperature is between 25 ℃ below zero and 35 ℃ below zero, the time is between 12 and 18 hours, and the vacuum degree is between 8 and 16pa;
step 3, heating 2: the temperature is between minus 10 ℃ and minus 20 ℃, the time is between 1.5 and 2.5 hours, and the vacuum degree is between 8 and 16pa;
step 4, heating up 3: the temperature is between-5 and 5 ℃, the time is between 1.5 and 2.5 hours, and the vacuum degree is between 8 and 16pa;
step 5, heating 4: the temperature is 20-30 ℃, the time is 2-3h, and the vacuum degree is 8-16pa;
step 6, heating up 5: the temperature is 25-35 ℃, the time is 0.5-1.5h, and the vacuum degree is 8-16pa;
and (5) finishing freeze-drying.
In another preferred embodiment, the freeze-drying comprises the steps of:
step 1, prefreezing: the temperature of the plate layer is-45 ℃ for 2-3 hours, and the vacuum degree is standard atmospheric pressure;
step 2, heating 1: the temperature is-30 ℃, the time is 15 hours, and the vacuum degree is 11.5pa;
Step 3, heating 2: the temperature is 15 ℃ below zero for 2 hours, and the vacuum degree is 11.5pa;
step 4, heating up 3: the temperature is 0 ℃, the time is 2 hours, and the vacuum degree is 11.5pa;
step 5, heating 4: the temperature is 25 ℃, the time is 2.5 hours, and the vacuum degree is 11.5pa;
step 6, heating up 5: the temperature is 28 ℃, the time is 1h, and the vacuum degree is 11.5pa;
and (5) finishing freeze-drying.
In a third aspect of the invention there is provided a rehydration of a lyophilized preparation of follicle stimulating hormone, the lyophilized preparation of follicle stimulating hormone being as described in the first aspect of the invention.
In a fourth aspect of the invention, a method for preparing a rehydration of a lyophilized preparation of follicle stimulating hormone according to the third aspect of the invention, said method comprising the steps of: rehydrating a lyophilized preparation of follicle stimulating hormone according to the first aspect of the invention with water, preferably at 37 ℃, thereby preparing said rehydration.
In a fifth aspect of the invention there is provided the use of a lyophilized formulation according to the first aspect of the invention or a rehydration according to the third aspect of the invention for the manufacture of a medicament for stimulating follicular development.
In another preferred embodiment, the follicle is a human follicle, preferably a human multiple follicle.
In a sixth aspect of the invention, there is provided a kit comprising:
(i) A container comprising a lyophilized formulation according to the first aspect of the invention or a rehydration according to the third aspect of the invention;
(ii) Optionally instructions for use.
In another preferred embodiment, the container is a penicillin bottle made of neutral borosilicate glass.
In another preferred embodiment, the stopper of the penicillin bottle is a chlorinated butyl rubber stopper.
In another preferred embodiment, the container is a penicillin bottle made of neutral borosilicate glass sealed with a chlorinated butyl rubber stopper.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 is a 40X histomorphometric chart (TM) of follicle stimulating hormone lyophilized powder for injection in a batch 140801 of example 1 of the present invention for rats.
FIG. 2 is a graph showing the 36 month purity (SEC-HPLC) test of recombinant follicle-stimulating hormone lyophilized powder for injection in a batch 140801 in example 1 of the present invention.
Detailed Description
The inventors have conducted extensive and intensive studies and have unexpectedly developed a lyophilized preparation of follicle stimulating hormone, which comprises a follicle stimulating hormone and a pharmaceutically acceptable carrier; the pharmaceutically acceptable carrier comprises 100-500 parts by weight of sugar alcohol, 0.1-10 parts by weight of amino acid, 0.05-5 parts by weight of surfactant and buffer. The follicle stimulating hormone freeze-dried preparation provided by the invention can ensure that the follicle stimulating hormone is stable in quality, high in biological activity and purity, and has excellent acceleration and long-term stability. The present invention has been completed on the basis of this finding.
Terminology
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, the terms "comprising," "including," and "containing" are used interchangeably, and include not only closed-form definitions, but also semi-closed-form and open-form definitions. In other words, the term includes "consisting of … …", "consisting essentially of … …".
The term "MOPS buffer" is used interchangeably with "3- (N-morpholino) propanesulfonic acid buffer".
The term "MES buffer" is used interchangeably with "2- (N-morpholino) ethanesulfonic acid buffer".
In the present invention, the term "HEPES buffer" is used interchangeably with "4- (2-hydroxyethyl) -1-piperazine ethane sulfonic acid buffer".
The term "rehydration" refers to the product of a lyophilized formulation after reconstitution with a liquid (e.g., water for injection, physiological saline for injection). Clinically, it is often necessary to reconstitute a lyophilized formulation with water for injection or physiological saline for injection to form a rehydration, and then administer the rehydration into a human.
As used herein, the terms "parts by weight" and "parts by weight" are used interchangeably and the parts by weight may be any fixed weight in milligrams, grams, or kilograms (e.g., 1mg, 1g, 2g, or 1kg, etc.). For example, a composition comprising 1 part by weight of component a and 9 parts by weight of component b may be a composition comprising 1 gram of component a+9 gram of component b, or 10 grams of component a+90 gram of component b, etc. In the composition, the percentage content of a certain component= (the parts by weight of the component/the sum of the parts by weight of all components) ×100%, and therefore, in the composition composed of 1 part by weight of component a and 9 parts by weight of component b, the content of component a is 10%, and the content of component b is 90%.
As used herein, "IU" is the international unit (International Unit). Follicle stimulating hormone is difficult to detect its quality specification by physicochemical methods, the potency is detected by biological experimental methods and comparison with standard substances, by which the minimum potency Unit with a certain biological potency is called "Unit" (U, unit), and the standard Unit defined by international negotiation is called "international Unit" (IU, international Unit).
Follicle stimulating hormone freeze-dried preparation
The invention provides a freeze-dried preparation, which comprises follicle stimulating hormone and a pharmaceutically acceptable carrier;
the pharmaceutically acceptable carrier comprises 100-500 parts by weight of sugar alcohol, 0.1-10 parts by weight of amino acid, 0.05-5 parts by weight of surfactant and buffer.
In a preferred embodiment of the present invention, the sum of the contents of all components of the lyophilized formulation is 100wt%.
In another preferred embodiment, the lyophilized preparation is a lyophilized preparation for injection, such as a lyophilized powder for injection.
In another preferred embodiment, the lyophilized formulation has a pH of 6.5-7.5, preferably 6.8-7.2, as measured after reconstitution with water.
In another preferred embodiment, the water is selected from the group consisting of: water for injection, ultrapure water, or a combination thereof.
Typically, the lyophilized formulation comprises 20-500IU of follicle stimulating hormone and a pharmaceutically acceptable carrier;
the pharmaceutically acceptable carrier comprises 100-500 parts by weight of sugar alcohol, 0.1-10 parts by weight of amino acid, 0.05-5 parts by weight of surfactant and 0.5-35 parts by weight of Na 2 HPO 4 And 0.5 to 30 parts by weight of NaH 2 PO 4 ·H 2 O。
In a preferred embodiment of the invention, the ratio of said follicle stimulating hormone to said sugar alcohol (IU: g) is 10-800:100-500, preferably 20-500:150-450, more preferably 20-400:150-400, more preferably 60-100:200-400, more preferably 75-85:250-350, optimally 77-83:280-320.
Follicle stimulating hormone
Follicle stimulating hormone, also known as follicle stimulating hormone (Follicle Stimulating Hormone, FSH), FSH is a heterodimeric glycoprotein consisting of non-covalently bound alpha and beta subunits. In the present invention, the follicle stimulating hormone is not particularly limited, and the follicle stimulating hormone may be a human follicle stimulating hormone, and may be a natural follicle stimulating hormone or a recombinant follicle stimulating hormone.
A preferred follicle stimulating hormone is recombinant human follicle stimulating hormone disclosed in Chinese patent CN 110305903A, wherein the amino acid sequence of alpha subunit gene of FSH is shown as SEQ ID NO. 1, and the amino acid sequence of beta subunit gene of FSH is shown as SEQ ID NO. 2.
MDYYRKYAAIFLVTLSVFLHVLHSAPDVQDCPECTLQENPFFSQPGAPILQ CMGCCFSRAYPTPLRSKKTMLVQKNVTSESTCCVAKSYNRVTVMGGFKVENH TACHCSTCYYHKS(SEQ ID NO:1)。
MKTLQFFFLFCCWKAICCNSCELTNITIAIEKEECRFCISINTTWCAGYCYTR DLVYKDPARPKIQKTCTFKELVYETVRVPGCAHHADSLYTYPVATQCHCGKCD SDSTDCTVRGLGPSYCSFGEMKE*(SEQ ID NO:2)。
In a preferred embodiment of the present invention, in the lyophilized formulation, the follicle stimulating hormone is 10-800IU, preferably 20-600IU, more preferably 20-500IU, more preferably 20-400IU, more preferably 20-300IU, more preferably 20-200IU, more preferably 20-160IU, more preferably 30-150IU, more preferably 40-120IU, more preferably 60-100IU, more preferably 70-90IU, more preferably 75-85IU, and most preferably 77-83IU.
Sugar alcohols
In the lyophilized formulation of the present invention, the sugar alcohols may include (but are not limited to): saccharides, sugar alcohols, or combinations thereof.
In another preferred embodiment, the saccharides include (but are not limited to): monosaccharides, disaccharides, polysaccharides, or combinations thereof.
In another preferred example, the monosaccharide is a monosaccharide having 3 to 6 carbon atoms in the molecular structure.
Preferably, the monosaccharide is a monosaccharide containing 5 to 6 carbon atoms in the molecular structure.
Preferably, the monosaccharides include (but are not limited to): five-carbon sugars, six-carbon sugars, or combinations thereof.
Representatively, the monosaccharides include (but are not limited to): arabinose, ribose, xylose, lyxose, glucose, mannose, fructose, galactose, or a combination thereof.
Representatively, the disaccharides include (but are not limited to): maltose, lactose, sucrose, trehalose, or a combination thereof.
Typically, the sugar alcohols include (but are not limited to): mannitol, xylitol, erythritol, sorbitol, isomalt, maltitol, lactitol, or a combination thereof.
In a preferred embodiment, the sugar alcohol comprises sucrose.
In another preferred embodiment of the present invention, in the lyophilized preparation, the sugar alcohol is 150 to 450 parts by weight, preferably 150 to 400 parts by weight, more preferably 200 to 400 parts by weight, still more preferably 250 to 350 parts by weight, and most preferably 280 to 320 parts by weight.
Amino acids
In the lyophilized preparation of the present invention, the sugar alcohol is preferably a natural amino acid.
In another preferred embodiment, the amino acid is an L-amino acid.
In another preferred embodiment, the L-amino acid includes (but is not limited to): glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, aspartic acid, glutamic acid, asparagine, glutamine, lysine, arginine, phenylalanine, tyrosine, tryptophan, histidine, proline, or a combination thereof.
In another preferred embodiment, the amino acid comprises methionine.
In another preferred embodiment of the present invention, the amino acid is present in the lyophilized formulation in an amount of 0.2 to 8 parts by weight, preferably 0.4 to 6 parts by weight, more preferably 0.5 to 5 parts by weight, still more preferably 0.5 to 2 parts by weight, still more preferably 0.5 to 1.5 parts by weight, and most preferably 0.8 to 1.2 parts by weight.
Surface active agent
In the lyophilized formulation of the present invention, the surfactant may include (but is not limited to): nonionic surfactants, or combinations thereof.
In another preferred embodiment, the nonionic surfactant includes (but is not limited to): cationic surfactants, anionic surfactants, or combinations thereof.
In another preferred embodiment, the nonionic surfactant includes (but is not limited to): polysorbate (tween), poloxamer, sorbitan fatty acid esters (span), or combinations thereof.
Representatively, the polysorbate (tween) includes (but is not limited to): polysorbate 20 (tween-20), polysorbate 40 (tween-40), polysorbate 60 (tween-60), polysorbate 80 (tween-80), or a combination thereof.
Typically, the polysorbate (tween) is polysorbate 20 (tween-20).
Representatively, the sorbitan fatty acid esters (span) include (but are not limited to): sorbitan fatty acid ester-20 (span-20), sorbitan fatty acid ester 80 (span-80), or a combination thereof.
Representatively, the poloxamers include (but are not limited to): poloxamer-188, poloxamer-407, or combinations thereof.
In another preferred embodiment, the surfactant is polysorbate (tween).
In another preferred embodiment of the present invention, the surfactant is 0.08 to 4 parts by weight, preferably 0.1 to 3 parts by weight, more preferably 0.1 to 2 parts by weight, still more preferably 0.1 to 1.5 parts by weight, still more preferably 0.1 to 1 part by weight, and most preferably 0.3 to 0.8 part by weight in the lyophilized preparation.
Buffering agents
In the lyophilized formulation of the present invention, the buffering agent may include (but is not limited to): acetic acid-acetate buffer, phosphate buffer, HEPES buffer, MES buffer, MOPS buffer, or a combination thereof.
Typically, the buffer comprises a phosphate buffer.
Typically, the phosphate buffer comprises Na 2 HPO 4 And NaH 2 PO 4 ·H 2 O。
In another preferred example, the Na 2 HPO 4 0.5 to 35 parts by weight, preferably 1 to 30 parts by weight, more preferably 2 to 25 parts by weight, still more preferably 2 to 20 parts by weight, more preferably 2 to 15 parts by weight, more preferably 3 to 12 parts by weight, most preferably 6 to 12 parts by weight.
In another preferred embodiment, the NaH 2 PO 4 ·H 2 O is 0.5 to 30 parts by weight, preferably 0.8 to 25 parts by weight, more preferably 1 to 20 parts by weight, still more preferably 1 to 15 parts by weight, more preferably 1 to 10 parts by weight, most preferably 3 to 8 parts by weight.
Preferably, the phosphate buffer comprises 0.5 to 35 parts by weight of Na 2 HPO 4 And 0.5 to 30 parts by weight of NaH 2 PO 4 ·H 2 O。
In another preferred embodiment, the buffering agent has a pH of 6.5 to 7.5, preferably 6.8 to 7.2, as measured after dissolution with water.
One preferred lyophilized formulation of the present invention comprises 20-500IU follicle stimulating hormone and a pharmaceutically acceptable carrier;
the pharmaceutically acceptable carrier comprises 200-400 parts by weight of sugar alcohol, 0.5-2 parts by weight of amino acid, 0.1-1.5 parts by weight of surfactant and buffer.
In another preferred embodiment, the buffering agent comprises 2-15 parts by weightNa 2 HPO 4 And 1 to 15 parts by weight of NaH 2 PO 4 ·H 2 O。
In another preferred embodiment, the lyophilized formulation comprises 20-500IU of follicle stimulating hormone and a pharmaceutically acceptable carrier;
the pharmaceutically acceptable carrier comprises 200-400 parts by weight of sugar alcohol, 0.5-2 parts by weight of amino acid, 0.1-1.5 parts by weight of surfactant and 2-15 parts by weight of Na 2 HPO 4 And 1 to 15 parts by weight of NaH 2 PO 4 ·H 2 O。
More preferably, the lyophilized formulation comprises 20-500IU of follicle stimulating hormone and a pharmaceutically acceptable carrier;
the pharmaceutically acceptable carrier comprises 200-400 parts by weight of sucrose, 0.5-2 parts by weight of methionine, 0.1-1.5 parts by weight of polysorbate and buffer.
In another preferred embodiment, the buffer comprises 2-15 parts by weight Na 2 HPO 4 And 1 to 15 parts by weight of NaH 2 PO 4 ·H 2 O。
In another preferred embodiment, the lyophilized formulation comprises 20-500IU of follicle stimulating hormone and a pharmaceutically acceptable carrier;
the pharmaceutically acceptable carrier comprises 200-400 parts by weight of sucrose, 0.5-2 parts by weight of methionine, 0.1-1.5 parts by weight of polysorbate and 2-15 parts by weight of Na 2 HPO 4 And 1 to 15 parts by weight of NaH 2 PO 4 ·H 2 O。
In another preferred embodiment of the present invention, the parts by weight are g.
In another preferred embodiment, the lyophilized formulation comprises 20-500IU of follicle stimulating hormone and a pharmaceutically acceptable carrier;
the pharmaceutically acceptable carrier comprises 100-500g sugar alcohol, 0.1-10g amino acid, 0.05-5g surfactant and buffer.
In another preferred embodiment, the phosphate buffer comprises 0.5-35g Na 2 HPO 4 And 0.5-30g NaH 2 PO 4 ·H 2 O。
Typically, the lyophilized formulation comprises:
(i) 10-800IU, preferably 20-600IU, more preferably 20-500IU, more preferably 20-400IU, more preferably 20-300IU, more preferably 20-200IU, more preferably 20-160IU, more preferably 30-150IU, more preferably 40-120IU, more preferably 60-100IU, more preferably 70-90IU, more preferably 75-85IU, and most preferably 77-83IU follicle stimulating hormone;
(ii) 100-500g, preferably 150-450g, preferably 150-400g, more preferably 200-400g, more preferably 250-350g, most preferably 280-320g sugar alcohols (preferably sucrose);
(iii) 0.1-10g, preferably 0.2-8g, preferably 0.4-6g, more preferably 0.5-5g, more preferably 0.5-2g, more preferably 0.5-1.5g, most preferably 0.8-1.2g of an amino acid (preferably methionine);
(iv) 0.05-5g, preferably 0.08-4g, preferably 0.1-3g, more preferably 0.1-2g, more preferably 0.1-1.5g, more preferably 0.1-1g, most preferably 0.3-0.8g of surfactant (preferably polysorbate); and
(v) A buffer.
In another preferred embodiment, the buffering agent includes (but is not limited to): acetic acid-acetate buffer, phosphate buffer, HEPES buffer, MES buffer, MOPS buffer, or a combination thereof.
In another preferred embodiment, the buffer comprises a phosphate buffer.
In another preferred embodiment, the phosphate buffer comprises Na 2 HPO 4 And NaH 2 PO 4 ·H 2 O。
In another preferred example, the Na 2 HPO 4 0.5-35g, preferably 1-30g, more preferably 2-25g, more preferably 2-20g, more preferably 2-15g, more preferably 3-12g, most preferably 6-12g.
In another preferred embodiment, the NaH 2 PO 4 ·H 2 O is 0.5-30g, preferably 0.8-25g, more preferably 1-20g, more preferably 1-15g, more preferably 1-10g, most preferably 3-8g.
In another preferred embodiment, the phosphate buffer comprises 0.5-35g Na 2 HPO 4 And 0.5-30g NaH 2 PO 4 ·H 2 O。
In the present invention, a buffer is formulated by adding a liquid (e.g., water) to the buffer.
Typically, the lyophilized formulation comprises:
(i) 10-800IU, preferably 20-600IU, more preferably 20-500IU, more preferably 20-400IU, more preferably 20-300IU, more preferably 20-200IU, more preferably 20-160IU, more preferably 30-150IU, more preferably 40-120IU, more preferably 60-100IU, more preferably 70-90IU, more preferably 75-85IU, and most preferably 77-83IU follicle stimulating hormone;
(ii) 100-500g, preferably 150-450g, preferably 150-400g, more preferably 200-400g, more preferably 250-350g, most preferably 280-320g sugar alcohols (preferably sucrose);
(iii) 0.1-10g, preferably 0.2-8g, preferably 0.4-6g, more preferably 0.5-5g, more preferably 0.5-2g, more preferably 0.5-1.5g, most preferably 0.8-1.2g of an amino acid (preferably methionine);
(iv) 0.05-5g, preferably 0.08-4g, preferably 0.1-3g, more preferably 0.1-2g, more preferably 0.1-1.5g, more preferably 0.1-1g, most preferably 0.3-0.8g of surfactant (preferably polysorbate);
(v) 0.5-35g, preferably 1-30g, more preferably 2-25g, more preferably 2-20g, more preferably 2-15g, more preferably 3-12g, most preferably 6-12g Na 2 HPO 4 The method comprises the steps of carrying out a first treatment on the surface of the And
(vi) 0.5-30g, preferably 0.8-25g, more preferably 1-20g, more preferably 1-15g, more preferably 1-10g, most preferably 3-8g NaH 2 PO 4 ·H 2 O。
Preparation method
The invention also provides a method for preparing the freeze-dried preparation, which comprises the following steps:
(1) Dissolving follicle stimulating hormone and a pharmaceutically acceptable carrier with water to obtain a solution to be freeze-dried;
(2) And (3) freeze-drying the solution to be freeze-dried obtained in the step (1) to obtain the freeze-dried preparation.
In a preferred embodiment, in the step (1), the pH of the solution to be lyophilized is from 6.5 to 7.5, preferably from 6.8 to 7.2.
In another preferred embodiment, the step (1) includes the steps of:
(1-1) dissolving a pharmaceutically acceptable carrier in water to form a diluent;
(1-2) adding follicle stimulating hormone to said dilution, optionally with water, to obtain a solution to be lyophilized.
In another preferred embodiment, in step (1-1), the pH of the diluent is from 6.5 to 7.5, preferably from 6.8 to 7.2.
In another preferred embodiment, in the step (1-2), the pH of the solution to be lyophilized is from 6.5 to 7.5, preferably from 6.8 to 7.2.
In another preferred embodiment, in the step (1-1), the temperature is maintained at 2-10 ℃ during the preparation of the diluent.
In another preferred embodiment, in the step (1-2), the temperature is maintained at 2-10 ℃ during the preparation of the solution to be lyophilized.
In another preferred embodiment, the volume of the solution to be lyophilized is 1-10L, preferably 2-8L, preferably 3-7L, more preferably 4-6L, and most preferably 5L.
In another preferred embodiment, the volume ratio of the dilution liquid in the step (1-1) to the solution to be freeze-dried in the step (1-2) is 2-6:3-7, preferably 3-5:4-6.
In another preferred embodiment, in the step (1-2), the solution to be lyophilized is placed in a penicillin bottle for lyophilization.
In another preferred example, the penicillin bottle is a penicillin bottle made of neutral borosilicate glass.
In another preferred embodiment, the bottle stopper of the penicillin bottle is a chlorinated butyl rubber stopper.
In another preferred embodiment, the surface area of the solution to be lyophilized is pi r 2 Where pi is the circumference (preferably 3.14) and r is 2-15mm, preferably 3-10mm, more preferably 5-9mm, most preferably 6-8mm.
In another preferred embodiment, in the step (2), the freeze-drying includes the steps of:
step 1, prefreezing: the temperature is-30 ℃ to-60 ℃;
step 2, heating 1: the temperature is between 20 ℃ below zero and 40 ℃ below zero, the time is between 10 and 25 hours, and the vacuum degree is between 4 and 40pa;
step 3, heating 2: the temperature is between-5 ℃ and-25 ℃, the time is between 1 and 4 hours, and the vacuum degree is between 4 and 40pa;
step 4, heating up 3: the temperature is between-10 ℃ and 10 ℃, the time is between 1 and 4 hours, and the vacuum degree is between 4 and 40pa;
Step 5, heating 4: the temperature is 15-35 ℃, the time is 1-5h, and the vacuum degree is 4-40pa;
step 6, heating up 5: the temperature is 20-40 ℃, the time is 0.4-3h, and the vacuum degree is 4-40pa;
and (5) finishing freeze-drying.
In another preferred embodiment, in the step 1 prefreezing, the vacuum is at a standard atmospheric pressure (e.g., 1 atm).
In another preferred embodiment, in said step 1 prefreezing, the temperature is from-35 ℃ to-55 ℃, preferably from-40 ℃ to-50 ℃, preferably from-42 ℃ to-47 ℃, most preferably-45 ℃.
In another preferred embodiment, in said step 1 pre-freezing, the time is 4-16h, preferably 4-12h, more preferably 4-8h, more preferably 5-8h, most preferably 5h.
In another preferred embodiment, in said step 2, the temperature is increased by 1, the temperature is from-20 ℃ to-35 ℃, preferably from-25 ℃ to-35 ℃, more preferably from-27 ℃ to-33 ℃, most preferably from-30 ℃.
In another preferred embodiment, in said step 2, temperature increase 1, the time is 10-20h, preferably 12-18h, more preferably 14-16h, most preferably 15h.
In another preferred embodiment, in said step 2 elevated temperature 1, the vacuum is 4-40pa, preferably 3-30pa, more preferably 5-25pa, more preferably 8-20pa, more preferably 8-16pa, most preferably 9-13pa.
In another preferred embodiment, in said 3 rd step of increasing the temperature 2, the temperature is from-10 ℃ to-20 ℃, preferably from-13 ℃ to-17 ℃, most preferably-15 ℃.
In another preferred embodiment, in said step 3, temperature increase 2, the time is 1-3 hours, preferably 1.5-2.5 hours, more preferably 1.7-2.3 hours, most preferably 2 hours.
In another preferred embodiment, in said 3 rd step of heating 2, the vacuum is 4-40pa, preferably 3-30pa, more preferably 5-25pa, more preferably 8-20pa, more preferably 8-16pa, most preferably 9-13pa.
In another preferred embodiment, in said 4 th step of increasing the temperature 3, the temperature is from-5 ℃ to 5 ℃, preferably from-3 ℃ to 3 ℃, more preferably from-1 ℃ to 1 ℃, most preferably 0 ℃.
In another preferred embodiment, in said 4 th step of heating 3, the time is 1 to 3 hours, preferably 1.5 to 2.5 hours, more preferably 1.7 to 2.3 hours, most preferably 2 hours.
In another preferred embodiment, in said 4 th step of heating up 3, the vacuum is 4 to 40pa, preferably 3 to 30pa, more preferably 5 to 25pa, more preferably 8 to 20pa, more preferably 8 to 16pa, most preferably 9 to 13pa.
In another preferred embodiment, in said 5 th step of increasing the temperature 4, the temperature is 20 ℃ to 30 ℃, preferably 23 ℃ to 27 ℃, more preferably 25 ℃.
In another preferred embodiment, in said step 5 warming 4, the time is 1-4 hours, preferably 2-3 hours, more preferably 2.3-2.7 hours, most preferably 2.5 hours.
In another preferred embodiment, in said step 5 warming 4, the vacuum is 4-40pa, preferably 3-30pa, more preferably 5-25pa, more preferably 8-20pa, more preferably 8-16pa, most preferably 9-13pa.
In another preferred embodiment, in said step 6 elevated temperature 5, the temperature is 20 ℃ to 35 ℃, preferably 25 ℃ to 35 ℃, more preferably 26 ℃ to 30 ℃, most preferably 28 ℃.
In another preferred embodiment, in said step 6, the temperature is raised 5 for a period of time ranging from 0.5 to 2 hours, preferably from 0.5 to 1.5 hours, more preferably from 0.8 to 1.2 hours, most preferably 1 hour.
In another preferred embodiment, in said step 6 heating 5, the vacuum is 4-40pa, preferably 3-30pa, more preferably 5-25pa, more preferably 8-20pa, more preferably 8-16pa, most preferably 9-13pa.
In another preferred embodiment, in the step (2), the freeze-drying includes the steps of:
step 1, prefreezing: the temperature is-40 ℃ to-50 ℃;
step 2, heating 1: the temperature is between 25 ℃ below zero and 35 ℃ below zero, the time is between 12 and 18 hours, and the vacuum degree is between 8 and 16pa;
step 3, heating 2: the temperature is between minus 10 ℃ and minus 20 ℃, the time is between 1.5 and 2.5 hours, and the vacuum degree is between 8 and 16pa;
step 4, heating up 3: the temperature is between-5 and 5 ℃, the time is between 1.5 and 2.5 hours, and the vacuum degree is between 8 and 16pa;
step 5, heating 4: the temperature is 20-30 ℃, the time is 2-3h, and the vacuum degree is 8-16pa;
Step 6, heating up 5: the temperature is 25-35 ℃, the time is 0.5-1.5h, and the vacuum degree is 8-16pa;
and (5) finishing freeze-drying.
In another preferred embodiment, the freeze-drying comprises the steps of:
step 1, prefreezing: the temperature of the plate layer is-45 ℃ and the time is 5 2-3 hours, and the vacuum degree is standard atmospheric pressure;
step 2, heating 1: the temperature is-30 ℃, the time is 15 hours, and the vacuum degree is 11.5pa;
step 3, heating 2: the temperature is 15 ℃ below zero for 2 hours, and the vacuum degree is 11.5pa;
step 4, heating up 3: the temperature is 0 ℃, the time is 2 hours, and the vacuum degree is 11.5pa;
step 5, heating 4: the temperature is 25 ℃, the time is 2.5 hours, and the vacuum degree is 11.5pa;
step 6, heating up 5: the temperature is 28 ℃, the time is 1h, and the vacuum degree is 11.5pa;
and (5) finishing freeze-drying.
Rehydration of lyophilized formulations
The present invention provides a rehydration of a lyophilized preparation of follicle stimulating hormone as described above.
In another preferred embodiment, the rehydration of the lyophilized formulation is prepared by a process comprising the steps of: the rehydration is prepared by rehydrating a lyophilized preparation of follicle stimulating hormone according to the invention with water, preferably at 37 ℃.
Use of the same
The invention also provides application of the freeze-dried preparation or the rehydration of the follicle stimulating hormone freeze-dried preparation, which is used for preparing medicines for stimulating the development of multiple follicles of a human.
Kit for detecting a substance in a sample
The invention also provides a kit comprising:
(i) A container comprising a lyophilized formulation according to the invention or a rehydration of a lyophilized formulation of follicle stimulating hormone according to the invention;
(ii) Optionally instructions for use.
In another preferred embodiment, the container is a penicillin bottle made of neutral borosilicate glass.
In another preferred embodiment, the stopper of the penicillin bottle is a chlorinated butyl rubber stopper.
In another preferred embodiment, the container is a penicillin bottle made of neutral borosilicate glass sealed with a chlorinated butyl rubber stopper.
The main advantages of the invention include:
1. the follicle stimulating hormone freeze-dried preparation can ensure that the follicle stimulating hormone quality is stable, has excellent acceleration and long-term stability, and can be placed for 36 months under the storage condition of 2-8 ℃.
2. The follicle-stimulating hormone freeze-dried preparation has short re-dissolution time and is convenient for quick administration.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.
Example 1
Preparation of recombinant follicle-stimulating hormone freeze-dried powder injection for injection
1. Prescription of prescription
Example 1 preparation of 3 batches of recombinant follicle-stimulating hormone lyophilized powder for injection, wherein the prescription (single prescription) information of 3 batches of recombinant follicle-stimulating hormone lyophilized powder for injection is shown in table 1 below:
table 1 3 prescription (single prescription) of recombinant follicle-stimulating hormone freeze-dried powder injection for injection
2. Preparation method
The recombinant follicle-stimulating hormone freeze-dried powder injection (10000 bottles in batch) for injection is prepared according to the general rule of the freeze-dried powder injection process, and is specifically as follows:
2.1 sucrose, methionine, polysorbate, na in the amounts prescribed in Table 1 2 HPO 4 And NaH 2 PO 4 ·H 2 O is added into 4L of injection water for dissolution, the mixture is mixed to obtain a diluent, the pH of the diluent is regulated to 7.0 by using 1mol/L NaOH solution or 1mol/L HCl, and the temperature is kept at 2-10 ℃ in the process of preparing the diluent.
2.2 adding recombinant follicle stimulating hormone (rhFSH) (amino acid sequence of FSH alpha subunit gene is shown as SEQ ID NO:1, amino acid sequence of FSH beta subunit gene is shown as SEQ ID NO: 2) containing the prescribed amount in Table 1 into the diluent prepared in the above 2.1, fixing volume to 5L with water for injection, adjusting the diluent to pH 7.0 with 1mol/L NaOH solution or 1mol/L HCl, mixing, stirring for 15 min, and then filtering and sterilizing with 0.22 μm cellulose acetate film (Sartobran 150 of Sartor IUs Co.) to obtain solution to be lyophilized, wherein the temperature is kept at 2-10deg.C during the preparation of the solution to be lyophilized.
Before 0.22 mu m cellulose acetate membrane filtration, the integrity test of the sterilizing filter is carried out by adopting a bubble point test method, and the integrity test is carried out before and after the filtration sample. The testing method comprises the following steps: the filter is fully soaked by ultrapure water (room temperature), the exhaust hole is closed, the liquid inlet end is connected with the pressure gauge and sterile compressed air by a high-strength pipeline, the air inlet valve is gradually opened to introduce air, the change of the number of the pressure gauge is observed, and after bubbles at the outlet of the rear part of the filter are uniform, the value of the pressure gauge is read, namely the bubble point. Filter integrity is satisfactory when the bubble point of the filter is not below the minimum bubble point specified in the product specification. If the filter is not qualified, the newly assembled filter is replaced and the integrity check is carried out, and the filtering can be started after the integrity check of the filter is qualified. After filtration, the filtration system should be qualified for integrity checking under the bubble point test method. And if the failure is treated according to the deviation treatment management system. The ingredients are made of stainless steel or disposable sterile liquid storage bags as much as possible. The time from preparation to filtration is controlled within 10 hours.
2.3 sub-packaging the solution to be freeze-dried prepared in the step 2.2 into a penicillin bottle made of neutral borosilicate glass in a sterile environment (the filling amount is 0.5 mL/bottle, the liquid level of the solution to be freeze-dried in the penicillin bottle is circular with the radius of 7 mm), and sealing the half-pressure chlorinated butyl rubber plug (W1T 13-A2-S).
2.4 placing the penicillin bottle prepared in the step 2.2 and filled with the solution to be freeze-dried on a plate layer of a freeze dryer for freeze drying, wherein the freeze drying parameters are as follows:
step 1, prefreezing: the temperature of the plate layer is-45 ℃ for 5 hours, and the vacuum degree is standard atmospheric pressure of 1atm;
step 2, heating 1: the temperature of the plate layer is minus 30 ℃ for 15 hours, and the vacuum degree is 11.5pa;
step 3, heating 2: the temperature of the plate layer is 15 ℃ below zero, the time is 2 hours, and the vacuum degree is 11.5pa;
step 4, heating up 3: the temperature of the plate layer is 0 ℃, the time is 2 hours, and the vacuum degree is 11.5pa;
step 5, heating 4: the temperature of the plate layer is 25 ℃, the time is 2.5 hours, and the vacuum degree is 11.5pa;
step 6, heating up 5: the temperature of the plate layer is 28 ℃, the time is 1h, and the vacuum degree is 11.5pa;
and (5) finishing freeze-drying.
2.5, after freeze drying, compacting a chlorinated butyl rubber plug (W1T 13-A2-S) in a sterile environment, sealing a penicillin bottle, rolling a cover by an aluminum-plastic combined cover to obtain a recombinant follicle stimulating hormone freeze-dried powder injection product for injection, and after light inspection, labeling, boxing and boxing the product, and packaging, 3 batches of recombinant follicle stimulating hormone freeze-dried powder injection for injection are obtained.
3. Quality standard
3.1 the product is white or off-white lyophilized cake or powder.
3.2pH
Taking 3-5 pieces of this product, respectively adding 3ml of water for injection, dissolving, mixing the solutions, and determining according to the method (Chinese pharmacopoeia 2015), wherein the determination pH should be 6.5-7.5.
3.3 purity of
The method uses ultrafiltration mode to remove each auxiliary material component, combines the molecular weight of the sample and each auxiliary material, adopts a 10K ultrafiltration tube of millipore, and according to the ultrafiltration principle, the method does not influence the content of target protein and protein related impurities, so that the analysis of the final result is not influenced.
1 bottle of sample is taken, and after re-dissolving with 500 μl of water for injection, the sample is collected after ultrafiltration and liquid exchange with water for injection for 3 times in a 10K ultrafiltration column, and then detection is carried out. SEC-HPLC purity detection is carried out by Agilent 1260 equipment, bio SEC-3 3 μmThe chromatographic column, the mobile phase adopts 0.1mol/L PB-0.1mol/L Na2SO4 (PH 7.0 + -0.5): acetonitrile=80:20, flow rate 0.4ml/min, detection wavelength 214nm.
The theoretical plate number is not less than 1000 calculated by rhFSH peak, and the separation degree of the rhFSH main peak and the adjacent impurity peak is not less than 1.0. In the chromatogram of the sample solution, the main peak area is not lower than 99.5% calculated by an area normalization method;
3.4 loss on drying
According to the Chinese pharmacopoeia 2010 edition, general rule "moisture determination method", the moisture loss weight is less than 3.0%;
3.5 reconstitution time to the time taken for the injection of the recombinant follicle-stimulating hormone lyophilized powder for injection to begin with the injection water until the powder for injection is completely dissolved.
3.6 abnormal toxicity: the freeze-dried powder injection is taken to be added with sodium chloride injection to prepare recombinant follicle-stimulating hormone solution, and the recombinant follicle-stimulating hormone freeze-dried powder injection for injection is subcutaneously injected into 12, 60 or 300IU/kg of the cynomolgus monkey 1 time per day for 30 continuous days, so that no animal death occurs, no obvious systemic toxic reaction or injection local inflammatory reaction exists, and the abnormal toxicity accords with pharmacopoeia requirements;
3.7 sterile assay
Taking the freeze-dried powder injection of the invention, adding 1-2ml of sterilized water for dissolution, and performing legal examination (Chinese pharmacopoeia) to meet the requirements;
3.8 bacterial endotoxin
Taking the product, and performing legal examination (Chinese pharmacopoeia), wherein the amount of endotoxin in each 1 bottle of rhFSH is less than 8EU;
3.9 visible foreign matter
Taking 5-8 parts of the product, and performing legal inspection (supplementary regulation of Chinese pharmacopoeia and visible foreign matter inspection) to meet the regulation;
3.10 insoluble particles
Taking the product, and detecting according to law (Chinese pharmacopoeia), wherein the product meets the regulations;
3.11 biological Activity recombinant follicle stimulating hormone according to the biological assay of follicle stimulating hormone (Chinese pharmacopoeia), should accord with the rule, the result of the assay should be 80% -125% of the marked amount.
3.12 polymers refer to organic molecules formed by addition polymerization or polycondensation reactions between organic molecules.
4. Quality inspection
4.1 example 1 3 batches of recombinant follicle-stimulating hormone freeze-dried powder products for injection are white loose bodies, have good appearance, become ideal loose bodies and dissolve quickly, wherein 140801 is taken as a test sample, the weight of ovaries is taken as a reaction value, and according to the second appendix of Chinese pharmacopoeia 2010 edition, the bioassay statistical method, the dosage of rats is 42IU/kg/d, and each injection is injected once every day for 3 days, and the number of large follicles is obviously increased as seen by histomorphology observation, and the figure 1 is shown in detail.
4.2 stability investigation
4.2.1 Accelerated test investigation at 25 DEG C
Three batches of finished products (140801, 140802 and 140901) are placed at 25 ℃ and are sampled and detected at 3 months and 6 months respectively, wherein zero data (0M) are data measured when the preparation of recombinant follicle-stimulating hormone freeze-dried powder injection for injection is completed, and the stability inspection result of the 25 ℃ accelerated test is shown in table 2.
TABLE 2 detection results of 25℃acceleration test
As can be seen from Table 2, the three batches of finished products are stored for 6 months at 25 ℃, and the redissolution time, visible foreign matters, insoluble particles, moisture, pH value, biological activity and sterile inspection and detection results of the samples are all unchanged obviously and accord with the quality standard.
4.2.2 Accelerated test at 37 DEG C
Three batches of finished products (140801, 140802 and 140901) are sampled and detected at 37 ℃ at 4 weeks and 8 weeks respectively, wherein zero data (0W) are data measured when the preparation of recombinant follicle stimulating hormone freeze-dried powder injection for injection is completed, and the stability examination result of the 37 ℃ accelerated test is shown in table 3.
TABLE 3 detection results of 37℃acceleration test
As can be seen from Table 3, the 3 batches of products were stored at 37℃for 8 weeks, and the appearance, reconstitution time, visible foreign matters, insoluble particles, moisture, pH, biological activity and sterility test results were not significantly changed, and were in accordance with the quality standards.
4.3. Long-term stability investigation
Three batches of finished products (140801, 140802 and 140901) are packaged according to the market, placed under the storage condition of 2-8 ℃ and sampled for 6 months, 12 months, 24 months and 36 months respectively, wherein zero point data (0M) are measured when the preparation of recombinant follicle stimulating hormone freeze-dried powder injection for injection is completed, and the long-term stability inspection result at 2-8 ℃ is shown in table 4. .
Table 4 2-8deg.C long-term stability test results (20140801 batch)
Table 5 2-8deg.C long term stability test results (20140802, 20140901 batch)
As can be seen from tables 4 and 5, the three batches of finished products (140801, 140802, 140901) are placed for 36 months under the storage condition of 2-8 ℃, and the indexes of appearance, redissolution time, moisture, pH, biological activity, purity, polymer and the like all meet the quality requirements, which indicate that the three batches of finished products (140801, 140802, 140901) have good stability in 36 months, wherein the SEC-HPLC graph of the purity measurement of the 140801 batch of products in 36 months is shown as fig. 2, and the 140801 batch of products still has very high purity after being placed for 36 months at 2-8 ℃ and almost no impurities exist as can be seen from fig. 2.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Claims (14)
1. A lyophilized preparation of follicle stimulating hormone, wherein the lyophilized preparation comprises follicle stimulating hormone and a pharmaceutically acceptable carrier;
the pharmaceutically acceptable carrier comprises 250-350 parts by weight of sugar alcohol, 0.5-1.5 parts by weight of amino acid, 0.1-1 parts by weight of surfactant and buffer;
the sugar alcohol is sucrose;
the amino acid is methionine;
the surfactant is polysorbate-20;
IU of said follicle stimulating hormone with said sugar alcohol: the g ratio is 75-85:250-350; and is also provided with
The pH value of the freeze-dried preparation measured after re-dissolving with water is 6.5-7.5;
the buffering agent is selected from the group consisting of: phosphate buffer;
the alpha subunit of the follicle-stimulating hormone has an amino acid sequence shown in SEQ ID NO. 1, and the beta subunit has an amino acid sequence shown in SEQ ID NO. 2:
SEQ ID NO:1：
MDYYRKYAAIFLVTLSVFLHVLHSAPDVQDCPECTLQENPFFSQPGAPILQCMG CCFSRAYPTPLRSKKTMLVQKNVTSESTCCVAKSYNRVTVMGGFKVENHTAC HCSTCYYHKS；
SEQ ID NO:2：
MKTLQFFFLFCCWKAICCNSCELTNITIAIEKEECRFCISINTTWCAGYCYTRDLV YKDPARPKIQKTCTFKELVYETVRVPGCAHHADSLYTYPVATQCHCGKCDSDS TDCTVRGLGPSYCSFGEMKE*；
The freeze-dried preparation is prepared by a method comprising the following steps:
(1) Dissolving follicle stimulating hormone and a pharmaceutically acceptable carrier with water to obtain a solution to be freeze-dried;
(2) Freeze-drying the solution to be freeze-dried obtained in the step (1), thereby obtaining the freeze-dried preparation;
the freeze drying in the step (2) comprises the following steps:
step 1, prefreezing: the temperature is-30 ℃ to-60 ℃;
step 2, heating 1: the temperature is between 20 ℃ below zero and 40 ℃ below zero, the time is between 10 and 25 hours, and the vacuum degree is between 4 and 40pa;
step 3, heating 2: the temperature is between-5 ℃ and-25 ℃, the time is between 1 and 4 hours, and the vacuum degree is between 4 and 40pa;
step 4, heating up 3: the temperature is between-10 ℃ and 10 ℃, the time is between 1 and 4 hours, and the vacuum degree is between 4 and 40pa;
step 5, heating 4: the temperature is 15-35 ℃, the time is 1-5h, and the vacuum degree is 4-40pa;
step 6, heating up 5: the temperature is 20-40 ℃, the time is 0.4-3h, and the vacuum degree is 4-40pa;
and (5) finishing freeze-drying.
2. The lyophilized formulation according to claim 1, wherein the lyophilized formulation has a pH of 6.8 to 7.2 as measured after reconstitution with water.
3. The lyophilized formulation of claim 1, wherein said follicle stimulating hormone is in combination with said sugar alcohol: the g ratio is 77-83:280-320.
4. The lyophilized formulation according to claim 1, wherein the lyophilized formulation has a pH of 7 as determined after reconstitution with water.
5. The lyophilized formulation of claim 1, wherein the lyophilized formulation comprises: (i) 77-83IU of follicle stimulating hormone;
(ii) 280-320g of sucrose;
(iii) 0.8-1.2g methionine;
(iv) 0.3-0.8g polysorbate; and
(v) A buffer.
6. The lyophilized formulation according to claim 1, wherein the amino acid is 0.8 to 1.2 parts by weight.
7. The lyophilized formulation according to claim 1, wherein the surfactant is 0.3-0.8 parts by weight.
8. The lyophilized formulation according to claim 1, wherein the sugar alcohol is 280-320 parts by weight.
9. A method of preparing the lyophilized formulation of claim 1, comprising the steps of:
(1) Dissolving follicle stimulating hormone and a pharmaceutically acceptable carrier with water to obtain a solution to be freeze-dried;
(2) Lyophilizing the solution to be lyophilized obtained in step (1) to obtain the lyophilized preparation according to claim 1.
And in the step (2), the freeze-drying includes the steps of:
step 1, prefreezing: the temperature is-30 ℃ to-60 ℃;
step 2, heating 1: the temperature is between 20 ℃ below zero and 40 ℃ below zero, the time is between 10 and 25 hours, and the vacuum degree is between 4 and 40pa;
Step 3, heating 2: the temperature is between-5 ℃ and-25 ℃, the time is between 1 and 4 hours, and the vacuum degree is between 4 and 40pa;
step 4, heating up 3: the temperature is between-10 ℃ and 10 ℃, the time is between 1 and 4 hours, and the vacuum degree is between 4 and 40pa;
step 5, heating 4: the temperature is 15-35 ℃, the time is 1-5h, and the vacuum degree is 4-40pa;
step 6, heating up 5: the temperature is 20-40 ℃, the time is 0.4-3h, and the vacuum degree is 4-40pa;
and (5) finishing freeze-drying.
10. The lyophilized formulation according to claim 9, wherein in step (2), the lyophilization comprises the steps of:
step 1, prefreezing: the temperature is-40 ℃ to-50 ℃;
step 2, heating 1: the temperature is between 25 ℃ below zero and 35 ℃ below zero, the time is between 12 and 18 hours, and the vacuum degree is between 8 and 16pa;
step 3, heating 2: the temperature is between minus 10 ℃ and minus 20 ℃, the time is between 1.5 and 2.5 hours, and the vacuum degree is between 8 and 16pa;
step 4, heating up 3: the temperature is between-5 and 5 ℃, the time is between 1.5 and 2.5 hours, and the vacuum degree is between 8 and 16pa;
step 5, heating 4: the temperature is 20-30 ℃, the time is 2-3h, and the vacuum degree is 8-16pa;
step 6, heating up 5: the temperature is 25-35 ℃, the time is 0.5-1.5h, and the vacuum degree is 8-16pa;
and (5) finishing freeze-drying.
11. The lyophilized formulation according to claim 9, wherein the lyophilization comprises the steps of:
Step 1, prefreezing: the temperature of the plate layer is-45 ℃ for 2-3 hours, and the vacuum degree is standard atmospheric pressure;
step 2, heating 1: the temperature is-30 ℃, the time is 15 hours, and the vacuum degree is 11.5pa;
step 3, heating 2: the temperature is 15 ℃ below zero for 2 hours, and the vacuum degree is 11.5pa;
step 4, heating up 3: the temperature is 0 ℃, the time is 2 hours, and the vacuum degree is 11.5pa;
step 5, heating 4: the temperature is 25 ℃, the time is 2.5 hours, and the vacuum degree is 11.5pa;
step 6, heating up 5: the temperature is 28 ℃, the time is 1h, and the vacuum degree is 11.5pa;
and (5) finishing freeze-drying.
12. A rehydration of a lyophilized preparation of follicle stimulating hormone, wherein the lyophilized preparation of follicle stimulating hormone is the lyophilized preparation of claim 1.
13. Use of a lyophilized formulation according to claim 1 or a rehydration according to claim 12 for the preparation of a medicament for stimulating follicular development.
14. A kit, comprising:
(i) A container containing the lyophilized formulation of claim 1 or the rehydration of claim 13;
(ii) Optionally instructions for use.
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| CN201911194805.3A CN111686079A (en) | 2019-11-28 | 2019-11-28 | Follicle stimulating hormone freeze-dried preparation and preparation method and application thereof |
| CN202310223677.0A CN116549403A (en) | 2019-11-28 | 2019-11-28 | Follicle-stimulating hormone freeze-dried preparation, and preparation method and application thereof |
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| CN115634284A (en) * | 2022-10-31 | 2023-01-24 | 景泽生物医药(合肥)有限公司 | Gonadotropin freeze-dried preparation and preparation method thereof |
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| CN110305903B (en) * | 2019-07-31 | 2021-10-01 | 江苏璟泽生物医药有限公司 | Recombinant human follicle stimulating hormone and preparation method thereof |
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