CN113274486A - Stable acidic fibroblast growth factor preparation and preparation method and application thereof - Google Patents
Stable acidic fibroblast growth factor preparation and preparation method and application thereof Download PDFInfo
- Publication number
- CN113274486A CN113274486A CN202110406503.9A CN202110406503A CN113274486A CN 113274486 A CN113274486 A CN 113274486A CN 202110406503 A CN202110406503 A CN 202110406503A CN 113274486 A CN113274486 A CN 113274486A
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- Prior art keywords
- growth factor
- fibroblast growth
- preparation
- acidic fibroblast
- stabilizer
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Classifications
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- 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
- A61K38/18—Growth factors; Growth regulators
- A61K38/1825—Fibroblast growth factor [FGF]
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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
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
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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
- A61K47/26—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
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- 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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A—HUMAN NECESSITIES
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- A61K9/00—Medicinal preparations characterised by special physical form
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- A—HUMAN NECESSITIES
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- A61K9/00—Medicinal preparations characterised by special physical form
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/50—Fibroblast growth factor [FGF]
- C07K14/501—Fibroblast growth factor [FGF] acidic FGF [aFGF]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
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- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
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- Inorganic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biomedical Technology (AREA)
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- General Engineering & Computer Science (AREA)
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- Plant Pathology (AREA)
- Microbiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Immunology (AREA)
- Toxicology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention relates to a stable acidic fibroblast growth factor preparation, a preparation method and application thereof. The preparation comprises acidic fibroblast growth factor, phosphate buffer solution and stabilizer; wherein the concentration of the acidic fibroblast growth factor is 1000-100000U/ml; the molar concentration of the phosphate buffer solution is 10-50 mmmol/L; the mass volume percentage content of the stabilizer is 0.01-30% (w/v); the liquid preparation has pH of 6.0-8.0. The protein in the acidic fibroblast growth factor preparation provided by the invention has good stability, and has high stability and biological activity when stored in a liquid state or stored after freeze-drying; in addition, the preparation method is simple, the cost is low, and the preparation can be popularized in large scale in the fields of wound skin healing, medical cosmetology and the like.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a stable acidic fibroblast growth factor preparation as well as a preparation method and application thereof.
Background
The addition of a stabilizer in a protein product is a key technology of the research of biological protein product preparations, each protein is called as a medicine, the biological activity of one protein is good, and if the stability of the protein is poor, the protein can be stored for a short time or the storage conditions are very harsh, the protein cannot become a medicine and is beneficial to human beings.
Fibroblast Growth Factors (FGFs) are a family of growth factors consisting of at least 9 structurally related proteins (FGF1-9), of which the most well known are acidic fibroblast growth factor (aFGF or FGF-1) and basic fibroblast growth factor (bFGF or FGF-2). Members of the FGF family are known to be mitogens for a variety of similar cells of mesodermal and neuroectodermal origin, and these factors also have a variety of biological functions such as promotion of angiogenesis, neurite extension, neuronal regeneration and survival, bone or cartilage tissue repair, and myoblast differentiation. In general, there is about 25-55% amino acid sequence homology between the core sequences of the members of the FGF family, and thus the FGF family may have common ancestor genes.
In general, the common chemical characteristics of aFGF and bFGF make these factors bind tightly to heparin sodium salt, but aFGF has less affinity for heparin sodium salt than bFGF, and the isoelectric point of aFGF is more acidic (pH 5-7). Heparin sodium salts are known to modulate the function of FGF molecules in several ways, e.g. heparin sodium salts or heparin sodium salt-like molecules can act directly on FGF, activating or enhancing the activity of aFGF and bFGF. In addition, the inventors have found that the synergistic effect of soluble sodium heparin salt appears to be selective for aFGF and that it potentiates aFGF activity to a 10-fold greater extent than bFGF.
Acidic fibroblast growth factor (aFGF) has previously been mainly extracted from bovine pituitary, known as bovine acidic fibroblast growth factor; a number of prior art documents have reported methods for the production of aFGF by recombinant DNA techniques (see, for example, Biotechnology 5: 960, 1987; J.Bio1. chem.263: 16471, 1988; EP 0219052 et al).
However, both extracted bovine acid fibroblast growth factor (HAFGF) and recombinant human acid fibroblast growth factor (rhaFGF) are very unstable in a solution state, easily precipitate out and lose activity, so that the freeze-dried preparation is usually required to be freeze-dried and stored, and the freeze-dried preparation is required to be re-dissolved into a solution and used as soon as possible to avoid activity loss. For a long time, because the stability can not meet the requirement, the liquid preparation product of aFGF is difficult to be popularized and applied as a pharmaceutical preparation. Therefore, the research on the stabilizing agent applicable to the aFGF is carried out to obtain the stable aFGF pharmaceutical composition, and the method has important significance; there is a pressing need in the art to provide liquid formulations that maintain the long-term stability of acidic fibroblast growth factors.
In addition, the unique angiogenic and cell proliferative activities of FGF make it particularly useful for promoting healing of tissue wounds, especially deep wounds. . There are also researchers proposing methods of protecting central neurons and improving brain function using aFGF active fragments. However, the application of aFGF to wound skin for healing and medical cosmetology has not been reported.
Disclosure of Invention
The invention provides a stable acidic fibroblast growth factor preparation, a preparation method and application thereof aiming at the defects of the prior art, researches are carried out on all components of the preparation, particularly different stabilizer combinations are selected and the proportions of all stabilizers are compared, and the stable preparation containing the acidic fibroblast growth factor is obtained through repeated experimental researches.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a stable liquid preparation of acidic fibroblast growth factor, which comprises an acidic fibroblast growth factor, a phosphate buffer and a stabilizer; wherein the concentration of the acidic fibroblast growth factor is 1000-100000U/ml; the molar concentration of the phosphate buffer solution is 10-50 mmmol/L; the mass volume percentage content of the stabilizer is 0.01-30% (w/v); the liquid preparation has pH of 6.0-8.0.
In a preferred embodiment of the present invention, the content of the stabilizer is 0.02 to 20% (w/v) by mass/volume; preferably 0.03-16% (w/v); more preferably 5-16% (w/v); more preferably 15-16% (w/v).
In a preferred embodiment of the present invention, the stabilizer is at least one selected from the group consisting of: mannitol, hydrolyzed gelatin, dextran, human serum albumin, sucrose, trehalose, glycerol, glycine, L-glutathione and heparin sodium salt; the stabiliser preferably comprises mannitol and one or more selected from: human serum albumin, glycerol, L-glutathione and heparin sodium salt; more preferably, the stabilizer comprises mannitol, human serum albumin, glycerol, L-glutathione and heparin sodium salt.
In a preferred embodiment of the present invention, the stabilizer comprises mannitol 1-5% (w/v) and human serum albumin 0.5-2% (w/v) based on the total volume of the liquid preparation, and one or more selected from the following: 10-15% (w/v) of glycerol, 0.01-0.05% (w/v) of L-glutathione and 0.05-0.1% (w/v) of heparin sodium salt; preferably, the stabilizer comprises mannitol 1-5% (w/v), human serum albumin 0.5-2% (w/v), glycerol 10-15% (w/v), L-glutathione 0.01-0.05% (w/v), and heparin sodium salt 0.05-0.1% (w/v); more preferably, the stabilizer comprises mannitol 4% (w/v), human serum albumin 1% (w/v), glycerol 10% (w/v), L-glutathione 0.03% (w/v) and heparin sodium salt 0.1% (w/v).
In a preferred embodiment of the present invention, the acidic fibroblast growth factor is a naturally extracted bovine acidic fibroblast growth factor or a recombinant human acidic fibroblast growth factor; preferably recombinant human acidic fibroblast growth factor.
In a preferred embodiment of the present invention, the method for preparing the recombinant human acidic fibroblast growth factor comprises the following steps:
step one, construction of engineering strains:
inserting the HAFGF gene obtained by PCR amplification into an expression empty vector, converting competent escherichia coli, and selecting positive clone containing a recon to obtain a recombinant plasmid; transforming the recombinant plasmid, and screening transformants on a resistant plate to obtain an engineering strain capable of intracellularly expressing the human acidic fibroblast growth factor;
step two, fermentation:
(1) preparing appropriate amount of fermentation culture medium according to volume of fermentation tank, sterilizing, inoculating seed solution, adopting continuous flow glucose-adding high density fermentation culture process, regulating rotation speed, ventilation amount, glucose flow rate, etc. to control dissolved oxygen content of culture solution to be not less than 20%, and when fermentation broth A is obtained600When the expression reaches about 25 hours, adding IPTG (isopropyl-beta-thiogalactoside) for induction expression, wherein the induction time is 3-5 hours;
(2) after fermentation is finished, centrifuging the culture solution by using a centrifuge; collecting thalli after centrifugation is finished, and weighing wet weight;
(3) crushing the harvested thalli by a pressure homogenizer, and performing microscopic examination on cell lysate until no complete thalli exist in a visual field; centrifuging the obtained cell lysate, collecting supernatant, and filtering to obtain crude rhaFGF;
step three, purification:
(1) and (3) crude purification: preliminarily purifying the obtained crude rhaFGF by using an SP-Sepharose Fast Flow ion exchange column, and collecting an active peak;
(2) and (3) fine purification: directly purifying the crude purification collection solution by a Heparin-Sepharose Fast Flow affinity chromatography column, and collecting an active peak; and (3) directly purifying the collected solution by a Superdex 75PrepGrade gel filtration chromatography column, collecting an active peak, and storing after filtration sterilization.
In a preferred embodiment of the present invention, in the method for preparing the recombinant human acidic fibroblast growth factor, the fermentation medium is composed of tryptone, yeast extract, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, sucrose, and glucose and magnesium sulfate are fed-batch.
In a preferred embodiment of the present invention, in the method for preparing the recombinant human acidic fibroblast growth factor, the seed solution is inoculated at a ratio of 1:10 to 1: 20.
In a preferred embodiment of the present invention, the phosphate buffer is a buffer solution composed of disodium hydrogen phosphate and sodium dihydrogen phosphate.
In a second aspect, the present invention provides a method for preparing the above liquid preparation, comprising the steps of:
mixing a proper amount of phosphate buffer solution, a stabilizer and water for injection to obtain a mixed solution;
regulating the pH value of the mixed solution to 6.0-8.0 by using phosphoric acid or NaOH;
adding a proper amount of acidic fibroblast growth factor into the mixed solution after the pH value is adjusted, and then fixing the volume by using water for injection;
and step four, sterile filtering the mixed solution after constant volume to obtain the stable acidic fibroblast growth factor liquid preparation.
In a third aspect, the invention provides a stable acidic fibroblast growth factor lyophilized preparation, which is obtained by further freezing the liquid preparation provided by the first aspect of the invention at low temperature and vacuum-pumping water.
In a fourth aspect, the invention provides the use of the above liquid formulation in the preparation of a medicament or medical cosmetic product for skin regeneration of minor second degree burns, ulcers and for promoting healing of wounded skin.
In a fifth aspect, the present invention provides the use of the above-described lyophilized formulation in the preparation of a medicament or medical cosmetic product for skin regeneration of minor second degree burns, ulcers and for promoting healing of wounded skin.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
the protein in the acidic fibroblast growth factor preparation provided by the invention has good stability, and has high stability and biological activity when stored in a liquid state or stored after freeze-drying; in addition, the preparation method is simple, the cost is low, and the preparation can be popularized in large scale in the fields of wound skin healing, medical cosmetology and the like.
Detailed Description
The inventor finds that the biological activity of the acidic fibroblast growth factor in the aqueous solution can be maintained for a long time by adding a certain type and quantity of stabilizing agents into the aqueous solution containing the acidic fibroblast growth factor to form a pharmaceutical preparation, and the biological activity of the acidic fibroblast growth factor can be maintained for a longer time after the pharmaceutical preparation is prepared into a freeze-dried preparation.
The specific mode is that the inventor adds one or more components of mannitol, hydrolyzed gelatin, dextran, human albumin, sucrose, trehalose, glycerol, glycine, L-glutathione, heparin sodium salt and the like as a stabilizer into a phosphate buffer system (10-50mmol/L, pH6-8) containing the acidic fibroblast growth factor to prepare the acidic fibroblast growth factor preparation, which can keep the biological activity for a long time and is convenient for clinical use; if the preparation is freeze-dried and vacuumized to prepare a freeze-dried preparation, the biological activity of the acidic fibroblast growth factor can be maintained for a longer time.
In the present invention, the "acidic fibroblast growth factor" may be obtained using methods well known to those skilled in the art, including "acidic fibroblast growth factor" prepared by a genetic recombination method and mutants thereof.
As used herein, a stabilizer refers to a substance that can maintain the activity of acidic fibroblast growth factor, and is selected from one or more of the following: mannitol, hydrolyzed gelatin, dextran, human serum albumin, sucrose, trehalose, glycerol, glycine, L-glutathione, heparin sodium salt, etc.; preferably one or more substances selected from mannitol, human serum albumin, glycerol, L-glutathione and heparin sodium salt; more preferably, the stabilizer comprises mannitol, human serum albumin, glycerol, L-glutathione and heparin sodium salt.
In the invention, the pH value of a phosphate buffer system selected by the stable acidic fibroblast growth factor preparation is 6-8, and the pH value is preferably 7.0; preferred buffer salts are disodium hydrogen phosphate and sodium dihydrogen phosphate.
In the invention, the acidic fibroblast growth factor preparation is placed at 4-25 ℃, the day of preparing the preparation is taken as a starting point (0), the biological activity of the acidic fibroblast growth factor in the preparation at 0 is taken as a reference value of the initial biological activity, and the biological activity of the acidic fibroblast growth factor is 80-150% of the reference value of the initial biological activity after the preparation is placed at 4-25 ℃ for 0-3 years, so that the stability of the preparation is achieved.
In the invention, the biological activity of the acidic fibroblast growth factor is measured by adopting an MTT method recorded in Chinese pharmacopoeia (current edition) to promote cell proliferation of mouse embryonic fibroblasts (Balb/c or NIH 3T 3).
The concentration of the stabilizer of the invention is based on the total volume of the formulation provided, and is used in a mass/volume percent concentration (w/v), e.g., 1% (w/v) means 0.1g of solute is dissolved in 100mL of solution.
The specific dosage of the stabilizer in the invention is as follows: 1-5% (w/v) of mannitol and 0.5-2% (w/v) of human serum albumin; 10-15% (w/v) of glycerol; 0.01-0.05% (w/v) of L-glutathione and 0.05-0.1% (w/v) of heparin sodium salt; the stabilizer may be any one of the above or a combination thereof.
In another preferred embodiment, the stable preparation containing acidic fibroblast growth factor provided by the invention can be prepared by the following method: weighing sodium dihydrogen phosphate, disodium hydrogen phosphate and protease stabilizer, adding appropriate amount of water for injection, dissolving, adding appropriate amount of acidic fibroblast growth factor, adjusting pH to appropriate value, adding water for injection to desired volume, filtering, sterilizing, pressing, and capping; if the solution is lyophilized and water is drained to obtain lyophilized powder, more stable preparation can be obtained. The water for injection is the water for pharmacy according with the regulations of Chinese pharmacopoeia, is prepared by distilling purified water and is a term in the pharmacopoeia.
The features mentioned above with reference to the invention, or the features mentioned with reference to the embodiments, can be combined arbitrarily. All the features mentioned herein can be combined in any combination and each feature disclosed in the specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, all features disclosed are merely representative of the same or similar features.
The present invention will be described in detail and specifically by the following examples to better understand the present invention, but the following examples do not limit the scope of the present invention, and the experimental methods without specifying the specific conditions in the following examples are according to the usual conditions or according to the experimental conditions recommended by the manufacturers.
Unless otherwise defined, 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. In addition, any similar or equivalent methods or materials may be used in the methods used in the present invention. The preferred methods and materials described herein are for exemplary purposes only.
The sources of the ingredients in the following examples are as follows:
sodium dihydrogen phosphate: from Jiuzhan pharmaceutical Co Ltd of Hunan province
Disodium hydrogen phosphate: from Jiuzhan pharmaceutical Co Ltd of Hunan province
Mannitol: from south-China-Guangxi chemical pharmaceuticals, Inc
Human serum albumin: from Olympic Okt enamel pharmaceutical Co Ltd
Glycerol: from Naokang pharmaceutical Co Ltd of lake
L-glutathione: from Biotechnology engineering (Shanghai) Ltd
Sodium salt of heparin: from Biotechnology engineering (Shanghai) Ltd
Example 1
This embodiment provides a method for preparing a recombinant human acidic fibroblast growth factor (taking intracellular expression of a human acidic fibroblast growth factor in an escherichia coli expression system as an example, other expression systems or expression modes are adopted, and the operation is basically the same), which includes the following steps:
1. construction of engineered Strain
The HAFGF gene obtained by PCR amplification is inserted into an expression empty vector pET3c, competent Escherichia coli DH5a is transformed, and a positive clone containing a recon is selected to obtain a recombinant plasmid pET3c-HAFGF (m). The recombinant plasmid pET3c-haFGF (m) was transformed into the host bacterium BL21(DE3) of the DE3 phage lysogen. And (4) screening transformants on the resistant plate to obtain the engineering strain capable of intracellularly expressing the human acidic fibroblast growth factor.
2. research on rhaFGF fermentation technology
2.1 seed liquid preparation
Inoculating qualified working seed batch strains in an LB culture medium for culturing for 16-20 hours to prepare a seed solution.
2.2 culture Medium for fermentation
Comprises tryptone, yeast extract, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, sucrose, and glucose and magnesium sulfate.
2.3 seed liquid inoculation and fermentation culture
Preparing appropriate amount of fermentation culture medium according to volume of fermentation tank, sterilizing, inoculating seed solution into the fermentation culture medium at a ratio of 1:10-1:20, adopting continuous fed-batch glucose high-density fermentation culture process, regulating rotation speed, ventilation amount, glucose feeding rate, etc. to control dissolved oxygen content of the culture solution to be not less than 20%, and allowing fermentation broth A to flow while regulating the flow rate, ventilation amount, glucose feeding rate, etc600When the expression reaches about 25 hours, IPTG is added for induction expression, and the induction time is 3-5 hours.
2.4 fermentation broth treatment
And (3) immediately centrifuging the culture solution by using a centrifuge at the rotating speed of more than 4000 rpm after the fermentation is finished, collecting thalli after the centrifugation is finished, and weighing the wet weight.
2.5 disruption of the cells
The harvested cells were suspended in 25mmol/L PB, 0.04% Tween-80, 2mmol/L EDTANA2In a buffer solution with pH of 7.0, a pressure homogenizer breaks the thalli (the pressure is not lower than 60MPa), and the cell cracking is examined by microscopyThe liquid is decomposed until no complete bacteria exist in the visual field. And centrifuging the obtained cell lysate, collecting supernatant, and filtering the supernatant by using a 0.22 mu m filter membrane to obtain the crude rhaFGF.
3. Study of purification Process
3.1 crude purification
Crude rhaFGF obtained by crushing thalli is firstly primarily purified by an SP-Sepharose Fast Flow ion exchange column to contain 25mmol/L PB, 0.6mol/L NaCl, 0.04% Tween-80 and 2mmol/L EDTANa2Elution was performed with a buffer solution of pH7.0, and the active peak was collected.
3.2 Fine purification
The crude purified collected solution was directly purified by a Heparin-Sepharose Fast Flow affinity column chromatography using 25mmol/L PB, 1.5mol/L NaCl, 0.04% Tween-80, 2mmol/L EDTANA2Eluting with pH7.0 buffer solution, and collecting active peak; purifying the collected solution directly with Superdex 75PrepGrade gel filtration chromatography column, using 10mmol/L PB, 0.15mol/L NaCl, 0.04% Tween-80, 2mmol/LEDTANa2Eluting with pH7.0 buffer solution, collecting active peak, filtering, sterilizing, and storing.
Example 2
The embodiment provides a recombinant human acidic fibroblast growth factor preparation I, and the formula and the preparation method thereof are as follows:
the formula is as follows: (preparation 1L as an example)
The preparation process comprises the following steps: weighing disodium hydrogen phosphate, monosodium phosphate monohydrate, mannitol and glycerol, adding a proper amount of water for injection to fully dissolve auxiliary materials, weighing a proper amount of human serum albumin and the recombinant human acidic fibroblast growth factor in the solution, fully and uniformly mixing, metering the volume to 1000ml by using the water for injection, sterilizing and filtering by using a 0.22 mu m filter membrane, filling, plugging and capping to obtain a recombinant human acidic fibroblast growth factor liquid preparation I; and (3) semi-tamponading the filled solution, freeze-drying, vacuumizing, tamponading and capping to obtain the recombinant human acidic fibroblast growth factor freeze-dried powder injection preparation I.
The invention carries out the accelerated stability investigation of the combined medicine at the temperature of 25 ℃. A liquid preparation prepared by taking 4% (w/v) mannitol as an excipient in a 10ml disodium hydrogen phosphate-sodium dihydrogen phosphate buffer system with pH7.0 and without any stabilizer has a white precipitate separated out after being placed at 25 ℃ for 2 weeks, and the activity is reduced by 50%; the activity of the solid preparation under the same condition is reduced by 30 percent after the solid preparation is placed at 25 ℃ for 4 weeks; meanwhile, the activity of the liquid preparation and the solid preparation which are simultaneously added with 1% (w/v) of human serum albumin and 10% (w/v) of glycerol as stabilizing agents is basically stable within 3 months, and the results are shown in Table 1.
TABLE 1 results of accelerated stability test at 25 ℃ for formulation I
The results show that the stabilizer combining 1% (w/v) human serum albumin and 10% (w/v) glycerol has good protection effect on the recombinant human acidic fibroblast growth factor.
Example 3
This example provides recombinant human acidic fibroblast growth factor preparations II and III and methods for preparing them, and the formulations of preparations II and III (taking preparation 1L as an example) are shown in Table 2 below.
TABLE 2 formulation compositions of formulations II, III
According to the above table 2, 15% (w/v) and 20% (w/v) glycerol and 1% (w/v) human serum albumin were used as stabilizers to prepare liquid and lyophilized formulations, and the activity was substantially stable within 6 months when accelerated stability examination of the formulations was performed at 25 ℃, and the results are shown in table 3.
TABLE 3 results of accelerated stability test at 25 ℃ for formulations II and III
The results show that the stabilizer comprising 1% (w/v) human serum albumin, 15% (w/v) glycerol and 20% (w/v) glycerol respectively has basically the same protective effect on the recombinant human acidic fibroblast growth factor, and the 15% (w/v) glycerol is considered to have a good protective effect by comparing the stability results with the stability results of the 10% (w/v) glycerol concentration in example 2.
Example 4
This example provides recombinant human acidic fibroblast growth factor preparations IV-VI and methods for their preparation, and the formulations of preparations IV-VI (taking preparation 1L as an example) are shown in Table 4 below.
TABLE 4 formulation compositions of formulations IV-VI
According to the above table 4, 15% (w/v) glycerol and 1% (w/v) human serum albumin are selected as the stabilizer, the concentrations of the recombinant human acidic fibroblast growth factor in the preparation are respectively adjusted to 5000U/ml, 12500U/ml and 100000U/ml to prepare the preparation IV-VI, and the accelerated stability of the preparation is examined at 25 ℃, so that the activity of the preparation is basically stable within 6 months, and the result is shown in table 5.
TABLE 5 results of accelerated stability test at 25 ℃ for formulations IV-VI
The results show that 1% (w/v) human albumin and 15% (w/v) glycerol are combined as a stabilizer, and the stabilizer has good protection effect on the recombinant human acidic fibroblast growth factor with the concentration of 5000U/ml-100000U/ml.
Example 5
This example provides recombinant human acidic fibroblast growth factor preparations VII-IX, the formulation of which (for example, formulation 1L) is shown in Table 6 below, and a method for preparing the same.
TABLE 6 formulation composition of preparations VII-IX
Recombinant human acidic fibroblast growth factor preparations VII-IX were prepared according to the above-mentioned recipe with reference to the preparation method of example 2. The preparation VII-IX was prepared by using a combination of 0.01% (w/v), 0.03% (w/v) and 0.05% (w/v) L-glutathione and 1% (w/v) human albumin as stabilizers, respectively, at a concentration of 50000U/ml recombinant human acidic fibroblast growth factor, and accelerated stability studies of the preparation were performed at 25 ℃ with the results shown in Table 7.
TABLE 7 results of accelerated stability examination of the preparations VII to IX at 25 ℃
The results show that the combination of L-glutathione with different concentrations and 1% (w/v) human serum albumin respectively serves as a stabilizer, the stabilizer has a certain protection effect on the recombinant human acidic fibroblast growth factor with the concentration of 50000U/ml, and the protection effects of 0.03% (w/v) and 0.05% (w/v) L-glutathione are not greatly different, so that the combination of 0.03% (w/v) L-glutathione and 1% (w/v) human serum albumin can be selected as the stabilizer.
Example 6
This example provides recombinant human acidic fibroblast growth factor preparations X-XII and a preparation method thereof, and the formula of preparations X-XII (taking preparation 1L as an example) is shown in Table 8 below.
TABLE 8 formulation composition of preparations X-XII
Recombinant human acidic fibroblast growth factor, themselves VII-IX, was prepared according to the above-described recipe with reference to the preparation method of example 2. Preparations X-XII were prepared by using 0.05% (w/v), 0.08% (w/v) and 0.1% (w/v) heparin sodium salt and 1% (w/v) human albumin as stabilizers, respectively, and the concentration of the recombinant human acidic fibroblast growth factor was 50000U/ml, and accelerated stability of the preparations was examined at 25 ℃ and the results are shown in Table 9.
TABLE 9 results of accelerated stability test at 25 ℃ for preparations X-XII
The results show that the combination of heparin sodium salts with different concentrations and 1% (w/v) human serum albumin is respectively used as a stabilizer, the stabilizer has a certain protection effect on recombinant human acidic fibroblast growth factor with the concentration of 50000U/ml, the protection effect of 0.1% (w/v) heparin sodium salt is better, the heparin sodium salt and the fibroblast growth factor have specific binding, the stabilizer has a certain promotion effect on promoting cell mitosis, the use concentration is not too high, and therefore the combination of 0.1% (w/v) heparin sodium salt and 1% (w/v) human serum albumin can be used as the stabilizer.
Example 7
This example provides recombinant human acidic fibroblast growth factor preparation XIII and a process for its preparation, the formulation of which (for example formulation 1L) is shown in Table 10 below.
TABLE 10 formulation composition of formulation XIII
Referring to the preparation method of example 2, three batches of the preparation (batch numbers: T2820151101, T2820151102 and T2820151103) were prepared at a concentration of 50000U/ml using 10% (w/v) glycerol, 0.03% (w/v) L-glutathione, 0.1% (w/v) heparin sodium salt and 1% (w/v) human serum albumin as a stabilizer, and accelerated stability of the combined drug was examined at 25 ℃, and the results are shown in Table 11.
TABLE 11 results of accelerated stability test of formulation XIII at 25 deg.C
The results show that the recombinant human acidic fibroblast growth factor preparation prepared by using the combination of 10% (w/v) glycerol, 0.03% (w/v) L-glutathione, 0.1% (w/v) heparin sodium salt and 1% (w/v) human albumin as the stabilizer can effectively protect the biological activity of the recombinant human acidic fibroblast growth factor, wherein the activity of the liquid preparation is slightly reduced after the liquid preparation is stored for 9 months at 25 ℃, while the activity of the freeze-dried powder injection is basically unchanged after the freeze-dried powder injection is stored for 12 months at 25 ℃, and the stability is better.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Claims (10)
1. A stable acidic fibroblast growth factor liquid preparation is characterized by comprising an acidic fibroblast growth factor, a phosphate buffer solution and a stabilizer; wherein, the concentration of the acidic fibroblast growth factor is 1000-100000U/ml; the molar concentration of the phosphate buffer solution is 10-50 mmmol/L; the mass volume percentage content of the stabilizer is 0.01-30% (w/v); the pH value of the liquid preparation is 6.0-8.0.
2. The liquid formulation of claim 1, wherein the stabilizer is present in an amount of 0.02 to 20% (w/v) by mass; preferably 0.03-16% (w/v); more preferably 5-16% (w/v); more preferably 15-16% (w/v).
3. The liquid formulation of claim 1, wherein the stabilizer is selected from at least one of the following: mannitol, hydrolyzed gelatin, dextran, human serum albumin, sucrose, trehalose, glycerol, glycine, L-glutathione and heparin sodium salt; preferably, the stabilizer comprises mannitol and one or more selected from the group consisting of: human serum albumin, glycerol, L-glutathione and heparin sodium salt; more preferably, the stabilizer comprises mannitol, human serum albumin, glycerol, L-glutathione and heparin sodium salt.
4. The liquid formulation of claim 3, wherein the stabilizer comprises mannitol 1-5% (w/v) and human serum albumin 0.5-2% (w/v) based on the total volume of the liquid formulation and one or more selected from the group consisting of: 10-15% (w/v) of glycerol, 0.01-0.05% (w/v) of L-glutathione and 0.05-0.1% (w/v) of heparin sodium salt; preferably, the stabilizer comprises mannitol 1-5% (w/v), human serum albumin 0.5-2% (w/v), glycerol 10-15% (w/v), L-glutathione 0.01-0.05% (w/v), and heparin sodium salt 0.05-0.1% (w/v); more preferably, the stabilizer comprises mannitol 4% (w/v), human serum albumin 1% (w/v), glycerol 10% (w/v), L-glutathione 0.03% (w/v) and heparin sodium salt 0.1% (w/v).
5. The liquid formulation of claim 1, wherein the acidic fibroblast growth factor is a naturally extracted bovine acidic fibroblast growth factor or a recombinant human acidic fibroblast growth factor; preferably recombinant human acidic fibroblast growth factor.
6. The liquid formulation of claim 1, wherein the phosphate buffer is a buffer solution consisting of disodium hydrogen phosphate and sodium dihydrogen phosphate.
7. A method for preparing a liquid formulation according to any one of claims 1 to 6, comprising the steps of:
mixing a proper amount of phosphate buffer solution, a stabilizer and water for injection to obtain a mixed solution;
regulating the pH value of the mixed solution to 6.0-8.0 by using phosphoric acid or NaOH;
adding a proper amount of acidic fibroblast growth factor into the mixed solution after the pH value is adjusted, and then fixing the volume by using water for injection;
and step four, sterile filtering the mixed solution after constant volume to obtain the stable acidic fibroblast growth factor liquid preparation.
8. A stable lyophilized preparation of acidic fibroblast growth factor, which is obtained by further freezing the liquid preparation of any one of claims 1-6 at low temperature and vacuum-drying the water.
9. Use of the liquid formulation according to any one of claims 1 to 6 for the preparation of a medicament or a medical cosmetic product for the skin regeneration of minor second degree burns, ulcers and for promoting the healing of wounded skin.
10. Use of the lyophilized formulation of claim 8 for the preparation of a medicament or medical cosmetic product for skin regeneration of minor second degree burns, ulcers and for promoting healing of wounded skin.
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| CN114010763A (en) * | 2021-10-12 | 2022-02-08 | 上海腾瑞制药股份有限公司 | Recombinant human acidic fibroblast growth factor gel and preparation method thereof |
| WO2022100643A1 (en) * | 2020-11-16 | 2022-05-19 | Eusol Biotech Co., Ltd. | Stabilized afgf compositions |
| CN117205162A (en) * | 2023-09-12 | 2023-12-12 | 上海腾瑞制药股份有限公司 | Preparation method of recombinant human acidic fibroblast growth factor freeze-dried preparation |
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| CN117205162A (en) * | 2023-09-12 | 2023-12-12 | 上海腾瑞制药股份有限公司 | Preparation method of recombinant human acidic fibroblast growth factor freeze-dried preparation |
| CN117205162B (en) * | 2023-09-12 | 2024-06-25 | 上海腾瑞制药股份有限公司 | Preparation method of recombinant human acidic fibroblast growth factor freeze-dried preparation |
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