CN112121150A - Fibroblast growth factor 10 freeze-dried powder - Google Patents
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- 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
- A61K38/18—Growth factors; Growth regulators
- A61K38/1825—Fibroblast growth factor [FGF]
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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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
- A61K47/06—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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- 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
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
Abstract
The invention aims to provide a fibroblast growth factor 10 freeze-dried powder, which contains recombinant human fibroblast growth factor 10, a freeze-drying protective agent and a pH regulator, wherein the freeze-drying protective agent is preferably dextran 40, more preferably the freeze-drying protective agent is composed of dextran 40, sorbitol and trehalose, and most preferably the freeze-drying protective agent is composed of dextran 40, sorbitol, trehalose and plant flavone. The freeze-dried powder does not contain albumin, avoids blood-borne pollution, has good protection effect and stability of the fibroblast growth factor 10, and has wide popularization value.
Description
Technical Field
The invention belongs to the field of active biological factors, and particularly relates to fibroblast growth factor 10 freeze-dried powder.
Background
Fibroblast growth factor is an important cytokine, which has various physiological functions and plays an important role in life activities. The fibroblast growth factor family is currently known to comprise 23 members, and the family members have 30-80% homology and can be divided into 7 subfamilies: FGF1/2/5, FGF3/4/6, FGF7/10/22, FGF8/17/18, FGF9/16/20, FGF11/12/13/14 and FGF 15/19/21/23.
FGF10 is a typical paracrine FGF, consisting of 208 amino acids, mediates mesenchymal and epithelial cell signaling in a paracrine manner, plays an important role in the development of multiple organs, and FGF10 is also involved in mitosis of cells, promotes movement, migration and differentiation of epithelial cells, and induces proliferation and differentiation of keratinocytes. In particular to diseases, evidence shows that FGF10 is involved in the occurrence of pancreatic cancer and breast cancer, in addition, FGF10 has an important role in the development of the brain, and has a good effect on ischemic injury and neuroprotection, and FGF10 is a potential target for myocardial repair. Therefore, FGF10 can provide new insights and therapeutic means for clinically solving related diseases, and it is necessary to prepare it into a preparation and further study its functions.
The cytokine preparation has the problems of poor thermal stability of liquid formulations, difficult long-term storage, long-distance transportation and the like in production practice, and the ideal solution is to carry out vacuum freeze drying on the preparation, so that the storage life can be prolonged by using simple conditions and the long-distance transportation is facilitated.
No stable freeze-dried formulation of FGF10 is currently available, and there is therefore still a need to provide a stable and effective freeze-dried formulation against FGF 10.
Disclosure of Invention
In order to overcome the defect of insufficient research on a freeze-dried preparation of FGF10 in the prior art, the invention provides a freeze-drying protective agent suitable for FGF10, FGF10 is prepared into freeze-dried powder, and the prepared freeze-dried powder has good FGF10 protective activity and excellent stability, and is beneficial to long-term storage of FGF 10.
Specifically, the invention provides a fibroblast growth factor 10(FGF10) freeze-dried powder which is prepared by freeze drying of recombinant human FGF10, a freeze-drying protective agent and a pH regulator.
Further, the lyoprotectant is dextran 40.
Further, the lyoprotectant consists of dextran 40, sorbitol, and trehalose.
Further, the lyoprotectant consists of dextran 40, sorbitol, trehalose and plant flavonoids.
Further, the pH regulator is PBS buffer.
Further, the pH adjuster is 50mM PBS buffer (pH 9.2).
Still further, the dextran 40 is present in an amount of 4-6 mg/vial (i.e., 4-6% (w/v concentration)).
Furthermore, the content of dextran 40 in the lyoprotectant is 4-6 mg/penicillin bottle (i.e. 4-6% (w/v)), the content of sorbitol is 0.5-2 mg/penicillin bottle (i.e. 0.5-2% (w/v)), and the content of trehalose is 0.5-2 mg/penicillin bottle (i.e. 0.5-2% (w/v)).
Furthermore, the content of dextran 40 in the cryoprotectant is 4-6 mg/penicillin bottle (i.e. 4-6% (w/v concentration)), the content of sorbitol is 0.5-2 mg/penicillin bottle (i.e. 0.5-2% (w/v concentration)), the content of trehalose is 0.5-2 mg/penicillin bottle (i.e. 0.5-2% (w/v concentration)), and the content of plant flavone is 0.1-0.4 mg/penicillin bottle (i.e. 0.1-0.4% (w/v concentration)).
Further, the plant flavone is one or more of daidzein, genistin and glycitin.
Furthermore, the content of FGF10 in the freeze-dried powder is 0.5-1 mg/bottle, and preferably 0.8-0.9 mg/bottle.
Most further, the content of dextran 40 in the freeze-drying protective agent is 5 mg/penicillin bottle, the content of sorbitol is 1 mg/penicillin bottle, the content of trehalose is 1 mg/penicillin bottle, and the content of plant flavone is 0.3 mg/penicillin bottle.
The invention also provides a preparation method of the FGF10 freeze-dried powder, which comprises the following steps: adding the freeze-drying protective agent into sterile double-distilled water for dissolving and filtering for sterilization to prepare protective agent diluent; dissolving the recombinant human FGF10 in a pH regulator solution to obtain a recombinant human FGF10 protein solution; adding the freeze-dried diluent and the recombinant human FGF10 protein solution into a penicillin bottle in a ratio of 1:1, wherein the initial volume is 1.0 mL/bottle; pre-freezing at-40 deg.C to-60 deg.C for 4-6h, naturally vacuum-drying at-40 deg.C to-7 deg.C for 24h, gradually heating to 4 deg.C, and maintaining for 10h to obtain FGF10 lyophilized powder.
Advantageous effects
The FGF10 freeze-dried powder can protect an active ingredient FGF10 from being damaged by freeze drying, can replace a conventional albumin freeze-drying protective agent, avoids blood-borne pollution, and is favorable for clinical popularization.
The FGF10 freeze-dried powder is very stable, and the storage life of FGF10 can be greatly prolonged.
Drawings
FIG. 1: expression and purification of recombinant human FGF 10.
Detailed Description
The present invention is described in more detail below to facilitate an understanding of the present invention.
It should be understood that the terms or words used in the specification and claims should not be construed as having meanings defined in dictionaries, but should be interpreted as having meanings that are consistent with their meanings in the context of the present invention on the basis of the following principles: the concept of terms may be defined appropriately by the inventors for the best explanation of the invention.
The experimental procedures, for which specific conditions are not noted in the following examples, are generally carried out according to the routine experimental procedures in the art or according to the conditions recommended by the manufacturers.
The experimental materials used in the present invention, except for recombinant human FGF10, were all commercially available.
Example 1: preparation of recombinant human FGF10 and activity determination thereof
Preparation of recombinant human FGF 10:
escherichia coli codon preference optimization is carried out according to the nucleotide sequence (accession number: AF508782.1) of FGF-10 of NCBI, a target gene is synthesized according to the 3 rd to 510bp nucleotide sequence of the target gene, and the target gene is connected into a cloning vector pUC57 through BamH I and Hind III enzyme cutting sites, so that a cloning vector pUC57-FGF10 is obtained.
And (2) carrying out plate streaking on a strain containing recombinant plasmid pUC57-FGF10 on a solid culture medium containing ampicillin resistance, then putting the strain into a thermostat at 37 ℃ for overnight culture, selecting a single escherichia coli colony the next day, inoculating the single escherichia coli colony into an LB liquid culture medium containing ampicillin, carrying out shake culture at 37 ℃ and 220r/min for 12-16h, extracting plasmid, carrying out double enzyme digestion, sequencing and identifying.
And selecting the strain with the correct identification result for the next prokaryotic expression step.
The prokaryotic expression vector pET28a and the extracted pUC57-FGF10 are subjected to double digestion by BamH I and Hind III respectively, the digested vector and FGF10 gene fragment are subjected to gel cutting and recovered respectively, pET28a and FGF10 are connected to obtain a vector pET28a-FGF10, and the vector pET28a-FGF10 is identified by double digestion and sequencing. Identifying a correct prokaryotic expression vector pET28a-FGF10 transformation competent escherichia coli E.coli DH5 alpha, selecting a recombinant expression vector pET28a-FGF10 bacterial colony for PCR identification, screening positive clones, culturing the screened positive clones in an ampicillin resistant liquid culture medium, and re-extracting plasmids for double enzyme digestion reaction and sequencing identification.
Selecting and identifying the correct positive clone to carry out prokaryotic expression and purification of FGF10, which is briefly described as follows:
transforming BL21(DE3) plysS into a recombinant plasmid pET28a-FGF10, picking a single colony to 5ml of LB liquid culture medium containing ampicillin resistance, carrying out overnight culture at 37 ℃ at 220r/min, transferring the single colony to 5ml of fresh LB liquid culture medium containing ampicillin resistance according to the ratio of 1:100 the next day, shaking the bacteria at 37 ℃ at 220r/min until OD600 is about 0.6, adding 0.5mmol/L IPTG (isopropyl-beta-thiogalactoside) to induce and culture at 20 ℃ for 4h, centrifuging at 12000r/min for 2min respectively to collect the bacteria, carrying out resuspension, carrying out ice bath ultrasonic disruption, centrifuging at 12000r/min for 2min at 4 ℃ to obtain a disrupted supernatant and a precipitate, taking the supernatant to carry out SDS-PAGE identification, and displaying that the supernatant contains a target protein FGF10 with a larger molecular weight of about 19 kDa.
The results are shown in FIG. 1.
Selecting recombinant human FGF-10(TONBO biosciences, cat # 21-7054) having an activity of about 2X 106Unit/mg, activity assay of recombinant human FGF10 was performed as a standard.
The activity determination method comprises the following steps:
BaF3 cells were washed and resuspended in DMEM medium (10% newborn bovine serum, L-glutamine) containing 2 μ g/ml heparin in medium plated at about 22500 cells per well in 96 well assay plates; the detection reagent was added to each well to make the volume of each well 200. mu.l, the cells were cultured at 37 ℃ for 2 days, 1. mu. Ci 3H-thymidine was added to each well in a volume of 50. mu.l, and after 4 to 5 hours, the cells were collected by filtration through glass fiber filter paper. The incorporated 3H-thymidine was counted using a Triathler liquid scintillation counter. And calculating the FGF-10 activity of the sample to be detected according to the counting results of the sample to be detected (such as freeze-dried powder renaturation solution) and the standard substance.
The activity of recombinant human FGF10 was determined according to the aforementioned FGF10 activity determination method, and it was found to be about 1.9X 106Unit/mg, equivalent to standard activity.
Example 2: preliminary screening of FGF10 lyophilized preparation
Freeze-drying can cause certain damage to the activity of the protein/polypeptide, the damage is mainly caused by two aspects, namely mechanical effect and solute effect, in order to protect the activity of the protein/polypeptide and facilitate the formation of the freeze-dried preparation, a freeze-drying protective agent is usually added, but different proteins/polypeptides have respective characteristics, and the required proper freeze-drying protective agents have respective differences. Therefore, there is a need to screen FGF10 for suitable lyoprotectants.
Selecting sucrose, trehalose, lactose, mannitol, dextran 40, albumin, glycine and sarcosine as single protective agents, preparing freeze-dried diluent in 50mM PBS buffer solution (pH9.2), freeze-drying to prepare freeze-dried powder, and determining activity after redissolving the freeze-dried powder.
The freeze-drying method comprises the following steps: adding a freeze-drying protective agent with a certain concentration into sterile double distilled water for dissolving, and filtering and sterilizing to prepare a protective agent diluent. In a clean bench, a certain amount of protecting group diluent prepared in advance and the recombinant human FGF10 protein solution prepared in example 1 are added into a penicillin bottle in a ratio of 1:1, the initial total volume is 1.0 mL/bottle, and the initial FGF10 activity is about 1.5 multiplied by 106unit/mL (protein content measured by absorptiometry, about 0.8mg per bottle), pre-freezing at-50 deg.C for 4-6h, naturally vacuum drying at-40 deg.C to-7 deg.C for 24h, gradually heating to 4 deg.C, and maintaining for 10 h.
The solubility determination method comprises the following steps: 3 samples were taken from each batch and 1ml of distilled water was added to each formulation and the time from the start to transparency and absence of insoluble material was recorded.
Biological activity assay method the activity assay method in example 1 was followed.
The results are shown in table 1:
as can be known from a single protectant screening test, 5mg of dextran 40 is added into each bottle (1mL) to obtain FGF10 activity protection effect basically equivalent to that obtained by adding 2mg of albumin, the prepared freeze-dried powder is milky and loose in appearance and meets the requirements of freeze-dried powder, and factors such as cost, blood-borne pollution of albumin and the like are comprehensively considered, so that 5mg of dextran 40 is initially selected to be added into each bottle to serve as FGF10 freeze-drying protectant.
Example 3: optimization of FGF10 lyophilized formulations
To further optimize the freeze-drying protective agent for FGF10, activity verification was performed with dextran 20 and dextran 70, and the results showed 5mg dextran 20 per vial (1.3. + -. 0.26X 10)6unit/mL) or 5mg per bottle of dextran 70(1.1 + -0.14X 10)6unit/mL), 5mg dextran 40 per bottle (1.65. + -. 0.16X 10)6unit/mL) has the best activity protection effect.
In addition, the concentration of dextran 40 was further optimized, and the results showed that the FGF10 activity was 1.3X 10 as compared to 2mg, 3mg, and 7mg of dextran 40 per vial (all at 1.3X 10)6Below unit/mL), 5mg of dextran 40 per vial had the best activity protection.
In the field of freeze drying, in order to obtain a better protection effect, two or more than two kinds of lyoprotectants are generally required to be compounded, so that the composite lyoprotectant containing dextran 40 is further discussed in the embodiment.
Lyophilization method and determination of solubility and biological Activity As in example 2, initial FGF10 Activity was about 1.5X 106units/mL (protein content determined by absorptiometry, which is about 0.8mg per vial).
The results are shown in table 2:
the results show that the composite lyophilized protectant consisting of dextran 40(5 mg/bottle), sorbitol (1 mg/bottle), trehalose (1 mg/bottle) can provide the best protective effect of FGF10 activity from the viewpoint of solubility, appearance and lyophilized powder FGF10 activity, and is easy to redissolve, therefore dextran 40(5 mg/bottle), sorbitol (1 mg/bottle), trehalose (1 mg/bottle) are preferred as the composite lyophilized protectant of FGF 10.
Protein/polypeptide is easy to oxidize and inactivate, and FGF10 contains a plurality of cysteine residues, which is high in risk of being easily oxidized, so in order to further improve the stabilizing and protecting effects of FGF10 freeze-dried preparation on FGF10, compounds with antioxidant activity are tried to be additionally added into a freeze-drying bottle, specifically anthocyanin, Daidzein (Daidzein), ginsenoside Rb2, curcumin and vitamin C are further added in an amount of 0.3 mg/bottle, namely 0.3 mg/bottle of the compounds with antioxidant activity are respectively added on the basis of a composite freeze-drying protective agent prepared from dextran, sorbitol and trehalose, and the activity of initial FGF10 is about 1.8 multiplied by 106units/mL (protein content determined by absorptiometry, which is about 0.9mg per vial) was repeated three times and lyophilized powder was assayed for FGF10 activity.
The result shows that the addition of other antioxidant active compounds has no obvious influence on the activity of FGF10 except that the addition of daidzein can obviously improve the activity of FGF 10.
Specific experimental data are shown in table 3:
as can be seen from the experimental data shown in table 3, the activity and stability of FGF10 can be significantly improved by the additional addition of daidzein.
Based on the experimental data in table 3, the protection effect of genistin and glycitin with similar structures to daidzein on FGF10 was further verified, and the results showed that 0.3 mg/bottle of genistin or glycitin and 0.3 mg/bottle of daidzein both have similar protection effect on FGF10 (FGF10 activity was maintained at about 2.20 × 106 units/mL).
Without being limited by theory, the specific structure of daidzein, genistin or glycitin can stabilize and activate FGF10, thereby significantly enhancing the activity of FGF 10.
Example 4: test examples
In this example, a composite lyophilized protectant containing dextran 40(5 mg/bottle), sorbitol (1 mg/bottle), and trehalose (1 mg/bottle) as FGF10 was used as an experimental group 1, dextran 40(5 mg/bottle) was used as an experimental group 2, a composite lyophilized protectant containing dextran 40(5 mg/bottle), sorbitol (1 mg/bottle), trehalose (1 mg/bottle), and daidzein (0.3 mg/bottle) as FGF10 was used as an experimental group 3, and an albumin lyophilized protectant (2 mg/bottle) was used as a control group to investigate the stability of lyophilized powder, wherein the initial biological activity of FGF10 was 1.4 × 106units/mL (protein content determined by absorptiometry, which is about 0.8mg per vial).
Accelerated stability testing: three batches of FGF10 freeze-dried powder prepared by an experimental group and a control group are respectively used for an accelerated stability experiment at 40 ℃, and freeze-dried powder which is frozen for 0 month and stored for 1-3 months is detected by the biological activity method of the embodiment 1.
The results are shown in Table 4:
as can be seen from the results in table 4, compared to the control group, the composite lyoprotectant of experiment group 1 and experiment group 3 and the dextran 40 lyoprotectant of experiment group 2 both have better protection effect on FGF10, and the protection effect of experiment group 3 is the best.
The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.
Claims (10)
1. A fibroblast growth factor 10(FGF10) freeze-dried powder is characterized in that: the freeze-dried powder is prepared by freeze drying of recombinant human FGF10, a freeze-drying protective agent and a pH regulator; the freeze-drying protective agent is dextran 40 or is composed of dextran 40, sorbitol and trehalose or is composed of dextran 40, sorbitol, trehalose and plant flavone.
2. Lyophilized powder according to claim 1, characterized in that: the pH regulator is PBS buffer solution.
3. Lyophilized powder according to claim 2, characterized in that: the pH adjusting agent was 50mM PBS buffer (pH 9.2).
4. Lyophilized powder according to claim 1, characterized in that: the dextran 40 content is 4-6 mg/vial (i.e. 4-6% (w/v concentration)).
5. Lyophilized powder according to claim 1, characterized in that: the content of dextran 40 in the freeze-drying protective agent is 4-6 mg/penicillin bottle (namely 4-6% (weight volume concentration)), the content of sorbitol is 0.5-2 mg/penicillin bottle (namely 0.5-2% (weight volume concentration)), and the content of trehalose is 0.5-2 mg/penicillin bottle (namely 0.5-2% (weight volume concentration)).
6. Lyophilized powder according to claim 1, characterized in that: the content of dextran 40 in the freeze-drying protective agent is 4-6 mg/penicillin bottle (namely 4-6% (weight volume concentration)), the content of sorbitol is 0.5-2 mg/penicillin bottle (namely 0.5-2% (weight volume concentration)), the content of trehalose is 0.5-2 mg/penicillin bottle (namely 0.5-2% (weight volume concentration)), and the content of plant flavone is 0.1-0.4 mg/penicillin bottle (namely 0.1-0.4% (weight volume concentration)).
7. Lyophilized powder according to claim 1, characterized in that: the plant flavone is one or more of daidzein, genistin and glycitin.
8. Lyophilized powder according to claim 6, characterized in that: the content of dextran 40 in the freeze-drying protective agent is 5 mg/penicillin bottle, the content of sorbitol is 1 mg/penicillin bottle, the content of trehalose is 1 mg/penicillin bottle, and the content of plant flavone is 0.3 mg/penicillin bottle.
9. A lyophilized powder according to any one of claims 1-8, wherein: the content of FGF10 in the freeze-drying protective agent is 0.5-1 mg/bottle, and preferably 0.8-0.9 mg/bottle.
10. The method for preparing the freeze-dried powder of claim 1, which is characterized in that: adding the freeze-drying protective agent into sterile double-distilled water for dissolving and filtering for sterilization to prepare protective agent diluent; dissolving the recombinant human FGF10 in a pH regulator solution to obtain a recombinant human FGF10 protein solution; adding the freeze-dried diluent and the recombinant human FGF10 protein solution into a penicillin bottle in a ratio of 1:1, wherein the initial volume is 1.0 mL/bottle; pre-freezing at-40 deg.C to-60 deg.C for 4-6h, naturally vacuum-drying at-40 deg.C to-7 deg.C for 24h, gradually heating to 4 deg.C, and maintaining for 10h to obtain FGF10 lyophilized powder.
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