CN103720642A - Bioprotein-containing solution for external use and preparation method thereof - Google Patents
Bioprotein-containing solution for external use and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a bioprotein-containing solution for external use and a preparation method thereof, in particular to a bioprotein active ingredient-containing solution for external use and a preparation method thereof, and bioprotein active ingredients such as fibroblast growth factors, epidermal growth factors and lecithin superoxide dismutase are used for treating burn injury, promoting healing of burn wounds including a superficial II wound, a deep II wound and a granulation wound, chronic wounds including chronic ulcer and the like and new wounds including trauma, a surgical wound and the like and also possibly treating fracture, periodontitis and diabetic skin ulcer in the future. According to the solution prepared by the method, any preservative or bacteriostat is not added, the infection of the wounds is avoided, the safety is high, a protecting agent in auxiliary materials is a special composition, a protein solution can be stored at low temperature for a long term, the defect that a common protein preparation needs to be freeze-dried and pulverized for stable storage is overcome, the solution is convenient to carry and use by patients, the use safety is improved, and high purity and high activity of protein in the solution can be guaranteed.
Description
Technical field
The invention belongs to field of pharmaceutical preparations, be specifically related to a kind of externally used solution that contains bioprotein active component and preparation method thereof.
Background technology
Due to the development of gene recombination technology, the commercialization of medical protein preparation is produced and is become a reality.Recently existing more than 140 kind of recombinant protein preparation carrying out I phase or clinical trial more than the I phase, has 12 kinds of approvals that obtained U.S. FDA.But, due to the physicochemical property of protein, it is existed when purification, separation, storage and sale exclusive because of difficulty.The degradation pathway of protein can be divided into two types, i.e. chemical instability and physical instability.The former means that protein is by becoming key or scission of link to generate new compound; Physical stability does not relate to the covalent bond variation of protein, but finger protein is changed into more senior structure (secondary or higher), comprises degeneration, surface adsorption, cohesion and precipitation.These physics and chemistry defects of pharmaceutical grade protein, have limited application and the development of its preparation.At present, in order to overcome the unstability of pharmaceutical grade protein, preserve after generally adopting Freeze Drying Technique to make freeze-dried powder, during use, redissolve again.Although lyophilized formulations is stable compared with liquid preparation, but lyophilization is a very complicated process, in first, second drying stage of pre-cooling and storage process, the physicochemical change that the structure of medicine may be regarded highly affects and changes, particularly two of protein and peptide class medicine, tertiary structure is easily damaged, lose activity and affect drug effect.During use, redissolve and also have all multi-risk Systems, as content heterogeneity, separate out impurity, have foreign body, cost is high, use is inconvenient etc.
Skin wound healing is complicated biological process, in this course, has many cells to participate in, and fibroblast is considered to one of main cell of repair in trauma.Recent study is found, various kinds of cell somatomedin, as b-FGF, aFGF, PDGF, TGF, IGF, epidermal growth factor etc. have huge effect in wound healing, their fibrosis, vascularization and re-epithelializations to skin wound have the effect of mediation and regulation and control, wound healing.Wherein, bFGF has to deriving from mesoderm and neuroectodermal cell (as epithelial cell, hypodermal cell, fibroblast, vascular endothelial cell etc.) effect that promotes reparation and regeneration, maintains and damage the aspects such as injury repairing to have significant curative effect in histo-differentiation, normal structure structure and physiological function.Research shows: it directly stimulates the albumen of fibroblast and extracellular matrix synthetic on the one hand, form collagen, on the other hand, can improve local microcirculation and histotrophic nutrition situation, promote into gratified cell, sarcoplast propagation, make granulation tissue ramp, edge of wound epithelium is creeped and wound surface is dwindled and heal to center rapidly.But because fibroblast growth factor, epidermal growth factor etc. is fusion rotein, easily be subject to the impact of various extraneous factors and reduce activity, the stability in preparation is very poor, affects therapeutic effect, high temperature or excessively cold environment also affect its biological activity, and albuminous degeneration easily occurs.
In current prior art; in order to solve the stability of protein formulation and the shortcoming of lyophilized formulations; Chinese patent application 200510036641.3 discloses a kind of externally used solution of recombination human basic fibroblast growth factor; wherein adopting heparin sodium is protective agent, realizes the cryopreservation of active polypeptide solution state.But, the main effectively activity index of ingredient r b-bFGF in externally used solution is not disclosed in this scheme, simultaneously, there is bibliographical information, heparin sodium can produce rare skin irritation as burn feeling, or anaphylaxis is as side effect such as erythra, prurituss, in the points for attention of the description of Hepudiod Cream, clearly write and understand that heparin sodium is not directly applied to and fester on wound and mucosal tissue, the healing of the wound surface of the skin ulcer that inapplicable and severe trauma, burn and scald and for example diabetes of some disease cause.From year January in mid-December, 2007 to 2008, special company of American Manufacturing Company hundred (Baxter International Inc.) receives continuously report and claims, nearly 350 people are after the heparin sodium (heparin) that has used the said firm to produce, produce serious anaphylaxis, wherein four people are dead.This is an in recent years larger heparin sodium Adverse Event.
In addition; Chinese patent application 201110342458.1 discloses bFGF bovine basic fibroblast growth factor externally used solution; wherein adopting human albumin is protein protective agent; in order to keep the stability of solution, a kind of in solution protective agent mannitol and Polyethylene Glycol or the combination of the two have also been added.Although this invention case has avoided employing heparin class material if heparin sodium is as protective agent; but the protective agent human albumin who adopts is blood products, although through processing, some materials wherein may make receiver produce anaphylaxis; or even infection virus, for example HIV (human immunodeficiency virus).For burn wound, particularly contain the wound surface festering and easily produce infection, also have human albumin to produce to shiver, the report of the symptom side effect such as heating, Blushing, erythra, nausea and vomiting.And mannitol is relevant to the protective effect of protein and its concentration, morphosis, the mannitol of crystalline state loses defencive function; 1% or the mannitol of lower concentration by the formation of undefined structure, stop the gathering of protein molecule, but the mannitol of high concentration is easy to form crystallization, can promote the gathering of protein, therefore, it adds can produce very large risk to protein denaturation.Studies show that, adding of human albumin and mannitol or Polyethylene Glycol, can reduce activity and the purity of activated protein after placement.
In view of current protein solution type preparation, especially for thering is burn, wound healing, the difficulty that the externally used solution agent at position such as fester exists and and not enough, the inventor has carried out research with great concentration, be surprised to find that with the form of calcium magnesium potassium salt and be extensively present in the acid of planting in plant seed, to maintaining the stable environment of protein solution agent, the cryopreservation of realizing protein solution agent has important function, planting acid is also present in animal erythroblast, can promote the release of oxygen in HbO2 Oxyhemoglobin, improve erythrocyte function, extend the existence of erythrocyte, the pharmacologically active itself having can play synergism with activated protein, particularly to relating to activated protein for wound healings such as burning as fibroblast growth factor, epidermal growth factor has synergism.Natural phytic acid is fabulous antioxygenic property also, has avoided as maintaining the stable antioxidant that adds other of protein active etc. in protein solution agent.
Summary of the invention
The object of this invention is to provide a kind of externally used solution that contains bioprotein and preparation method thereof; in this externally used solution, do not contain antiseptic and antibacterial; guaranteed with the wound surface of wound and the safety of the wound surface use of festering; avoid side effect and the defect of in prior art, using heparin class material and human albumin to produce as protective agent simultaneously, realized the cryopreservation of protein solution without lyophilizing.The preparation method such as aseptic thin-film filtering, best pH regulator step is simple, favorable reproducibility, is suitable for industrialized great production.Externally used solution agent sterile filling, in spray bottle, facilitates patient to use, and prevents from producing pollution to remaining medicinal liquid in bottle.
In conjunction with existing achievement in research, the present invention seeks to be achieved through the following technical solutions:
An externally used solution that contains bioprotein, volume ratio (g/100ml) is calculated by weight, and the amount that contains bioprotein is that 5-50mg/100mL, buffer system are 0.5-2g/100mL, stabilizing agent, protective agent, and surplus is water for injection.
Wherein, contained bioprotein is any one in fibroblast growth factor, epidermal growth factor, superoxide dismutase, Lecithinized superoxide dismutase, laminin，LN, human complement factor H, lipoprotein lipase, amphiregulin, is wherein preferably fibroblast growth factor, epidermal growth factor, Lecithinized superoxide dismutase.
Wherein, described fibroblast growth factor is acid fibroblast growth factor (aFGF) or basic fibroblast growth factor (bFGF).
Wherein, contained buffer system is selected from citric acid/sodium citrate system or sodium dihydrogen phosphate/sodium hydrogen phosphate system or phosphate buffer or citric acid/sodium dihydrogen phosphate system or citric acid/sodium hydrogen phosphate system.
Wherein, contained stabilizing agent is the compositions that 5-30mg/100mL surfactant and 10-50mg/100mL are selected from low molecular dextran or ethylenediaminetetraacetic acid or disodiumedetate or sodium ethylene diamine tetracetate calcium any one.
Wherein, surfactant be selected from that Tween 80, amino acid pattern, fatty acid Pyrusussuriensis are smooth, any one in dodecyl sodium sulfate.
Wherein, contained protective agent is that 1-10g/100mL glycerol and 5-50mg/100mL are selected from any one the compositions in sucrose octasulfate sodium, dextran sulfate, phytic acid, matter acid sodium, phytic acid calcium, phytic acid magnesium, Monopotassium phytate, phosvitin, ammonium polyphosphate, inositol six sulfuric esters.
Wherein, prepare the method for bioprotein externally used solution, step is as follows:
(1) in the clean area of local laminar flow, the buffer system of recipe quantity, stabilizing agent, protective agent are dissolved in 100ml water for injection, use 0.5-2mol/L hydrochloric acid or sodium hydrate regulator solution pH value to 4-6;
(2) with the degerming of 0.22um membrane filtration, collect filtrate, add and inject water to 1L;
(3) the sterilized bio albumen of interpolation recipe quantity mixes;
(4) press every bottle of 2.5ml loading amount sterile filling to spray bottle, fill aseptic nitrogen;
(5) use plastic bag to complete packing in ten thousand grades of clean areas, be stored in 2-8 ℃.
The beneficial effect that technical scheme of the present invention produces comprises:
(1) having overcome activated protein preparation needs freeze drying process could stablize the defect of preserving, and has realized activated protein with solution state cryopreservation;
(2) overcome and in prior art, reassembled into the side effect that fibroblast growth factor externally used solution adopts heparin sodium to produce for protective agent;
(3) human albumin who has overcome available technology adopting blood source for activated protein solution protective agent produce batch between poor repeatability, have the defect of irritated risk;
(4), not containing any antiseptic, antibacterial, burn wound, the wound surface that festers are used to safety;
(5) preparation method is simple, has guaranteed the technique of sterile working, and fill is in spray bottle, easy to use, with after do not pollute residue medicinal liquid;
(6) take natural origin, plant acid as protein protective agent, be conducive to maintaining of activated protein stable environment, and phytic acid itself is exactly the nutriment useful to human body, phytic acid hydrolyzate in human body is inositol and phospholipid, the former has anti-aging effects, and the latter is human body cell important component part;
(7) plant acid most metal ions are had to extremely strong complexing power, complexation power is similar to EDTA, but the range of application than EDTA is wider, the ion that adsorbable various containers or filling bottle are separated out due to long-term placement etc., the stability of assurance solution environmental
(8) each phytic acid molecule can provide the electronics that six pairs of hydrogen atoms make free radical to form rock-steady structure, thereby replaces by fresh-keeping thing molecule as for oxygen molecule, can be used as natural antioxidant and avoids activated protein oxidation inactivation, avoids adding more adjuvant.
Accompanying drawing explanation
Fig. 1 is the purity liquid chromatogram that the embodiment of the present invention 1 makes externally used solution bFGF.
Fig. 2 is the purity liquid chromatogram take human albumin as protectant externally used solution bFGF in comparative example 1.
The specific embodiment
The specific embodiment of form by the following examples, is described in further detail content of the present invention.But should not be understood as scope of the present invention and only limit to following examples.All technical schemes realizing based on content of the present invention all belong to scope of the present invention.Obviously, according to content of the present invention, according to ordinary skill knowledge and the customary means of this area, do not departing under the prerequisite of basic fundamental thought of the present invention, can also make modification, replacement and the change of other various ways.
The preparation prescription of embodiment 1:rh-bFGF externally used solution:
Citric acid 2.9g
Sodium citrate 10.7g
Tween80 0.1g
Dextran 0.185g
Glycerol 10g
Phytic acid 0.05g
Water for injection adds to 1L recombination human basic fibroblast growth factor 100mg and makes altogether 400.
Concrete preparation method is as follows:
(1) in the clean area of local laminar flow, the buffer system of recipe quantity, stabilizing agent, protective agent are dissolved in 100ml water for injection, use 0.5-2mol/L hydrochloric acid or sodium hydrate regulator solution pH value to 4-6;
(2) with the degerming of 0.22um membrane filtration, collect filtrate, add and inject water to 1L;
(3) the sterilized bio albumen of interpolation recipe quantity mixes;
(4) press every bottle of 2.5ml loading amount sterile filling to spray bottle, fill aseptic nitrogen;
(5) use plastic bag to complete packing in ten thousand grades of clean areas, be stored in 2-8 ℃.
Purity test: adopt liquid chromatography to carry out the detection of activated protein purity.
Detection method:
Detect wavelength: 215nm flow velocity 1.0 column temperatures: 40 ℃ of chromatographic columns: C-18 sampling volume: 200 μ l
Mobile phase A: water-0.1% trifluoroacetic acid Mobile phase B: acetonitrile-0.08% trifluoroacetic acid
Gradient:
Time B%
0.1 10
5 20
30 50
40 70
45 10
60 10
Active inspection: the method derives from the 3rd appendix of version Chinese Pharmacopoeia in 2010.
Test principle: the bFGF of debita spissitudo has promotion proliferation function to BALB/c 3T3 cell, MTT can be reduced to quantitatively MTT Formazan(first a ceremonial jade-ladle, used in libation in the mitochondrion of living cells), amount by colorimetric method for determining MTT Formazan can direct representation BALB/c 3T3 cell growth conditions, by the extension rate of 50% ceiling effect point, can convert as tiring in product to be checked, measurement result is proofreaied and correct with determination of activity standard substance.
Test method:
First day:
(1) bed board: digestion and collection BALB/c 3T3 cell, by complete medium 1(20%BCS DMEM culture medium) be made into 8.0 × 10
4the cell suspension of/ml, is inoculated in 96 porocyte culture plates, every hole 100 μ l, 37 ℃, 5%CO
2under condition, cultivate 18-24 hour.
Second day:
(2) hunger: get Tissue Culture Plate prepared by previous step, suck each hole supernatant, add complete medium 2(0.4% BCS DMEM culture medium), every hole 100 μ l.37 ℃, 5%CO
2under condition, cultivate 18-24 hour.
The 3rd day:
(3) prepare sample solution: sample thief is according to its protein concentration, and with complete medium 2, diluting in advance certain multiple to protein concentration is 1 μ g/ml left and right, then in 96 orifice plates, by 4 times, carries out gradient dilution, and each dilution factor does 2-3 multiple hole.
(4) application of sample: get Tissue Culture Plate prepared by the 2nd step, suck each hole supernatant, the sample having diluted is joined in each hole, every hole adds 100 μ l.37 ℃, 5%CO
2under condition, cultivate 64-72 hour.
The 6th day:
(5) add MTT solution and cultivate: every hole adds 20 μ l MTT solution (5.0mg/ml), 37 ℃, 5%CO
2under condition, cultivate 4-5 hour.
(6) add lysate measurement result: suck each hole supernatant, add lysate (DMSO) 100 μ l, after dissolving mixes, with microplate reader mensuration 570/630nm absorbance.
Result is calculated: adopt four parametric methods to carry out matching to result, that calculates respectively each sample partly imitates extension rate, then calculation sample IC50 etc. respectively.
Embodiment 2:
The preparation prescription of aFGF externally used solution:
Sodium dihydrogen phosphate 2.5g
Sodium hydrogen phosphate 2.5g
Dodecyl sodium sulfate 0.05g
EDTA 0.1g
Glycerol 20g
Monopotassium phytate 0.1g
Water for injection adds to 1L
AFGF 100mg makes 400 altogether.
Concrete preparation method is as follows:
(1) in the clean area of local laminar flow, the buffer system of recipe quantity, stabilizing agent, protective agent are dissolved in 100ml water for injection, use 0.5-2mol/L hydrochloric acid or sodium hydrate regulator solution pH value to 4-6;
(2) with the degerming of 0.22um membrane filtration, collect filtrate, add and inject water to 1L;
(3) the sterilized bio albumen of interpolation recipe quantity mixes;
(4) press every bottle of 2.5ml loading amount sterile filling to spray bottle, fill aseptic nitrogen;
(5) use plastic bag to complete packing in ten thousand grades of clean areas, be stored in 2-8 ℃.
Purity test and the active method checking are with embodiment 1.
Embodiment 3:
The preparation prescription of bFGF externally used solution:
Citric acid 3g
Sodium citrate 7g
Tween80 0.1g
EDTA-2Na 0.215g
Glycerol 50g
Phytic acid 0.15g
Water for injection adds to 1L
BFGF 50mg makes 400 altogether.
Concrete preparation method is as follows:
(1) in the clean area of local laminar flow, the buffer system of recipe quantity, stabilizing agent, protective agent are dissolved in 100ml water for injection, use 0.5-2mol/L hydrochloric acid or sodium hydrate regulator solution pH value to 4-6;
(2) with the degerming of 0.22um membrane filtration, collect filtrate, add and inject water to 1L;
(3) the sterilized bio albumen of interpolation recipe quantity mixes;
(4) press every bottle of 2.5ml loading amount sterile filling to spray bottle, fill aseptic nitrogen;
(5) use plastic bag to complete packing in ten thousand grades of clean areas, be stored in 2-8 ℃.
Purity test and the active method checking are with embodiment 1.
Embodiment 4:
The preparation prescription of epidermal growth factor (EGF) externally used solution:
Citric acid 8g
Sodium dihydrogen phosphate 7g
Cysteine 0.3g
EDTA-2Na 0.3g
Glycerol 100g
Sodium phytate 0.2g
Water for injection adds to 1L
Epidermal growth factor 300mg makes 400 altogether.
Concrete preparation method is as follows:
(1) in the clean area of local laminar flow, the buffer system of recipe quantity, stabilizing agent, protective agent are dissolved in 100ml water for injection, use 0.5-2mol/L hydrochloric acid or sodium hydrate regulator solution pH value to 4-6;
(2) with the degerming of 0.22um membrane filtration, collect filtrate, add and inject water to 1L;
(3) the sterilized bio albumen of interpolation recipe quantity mixes;
(4) press every bottle of 2.5ml loading amount sterile filling to spray bottle, fill aseptic nitrogen;
(5) use plastic bag to complete packing in ten thousand grades of clean areas, be stored in 2-8 ℃.
Purity test and the active method checking are with embodiment 1.
Embodiment 5:
The preparation prescription of bFGF externally used solution:
Citric acid 4g
Sodium hydrogen phosphate 16g
The smooth 0.2g of fatty acid Pyrusussuriensis
EDTA 0.42g
Glycerol 80g
Phosvitin 0.3g
Water for injection adds to 1L bFGF 400mg and makes altogether 400.
Concrete preparation method is as follows:
(1) in the clean area of local laminar flow, the buffer system of recipe quantity, stabilizing agent, protective agent are dissolved in 100ml water for injection, use 0.5-2mol/L hydrochloric acid or sodium hydrate regulator solution pH value to 4-6;
(2) with the degerming of 0.22um membrane filtration, collect filtrate, add and inject water to 1L;
(3) the sterilized bio albumen of interpolation recipe quantity mixes;
(4) press every bottle of 2.5ml loading amount sterile filling to spray bottle, fill aseptic nitrogen;
(5) use plastic bag to complete packing in ten thousand grades of clean areas, be stored in 2-8 ℃.
Purity test and the active method checking are with embodiment 1.
Embodiment 6:
The preparation prescription of aFGF externally used solution:
Sodium dihydrogen phosphate 8g
Sodium hydrogen phosphate 9g
Arginine 0.3g
EDTANaCa 0.5g
Glycerol 30g
Dextran phosphate 0.5g
Water for injection adds to 1L
AFGF 500mg makes 400 altogether.
Concrete preparation method is as follows:
(1) in the clean area of local laminar flow, the buffer system of recipe quantity, stabilizing agent, protective agent are dissolved in 100ml water for injection, use 0.5-2mol/L hydrochloric acid or sodium hydrate regulator solution pH value to 4-6;
(2) with the degerming of 0.22um membrane filtration, collect filtrate, add and inject water to 1L;
(3) the sterilized bio albumen of interpolation recipe quantity mixes;
(4) press every bottle of 2.5ml loading amount sterile filling to spray bottle, fill aseptic nitrogen;
(5) use plastic bag to complete packing in ten thousand grades of clean areas, be stored in 2-8 ℃.
Purity test and the active method checking are with embodiment 1.
Comparative example
According to the prescription of Chinese patent application 201110342458.1, the externally used solution of preparing take human albumin as protective agent.Adopt liquid chromatography and active method to detect.
Test example: accelerate the comparison of experiment stability
At 25 ℃, lucifuge is placed 60 days results
Embodiment purity actives
Embodiment 1 96.5 % 9.86 × 105U/mg
Embodiment 2 97.1 % 9.69 × 105U/mg
Embodiment 3 98.4 % 1.09 × 106U/mg
Embodiment 4 96.6 % 9.60 × 105U/mg
Embodiment 5 96.0 % 9.12 × 105U/mg
Embodiment 6 96.2 % 9.03 × 105U/mg
Comparative example 77.2 % 5.06 × 104U/mg
Test example: long-term experiment stability comparison
At 4 ℃, lucifuge is placed 360 days results
Embodiment purity actives
Embodiment 1 97.1 % 1.01 × 106U/mg
Embodiment 2 97.7 % 9.72 × 105U/mg
Embodiment 3 97.8 % 1.04 × 106U/mg
Embodiment 4 96.6 % 9.34 × 105U/mg
Embodiment 5 96.8 % 9.14 × 105U/mg
Embodiment 6 96.3 % 8.89 × 105U/mg
Comparative example 76.4 % 9.02 × 103U/mg
From accelerated test and long term test, comparative example purity and active decline obviously, be not suitable for long-term preservation, and embodiment can keep most purity and activity substantially, illustrate that the prescription formation that this patent relates to can keep the stable of active component effectively.
Trauma model efficiency assay
1, animal model is chosen:
Select the Chinese miniature pig of three 25kg to carry out animal experiment, after anesthesia, at each back, cause 8 of the dark 11 ° of burn woundes of diameter 3cm respectively, with the normal saline that does not contain active component, do negative control, with the positive contrast of comparative example.
2, administering mode:
After debridement, the wound surface of positive controls directly sprays comparative example solution.All the other each group is undertaken by double-blind method, in the mode of three parallel tests, respectively by example 1 to the externally used solution of gained in example 6 from wound surface 5cm part spray 5 times, with clean gauze, cover.With same method spray medicine once, share medicine 13 days every day later.
3, wound healing index:
When changing dressings, measure wound surface diameter, diameter is less shows that promoting healing effect is better at every turn.
4, result:
Each sample average wound surface diameter (cm)
Natural law embodiment comparative example negative control
1 2 3 4 5 6
0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0
2 2.6 2.8 2.7 2.8 2.7 2.7 2.8 3.0
4 2.3 2.5 2.1 2.5 2.4 2.4 2.5 2.9
6 1.7 1.5 1.3 1.6 1.8 1.4 2.4 2.5
9 1.1 1.3 0.9 1.2 1.5 1.1 2.0 2.2
13 0.6 0.4 0.1 0.6 0.8 0.5 1.1 2.1
As can be seen from the results, embodiment is better than comparative example, illustrates that the prescription formation that this patent relates to can not affect drug effect, can promote albumen more to play consistently physiological activity, without any stimulation and sensitization.
Claims (8)
1. contain an externally used solution for bioprotein, it is characterized in that volume ratio (g/100ml) is calculated by weight, the amount that contains bioprotein is that 5-50mg/100mL, buffer system are 0.5-2g/100mL, stabilizing agent, protective agent, and surplus is water for injection.
2. a bioprotein externally used solution according to claim 1, it is characterized in that contained bioprotein is any one in fibroblast growth factor, epidermal growth factor, superoxide dismutase, Lecithinized superoxide dismutase, laminin，LN, human complement factor H, lipoprotein lipase, amphiregulin, is wherein preferably fibroblast growth factor, epidermal growth factor, Lecithinized superoxide dismutase.
3. a bioprotein externally used solution according to claim 2, is characterized in that described fibroblast growth factor is acid fibroblast growth factor (aFGF) or basic fibroblast growth factor (bFGF).
4. a bioprotein externally used solution according to claim 1, is characterized in that contained buffer system is selected from citric acid/sodium citrate system or sodium dihydrogen phosphate/sodium hydrogen phosphate system or citric acid/sodium dihydrogen phosphate system or citric acid/sodium hydrogen phosphate system.
5. a bioprotein externally used solution according to claim 1, is characterized in that contained stabilizing agent is the compositions that 5-30mg/100mL surfactant and 10-50mg/100mL are selected from low molecular dextran or ethylenediaminetetraacetic acid or disodiumedetate or sodium ethylene diamine tetracetate calcium any one.
6. a bioprotein externally used solution according to claim 4, is characterized in that, wherein surfactant be selected from that Tween 80, amino acid pattern, fatty acid Pyrusussuriensis are smooth, any one in dodecyl sodium sulfate.
7. a bioprotein externally used solution according to claim 1, is characterized in that contained protective agent is that 1-10g/100mL glycerol and 5-50mg/100mL are selected from any one the compositions in sucrose octasulfate sodium, dextran sulfate, phytic acid, matter acid sodium, phytic acid calcium, phytic acid magnesium, Monopotassium phytate, phosvitin, ammonium polyphosphate, inositol six sulfuric esters.
8. a method of preparing bioprotein externally used solution according to claim 1, step is as follows:
(1) in the clean area of local laminar flow, the buffer system of recipe quantity, stabilizing agent, protective agent are dissolved in 100ml water for injection, use 0.5-2mol/L hydrochloric acid or sodium hydrate regulator solution pH value to 4-6;
(2) with the degerming of 0.22um membrane filtration, collect filtrate, add and inject water to 1L;
(3) the sterilized bio albumen of interpolation recipe quantity mixes;
(4) press every bottle of 2.5ml loading amount sterile filling to spray bottle, fill aseptic nitrogen;
(5) use plastic bag to complete packing in ten thousand grades of clean areas, be stored in 2-8 ℃.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104645313A (en) * | 2015-01-28 | 2015-05-27 | 南京航空航天大学 | Mussel mucoprotein gel for repairing and reliving itching and preparation method of mussel mucoprotein gel |
| CN112569346A (en) * | 2019-09-29 | 2021-03-30 | 国家卫生健康委科学技术研究所 | Hydrogel for promoting wound healing and preparation method thereof |
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| US5348941A (en) * | 1992-04-01 | 1994-09-20 | Merck & Co., Inc. | Stabilizers for fibroblast growth factors |
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| 李国辉: "重组人碱性成纤维细胞生长因子体外稳定性的研究", 《中国优秀博硕士学位论文全文数据库（硕士） 医药卫生科技辑》, 15 January 2005 (2005-01-15), pages 059 - 25 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104645313A (en) * | 2015-01-28 | 2015-05-27 | 南京航空航天大学 | Mussel mucoprotein gel for repairing and reliving itching and preparation method of mussel mucoprotein gel |
| CN112569346A (en) * | 2019-09-29 | 2021-03-30 | 国家卫生健康委科学技术研究所 | Hydrogel for promoting wound healing and preparation method thereof |
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| CN103720642B (en) | 2015-09-16 |
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