CN101224296A - Stable recombinant human endostain preparation and preparing method thereof - Google Patents
Stable recombinant human endostain preparation and preparing method thereof Download PDFInfo
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- CN101224296A CN101224296A CNA2008100060577A CN200810006057A CN101224296A CN 101224296 A CN101224296 A CN 101224296A CN A2008100060577 A CNA2008100060577 A CN A2008100060577A CN 200810006057 A CN200810006057 A CN 200810006057A CN 101224296 A CN101224296 A CN 101224296A
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Abstract
The invention discloses a stable preparation of a recombinant human vascular endothelial inhibin and a preparation technique thereof; the preparation contains 0.05-65 percent of the recombinant human vascular endothelial inhibin and 1-90 percent of protective agent by weight percentage. The recombinant human vascular endothelial inhibin is carried out ultrafiltration dialysis in a PH value buffer system; the protective agent is dissolved in the water for injection, and is carried out ultrafiltration dialysis after being filtered; the dialysis fluids are mixed, filtered, sterilized, frozen dried, and then separated into a sterile container. The freeze dried preparation produced by the invention is loose, porous and high solubility; under the action of the optimized protective agent, the invention can keep stable in long-term storage.
Description
Technical field
The present invention relates to the biological preparation field, be specifically related to stable recombinant human endostain preparation and preparation technology.Improve recombinant human endothelial inhibin stability of formulation by adding protective agent; can be through the stable higher recombinant human endothelial inhibin preparation that process optimization obtains for clinical use, said preparation can be used for treatment of diseases such as malignant tumor, angiogenic retinopathy and endometriosis.
Background technology:
Human Endostatin (endostatin) is studied one of angiogenesis inhibitor the most widely.It is the segment that collagen XVIII carboxyl (C) end molecule amount is about 20KD, has the function of inhibition of endothelial cell proliferation and migration.
Recombinant human vascular endothelial inhibin uses genetic engineering means to obtain mostly at present, by people's vascellum esoderma inhibin is gene constructed in escherichia coli (E.Coli) or Pichia sp. (Pichiapastoris) expression system, by fermentation, means such as separation, the purification vascellum esoderma inhibin that obtains having biologic activity.Also have with through changing vascellum esoderma inhibin structure, amino acid modified gene constructed in the expression system to reach the purpose of simplifying technology, reducing cost, obtain high stability and bioactive vascellum esoderma inhibin.
(patent publication No. is CN1324818A to Chinese invention patent, it open day was calendar year 2001 December 5 days, denomination of invention is " method of producing endostatin ") a kind of improvement is provided, with the method for higher productive rate and simpler purifying process production recombinant human endothelial inhibin.This invention utilizes DNA recombined engineering technology, with escherichia expression system, produce the human Endostatin (wherein Xaa represents neutral amino acid or do not exist) that N-terminal has the additional aminoacid sequence of (Met) GlyGlyXaaHisHisHisHisHis in enormous quantities with simple method and lower cost, not only kept the biologic activity completely of endostatin, and do not produced because of adding immunity in the body due to the additional N terminal sequence.Above-mentioned additional aminoacid sequence has not only improved transcribing and translation initiation efficient of endostatin structural gene, helps the purification of recombiant protein.
Recombinant human vascular endothelial inhibin has been applied to clinical and has demonstrated vast market prospect so far, has reported that the injected dose of clinical practice vascellum esoderma inhibin is generally 7.5-15mg/m
2, even higher.Therefore this minimum package unit that just requires every medication is 15-30mg, and to need stable formulation to be applied to clinical for the biological activity that keeps endostatin.Existing recombinant human vascular endothelial inhibin aqueous injection is that purifying protein is dissolved in the water for injection, and its stability is not ideal enough, and the stability that improves existing recombinant human endostain preparation is to guarantee that clinical therapeutic efficacy is that a technical problem to be solved is arranged.Research about recombinant human vascular endothelial inhibin stabilization formulations aspect at present has not yet to see relevant report.
Summary of the invention:
For this class biopharmaceutical macromolecular drug of protein, determine its biologic activity by higher structure, and the factor that influences its structure has a lot, as: pH, temperature, ionic strength, metal ion, oxidant Reducing agent etc.These influences single or composite factor will influence proteinic physical stability and chemical stability.Influence to physical stability shows: higher structure degeneration, protein aggregation and albumen precipitation, surface adsorption etc.Influence to chemical stability then shows: deaminizating, hydrolysis, racemization, disulfide bond mispairing, oxidation or the like.The inventor is through a large amount of screening tests, and the protective agent that adds the stabilize proteins native conformation has obtained stable ideal recombinant human endostain preparation, makes it can be convenient to transportation and long-term storage under suitable storage requirement.
The recombinant human endostain preparation that one of purpose of the present invention provides a kind of steady quality, easily stores, is convenient to transport.
Another object of the present invention provides a kind of preparation technology of stable recombinant human endostain preparation.
Stable recombinant human endostain preparation of the present invention contains the recombinant human vascular endothelial inhibin of 0.05%-65% and the protective agent of 1%-90% by weight percentage.
Described herein recombinant human vascular endothelial inhibin has kept the biologic activity completely of endostatin, can use genetic engineering means to obtain, it is gene constructed in escherichia coli (E.Coli) or Pichia sp. (Pichia pastoris) expression system maybe will to change human Endostatin structure, amino acid modified by the vascellum esoderma inhibin gene with the people, by fermentation, means such as separation, purification obtain.
Preferred recombinant human vascular endothelial inhibin is to add 0-4 arbitrary amino acid and 2-8 histidine sequence behind the initial amino acid Met of human Endostatin encoding amino acid sequence successively among the present invention.The same Chinese invention patent of its preparation method (patent publication No. is CN1324818A, open day be calendar year 2001 December 5 days, and denomination of invention is " method of production endostatin ").
The initial amino acid Met of above-mentioned recombinant human vascular endothelial inhibin may be cut, and reason is may be deleted in the protein translation post-treatment process in expressive host.
The aminoacid sequence of the preferred recombinant human vascular endothelial inhibin of the present invention is shown in SEQ ID NO:1, this sequence is identical with the disclosed SEQ IDNO:2 of Chinese invention patent (patent publication No. is CN1324818A), and the proteinic preparation method that this sequence constitutes is identical with the preparation embodiment of above-mentioned publication.
In the stabilization formulations of the invention described above, also contain the cosolvent of 0.05%-35% by weight percentage.Wherein, recombinant human vascular endothelial inhibin further is optimized for 0.1%-60%, and protective agent is 5%-80% more preferably.Protective agent among the present invention can be stablized the native conformation of recombinant human vascular endothelial inhibin, and cosolvent can increase it at the dissolubility of aqueous phase and improve clarity.
The stabilization formulations of the invention described above can be injection or lyophilized formulations.The stable injection or the preparation technology of lyophilized formulations describe in detail in the embodiment of this paper.
The recombinant human vascular endothelial inhibin of the invention described above can be the recombinant human vascular endothelial inhibin of chemical purification, recombinant human vascular endothelial inhibin or the recombinant human vascular endothelial inhibin microsphere that PEG modifies.The total average molecular weight of wherein said PEG is 5000-30000, and PEG links to each other with the recombinant human vascular endothelial inhibin molecule by covalent bond; Wherein said recombinant human vascular endothelial inhibin microsphere is to be mixed with by host material and recombinant human vascular endothelial inhibin to form, and its host material can be polylactic-co-glycolic acid block copolymer (PLGA), polyglycolic acid (PLA) etc.
The stabilization formulations of the invention described above also can add the PH regulator, and its scope of regulating pH value is 4.0-8.5.Described PH regulator can be one or more in dibastic sodium phosphate, potassium hydrogen phosphate, Tris-HCl, sodium citrate-citric acid, potassium citrate-citric acid, sodium acetate-acetic acid, the sodium borate-boric acid.
Protective agent in the invention described above comprises at least a in polyhydric alcohol, saccharide, aminoacid and the albumin, and wherein said polyhydric alcohol comprises at least a in glycerol, mannitol, the sorbitol; Described saccharide comprises at least a in glucose, sucrose, dextran, trehalose, lactose, the beta-schardinger dextrin-; Described aminoacid comprises at least a in arginine, glycine, proline, aspartic acid, threonine, serine, glutamic acid, the leucine.Above-mentioned protective agent preferably includes at least a in mannitol, trehalose, sucrose, glycine, threonine and the human albumin.
Cosolvent in the invention described above comprises surfactant and/or salt, and wherein said surfactant comprises at least a among tween 80 and the pluronic F-68; Described salt comprises at least a among EDTA and the NaCl.Above-mentioned cosolvent preferably includes at least a among tween 80 and the EDTA.
The preparation technology of the recombinant human endostain preparation of aforementioned stable is:
1), the recombinant human vascular endothelial inhibin protein solution and the protective agent of prepare purifying, wherein by accounting for final formulation products weight percent meter, recombinant human vascular endothelial inhibin accounts for 0.05%-65%, protective agent accounts for 1%-90%;
2), use pH buffer system is carried out ultrafiltration dialysis to the recombinant human vascular endothelial inhibin protein solution of above-mentioned purification;
3), above-mentioned protective agent is dissolved in the injection water ultrafiltration dialysis after filtering;
4), with step 2) and 3) dialysis solution mixes, filtration sterilization is sub-packed in the sterile chamber.
Can also increase following step among the above-mentioned preparation technology: take by weighing the cosolvent of 0.05%-35% by weight percentage, above-mentioned cosolvent is dissolved in the water for injection, filter the back ultrafiltration dialysis, again with step 2) and 3) the dialysis solution mixing.
Above-mentioned steps 2) molecular cut off of the used ultrafilter membrane of ultrafiltration dialysis is 5-6kD in; The scope that the PH buffer system is regulated pH value is 4.0-8.5.
After the step of above-mentioned preparation technology's the described heat sterilization of step 4), can increase following freeze-dry process step: the medicinal liquid after the degerming is sub-packed in the container, the container that branch is installed medicinal liquid is positioned in advance and is cooled to-the freezing in the case of 35--45 ℃, be incubated after 3-6 hour, evacuation, after the secondary temperature elevation distillation, take out behind the vacuum gland.
Above the distillation of described secondary temperature elevation be meant that for the first time sublimation temperature is-30--10 ℃, persistent period 8-12 hour, distil 25-30 ℃ for the second time, continue 4-10 hour.
According to the needs of clinical practice, the stabilization formulations among the present invention can be made into the specification that 1-50mg/ props up, and wherein preferred specification comprises following several:
Liquid preparation: 1mg/ml/ props up, and 10mg/2.5ml/ props up, and 10mg/2ml/ props up, and 15mg/3.75ml/ props up, and 15mg/3ml/ props up, and 15mg/1.5ml/ props up, and 30mg/3ml/ props up, and 50mg/5ml/ props up.
Lyophilized formulations: 1mg/ props up, and 2mg/ props up, and 5mg/ props up, and 10mg/ props up, and 15mg/ props up; 30mg/ props up; 60mg/ props up.
Endostatin preparation of the present invention can be for clinical using method: liquid preparation can add and carries out intravenous drip in the 250-500ml normal saline, also can carry out subcutaneous injection or focus local injection.Dissolubility after lyophilized formulations provided by the invention redissolves reaches as high as 50-80mg/ml, presses the liquid preparation method after freeze-dried powder preparation redissolves with normal saline or injection water and uses.Stabilization formulations of the present invention also can be according to therapeutic purposes with other medicines or cooperate other Therapeutic Method use in conjunction except that independent use.
The reasonable recipe of stable recombinant human endostain preparation of the present invention, preparation technology is simple, through quickening and long-time stability are investigated the product stability height.The present invention's recombinant human vascular endothelial inhibin activity under preferred prescription and process conditions is not destroyed, and does not produce polymer and degradation product.The lyophilized formulations product that the present invention makes, loose porous, the dissolubility height, the dissolubility after the redissolution reaches as high as 50-80mg/ml, and has kept the stability of long-term storage under preferred protective agent effect.
The specific embodiment
For better explanation the present invention, below in conjunction with preparation embodiment and stable recombinant human endostain preparation and the preparation technology thereof of experimental example explanation the present invention.In addition, if no special instructions, hereinafter related experiment all can obtain by commercial sources with material.Employed recombinant human vascular endothelial inhibin has the aminoacid sequence shown in the SEQ ID NO:1 among experimental example hereinafter and the preparation embodiment, disclosed SEQ ID NO:2 is identical for the same Chinese invention patent of its preparation method (patent publication No. is CN1324818A), specifically sees the preparation embodiment of above-mentioned open source literature.
Preparation embodiment 1
The recombinant human vascular endothelial inhibin protein solution of acetic acid-sodium acetate buffer system of using 30mM pH5.5 ± about 0.5 after to purification carries out ultrafiltration dialysis, being mixed with 151.5ml concentration is the recombinant human vascular endothelial inhibin solution of 9.9mg/ml, add 20% mannitol 60ml and 20% sucrose solution 7.5ml, add the acetic acid-sodium-acetate buffer 2.98ml about 1.5M pH5.5, add injection water to 300ml.Through 0.22 μ m microporous filter membrane aseptic filtration, be sub-packed in the pre-encapsulated injector, in 4 ℃ of preservations.
Preparation embodiment 2:
The recombinant human vascular endothelial inhibin protein solution of acetic acid-sodium acetate buffer system of using 30mM pH5.5 ± about 0.5 after to purification carries out ultrafiltration dialysis, being mixed with 90.9ml concentration is the recombinant human vascular endothelial inhibin solution of 9.9mg/ml, add 20% mannitol 60ml, add the acetic acid-sodium-acetate buffer 4.20ml about 1.5MpH5.5, add injection water to 300ml.Through 0.22 μ m microporous filter membrane aseptic filtration, be sub-packed in the cillin bottle, liquid level apart from bottle at the bottom of the 1-1.5cm height, add butyl rubber plug, medicinal liquid places in the freeze drying box, and products temperature drops to-40 ℃, kept evacuation, dividing plate heating 3-4 hour, make products temperature be increased to-20 ℃, kept 8 hours, continue heating elevated temperature to 25 ℃, kept 6 hours, change when little to vacuum, take out after the vacuum tamponade, roll lid.
Preparation embodiment 3:
The recombinant human vascular endothelial inhibin protein solution of acetic acid-sodium acetate buffer system of using 30mM pH5.5 ± about 0.5 after to purification carries out ultrafiltration dialysis, being mixed with 151.5ml concentration is the recombinant human vascular endothelial inhibin solution of 9.9mg/ml, add 20% mannitol 60ml and 20% sucrose solution 15ml, add the acetic acid-sodium-acetate buffer 2.98ml about 1.5M pH5.5, add injection water to 300ml.Through 0.22 μ m microporous filter membrane aseptic filtration, be sub-packed in the cillin bottle, liquid level apart from bottle at the bottom of the 1-1.5cm height, add butyl rubber plug, medicinal liquid places in the freeze drying box, and products temperature drops to-40 ℃, kept evacuation, dividing plate heating 4-5 hour, make products temperature be increased to-25 ℃, kept 10 hours, continue heating elevated temperature to 30 ℃, kept 4 hours, change when little to vacuum, take out after the vacuum tamponade, roll lid.
Preparation embodiment 4:
The recombinant human vascular endothelial inhibin protein solution of acetic acid-sodium acetate buffer system of using 30mM pH5.5 ± about 0.5 after to purification carries out ultrafiltration dialysis, being mixed with 97.8ml concentration is the recombinant human vascular endothelial inhibin solution of 30.61mg/ml, add 20% mannitol 60ml and 20% sucrose solution 60ml, add the acetic acid-sodium-acetate buffer 4.04ml about 1.5M pH5.5, add injection water to 300ml.Through 0.22 μ m microporous filter membrane aseptic filtration, be sub-packed in the cillin bottle, liquid level apart from bottle at the bottom of the 1-1.5cm height, add butyl rubber plug, medicinal liquid places in the freeze drying box, and products temperature drops to-45 ℃, kept evacuation, dividing plate heating 6 hours, make products temperature be increased to-30 ℃, kept 6 hours, the reheat elevated temperature is to-25 ℃, kept 6 hours, and continued heating elevated temperature to 25 ℃, kept 8 hours, change when little to vacuum and to take out after the vacuum tamponade, roll lid.
Preparation embodiment 5:
The recombinant human vascular endothelial inhibin protein solution of acetic acid-sodium acetate buffer system of using 30mM pH5.5 ± about 0.5 after to purification carries out ultrafiltration dialysis, being mixed with 147ml concentration is the recombinant human vascular endothelial inhibin solution of 30.61mg/ml, add 20% mannitol 60ml, 20% sucrose solution 60ml and 20% aqueous trehalose 30ml, add the acetic acid-sodium-acetate buffer 3.06ml about 1.5M pH5.5, add injection water to 300ml.Through 0.22 μ m microporous filter membrane aseptic filtration, be sub-packed in the cillin bottle, liquid level apart from bottle at the bottom of the 1-1.5cm height, add butyl rubber plug, medicinal liquid places in the freeze drying box, and products temperature drops to-45 ℃, kept evacuation, dividing plate heating 2-4 hour, make products temperature be increased to-30 ℃, kept 12 hours, continue heating elevated temperature to 30 ℃, kept 6 hours, change when little to vacuum, take out after the vacuum tamponade, roll lid.
Preparation embodiment 6:
The recombinant human vascular endothelial inhibin protein solution of the citric acid-sodium citrate buffer body that uses 10mM pH6.0 ± about 0.5 after to purification carries out ultrafiltration dialysis, being mixed with 30ml concentration is the recombinant human vascular endothelial inhibin solution of 9.9mg/ml, add 2% glycine 15ml, tween 80 0.15ml, add the citric acid-sodium citrate buffer 2.70ml about 1.0M pH6.5, add injection water to 300ml.Through 0.22 μ m microporous filter membrane aseptic filtration, be sub-packed in the cillin bottle, liquid level apart from bottle at the bottom of the 1-1.5cm height, add butyl rubber plug, medicinal liquid places in the freeze drying box, and products temperature drops to-40 ℃, kept evacuation, dividing plate heating 2-4 hour, make products temperature be increased to-30 ℃, kept 8 hours, continue heating elevated temperature to 30 ℃, kept 8 hours, change when little to vacuum, take out after the vacuum tamponade, roll lid.
Preparation embodiment 7:
The recombinant human vascular endothelial inhibin protein solution of sodium dihydrogen phosphate-sodium hydrogen phosphate buffer system of using 10mM pH7.0 ± about 0.5 after to purification carries out ultrafiltration dialysis, being mixed with 73.5ml concentration is the recombinant human vascular endothelial inhibin solution of 30.61mg/ml, add 2%EDTA 30ml, 20% mannitol solution 30ml and 20% sucrose solution 7.5ml, add the sodium dihydrogen phosphate-sodium hydrogen phosphate buffer 2.26ml about 1.5M pH7.0 ± 0.5, add injection water to 300ml.Through 0.22 μ m microporous filter membrane aseptic filtration, be sub-packed in the cillin bottle, liquid level apart from bottle at the bottom of the 1-1.5cm height, add butyl rubber plug, medicinal liquid places in the freeze drying box, and products temperature drops to-45 ℃, kept evacuation, dividing plate heating 8 hours, make products temperature be increased to-30 ℃, kept 12 hours, continue heating elevated temperature to 25 ℃, kept 10 hours, change when little to vacuum, take out after the vacuum tamponade, roll lid.
Preparation embodiment 8:
The recombinant human vascular endothelial inhibin protein solution of acetic acid-sodium acetate buffer system of using 30mM pH5.5 ± about 0.5 after to purification carries out ultrafiltration dialysis, being mixed with 15ml concentration is the recombinant human vascular endothelial inhibin solution of 9.9mg/ml, add 10% glutamic acid 15ml and 10% threonine 15ml, add the acetic acid-sodium-acetate buffer 5.7ml about 1.5M pH5.5, add injection water to 300ml.Through 0.22 μ m microporous filter membrane aseptic filtration, be sub-packed in the cillin bottle, liquid level apart from bottle at the bottom of the 1-1.5cm height, add butyl rubber plug, medicinal liquid places in the freeze drying box, and products temperature drops to-40 ℃, kept evacuation, dividing plate heating 4-5 hour, make products temperature be increased to-25 ℃, kept 10 hours, continue heating elevated temperature to 30 ℃, kept 4 hours, change when little to vacuum, take out after the vacuum tamponade, roll lid.
Preparation embodiment 9:
The recombinant human vascular endothelial inhibin protein solution of acetic acid-sodium acetate buffer system of using 30mM pH5.5 ± about 0.5 after to purification carries out ultrafiltration dialysis, being mixed with 151.5ml concentration is the recombinant human vascular endothelial inhibin solution of 9.9mg/ml, add 20% human albumin's solution 30ml, add the acetic acid-sodium-acetate buffer 2.98ml about 1.5M pH5.5, add injection water to 300ml.Through 0.22 μ m microporous filter membrane aseptic filtration, be sub-packed in the cillin bottle, liquid level apart from bottle at the bottom of the 1-1.5cm height, add butyl rubber plug, medicinal liquid places in the freeze drying box, and products temperature drops to-40 ℃, kept evacuation, dividing plate heating 4-5 hour, make products temperature be increased to-15 ℃, kept 10 hours, continue heating elevated temperature to 20 ℃, kept 10 hours, change when little to vacuum, take out after the vacuum tamponade, roll lid.
Preparation embodiment 10:
Take by weighing recombinant human vascular endothelial inhibin microsphere (PLGA enclose) 30g, interior enclose recombinant human vascular endothelial inhibin 3g adds 20% mannitol 60ml, and 20% sucrose solution 15ml adds the acetic acid-sodium-acetate buffer 6.0ml about 1.5MpH5.5, add injection water to 300ml, be sub-packed in the cillin bottle, liquid level apart from bottle at the bottom of the 1-1.5cm height, add butyl rubber plug, medicinal liquid places in the freeze drying box, products temperature drops to-40 ℃, keeps evacuation 4-5 hour, the dividing plate heating, make products temperature be increased to-25 ℃, kept 10 hours, continue heating elevated temperature to 30 ℃, kept 4 hours, change when little to vacuum, take out after the vacuum tamponade, roll lid.
Test example 1
The recombinant human vascular endothelial inhibin content assaying method can adopt reversed phase high-performance liquid chromatography (RP-HPLC) among the present invention:
A liquid, the aqueous solution of 0.05% trifluoroacetic acid.
B liquid, the acetonitrile solution of 0.05% trifluoroacetic acid.
The ultraviolet detection wavelength is 214nm
Balance: 64%A, 5-10 minute
Chromatographic condition: gradient elution 64%A-40%A, 25 minutes.
Precision is measured the physics and chemistry reference substance solution, and accurate sample introduction 1,10,20,30,40,50 μ g albumen record peak area respectively, and (μ g) is abscissa with sample size, and peak area (A) is a vertical coordinate, carries out linear regression, obtains linear regression equation.
Accurate amount takes by weighing need testing solution, injects instrument, and elution process as above records peak area, and METHOD FOR CONTINUOUS DETERMINATION 3 times is tried to achieve meansigma methods.
The result calculates:
With the above-mentioned linear regression equation of peak area meansigma methods substitution of the need testing solution that records, promptly get the absolute sample size of albumen of need testing solution, calculate protein content in the test sample according to following formula again, should be the 85%-115% of labelled amount.
Migration suppresses experiment to test example 2 recombinant human vascular endothelial inhibin albumen to human microvascular endothelial cell (mvec)
The trophophase human microvascular endothelial cell (mvec) (HMEC) of taking the logarithm is adjusted cell suspension density to 5 * 10 with the DMEM-0.2%FBS culture fluid
5Individual/ml.Formulation products of the present invention is diluted with physiological saline solution, make the recombinant human vascular endothelial inhibin final concentration of protein be respectively 5mg/ml, 0.5mg/ml, 5 μ g/ml, 50ng/ml, 5ng/ml.Get 630 μ l cell suspension and add the 70 μ l test sample (recombinant human vascular endothelial inhibins of above-mentioned dilution, do blank with physiological saline solution, the recombinant human vascular endothelial inhibin final concentration of protein will be respectively 0.5mg/ml, 0.05mg/ml, 0.5 μ g/ml, 5ng/ml, 0.5ng/ml), mixing, the hole adds 200ul on each, do 3 parallel, in 37 ℃, 5%CO
2After cultivating 16h in the incubator, hematoxylin-eosin staining, cell scraping, film-making, the microscopically counting, and calculate the cell migration suppression ratio.Suppression ratio=1-(cell number of the cell number of experimental group migration/matched group migration).The result shows that the vascellum esoderma inhibin albumen of each extension rate all can effectively suppress the propagation of vascular endothelial cell, and the highest suppression ratio can reach 95% (seeing Table 1).
Table 1: recombinant human vascular endothelial inhibin is to the suppression ratio of HMEC
| Recombinant human vascular endothelial inhibin dosage | Suppression ratio (%) |
| 0.5ng/ml 5ng/ml 0.5μg/ml 50μg/ml 500μg/ml | 16 34 66 86 95 |
Freeze dried recombinant human endostain preparation moisture determination method is measured by official method among the present invention.
But medium vessels endostatin method for detecting purity reversed phase high-performance liquid chromatography of the present invention (RP-HPLC) and 15%SDS-PAGE method, RP-HPLC purity testing method chromatographic condition is with above-mentioned concentration determination method, and it is conventional analysis condition that the SDS-PAGE method is measured purity of protein.
Test example 3:
Recombinant human vascular endothelial inhibin lyophilized injectable powder stability test result of the present invention simultaneously compares (seeing Table 2) with the aqueous injection that has gone on the market the result that keeps sample for a long time, and effect duration can be prolonged at least to 24 months by 18 months of aqueous injection.
Table 2 injection-use recombinant human Endostatin lyophilized powder stability test is investigated the result
| Experimental temperature | Standing time (moon) | Outward appearance | Moisture≤3% | Content (%) | Purity detecting (＞95%) | Active 1.2 * 10 5-4.8 ×10 5U/ props up | |||
| SDS-PAGE | HPLC | ||||||||
| Character | The redissolution time | ||||||||
| Reduction | Non-reduced | ||||||||
| 37℃± 2℃ | 0 | The white loose body | ＜1min | 1.87％ | 98.6 | ＞97％ | ＞97％ | ＞97％ | 3.3× 10 5 |
| 1 | The white loose body | ＜1min | 1.92％ | 95.4 | ＞97％ | ＞97％ | ＞97％ | 2.9× 10 5 | |
| 2 | The white loose body | ＜1min | 2.03％ | 103.8 | ＞97％ | ＞97％ | ＞97％ | 3.1× 10 5 | |
| 3 | The white loose body | ＜1min | 1.57％ | 100.4 | ＞97％ | ＞97％ | ＞97％ | 2.8× 10 5 | |
| 2 5 ℃ ± 2 ℃ | 1 | The white loose body | ＜1min | 1.92％ | 92.6 | ＞97％ | ＞97％ | ＞97％ | 2.7× 10 5 |
| 2 | The white loose body | ＜1min | 2.11％ | 97.8 | ＞97％ | ＞97％ | ＞97％ | 3.1× 10 5 | |
| 3 | The white loose body | ＜1min | 2.13％ | 106.4 | ＞97％ | ＞97％ | ＞97％ | 3.4× 10 5 | |
| 6 | The white loose body | ＜1min | 1.75％ | 102.7 | ＞97％ | ＞97％ | ＞97％ | 2.9× 10 5 | |
| 6 ℃ ± 2 ℃ | 3 | The white loose body | ＜1min | 1.92％ | 100.4 | ＞97％ | ＞97％ | ＞97％ | 2.9× 10 5 |
| 6 | The white loose body | ＜1min | 2.05％ | 97.6 | ＞97％ | ＞97％ | ＞97％ | 3.1× 10 5 | |
| 9 | The white loose body | ＜1min | 1.38％ | 94.3 | ＞97％ | ＞97％ | ＞97％ | 2.4× 10 5 | |
| 12 | The white loose body | ＜1min | 2.31％ | 101.6 | ＞97％ | ＞97％ | ＞97％ | 2.6× 10 5 | |
| 18 | The white loose body | ＜1min | 2.48％ | 108.4 | ＞97％ | ＞97％ | ＞97％ | 2.5× 10 5 | |
| 24 | The white loose body | ＜1min | 2.54％ | 99.4 | ＞97％ | ＞97％ | ＞97％ | 3.3× 10 5 |
Test example 4: prescription screening test (seeing Table 3)
Prepare recombinant human vascular endothelial inhibin protein solution, protective agent and the cosolvent of purification.PH buffer system in the use table is carried out ultrafiltration dialysis to the recombinant human vascular endothelial inhibin solution of purifying, and each protective agent, cosolvent in will tabulating are dissolved in the injection water ultrafiltration dialysis after filtering; With protein solution and protective agent and/or cosolvent mixing, filtration sterilization is sub-packed in the sterile chamber according to the content in the prescription table.
Table 3 stabilization formulations prescription of the present invention screening test related raw material proportioning
| Preparation recipe (every ml) | Recombinant human vascular endothelial inhibin 0.5-20 mg | Protective agent (mg) | Cosolvent (mg) | Buffer system (mg) | |||||||||||
| Mannitol 10-80 | Dextran 5-30 | Sucrose 5-80 | Trehalose 5-80 | Glutamic acid 0.5-20 | Glycine 0.5-20 | Threonine 0.5-20 | Human albumin 1-40 | Sodium chloride 10-50 | E D T A 0.1 -10 | Tween 80 0.1-10 | Acetic acid-sodium acetate 0.6-12 | Citric acid-sodium citrate 2-20 | Sodium ascorbyl phosphate 2-20 | ||
| 1 | 5 | 40 | 5 | 3.7 | |||||||||||
| 2 | 5 | 40 | 3.7 | ||||||||||||
| 3 | 5 | 40 | 10 | 3.7 | |||||||||||
| 4 | 10 | 40 | 40 | 3.7 | |||||||||||
| 5 | 15 | 40 | 40 | 20 | 3.7 | ||||||||||
| 6 | 1 | 1 | 0.5 | 3.4 | |||||||||||
| 7 | 7.5 | 20 | 5 | 2 | 2.7 | ||||||||||
| 8 | 0.5 | 5 | 5 | 3.7 | |||||||||||
| 9 | 5 | 20 | 3.7 | ||||||||||||
| 10 | 10 | 40 | 10 | 3.7 | |||||||||||
| 11 | 20 | 40 | 20 | 0.1 | 3.7 | ||||||||||
| 12 | 7.5 | 80 | 10 | 0.5 | 3.7 | ||||||||||
| 13 | 7.5 | 10 | 5 | 20 | 0.5 | 3.7 | |||||||||
| 14 | 7.5 | 30 | 20 | 0.5 | 3.7 | ||||||||||
| 15 | 7.5 | 10 | 80 | 1 | 3.7 | ||||||||||
| 16 | 7.5 | 10 | 20 | 10 | 3.7 | ||||||||||
| 17 | 7.5 | 10 | 80 | 0.1 | 12 | ||||||||||
| 18 | 7.5 | 20 | 5 | 20 | 10 | 0.6 | |||||||||
| 19 | 7.5 | 40 | 20 | 10 | 2 | ||||||||||
| 20 | 7.5 | 5 | 0.5 | 50 | 20 | ||||||||||
| 21 | 7.5 | 5 | 20 | 20 | 2 | ||||||||||
| 22 | 7.5 | 40 | 40 | 0.5 | 5 | 6 | |||||||||
| 23 | 7.5 | 40 | 0.5 | 10 | |||||||||||
| 24 | 7.5 | 5 | 10 | 10 | 5 | 0.6 | |||||||||
| 25 | 7.5 | 20 | 10 | 0.5 | 20 | 10 |
The above, it only is better embodiment of the present invention, be not that the present invention is done any pro forma restriction, therefore be not in order to limit the present invention, any those skilled in the art, in not breaking away from the technical solution of the present invention scope, to any simple modification, equivalent variations and modification that above embodiment did, all still belong in the scope of technical solution of the present invention according to technical spirit of the present invention.
Sequence table
＜110〉Shandong Xiansheng Maidejin Biological Pharmaceutical Co., Ltd.
＜120〉a kind of stable recombinant human endostain preparation and preparation technology thereof
<130>MP080024
<160>1
<170>PatentIn version 3.3
<210>1
<211>192
<212>PRT
<213>Artificial
<220>
＜223〉recombinant human vascular endothelial inhibin
<400>1
Met Gly Gly Ser His His His His His His Ser His Arg Asp Phe Gln
1 5 10 15
Pro Val Leu His Leu Val Ala Leu Asn Ser Pro Leu Ser Gly Gly Met
20 25 30
Arg Gly Ile Arg Gly Ala Asp Phe Gln Cys Phe Gln Gln Ala Arg Ala
35 40 45
Val Gly Leu Ala Gly Thr Phe Arg Ala Phe Leu Ser Ser Arg Leu Gln
50 55 60
Asp Leu Tyr Ser Ile Val Arg Arg Ala Asp Arg Ala Ala Val Pro Ile
65 70 75 80
Val Asn Leu Lys Asp Glu Leu Leu Phe Pro Ser Trp Glu Ala Leu Phe
85 90 95
Ser Gly Ser Glu Gly Pro Leu Lys Pro Gly Ala Arg Ile Phe Ser Phe
100 105 110
Asp Gly Lys Asp Val Leu Arg His Pro Thr Trp Pro Gln Lys Ser Val
115 120 125
Trp His Gly Ser Asp Pro Asn Gly Arg Arg Leu Thr Glu Ser Tyr Cys
130 135 40
Glu Thr Trp Arg Thr Glu Ala Pro Ser Ala Thr Gly Gln Ala Ser Ser
145 150 155 160
Leu Leu Gly Gly Arg Leu Leu Gly Gln Ser Ala Ala Ser Cys His His
165 170 175
Ala Tyr Ile Val Leu Cys Ile Glu Asn Ser Phe Met Thr Ala Ser Lys
180 185 190
Claims (18)
1. a stable recombinant human endostain preparation wherein contains the recombinant human vascular endothelial inhibin of 0.05%-65% and the protective agent of 1%-90% by weight percentage.
2. the described preparation of claim 1, wherein said recombinant human vascular endothelial inhibin is to add 0-4 arbitrary amino acid and 2-8 histidine sequence behind the initial amino acid Met of human Endostatin encoding amino acid sequence successively.
3. the described preparation of claim 2, the initial amino acid Met of wherein said recombinant human vascular endothelial inhibin is cut.
4. the described application of claim 2, the aminoacid sequence of wherein said recombinant human vascular endothelial inhibin is shown in SEQ ID NO:1.
5. each described preparation of claim 1-4 wherein also contains the cosolvent of 0.05%-35% by weight percentage.
6. the described preparation of claim 5 wherein also contains the PH regulator, and its scope of regulating pH value is 4.0-8.5.
7. the described preparation of claim 5, wherein said preparation can be injection or lyophilized formulations.
8. the described preparation of claim 5, wherein said recombinant human vascular endothelial inhibin can be by genetic engineering means express, the recombinant human vascular endothelial inhibin of purification, recombinant human vascular endothelial inhibin or the recombinant human vascular endothelial inhibin microsphere that PEG modifies.
9. the described preparation of claim 5, wherein said protective agent comprise at least a in polyhydric alcohol, saccharide, aminoacid and the albumin, and wherein said polyhydric alcohol comprises at least a in glycerol, mannitol, the sorbitol; Described saccharide comprises at least a in glucose, sucrose, dextran, trehalose, lactose, the beta-schardinger dextrin-; Described aminoacid comprises at least a in arginine, glycine, proline, aspartic acid, threonine, serine, glutamic acid, the leucine.
10. the described preparation of claim 5, wherein said cosolvent comprises surfactant and/or salt, wherein said surfactant comprises at least a among tween 80 and the pluronic F-68; Described salt comprises at least a among EDTA and the NaCl.
11. the described preparation of claim 6, wherein said PH regulator can be one or more in dibastic sodium phosphate, potassium hydrogen phosphate, Tris-HCl, sodium citrate-citric acid, potassium citrate-citric acid, sodium acetate-acetic acid, the sodium borate-boric acid.
12. the described preparation of claim 8, the total average molecular weight of wherein said PEG is 5000-30000, and PEG links to each other with the recombinant human vascular endothelial inhibin molecule by covalent bond.
13. the described preparation of claim 8, wherein said recombinant human vascular endothelial inhibin microsphere are to be mixed with by host material and recombinant human vascular endothelial inhibin to form.
14. the described preparation of claim 9, wherein said protective agent comprise at least a in mannitol, trehalose, sucrose, glycine, threonine and the human albumin.
15. the described preparation of claim 10, wherein said cosolvent comprise at least a among tween 80 and the EDTA.
16. the preparation method of a stable recombinant human endostain preparation comprises the steps:
1), the recombinant human vascular endothelial inhibin protein solution and the protective agent of prepare purifying, wherein by accounting for final formulation products weight percent meter, recombinant human vascular endothelial inhibin accounts for 0.05%-65%, protective agent accounts for 1%-90%;
2), use pH buffer system is carried out ultrafiltration dialysis to the recombinant human vascular endothelial inhibin protein solution of above-mentioned purification;
3), above-mentioned protective agent is dissolved in the injection water ultrafiltration dialysis after filtering;
4), with step 2) and 3) dialysis solution mixes, filtration sterilization is sub-packed in the sterile chamber.
17. the described preparation method of claim 6, wherein after described filtration sterilization, increase the freeze-dry process step, described freeze-dry process step is: the medicinal liquid after the degerming is sub-packed in the container, the container that branch is installed medicinal liquid is positioned in advance and is cooled to-the freezing in the case of 35--45 ℃, be incubated after 3-6 hour, evacuation after the secondary temperature elevation distillation, takes out behind the vacuum gland.
18. the described preparation method of claim 17, the distillation of described secondary temperature elevation are meant that for the first time sublimation temperature is-30--10 ℃, persistent period 8-12 hour, distil 25-30 ℃ for the second time, and continue 4-10 hour.
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| CNA2008100060577A CN101224296A (en) | 2008-02-01 | 2008-02-01 | Stable recombinant human endostain preparation and preparing method thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104548067A (en) * | 2014-12-23 | 2015-04-29 | 浙江省人民医院 | Application of recombinant human endostatin in preparing drugs for treating ocular neovascular diseases |
| CN105218660A (en) * | 2014-07-03 | 2016-01-06 | 苏州方舟基因药业有限公司 | The novel purification renaturation method of Recombinant Endostatin and antitumor application thereof |
| CN107115522A (en) * | 2016-02-24 | 2017-09-01 | 山东先声生物制药有限公司 | A kind of recombinant human vascular endothelial inhibin pharmaceutical composition |
| CN107865824A (en) * | 2016-09-28 | 2018-04-03 | 山东先声生物制药有限公司 | A kind of recombinant human vascular endothelial inhibin subcutaneous composition of stabilization |
| CN111346220A (en) * | 2018-12-24 | 2020-06-30 | 山东先声生物制药有限公司 | Polyethylene glycol modified vascular endothelial inhibin preparation composition |
| CN113633634A (en) * | 2016-01-08 | 2021-11-12 | 细胞基因公司 | Formulations of2- (4-chlorophenyl) -N- ((2- (2,6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2,2-difluoroacetamide |
-
2008
- 2008-02-01 CN CNA2008100060577A patent/CN101224296A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105218660A (en) * | 2014-07-03 | 2016-01-06 | 苏州方舟基因药业有限公司 | The novel purification renaturation method of Recombinant Endostatin and antitumor application thereof |
| CN105218660B (en) * | 2014-07-03 | 2019-02-12 | 苏州方舟基因药业有限公司 | The novel purification renaturation method and its antitumor application thereof of Recombinant Endostatin |
| CN104548067A (en) * | 2014-12-23 | 2015-04-29 | 浙江省人民医院 | Application of recombinant human endostatin in preparing drugs for treating ocular neovascular diseases |
| CN113633634A (en) * | 2016-01-08 | 2021-11-12 | 细胞基因公司 | Formulations of2- (4-chlorophenyl) -N- ((2- (2,6-dioxopiperidin-3-yl) -1-oxoisoindolin-5-yl) methyl) -2,2-difluoroacetamide |
| CN107115522A (en) * | 2016-02-24 | 2017-09-01 | 山东先声生物制药有限公司 | A kind of recombinant human vascular endothelial inhibin pharmaceutical composition |
| CN107865824A (en) * | 2016-09-28 | 2018-04-03 | 山东先声生物制药有限公司 | A kind of recombinant human vascular endothelial inhibin subcutaneous composition of stabilization |
| WO2018059193A1 (en) * | 2016-09-28 | 2018-04-05 | 山东先声生物制药有限公司 | Stable recombinant human endostatin subcutaneous injection composition |
| CN111346220A (en) * | 2018-12-24 | 2020-06-30 | 山东先声生物制药有限公司 | Polyethylene glycol modified vascular endothelial inhibin preparation composition |
| CN111346220B (en) * | 2018-12-24 | 2022-12-09 | 山东先声生物制药有限公司 | Polyethylene glycol modified vascular endothelial inhibin preparation composition |
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Application publication date: 20080723 |