CN101618023B - Method for preparing micronized protein - Google Patents
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- CN101618023B CN101618023B CN2008101227657A CN200810122765A CN101618023B CN 101618023 B CN101618023 B CN 101618023B CN 2008101227657 A CN2008101227657 A CN 2008101227657A CN 200810122765 A CN200810122765 A CN 200810122765A CN 101618023 B CN101618023 B CN 101618023B
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- 238000004108 freeze drying Methods 0.000 claims abstract description 26
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- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 4
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- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to a method for preparing micronized protein. The method comprises the following steps: (1) adding soluble metal salts to a buffer salt solution containing soluble protein, adjusting the pH of the solution to the isoelectric point (pI) of protein, obtaining fine granular precipitate of the protein and turning a protein solution into a suspension; (2) mixing the suspension with a freeze-drying protective agent well and then freeze-drying the mixture; and (3) washing freeze-dried solid powder masses with a solvent, removing the freeze-drying protective agent and obtaining the micronized protein.
Description
Technical field
The present invention relates to prepare the method for the micronized human vascular endostatin of solid form by the material solution that contains human Endostatin.Be particularly related to a kind of method that can obtain highly active micronized human vascular endostatin.
Background technology
The innovation of biotechnology and development make recombiant protein and polypeptide drugs be achieved large-scale production and application.Albumen and polypeptide are unstable in aqueous solution, and easily inactivation has hindered this type of medication preparation patent medicine preparation.For example with protein solution with after organic solvent contacts, because the capillary existence of oil-water interfaces changes and inactivation protein conformation.The existing freeze-drying that directly adds freeze drying protectant in albumen stock solution also can obtain exsiccant solid-state protein, but the protein body that obtains is big, and the ratio that adjuvant accounts for is great, is used for making lyophilized injectable powder more, and purposes is not extensive.
In the research of albumen and polypeptide drug drug-supplying system, the dosage form that turns to prerequisite with drug powder is increasing, the application of these dosage forms and development, and its prerequisite and key are the micronizations of medicine.But the technology of existing production solid-state protein can't be produced the micronized protein of particle diameter less than 3um, so can not satisfy the needs of novel form research and development.The advantage of uniqueness of the present invention is: one, protein Preparation is become solid-state, improved the stability that albumen is prepared to other dosage forms; Two, solid-state albumen, its particle is minimum, is micronized solid-state protein, has expanded its preparation range of application, not only can be used for preparing injectable powder, because its particle diameter minimum (1um is following) also can be used for preparing Foradil Aerolizer formoterol fumarate, injectable powder or the like.
The method of existing micronized protein has had a lot, and overall conclusion can be divided into following several: spray drying method, freeze-drying, atomizing freeze drying method, supercritical fluid technology.Spray drying is to utilize nebulizer that drug solution is separated into tiny droplet, and in the heated drying medium rapidly evaporating solvent form the process of dry powder, the grain size of micropowder that this technology makes is even, but this method need be used hot-air, is not suitable for dry temperature-resistant albumen and polypeptide drugs.The atomizing freeze drying method combines the atomization steps in the spray drying with lyophilization, step is that the pharmaceutical aqueous solution that will atomize sprays in the liquid nitrogen, is dried with cryodesiccated step, obtains the uniform drug powder of particle diameter.This method batch processing amount is big, but cost is higher, and the grain size of micropowder that obtains is bigger.Supercritical fluid technology is the technology of newly-developed, the character that can be used for supercritical fluid is extracted solvent, desiccation protein, but, the problem that still there are many needs researchs in this technology and solve: the dissolubility in organic solvent is generally less as albumen and polypeptide drug, how increase the dissolubility of medicine by changing solvent composition, and the relation between different operating conditions and the heterogeneity micropowder that finally obtains.
Summary of the invention
To the objective of the invention is the problem that exists in the existing micronized protein method in order solving, a kind of active keep good and the high micronized protein method of micronization efficient to be provided.
The micronized protein of the present invention's preparation can be applicable to multiple use: Foradil Aerolizer formoterol fumarate, injectable powder and microsphere microcapsule controlled-release preparation etc.
The invention discloses a kind of method for preparing micronized protein, it may further comprise the steps:
(1) add soluble metallic salt in containing the buffer salt solution of soluble protein, the pH that adjusts this solution then obtains proteic fine particle precipitation to proteic isoelectric point, IP (pI), and protein solution becomes suspension;
(2) with behind suspension and the freeze drying protectant mix homogeneously, lyophilizing;
(3) with the solid powder agglomates solvent wash after the lyophilizing, remove freeze drying protectant, obtain micronized protein.
The principle of this technology utilization is that " isoelectric point, IP+slaine " two kinds of method combineds effect obtain the tiny deposit seed of albumen, is applicable to most soluble proteins.
Above-mentioned soluble protein can be recombinant human vascular endothelial inhibin Endostar, recombinant human somatropin, interferon-ALPHA
2-b (IFN α
2-b), recombinant human insulin etc.
Utilize this polymer of Polyethylene Glycol as cryodesiccated protective agent among the present invention, play space figuration and stable effect in lyophilizing, the protein body that precipitates can be adsorbed on the skeleton of Polyethylene Glycol.
Above-mentioned molecular weight polyethylene glycol scope is 1000 to 8000 dalton.Polyethylene Glycol has good suspension stability, and molecular weight ranges keeps it not assemble in lyophilizing at the equal good stabilize proteins granule of 1000 to 8000 dalton, does not sink.
The volume ratio of suspension and freeze drying protectant is 1: 99～99: 1 in the preparation method of above-mentioned micronized protein, step (2), preferred 15: 85～90: 10; Protein concentration in the suspension is 0.01mg/ml～500mg/ml, and the concentration of freeze drying protectant is 1%～50% (w/v).The concentration of freeze drying protectant can not be less than 1%, otherwise suspension stability is very poor, and albumen precipitation can be assembled in very fast sinking; The excessive concentration of freeze drying protectant also is not suitable for because after the lyophilizing in order to obtain the albumen micropowder, need washing with an organic solvent to remove this protective agent, if the ratio that protective agent accounts for is too high, need to consume a large amount of organic solvents, clean repeatedly, make troubles to operation.Keep the concentration of 1%～50% (w/v) more suitable.
The preparation method of above-mentioned micronized protein, the cleaning solvent in the step (3) is the solvent of solubilized Polyethylene Glycol, for example: acetone, ethyl acetate, dichloromethane, acetonitrile.Ethyl acetate, dichloromethane, acetonitrile.
The invention belongs to technology with albumen precipitation technology and the bonded new preparation albumen micropowder of Freeze Drying Technique.Technology of the present invention is gentle simple, and cost is little, and the grain size of micropowder that obtains is less than 1um, and the protein active maintenance is good, is a micronized protein method that extensive medical applications prospect is arranged.
Advantage of the present invention has:
One the present invention is with albumen precipitation granule and freeze drying protectant lyophilization, and proteic deposit seed has kept the dispersibility of albumen micropowder and the integrity of albumen multilevel hierarchy attached on protectant skeleton.The micronized protein of preparation can keep the preceding activity more than 85% of preparation.
After two lyophilizing, it is solid-state that albumen is, not moisture, when this moment, with an organic solvent Polyethylene Glycol was removed in washing, can not make the degeneration on the solid-state protein generation oil-water interfaces, and exsiccant albumen micropowder stable in properties is convenient to next step preparation or long term storage.
Three albumen have been expanded its preparation range of application through behind the micronization, not only can be used for preparing microball preparation, because its particle diameter minimum (1um is following) also can be used for preparing Foradil Aerolizer formoterol fumarate, injectable powder, implantable pump preparation.
Description of drawings
The SEM stereoscan photograph of albumen micropowder among Fig. 1 embodiment one
The scale of left figure is 5 μ m, and the scale of right figure is 10 μ m, and the diameter of visible protein particle is less than 1 μ m.
The SEM stereoscan photograph of albumen micropowder among Fig. 2 embodiment two
The scale of left figure is 5 μ m, and the scale of right figure is 1 μ m, and the diameter of visible protein particle is less than 1 μ m.
The albumen micropowder is to the HUVEC cell-proliferation activity among Fig. 3 embodiment two
The specific embodiment
The present invention is for a more detailed description by following examples, but the protection domain that it can not be construed as limiting the invention.
Embodiment one
The isoelectric focusing electrophoresis method is measured the proteic isoelectric point, IP of recombinant human vascular endothelial inhibin, measurement result is PI=8.8～9.3, measure 50ml recombinant human vascular endothelial inhibin protein solution (9.1462mg/ml, PH=5.5), the zinc acetate of adding 0.004%, after dissolving fully to zinc salt, transfer with sodium hydroxide solution (1mol/L) in this interval of PH to 8.8-9.3 of protein solution.At this moment, a large amount of albumen precipitations, solution presents milky, and adding 50ml concentration is the PEG-8000 solution of 50% (w/v), and magnetic agitation 5min makes it mix homogeneously.
The above-mentioned solution for preparing is divided in the cillin bottle of specification 30ml, and the loading amount specification is the 5ml/ bottle, lyophilizing.Dissolve lyophilized products with dichloromethane, PEG-8000 dissolves in dichloromethane, centrifugal after, obtain containing the micronized protein of dichloromethane in centrifuge tube bottom, the room temperature drying under reduced pressure it, obtain exsiccant micronized protein, between the particle diameter 0.01-1um.(Fig. 1)
Embodiment two
The isoelectric focusing electrophoresis method is measured the proteic isoelectric point, IP of recombinant human vascular endothelial inhibin, measurement result is PI=8.8～9.3, measure 50ml protein solution (102.8mg/ml, PH=6.5), the magnesium sulfate of adding 0.001%, after dissolving fully to magnesium salt, transfer with sodium hydroxide solution (1mol/L) in this interval of PH to 8.8-9.3 of protein solution.At this moment, a large amount of albumen precipitations, solution presents milky, and adding 50ml concentration is the PEG-6000 solution of 20% (w/v), and magnetic agitation 5min makes it mix homogeneously.
The above-mentioned solution for preparing is divided in the cillin bottle of specification 30ml, and the loading amount specification is the 5ml/ bottle, lyophilizing.Freeze-drying time is one day.Dissolve lyophilized products with dichloromethane, PEG-6000 dissolves in dichloromethane, centrifugal after, obtain containing the micronized protein of dichloromethane in centrifuge tube bottom, the room temperature drying under reduced pressure it, obtain exsiccant micronized recombinant human vascular endothelial inhibin albumen.
Between the particle diameter 0-1um.(Fig. 2)
Embodiment three
The isoelectric focusing electrophoresis method is measured recombinant human somatropin's isoelectric point, IP, measurement result is PI=5.30～5.80, measure 50ml protein solution (86.90mg/ml, PH=7.4), the calcium sulfate of adding 0.04%, after dissolving fully to calcium salt, transfer with acetum (1mol/L) in this interval, PH to 5.30～5.80 of protein solution.At this moment, a large amount of albumen precipitations, solution presents milky, and adding 50ml concentration is the PEG-4000 solution of 30% (w/v), and magnetic agitation 8min makes it mix homogeneously.
The above-mentioned solution for preparing is divided in the cillin bottle of specification 30ml, and the loading amount specification is the 5ml/ bottle, lyophilizing.Freeze-drying time is one day.Dissolve lyophilized products with dichloromethane, PEG-4000 dissolves in dichloromethane, centrifugal after, obtain containing the micronization recombinant human somatropin of dichloromethane in centrifuge tube bottom, the room temperature drying under reduced pressure it, obtain exsiccant micronized recombinant human somatropin.
Embodiment four
The isoelectric focusing electrophoresis method is measured interferon-ALPHA
2-b (IFN α
2-b) isoelectric point, IP, measurement result is PI=5.20～6.10, measures 50ml protein solution (117.2mg/ml, PH=7.4), add 0.025% zinc acetate, after dissolving fully to zinc salt, transfer with acetum (1mol/L) in this interval, PH to 5.20～6.10 of protein solution.At this moment, a large amount of albumen precipitations, solution presents milky, and adding 50ml concentration is the PEG-8000 solution of 8% (w/v), and magnetic agitation 5min makes it mix homogeneously.
The above-mentioned solution for preparing is divided in the cillin bottle of specification 30ml, and the loading amount specification is the 5ml/ bottle, lyophilizing.Freeze-drying time is one day.Dissolve lyophilized products with dichloromethane, PEG-8000 dissolves in dichloromethane, centrifugal after, obtain containing the micronization interferon-ALPHA of dichloromethane in centrifuge tube bottom
2-b, the room temperature drying under reduced pressure it, obtain exsiccant micronization interferon-ALPHA
2-b.
Embodiment five
The isoelectric focusing electrophoresis method is measured recombinant human insulin's isoelectric point, IP, measurement result is PI=5.30～5.50, measure 50ml protein solution (98.36mg/ml, PH=7.4), the calcium sulfate of adding 0.04%, after dissolving fully to calcium salt, transfer with acetum (1mol/L) in this interval, PH to 5.30～5.50 of protein solution.At this moment, a large amount of albumen precipitations, solution presents milky, and adding 50ml concentration is the PEG-6000 solution of 40% (w/v), and magnetic agitation 10min makes it mix homogeneously.
The above-mentioned solution for preparing is divided in the cillin bottle of specification 30ml, and the loading amount specification is the 5ml/ bottle, lyophilizing.Freeze-drying time is one day.Dissolve lyophilized products with dichloromethane, PEG-4000 dissolves in dichloromethane, centrifugal after, obtain containing the micronization recombinant human insulin of dichloromethane in centrifuge tube bottom, the room temperature drying under reduced pressure it, obtain exsiccant micronization recombinant human insulin,
Embodiment six
Precision takes by weighing the micronized protein of preparation among the embodiment one, redissolves with buffer, is diluted to 1500ng/ml, 1200ng/ml, 1000ng/ml, 800ng/ml, 600ng/ml, 400ng/ml, 200ng/ml, 100ng/ml, 50ng/ml.With its immunologic competence of commercially available ELISA (enzyme-linked immunoassay) kit measurement.Method: do one without micronized protein standard curve, remake the standard curve after a micronized protein redissolves, relatively two curves get the immunologic competence conservation rate in light absorption value (OD) difference of 100ng/ml-1000ng/ml.Active conservation rate computational methods are: active conservation rate=100%-(not albumen OD behind the micronized protein OD-micronization)/(not micronized protein OD-blank value 0.0392) * 100%.Table one shows that the protein active conservation rate behind the micronization is greater than 90% (table one)
Embodiment seven
Precision takes by weighing the micronized protein of preparation among the embodiment two, redissolves with buffer, carries out the HUVEC Determination of biological activity.The HUVEC cell is cultivated based on 37 ℃ with the ECM that adds 5%FBS, 1%ECGS, 1%P/O Solution, 5%CO
2Incubator in cultivate, inoculate after treating that cell state is good and entering exponential phase, select for use the 4th generation cell inoculation.Cell 0.25% trypsinization, centrifugal 5 minutes of 1000rpm abandons supernatant, with culture medium suspendible again, microscopically blood cell counting plate living cell counting.Transferring cell density is 5000/ml, and every hole adds 160 μ l cell suspension.Dosing: add the every hole 40 μ l of medicine after the inoculation at once, the medicine final concentration: 200,100,50,25,12.5,6.25,0 μ g/ml, each concentration is provided with 3 multiple holes, 37 ℃ of 5%CO
2Cultivated 96 hours in the incubator.Add MTT reagent, every hole 20 μ l put 37 ℃ of 5%CO
2Cultivated 4 hours in the incubator, add three joint-trial agent and put 37 ℃ of 5%CO
2Overnight incubation in the incubator uses microplate reader 570nm wavelength to measure the OD value down.Try to achieve suppression ratio by following formula: the suppression ratio %=of sample (negative control OD value-medicine OD value)/(negative control OD value one acellular control wells OD value) * 100%.(Fig. 3)
Claims (10)
1. method for preparing micronized protein, it comprises:
(1) add soluble metallic salt in containing the buffer salt solution of soluble protein, the pH that adjusts this solution then obtains proteic fine particle precipitation to proteic isoelectric point, IP, and protein solution becomes suspension;
(2) with behind suspension and the freeze drying protectant mix homogeneously, lyophilizing;
(3) with the solid powder agglomates solvent wash after the lyophilizing, remove freeze drying protectant, obtain micronized protein.
2. preparation method according to claim 1 is characterized in that described soluble protein is Endostar, recombinant human somatropin, interferon-ALPHA
2-b, the recombinant human insulin; Buffer salt solution is a sodium acetate solution.
3. preparation method according to claim 1 and 2 is characterized in that protein concentration is 0.01mg/ml ~ 500mg/ml in the buffer salt solution of described soluble protein; Buffer salt solution concentration is 0.0001% (w/v) ~ 30% (w/v).
4. preparation method according to claim 1 is characterized in that described soluble metallic salt is selected from mantoquita, zinc salt, cadmium salt, calcium salt, magnesium salt, potassium salt, sodium salt, silver salt, aluminum salt.
5. preparation method according to claim 4 is characterized in that described soluble metallic salt is selected from zinc salt, magnesium salt, calcium salt.
6. preparation method according to claim 1, the addition that it is characterized in that described soluble metallic salt are 0.001% (w/v) ~ 10% (w/v).
7. preparation method according to claim 6, the addition that it is characterized in that described soluble metallic salt are 0.001% (w/v) ~ 3% (w/v).
8. preparation method according to claim 1 is characterized in that employed freeze drying protectant is a molecular weight ranges at 1000 to 9000 daltonian Polyethylene Glycol or molecular weight ranges at 2000 to 20000 daltonian polyvinylpyrrolidones.
9. preparation method according to claim 1, the volume ratio that it is characterized in that suspension and freeze drying protectant is 15:85 ~ 90:10.
10. method according to claim 1 is characterized in that described washing organic solvent is one or more in acetone, ethyl acetate, dichloromethane, the acetonitrile.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1171049A (en) * | 1994-12-22 | 1998-01-21 | 阿斯特拉公司 | Powder formulations containing melezitose as diluent |
| CN1972670A (en) * | 2004-03-23 | 2007-05-30 | 诺瓦提斯公司 | Pharmaceutical composition |
| CN101045158A (en) * | 2006-03-31 | 2007-10-03 | 北京大学 | Insulin analog dry powder composition and its preparing method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1171049A (en) * | 1994-12-22 | 1998-01-21 | 阿斯特拉公司 | Powder formulations containing melezitose as diluent |
| CN1972670A (en) * | 2004-03-23 | 2007-05-30 | 诺瓦提斯公司 | Pharmaceutical composition |
| CN101045158A (en) * | 2006-03-31 | 2007-10-03 | 北京大学 | Insulin analog dry powder composition and its preparing method |
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