CN105348382A - Method for preparing high-purity human coagulation factor VIII - Google Patents
Method for preparing high-purity human coagulation factor VIII Download PDFInfo
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Abstract
The invention discloses a method for preparing a high-purity human coagulation factor VIII from a cryoprecipitate of a human plasma fraction. The method comprises the following steps: (1) cryoprecipitate dissolution; (2) aluminum hydroxide gel adsorption and filter pressing; (3) S/D virus inactivation; (4) anion exchange resin column chromatography; (5) hydrophobic column chromatography; (6) ultrafiltration dialysis and concentration; (7) addition of one or more stabilizers and titer adjustment; (8) nano-membrane virus-removing filtration; (9) sterile filtration and sub-packaging; (10) freeze-drying; (11) dry-heat virus inactivation. The method has the advantages that a human coagulation factor VIII is purified through the two column chromatography steps, so that the prepared high-purity product can reach a specific activity of about 300 IU/mg, considerably higher than about 50 IU/mg in the prior art, and the product appearance and the heat stability are obviously improved; in the preparation process, three virus removing modes are adopted, so that the clinical use safety of the product can be greatly improved.
Description
Technical field
The invention belongs to field of biological pharmacy, relate to a kind of preparation of blood products, specifically relate to the preparation method of a kind of high-purity human blood coagulation factor VII I.
Background technology
Human blood coagulation factor VII I (FVIII) is one of most important thrombin of human body, synthesizes in liver, has 2332 amino-acid residues, is made up of two peptide chains, and heavy chain molecule amount is 90-200kDa; Light chain molecule amount is 80kDa, and two peptide interchains are by Ca
2+connect.Transformation period 8-12 hour in human body, the content in blood plasma is only 0.05-0.lmg/L.
FVIII shortage can cause blood coagulation disorders, hemophilia A is caused to occur, it is that the sudden change of FVIII encoding gene causes a kind of coagulation disorders sex-controlled inheritance caused by this thrombin functional defect sick, the heredity in x linked recessive, transmitted by women, the male sex falls ill, and global incidence 15-20 people/100,000 people, Chinese sickness rate is about 2-3 people/100,000 people.The cardinal symptom of this disease has long-time bleeding tendency after slight damage or minor operation.Hemorrhage performance the following aspects: 1, skin, mucosal bleeding, child is hemorrhage and hemotoncus after being also common in head impact; 2, hemarthrosis, shows as recurrent arthrorrhagia and hemophilic arthosis, is one of modal clinical manifestation of hemophilia, is more common in knee joint, is secondly the places such as ankle, hip, elbow, shoulder joint; 3, hemorrhage of muscle and hemotoncus; 4, wound or postoperative hemorrhage; 5, other positions hemorrhage as internal organs and encephalic; In addition, there is blood urine in the patient of 2/3rds.Hemophilia A belongs to heredopathia, can only carry out surrogate therapeutic at present by infusion FVIII preparation, as long as but regular injections FIII preparation, hemophilia A patients can live completely as normal people.External recombinant human blood coagulation factor VII I entered Chinese market in 2007, but the price of its costliness allows general patient hang back, and, also increasing clinical data is had to show, can inhibition be produced in the patient body of frequent injection recombinant human blood coagulation factor VII I, coagulating effectiveness is obviously declined.
It is very limited that domestic current blood product company supplies clinical FVIII product amount, is difficult to the service requirements meeting domestic hemophilia A patients.Trace it to its cause, mainly traditional FVIII production technique is unstable, and cause product yield low, in addition because foreign protein content is higher, FVIII specific activity is low, and product is unstable, and appearance poor, thermostability is bad, often causes product rejection.
This invention exploits the novel method of a kind of separation and purification FVIII from cryoprecipitate, optimize the parameter of preparation technology, particularly have employed two step chromatography purification techniques, overcome the ubiquitous specific activity of current production technique low (generally lower than 50IU/mg), yield low (generally low 100,000 IU/ ton blood plasma) and product appearance poor (lyophilized powder redissolve liquid have precipitate, opalescence is obvious) shortcoming, have that specific activity is high, steady quality, the advantage that qualification rate is high, can produce on a large scale.
Summary of the invention
Technical problem to be solved by this invention is to provide the preparation method of the human blood coagulation factor VII I of high, the superior in quality and use safety of a kind of process stabilizing, conforming product rate.
A preparation method of high-purity human blood coagulation factor VII I, comprises the steps:
(1) cryoprecipitate is dissolved: by the weight ratio of 1:4-10, cryoprecipitate dropped in the dissolving damping fluid prepared in advance, temperature controls at 15-30 DEG C, stirs 1.5-3 hour, makes it fully dissolve;
(2) aluminum hydroxide gel absorption and press filtration: add Al (OH) 3 gel in the lysate that step (1) obtains, abundant stirring 0.5-1.5 hour, then to connect 1.0 μm of filter core press filtrations with the Supradur50P filter plate that Solution produces, collect clear filtrate, filter front step (1) described dissolving damping fluid prewashing filter plate and filter core;
(3) S/D inactivation of virus: add Tween80 to 1.0% (wt%) in the filtrate collected by step (2), TNBP(tributyl phosphate) to 0.3% (wt%), 24-26 DEG C is warming up to after stirring evenly, rear insulation 6-8 hour, then use 0.45 μm of filter core clarification filtration, collect filtrate;
(4) anion exchange chromatography: the upper anion-exchange column of the filtrate collected by step (3), pillar fully balances with level pad A in advance, after upper prop terminates, pillar is rinsed with level pad A, wash pillar with lavation buffer solution A afterwards, then use elution buffer A wash-out pillar, collect the FVIII solution that elutriant is purifying, timely use 0.45 μm of filter core filters, and collects filtrate;
(5) hydrophobic chromatography: add sodium-chlor to 1.8M-2.0M in the filtrate collected by step (4), then drainage column is gone up, pillar fully balances with level pad B in advance, upper prop terminates rear level pad B and rinses pillar, pillar is washed afterwards with lavation buffer solution B, use elution buffer B wash-out pillar again, collect elutriant and be high-purity FVIII solution; With 0.45 μm or 0.22 μm of above elutriant of filter element filtering;
(6) ultrafiltration: concentrate above filtrate with ultra-filtration membrane bag, then constant volume dialysis 5-7 doubly, tire higher than 35IU/ml to blood coagulation FVIII by reconcentration; After shift out ultra-filtration membrane Bao Binghou and wash film bag;
(7) stablizer and adjustment is added: FVIII content is diluted to desirable value (general requirements is 20-30IU/ml), add stablizer, rear tune pH value is to 6.50-7.50;
(8) nanometer film is except virus filtration: carry out except virus filtration with 0.1 μm of filter core series connection 15-20 nanometer filter core;
(9) Sterile Filtration and packing: Sterile Filtration is carried out and packing to product with 0.22 μm of filter core;
(10) freeze-drying;
(11) xeothermic inactivation of virus: be incubated 0.5-1 hour in 100 DEG C of boiling water baths, carries out xeothermic inactivation of virus.
Dissolving damping fluid described in step (1) is by TRIS(Tutofusin tris), sodium-chlor, glycine, heparin sodium and water forms, in described dissolving damping fluid, the concentration of TRIS is 0.0lM-0.02M, the concentration of sodium-chlor is 0.1M-0.15M, the concentration of glycine is 0.5-1.5% (wt%), the add-on of heparin sodium is 2000-8000IU/ liter, and the pH value of described dissolving damping fluid is 6.50-7.50.
The add-on of the aluminum hydroxide gel described in step (2) is 80-160 gram of/kilogram of suspension.
The resin of the anion-exchange column filling described in step (4) is any one in DEAESepharoseFF, CaptoDEAE, QsepharoseFF, CaptoQ and QSepharoseHP; Described level pad A, lavation buffer solution A and elution buffer A form by Trisodium Citrate, sodium-chlor, glycine, calcium chloride and water; In described level pad A, the concentration of Trisodium Citrate is 0.0lM-0.02M, and the concentration of sodium-chlor is 0.075M-0.15M, and the concentration of glycine is 0.5-1.5%, and the concentration of calcium chloride is 0.005M-0.0l5M, and pH value is PH6.50-7.50; In described lavation buffer solution A, the concentration of Trisodium Citrate is 0.0lM-0.02M, and the concentration of sodium-chlor is 0.2M-0.3M, and the concentration of glycine is 0.5-1.5% (wt%), and the concentration of calcium chloride is 0.005M-0.0l5M, and pH value is PH6.50-7.50; In described elution buffer A liquid, the concentration of Trisodium Citrate is 0.0lM-0.02M, and the concentration of sodium-chlor is 0.5M-2.0M, and the concentration of glycine is 0.5-1.5% (wt%), and the concentration of calcium chloride is 0.001M-0.005M, and pH value is PH6.50-7.50.
The filler of the drainage column described in step (5) is CaptoPhenyl (LS), PhenylSepharose6FF (LS), any one in OctylSepharose4FF; Described level pad B, lavation buffer solution B form by Trisodium Citrate, sodium-chlor, glycine, calcium chloride and water; In described level pad B, the concentration of Trisodium Citrate is 0.01M-0.02M, and the concentration of sodium-chlor is 1.8M-2M, and the concentration of glycine is 0.5-1.5% (wt%), and the concentration of calcium chloride is 0.0lM-0.02M, and pH value is PH6.50-7.50; In described lavation buffer solution B, the concentration of Trisodium Citrate is 0.0lM-0.02M, and the concentration of sodium-chlor is 0.6M-1.6M, and the concentration of glycine is 0.5-1.5% (wt%), and the concentration of calcium chloride is 0.0lM-0.02M, and pH value is PH6.50-7.50; Described elution buffer B is made up of Trisodium Citrate, glycine, calcium chloride and water; In described elution buffer B, the concentration of Trisodium Citrate is 0.005M-0.0lM, and the concentration of glycine is 0.1-1.5% (wt%), and the concentration of calcium chloride is 0.00lM-0.003M, and pH value is PH6.50-7.50.
The ultra-filtration membrane molecular weight cut-off of the ultra-filtration membrane bag described in step (6) is 10K-30K.
Stablizer described in step (7) is the one or more combination in glycine, Histidine, leucine and L-glutamic acid, the add-on of described glycine is 0.5-3% (wt%), the add-on of described Histidine is 0.5-3% (wt%), described leucic add-on is 0.1-3% (wt%), and the add-on of described L-glutamic acid is 0.1-3% (wt%).
Advantage of the present invention is: (1) the present invention adopts anion-exchange chromatography bonded hydrophobic layer to analyse purifying FVIII, and gained FVIII specific activity can reach about 300IU/mg, far above product prepared by current traditional technology; (2) the present invention adopts filter press technique to replace centrifuging and obtains column chromatography upper prop liquid, avoids in centrifugal process that Strong shear power is to the damage of albumen, and reduce the deactivation of FVlll, product is more stable; (3) product prepared of production technique of the present invention, lyophilized powder is that white uniformity is spongy, and the redissolution time is exceedingly fast, and the liquid that redissolves is clear and bright, without albumen precipitation, almost without opalescence; (4) high-purity FVIII product of preparing of the present invention, after xeothermic inactivation of virus, tire can reach xeothermic before about 95%, vigor loss is seldom; (5) have employed S/D inactivation of virus in preparation technology's flow process of the present invention altogether, nano-film filtration virus removal and xeothermic inactivation of virus are sick triple goes out except viral measure, carry out effectively going out removing to lipid-coated virus, non-lipid-coated virus and parvovirus, greatly improved the safety in utilization of product.
accompanying drawing illustrates:
Fig. 1 is preparation technology's schema of high-purity human blood coagulation factor VII I.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail; but can not think that embodiments of the present invention are only limitted to this; for general technical staff of the technical field of the invention; some simple change made without departing from the inventive concept of the premise or replacement, all should be considered as belonging to the scope of patent protection that claims of the present invention is determined.
Embodiment one
1, cryoprecipitate is dissolved: 1kg cryoprecipitate is dropped into 5kg and dissolves (0.02MTRIS-HCL in damping fluid, 0.1MNacL, glycine 1.5% (wt%), PH6.50-6.60), dissolve in damping fluid and add heparin in advance to 2000IU/kg, temperature controls at 15-20 DEG C, stir 3 hours, make it fully dissolve;
2, Al (OH) 3 adsorbs and press filtration: Al (OH) 3 gel adding 0.6kg in above lysate, abundant stirring 1 hour, then to connect 1.0 μm of filter element filterings with the Supradur50P filter plate that Solution produces, collect clear filtrate, with dissolving damping fluid prewashing filter plate and filter core described in step 1 before filtering;
3, S/D inactivation of virus: add Tween80 to 1.0% (wt%) in above filtrate, TNBP(tributyl phosphate) to 0.3% (wt%), after stirring evenly, be warming up to 24-26 DEG C, rear insulation 6 hours, then use 0.45 μm of filter core clarification filtration;
4, anion exchange chromatography: the upper CaptoQ post of the filtrate after above inactivation of virus, pillar fully balances with level pad in advance, and level pad consists of 0.02MTRIS-HCL, 0.1MNaCL, glycine 1.5% (wt%), 0.005MCaCL
2, PH6.50-6.60; After upper prop terminates, use Equilibration buffer wash pillar, rear lavation buffer solution (0.02MTRIS-HCL, 0.25MNaCL, glycine 1.5% (wt%), 0.005MCaCL
2, PH6.50-6.60) and wash pillar, then use elution buffer (0.02MTRIS-HCL, 2.0MNaCL, glycine 1.5% (wt%), 0.00lMCaCL
2, PH6.50-6.60) and wash-out, collect the FVIII solution of purifying;
5, hydrophobic chromatography: CaptoPhenyl (LS) drainage column on above elutriant, pillar fully balances with level pad in advance, level pad consists of 0.01MNa-Citrate, 2.0MNacL, glycine 1.5% (wt%), 0.01MCaCL2, PH6.50-6.60; After upper prop terminates, use Equilibration buffer wash pillar, rear lavation buffer solution (0.0lMNa-Citrate, 1.0MNaCL, glycine 1.5% (wt%), 0.0lMCacL
2, PH6.50-6.60) and wash pillar, then use elution buffer (0.005MNa-Citrate, glycine 0.5% (wt%), 0.001MCaCL
2, PH6.50-6.60) and wash-out, collect high-purity FVIII solution; With 0.45 μm of above elutriant of filter element filtering;
6, with the ultra-filtration membrane (0.1M of 10K molecular weight
2) concentrated above filtrate, then constant volume dialyses 4 times, again concentrates; After shift out ultra-filtration membrane Bao Binghou and wash film bag, record FVIII and tire as 38.4IU/ml;
7, above concentrated solution adds WFI, and being tired by FVIII is adjusted to 30IU/ml, after add glycine to 1.5% (wt%), Histidine to 1.5% (wt%), rear tune pH value is to 6.90-7.10;
8, carry out except virus filtration with 0.1 μm of filter core, 20 nanometer filter cores of connecting;
9, with 0.22 μm of filter core, Sterile Filtration is carried out and packing to product;
10, freeze-drying;
11, xeothermic inactivation of virus: be incubated 30 minutes in 100 DEG C of boiling water baths, carry out xeothermic inactivation of virus.
Embodiment two
1, cryoprecipitate is dissolved: Ikg cryoprecipitate is dropped into (0.02MTRIS-HcL in 10 female dissolving damping fluids, glycine 0.5%, (wt%), 0.1MNaCL, PH7.30-7.40), dissolve in damping fluid and add heparin in advance to 10000IU/kg, temperature controls at 25-30 DEG C, stirs 1.5 hours, makes it fully dissolve;
2, Al (OH) 3 adsorbs and press filtration: Al (OH) 3 gel adding 1.65kg in above lysate, abundant stirring 1.5 hours, then to connect 1.0 μm of filter element filterings with the Supradur50P filter plate that Solution produces, collect clear filtrate, with dissolving damping fluid prewashing filter plate and filter core described in step 2 before filtering;
3, S/D inactivation of virus: with embodiment one;
4, anion exchange chromatography: the upper CaptoDEAE post of the filtrate after above inactivation of virus, pillar fully balances with level pad in advance, and level pad consists of 0.02MTRIS-HCL, 0.1MNacL, glycine 0.5% (wt%), 0.0l5MCaCL
2, PH7.30-7.40; After upper prop terminates, use Equilibration buffer wash pillar, rear lavation buffer solution (0.02MTRIS-HCL, 0.2MNaCL, glycine 0.5% (wt%), 0.015MCaCL
2, PH7.30-7.40) and wash pillar, then use elution buffer (0.02MTRIS-HCL, 0.5MNaCL, glycine 0.5% (wt%), 0.005MCaCL
2, PH7.30-7.40) and wash-out, collect the FVIII solution of purifying;
5, hydrophobic chromatography: add sodium-chlor in above elutriant to 1.8M, then go up OctylSePharose4FF drainage column, pillar fully balances with level pad in advance, and level pad consists of 0.02MNa-Citrate, 1.8MNaCL, glycine 0.5% (wt%), 0.02MCaCL
2, PH7.30-7.40); After upper prop terminates, use Equilibration buffer wash pillar, and rear lavation buffer solution (0.02MNa-Citrate, l.4MNaCL, glycine 0.5% (wt%), 0.02MCaCL
2, PH7.30-7.40) and wash pillar, then use elution buffer (0.01MNa-citrate, glycine 0.2% (wt%), 0.003MCaCL
2, PH7.30-7.40) and wash-out, collect high-purity FVIII solution; With 0.45 μm of above elutriant of filter element filtering;
6, with the ultra-filtration membrane (0.1M of 30K molecular weight
2) concentrated above filtrate, then constant volume dialyses 6 times, again concentrates; After shift out ultra-filtration membrane Bao Binghou and wash film bag, record FVIII and tire as 40.1IU/ml;
7, above concentrated solution adds WFI, and being tired by FVlll is adjusted to 30IU/ml, after add glycine to 3% (wt%), rear tune pH value is to 6.90-7.10;
8-11, with embodiment one.
Embodiment three
1, cryoprecipitate is dissolved: 1kg cryoprecipitate is dropped into 7kg and dissolves (0.02MTRIS-HCL in damping fluid, 0.15MNaCL, glycine 1.0% (wt%), PH6.90-7.10), dissolve in damping fluid and add heparin in advance to 6000IU/kg, temperature controls at 20-25 DEG C, stir 2 hours, make it fully dissolve;
2, Al (OH) 3 adsorbs and press filtration: Al (OH) 3 gel adding 0.7kg in above lysate, abundant stirring 0.5 hour, then to connect 1.0 μm of filter element filterings with the Supradur50P filter plate that Solution produces, collect clear filtrate, with dissolving damping fluid prewashing filter plate and filter core described in step 2 before filtering;
3, S/D inactivation of virus: with embodiment one;
4, anion exchange chromatography: the upper QSePharose4FF post of the filtrate after above inactivation of virus, pillar fully balances with level pad in advance, level pad consists of 0.02MTRIS-HcL, 0.15MNaCL, glycine 1.0% (wt%), 0.01MCacL2, PH6.90-7.10; After upper prop terminates, use Equilibration buffer wash pillar, rear lavation buffer solution (0.02MTRIS-HCL, 0.3MNacL, glycine 1.0% (wt%), 0.0lMCacL
2, PH6.90-7.10) and wash pillar, then use elution buffer (0.02MTRIS-HCL, 1.0MNaCL, glycine 1.0% (wt%), 0.003MCacL
2, PH6.90-7.10) and wash-out, collect the FVIII solution of purifying;
5, hydrophobic chromatography: add sodium-chlor in above elutriant to 2.0M, then Phenylsepharose6FF (LS) drainage column is gone up, pillar fully balances with level pad in advance, level pad consists of 0.02MNa-citrate, 2.0MNaCL, glycine 1.0% (wt%), 0.015MCacL
2, PH6.90-7.10; After upper prop terminates, use Equilibration buffer wash pillar, rear lavation buffer solution (0.02MNa-Citrate, 0.8MNaCL, glycine 1.0% (wt%), 0.0l5MCaCL
2, PH6.90-7.10) and wash pillar, then use elution buffer (0.0lMNa-Citrate, glycine 0.1% (wt%), 0.002MCaCL
2, PH6.90-7.10) and wash-out, collect high-purity FVIII solution; With 0.45 μm of above elutriant of filter element filtering;
6, with the ultra-filtration membrane (0.1M of 10K molecular weight
2) concentrated above filtrate, then constant volume dialyses 5 times, again concentrates; After shift out ultra-filtration membrane Bao Binghou and wash film bag, record FVIII and tire as 35.3IU/ml;
7, above concentrated solution adds WFI, and being tired by FVIII is adjusted to 30IU/ml, after add glycine to 2% (wt%), rear tune pH value is to 6.90-7.10;
8-11, with embodiment one.
Table FVIII preproduction yield and specific activity data statistics
Claims (10)
1. a preparation method of high-purity human blood coagulation factor VII I, is characterized in that comprising the steps:
(1) cryoprecipitate is dissolved
By the weight ratio of 1:4-10, cryoprecipitate dropped in the dissolving damping fluid prepared in advance, temperature controls at 15-30 DEG C, stirs 1.5-3 hour, makes it fully dissolve;
(2) aluminum hydroxide gel absorption and press filtration
Al (OH) 3 gel is added in the lysate that step (1) obtains, abundant stirring 0.5-1.5 hour, then to connect 1.0 μm of filter element filterings with the Supradur50P filter plate that Solution produces, collect clear filtrate, filter front step (1) described dissolving damping fluid prewashing filter plate and filter core;
(3) S/D inactivation of virus
Tween80 to 1.0% (wt%) is added in filtrate collected by step (2), TNBP(tributyl phosphate) to 0.3% (wt%), after stirring evenly, be warming up to 24-26 DEG C, rear insulation 6-8 hour, then use 0.45 μm of filter core clarification filtration, collect filtrate;
(4) anion exchange chromatography
The upper anion-exchange column of filtrate collected by step (3), pillar fully balances with level pad A in advance, after upper prop terminates, pillar is rinsed with level pad A, wash pillar with lavation buffer solution A afterwards, then use elution buffer A wash-out pillar, collect the FVIII solution that elutriant is purifying, timely use 0.45 μm of filter core filters, and collects filtrate;
(5) hydrophobic chromatography
Sodium-chlor is added to 1.8M-2.0M in filtrate collected by step (4), then drainage column is gone up, pillar fully balances with level pad B in advance, upper prop terminates rear level pad B and rinses pillar, pillar is washed afterwards with lavation buffer solution B, use elution buffer B wash-out pillar again, collect elutriant and be high-purity FVIII solution; With 0.45 μm or 0.22 μm of above elutriant of filter element filtering;
(6) ultrafiltration
Concentrate above filtrate with ultra-filtration membrane bag, then constant volume dialysis 5-7 doubly, tire higher than 35IU/ml to FVIII by reconcentration; After shift out ultra-filtration membrane Bao Binghou and wash film bag;
(7) stablizer and adjustment is added
Tired by FVIII and be adjusted to desirable value (general requirements is 20-30IU/ml), add stablizer, rear tune pH value is to 6.50-7.50;
(8) nanometer film is except virus filtration
Carry out except virus filtration with 0.1 μm of filter core series connection 15-20 nanometer filter core;
(9) Sterile Filtration and packing
With 0.22 μm of filter core, Sterile Filtration is carried out and packing to product;
(10) freeze-drying;
(11) xeothermic inactivation of virus
In 100 DEG C of boiling water baths, be incubated 0.5-1 hour, carry out xeothermic inactivation of virus.
2. the preparation method of a kind of high-purity human blood coagulation factor VII I according to claim 1, it is characterized in that: the dissolving damping fluid described in step (1) is by TRIS(Tutofusin tris), sodium-chlor, glycine, heparin sodium and water forms, in described dissolving damping fluid, the concentration of TRIS is 0.0lM-0.02M, the concentration of sodium-chlor is 0.1M-0.15M, the concentration of glycine is 0.5-1.5% (wt%), the add-on of heparin sodium is 2000-8000IU/ liter, and the pH value of described dissolving damping fluid is 6.50-7.50.
3. the preparation method of a kind of high-purity human blood coagulation factor VII I according to claim 1, is characterized in that: the add-on of the aluminum hydroxide gel described in step (2) is 80-160 gram of/kilogram of suspension.
4. the preparation method of a kind of high-purity human blood coagulation factor VII I according to claim 1, is characterized in that: the resin of the anion-exchange column filling described in step (4) is any one in DEAESepharoseFF, CaptoDEAE, QsepharoseFF, CaptoQ and QSepharoseHP.
5. the preparation method of a kind of high-purity human blood coagulation factor VII I according to claim 1, is characterized in that: the level pad A described in step (4), lavation buffer solution A and elution buffer A form by Trisodium Citrate, sodium-chlor, glycine, calcium chloride and water; In described level pad A, the concentration of Trisodium Citrate is 0.0lM-0.02M, and the concentration of sodium-chlor is 0.075M-0.15M, and the concentration of glycine is 0.5-1.5% (wt%), and the concentration of calcium chloride is 0.005M-0.0l5M, and pH value is PH6.50-7.50; In described lavation buffer solution A, the concentration of Trisodium Citrate is 0.0lM-0.02M, and the concentration of sodium-chlor is 0.2M-0.3M, and the concentration of glycine is 0.5-1.5% (wt%), and the concentration of calcium chloride is 0.005M-0.0l5M, and pH value is PH6.50-7.50; In described elution buffer A liquid, the concentration of Trisodium Citrate is 0.0lM-0.02M, and the concentration of sodium-chlor is 0.5M-2.0M, and the concentration of glycine is 0.5-1.5% (wt%), and the concentration of calcium chloride is 0.001M-0.005M, and pH value is PH6.50-7.50.
6. the preparation method of a kind of high-purity human blood coagulation factor VII I according to claim 1, it is characterized in that: the filler of the drainage column described in step (5) is CaptoPhenyl (LS), PhenylSepharose6FF (LS), any one in OctylSepharose4FF.
7. the preparation method of a kind of high-purity human blood coagulation factor VII I according to claim 1, is characterized in that: the described level pad B of step (5), lavation buffer solution B form by Trisodium Citrate, sodium-chlor, glycine, calcium chloride and water; In described level pad B, the concentration of Trisodium Citrate is 0.01M-0.02M, and the concentration of sodium-chlor is 1.8M-2M, and the concentration of glycine is 0.5-1.5% (wt%), and the concentration of calcium chloride is 0.0lM-0.02M, and pH value is PH6.50-7.50; In described lavation buffer solution B, the concentration of Trisodium Citrate is 0.0lM-0.02M, and the concentration of sodium-chlor is 0.6M-1.6M, and the concentration of glycine is 0.5-1.5% (wt%), and the concentration of calcium chloride is 0.0lM-0.02M, and pH value is PH6.50-7.50; Described elution buffer B is made up of Trisodium Citrate, glycine, calcium chloride and water; In described elution buffer B, the concentration of Trisodium Citrate is 0.005M-0.0lM, and the concentration of glycine is 0.1-1.5% (wt%), and the concentration of calcium chloride is 0.00lM-0.003M, and pH value is PH6.50-7.50.
8. the preparation method of a kind of high-purity human blood coagulation factor VII I according to claim 1, is characterized in that: the ultra-filtration membrane molecular weight cut-off of the ultra-filtration membrane bag described in step (6) is 10K-30K.
9. the preparation method of a kind of high-purity human blood coagulation factor VII I according to claim 1, it is characterized in that: the stablizer described in step (7) is the one or more combination in glycine, Histidine, leucine and L-glutamic acid, the add-on of described glycine is 0.5-3% (wt%), the add-on of described Histidine is 0.5-3% (wt%), described leucic add-on is 0.1-3% (wt%), and the add-on of described L-glutamic acid is 0.1-3% (wt%).
10. the preparation method of a kind of high-purity human blood coagulation factor VII I according to claim 1-9, is characterized in that: human blood coagulation factor VII I product goes out except step through S/D, nano-film filtration and xeothermic three step viruses in whole preparation flow.
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