CN105541997A - Preparation process of high-purity and high-activity von Willebrand factors - Google Patents
Preparation process of high-purity and high-activity von Willebrand factors Download PDFInfo
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- CN105541997A CN105541997A CN201610077518.4A CN201610077518A CN105541997A CN 105541997 A CN105541997 A CN 105541997A CN 201610077518 A CN201610077518 A CN 201610077518A CN 105541997 A CN105541997 A CN 105541997A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/745—Blood coagulation or fibrinolysis factors
- C07K14/7455—Thrombomodulin
Abstract
The invention discloses a preparation process of high-purity and high-activity von Willebrand factors. According to the preparation process, waste, namely a liquid flowing out from a chromatographic column in the preparation of cryoprecipitation extracted blood coagulation factors VIII, of the cryoprecipitation extracted blood coagulation factors VIII is collected as a raw material; glycine is added into a chromatographic buffer solution of a protein fluid after being subjected to chromatography, so as to protect vWF activity; preferably, lysine, glycine and albumin are added into the protein fluid after being subjected to chromatography before freeze drying as a freeze-drying protecting agent, so as to protect vWF activity. The high-purity and high-activity von Willebrand factors prepared by virtue of the preparation process are reliable in quality, can meet urgent therapeutic medication requirements of patients with von Willebrand on clinic and are simultaneously significant for the comprehensive utilization of cryoprecipitation and the indirect saving of the scarce plasma resource.
Description
Technical field
The present invention relates to a kind of preparation technology extracting human von willebrand disease factor from the waste material of human plasma cryoprecipitate extraction platelet cofactor Ⅰ, particularly the preparation technology of a kind of high purity and high reactivity vWF ELISA, belongs to field of biological pharmacy.
Background technology
VWF is that one has polymeric plasma glycoprotein, and its molecular weight from 250kDa to 2,000 ten thousand kDa not etc., forms maximum molecular weight soluble proteins known in blood plasma.Blood plasma small molecular amount vWF mainly dimeric forms, molecular weight is about 500kDa.The polymer differed in size, significant to maintenance vWF normal biological activity.
Plasma vWF plays an important role in primary hemostasis, and it is responsible for the adhesion of thrombocyte and damaged blood vessel surface, and therefore forms platelet thrombus, contributes to the formation that scleroproein is crosslinked.In addition, plasma vWF plays a part transport agent and the stablizer of blood coagulation factor VIII.
Von Willebrand disease (vWD) is modal hereditary hemorrhagic disease, and patient's vWF (vonWillebrandfactor, vWF) transgenation or the abnormal plasma vWF quantity that causes of regulation and control reduce or abnormal quality.It is hemorrhage that vWD Clinical symptoms is mainly skin mucosa, and the hemorrhage and muscle hemotoncus of the joint cavity similar to hemophilia A also can occur for 2N type and 3 type patients.According to external, vWD sickness rate (2.3 ~ 11.0)/100,000 population, it is generally acknowledged that, in hereditary hemorrhagic disease, vWD occupies second.
The activity Ristocetin co-factor activity (vWF:RCo) of vWF characterizes clinically, is mainly used in treating congenital, Of Acquired von Willebrand Disease (vWD), generally carrys out administration with 20 ~ 60UvWF:RCo/kg dosage.
Research shows to be rich in the blood clotting factors materials such as vWF in cryoprecipitate.In today that blood plasma raw material is increasingly in short supply, prepare vWF by cryoprecipitate, to the comprehensive utilization of cryoprecipitate, save rare blood plasma resource, improve the market competitiveness and be significant.
In prior art, technique comparatively early uses the antibody coupling of vWF on sepharose, by immunoaffinity chromatography purifying vWF, but its antibody preparation cycle is long, gel carrying capacity is low, the extensive preparation of uncomfortable cooperation, as " PurificationofvonWillebrandFactorsolutionsusinggelpermea tionchromatography " (UP4774323).
There is patent DEAEFractogelTSK650M (Merck) purifying vWF factor component, purity is high, but technics comparing is complicated, need two-step solution and a step affinity chromatography, as " Processforanindustrial-scalepreparationofastandardizedhu manvonWillebrandfactorconcentrateofveryhighpurityandsuit ablefortherapeuticuse " (US5408039).Also report CHT chromatography is had to be separated vWF component in conjunction with PH5.4 Acid precipitation, its specific activity is not high, and while Acid precipitation removal Fiberonectin, the biologic activity of vWF may be affected, as " Productionofavonwillebrandfactorprepartionhavingagreatsp ecificactivity " (US20070135619A1).
Some technique EMDFractogelTMAE (Merck) are also had to carry out purifying VIII/vWF factor coniplexes in addition, but all cannot obtain separately highly purified vWF, treatment specific aim is not strong, as " preparation containing vWF with styptic activity and preparation method thereof " (CN1425024A), " AProcessforrecoveringahigh-purityvirus-inactivatedfactor VIIIbyanionexchangerchromatography " (US5714590).
Also has method (monkey-kidney cells or Chinese hamster ovary celI) the purifying vWF of some company's gene recombination, they are purified from animal cell culture fluid by anion exchange chromatography, but due to the existence of animal originality antigen, human body may have repulsive interaction to it, as " MethodforisolationofhighlypurevonWillebrandFactor " (US5854403).
Separately have the report adopting two-step chromatography purification vWF, but its purity is not high, specific activity is relatively low, only 50IU/mg, as " ProcessformanufacturingvonWillebrandfactor " (US5252710).
Visible, still there are some problems in existing vWF purifying process: (1) preparation cycle is long, is not suitable for extensive preparation, and when especially adopting immune-affinity chromatography purifying, its antibody preparation cycle is long, and gel carrying capacity is low.(2) VIII/vWF composite product is in more difficult Controlling Technology stability; (3) may there is heterogeneic antigen in recombination method gained vWF product; (4) separation purifying technique is loaded down with trivial details.
At present, von Willebrand disease can use cryoprecipitate, VIII/vWF factor coniplexes and DDAVP (DDAVP) to treat, but specific aim is not strong, the purer vWF factor can the various types of von Willebrand disease of specific therapeutic, has greater advantage with front several method ratio.
Summary of the invention
The object of the present invention is to provide the preparation technology of a kind of high purity and high reactivity vWF ELISA.
Major technique design of the present invention is as follows:
The present invention adds glycine protection vWF activity to the protein liquid after chromatography in chromatography buffer; Add Methionin, glycine and albumin in protein liquid after preferred chromatography before freeze-drying as lyophilized vaccine, protection vWF is active; Glycine mass concentration 0.6% ~ 1% in preferred chromatography buffer, Methionin mass concentration 2 ~ 3% in lyophilized vaccine, glycine mass concentration 0.2%, albumin mass concentration 0.8 ~ 1.3%.
The present invention optimizes interpolation 2% aluminum hydroxide gel and carries out Adsorption II, VII, IX, X factor before S/D inactivation of virus; The filter core that before S/D deactivation, cascade filtration uses is of a size of the filter core of 1.0 μm and 0.45 μm.
The present invention one step Q-Sepharose anion-exchange chromatography and a step affinitive layer purification obtain the higher vWF of purity.Namely the liquid of the waste material of collection cryoprecipitate extraction platelet cofactor Ⅰ namely through chromatographic column effluent in the preparation of cryoprecipitate extraction platelet cofactor Ⅰ is as raw material; After Q-Sepharose ion-exchange chromatography, collect elutriant, then through gelatin affinity chromatography, remove remaining Fiberonectin and impurity, obtain vWF ELISA.
In preparation technology of the present invention:
(1) by the salt concn in adjustment lavation buffer solution B and elution buffer B; Q-Sepharose column chromatography is adopted to remove Fibrinogen and Fiberonectin and other foreign protein;
(2) elutriant after Q-Sepharose column chromatography, then through gelatin chromatography, by adjusting the salt concn of elution buffer B and dialyzate, remove remaining Fiberonectin and impurity, the stream obtained containing high purity vWF wears liquid.
Preparation technology of the present invention, it comprises successively: cryoprecipitate is dissolved, 2% aluminum hydroxide gel adsorbs, regulate ionic strength, cascade filtration, S/D inactivation of virus, ion exchange chromatography; Ion exchange chromatography must collect elutriant, and elutriant, through ultrafiltration, dialysis, filtration, for the production of blood coagulation factor VIII, then extracts the waste material of platelet cofactor Ⅰ from cryoprecipitate and namely collects the liquid passed through chromatography column stream and extract vWF; Q-Sepharose anion-exchange chromatography, gelatin affinity chromatography, Sterile Filtration packing, lyophilize, xeothermic deactivation.After ion exchange chromatography stream wears liquid collection, first concentrated one times, then use lavation buffer solution B equal-volume ultrafiltration 6 times, then through Q-Sepharose chromatography column, collect the protein liquid eluted with elution buffer B.
The present invention is achieved like this, and its concrete technology scheme is as follows:
(1), after qualified human plasma of quarantining quarantine is got, 75% ethanol plasma bags surface, rinses with water for injection, and be merged into and melt in slurry tank, melt with less than 30 ~ 35 DEG C recirculated waters, blood plasma temperature controls not higher than 4 DEG C; After melting, centrifugal, go out liquid temp and control at 0 ~ 4 DEG C, collect cryoprecipitate;
(2) added in 3IU/ml heparin sodium aqua by the obtained thing of step (1), be stirred to cryoprecipitate and dissolve completely, the temperature of recirculated water controls at 28 ~ 37 DEG C; Start centrifugal, collect supernatant liquor, weigh;
(3) the obtained thing supernatant liquor 1mol/L hydrochloric acid of step (2) is regulated PH to 6.0 ~ 7.0; Add 2% aluminum hydroxide gel, stir; Start centrifugal, collect supernatant liquor, weigh;
(4) calculate by " W (ionic strength buffer liquid)=supernatant liquor weight/10 ", take the ionic strength buffer liquid of calculated amount in the obtained thing supernatant liquor of step (3); Regulate supernatant liquor PH to 6.0 ~ 7.0; 10g/L is not more than by protein concentration damping fluid Function protein concentration; With the filter element filtering of 1.0 μm, collect filtered liquid, weigh;
(5) by step (4) obtained thing filtered liquid volume 1/10 add S/D solution, stir, temperature controls at 24 ~ 26 DEG C, continuously insulation 6 hours; 0.45 μm of filter element filtering; Filtered liquid 30KD ultra-filtration membrane is concentrated into protein concentration 2%, then uses more than the 2 times lavation buffer solution A constant volumes ultrafiltration of protein liquid weight, obtains ultrafiltrated, weigh;
(6) the obtained thing ultrafiltrated ion exchange column of step (5) is carried out chromatography purification, pillar is washed until become baseline with lavation buffer solution A, with elution buffer A wash-out, collect elutriant (elutriant through ultrafiltration, dialysis, filtration, the production for blood coagulation factor VIII).
(7) collect the liquid that step (6) is come through chromatographic column effluent, concentrated one times, lavation buffer solution B equal-volume ultrafiltration 6 times, obtains ultrafiltrated, weighs.
(8) the obtained thing ultrafiltrated Q-Sepharose anion-exchange chromatography post of step (7) is carried out chromatography purification, wash pillar until become baseline with lavation buffer solution B, with elution buffer B wash-out, collect elutriant, weigh.
(9) the obtained thing elutriant gelatin affinity chromatography of step (8) is carried out purifying, wash pillar until become baseline with elution buffer B, the liquid come through chromatographic column effluent after collecting loading, with dialyzate dialysis, be diluted to the 100IU/ml that tires, obtain protein liquid, weigh.
(10) by the obtained thing protein liquid of step (9) through 0.2 μm of degerming filter element filtering packing; Divide the goods installed to import freeze-drying cabinet into and carry out lyophilize; Offer for sale Zha Gai; 99 ~ 100 DEG C, 30 minutes xeothermic inactivation of virus; Censorship, packs after the assay was approved, puts in storage.
The compound method of ionic strength buffer liquid: 48.5g Tutofusin tris, 10g calcium chloride, 100g sodium-chlor, 28.1g glycine, adds appropriate water for injection and fully dissolves, and mends and injects water to 1L, regulates PH to be 6.0 ~ 7.0; The compound method of protein concentration damping fluid: measure 0.05L and regulate ionic strength buffer liquid, inject water to 1L, regulate PH to be 6.0 ~ 7.0.
Lavation buffer solution A fills a prescription: 2.5g Tutofusin tris, 10g calcium chloride, 25g sodium-chlor, adds appropriate water for injection and fully dissolves, and mends and injects water to 1L, regulates PH to be 6.0 ~ 7.0.
Elution buffer A fills a prescription: 2.5g Tutofusin tris, 15g calcium chloride, 40.9g sodium-chlor, adds appropriate water for injection and fully dissolves, and mends and injects water to 1L, regulates PH to be 6.0 ~ 7.0.
The formula of lavation buffer solution B: 4g Sodium Citrate, 11.4g sodium-chlor, 3g calcium chloride, 6g glycine, adds appropriate water for injection and fully dissolves, and mends and injects water to 1L, regulates PH to be 5.8 ~ 6.8.
Elution buffer B fills a prescription: 4g Sodium Citrate, 16.38g sodium-chlor, 3g calcium chloride, 6g glycine, adds appropriate water for injection and fully dissolves, and mends and injects water to 1L, regulates PH to be 5.8 ~ 6.8.
The formula of dialyzate: 2.94g Sodium Citrate, 25g sodium-chlor, 0.111g calcium chloride, 30g glycine, 2g lysine hydrochloride, human serum albumin 8g, adds appropriate water for injection and fully dissolves, and mends and injects water to 1L, regulates PH to be 6.8 ~ 7.2.
Described percentage ratio is apart from outside restriction, and all the other are mass percent.
Positively effect of the present invention:
1, in today that blood plasma raw material is increasingly in short supply, prepare vWF by cryoprecipitate, making full use of cryoprecipitate, improve the market competitiveness and have great significance, in addition also can the rare blood plasma resource of indirect saving.
2, in the process of ion exchange chromatography purification vWF, the solution got off with suitable elution, then through operations such as ultrafiltration, dialysis, filtrations, can be used for producing platelet cofactor Ⅰ.
The present invention is compared to the preparation technology of traditional vWF, and innovative point is:
1. make full use of blood plasma resource, wear waste liquid with the stream of blood coagulation factor VIII ion-exchange and prepare high purity vWF.Waste material can be produced as vWF, Fibrinogen etc. when producing blood coagulation factor VIII, by the Component seperation in waste material out, turn waste into wealth, utilize blood plasma resource in short supply to greatest extent, improve blood plasma utilization ratio.
2, add glycine to chromatography buffer, add Methionin, glycine and albumin in the protein liquid after chromatography before freeze-drying, protection vWF is active.Add glycine in chromatography buffer and in chromatographic separation purge process, the loss of activity of vWF can be reduced.Albumin, Methionin, glycine is added as lyophilized vaccine, Absorbable organic halogens vWF molecule in albumen dialyzate.Albumin is excellent protein stabiliser, simultaneously can the absorption of effective protein surface.Methionin, glycine are small molecules amino acid, can protected protein matter structure, can raise the collapse temperature of finished product, stop the Protein Damage caused because subsiding in freeze-drying process.Keep biologic activity.
3, ion exchange chromatography is separated the salt ionic concentration that the most critical factor of vWF is washings and elutriant.The too low meeting of washings salt concn causes the impurity such as Fibrinogen, Fiberonectin remaining, and washings salt concn is too high, will the rate of recovery of vWF be caused to reduce, and the vWF amount that wash-out obtains reduces.Find after test of many times, 195mM (11.4g/L) sodium-chlor is best washings salt concn.Eluent salt concentration also need in optimum range in addition, and the too high too low acquisition being also unfavorable for vWF, we are defined as 280mM (16.38g/L) by test.The proportioning of these two kinds of salt concn can take into account activity and the specific activity of vWF, is a pair best matched proportion density.
4, this technique adopts a step ion-exchange and a step affinity chromatography technique, can ensure purity and the yield of vWF, also can simplify production technique, fully reduces manpower and time cost.Ion exchange chromatography plays the effect of preliminary purification, and affinity chromatography filler has the feature of binding proteins specific, carries out polishing purification further to albumen.
5, adopt the cryoprecipitate of humanized as starting raw material, avoid the generation of the heterogeneic antigen rejection of recombinant protein, improve Clinical practice security.
Accompanying drawing explanation
Fig. 1 is present invention process schema.
Concrete case study on implementation
The present invention is by the following examples can the invention will be further described, but scope of the present invention is not limited to following embodiment.
For 6000L blood plasma, concrete preparation technology is as follows:
(1), after qualified human plasma of quarantining quarantine is got, 75% ethanol plasma bags surface, rinses with water for injection, and be merged into and melt in slurry tank, melt with less than 30 ~ 35 DEG C recirculated waters, blood plasma temperature controls not higher than 4 DEG C; After melting, centrifugal, go out liquid temp and control, at 0 ~ 4 DEG C, to collect to obtain cryoprecipitate 45.2kg;
(2) the obtained thing cryoprecipitate of step (1) added in 3IU/ml heparin sodium aqua, be stirred to cryoprecipitate and dissolve completely, the temperature of recirculated water controls at 28 ~ 37 DEG C; Start centrifugal, collect supernatant liquor, weigh to obtain 169.5kg;
(3) the obtained thing supernatant liquor 1mol/LHCL of step (2) is regulated PH to 6.0 ~ 7.0; Add 2% aluminum hydroxide gel, stir; Start centrifugal, collect supernatant liquor, weigh to obtain 178.6kg;
(4) calculate by " W (ionic strength buffer liquid)=supernatant liquor weight/10 ", take the ionic strength buffer liquid of calculated amount in the obtained thing supernatant liquor of step (3); Regulate supernatant liquor PH to 6.0 ~ 7.0; 10g/L is not more than by protein concentration damping fluid Function protein concentration; Filter afterwards with the filter core series connection of the filter cores of 1.0 μm and 0.45 μm, collect filtered liquid, weigh to obtain 189.9kg;
(5) by step (4) obtained thing filtered liquid volume 1/10 add S/D solution, stir, temperature controls at 24 ~ 26 DEG C, continuously insulation 6 hours; 0.45 μm of filter element filtering; Filtered liquid 30KD ultra-filtration membrane is concentrated into protein concentration about 2%, and then use more than the 2 times lavation buffer solution A constant volumes ultrafiltration of protein liquid weight, i.e. ultrafiltrated, weigh to obtain 201.4L;
(6) the obtained thing ultrafiltrated ion exchange column of step (5) is carried out chromatography purification, wash pillar until become baseline with lavation buffer solution A, with elution buffer A wash-out, collect elutriant.
(7) collect the liquid that step (6) is come through chromatographic column effluent, concentrated one times, lavation buffer solution B equal-volume ultrafiltration 6 times, obtains ultrafiltrated, and weigh to obtain 120.5L;
(8) the obtained thing ultrafiltrated Q-Sepharose anion-exchange chromatography post of step (7) is carried out chromatography purification, wash pillar until become baseline with lavation buffer solution B, with elution buffer B wash-out, collect elutriant, weigh to obtain 48.5L;
(9) the obtained thing elutriant gelatin affinity chromatography of step (8) is carried out purifying, wash pillar until become baseline with elution buffer B, the liquid come through chromatographic column effluent after collecting loading, ultrafiltration and concentration is to 5L, dialyse 6 times, obtain ultrafiltrated, weigh to obtain 5.2L; 6.1L is assigned to by the 100IU/ml that tires is rare;
(10) by the obtained thing filtrate of step (9) through 0.2 μm of degerming filter element filtering packing, packing loading amount is every bottle of 5mL, and packing quantity is 1196 bottles; Divide the goods installed to import freeze-drying cabinet into and carry out lyophilize; Offer for sale Zha Gai; 99 ~ 100 DEG C, 30 minutes xeothermic inactivation of virus; Censorship, packs after the assay was approved, puts in storage;
Described percentage ratio is apart from outside restriction, and all the other are mass percent.
The contrast of the vWF Key Quality Indicator that product prepared by the inventive method describes with " in European Pharmacopoeia " is as following table 1.
Table 1:
| Project | The present invention | " European Pharmacopoeia " |
| PH | 6.8～7.2 | 6.5～7.5 |
| Active | Should >=100IU/ml | ≥1IU/ml |
| Specific activity | Should >=200IU/mg | ≥20IU/mg |
| Blood coagulation factor VIII content | VIII:C 2 ~ 4IU/ml in every 100IU vWF:RCo | VIII:C≤10IU/ml in every 100IUvWF:RCo |
| Osmotic pressure molar density | 240～1000mOsmol/kg | 240mOsmol/kg should be not less than |
In product prepared by the inventive method, different ion-exchange salt concn is studied, as following table 2 lab scale of the impact of quality product.
Table 2:
Illustrate: in anion exchange procedures, vWF and Fiberonectin are in this salt concn interval of 150mMNaCl to 300mMNaCl, to salt concn and ionic strength very responsive, through Experimental Comparison analysis, screening obtains the minimum column buffer liquid formula of two kinds of major impurities (Fiberonectin and Fibrinogen).Preferred washings is 195mMNaCl (11.4g/L) and elutriant is 280mMNaC (16.38g/L) l, can obtain active and that specific activity is all higher vWF.
In product prepared by the inventive method, in chromatography buffer, glycine is on the impact of product, as table 3.
Table 3:
| Glycine concentration (%) | Washings B salt concn (%) | Elutriant B salt concn (%) | VWF:RCo activity (IU/ml) |
| 0 | 195 | 280 | 101.5 |
| 0.2 | 195 | 280 | 102.1 |
| 0.6 | 195 | 280 | 104.8 |
| 1.0 | 195 | 280 | 104.81 |
Illustrate: the glycine in chromatography buffer can in chromatography process, and the effect in protection vWF, mass concentration 0.6% ~ 1%, the provide protection of glycine concentration to chromatography process vWF of this scope is more or less the same, and selects lower, it is still further preferred that 0.6%.
In product prepared by the inventive method, dialyzate (frozen-dried protective liquid) various heterogeneity on the impact of product, as table 4.
Table 4:
Illustrating: glycine mass concentration 2 ~ 3%, Methionin mass concentration 0.2%, albumin mass concentration 0.8 ~ 1.3%, is the good scope of application, and outward appearance slightly or without atrophy, and has slight or without opalescence, activity is also better protected.The difference of the mass concentration of Methionin is less on frozen-dried protective impact, selects 0.2%.Best dialysis buffer liquid (frozen-dried protective liquid) is filled a prescription as glycine concentration is 3%, and albumin concentration is 0.8%, and outward appearance and activity are the best.
Claims (10)
1. a preparation technology for high purity and high reactivity vWF ELISA, is characterized in that, collect cryoprecipitate extract platelet cofactor Ⅰ waste material namely cryoprecipitate extract platelet cofactor Ⅰ preparation in through chromatographic column effluent come liquid as raw material; In the preparation technology of vWF ELISA, glycine protection vWF activity is added in chromatography buffer to the protein liquid after chromatography; Add Methionin, glycine and albumin in protein liquid after preferred chromatography before freeze-drying as lyophilized vaccine, protection vWF is active; Glycine mass concentration 0.6% ~ 1% in preferred chromatography buffer, Methionin mass concentration 2 ~ 3% in lyophilized vaccine, glycine mass concentration 0.2%, albumin mass concentration 0.8 ~ 1.3%.
2. the preparation technology of a kind of high purity according to claim 1 and high reactivity vWF ELISA, it is characterized in that, obtain the higher vWF of purity with a step Q-Sepharose anion-exchange chromatography and a step affinitive layer purification: collect cryoprecipitate extract platelet cofactor Ⅰ waste material namely cryoprecipitate extract platelet cofactor Ⅰ preparation in through chromatographic column effluent come liquid as raw material; After Q-Sepharose ion-exchange chromatography, collect elutriant, then through gelatin affinity chromatography, remove remaining Fiberonectin and impurity, obtain vWF ELISA.
3. the preparation technology of a kind of high purity according to claim 1 and high reactivity vWF ELISA, it is characterized in that, cryoprecipitate extract platelet cofactor Ⅰ preparation technology comprise: cryoprecipitate dissolve, 2% aluminum hydroxide gel absorption, regulate ionic strength, cascade filtration, S/D deactivation, ion exchange chromatography; Ion exchange chromatography collect elutriant, elutriant through ultrafiltration, dialysis, filtration, for the production of blood coagulation factor VIII.
4. a kind of high purity according to claim 1,2 or 3 and the preparation technology of high reactivity vWF ELISA, is characterized in that,
(1) by the salt concn in adjustment lavation buffer solution B and elution buffer B; Q-Sepharose column chromatography is adopted to remove Fibrinogen and Fiberonectin and other foreign protein;
(2) elutriant after Q-Sepharose column chromatography, then through gelatin chromatography, by adjusting the salt concn of elution buffer B and dialyzate, remove remaining Fiberonectin and impurity, the stream obtained containing high purity vWF wears liquid.
5. the preparation technology of a kind of high purity according to claim 3 and high reactivity vWF ELISA, is characterized in that, the filter core that before S/D deactivation, cascade filtration uses is of a size of the filter core of 1.0 μm and 0.45 μm.
6. the preparation technology of a kind of high purity according to claim 3 and high reactivity vWF ELISA, it is characterized in that, after ion exchange chromatography stream wears liquid collection, first concentrated one times, use lavation buffer solution B equal-volume ultrafiltration 6 times again, again through Q-Sepharose chromatography column, collect the protein liquid eluted with elution buffer B.
7. the preparation technology of a kind of high purity according to claim 4 and high reactivity vWF ELISA, it is characterized in that, the formula of described lavation buffer solution B: 4g Sodium Citrate, 11.4g sodium-chlor, 3g calcium chloride, 6g glycine, adds appropriate water for injection and fully dissolves, benefit injects water to 1L, regulates pH to be 5.8 ~ 6.8.
8. the preparation technology of a kind of high purity according to claim 4 and high reactivity vWF ELISA, it is characterized in that, described elution buffer B fills a prescription: 4g Sodium Citrate, 16.38g sodium-chlor, 3g calcium chloride, 6g glycine, adds appropriate water for injection and fully dissolves, benefit injects water to 1L, regulates pH to be 5.8 ~ 6.8.
9. the preparation technology of a kind of high purity according to claim 4 and high reactivity vWF ELISA, it is characterized in that, described dialyzate formula: 2.94g Sodium Citrate, 25g sodium-chlor, 0.111g calcium chloride, 30g glycine, 2g lysine hydrochloride, human serum albumin 8g, adds appropriate water for injection and fully dissolves, benefit injects water to 1L, regulates pH to be 6.8 ~ 7.2.
10. the preparation technology of a kind of high purity according to claim 1 and high reactivity vWF ELISA, it is characterized in that, preparation technology is as follows:
(1) cryoprecipitate is collected;
(2) cryoprecipitate of step (1) added in 3IU/ml heparin sodium aqua, be stirred to cryoprecipitate and dissolve completely, the temperature of recirculated water controls at 28 ~ 37 DEG C; Start centrifugal, collect supernatant liquor, weigh;
(3) the obtained thing supernatant liquor 1mol/LHCL of step (2) is regulated pH to 6.0 ~ 7.0; Add 2% aluminum hydroxide gel, stir; Start centrifugal, collect supernatant liquor, weigh;
(4) calculate by " regulating ionic strength buffer liquid W=supernatant liquor weight/10 ", take the adjustment ionic strength buffer liquid of calculated amount in the obtained thing supernatant liquor of step (3); Regulate supernatant liquor pH to 6.0 ~ 7.0; 10g/L is not more than by Function protein concentration buffer liquid Function protein concentration; With the filter core cascade filtration of the filter cores of 1.0 μm and 0.45 μm, collect filtered liquid, weigh;
(5) by step (4) obtained thing filtered liquid volume 1/10 add S/D solution, stir, temperature controls at 24 ~ 26 DEG C, continuously insulation 6 hours; 0.45 μm of filter element filtering; Filtered liquid 30KD ultra-filtration membrane is concentrated into protein concentration 2%, then uses more than the 2 times lavation buffer solution A constant volumes ultrafiltration of protein liquid weight, obtains ultrafiltrated, weigh;
(6) the obtained thing ultrafiltrated ion exchange column of step (5) is carried out chromatography purification, wash pillar until become baseline with lavation buffer solution A, with elution buffer A wash-out, collect elutriant;
(7) collect the liquid that step (6) is come through chromatographic column effluent, concentrated one times, lavation buffer solution B equal-volume ultrafiltration 6 times, obtains ultrafiltrated, weighs;
(8) the obtained thing ultrafiltrated Q-Sepharose anion-exchange chromatography post of step (7) is carried out chromatography purification, wash pillar until become baseline with lavation buffer solution B, with elution buffer B wash-out, collect elutriant, weigh;
(9) the obtained thing elutriant gelatin affinity chromatography of step (8) is carried out purifying, wash pillar until become baseline with elution buffer B, the liquid come through chromatographic column effluent after collecting loading, with dialyzate dialysis, be diluted to the 100IU/ml that tires, obtain protein liquid, weigh;
(10) by the obtained thing protein liquid of step (9) through 0.2 μm of degerming filter element filtering packing; Divide the goods installed to import freeze-drying cabinet into and carry out lyophilize; Offer for sale Zha Gai; 99 ~ 100 DEG C, 30 minutes xeothermic inactivation of virus; Censorship, packs after the assay was approved, puts in storage;
Described percentage ratio is apart from outside restriction, and all the other are mass percent;
Preferred: the formula of the adjustment ionic strength buffer liquid described in step (4): the compound method of ionic strength buffer liquid: 48.5g Tutofusin tris, 10g calcium chloride, 100g sodium-chlor, 28.1g glycine, add appropriate water for injection fully to dissolve, benefit injects water to 1L, regulates pH to be 6.0 ~ 7.0; The compound method of protein concentration damping fluid: measure 0.05L and regulate ionic strength buffer liquid, inject water to 1L, regulate pH to be 6.0 ~ 7.0;
Preferred: the formula of the lavation buffer solution A described in step (5): 2.5g Tutofusin tris, 10g calcium chloride, 25g sodium-chlor, adds appropriate water for injection and fully dissolves, and mends and injects water to 1L, regulates pH to be 6.0 ~ 7.0;
Preferred: the elution buffer A described in step (6) fills a prescription: 2.5g Tutofusin tris, 15g calcium chloride, 40.9g sodium-chlor, add appropriate water for injection and fully dissolve, mend and inject water to 1L, regulate pH to be 6.0 ~ 7.0.
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| CN109705208A (en) * | 2018-12-29 | 2019-05-03 | 山东泰邦生物制品有限公司 | A kind of technique of single step chromatography preparation high-purity vWF ELISA |
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| CN109705208A (en) * | 2018-12-29 | 2019-05-03 | 山东泰邦生物制品有限公司 | A kind of technique of single step chromatography preparation high-purity vWF ELISA |
| CN109705208B (en) * | 2018-12-29 | 2022-04-26 | 山东泰邦生物制品有限公司 | Process for preparing high-purity von willebrand factor by single-step chromatography |
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