CN102295696B - Method for preparing coagulation factor VIII, fibrinogen and fibronectin from cryoprecitation - Google Patents
Method for preparing coagulation factor VIII, fibrinogen and fibronectin from cryoprecitation Download PDFInfo
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Abstract
The invention relates to the field of blood products, in particular to a process method for preparing three kinds of blood products from cryoprecitation, and aims to provide a complete separation and purification process for respectively and sequentially preparing the three kinds of blood products, i.e. a coagulation factor VIII, fibrinogen and fibronectin, from the cryoprecitation. The process method is implemented by the following technical scheme that the process method comprises the preparation steps of cryoprecitation dissolution, gel adsorption, precipitation of the fibrinogen and the fibronectin, two-step ion exchange chromatography and glycin/sodium chloride precipitation and inactivation of virus. The process method has the beneficial effects that the three kinds of useful blood products, i.e. the coagulation factor VIII, the fibrinogen and the fibronectin, can be separated and extracted from the cryoprecitation sequentially; and the utilization rate of blood plasma is greatly improved.
Description
Technical field
The present invention relates to the blood products field, particularly a kind ofly can prepare from cryoprecipitate the processing method of three kinds of blood productss.
Technical background
A kind of deposited components that cryoprecipitate fresh frozen plasma low temperature is separated out after melting is rich in platelet cofactor Ⅰ, Fibrinogen and Fiberonectin.The approximately platelet cofactor Ⅰ of half and Fibrinogen in blood plasma, and the Fiberonectin of the overwhelming majority can be enriched in the middle of cryoprecipitate.
Platelet cofactor Ⅰ is the unique specifics for the treatment of hemophilia A, and huge using value is arranged clinically.Fibrinogen is the choice drug for the treatment of congenital and acquired afibrinogenemia.Fiberonectin is also having higher clinical value aspect the treatment herpetic ulcer,corneal.
At present nearly all plasma-derived coagulation factorⅧ all prepares from cryoprecipitate in the world; The Fibrinogen majority is prepared by the CohnShi components I, also has small part to be prepared by cryoprecipitate; Fiberonectin is directly replaced by cryoprecipitate clinically.
Prepare platelet cofactor Ⅰ or fibrinogenic report is very many about cryoprecipitate, and many technical schemes are implemented commercially.The technical scheme of preparation Fiberonectin is also very many, and majority is the gelatin affinity chromatography.But due to the limitation of technology, also can successively prepare platelet cofactor Ⅰ, Fibrinogen and three kinds of blood productss of Fiberonectin by cryoprecipitate without any a kind of technical scheme.Because in present existing technical scheme, all be conceived to a kind of separation and Extraction and purifying of goods, other compositions all are removed as foreign protein, are difficult to be utilized again.
In cryoprecipitate, platelet cofactor Ⅰ is a kind of trace of albumin, and Fibrinogen will relative enrich many with Fibronectin Content.Therefore if take into account the yield of Fibrinogen and Fiberonectin, the yield of platelet cofactor Ⅰ will be easy to be affected so, this be also at present without any the technique of having reported can these three kinds of protein articles of Simultaneous purification one of reason.
In the middle of existing blood products technique, cryoprecipitate is multiplex in the preparation of platelet cofactor Ⅰ, because platelet cofactor Ⅰ is a kind of more precious, protein that economic worth is higher.Due to the restriction of technology, Fibrinogen and Fiberonectin all can be abandoned.In the process of purifying blood coagulation I2GdBN, separate out from solution by precipitation technology Fibrinogen and Fiberonectin that content is relatively much higher, thus the purity of raising platelet cofactor Ⅰ.Precipitation technology commonly used has: glycine precipitation, PEG precipitation, ethanol precipitation, low pH precipitation etc.The glycine precipitator method need to consume a large amount of glycine, and cost is higher, and platelet cofactor Ⅰ is understood co-precipitation and cause yield to reduce; There are the problem of the residual or protein denaturation of PEG in the PEG precipitator method and ethanol precipitation; Low pH method can cause Fibrinogen and Fiberonectin precipitation not exclusively, and purification effect is bad.
For example patent of invention (application publication number 101703763A) discloses a kind of fibrinogenic method for preparing, utilize cryoprecipitate to prepare Fibrinogen through anion exchange chromatography and the glycine precipitator method, can't obtain platelet cofactor Ⅰ and Fiberonectin; Patent of invention (patent No. 03115784.X) discloses a kind of method for preparing Fiberonectin, precipitate and anion-exchange chromatography through alcohol precipitation, PEG take cryoprecipitate as raw material, make Fiberonectin, but platelet cofactor Ⅰ, the Fibrinogen that also can't obtain to be rich in cryoprecipitate.
Blood plasma (particularly human plasma) or plasma component are very valuable and rare resources, only have separation and purification as much as possible to go out to have the protein component of medical value, could improve the effective rate of utilization of blood plasma.The technology of preparing of this class obviously can't make the various useful components in cryoprecipitate be fully utilized, and realizes comprehensive exploitation and the utilization of multiple plasma proteins in cryoprecipitate.
Summary of the invention
The invention provides a complete separation purifying technique of cover, can successively prepare respectively platelet cofactor Ⅰ, Fibrinogen and Fiberonectin, totally three kinds of blood productss from cryoprecipitate.
A kind of method that is prepared platelet cofactor Ⅰ, Fibrinogen and Fiberonectin by cryoprecipitate, preparation process comprises: cryoprecipitate dissolving, gel absorption, Fibrinogen and Fiberonectin Precipitation, two step ion exchange chromatography and glycine/Sodium chloride deposit methods, and inactivation of virus.
A kind of method that is prepared platelet cofactor Ⅰ, Fibrinogen and Fiberonectin by cryoprecipitate, preparation process comprises: after cryoprecipitate is dissolved with water for injection, adsorb with DEAE Sephadex A50 gel under suitable condition, solution after absorption is adjusted to suitable pH, specific conductivity and temperature, Fibrinogen and Fiberonectin form Precipitation, centrifugation precipitation and supernatant liquor.Supernatant liquor obtains platelet cofactor Ⅰ through S/D inactivation of virus and ion exchange chromatography purifying.Precipitation is divided into stream through ion exchange chromatography and wears liquid and elutriant after dissolving again.Stream is worn liquid and is obtained the Fibrinogen of purifying through S/D inactivation of virus and 2 hypoglycins precipitation; Elutriant obtains Fiberonectin through pasteurization.
A kind of method that is prepared platelet cofactor Ⅰ, Fibrinogen and Fiberonectin by cryoprecipitate comprises following concrete steps:
(1) cryoprecipitate water for injection stirring and dissolving in 15 ~ 35 ℃ of scopes, second acid for adjusting pH value with 0.1mol/L is 6.6 ~ 7.0, and specific conductivity adds the good DEAE Sephadex A50 gel of balance less than 4 mS/cm, whip attachment 40 ~ 60 minutes, centrifugal gel and the undissolved precipitation of removing;
(2) supernatant liquor after centrifugal in step 1 is 6.2 ~ 6.5 with the second acid for adjusting pH value of 0.1mol/L again, and reduce temperature to 12 ~ 18 ℃, Fibrinogen and Fiberonectin can form precipitation, can precipitate by centrifugation, and platelet cofactor Ⅰ is stayed in the middle of supernatant liquor;
(3) supernatant liquor after centrifugal in step 2 is rich in platelet cofactor Ⅰ, through after clarification filtration, adds 1% polysorbate-80 and 0.3% tributyl phosphate, hatches 6 hours for 24 ℃, and the effective potential lipid-coated virus of deactivation is as HBV etc.;
(4) the solution process weak anionic displacement chromatography of process inactivation of virus in step 3, can adsorb platelet cofactor Ⅰ wherein, most of foreign protein is worn from the chromatography column upper reaches, chromatography column after absorption can be removed residual foreign protein and polysorbate-80, tributyl phosphate through washing, can wash platelet cofactor Ⅰ from chromatography column with suitable elutriant, through concentrated, preparation, degerming, packing, freeze-drying, the xeothermic inactivation of virus of 100 ℃/30min, can obtain the platelet cofactor Ⅰ goods;
(5) precipitation fibrinogen and the Fiberonectin of centrifugal gained in step 2, after damping fluid dissolving with sodium chloride-containing, Sodium Citrate and arginine hydrochloride, through clarification filtration, add 1% polysorbate-80 and 0.3% tributyl phosphate, hatched 6 hours for 24 ℃, the effective potential lipid-coated virus of deactivation is as HBV etc.;
(6) the solution process weak anionic displacement chromatography of process inactivation of virus in step 5, can adsorb Fiberonectin wherein, most of scleroproein principle is worn from the chromatography column upper reaches, chromatography column after absorption can be washed the Fibrinogen that is adsorbed on a small quantity and residual polysorbate-80, tributyl phosphate off through washing, can wash Fiberonectin from chromatography column with suitable elutriant, after 6 ℃/10h pasteurization, then can make injection liquid or eye drop through concentrated, preparation, degerming, packing;
(7) in step 6, the stream on chromatography column is worn liquid and washings fiber-enriched proteinogen, add wherein glycine (0.5 ~ 1.5mol/L) and sodium-chlor (1.0 ~ 2.1mol/L), Fibrinogen forms precipitation, precipitate by centrifugation, polysorbate-80, tributyl phosphate are stayed in the middle of supernatant liquor;
(8) with after the dissolving of the fibrinogen deposition of gained in step 7, then add glycine and sodium-chlor, the recentrifuge precipitation separation further reduces the residual quantity of polysorbate-80 and tributyl phosphate;
(9) fibrinogen deposition of gained dissolving in step 8 through concentrated, preparation, degerming, packing, freeze-drying, the xeothermic inactivation of virus of 100 ℃/30min, can obtain the Fibrinogen goods.
" centrifugal " described in above step is solid-liquid separation technique general in the blood products industry, generally adopts continuous flow centrifuge (tubular-bowl centrifuge), and rotating speed is common 14000 rev/mins, and feed liquor speed is generally 1 ~ 2.5L/min.Described lipid-coated virus deactivation, i.e. usually said organic solvent/stain remover method inactivation of virus technology, organic solvent generally adopts tributyl phosphate, and stain remover generally adopts polysorbate-80 or Triton X-100.Hatched 6 hours at 24 ℃, the inactivating efficacy of lipid-coated virus is effectively verified.Described " the xeothermic inactivation of virus of 100 ℃/30min " and " pasteurization " are also the inactivation of virus technology of blood products industry universal, effectively the multiple fat coating of deactivation and non-lipid-coated virus.Described " clarification filtration " is Depth Filtration or the membrane filtration technique that the bio-pharmaceuticals industry is known.Described " concentrated, preparation, degerming, packing, freeze-drying " is all technology or the working method that pharmaceutical industry is known.
The ratio of cryoprecipitate described in step 1 and water for injection is between 1:2 ~ 1:5, and is best with 1:3.Described ratio refers to mass ratio.
The addition of the Sephadex of DEAE described in step 1 A50 gel is the dried glue of every kilograin precipitation 1 ~ 2g.Dried glue need be through using after swelling and balance.Swelling method is with reference to DEAE Sephadex A50 gel working instructions.Balance method is: will contain 10 ~ 30mmol/L Sodium Citrate, 30 ~ 80mmol/L sodium-chlor, pH and be 6.6 ~ 7.0 balance liquid and add in the good gel of swelling, stir and balance 10 ~ 20 minutes, suction filtration is removed balance liquid, then adds new balance liquid, and balance is 3 ~ 5 times repeatedly.
Regulate the pH process described in step 2, the speed that adds acetic acid is 100 ~ 300ml/min, uses the mode of spraying best.
Clarification filtration described in step 3 and step 5 requires filter membrane to have certain filtering accuracy.General requirement≤0.5 micron.
Weak anionic displacement chromatography described in step 4 can adopt Toyopeal DEAE-650M gel or DEAE-Sepharase Fast Flow gel.The chromatography column scale is calculated according to every setting prop bed volume 1 ~ 3 kilograin precipitation.Gel is packed into chromatography column, needs through overbalance.Balance liquid contains 10 ~ 30mmol/L Sodium Citrate, 50 ~ 80mmol/L sodium-chlor, 80 ~ 120mmol/L glycine, and pH is 6.6 ~ 7.0.Need 3 ~ 5 column volumes of balance.Solution through deactivation in step 3 is pumped into the good chromatography column of balance.Then rinse chromatography column with washings and elutriant successively.Washings contains 10 ~ 30mmol/L Sodium Citrate, 80 ~ 150mmol/L sodium-chlor, 80 ~ 120mmol/L glycine, and pH is 6.6 ~ 7.0.Wash at least 3 column volumes, until the ultraviolet absorption value of wavelength 280nm recovers or near baseline values.Elutriant contains 10 ~ 30mmol/L Sodium Citrate, 200 ~ 250mmol/L sodium-chlor, 80 ~ 120mmol/L glycine, and pH is 6.6 ~ 7.0.Collect elution peak according to the ultraviolet absorption value of wavelength 280nm, the platelet cofactor Ⅰ enriched material of results purifying.The linear flow speed of chromatography process can be with reference to the specification sheets of the gel that uses, and general control is advisable at 100 ~ 150cm/h.
Lysate described in step 5 contains 10 ~ 30mmol/L Sodium Citrate, 50 ~ 80mmol/L sodium-chlor, 10 ~ 30mmol/L arginine hydrochloride, pH8.0 ~ 8.5.The dissolution process temperature is advisable with 20 ~ 35 ℃.The per kilogram precipitation is added 5 ~ 15 kilograms of lysates and is advisable.After dissolving, specific conductivity is controlled at 6.0 ~ 9.0 mS/cm, and pH is controlled at 7.5 ~ 8.0.
Weak anionic displacement chromatography described in step 6 can adopt Toyopeal DEAE-650M gel or DEAE-Sepharase Fast Flow gel.The chromatography column scale is calculated according to every setting prop bed volume 0.8 ~ 2 kilograin precipitation.Gel is packed into chromatography column, needs through overbalance.Balance liquid contains 10 ~ 30mmol/L Sodium Citrate, 50 ~ 80mmol/L sodium-chlor, 10 ~ 30mmol/L arginine hydrochloride, and pH is 7.5 ~ 8.0, and specific conductivity is controlled at 6.0 ~ 9.0 mS/cm.Need 3 ~ 5 column volumes of balance.Solution through deactivation in step 5 is pumped into the good chromatography column of balance, collect stream and wear liquid.Then rinse chromatography column (washing process) with balance liquid.Wash at least 3 column volumes, until the ultraviolet absorption value of wavelength 280nm recovers or near baseline values.Collect washings, and wear liquid with stream and mix.Elutriant contains 10 ~ 30mmol/L Sodium Citrate, 250 ~ 350mmol/L sodium-chlor pH is 6.5 ~ 7.5.Collect elution peak according to the ultraviolet absorption value of wavelength 280nm, the Fiberonectin enriched material of results purifying.The linear flow speed of chromatography process can be with reference to the specification sheets of the gel that uses, and general control is advisable at 100 ~ 150cm/h.
Glycine described in step 7 and 8 and sodium-chlor can be mixed with the mother liquor of high density in advance, then pump in goods, make the ultimate density of glycine and sodium-chlor reach respectively 0.5 ~ 1.5mol/L and 1.0 ~ 2.1mol/L, the glycine and the sodium-chlor that also can be directly add Powdered or fine particulate in the goods make its ultimate density reach 0.5 ~ 1.5mol/L respectively and 1.0 ~ 2.1mol/L gets final product.
The present invention has the following advantages:
1, can be according to sequencing separation and Extraction platelet cofactor Ⅰ, Fibrinogen and Fiberonectin totally 3 kinds of useful blood productss from cryoprecipitate respectively.Greatly improved the utilization ratio of blood plasma.
2, the production of three kinds of goods can utilize identical equipment, as sharing precipitin reaction tank and whizzer with fibrinogenic production; The production of platelet cofactor Ⅰ and Fiberonectin can share chromatography column (two kinds of goods use identical ion-exchange gel).
3, three kinds of goods can obtain higher yield and purity simultaneously.
4, replacing aluminum hydroxide gel in traditional technology to adsorb thrombogen residual in cryoprecipitate, VII, IX, X with DEAE Sephadex A50 gel has the following advantages: the specificity of A50 gel absorption thrombogen, VII, IX, X is better than aluminum hydroxide gel; The A50 gel is provided by the supplier of specialty, and quality controllability is good, and differences between batches are little; The hydroxide gel needs in situ preparation, and the quality controllability such as the corresponding concentration of the gel of in situ preparation, purity are not high, and easily sneaks into the metal ions (thrombin activator) such as calcium, magnesium with raw materials.
5, the inventor is through fully research confirmation, and the adsorption effect of various thrombin depends on mutually combining of the factors such as different adsorption methods and adsorption conditions, just can reach adsorption effect required for the present invention, thereby optimum process condition has been determined in screening.
6, the inventor is through fully research confirmation, three kinds of protein (particularly platelet cofactor Ⅰ) high yields and purification effect depend on intermediate processing, the best of breed of temperature, pH, conductivity value, can guarantee that just three kinds of protein (particularly platelet cofactor Ⅰ) have higher yield and purification effect, thereby optimum process condition has been determined in screening.
7, the inventor is through fully research confirmation, and two kinds of protein purifications depend on the ion exchange chromatography condition, and specific conductivity and pH value best of breed just can reach effect required for the present invention, thereby optimum process condition has been determined in screening.
Embodiment
Example 1:
The first step: get cryoprecipitate 10kg, add the water for injection of 30kg30 ℃, stirring and dissolving 1 hour.Add the acetic acid of 0.1mol/L with the mode of spraying, the speed that adds acetic acid is 120ml/min, and regulating the pH value is 6.9, regulates specific conductivity less than 4 mS/cm, spends approximately 1.4L of acetic acid.Add the good DEAE Sephadex A50 gel of balance, whip attachment 40 minutes adopts centrifugal gel and a small amount of undissolved precipitation of removing of continuous flow centrifuge (tubular-bowl centrifuge), collects supernatant liquor.
Should do following preparation before the first step: take DEAE Sephadex A50 gel (dried glue) 20g.Dried glue is placed in beaker, adds 80mmol/L sodium chloride solution 1L swelling 6 hours.The preparation balance liquid: 20mmol/L Sodium Citrate, 60mmol/L sodium-chlor, pH are 6.9.Discard beaker swelling solution at the middle and upper levels, keep lower floor's gel, then add the 1L balance liquid, stir, and balance 10 ~ 20 minutes, suction filtration is removed balance liquid, then adds new balance liquid, and balance is 3 ~ 5 times repeatedly.
Second step: collect the supernatant liquor 40.5kg in the first step.Continue approximately 1.8L of acetic acid that spraying adds 0.1mol/L with the speed of 200 ml/min, regulate pH to 6.2.Lower the temperature with ice-water bath when adding acetic acid, adjust the temperature to 16 ℃.After temperature and pH reach requirement, centrifugation precipitates (being called Acid precipitation) and supernatant liquor.
The 3rd step: collect second step gained supernatant liquor 35kg, with Milligard 1.2/0.5 μ m filter element filtering.Add 1% polysorbate-80 and 0.3% tributyl phosphate in the filtrate, mix, hatched 6 hours inactivation of viruses for 24 ℃.
The 4th step: with Toyopeal DEAE-650M gel filling chromatography column, column volume is 5L(post bed height 10cm, diameter 25cm).The preparation balance liquid: 10mmol/L Sodium Citrate, 80mmol/L sodium-chlor, 80mmol/L glycine, pH are 6.8.Need 5 column volumes of balance.Solution after deactivation in the 3rd step is pumped into chromatography column with peristaltic pump, and flow velocity is 0.5L/min.Then rinse chromatography column with washings and elutriant successively.Washings contains 10mmol/L Sodium Citrate, 150mmol/L sodium-chlor, 120mmol/L glycine, and pH is 6.8.Wash at least 3 column volumes, until the ultraviolet absorption value of wavelength 280nm recovers or near baseline values.Elutriant contains 10mmol/L Sodium Citrate, 250mmol/L sodium-chlor, 120mmol/L glycine, and pH is 6.8.Collect elution peak according to the ultraviolet absorption value of wavelength 280nm, the platelet cofactor Ⅰ enriched material of results purifying is 2.3L approximately, measures contained platelet cofactor Ⅰ and tires and be that 48IU/ml, specific activity are 115IU/mg protein.Through preparation, degerming, packing, freeze-drying, the xeothermic inactivation of virus of 100 ℃/30min, obtain 440 bottles, platelet cofactor Ⅰ goods (specification is 200IU/10ml/ bottle).
The 5th step: preparation 45kg lysate: 20mmol/L Sodium Citrate, 50mmol/L sodium-chlor, 20mmol/L arginine hydrochloride, pH8.5.Add the 4.95kg Acid precipitation collected in second step (" the per kilogram precipitation is added 5 ~ 15 kilograms of lysates and is advisable), 30 ~ 35 ℃ of stirring and dissolving 2 hours.After dissolving, specific conductivity is 8.5 mS/cm, and pH is 8.0.Solution after dissolving filters with the Milligard 1.2/0.5 μ m filter core (Milligard is the filtering product of milipore company) of filtering accuracy≤0.5 micron.Add 1% polysorbate-80 and 0.3% tributyl phosphate in the filtrate, mix, hatched 6 hours inactivation of viruses for 24 ℃.
The 6th step: adopt the chromatography column that fills in the 4th step, after gel specification sheets manipulation of regeneration, adopt new balance liquid rebalancing.Balance liquid: 20mmol/L Sodium Citrate, 50mmol/L sodium-chlor, 20mmol/L arginine hydrochloride, pH are 8.0, specific conductivity 8.5 mS/cm.5 column volumes of balance.Solution after deactivation in the 5th step is pumped into chromatography column with peristaltic pump, and flow velocity is 0.5L/min.Collect stream and wear liquid.Then rinse chromatography column (washing process) with balance liquid.Wash at least 3 column volumes.Collect the washings of first column volume (approximately 5L), and wear liquid with stream and mix.Elutriant contains the 20mmol/L Sodium Citrate, 300mmol/L sodium-chlor pH is 7.0.Collect elution peak according to the ultraviolet absorption value of wavelength 280nm, the Fiberonectin enriched material 4.1L of results purifying, fibre-bearing is in conjunction with albumen 6.5g/L, and purity is 85.3%.After 6 ℃/10h pasteurization, then can make injection liquid or eye drop through concentrated, preparation, degerming, packing.
The 7th step: the stream of collecting in the 6th step wears liquid and washings is total to 54kg, when stirring, slowly adds wherein pulverous glycine 5.4kg and sodium-chlor 7.02kg, continues to stir 30min.Centrifugal and collecting precipitation obtains 5.2kg.
The 8th step: add 50kg water for injection, 25 ℃ of stirrings dissolved precipitation in 1 hour fully again.When stirring, slowly add wherein pulverous glycine 5.5kg and sodium-chlor 7.2kg, continue to stir 30min.Centrifugal and collecting precipitation obtains 4.9kg.
The 9th step: precipitation through concentrated, preparation, degerming, packing, freeze-drying, the xeothermic inactivation of virus of 100 ℃/30min, obtains 528 bottles, Fibrinogen goods (specification is the 0.5g/25ml/ bottle) after dissolving with water for injection.Through detecting, Fibrinogen purity is 98.2%.
In this example, the product for preparing of 10kg cryoprecipitate and quality standard thereof statistics is as following table:
| ? | Purity (specific activity) | Output | Yield |
| Human blood coagulation factors VIII | 115IU/mg protein | 440 bottles (200IU/bottle) | 44 bottles/kg cryoprecipitate |
| People's Fiberonectin | 85.3% | 26.65g(not packing) | 2.6g/kg cryoprecipitate |
| The human fibrinogen | 98.2% | 528 bottles (0.5g/ bottle) | 52.8 bottle/kg cryoprecipitate |
Example 2:
The first step: get cryoprecipitate 10kg, add the water for injection of 50kg35 ℃, stirring and dissolving 1 hour; Speed spray pattern take 100 ml/min adds the second acid for adjusting pH value as 6.7.Add the good DEAE Sephadex A50 gel of balance, whip attachment 50 minutes;
Should do following preparation before the first step: take DEAE Sephadex A50 gel (dried glue) 15g.The preparation balance liquid: 10mmol/L Sodium Citrate, 80mmol/L sodium-chlor, pH are 6.7;
All the other operation stepss are with embodiment 1 the first step.
Second step: the speed with 100 ml/min continues the acetic acid that spray pattern adds 0.1mol/L, regulates pH to 6.5.Lower the temperature with ice-water bath when adding acetic acid, adjust the temperature to 12 ℃.
All the other operation stepss are with embodiment 1 second step.
The 3rd step: collect second step gained supernatant liquor 55kg, operation steps is with the 3rd step of embodiment 1.
The 4th step: with DEAE-Sepharase Fast Flow gel filling chromatography column, column volume is 6L(post bed height 6cm, diameter 25cm).The preparation balance liquid: 30mmol/L Sodium Citrate, 50mmol/L sodium-chlor, 120mmol/L glycine, pH are 6.6.Need 3 column volumes of balance.Washings contains 20mmol/L Sodium Citrate, 80mmol/L sodium-chlor, 100mmol/L glycine, and pH is 7.0.Wash at least 3 column volumes, until the ultraviolet absorption value of wavelength 280nm recovers or near baseline values.Elutriant contains 30mmol/L Sodium Citrate, 200mmol/L sodium-chlor, 80mmol/L glycine, and pH is 6.6.Solution after deactivation in the 3rd step is pumped into chromatography column with peristaltic pump, and flow velocity is 0.6L/min.Then rinse chromatography column with washings and elutriant successively.
All the other operation stepss are with the 4th step of embodiment 1.
The about 1.6L of platelet cofactor Ⅰ enriched material of results purifying measures contained platelet cofactor Ⅰ and tires and be that 64IU/ml, specific activity are 102IU/mg protein.Through preparation, degerming, packing, freeze-drying, the xeothermic inactivation of virus of 100 ℃/30min, obtain 409 bottles, platelet cofactor Ⅰ goods (specification is 200IU/10ml/ bottle).
The 5th step: preparation 45kg lysate: 30mmol/L Sodium Citrate, 60mmol/L sodium-chlor, 10mmol/L arginine hydrochloride, pH8.0.Add the precipitation 3.8kg that collects in second step, 20 ~ 25 ℃ of stirring and dissolving 2 hours.
All the other operation stepss are with the 5th step of embodiment 1.
The 6th step: adopt the chromatography column that fills in the 4th step.Balance liquid: 10mmol/L Sodium Citrate, 70mmol/L sodium-chlor, 30mmol/L arginine hydrochloride, pH are 7.9.4 column volumes of balance.It is 7.5 that elutriant contains 10mmol/L Sodium Citrate, 350mmol/L sodium-chlor, pH.
All the other operation stepss are with the 6th step of embodiment 1.
The Fiberonectin enriched material 2.9L of results purifying, fibre-bearing binding protein 28 .9g/L, purity is 86.1%.
The 7th step: slowly add the glycine, the sodium chloride solution that prepare in advance.
All the other operation stepss are with the 7th step of embodiment 1.
The 8th step: slowly add the glycine, the sodium chloride solution that prepare in advance.
All the other operation stepss are with the 8th step of embodiment 1.
The 9th step: operation steps is with the 9th step of embodiment 1.
Obtain 528 bottles, Fibrinogen goods (specification is the 0.5g/25ml/ bottle).
In this example, the product for preparing of 10kg cryoprecipitate and quality standard thereof statistics is as following table:
| ? | Purity (specific activity) | Output | Yield |
| Human blood coagulation factors VIII | 102IU/mg protein | 409 bottles (200IU/bottle) | 41 bottles/kg cryoprecipitate |
| People's Fiberonectin | 86.1% | 28.65g(not packing) | 2.8g/kg cryoprecipitate |
| The human fibrinogen | 99.1% | 506 bottles (0.5g/ bottle) | 50.6 bottle/kg cryoprecipitate |
Embodiment 3: different adsorption methods and adsorption conditions research test
Many about all having comprised the step of aluminum hydroxide gel absorption in the middle of platelet cofactor Ⅰ, fibrinogenic purifying process.Aluminum hydroxide gel can adsorb multiple thrombin, but it is a kind of non-specific adsorption agent, and adsorptive power is limited.And the preparation of aluminum hydroxide gel and quality control more complicated, be not easy to control.Why existing technique can not well utilize the plurality of active ingredients in cryoprecipitate, a chief reason is, when thrombogen, VII, IX, X are removed not completely, because the multiple protein extraction process is complicated, technical process and consuming time long, isolated a kind of component from cryoprecipitate (for example component of fiber-enriched proteinogen and Fiberonectin) thrombin can occur and activate when being used for other protein of purifying, the phenomenon of aggregation occurs.Even if obvious aggregation does not occur in preparation process, due to the effect of the thrombin that activates, often cause cotton-shaped protein precipitation occurring, even gluey condensation product after the redissolution of platelet cofactor Ⅰ and fibrinogen product.
The DEAE Sephadex A50 gel that General Electric Corporation (GE) produces is a kind of ion-exchange gel, thrombogen, VII, IX, X that can specific adsorption zone negative charge.Also just because of this characteristic, this gel is by for a long time for the production of Human Factor Ⅸ Complex (main component is thrombogen, VII, IX, X).This investigator finds, adopt DEAE Sephadex A50 gel to replace aluminum hydroxide gel absorption, can more effectively remove thrombogen, VII, IX, X in cryoprecipitate, can prevent that platelet cofactor Ⅰ and Fibrinogen are activated, and make goods more stable.Yet, platelet cofactor Ⅰ and Fibrinogen are also electronegative protein, also can be adsorbed by DEAE Sephadex A50 gel, key is to find suitable condition (pH, specific conductivity, damping fluid composition, temperature, adsorption time and amount of gel), thrombogen, VII, IX, X are adsorbed fully, and platelet cofactor Ⅰ and scleroproein principle seldom are adsorbed.
The present invention replaces aluminum hydroxide gel as sorbent material with DEAE Sephadex A50 gel, improved widely the specificity of absorption, and found best adsorption conditions by simultaneous test, can remove completely thrombogen, VII, IX, X, and keep preferably the effective constituents such as platelet cofactor Ⅰ and Fibrinogen.Concrete experimental technique is: gets the 300g cryoprecipitate, adds 750ml water for injection, and 30 ℃ of stirring and dissolving, in 9 beakers that are placed in, each beaker is contained 100ml; With the second acid for adjusting pH value of 0.1mol/L, the NaCl solution of 2mol/L is transferred specific conductivity; Proportionally taking DEAE Sephadex A50 gel (dried glue), is 6.8 balance liquid balance 3 ~ 5 times repeatedly with containing 20mmol/L Sodium Citrate, 30mmol/L sodium-chlor, pH, adds in above-mentioned beaker, adsorbs the different time.Aluminum hydroxide gel adopts aluminum trichloride solution and sodium hydroxide solution preparation.Tire with the rear various thrombin of absorption before mensuration absorption." carry out according to 2010 editions by three ones of Chinese pharmacopoeia for thrombin titration method.
Different adsorption methods and the adsorption conditions adsorption effect to various thrombin
Annotate: (a) being illustrated in the maintenance specific conductivity is that 3.1mS/cm, adsorption time are that 40min, amount of gel are in the constant situation of condition of 1.5g/kg cryoprecipitate, under the research condition of different pH, and the variation (every gram cryoprecipitate) that before and after absorption, various thrombin are tired.
(b) be illustrated in and keep that pH is 6.8, adsorption time is that 40min, amount of gel are in the constant situation of condition of 1.5g/kg cryoprecipitate, study under different specific conductivity conditions the variation (every gram cryoprecipitate) that the various thrombin in absorption front and back are tired.
(c) be illustrated in and keep that pH is 6.8, specific conductivity is that 3.1mS/cm, amount of gel are in the constant situation of condition of 1.5g/kg cryoprecipitate, study under different adsorption times the variation (every gram cryoprecipitate) that the various thrombin in absorption front and back are tired.
(d)) be illustrated in and keep that pH is 6.8, specific conductivity is that 3.1mS/cm, adsorption time are in the constant situation of condition of 40min, study under different amount of gel conditions the variation (every gram cryoprecipitate) that the various thrombin in absorption front and back are tired.
(e) the expression adsorption conditions is: pH is 6.8, specific conductivity is that 3.1mS/cm, adsorption time are the aluminum hydroxide gel that 40min, per kilogram cryoprecipitate are added 108g 20%.(variation of every gram Cryoprecipitated Antihemophilic Factors content before and after absorption)
Can draw to draw a conclusion from result of study:
1, pH is proper near 6.8.PH can not be adsorbed fully near 6.0 o'clock thrombogens, VII, IX, X, and pH can be adsorbed near 7.6 o'clock part platelet cofactor Ⅰs;
2, specific conductivity is proper near 3.1mS/cm.Specific conductivity 8.3 and the adsorptive capacity of 15.2 o'clock thrombogens, VII, IX, X obviously reduce;
3, adsorption time is most suitable between 40 ~ 60min, and only adsorbing 20min, to have a small amount of thrombogen, VII, IX, X residual;
4, amount of gel is proper in the 1.5g/kg cryoprecipitate.This value is residual less than having part thrombogen, VII, IX, X at 0.5 o'clock, and this value can cause a small amount of platelet cofactor Ⅰ to be adsorbed greater than 3 o'clock.
5, optimal adsorption conditions is: pH6.8, specific conductivity 3.1mS/cm, adsorption time 40 ~ 60min, amount of gel 1.5g/kg cryoprecipitate.
6,, under suitable condition, adopt the adsorption effect of DEAE Sephadex A50 gel will obviously be better than traditional aluminum hydroxide gel: the absorption of thrombogen, VII, IX, X is more complete, and the absorption of platelet cofactor Ⅰ still less.
Embodiment 4: different deposition condition research tests
Another key problem in technology point that affects the cryoprecipitate comprehensive development and utilization is the intermediate processing of Fibrinogen and Fiberonectin.Fibrinogen and Fiberonectin can be separated out with the form of precipitation at low temperatures, and Fibrinogen also can solubleness reduce under slightly acidic or ground ionic strength (low conductivity value) condition.But under cold condition, platelet cofactor Ⅰ also can form precipitation, as the blood plasma cryoprecipitate.The best of breed of temperature, pH, conductivity value can guarantee that three kinds of protein (particularly platelet cofactor Ⅰ) have higher yield and purification effect.Following table is the rate of recovery (platelet cofactor Ⅰ is measured the rate of recovery in centrifugal rear supernatant liquor, and Fibrinogen and Fiberonectin are measured the rate of recovery in centrifuged deposit) of the lower three kinds of protein of different condition.
The impact of different deposition conditions on platelet cofactor Ⅰ, Fibrinogen and Fiberonectin yield
Annotate: a represents that specific conductivity is 3.0, temperature is under 14 ℃ of conditions, and different pH are for the impact of platelet cofactor Ⅰ, Fibrinogen and Fiberonectin yield.
B represents that pH is 6.3, temperature is under 14 ℃ of conditions, and different specific conductivity are for the impact of platelet cofactor Ⅰ, Fibrinogen and Fiberonectin yield.
C represents that pH is 6.3, specific conductivity is under 3.0 conditions, and differing temps is for the impact of platelet cofactor Ⅰ, Fibrinogen and Fiberonectin yield.
Can draw to draw a conclusion from result of study:
1, pH is proper near 6.3, and yield relative equilibrium, particularly platelet cofactor Ⅰ, the fibrinogenic yield of platelet cofactor Ⅰ, Fibrinogen and Fiberonectin are relatively high.PH obviously reduces near 5.8 o'clock platelet cofactor Ⅰ yields; PH obviously reduces near 6.8 o'clock fibrinogenic yields.
2, conductivity value is proper near 3.0.Along with the rising of specific conductivity, fibrinogenic yield sharply reduces.
3, temperature is proper near 14 ℃.Temperature platelet cofactor Ⅰ yield in the time of 8 ℃ sharply reduces; Near 20 ℃, fibrinogenic yield obviously reduces, and the yield of Fiberonectin sharply reduces.
4, best deposition condition is 14 ℃ of pH6.3, conductivity value 3.0mS/cm, temperature.
Embodiment 5: different chromatography condition research tests
After the resolution of precipitate of fiber-enriched proteinogen and Fiberonectin, can effectively make two kinds of protein obtain respectively purifying through ion exchange chromatography, particularly fibrinogenic purity can reach 98%.Specific conductivity and pH affect the yield of Fibrinogen and Fiberonectin and the key factor of purity.Specific conductivity is lower, pH is higher, and Fibrinogen and Fiberonectin more easily are attracted on chromatography media.Under certain conditions, Fiberonectin is always high than Fibrinogen with the binding ability of chromatography media.Chromatography condition is different, and they are also different from the difference of the binding ability of chromatography media.Under suitable condition, the difference of binding ability is maximum, and Fibrinogen can better be separated with Fiberonectin, keeps respectively higher purity and yield.Following table is about the comparative study of two kinds of protein yields and purity under different chromatography conditions.The pH of lysate regulates with the NaOH solution of 0.1mol/L, according to NaCl concentration different adjustment specific conductivity contained in lysate.
Yield and the purity of Fibrinogen and Fiberonectin under different chromatography conditions
Annotate: when a represented to study affecting of pH, specific conductivity was 8.0mS/cm; When b represented to study affecting of specific conductivity, pH was 7.5.
Can draw to draw a conclusion from result of study: be 8.0 at pH, when specific conductivity was 8.0mS/cm, the yield of Fibrinogen and Fiberonectin and purity can reach a comparatively balanced state.
Claims (2)
1. method that is prepared platelet cofactor Ⅰ, Fibrinogen and Fiberonectin by cryoprecipitate is characterized in that comprising the following steps:
(1) cryoprecipitate water for injection stirring and dissolving in 15 ~ 35 ℃ of scopes, second acid for adjusting pH value with 0.1mol/L is 6.6 ~ 7.0, and specific conductivity adds the good DEAE Sephadex A50 gel of balance less than 4 mS/cm, whip attachment 40 ~ 60 minutes, centrifugal gel and the undissolved precipitation of removing; The ratio of described cryoprecipitate and water for injection is between 1:2 ~ 1:5, and described ratio refers to mass ratio; The addition of described DEAE Sephadex A50 gel is the dried glue of every kilograin precipitation 1 ~ 2g, dried glue need be through using after swelling and balance, balance method is: will contain 10 ~ 30mmol/L Sodium Citrate, 30 ~ 80mmol/L sodium-chlor, pH and be 6.6 ~ 7.0 balance liquid and add in the good gel of swelling, stir and balance 10 ~ 20 minutes, suction filtration is removed balance liquid, add new balance liquid, balance is 3 ~ 5 times repeatedly again;
(2) supernatant liquor after centrifugal in step 1 is 6.2 ~ 6.5 with the second acid for adjusting pH value of 0.1mol/L again, and reduce temperature to 12 ~ 18 ℃, Fibrinogen and Fiberonectin form precipitation, precipitate by centrifugation, and platelet cofactor Ⅰ is stayed in the middle of supernatant liquor; Described adjusting pH process, the speed that adds acetic acid is 100 ~ 300ml/min, uses the mode of spraying;
(3) supernatant liquor after centrifugal in step 2 is rich in platelet cofactor Ⅰ, through after clarification filtration, adds 1% polysorbate-80 and 0.3% tributyl phosphate, hatches 6 hours for 24 ℃, effectively the potential lipid-coated virus of deactivation; Described clarification filtration requires the filtering accuracy of filter membrane≤0.5 micron;
(4) the solution process weak anionic displacement chromatography of process inactivation of virus in step 3, absorption platelet cofactor Ⅰ wherein, most of foreign protein is worn from the chromatography column upper reaches, chromatography column after absorption is removed residual foreign protein and polysorbate-80, tributyl phosphate through washing, wash platelet cofactor Ⅰ with elutriant from chromatography column, through concentrated, preparation, degerming, packing, freeze-drying, the xeothermic inactivation of virus of 100 ℃/30min, can obtain the platelet cofactor Ⅰ goods, described weak anionic displacement chromatography adopts Toyopeal DEAE-650M gel or DEAE-Sepharase Fast Flow gel, the chromatography column scale is calculated according to every setting prop bed volume 1 ~ 3 kilograin precipitation, gel is packed into chromatography column, need through overbalance, balance liquid contains 10 ~ 30mmol/L Sodium Citrate, 50 ~ 80mmol/L sodium-chlor, 80 ~ 120mmol/L glycine, pH is 6.6 ~ 7.0, need 3 ~ 5 column volumes of balance, solution through deactivation in step 3 is pumped into the good chromatography column of balance, then rinse chromatography column with washings and elutriant successively, washings contains 10 ~ 30mmol/L Sodium Citrate, 80 ~ 150mmol/L sodium-chlor, 80 ~ 120mmol/L glycine, pH is 6.6 ~ 7.0, wash at least 3 column volumes, until the ultraviolet absorption value of wavelength 280nm recovers or near baseline values, elutriant contains 10 ~ 30mmol/L Sodium Citrate, 200 ~ 250mmol/L sodium-chlor, 80 ~ 120mmol/L glycine, pH is 6.6 ~ 7.0, collect elution peak according to the ultraviolet absorption value of wavelength 280nm, the platelet cofactor Ⅰ enriched material of results purifying,
(5) precipitation fibrinogen and the Fiberonectin of centrifugal gained in step 2, after damping fluid dissolving with sodium chloride-containing, Sodium Citrate and arginine hydrochloride, through clarification filtration, add 1% polysorbate-80 and 0.3% tributyl phosphate, hatched 6 hours for 24 ℃, effectively the potential lipid-coated virus of deactivation; Described clarification filtration requires the filtering accuracy of filter membrane≤0.5 micron; Described lysate contains 10 ~ 30mmol/L Sodium Citrate, 50 ~ 80mmol/L sodium-chlor, 10 ~ 30mmol/L arginine hydrochloride, pH8.0 ~ 8.5, the dissolution process temperature is advisable with 20 ~ 35 ℃, the per kilogram precipitation is added 5 ~ 15 kilograms of lysates and is advisable, after dissolving, specific conductivity is controlled at 6.0 ~ 9.0 mS/cm, and pH is controlled at 7.5 ~ 8.0;
(6) the solution process weak anionic displacement chromatography of process inactivation of virus in step 5, absorption Fiberonectin wherein, most of scleroproein principle is worn from the chromatography column upper reaches, chromatography column after absorption is through washing the Fibrinogen that is adsorbed on a small quantity and residual polysorbate-80, tributyl phosphate off, wash Fiberonectin with elutriant from chromatography column, after 6 ℃/10h pasteurization, then make injection liquid or eye drop through concentrated, preparation, degerming, packing, described weak anionic displacement chromatography adopts Toyopeal DEAE-650M gel or DEAE-Sepharase Fast Flow gel, the chromatography column scale is calculated according to every setting prop bed volume 0.8 ~ 2 kilograin precipitation, gel is packed into chromatography column, need through overbalance, balance liquid contains 10 ~ 30mmol/L Sodium Citrate, 50 ~ 80mmol/L sodium-chlor, 10 ~ 30mmol/L arginine hydrochloride, pH is 7.5 ~ 8.0, specific conductivity is controlled at 6.0 ~ 9.0 mS/cm, need 3 ~ 5 column volumes of balance, solution through deactivation in step 5 is pumped into the good chromatography column of balance, collect stream and wear liquid, then rinse chromatography column with balance liquid, wash at least 3 column volumes, until the ultraviolet absorption value of wavelength 280nm recovers or near baseline values, collect washings, and wear liquid with stream and mix, elutriant contains 10 ~ 30mmol/L Sodium Citrate, 250 ~ 350mmol/L sodium-chlor pH is 6.5 ~ 7.5, collects elution peak according to the ultraviolet absorption value of wavelength 280nm, the Fiberonectin enriched material of results purifying,
(7) in step 6, the stream on chromatography column is worn liquid and washings fiber-enriched proteinogen, adds wherein glycine and sodium-chlor, and Fibrinogen forms precipitation, precipitates by centrifugation, and polysorbate-80, tributyl phosphate are stayed in the middle of supernatant liquor; Described glycine and sodium-chlor can be mixed with the mother liquor of high density in advance, then pump in goods, make the ultimate density of glycine and sodium-chlor reach respectively 0.5 ~ 1.5mol/L and 1.0 ~ 2.1mol/L, the glycine and the sodium-chlor that also can be directly add Powdered or fine particulate in the goods make its ultimate density reach 0.5 ~ 1.5mol/L respectively and 1.0 ~ 2.1mol/L gets final product;
(8) with after the dissolving of the fibrinogen deposition of gained in step 7, then add glycine and sodium-chlor, the recentrifuge precipitation separation further reduces the residual quantity of polysorbate-80 and tributyl phosphate; Described glycine and sodium-chlor can be mixed with the mother liquor of high density in advance, then pump in goods, make the ultimate density of glycine and sodium-chlor reach respectively 0.5 ~ 1.5mol/L and 1.0 ~ 2.1mol/L, the glycine and the sodium-chlor that also can be directly add Powdered or fine particulate in the goods make its ultimate density reach 0.5 ~ 1.5mol/L respectively and 1.0 ~ 2.1mol/L gets final product;
(9) fibrinogen deposition of gained dissolving in step 8 through concentrated, preparation, degerming, packing, freeze-drying, the xeothermic inactivation of virus of 100 ℃/30min, can obtain the Fibrinogen goods.
2. the method that is prepared platelet cofactor Ⅰ, Fibrinogen and Fiberonectin by cryoprecipitate according to claim 1, it is characterized in that: described in step (1), the ratio of cryoprecipitate and water for injection is 1:3.
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