CN104231072A - Preparation process for extracting human fibrinogens from waste for extracting cryoprecipitated blood coagulation factor VIII - Google Patents
Preparation process for extracting human fibrinogens from waste for extracting cryoprecipitated blood coagulation factor VIII Download PDFInfo
- Publication number
- CN104231072A CN104231072A CN201410524351.2A CN201410524351A CN104231072A CN 104231072 A CN104231072 A CN 104231072A CN 201410524351 A CN201410524351 A CN 201410524351A CN 104231072 A CN104231072 A CN 104231072A
- Authority
- CN
- China
- Prior art keywords
- cryoprecipitate
- liquid
- waste material
- filtration
- human fibrinogen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- C07K14/75—Fibrinogen
-
- 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
- C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Hematology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a preparation process for extracting human fibrinogens from waste for extracting cryoprecipitated blood coagulation factor VIII. The preparation process is characterized by comprising the steps of cryoprecipitation dissolution, 2% aluminium hydroxide gel absorption, ion strength adjustment, series connection filtering, S/D viral inactivation, ion-exchange chromatography, EDTA Ca2+ removal, glycine precipitation, primary low-temperature ethanol precipitation, AT-III thrombin inhibition, secondary low-temperature ethanol precipitation, nanofilm filtering and dry-heat inactivation. For ensuring the safety, a nanofilm ia added to filter virus except S/D and dry-heat inactivation. By means of an added AT-III inactivated thrombin and EDTA Ca2+ removal process, fibrinogen in the production process is effectively prevented from being activated into fibrous protein. The glycine precipitation is utilized to remove fibrous protein monomers and polymers in products so as obtain high-purity human fibrinogen. The preparation products are safe and reliable, redissolution time is short, the clinic first-aid demand is met, and meanwhile the preparation process has important significance on indirect saving of scarce plasma resources.
Description
Technical field
The present invention relates to a kind of preparation technology extracting human fibrinogen from the waste material of cryoprecipitate extraction platelet cofactor Ⅰ, belong to field of biological pharmacy.
Background technology
Fibrinogen (Fg), is synthesized by liver cell and secretes, and is thrombin important in body hemostasis physiology.Package stability is better, but poor heat stability, can form irreversible precipitation at 56 DEG C, in human normal plasma, Fg concentration is 2.4 ~ 4.0g/L.
Fg is as a kind of Acute reaction protein, and its blood content is the risk factor of the diseases such as ischemic angiocardiopathy and cerebrovascular disease extremely.Fg increases a kind of non-specific responding of body often, is common in the aseptic inflammation such as the infection such as toxicaemia, pneumonia, cholecystitis, pulmonary tuberculosis and nephrotic syndrome, rheumatic fever, malignant tumour, cerebral thrombosis, myocardial infarction; In addition as surgical operation, radiotherapy, the Gestation period also show Fg and slightly increase.Fg reduces more rare, but when it is lower than 1.0g/L, hemorrhage sign can appear in body.Congenital afibrinogenemia is a very rare heredopathia, and by autosomal recessive gene heredity, this patient's liver can not synthesize Fg; The fibrinogenopenic reason of Secondary cases is caused by fibrinolytic enzymes scleroproein, as placental abruption, during childbirth, amniotic fluid intravasation forms thrombus, cause disseminated intravascular coagulation, activate plasminogen, fibrinoclase vigor in blood is increased, solution fibrin, original Fg in consumer, makes its content reduce; , also can there is the minimizing of Fg in the hepatic necrosis caused as a variety of causes, chronic hepatopathy late period etc. in serious liver parenchyma lesion.
Fg is mainly used in congenital, the acquired Fibrinogen for the treatment of and reduces or deficiency disease clinically, and Severe Hepatic Injury, liver cirrhosis, disseminated inravascular coagulation, postpartum hemorrhage and the blood coagulation disorders that causes because of major operation, wound or internal hemorrhage etc.
Research to show in cryoprecipitate the blood clotting factors materials such as fiber-enriched proteinogen, Fibrinogen >=150mg in the cryoprecipitate that 400ml whole blood obtains.In today that blood plasma raw material is increasingly in short supply, prepare human fibrinogen by cryoprecipitate, to the comprehensive utilization of cryoprecipitate, save rare blood plasma resource, improve the market competitiveness and be significant.
In prior art, the gel adsorption removal of impurity is not carried out as traditional fibre proteinogen preparation technology is last in S/D deactivation, as patent of invention " production method of human fibrinogen " (application publication number: 101703763A) etc., or use DEAE Sephadex A50 gel adsorption, as patent of invention " being prepared the method for platelet cofactor Ⅰ, Fibrinogen and Fiberonectin by cryoprecipitate " (application publication number: 102295696A).Only find that (application publication number: 103351432 A) uses aluminum hydroxide gel to patent of invention " extracting the technique of human blood coagulation factors VIII and human fibrinogen from plasma component precipitation " at present, and aluminium hydroxide adding proportion is 3g/Kg.The physical strength of DEAE Sephadex A50 gel when swelling state is poor, and in column chromatography, volume and flow velocity can change.
Secondly, conventional preparation techniques generally only adopts scleroproein stoste after the glycine precipitator method or chilled alcohol precipitation method purifying chromatography, as " extracting the technique of human blood coagulation factors VIII and human fibrinogen from plasma component precipitation " (application publication number: 103351432 A), " being prepared the method for platelet cofactor Ⅰ, Fibrinogen and Fiberonectin by cryoprecipitate " (application publication number: 102295696A) adopt two step glycine precipitator method purifying to obtain Fibrinogen; " fibrinogenic preparation method " (application publication number: 102286095A) adopts the plasma supernatant of two step chilled alcohol precipitation method purifying after deactivation.
Moreover, in preparation technology, it should further be appreciated that the problem such as solvability and redissolution time.Dissolving after difficulty is even dissolved has a large amount of metaprotein to separate out, and is activated cause by Fibrinogen.Fibrinogen is easy to activate, especially at Ca
2+deposit in case with zymoplasm, Fibrinogen that originally can be water-soluble is activated fibroblast cells monomer by zymoplasm, the latter through polymerization, and at Ca
2+exist and lower form water-fast scleroproein, scleroproein is twisted together agglomerating again, forms Fibrin Glue, therefore, easily causes filtration difficulty and goods to redissolve the time long in technique preparation process.
Visible, still there are some problems in existing human fibrinogen: (1) purity is not high, and product foreign matter content is on the high side, and the untoward reactions such as disseminated inravascular coagulation, fash, tachycardia, heating easily occur Clinical practice; (2) solvability is low, redissolves overlong time in use, is not easy to use, particularly in emergency situations.Some even has a large amount of metaprotein and separates out after dissolving.Major part product redissolves the time at about 20 minutes, and some even arrives about 30 minutes; (3) yield is not high, particularly from cryoprecipitate, extracts human fibrinogen.
At present, Fibrinogen market is still in short situation, and the human fibrinogen's extraction process developing high purity and the high resolution made new advances seems particularly necessary.
Summary of the invention
The object of the present invention is to provide a kind of high purity, redissolution time short extract from cryoprecipitate the preparation technology extracting human fibrinogen the waste material of platelet cofactor Ⅰ.
Major technique design of the present invention is as follows:
The present invention optimizes interpolation 2% aluminum hydroxide gel before S/D inactivation of virus adsorbs;
The present invention removes after fibrin monomer, fibrin complex and scleroproein lysate through step glycine precipitator method the protein liquid after chromatography, then through two step cold ethanol method purifying, removes impurity protein, guarantee that product purity improves further;
The present invention's newly-increased interpolation AT-III deactivation zymoplasm, EDTA remove Ca
2+, the step such as centrifugal segregation fibrin monomer impurity, to obtain high purity, redissolution time short human fibrinogen.
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 come through chromatographic column effluent and extract human fibrinogen; Comprise successively: EDTA is except Ca
2+, glycine precipitation, first time chilled alcohol precipitation, AT-III deactivation zymoplasm, second time chilled alcohol precipitation, nano-film filtration and xeothermic deactivation.
The present invention is achieved like this, and its concrete technology scheme is as follows:
(1), quarantine after qualified human plasma gets quarantine, 75% ethanol plasma bags surface, rinses with water for injection, is merged into and melts in slurry tank, melt with less than 30 ~ 35 DEG C recirculated waters, and the control of blood plasma temperature is not higher than 4 DEG C; After melting, centrifugal, go out liquid temp and control at 0 ~ 4 DEG C, collect cryoprecipitate;
(2), by the obtained thing of step (1) add in 3IU/ml heparin sodium aqua, be stirred to cryoprecipitate and dissolve completely, the temperature of recirculated water controls at 20 ~ 28 DEG C; Start centrifugal, collect supernatant liquor, weigh;
(3), the obtained thing supernatant liquor 0.5mol/L HCL of step (2) is regulated PH to 6.6 ~ 7.2; Add 2% aluminum hydroxide gel, stir; Start centrifugal, collect supernatant liquor, weigh;
(4), by " W(regulates ionic strength buffer liquid)=supernatant liquor weight/19 " calculate, take the adjustment ionic strength buffer liquid of calculated amount in the obtained thing supernatant liquor of step (3); Regulate supernatant liquor PH to 6.8 ~ 7.5; 15.0g/L is not more than by Function protein concentration buffer liquid Function protein concentration; Filter with after the filter core of 1.0um and the filter core series connection of 0.45um, collect filtered liquid, weigh; Regulate the compound method of ionic strength buffer liquid: 48.5g Tutofusin tris, 1.7g calcium chloride, 69.0g sodium-chlor, 28.1g glycine, adds appropriate water for injection and fully dissolves, and mends and injects water to 1L, regulates PH to be 6.8 ~ 7.2; The compound method of Function protein concentration buffer liquid: measure 0.05L and regulate ionic strength buffer liquid, inject water to 1L, regulate PH to be 6.4 ~ 6.8;
(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; 0.45um filter element filtering; Filtered liquid 100KD ultra-filtration membrane is concentrated into protein concentration 1.5%, then uses more than the 2 times lavation buffer solution constant volumes ultrafiltration of protein liquid weight, obtains ultrafiltrated, weigh; The compound method of lavation buffer solution: 2.5g Tutofusin tris, 1.5g calcium chloride, 14.5g sodium-chlor, adds appropriate water for injection and fully dissolves, and mends and injects water to 1L, regulates PH to be 6.4 ~ 6.8;
(6), the obtained thing ultrafiltrated ion exchange column of step (5) is carried out chromatography purification, with washings washing pillar until become baseline, with elution, collect elutriant (elutriant through ultrafiltration, dialysis, filtration, the production for blood coagulation factor VIII); Collect the liquid come through chromatographic column effluent, weigh; Add appropriate 0.01mol/L ethylenediamine tetraacetic acid (EDTA) (EDTA) liquid dissolves, gentle agitation 10 hours, temperature controls at 2 ~ 4 DEG C, and PH controls 7.1 ~ 7.3; Centrifugal, collect supernatant liquor; The compound method of elution buffer: 2.4g Tutofusin tris, 0.09g 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 6.4 ~ 6.8;
(7), in the liquid, in step (6) collected, add appropriate glycine, make whole glycine concentration be 6%, temperature controls, at-1 ~-3 DEG C, to stir centrifugal, collecting precipitation; Precipitation adds in 10 times amount Sodium Citrates, sodium chloride buffer, and stirring and dissolving is about 30min and carries out compression filtration, collects filtrate, by filtrate greenhouse cooling to 0 ~ 1 DEG C; Add 95% ethanol of less than-15 DEG C, make ethanol final concentration be 8%(V/V), temperature controls at-1 ~-3 DEG C, and after adding ethanol, pH value is 6.95 ~ 7.15; Stir 30 minutes; Centrifugal, go out liquid temp in centrifugal process and control at-1 ~-3 DEG C, collect centrifuged deposit, weigh; The compound method of Sodium Citrate, sodium chloride buffer: 14g Sodium Citrate, 9g sodium-chlor, adds appropriate water for injection and fully dissolves, and mends and injects water to 1L, regulates PH to be 7.00 ~ 7.10;
(8), measure Sodium Citrate, sodium chloride buffer described in 10 times amount steps (7), add AT-III and reach 1mU/ml, add in lysate by the obtained thing precipitation of step (7), stirring and dissolving is about 30min and carries out compression filtration; Collect filtrate, by filtrate greenhouse cooling to 0 ~ 1 DEG C; Add 95% ethanol of less than-15 DEG C, make ethanol final concentration be 8%(V/V), temperature controls at-1 ~-3 DEG C, and after adding ethanol, pH value is 6.95 ~ 7.15; Stir 30 minutes; Centrifugal, go out liquid temp in centrifugal process and control at-1 ~-3 DEG C, collect centrifuged deposit, weigh;
(9), add in 5 times amount Sodium Citrates, sodium-chlor, arginine hydrochloride damping fluid by the obtained thing precipitation of step (8), stirring and dissolving is carried out compression and is filtered; Collect filtrate, metering; Rarely join, adjustment protein content is 2.0 ~ 3.0 ℅, and adjustment pH is 7.0 ± 0.1; Nano-film filtration (35nm), collects filtrate; Compound method in Sodium Citrate, sodium-chlor, arginine hydrochloride damping fluid: 15.5 ± 0.5g Sodium Citrate, 8.5g sodium-chlor, 45g arginine hydrochloride, adds appropriate water for injection and fully dissolves, and mends and injects water to 1L, regulates PH to be 6.90 ~ 7.10;
(10), by the obtained thing filtrate 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.
Positively effect of the present invention:
1, in today that blood plasma raw material is increasingly in short supply, prepare human fibrinogen by cryoprecipitate, to the comprehensive utilization of cryoprecipitate, improve the market competitiveness and be significant, on the other hand also can the rare blood plasma resource of indirect saving.
2, purify in fibrinogenic process at ion exchange chromatography, with suitable elutriant (2.4g Tutofusin tris, 0.09g calcium chloride, 40.9g sodium-chlor, add appropriate water for injection fully to dissolve, mend and inject water to 1L, regulate PH to be 6.4 ~ 6.8) solution that elutes, again 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 fibre proteinogen, and innovative point is:
1, Ca
2+under there is situation, can become zymoplasm by catalyze prothrombin, Fibrinogen activates into by zymoplasm
scleroproein, cause filtration difficulty and the redissolution time long.For this reason, before the present invention's second time chilled alcohol precipitation, newly-increased AT-III deactivation zymoplasm and ion-exchange loading stream are worn liquid interpolation EDTA and are removed Ca
2+technique, and through impurity such as centrifugal segregation fibrin monomers, to obtain high purity, redissolution time short human fibrinogen, can at room temperature rapid solution, meet the need of emergency clinically.Present invention process, the redissolution time of human fibrinogen, by about the 30min of traditional technology, shortens within 15min.
2, in order to ensure Product Safety, before S/D deactivation, optimize interpolation 2% aluminum hydroxide gel adsorb; Except S/D and xeothermic deactivation, after rare joining, before degerming packing, newly-increased nanometer film DV35 crosses filtering virus.
3, remove after fibrin monomer, fibrin complex and scleroproein lysate to the protein liquid after chromatography through step glycine precipitator method, again through two step cold ethanol method purifying, remove impurity protein, ensure that product purity improves further, reduce adverse reaction rate.
4, the present invention by test two steps less than-15 DEG C cold ethanol precipitation purifying fibrinogen time, the optimum final concentration of ethanol is 8%(V/V).
The human fibrinogen described in the product prepared of the inventive method and " Chinese Pharmacopoeia " (version in 2010, three) in the contrast of Key Quality Indicator as following table 1.
Table 1
| project | the present invention | " Chinese Pharmacopoeia " (version in 2010, three) |
| purity | answer>=85.0% | be not less than 70.0% |
| the redissolution time | should dissolve completely in 15 minutes | add 30 ~ 37 DEG C of sterilizeds water for injection, shake gently, should dissolve completely in 30 minutes |
| thermostability | 57 ± 0.5 DEG C 4 hours, gel-free or floss are answered in visual inspection | to redissolve in rearmounted 30 ~ 37 DEG C of water-baths insulation 60 minutes, should without grumeleuse or fibrin deposition. |
| solidify vigor | 55 seconds should be no more than | secondary measurement result mean value should be no more than 60 seconds. |
| osmotic pressure molar density | 240 ~ 1000mOsmol/kg | 240mOsmol/kg should be not less than |
In a word, the present invention is in order to ensure security, and except S/D and xeothermic deactivation, newly-increased nano-film filtration is except virus; Newly-increased AT-III deactivation zymoplasm and EDTA are except Ca
2+technique, effectively stops Fibrinogen in process of production to activate fibroblast cells; With the fibrin monomer in glycine precipitation removal goods and polymer, to obtain highly purified human fibrinogen; Gained formulation products is safe and reliable, and the time of redissolving is short, meets the need of emergency clinically.For the comprehensive utilization of cryoprecipitate, indirect saving rare blood plasma resource is significant.
Accompanying drawing explanation
Fig. 1 is present invention process schema.
Embodiment
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.
Embodiment 1: for 20000 liters of blood plasma, concrete preparation technology is as follows:
(1), quarantine after qualified human plasma gets quarantine, 75% ethanol plasma bags surface, rinses with water for injection, is merged into and melts in slurry tank, melt with less than 30 ~ 35 DEG C recirculated waters, and the control of blood plasma temperature is 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 136.9kg;
(2), by the obtained thing cryoprecipitate of step (1) add in 3IU/ml heparin sodium aqua, be stirred to cryoprecipitate and dissolve completely, the temperature of recirculated water controls at 20 ~ 28 DEG C; Start centrifugal, collect supernatant liquor, weigh to obtain 507.8kg;
(3), the obtained thing supernatant liquor 0.5mol/L HCL of step (2) is regulated PH to 6.6 ~ 7.2; Add 2% aluminum hydroxide gel, stir; Start centrifugal, collect supernatant liquor, weigh to obtain 540.2kg;
(4), by " W(regulates ionic strength buffer liquid)=supernatant liquor weight/19 " calculate, take the adjustment ionic strength buffer liquid of calculated amount in the obtained thing supernatant liquor of step (3); Regulate supernatant liquor PH to 6.8 ~ 7.5; 15.0g/L is not more than by Function protein concentration buffer liquid Function protein concentration; Filter with after the filter core of 1.0um and the filter core series connection of 0.45um, collect filtered liquid, weigh to obtain 568.7kg;
(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; 0.45um filter element filtering; Filtered liquid 100KD ultra-filtration membrane is concentrated into protein concentration about 1.5%, and then use more than the 2 times lavation buffer solution constant volumes ultrafiltration of protein liquid weight, i.e. ultrafiltrated, weigh to obtain 611.6L;
(6), the obtained thing ultrafiltrated ion exchange column of step (5) is carried out chromatography purification, with washings washing pillar until become baseline, with elution, collect elutriant (elutriant through ultrafiltration, dialysis, filtration, the production for blood coagulation factor VIII); Collect the liquid come through chromatographic column effluent, weigh; Add appropriate 0.01mol/L ethylenediamine tetraacetic acid (EDTA) (EDTA) liquid dissolves, gentle agitation 10 hours, temperature controls at 2 ~ 4 DEG C, and PH controls 7.1 ~ 7.3; Centrifugal, collect supernatant liquor, weigh to obtain 614.4L;
(7), in the liquid, in step (6) collected, add appropriate glycine, make whole glycine concentration be 6%, temperature controls, at-1 ~-3 DEG C, to stir centrifugal, collecting precipitation; Precipitation adds in 10 times amount Sodium Citrates, sodium chloride buffer, and stirring and dissolving is about 30min and carries out compression filtration, collects filtrate, by filtrate greenhouse cooling to 0 ~ 1 DEG C; Add 95% ethanol of less than-15 DEG C, make ethanol final concentration be 8%(V/V), temperature controls at-1 ~-3 DEG C, and after adding ethanol, pH value is 7.00 ~ 7.10; Stir 30 minutes; Centrifugal, go out liquid temp and control at-1 ~-3 DEG C, collect centrifuged deposit, weigh to obtain 31.5kg;
(8), measure Sodium Citrate, sodium chloride buffer described in 10 times amount steps (8), add AT-III and reach 1mU/ml, add in lysate by the obtained thing precipitation of step (7), stirring and dissolving is about 30min and carries out compression filtration; Collect filtrate, by filtrate greenhouse cooling to 0 ~ 1 DEG C; Add 95% ethanol of less than-15 DEG C, make ethanol final concentration be 8%(V/V), temperature controls at-1 ~-3 DEG C, and after adding ethanol, pH value is 7.00 ~ 7.10; Stir 30 minutes; Centrifugal, go out liquid temp and control at-1 ~-3 DEG C, collect centrifuged deposit, weigh to obtain 23.1kg;
(9), add in 5 times amount Sodium Citrates, sodium-chlor, arginine hydrochloride damping fluid by the obtained thing precipitation of step (8), stirring and dissolving is carried out compression and is filtered; Collect filtrate, measure to obtain 135L; Rarely join, adjustment protein content is 2.0 ~ 3.0 ℅, and adjustment pH is 7.0 ± 0.1; Nano-film filtration (35nm), collects filtrate, weighs to obtain 173.2L;
(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 25mL, and packing quantity is 6765 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 verification result of the product prepared through the present embodiment sees table 2 ~ table 6.
table 2: redissolution timing
table 3, osmotic pressure molar density measure
table 4: stability test
table 5, solidify vitality test
table 6: purity and the total flow measurement of Fibrinogen
1, sample protein matter assay:
2, sample solidifiable protein content determination: get sample after redissolving
10.0ml, adds physiological sodium chloride and is diluted to
50ml, gets the rear sample of dilution
5.0ml, adds physiological sodium chloride
5.0ml, adds every ml containing 3IU thrombin solution (containing 0.05mmol/L calcium chloride)
10.0ml.Mixing.Put 37 DEG C of water-baths
20minute, separating coagulated protein, then by solidifying egg white access alimentary canal.
3, result calculates:
3.1 Fibrinogen purity (%)=solidifiable protein content/protein content × 100
=2.16%?/2.54?×100=85.1%
3.2 Fibrinogen total amounts (g/ bottle)=solidifiable protein content × sample marker volume of dissolution
=2.16% × 25=0.5g/ bottle.
Claims (15)
1. one kind is extracted from cryoprecipitate the preparation technology extracting human fibrinogen the waste material of platelet cofactor Ⅰ, it is characterized in that, 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 come through chromatographic column effluent and extract human fibrinogen; Comprise successively: EDTA is except Ca
2+, glycine precipitation, first time chilled alcohol precipitation, AT-III deactivation zymoplasm, second time chilled alcohol precipitation, nano-film filtration and xeothermic deactivation.
2. extract from cryoprecipitate the preparation technology extracting human fibrinogen the waste material of platelet cofactor Ⅰ, it comprises: cryoprecipitate dissolving, gel adsorption, adjustment ionic strength, cascade filtration, S/D deactivation, 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 come through chromatographic column effluent and extract human fibrinogen; Comprise successively: glycine precipitation, chilled alcohol precipitation, nano-film filtration and xeothermic deactivation, is characterized in that, chilled alcohol precipitation adopts secondary chilled alcohol precipitation, is provided with AT-III deactivation zymoplasm before second time chilled alcohol precipitation; Wear in liquid at ion-exchange loading stream and add EDTA removing Ca
2+technique.
3. extract from cryoprecipitate the preparation technology extracting human fibrinogen the waste material of platelet cofactor Ⅰ, it comprises: cryoprecipitate dissolving, gel adsorption, adjustment ionic strength, cascade filtration, S/D deactivation, 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 come through chromatographic column effluent and extract human fibrinogen; Comprise successively: glycine precipitation, chilled alcohol precipitation, nano-film filtration and xeothermic deactivation, it is characterized in that, remove after fibrin monomer, fibrin complex and scleroproein lysate to the protein solution after ion exchange chromatography through step glycine precipitator method, again through two step cold ethanol method purifying, remove impurity protein.
4. a kind of preparation technology extracting human fibrinogen from the waste material of cryoprecipitate extraction platelet cofactor Ⅰ according to Claims 2 or 3, it is characterized in that, the gel adsorption before S/D deactivation is that interpolation 2% aluminum hydroxide gel adsorbs.
5. extract from cryoprecipitate the preparation technology extracting human fibrinogen the waste material of platelet cofactor Ⅰ, it comprises: cryoprecipitate dissolving, gel adsorption, adjustment 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 come through chromatographic column effluent and extract human fibrinogen; Comprise successively: glycine precipitation, chilled alcohol precipitation, nano-film filtration and xeothermic deactivation, is characterized in that,
(1) gel adsorption, before S/D inactivation of virus is that interpolation 2% aluminum hydroxide gel adsorbs;
(2), to the protein solution after ion exchange chromatography remove after fibrin monomer, fibrin complex and scleroproein lysate through step glycine precipitator method, then through two step cold ethanol method purifying, remove impurity protein;
(3), ion-exchange loading stream wear in liquid add EDTA remove Ca
2+technique; AT-III deactivation zymoplasm is provided with before second time chilled alcohol precipitation.
6. a kind of preparation technology extracting human fibrinogen from the waste material of cryoprecipitate extraction platelet cofactor Ⅰ according to 1-5 arbitrary claims, it is characterized in that, the filter core that before S/D deactivation, cascade filtration uses is of a size of the filter core of 1.0um and 0.45um.
7. a kind of preparation technology extracting human fibrinogen from the waste material of cryoprecipitate extraction platelet cofactor Ⅰ according to 1-5 arbitrary claims, it is characterized in that, the loading stream collected through ion exchange column wears liquid, weighs; Add appropriate 0.01mol/L ethylenediamine tetraacetic acid (EDTA) (EDTA) liquid dissolves, gentle agitation 10 hours, temperature controls at 2 ~ 4 DEG C, and PH controls 7.1 ~ 7.3; Centrifugal, collect supernatant liquor.
8. a kind of preparation technology extracting human fibrinogen from the waste material of cryoprecipitate extraction platelet cofactor Ⅰ according to 1-5 arbitrary claims, it is characterized in that, before second time chilled alcohol precipitation, in lysate, add AT-III deactivation zymoplasm reach 1mU/ml, precipitation adds in lysate, and stirring and dissolving is about 30min and filters; In filtration procedure, keep liquid temp to be-5 ~-7 DEG C, collect filtrate, by filtrate greenhouse cooling to 0 ~ 1 DEG C.
9. a kind of preparation technology extracting human fibrinogen from the waste material of cryoprecipitate extraction platelet cofactor Ⅰ according to 1-5 arbitrary claims, is characterized in that, the front rare nanometer film of joining rear nano-film filtration employing 35nm of degerming packing, collects filtrate.
10. a kind of preparation technology extracting human fibrinogen from the waste material of cryoprecipitate extraction platelet cofactor Ⅰ according to 1-5 arbitrary claims, it is characterized in that, preparation technology is as follows:
(1), cryoprecipitate is collected;
(2), by the cryoprecipitate of step (1) add in 3IU/ml heparin sodium aqua, be stirred to cryoprecipitate and dissolve completely, the temperature of recirculated water controls at 20 ~ 28 DEG C; Start centrifugal, collect supernatant liquor, weigh;
(3), the obtained thing supernatant liquor 0.5mol/L HCL of step (2) is regulated PH to 6.6 ~ 7.2; Add 2% aluminum hydroxide gel, stir; Start centrifugal, collect supernatant liquor, weigh;
(4), by " regulating ionic strength buffer liquid W=supernatant liquor weight/19 " calculate, take the adjustment ionic strength buffer liquid of calculated amount in the obtained thing supernatant liquor of step (3); Regulate supernatant liquor PH to 6.8 ~ 7.5; 15.0g/L is not more than by Function protein concentration buffer liquid Function protein concentration; With the filter core of 1.0um and the filter core cascade filtration of 0.45um, 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; 0.45um filter element filtering; Filtered liquid 100KD ultra-filtration membrane is concentrated into protein concentration 1.5%, then uses more than the 2 times lavation buffer solution constant volumes ultrafiltration of protein liquid weight, obtains ultrafiltrated, weigh;
(6), by the obtained thing ultrafiltrated ion exchange column of step (5) carry out chromatography purification, with washings washing pillar until become baseline, with elution, collect elutriant, elutriant through ultrafiltration, dialysis, filtration, for the production of blood coagulation factor VIII;
Namely collect the liquid come through chromatographic column effluent from the waste material of cryoprecipitate extraction platelet cofactor Ⅰ again and extract human fibrinogen; The liquid come through chromatographic column effluent weighs; Add appropriate 0.01mol/L ethylenediamine tetraacetic acid (EDTA) (EDTA) liquid dissolves, gentle agitation 10 hours, temperature controls at 2 ~ 4 DEG C, and PH controls 7.1 ~ 7.3; Centrifugal, collect supernatant liquor;
(7), in the liquid, in step (6) collected, add appropriate glycine, make whole glycine concentration be 6%, temperature controls, at-1 ~-3 DEG C, to stir centrifugal, collecting precipitation; Precipitation adds in 10 times amount Sodium Citrates, sodium chloride buffer, and stirring and dissolving is about 30min and carries out compression filtration, collects filtrate, by filtrate greenhouse cooling to 0 ~ 1 DEG C; Add 95% ethanol of less than-15 DEG C, make ethanol final concentration be 8%(V/V), temperature controls at-1 ~-3 DEG C, and after adding ethanol, pH value is 6.95 ~ 7.15; Stir 30 minutes; Centrifugal, go out liquid temp in centrifugal process and control at-1 ~-3 DEG C, collect centrifuged deposit, weigh;
(8) Sodium Citrate, the sodium chloride buffer described in 10 times amount steps (7), is measured, add AT-III deactivation zymoplasm and reach 1mU/ml, the obtained thing precipitation of step (7) is added in lysate, stirring and dissolving is about 30min and carries out compression filtration, collect filtrate, by filtrate greenhouse cooling to 0 ~ 1 DEG C; Add 95% ethanol of less than-15 DEG C, make ethanol final concentration be 8%(V/V), temperature controls at-1 ~-3 DEG C, and after adding ethanol, pH value is 6.95 ~ 7.15; Stir 30 minutes; Centrifugal, go out liquid temp in centrifugal process and control at-1 ~-3 DEG C, collect centrifuged deposit, weigh;
(9), add in 5 times amount Sodium Citrates, sodium-chlor, arginine hydrochloride damping fluid by the obtained thing precipitation of step (8), stirring and dissolving is carried out compression and is filtered; Collect filtrate, metering; Rarely join, adjustment protein content is 2.0 ~ 3.0 ℅, and adjustment pH is 7.0 ± 0.1; 35nm nano-film filtration, collects filtrate;
(10), by the obtained thing filtrate 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.
11. a kind of preparation technologies extracting human fibrinogen from the waste material of cryoprecipitate extraction platelet cofactor Ⅰ according to claim 10, it is characterized in that, the compound method of the adjustment ionic strength buffer liquid described in step (4): 48.5g Tutofusin tris, 1.7g calcium chloride, 69.0g sodium-chlor, 28.1g glycine, adds appropriate water for injection and fully dissolves, benefit injects water to 1L, regulates PH to be 6.8 ~ 7.2; The compound method of described Function protein concentration buffer liquid: measure 0.05L and regulate ionic strength buffer liquid, inject water to 1L, regulate PH to be 6.4 ~ 6.8.
12. a kind of preparation technologies extracting human fibrinogen from the waste material of cryoprecipitate extraction platelet cofactor Ⅰ according to claim 10, it is characterized in that the compound method of the lavation buffer solution described in step (5): 2.5g Tutofusin tris, 1.5g calcium chloride, 14.5g sodium-chlor, add appropriate water for injection fully to dissolve, benefit injects water to 1L, regulates PH to be 6.4 ~ 6.8.
13. according to a kind of preparation technology extracting human fibrinogen from the waste material of cryoprecipitate extraction platelet cofactor Ⅰ according to claim 10, it is characterized in that the compound method of the elutriant described in step (6): 2.4g Tutofusin tris, 0.09g calcium chloride, 40.9g sodium-chlor, add appropriate water for injection fully to dissolve, benefit injects water to 1L, regulates PH to be 6.4 ~ 6.8.
14. a kind of preparation technologies extracting human fibrinogen from the waste material of cryoprecipitate extraction platelet cofactor Ⅰ according to claim 10, it is characterized in that, Sodium Citrate described in step (7), the compound method of sodium chloride buffer: 14g Sodium Citrate, 9g sodium-chlor, add appropriate water for injection fully to dissolve, benefit injects water to 1L, regulates PH to be 7.00 ~ 7.10.
15. a kind of preparation technologies extracting human fibrinogen from the waste material of cryoprecipitate extraction platelet cofactor Ⅰ according to claim 10, it is characterized in that, the compound method of the Sodium Citrate described in step (9), sodium-chlor, arginine hydrochloride damping fluid: 15.5 ± 0.5g Sodium Citrate, 8.5g sodium-chlor, 45g arginine hydrochloride, add appropriate water for injection fully to dissolve, benefit injects water to 1L, regulates PH to be 6.90 ~ 7.10.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410524351.2A CN104231072B (en) | 2014-10-09 | 2014-10-09 | Preparation process for extracting human fibrinogens from waste for extracting cryoprecipitated blood coagulation factor VIII |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410524351.2A CN104231072B (en) | 2014-10-09 | 2014-10-09 | Preparation process for extracting human fibrinogens from waste for extracting cryoprecipitated blood coagulation factor VIII |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104231072A true CN104231072A (en) | 2014-12-24 |
| CN104231072B CN104231072B (en) | 2017-03-22 |
Family
ID=52220056
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410524351.2A Active CN104231072B (en) | 2014-10-09 | 2014-10-09 | Preparation process for extracting human fibrinogens from waste for extracting cryoprecipitated blood coagulation factor VIII |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104231072B (en) |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104560925A (en) * | 2014-12-30 | 2015-04-29 | 山东泰邦生物制品有限公司 | Method for preparing human thrombin by activating human coagulation factor IX chromatography waste liquor |
| CN105315360A (en) * | 2015-11-06 | 2016-02-10 | 上海洲跃生物科技有限公司 | Method for simultaneously preparing high-purity human coagulation factor VIII and human fibrinogen |
| CN105504046A (en) * | 2015-12-23 | 2016-04-20 | 同路生物制药有限公司 | Preparation method of human fibrinogen |
| CN105541997A (en) * | 2016-02-04 | 2016-05-04 | 江西博雅生物制药股份有限公司 | Preparation process of high-purity and high-activity von Willebrand factors |
| CN105622746A (en) * | 2016-02-04 | 2016-06-01 | 江西博雅生物制药股份有限公司 | Preparation process for extracting human von Willebrand factor from waste of cryoprecipitate extraction blood coagulation factor VIII |
| CN105622747A (en) * | 2016-02-04 | 2016-06-01 | 江西博雅生物制药股份有限公司 | vWF (von Willebrand factor) activity protection fluid |
| WO2017123012A1 (en) * | 2016-01-12 | 2017-07-20 | 주식회사 녹십자홀딩스 | Method for purifying fibrinogen |
| CN107226859A (en) * | 2017-08-10 | 2017-10-03 | 博雅生物制药集团股份有限公司 | A kind of preparation method of human blood coagulation factors VIII |
| CN111518197A (en) * | 2020-03-30 | 2020-08-11 | 哈尔滨派斯菲科生物制药股份有限公司 | Production method of fibrinogen |
| CN111848783A (en) * | 2020-07-29 | 2020-10-30 | 同路生物制药有限公司 | Preparation method of human fibrinogen |
| CN113577295A (en) * | 2021-09-06 | 2021-11-02 | 成都蓉生药业有限责任公司 | Human fibrinogen dry heat treatment stabilizer and application thereof |
| CN113754757A (en) * | 2021-07-01 | 2021-12-07 | 华兰生物工程股份有限公司 | Preparation method of human fibrinogen |
| CN115521370A (en) * | 2021-06-25 | 2022-12-27 | 上海利康瑞生物工程有限公司 | Preparation process of fibrinogen with high blood coagulation factor XIII titer |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101703763A (en) * | 2009-11-05 | 2010-05-12 | 绿十字(中国)生物制品有限公司 | Production method of human fibrinogen |
| CN102286095A (en) * | 2011-07-06 | 2011-12-21 | 大田华灿生物科技有限公司 | Preparation method for fibrinogen |
| WO2013135684A1 (en) * | 2012-03-13 | 2013-09-19 | Octapharma Ag | Improved process for production of fibrinogen and fibrinogen produced thereby |
| CN103351432A (en) * | 2013-07-09 | 2013-10-16 | 武汉中原瑞德生物制品有限责任公司 | Technology for extracting human blood coagulation factor VIII and human fibrinogen from plasma constituent precipitation |
-
2014
- 2014-10-09 CN CN201410524351.2A patent/CN104231072B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101703763A (en) * | 2009-11-05 | 2010-05-12 | 绿十字(中国)生物制品有限公司 | Production method of human fibrinogen |
| CN102286095A (en) * | 2011-07-06 | 2011-12-21 | 大田华灿生物科技有限公司 | Preparation method for fibrinogen |
| WO2013135684A1 (en) * | 2012-03-13 | 2013-09-19 | Octapharma Ag | Improved process for production of fibrinogen and fibrinogen produced thereby |
| CN103351432A (en) * | 2013-07-09 | 2013-10-16 | 武汉中原瑞德生物制品有限责任公司 | Technology for extracting human blood coagulation factor VIII and human fibrinogen from plasma constituent precipitation |
Cited By (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104560925A (en) * | 2014-12-30 | 2015-04-29 | 山东泰邦生物制品有限公司 | Method for preparing human thrombin by activating human coagulation factor IX chromatography waste liquor |
| CN104560925B (en) * | 2014-12-30 | 2017-11-03 | 山东泰邦生物制品有限公司 | A kind of method that activation prepares human thrombin in chromatography waste liquid from humanclottingfactorⅨ |
| CN105315360A (en) * | 2015-11-06 | 2016-02-10 | 上海洲跃生物科技有限公司 | Method for simultaneously preparing high-purity human coagulation factor VIII and human fibrinogen |
| CN105504046A (en) * | 2015-12-23 | 2016-04-20 | 同路生物制药有限公司 | Preparation method of human fibrinogen |
| WO2017123012A1 (en) * | 2016-01-12 | 2017-07-20 | 주식회사 녹십자홀딩스 | Method for purifying fibrinogen |
| US10815293B2 (en) | 2016-01-12 | 2020-10-27 | Green Cross Holdings Corporation | Method for purifying fibrinogen |
| CN105622746A (en) * | 2016-02-04 | 2016-06-01 | 江西博雅生物制药股份有限公司 | Preparation process for extracting human von Willebrand factor from waste of cryoprecipitate extraction blood coagulation factor VIII |
| CN105622747A (en) * | 2016-02-04 | 2016-06-01 | 江西博雅生物制药股份有限公司 | vWF (von Willebrand factor) activity protection fluid |
| CN105541997A (en) * | 2016-02-04 | 2016-05-04 | 江西博雅生物制药股份有限公司 | Preparation process of high-purity and high-activity von Willebrand factors |
| CN107226859A (en) * | 2017-08-10 | 2017-10-03 | 博雅生物制药集团股份有限公司 | A kind of preparation method of human blood coagulation factors VIII |
| CN107226859B (en) * | 2017-08-10 | 2020-11-24 | 博雅生物制药集团股份有限公司 | Preparation method of human blood coagulation factor VIII |
| CN111518197A (en) * | 2020-03-30 | 2020-08-11 | 哈尔滨派斯菲科生物制药股份有限公司 | Production method of fibrinogen |
| CN111518197B (en) * | 2020-03-30 | 2024-01-05 | 哈尔滨派斯菲科生物制药有限公司 | Production method of fibrinogen |
| CN111848783A (en) * | 2020-07-29 | 2020-10-30 | 同路生物制药有限公司 | Preparation method of human fibrinogen |
| CN111848783B (en) * | 2020-07-29 | 2023-07-04 | 同路生物制药有限公司 | Preparation method of human fibrinogen |
| CN115521370A (en) * | 2021-06-25 | 2022-12-27 | 上海利康瑞生物工程有限公司 | Preparation process of fibrinogen with high blood coagulation factor XIII titer |
| CN113754757A (en) * | 2021-07-01 | 2021-12-07 | 华兰生物工程股份有限公司 | Preparation method of human fibrinogen |
| CN113577295A (en) * | 2021-09-06 | 2021-11-02 | 成都蓉生药业有限责任公司 | Human fibrinogen dry heat treatment stabilizer and application thereof |
| CN113577295B (en) * | 2021-09-06 | 2023-02-28 | 成都蓉生药业有限责任公司 | Human fibrinogen dry heat treatment stabilizer and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104231072B (en) | 2017-03-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104231072A (en) | Preparation process for extracting human fibrinogens from waste for extracting cryoprecipitated blood coagulation factor VIII | |
| CN107226859B (en) | Preparation method of human blood coagulation factor VIII | |
| CN104231073B (en) | Preparation method of human coagulation factor VIII | |
| CN105330736A (en) | Method for preparing human blood coagulation factors IX and VII subcutaneously from cold-glue-removed blood plasma | |
| NO863902L (en) | PROCEDURE FOR PURIFICATION OF PROTEINS. | |
| CN111560064A (en) | Preparation process of high-concentration human fibrinogen | |
| CN103160486A (en) | Preparation method of porcine thrombin | |
| CN104513307B (en) | A method of recycling Kunitz and bowman-Birk trypsin inhibitor from soy-bean whey | |
| CN105622746A (en) | Preparation process for extracting human von Willebrand factor from waste of cryoprecipitate extraction blood coagulation factor VIII | |
| CN103351432A (en) | Technology for extracting human blood coagulation factor VIII and human fibrinogen from plasma constituent precipitation | |
| CN105175486A (en) | Preparation method of high-purity human coagulation factor IX | |
| CN108017710B (en) | Preparation method of human fibrinogen | |
| CN105315360A (en) | Method for simultaneously preparing high-purity human coagulation factor VIII and human fibrinogen | |
| CN107337727A (en) | A kind of haematogenous human blood coagulation factors VIII preparation method | |
| CN102993298B (en) | Method for preparing alpha 1-antitrypsin | |
| CN103285031B (en) | The application of depolymerization glycosaminoglycan extracted from sea cucumber in preparation control thromboembolic disorders medicine | |
| CN104225601B (en) | Human blood coagulation factor VII I is freezed and dry heat treatment protective agent | |
| CN103060297B (en) | Method for separating and purifying trypsin | |
| CN107540743A (en) | A kind of method that bilayer chromatography prepares human fibrinogen | |
| CN105622747A (en) | vWF (von Willebrand factor) activity protection fluid | |
| CN102212129B (en) | Method for extracting human fibrinogen from component I through column chromatography | |
| CN107058270A (en) | The preparation method and its production system of pig thrombiase | |
| CN103725665A (en) | Method for extracting chymotrypsin from sheep pancreas | |
| CN115947825A (en) | Fibrinogen preparation process based on chromatography method | |
| CN113652414B (en) | Preparation method of high-purity human thrombin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 344000 Jiangxi city of Fuzhou province high tech Industrial Park Hui Road No. 333 Patentee after: BOYA BIO-PHARMACEUTICAL GROUP CO.,LTD. Address before: 344000 Jiangxi city of Fuzhou province high tech Industrial Park Hui Road No. 333 Patentee before: JIANGXI BOYA BIOLOGICAL PHARMACEUTICAL CO.,LTD. |
|
| CP03 | Change of name, title or address | ||
| CP03 | Change of name, title or address |
Address after: 344000 No. 333, Huiquan Road, Fuzhou high tech Industrial Development Zone, Fuzhou City, Jiangxi Province Patentee after: China Resources Boya biopharmaceutical Group Co.,Ltd. Address before: 344000 No. 333 Huiquan Road, Fuzhou High-tech Industrial Park, Jiangxi Province Patentee before: BOYA BIO-PHARMACEUTICAL GROUP CO.,LTD. |