CN105504046A - Preparation method of human fibrinogen - Google Patents
Preparation method of human fibrinogen Download PDFInfo
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- CN105504046A CN105504046A CN201510992448.0A CN201510992448A CN105504046A CN 105504046 A CN105504046 A CN 105504046A CN 201510992448 A CN201510992448 A CN 201510992448A CN 105504046 A CN105504046 A CN 105504046A
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- 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
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Abstract
The invention discloses a preparation method of human fibrinogen. The method comprises the following steps that 1, cryoprecipitate serves as a staring material and is dissolved; 2, clarification and filtration are conducted; 3, solvent/detergent (S/D) inactivation is conducted; 4, Q Sepharose fastflow gel adsorption chromatographic processing is conducted; 5, centrifugation is conducted, precipitation is collected, sterilizing, subpackaging, freeze-drying, capping and dry-heating inactivating are conducted, and the product is obtained. The preparation method of the human fibrinogen is simple, easy to operate, low in cost and capable of increasing the comprehensive utilization ratio of blood plasma, and the obtained product is high in purity, activity and safety.
Description
Technical field
The invention belongs to field of biological pharmacy, be specifically related to a kind of fibrinogenic preparation method.
Background technology
Human fibrinogen is mainly rich in cryoprecipitate and components I, the domestic producer possessing human fibrin original production mainly contains several families such as China orchid is biological, Jiangxi is learned at present, overwhelming majority manufacturing enterprise all adopts and extracts through twice ethanol precipitation from components I, production craft step is numerous and diverse, and the finished product purity and biological activity are not all ideal.
Human fibrinogen is thrombin, clinical application is mainly treated acquired Fibrinogen and reduces disease: Severe Hepatic Injury; Liver cirrhosis; Disseminated inravascular coagulation; Postpartum hemorrhage and the fibrinogenopenia that causes because of major operation, wound or internal hemorrhage etc. and the blood coagulation disorders caused.Human fibrinogen is humanized's Blood Preparations, and clinical side effects is few, the virus inactivation technology that its preparation is special in producing, and security has reliable guarantee, as the indispensable preparation for the treatment of hemorrhagic surgery, and current domestic genus medicine in short supply.
In the face of the shortage of domestic human fibrinogen preparation, how present stage improves the comprehensive utilization ratio of blood plasma, develops high purity, high reactivity, security are high, and concise in technology human fibrinogen preparation easy to operate, with low cost has positive effect.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of comprehensive utilization ratio how improving blood plasma, develops high purity, high reactivity, security are high, the preparation method of the human fibrinogen that concise in technology is easy to operate, with low cost.
A human fibrinogen's preparation method, take cryoprecipitate as starting raw material, comprises the steps:
(1) cryoprecipitate is dissolved;
(2) clarification filtration;
(3) S/D deactivation;
(4) QSepharosefastflow gel adsorption Image processing;
(5) centrifugal collecting precipitation, degerming packing, freeze-drying, roll lid, xeothermic deactivation after obtain product.
The preparation method of human fibrinogen of the present invention, wherein, step (1) specifically comprises the steps: the cryoprecipitate of fresh preparation to be cut into 1-3 centimetre of bulk, dissolve under 24 ~ 26 DEG C of conditions with the Tris-HCl lysate that the concentration of 3 times of volumes is 0.01-0.05mol/L, adding heparin sodium aqua to ultimate density is 0.005-0.065%, adding aluminum hydroxide gel suspension to ultimate density is 5%, stirs collected by centrifugation supernatant liquor after 30 minutes.
The preparation method of human fibrinogen of the present invention, wherein, step (2) specifically comprises the steps: to carry out clarification filtration with the filter core in pretreated 1 μm of aperture, with balance liquid filter cartridge flushing to cryoprecipitate 4 times of volumes.
The preparation method of human fibrinogen of the present invention, wherein, step (3) specifically comprises the steps: accurate-metering filtered liquid volume, the S/D solution prepared under agitation slowly is added by the volume ratio of 1:10, Polysorbate 80 ultimate density in protein liquid is made to be 1%, tbp ultimate density is 0.3%, is warming up to 24 ~ 26 DEG C, and the filter core be incubated through 0.6 μm of aperture after 6 hours carries out clarification filtration.
The preparation method of human fibrinogen of the present invention, wherein, step (4) specifically comprises the steps: QSepharosefastflow gel adsorption Image processing: by the solution loading after clarification filtration, rinse with the balance liquid of 3 times of volumes after terminating, rinse with the washings of 3 times of volumes, collect elutriant in processed aseptic, pyrogen-free specified containers with the elution of 3 times of volumes, rinse envrionment temperature and keep 20-25 DEG C.
The preparation method of human fibrinogen of the present invention, wherein, step (5) specifically comprises the steps: accurate-metering effluent volume, adding glycine to ultimate density is 15%, stir centrifugal after 30 minutes, collecting precipitation, dissolves with the lysate of 3-4 times of volume, and the assay according to protein content regulates adjustment protein concentration to 3.5 ± 0.5% with dialyzate; Work in-process after preparation, through degerming packing, freeze-drying, roll lid, obtain product after xeothermic deactivation in 100 DEG C, 30 minutes.
The preparation method of human fibrinogen of the present invention, wherein, the balance liquid in step (2) and step (4) is Sodium Citrate 0.01mol/L, and glycine 0.12mol/L, sodium-chlor 0.12mol/L, pH value is 6.9-7.1.
The preparation method of human fibrinogen of the present invention, wherein, described washings is: Sodium Citrate 0.01mol/L, glycine 0.12mol/L, sodium-chlor 0.16mol/L, and pH value is 6.9-7.1; Elutriant is: Sodium Citrate 0.01mol/L, glycine 0.12mol/L, sodium-chlor 0.25mol/L, and pH value is 6.9-7.1.
The human fibrinogen that the preparation method of human fibrinogen of the present invention prepares.
Human fibrinogen of the present invention, wherein, described human fibrinogen only produces precipitation line with anti-human blood plasma, does not produce precipitation line with anti-horse, ox, pig, sheep blood plasma.
The fibrinogenic preparation method's difference from prior art of the present inventor is:
The fibrinogenic preparation method of the present inventor has following beneficial effect:
1, adopt cryoprecipitate to be raw material, this technique can not only prepare human fibrinogen, and can better extract human blood coagulation factor VII I after extracting perfect person's Fibrinogen, substantially increases the comprehensive utilization ability of valuable human plasma, increases economic efficiency.
2, adopt chromatogram gel in conjunction with glycine extraction purification production technique, not only reduce production stage, technological process quality is more easily monitored, and reduces the requirement to main equipment, with low cost, and biological activity and purity high, increase economic efficiency greatly.
Below in conjunction with specific embodiment, the preparation method to human fibrinogen of the present invention is described further.
Embodiment
Embodiment 1
Low-temperature centrifugation partition method is adopted to prepare cryoprecipitate from human plasma.
A, blood plasma thawing
Blood plasma thawing should carry out in 100,000 grades of clean rooms.Method is melted in water-bath.The water temperature that method is melted in water-bath should control below 37 DEG C.
B, blood plasma merge
Blood plasma merges and should carry out under 100,000 grades of ventilation openings, and before merging, reply plasma bags disinfection, namely uses 75% alcohol-pickled more than three minutes, could merge after being cleaned by outside for plasma bags alcohol with clean gauze.When merging blood plasma, should notice that drop reclaims.
C, cryoprecipitate centrifugation
Pooled plasma temperature is down to less than 0 DEG C, with 3 ± 1L/ platform/point carry out centrifugation, goes out liquid temp and control at 0-4 DEG C.
Attached cryoprecipitate raw materials quality standard
1. cryoprecipitate raw material is the plasma component adopting low-temperature centrifugation partition method.Blood plasma raw material used should meet " blood products production human plasma " regulation
2. cryoprecipitate should be use up and can keep aseptic and cryogenic freezing preservation, and storage temperature must not exceed-30 DEG C, and preservation period should be no more than 1 year.
3. the calibrating of cryoprecipitate
Accurately take cryoprecipitate 10g, be diluted to 100ml with physiological sodium chloride solution, stir at 1 ~ 3 DEG C and fully dissolve rear centrifugal or filtration, get supernatant liquor and carry out following items detection.
3.1 human fibrinogens solidify viability examination
In reaction tubes, add thrombin solution (3IU/ml) 0.5ml being preheated to 37 DEG C, then add the need testing solution 0.5ml being diluted to 3mg/ml with physiological sodium chloride solution, shake up.Put 37 DEG C of record setting times.Twice measurement result mean value should be no more than 60 seconds.
3.2 bacterial endotoxin
Detect with the test kit through approval, should be negative.
3.3HBsAg
Detect with the test kit through approval, should be negative.
3.4HIV antibody
Detect with the test kit through approval, should be negative.
3.5HCV antibody
Detect with the test kit through approval, should be negative.
Embodiment 2
The technique of human fibrinogen is extracted from cryoprecipitate
(1) cryoprecipitate is dissolved: the cryoprecipitate of fresh preparation is cut into 1-3 centimetre of bulk, dissolve under 24 ~ 26 DEG C of conditions with the Tris-HCl lysate that the concentration of 3 times of volumes is 0.01-0.05mol/L, adding heparin sodium aqua to ultimate density is 0.005-0.065%, adding aluminum hydroxide gel suspension to ultimate density is 5%, stirs collected by centrifugation supernatant liquor after 30 minutes.
(2) clarification filtration: carry out clarification filtration with the filter core in pretreated 1 μm of aperture, with balance liquid filter cartridge flushing to cryoprecipitate 4 times of volumes.
(3) S/D deactivation: accurate-metering filtered liquid volume, the S/D solution prepared under agitation slowly is added by the volume ratio of 1:10, Polysorbate 80 ultimate density in protein liquid is made to be 1%, tbp ultimate density is 0.3%, be warming up to 24 ~ 26 DEG C, the filter core be incubated through 0.6 μm of aperture after 6 hours carries out clarification filtration.
(4) QSepharosefastflow gel adsorption Image processing: QSepharosefastflow gel adsorption Image processing: by the solution loading after clarification filtration, rinse with the balance liquid of 3 times of volumes after terminating, rinse with the washings of 3 times of volumes, collect elutriant in processed aseptic, pyrogen-free specified containers with the elution of 3 times of volumes, rinse envrionment temperature and keep 20-25 DEG C.
(5) accurate-metering effluent volume, adding glycine to ultimate density is 15%, stirs centrifugal after 30 minutes, collecting precipitation, dissolve with the lysate of 3-4 times of volume, the assay according to protein content regulates adjustment protein concentration to 3.5 ± 0.5% with dialyzate; Work in-process after preparation, through degerming packing, freeze-drying, roll lid, obtain product after xeothermic deactivation in 100 DEG C, 30 minutes.
Wherein, balance liquid in step (2) and step (4) is Sodium Citrate 0.01mol/L, glycine 0.12mol/L, sodium-chlor 0.12mol/L, pH value is 6.9-7.1, and described washings is: Sodium Citrate 0.01mol/L, glycine 0.12mol/L, sodium-chlor 0.16mol/L, pH value is 6.9-7.1; Elutriant is: Sodium Citrate 0.01mol/L, glycine 0.12mol/L, sodium-chlor 0.25mol/L, and pH value is 6.9-7.1.
Finished product detection is reported as follows table:
The general comment of human fibrinogen's finished product verification result
Above-described embodiment is only be described the preferred embodiment of the present invention; not scope of the present invention is limited; under not departing from the present invention and designing the prerequisite of spirit; the various distortion that those of ordinary skill in the art make technical scheme of the present invention and improvement, all should fall in protection domain that claims of the present invention determines.
Claims (10)
1. a human fibrinogen's preparation method, is characterized in that: be starting raw material with cryoprecipitate, comprises the steps:
(1) cryoprecipitate is dissolved;
(2) clarification filtration;
(3) S/D deactivation;
(4) QSepharosefastflow gel adsorption Image processing;
(5) centrifugal collecting precipitation, degerming packing, freeze-drying, roll lid, xeothermic deactivation after obtain product.
2. the preparation method of human fibrinogen according to claim 1, it is characterized in that: step (1) specifically comprises the steps: the cryoprecipitate of fresh preparation to be cut into 1-3 centimetre of bulk, dissolve under 24 ~ 26 DEG C of conditions with the Tris-HCl lysate that the concentration of 3 times of volumes is 0.01-0.05mol/L, adding heparin sodium aqua to ultimate density is 0.005-0.065%, adding aluminum hydroxide gel suspension to ultimate density is 5%, stirs collected by centrifugation supernatant liquor after 30 minutes.
3. the preparation method of human fibrinogen according to claim 2, it is characterized in that: step (2) specifically comprises the steps: to carry out clarification filtration with the filter core in pretreated 1 μm of aperture, with balance liquid filter cartridge flushing to cryoprecipitate 4 times of volumes.
4. the preparation method of human fibrinogen according to claim 3, it is characterized in that: step (3) specifically comprises the steps: accurate-metering filtered liquid volume, the S/D solution prepared under agitation slowly is added by the volume ratio of 1:10, Polysorbate 80 ultimate density in protein liquid is made to be 1%, tbp ultimate density is 0.3%, be warming up to 24 ~ 26 DEG C, the filter core be incubated through 0.6 μm of aperture after 6 hours carries out clarification filtration.
5. the preparation method of human fibrinogen according to claim 4, it is characterized in that: step (4) specifically comprises the steps: QSepharosefastflow gel adsorption Image processing: by the solution loading after clarification filtration, rinse with the balance liquid of 3 times of volumes after terminating, rinse with the washings of 3 times of volumes, collect elutriant in processed aseptic, pyrogen-free specified containers with the elution of 3 times of volumes, rinse envrionment temperature and keep 20-25 DEG C.
6. the preparation method of human fibrinogen according to claim 5, it is characterized in that: step (5) specifically comprises the steps: accurate-metering effluent volume, adding glycine to ultimate density is 15%, stir centrifugal after 30 minutes, collecting precipitation, dissolve with the lysate of 3-4 times of volume, the assay according to protein content regulates adjustment protein concentration to 3.5 ± 0.5% with dialyzate; Work in-process after preparation, through degerming packing, freeze-drying, roll lid, obtain product after xeothermic deactivation in 100 DEG C, 30 minutes.
7. the preparation method of human fibrinogen according to claim 6, it is characterized in that: the balance liquid in step (2) and step (4) is Sodium Citrate 0.01mol/L, glycine 0.12mol/L, sodium-chlor 0.12mol/L, pH value is 6.9-7.1.
8. the preparation method of human fibrinogen according to claim 7, is characterized in that: described washings is: Sodium Citrate 0.01mol/L, glycine 0.12mol/L, sodium-chlor 0.16mol/L, and pH value is 6.9-7.1; Elutriant is: Sodium Citrate 0.01mol/L, glycine 0.12mol/L, sodium-chlor 0.25mol/L, and pH value is 6.9-7.1.
9. the human fibrinogen that the preparation method of the human fibrinogen in claim 1 ~ 8 described in any one prepares.
10. human fibrinogen according to claim 9, is characterized in that: described human fibrinogen only produces precipitation line with anti-human blood plasma, does not produce precipitation line with anti-horse, ox, pig, sheep blood plasma.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107540743A (en) * | 2017-10-11 | 2018-01-05 | 南岳生物制药有限公司 | A kind of method that bilayer chromatography prepares human fibrinogen |
| CN111848783A (en) * | 2020-07-29 | 2020-10-30 | 同路生物制药有限公司 | Preparation method of human fibrinogen |
| CN113698470A (en) * | 2021-09-02 | 2021-11-26 | 成都蓉生药业有限责任公司 | Purification method of human fibrinogen |
| CN115197315A (en) * | 2022-09-02 | 2022-10-18 | 同路生物制药有限公司 | Method for preparing human fibrinogen by using cryoprecipitate as raw material and using chromatographic method |
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2015
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107540743A (en) * | 2017-10-11 | 2018-01-05 | 南岳生物制药有限公司 | A kind of method that bilayer chromatography prepares human 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 |
| CN113698470A (en) * | 2021-09-02 | 2021-11-26 | 成都蓉生药业有限责任公司 | Purification method of human fibrinogen |
| CN115197315A (en) * | 2022-09-02 | 2022-10-18 | 同路生物制药有限公司 | Method for preparing human fibrinogen by using cryoprecipitate as raw material and using chromatographic method |
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Inventor after: Hu Huiheng Inventor after: Yang Jingpeng Inventor after: Xu Xiaonan Inventor after: Deng Kun Inventor after: Wei Cheng Inventor before: Hu Huiheng Inventor before: Deng Kun Inventor before: Wei Cheng |
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Application publication date: 20160420 |