CN107540743A - A kind of method that bilayer chromatography prepares human fibrinogen - Google Patents
A kind of method that bilayer chromatography prepares human fibrinogen Download PDFInfo
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Abstract
The invention discloses a kind of method that bilayer chromatography prepares human fibrinogen, this method comprises the following steps：The dissolving of components I precipitation, filtering；S/D inactivation of virus；Ion-exchange chromatography；Once it is concentrated by ultrafiltration；Heparin affinity chromatography；Second ultrafiltration concentrates；Detection is prepared；Packing is lyophilized；Roll lid and xeothermic inactivation.The present invention uses ion-exchange chromatography heparin-binding affinity chromatography, and whole to be produced in normal temperature environment, technological process is short, and product purity is high, high income, and it is short to redissolve the time.
Description
Technical field
The invention belongs to biopharmaceutical technology, is related to a kind of method that bilayer chromatography prepares human fibrinogen.
Background technology
Human fibrinogen (fibrinogen, Fg), i.e. factor I, it is content highest clotting factor in blood plasma,
It is " the " center " protein in blood coagulation system.Human fibrinogen be it is a kind of synthesized by liver, be fibrinous precursor.Point
Son amount 340,000,4~6 days half-life period.2~4 g/l of reference value in blood plasma.Fibrin reason α, β, γ tri- are to not homopolypeptide
Chain is formed, and polypeptide interchain is connected with disulfide bond.It is relevant with the clotting of plasma.Under thrombin action, α chains are released respectively with β chains
A peptides and B peptides are released, generates fibrin monomer.It is mainly used in congenital human fibrinogen's reduction or deficiency disease；It is acquired
Fibrinogen reduces disease, including：Severe Hepatic Injury；Hepatic sclerosis；Disseminated intravascular coagulation；Postpartum hemorrhage and because of big hand
Fibrinogenopenia caused by art, wound or internal haemorrhage etc. and caused by blood coagulation disorders.Be clinically for massive haemorrhage must
One of standby first-aid medicine.
Human fibrinogen is the blood product of obtained official standard, is embodied in 2015 editions《Chinese Pharmacopoeia》, should in three
For many years, its technics comparing is ripe for Clinical practice at home for medicine.Mainly there are several families such as magnificent orchid is biological, Jiangxi is learned in producer,
Most manufacturing enterprises extract using from components I through ethanol precipitation twice, also have been reported that with glycine precipitation method knot
Ion exchange chromatography or other similar approach production human fibrinogen are closed, biology of such as going the same way is with Q Sepharose
The fastflow gel adsorptions combination glycine precipitation method extract human fibrinogen, application number 201510992448.0 from components I.
Green cross (China) extracts human fibrinogen, application number with DEAE-650 combinations glycine precipitation from cryoprecipitate
200910237204.6.Have no that report prepares human fibrinogen using bilayer chromatography.
The content of the invention
It is an object of the invention to overcome defect present in prior art, there is provided a kind of high-purity, in high yield, finished product it is multiple
The method that short bilayer chromatography of molten time prepares human fibrinogen, this method use the affine layer of ion-exchange chromatography heparin-binding
Analysis, whole to be produced in normal temperature environment, technological process is short, and product purity is high, high income, and it is short to redissolve the time.
Its concrete technical scheme is：
A kind of method that bilayer chromatography prepares human fibrinogen, comprises the following steps：
(1) dissolving of components I precipitation, filtering：Components I precipitation is with 10~15 times of lysates, and 20~26 DEG C of stirring in water bath are to molten
Solution is complete, plate-frame filtering, collects filtrate.The lysate is：Per 1000ml water for injection 10~15g containing sodium citrate, chlorination
8~12g of sodium, 3~6g of lysine hydrochloride, 3000~5000IU of liquaemin, pH6.7~7.0,20~26 DEG C of temperature are adjusted with 1MHCl.
(2) S/D inactivation of virus：S/D solution is added by the 1/10 of step (1) filtered fluid weight, makes poly- sorb in mixed liquor
The content of ester 80 is 1.0%, and tributyl phosphate content is 0.3%, is to slowly warm up to 24.0~26.0 DEG C, temperature reaches 24.0 DEG C simultaneously
Start timing, constant temperature 6 hours after constant.
(3) ion-exchange chromatography：By step (2) resulting solution Q Sepharose Fast Flow ion-exchange chromatographies
Post adsorbs, and collection flows through liquid, after absorption terminates, washs chromatographic column with 3~5 times of column volume lysates, cleaning solution, which is incorporated to, flows through liquid.
(4) once it is concentrated by ultrafiltration：Liquid will be flowed through obtained by step (3) to be concentrated by ultrafiltration to 8~10 times of components I weight, 5~8
Times dialyzate is dialysed to protein concentration 3.0%~3.5% (g/ml), pH6.8~7.2.The dialyzate is：1000ml injections
8~12g containing sodium citrate in water, 3~6g of sodium chloride, 30~36g of R-gene, 500~1000IU of liquaemin, use
0.5MNaOH adjusts pH 6.8~7.2,20~26 DEG C of temperature.
(5) heparin affinity chromatography：Gel uses UniGel-DEAE gels, first balances chromatographic column with a ultrafiltrate, then will
Step (4) resulting solution upper prop purifies, and the ultrafiltrate washing of 3~5 times of column volumes, 3~6 times of column volume elutions, receives
Collect eluent.The eluent is：10~20g containing sodium citrate in per 1000ml waters for injection, 55~100g of sodium chloride, sweet ammonia
20~24g of acid, pH 6.8~7.2,20~26 DEG C of liquid temperature are adjusted with 1M hydrochloric acid.
(6) second ultrafiltration concentrates：Eluent obtained by step (5) is concentrated by ultrafiltration, 5~8 times of dialyzates are dialysed dense to albumen
Degree >=2.5%g/ml.The dialyzate is：8~12g containing sodium citrate in 1000ml waters for injection, 3~6g of sodium chloride, sweet ammonia
20~24g of acid, pH 6.8~7.2,20~26 DEG C of temperature are adjusted with 0.5MNaOH.
(7) detection is prepared：Ultrafiltrate protein content obtained by detecting step (6), matched somebody with somebody with step (6) the dialyzate dilution
System, the protein content for making product is 2.2~2.4%g/ml.
(8) packing is lyophilized：Solution, which is dispensed to 50ml, after step (7) is prepared freezes in vial, per bottled 25ml, then
Freeze-drying.
(9) lid and xeothermic inactivation are rolled：Product rolls lid after step (8) is freeze-dried, and then puts 99.5~100.5 DEG C of water-baths
In xeothermic inactivation 30min.
Further, in step (1), the filter plate aperture that plate-frame filtering uses is 0.7 μm~1.0 μm.
Compared with prior art, beneficial effects of the present invention：
1st, the preparation method of human fibrinogen of the invention, appropriate liquaemin is added in preparation process, substitutes and pass
Sucrose in system cold ethanol method, both effectively protects the activity of human fibrinogen, improved finished appearance quality again.
2nd, the preparation method of human fibrinogen of the invention, using ion-exchange chromatography and the double-deck analysis of heparin affinity chromatography
Method, the purity of product is substantially increased, product purity is up to more than 96%, and product purity is high, and clinical side effects are small.
3rd, the production method of human fibrinogen of the invention, technological process is short, production process whole process ordinary temperature production (20~
26 DEG C), stable production process is controllable, and target protein loss is few, high income, and human fibrin 150 can be obtained per 1kg components Is precipitation
~160 bottles (0.5g).The finished product redissolution time is short, was foreshortened to by original 20 minutes or so within 10 minutes, is more beneficial for clinic
Use.
Brief description of the drawings
Fig. 1 is the process chart for the method that bilayer chromatography of the present invention prepares human fibrinogen.
Embodiment
Technical scheme is described in more detail with specific embodiment below in conjunction with the accompanying drawings.
Components I of the present invention is the art adopted name, its main component be fibrinogen 80%, γ-
Globulin 10%, factor 5%, betaglobulin 2%, iodine associated proteins 2% etc..
Embodiment 1
(1) components I precipitation dissolving：Components I precipitation 2.0kg is weighed, is crushed after being cut into fritter by knife, adds 20kg dissolvings
Liquid, 20~26 DEG C of stirring in water bath, 1.5 hours, plate-frame filterings (filter plate aperture 0.7 μm~1.0 μm) complete to dissolving, collection filtrate
20.2kg.The lysate is：Per 1000ml water for injection 12g containing sodium citrate, sodium chloride 8.5g, lysine hydrochloride 4.5g,
Liquaemin 3000IU, pH6.9,23 DEG C of temperature are adjusted with 1MHCl.
(2) S/D inactivation of virus：S/D solution 2.0L are added in step (1) filtrate, contain polyoxyethylene sorbitan monoleate in mixed liquor
To measure as 1.0%, tributyl phosphate content is 0.3%, carries out S/D inactivations in special S/D inactivates tank, sets 24.5 DEG C of temperature,
Mixing speed 100rmp, temperature reach 24.0 DEG C and constant rear beginning timing, constant temperature 6 hours.
(3) ion-exchange chromatography：Solution is through Q Sepharose Fast Flow ion exchange columns after S/D is inactivated
Absorption, collection flow through liquid, after absorption terminates, chromatographic column are washed with 60L lysates, and cleaning solution, which is incorporated to, flows through liquid, collect flow through altogether
Liquid 81.5kg.
(4) once it is concentrated by ultrafiltration：Step (3) gained is flowed through into liquid to be concentrated by ultrafiltration to 20kg, 120kg (6 times) dialyzate perseverance
Volume is dialysed to protein concentration 3.18% (g/ml).The dialyzate is：12g containing sodium citrate in 1000ml waters for injection, chlorine
Change sodium 6g, R-gene 36g, liquaemin 1000IU, pH 6.9,23 DEG C of temperature are adjusted with 0.5MNaOH.
(5) heparin affinity chromatography：UniGel-DEAE chromatographic columns first are balanced with a ultrafiltrate, then will be super obtained by step (4)
Filtrate upper prop purifies, and ultrafiltrate washing of 60kg, 80kg elutions, collects eluent 79.5kg.Eluent is：Often
12g containing sodium citrate in 1000ml waters for injection, sodium chloride 100g, glycine 24g, pH 6.9, liquid temperature 23 are adjusted with 1M hydrochloric acid
℃。
(6) it is concentrated by ultrafiltration：Eluent obtained by step (5) is concentrated by ultrafiltration to 8kg, 50kg dialyzates dialysis protein concentration is extremely
2.8% (g/ml), gained ultrafiltrate are 7.5kg.The dialyzate is：10g containing sodium citrate in 1000ml waters for injection, chlorination
Sodium 6g, glycine 24g, pH 6.9,23 DEG C of temperature are adjusted with 0.5MNaOH.
(7) detection is prepared：Ultrafiltrate protein content obtained by detecting step (6), matched somebody with somebody with step (6) the dialyzate dilution
System, the protein content for making product is 2.3% (g/ml).
(8) packing is lyophilized：Solution, which is dispensed to 50ml, after step (7) is prepared freezes in vial, per bottled 25ml, then
Freeze-drying.
(9) lid and xeothermic inactivation are rolled：Product rolls lid after step (8) is freeze-dried, and then puts 99.5~100.5 DEG C of water-baths
In xeothermic inactivation 30min.
Embodiment 2
(1) components I precipitation dissolving：Components I precipitation 2.0kg is weighed, is crushed after being cut into fritter by knife, adds 30kg dissolvings
Liquid, 20~26 DEG C of stirring in water bath, 1.5 hours, plate-frame filterings (filter plate aperture 0.7 μm~1.0 μm) complete to dissolving, collection filtrate
30.5kg.The lysate is：Per 1000ml water for injection 12g containing sodium citrate, sodium chloride 10g, lysine hydrochloride 6g, heparin
Sodium 5000IU, pH6.9~7.1,20~26 DEG C of temperature are adjusted with 1MHCl.
(2) S/D inactivation of virus：S/D solution 3.05L are added in step (1) filtrate, contain polyoxyethylene sorbitan monoleate in mixed liquor
To measure as 1.0%, tributyl phosphate content is 0.3%, carries out S/D inactivations in special S/D inactivates tank, sets 24.5 DEG C of temperature,
Mixing speed 100rmp, temperature reach 24.0 DEG C and constant rear beginning timing, constant temperature 6 hours.
(3) ion-exchange chromatography：Solution is through Q Sepharose Fast Flow ion exchange columns after S/D is inactivated
Absorption, collection flow through liquid, after absorption terminates, chromatographic column are washed with 60L lysates, and cleaning solution, which is incorporated to, flows through liquid, collect flow through altogether
Liquid 80.0kg
(4) once it is concentrated by ultrafiltration：Step (3) gained is flowed through into liquid to be concentrated by ultrafiltration to 20kg, 120kg (6 times) dialyzate perseverance
Volume is dialysed to protein concentration 3.2% (g/ml).The dialyzate is：12g containing sodium citrate in 1000ml waters for injection, chlorination
Sodium 6g, R-gene 30g, liquaemin 600IU, pH 6.9~7.1,20~26 DEG C of temperature are adjusted with 0.5MNaOH.
(5) heparin affinity chromatography：UniGel-DEAE chromatographic columns first are balanced with a ultrafiltrate, then will be super obtained by step (4)
Filtrate upper prop purifies, and ultrafiltrate washing of 60kg, 80kg elutions, collects eluent 80.0kg.Eluent is：Often
12g containing sodium citrate in 1000ml waters for injection, sodium chloride 70g, glycine 20g, pH6.9~7.1, liquid temperature are adjusted with 1M hydrochloric acid
20~26 DEG C.
(6) it is concentrated by ultrafiltration：Eluent obtained by step (5) is concentrated by ultrafiltration to 8kg, 50kg dialyzates dialysis protein concentration is extremely
2.7% (g/ml), gained ultrafiltrate are 8.0kg.The dialyzate is：10g containing sodium citrate in 1000ml waters for injection, chlorination
Sodium 6g, glycine 24g, pH 6.9~7.1,20~26 DEG C of temperature are adjusted with 0.5MNaOH.
(7) detection is prepared：Ultrafiltrate protein content obtained by detecting step (6), matched somebody with somebody with step (6) the dialyzate dilution
System, the protein content for making product is 2.3% (g/ml).
(8) packing is lyophilized：Solution, which is dispensed to 50ml, after step (7) is prepared freezes in vial, per bottled 25ml, then
Freeze-drying.
(9) lid and xeothermic inactivation are rolled：Product rolls lid after step (8) is freeze-dried, and then puts 99.5~100.5 DEG C of water-baths
In xeothermic inactivation 30min.
Embodiment 3：Comparative example, Conventional cryogenic Ethanol Method, specific preparation technology are as follows：
(1) component I is once suspended, filtered：Components I precipitation 2.0kg is weighed, crushes, is added once after being cut into fritter by knife
Suspension 20kg, 24 DEG C~26 DEG C stirring in water bath, 1.5 hours, plate-frame filtering (filter plate aperture 0.7 μm~1.0 μs complete to dissolving
m).Suspension is：1000ml water for injection 12g containing sodium citrate, sodium chloride 8.5g, Tris 2.7g, sucrose 11g,
Lysine hydrochloride 4.4g, pH6.7~7.0 are adjusted with 1MHCl.
(2) S/D inactivation of virus：S/D solution is added by 1/10 of filtrate weight obtained by step (1), makes poly- mountain in mixed liquor
The content of pear ester 80 is 1.0%, and tributyl phosphate content is 0.3%, carries out S/D inactivations in special S/D inactivates tank, sets temperature
24.5 DEG C, mixing speed 100rmp, temperature reaches 24.0 DEG C and constant rear beginning timing, constant temperature 6 hours.
(3) 8% ethanol precipitations, centrifugation：By step (2) resulting solution with a suspension doubling dilution, by solution temperature
0~2 DEG C is down to, less than -15 DEG C 50% of ethanol is then added with speed of the flow velocity no more than 0.5L/min, makes ethanol finally dense
Spend for 8%, 2~-3 DEG C of products temperature, stirring centrifuges after 30 minutes.Centrifuging temperature：2~-3 DEG C, the separation speed per desk centrifuge
Degree was controlled in 1000~1500ml/ minutes, went out liquid temperature degree：2~-3 DEG C, collect precipitation.
(4) secondary suspension, filtering：Add the secondary suspension of 15 times of step (3) precipitation capacities, 24 DEG C~26 DEG C stirring in water bath
To dissolving 1.5 hours, plate-frame filtering (0.2 μm~0.45 μm of filter plate aperture), the secondary suspension was：Sodium citrate 18g, chlorine
Change sodium 10g, Tris3g, sucrose 11g, pH6.7~7.0 are adjusted with 1MHCl.
(5) 8% ethanol precipitations, centrifugation：Step (4) solution temperature is down to 0~2 DEG C, 0.5L/ is then not more than with flow velocity
Min speed adds less than -15 DEG C 50% of ethanol, and it is 8% to make final ethanol concentration, 2~-3 DEG C of products temperature, stirs 30 points
Centrifuged after clock.Centrifuging temperature：2~-3 DEG C, the separating rate per desk centrifuge was controlled in 1000~1500ml/ minutes, went out liquid temperature
Degree：2~-3 DEG C.Collect precipitation.
(6) third time is suspended, filtered：The suspension three times of 5 times of step (5) precipitation capacities is added, 24 DEG C~26 DEG C water-baths are stirred
Mix to dissolving 1.5 hours, plate-frame filtering (0.2 μm~0.45 μm of filter plate aperture), the suspension three times is：Sodium citrate 12g,
Sodium chloride 2.4g, R-gene 36g, glycine 24g, pH 6.9~7.3 is adjusted with 0.5MNaOH
(7) filtrate protein content obtained by detecting step (6), with suspension dilution is prepared three times step (6) Suo Shu, product is made
Protein content be 2.8% (g/ml).
(8) packing is lyophilized：Solution, which is dispensed to 50ml, after step (7) is prepared freezes in vial, per bottled 25ml, then
Freeze-drying.
(9) lid and xeothermic inactivation are rolled：Product rolls lid after step (8) is freeze-dried, and then puts 99.5~100.5 DEG C of water-baths
In xeothermic inactivation 30min.
Embodiment 4：Comparative example, monolayer chromatography
(1) components I precipitation dissolving：Components I precipitation 2.0kg is weighed, is crushed after being cut into fritter by knife, adds 20kg dissolvings
Liquid, 20~26 DEG C of stirring in water bath, 1.5 hours, plate-frame filterings (filter plate aperture 0.7 μm~1.0 μm) complete to dissolving, collection filtrate
30.5kg.The lysate is：Per 1000ml water for injection 12g containing sodium citrate, sodium chloride 10g, lysine hydrochloride 6g, heparin
Sodium 5000IU, pH6.9~7.1,20~26 DEG C of temperature are adjusted with 1MHCl.
(2) S/D inactivation of virus：S/D solution 3.05L are added in step (1) filtrate, contain polyoxyethylene sorbitan monoleate in mixed liquor
To measure as 1.0%, tributyl phosphate content is 0.3%, carries out S/D inactivations in special S/D inactivates tank, sets 24.5 DEG C of temperature,
Mixing speed 100rmp, temperature reach 24.0 DEG C and constant rear beginning timing, constant temperature 6 hours.
(3) ion-exchange chromatography：Solution is through Q Sepharose Fast Flow ion exchange columns after S/D is inactivated
Absorption, collection flow through liquid, after absorption terminates, chromatographic column are washed with 60L lysates, and cleaning solution, which is incorporated to, flows through liquid, collect flow through altogether
Liquid 80.0kg
(4) glycine precipitates：Liquid will be flowed through obtained by step (3) and is cooled to 2~8 DEG C, adding glycine makes final glycine
Concentration is 15%, is centrifuged after continuing stirring after dissolving completely 30 minutes.Centrifuging temperature：2~-3 DEG C, the separation speed per desk centrifuge
Degree was controlled in 1000~1500ml/ minutes, went out liquid temperature degree：2~-3 DEG C.Collect precipitation.
(5) dissolving is prepared：Precipitation obtained by step (4) is dissolved with secondary lysate, protein concentration is detected, with secondary dissolving
Liquid adjusts protein concentration to 2.5% (g/ml).The secondary lysate is：10g containing sodium citrate in 1000ml waters for injection, chlorine
Change sodium 6g, glycine 24g, pH 6.9~7.1,20~26 DEG C of temperature are adjusted with 0.5MNaOH.
(6) packing is lyophilized：Solution, which is dispensed to 50ml, after step (5) is prepared freezes in vial, per bottled 25ml, then
Freeze-drying.
(7) lid and xeothermic inactivation are rolled：Product rolls lid after step (6) is freeze-dried, and then puts 99.5~100.5 DEG C of water-baths
In xeothermic inactivation 30min.
Human fibrinogen obtained by the present invention, its key detection data, such as outward appearance, visible foreign matters, redissolves the time, pure
Degree, solidification vigor etc., with cold ethanol method, monolayer chromatography compares, and has a clear superiority.
The crucial detection test data of human fibrinogen prepared by 1 three kinds of methods of table
The foregoing is only a preferred embodiment of the present invention, protection scope of the present invention not limited to this, any ripe
Those skilled in the art are known in the technical scope of present disclosure, the letter for the technical scheme that can be become apparent to
Altered or equivalence replacement are each fallen within protection scope of the present invention.
Claims (2)
1. a kind of method that bilayer chromatography prepares human fibrinogen, it is characterised in that comprise the following steps：
(1) dissolving of components I precipitation, filtering：Components I precipitation is with 10~15 times of lysates, and 20~26 DEG C of stirring in water bath are to having dissolved
Entirely, plate-frame filtering, filtrate is collected；The lysate is：Per 1000ml water for injection 10~15g containing sodium citrate, sodium chloride 8~
12g, 3~6g of lysine hydrochloride, 3000~5000IU of liquaemin, pH6.7~7.0,20~26 DEG C of temperature are adjusted with 1MHCl；
(2) S/D inactivation of virus：S/D solution is added by the 1/10 of step (1) filtered fluid weight, makes polyoxyethylene sorbitan monoleate in mixed liquor
Content is 1.0%, and tributyl phosphate content is 0.3%, is to slowly warm up to 24.0~26.0 DEG C, and temperature reaches 24.0 DEG C and constant
After start timing, constant temperature 6 hours；
(3) ion-exchange chromatography：Step (2) resulting solution is inhaled with Q Sepharose Fast Flow ion exchange columns
Attached, collection flows through liquid, after absorption terminates, washs chromatographic column with 3~5 times of column volume lysates, cleaning solution, which is incorporated to, flows through liquid；
(4) once it is concentrated by ultrafiltration：Liquid will be flowed through obtained by step (3) to be concentrated by ultrafiltration to 8~10 times of components I weight, 5~8 times thoroughly
Analysis liquid is dialysed to protein concentration 3.0%~3.5% (g/ml), pH6.8~7.2；The dialyzate is：In 1000ml waters for injection
Containing 8~12g of sodium citrate, 3~6g of sodium chloride, 30~36g of R-gene, 500~1000IU of liquaemin, adjusted with 0.5MNaOH
PH 6.8~7.2,20~26 DEG C of temperature；
(5) heparin affinity chromatography：Gel uses UniGel-DEAE gels, first balances chromatographic column with ultrafiltrate, then by step
(4) resulting solution upper prop is purified, and ultrafiltrate washing of 3~5 times of column volumes, 3~6 times of column volume elutions, collection is washed
De- liquid；The eluent is：10~20g containing sodium citrate in per 1000ml waters for injection, 55~100g of sodium chloride, glycine 20
~24g, pH 6.8~7.2,20~26 DEG C of liquid temperature are adjusted with 1M hydrochloric acid；
(6) second ultrafiltration concentrates：Eluent obtained by step (5) is concentrated by ultrafiltration, 5~8 times of dialyzates dialyse to protein concentration >=
2.5%g/ml；The dialyzate is：8~12g containing sodium citrate in 1000ml waters for injection, 3~6g of sodium chloride, glycine 20
~24g, pH 6.8~7.2,20~26 DEG C of temperature are adjusted with 0.5MNaOH；
(7) detection is prepared：Ultrafiltrate protein content obtained by detecting step (6), prepared, made with step (6) the dialyzate dilution
The protein content of product is 2.2~2.4%g/ml；
(8) packing is lyophilized：Solution, which is dispensed to 50ml, after step (7) is prepared freezes in vial, per bottled 25ml, then freezes
Dry；
(9) lid and xeothermic inactivation are rolled：Product rolls lid after step (8) is freeze-dried, and then puts in 99.5~100.5 DEG C of water-baths and does
Heat inactivation 30min.
2. the method that bilayer chromatography according to claim 1 prepares human fibrinogen, it is characterised in that in step (1),
The filter plate aperture that plate-frame filtering uses is 0.7 μm~1.0 μm.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111560064A (en) * | 2020-06-05 | 2020-08-21 | 博雅生物制药集团股份有限公司 | Preparation process of high-concentration human fibrinogen |
| 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 |
| CN114249817A (en) * | 2020-09-22 | 2022-03-29 | 四川远大蜀阳药业有限责任公司 | Method for separating and purifying human antithrombin III |
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| CN111560064A (en) * | 2020-06-05 | 2020-08-21 | 博雅生物制药集团股份有限公司 | Preparation process of high-concentration human fibrinogen |
| CN114249817A (en) * | 2020-09-22 | 2022-03-29 | 四川远大蜀阳药业有限责任公司 | Method for separating and purifying human antithrombin III |
| CN114249817B (en) * | 2020-09-22 | 2023-11-14 | 四川远大蜀阳药业有限责任公司 | Method for separating and purifying human antithrombin III |
| 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 |
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