CN108191968A - A kind of preparation method of human fibrinogen - Google Patents
A kind of preparation method of human fibrinogen Download PDFInfo
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- CN108191968A CN108191968A CN201810199160.1A CN201810199160A CN108191968A CN 108191968 A CN108191968 A CN 108191968A CN 201810199160 A CN201810199160 A CN 201810199160A CN 108191968 A CN108191968 A CN 108191968A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/745—Blood coagulation or fibrinolysis factors
- C07K14/75—Fibrinogen
Abstract
The present invention is using people's blood as raw material,Prepare human fibrinogen,Its crude extract is handled through lysine,Lysine addition PEG 1000 is precipitated into extract,With the cold ethyl alcohol primary deposition of the ethyl alcohol of 1.2mol/L,Obtain semifinished product,It is precipitated again through acid,Improve bioactivity and dissolution rate,Through gel adsorption twice,It is first to cross 6 resins of CG,Human blood coagulation factor VII I in adsorption precipitation,It is chromatographed again through gel column Toyopeal DEAE 650M,Adsorb foreign protein,Improve the purity of fibrinogen,Most precipitated afterwards through glycine twice,Obtain human fibrinogen's product,Through lipid-coated virus inactivation and the processing of xeothermic two step viral inaction steps of inactivation of virus,It ensure that safety of the blood product in terms of viral transmission,Add in triglycerides,Lecithin,Micella is formed after redissolution in the solution,Increase the solubility of human fibrinogen,Aqueous trehalose is as impermeability protective agent,Dry damage when protecting the human fibrinogen to be lyophilized,Also product can be made to be loaded into cell well in human body,Promote blood coagulation.
Description
Technical field
The invention belongs to field of biological pharmacy, are related to the preparation process of blood product, and in particular to a kind of human fibrinogen.
Background technology
Human fibrinogen（Human fibrinogen, referred to as fine former or Fg）It is the macromolecular sugar containing 2964 amino acid
Albumen is a kind of protein with coagulation function synthesized by liver, it is that content is maximum in 13 kinds of coagulation factors of human body
A kind of coagulation factor, also known as factor I.Human fibrinogen is that one kind clinically uses very frequent blood system
Stop blooding indispensable first-aid medicine on product and battlefield, however fibrinogen is clinically constantly in that supply falls short of demand shape
State.Particularly in recent years, a kind of surgery blood product with efficient hemostatic function -- human fibrin adhesive is increasingly
To be favored by doctor and patient, the demand of the high concentration human fibrinogen as one of the drug compatibility increases, so as to
The product is made to face shorter situation.Human fibrinogen and conventional venoclysis used in human fibrin adhesive
Human fibrinogen is very different, and is mainly reflected in the former with very high protein concentration, at least the latter（Generally
2.5%）Two times or higher i.e. 5 ~ 7%.It is long and molten that the problem of human fibrinogen's product is maximum is that freeze-dried powder redissolves the time
Xie Shixu is carried out in 30 ~ 37 DEG C of water-bath, another problem is easy precipitation albuminate and has protein body suspension, this gives
Clinical practice brings inconvenience, must for example shift to an earlier date to take out being placed in water-bath in refrigerator, and when venoclysis need to be equipped with filtering dress
It puts.Low concentration human fibrinogen is even in this way, the redissolution of high concentration human fibrinogen is just more difficult.Therefore with routine
Human fibrin original production process is difficult to prepare qualified concentration cellulose albumen original product, for that purpose it is necessary to from the source of technique
Head starts to include the formula of stoste in links or even last freeze-drying process is both needed to take targetedly measure.
At present, there are mainly three types of the raw materials for preparing human fibrinogen, first, cryoprecipitate, is mainly used for preparation people and coagulates
Blood factor FVIII's, during because detaching cold glue from blood plasma, due to the coprecipitation phenomena of albumen, the part human fibrin in blood plasma
Former and other oroteins are precipitated out simultaneously with cold glue（Fibrinogen content therein is limited, only accounts for entire plasma Fg very
Small scale）；Second is that component I is precipitated, which is to detach to receive after adding in certain density ethyl alcohol in removing cold glue blood plasma and cooling down
The precipitation of collection, the Fg containing maximum ratio in blood plasma；Third, cold glue is co-precipitated with component I, i.e., added in blood plasma melting certain
Make cold glue and component I coprecipitations after the ethyl alcohol of concentration and cooling.
Using cold glue as the former preparation process of the fibre of raw material, because Fg contained by raw material the inside is very few, by series of operation steps, layer
Layer loss, last yield is very limited, lacks the value of commodity production；And the fibre for raw material is co-precipitated with cold glue and component I
Original production process, there is also it is following the problem of, first, containing 8% ethyl alcohol in precipitation, to the human blood coagulation factor VII I in precipitation
Stability for, be a very unfavorable factor, the storage of precipitation must very with caution, and the holding time can not be long, when feeding intake
Ethyl alcohol must be handled with great care to act on the degenerative lesion of human blood coagulation factor VII I, so the presence of ethyl alcohol undoubtedly coagulates people in raw material
The production technology of blood factor VIII proposes harsh requirement；Secondly as precipitation in human blood coagulation factor VII I a large amount of presence,
Hidden danger is also brought to fine former production, production is caused to fail because of fine former easy be activated, fine former the reason of activating compares
Complexity, but the reason of fibrin ferment is wherein most direct, fibrin ferment is factor in Ca2+Ion and other coagulation factors include solidifying
Blood factor VIII participates in what the lower chain reaction by a series of complex was activated, so in the preparation process of fine original, people coagulates
As soon as the presence of blood factor VIII is also a unfavorable factor, should thoroughly be removed in the initial process of technique as far as possible；Separately
Outside, in terms of fine former preparation process, some technique processes are various, and especially dissolving is repeated with precipitation, thus causes frequently
Heating, cooling, addition ethyl alcohol, the operations such as soda acid, these operations all generate damage to albumen to some extent for albumen,
Once there is the situation of acute variation in operation, it is likely that may result in final products and generate opalescence or even precipitation and redissolve the time
Long the reason of waiting.In addition, excessively high pursue fine former purity and lead to that some processes are various, and the production cycle is long
Reason much it was verified that excessively high product purity is unfavorable instead to stability, is easy to cause fine former precipitation.Some albumen examples
If albumin is famous protein protective agent and the indispensable protective agent of some biological products such as vaccine.So for fibre
For former product, stability, the instant capacity target of product should be more pursued.
Invention content
The technical problems to be solved by the invention：It is fine former for being prepared at present using cold glue as raw material, containing ethyl alcohol, shadow in precipitation
The stability of the human blood coagulation factor VII I in precipitation is rung, the excessively high purity for pursuing fine original is unfavorable to stability, is easy to cause fine original
The problem of precipitation, provides a kind of preparation method of human fibrinogen.
In order to solve the above technical problems, the present invention is using technical solution as described below：
A kind of preparation method of human fibrinogen, the preparation method include the following steps：
（1）Take fresh human plasma in mass ratio 10：1 adds in the polyethylene glycol-1000 of a concentration of 0.1mol/L, is stirred in 35 ~ 37 DEG C
Mixing is mixed, in 4 DEG C of centrifugations, takes precipitation in mass ratio 1：8 add in citrate buffer extracting, take extract in mass ratio 10：1
Lysine is added in, is stirred in 2 ~ 4 DEG C, obtains stirring mixture；
（2）Take stirring mixture in mass ratio 1：7 ~ 10 add in the ethyl alcohol of a concentration of 1.2mol/L, centrifuge, take in 1000r/min
Precipitate A according to the mass fraction, takes 10 ~ 15 parts of precipitate As, 3 ~ 5 parts of sodium citrates, 4 ~ 6 parts of R-genes, 0.4 ~ 0.9 part of chlorination
Sodium, the mixing of 70 ~ 90 parts of waters for injection, are stirred 1 ~ 2h in 25 ~ 30 DEG C, obtain mixed liquor, take mixed liquor prior to pH7.0 ~ 7.5,
10 ~ 15 DEG C are crossed CG-6 resins, and oscillation 2 ~ 3h of absorption collects primary and flows through liquid, then primary is taken to flow through liquid in pH7 ~ 8, sodium chloride is molten
Toyopeal DEAE-650M chromatographic columns, 3 ~ 5h of gel adsorption are crossed under conditions of a concentration of 40 ~ 60mmol/L of liquid, collection flows through
Liquid；
（3）It takes and flows through liquid in mass ratio 70 ~ 80：1：0.3 ~ 0.5 adds in Tween-80, tributyl phosphate, is stirred in 23 ~ 26 DEG C
Mixing is mixed, liquid must be stirred, takes and is stirred liquid in mass ratio 10：2：3 addition glycine, a concentration of 2mol/L sodium chloride are molten
Liquid mixes, and in 10 ~ 15 DEG C of standings, centrifugation takes precipitation, repeats to add in glycine, sodium chloride solution, stands centrifugal process, take most
Precipitation eventually；
（4）Take final precipitation in mass ratio 1：6 add in citrate buffer, are stirred in 25 ~ 30 DEG C, ultrafiltration takes ultrafiltration
Liquid takes ultrafiltrate in mass ratio 100：0.25：1 adds in R-gene mixing, pH to 6.5 ~ 7.5 is adjusted, through 0.22 μm of micropore
Membrane filtration takes filtrate to concentrate, and obtains concentrate；
（5）According to the mass fraction, take 20 ~ 30 parts of concentrates, 15 ~ 20 parts of triglycerides, 1 ~ 3 part of lecithin, 120 ~ 150 parts of water,
12 ~ 15 parts of a concentration of 50mmol/L aqueous trehaloses in 280W ultrasounds, take ultrasonic liquid to be freeze-dried, xeothermic then at 98 ~ 100 DEG C
20 ~ 30min is inactivated, obtains human fibrinogen.
Compared with other methods, advantageous effects are the present invention：
（1）The present invention prepares fibrinogen using fresh people's blood as raw material, due to itself and the original high affinity of fibrinolysin, according to
Binding site of its space structure containing lysine, crude extract are handled through lysine, and through gel filtration, lysine is added in PEG-
1000 precipitate in extracts, then carry out primary deposition with cold ethyl alcohol, are recycled a large amount of fibrinogens, obtain semifinished product,
And the ethyl alcohol of 1.2mol/L does not destroy the space structure of fibrinogen, ensure that its bioactivity；
（2）Present invention semifinished product is precipitated through acid, improves bioactivity and dissolution rate, and blood plasma is in freezing, dissolution in low temperature
With handled under the conditions of isoelectric precipitation etc., belong to reversible degenerative process, through gel adsorption process twice, first mistake
CG-6 resins adsorb human blood coagulation factor VII I in precipitation, remove its interference to human fibrinogen, and it is solidifying to reduce fibrin ferment
Hemase original is in Ca2+Ion and other coagulation factors include blood coagulation factor VIII and participate in the lower chain reaction quilt by a series of complex
The possibility of activation, then chromatographed through gel column Toyopeal DEAE-650M, other foreign proteins can be adsorbed on gel column,
And fibrinogen is stayed in and is flowed through in liquid, improves the purity of fibrinogen, is most precipitated afterwards through glycine twice, obtains people's fibre
Fibrillarin original product, improves whole stability, the precipitation of fiber original is not tended to have after dissolving, through Tween-80, tricresyl phosphate
Butyl ester carries out lipid-coated virus inactivation and xeothermic two step viral inaction steps of the inactivation of virus processing of 100 DEG C/30min, ensure that blood
Safety in terms of the potential viral transmission of liquid product；
（3）The present invention using ultrasonication add in triglycerides, lecithin, enhance its amphipathic property energy, after redissolution
Micella can be formed in solution, so as to increase the solubility of human fibrinogen, solute is whole with micelle forma-tion after addition, is formed
Micellar solution for thermodynamic stable system, no layering, transparent and stable, aqueous trehalose is as impermeability protective agent, it is impossible to
After birth is freely passed through to enter intracellular, using this characteristic, dry damage when protecting the human fibrinogen to be lyophilized, seaweed
Sugar is soluble easily in water, and stability is high, and protection is played to product, and product can also be made to be loaded into cell well in human body, promotes blood coagulation.
Specific embodiment
CG-6 resins：Polymethyl acrylate synthetic resin, middle strong basicity are purchased from Shanghai vast of heaven honor Industrial Co., Ltd..
A kind of preparation method of human fibrinogen, includes the following steps：
（6）Take fresh human plasma in mass ratio 10：1 adds in the polyethylene glycol-1000 of a concentration of 0.1mol/L, is stirred in 35 ~ 37 DEG C
20 ~ 30min of mixing is mixed, 15 ~ 20min is centrifuged in 4 DEG C, takes precipitation in mass ratio 1：8 add in citrate buffer extracting 2 ~ 3
It is secondary, take extract in mass ratio 10：1 adds in lysine, is stirred 2 ~ 3h in 2 ~ 4 DEG C, obtains stirring mixture；
（7）Take stirring mixture in mass ratio 1：7 ~ 10 add in the ethyl alcohol of a concentration of 1.2mol/L, 10 are centrifuged in 1000r/min ~
20min takes precipitate A, according to the mass fraction, take 10 ~ 15 parts of precipitate As, 3 ~ 5 parts of sodium citrates, 4 ~ 6 parts of R-genes, 0.4 ~
0.9 part of sodium chloride, the mixing of 70 ~ 90 parts of waters for injection, are stirred 1 ~ 2h in 25 ~ 30 DEG C, obtain mixed liquor, take mixed liquor prior to
PH7.0 ~ 7.5,10 ~ 15 DEG C cross CG-6 resins, oscillation absorption 2 ~ 3h, collect primary flow through liquid, then take primary flow through liquid in pH7 ~
8, mistake Toyopeal DEAE-650M chromatographic columns under conditions of concentration of sodium chloride solution is 40 ~ 60mmol/L, 3 ~ 5h of gel adsorption,
Collection flows through liquid；
（8）It takes and flows through liquid in mass ratio 70 ~ 80：1：0.3 ~ 0.5 adds in Tween-80, tributyl phosphate, is stirred in 23 ~ 26 DEG C
5 ~ 6h of mixing is mixed, liquid must be stirred, takes and is stirred liquid in mass ratio 10：2：3 add in glycine, a concentration of 2mol/L chlorinations
Sodium solution mixes, and 2 ~ 4h is stood in 10 ~ 15 DEG C, and centrifugation takes precipitation, repeats to add in glycine, sodium chloride solution, standing centrifuged
Journey takes final precipitation；
（9）Take final precipitation in mass ratio 1：6 add in citrate buffer, and 30 ~ 40min is stirred in 25 ~ 30 DEG C, surpass
Filter, takes ultrafiltrate, takes ultrafiltrate in mass ratio 100：0.25：1 adds in R-gene mixing, adjusts pH to 6.5 ~ 7.5, warp
0.22 μm of filtering with microporous membrane, takes filtrate to concentrate, obtains concentrate；
（10）According to the mass fraction, take 20 ~ 30 parts of concentrates, 15 ~ 20 parts of triglycerides, 1 ~ 3 part of lecithin, 120 ~ 150 parts of water,
12 ~ 15 parts of a concentration of 50mmol/L aqueous trehaloses are ultrasonically treated 3 ~ 5min in 280W, ultrasonic liquid are taken to be freeze-dried, then at 98 ~
100 DEG C of 20 ~ 30min of xeothermic inactivation, obtain human fibrinogen.
Embodiment 1
CG-6 resins：Polymethyl acrylate synthetic resin, middle strong basicity are purchased from Shanghai vast of heaven honor Industrial Co., Ltd..
A kind of preparation method of human fibrinogen, includes the following steps：
（1）Take fresh human plasma in mass ratio 10：1 adds in the polyethylene glycol-1000 of a concentration of 0.1mol/L, mixed in 35 DEG C of stirrings
20min is closed, 15min is centrifuged in 4 DEG C, takes precipitation in mass ratio 1：8 add in citrate buffers extract 2 times, take extract by
Mass ratio 10：1 adds in lysine, is stirred 2h in 2 DEG C, obtains stirring mixture；
（2）Take stirring mixture in mass ratio 1：7 add in the ethyl alcohol of a concentration of 1.2mol/L, and 13min is centrifuged in 1000r/min,
Precipitate A is taken, according to the mass fraction, takes 12 parts of precipitate As, 3 parts of sodium citrates, 4 parts of R-genes, 0.5 part of sodium chloride, 70 parts of notes
It penetrates and is mixed with water, be stirred 1h in 26 DEG C, obtain mixed liquor, take mixed liquor prior to pH7.0,10 DEG C are crossed CG-6 resins, and oscillation is inhaled
Attached 2h collects primary and flows through liquid, then primary is taken to flow through liquid in pH7.0, and concentration of sodium chloride solution is mistake under conditions of 40mmol/L
Toyopeal DEAE-650M chromatographic columns, gel adsorption 3h, collection flow through liquid；
（3）It takes and flows through liquid in mass ratio 70：1：0.3 adds in Tween-80, tributyl phosphate, is stirred 5h in 23 DEG C, obtains
Liquid is stirred, takes and is stirred liquid in mass ratio 10：2：3 add in glycine, the mixing of a concentration of 2mol/L sodium chloride solutions, in
10 DEG C of standing 2h, centrifugation take precipitation, repeat to add in glycine, sodium chloride solution, stand centrifugal process, take final precipitation；
（4）Take final precipitation in mass ratio 1：6 add in citrate buffer, 300min are stirred in 25 DEG C, ultrafiltration takes super
Filtrate takes ultrafiltrate in mass ratio 100：0.25：1 adds in R-gene mixing, adjusts pH to 6.5, is filtered through 0.22 μm of micropore
Membrane filtration takes filtrate to concentrate, and obtains concentrate；
（5）According to the mass fraction, take 20 parts of concentrates, 15 parts of triglycerides, 1 part of lecithin, 120 parts of water, 12 parts it is a concentration of
50mmol/L aqueous trehaloses are ultrasonically treated 3min in 280W, ultrasonic liquid are taken to be freeze-dried, then at 98 DEG C of xeothermic inactivation 20min,
Obtain human fibrinogen.
Embodiment 2
CG-6 resins：Polymethyl acrylate synthetic resin, middle strong basicity are purchased from Shanghai vast of heaven honor Industrial Co., Ltd..
A kind of preparation method of human fibrinogen, includes the following steps：
（1）Take fresh human plasma in mass ratio 10：1 adds in the polyethylene glycol-1000 of a concentration of 0.1mol/L, mixed in 36 DEG C of stirrings
25min is closed, 18min is centrifuged in 4 DEG C, takes precipitation in mass ratio 1：8 add in citrate buffers extract 3 times, take extract by
Mass ratio 10：1 adds in lysine, is stirred 2.5h in 3 DEG C, obtains stirring mixture；
（2）Take stirring mixture in mass ratio 1：9 add in the ethyl alcohol of a concentration of 1.2mol/L, and 15min is centrifuged in 1000r/min,
Precipitate A is taken, according to the mass fraction, takes 13 parts of precipitate As, 4 parts of sodium citrates, 5 parts of R-genes, 0.7 part of sodium chloride, 80 parts of notes
It penetrates and is mixed with water, be stirred 1.5h in 28 DEG C, obtain mixed liquor, take mixed liquor prior to pH7.3,13 DEG C are crossed CG-6 resins, oscillation
2.5h is adsorbed, primary is collected and flows through liquid, then primary is taken to flow through liquid in pH7.5, concentration of sodium chloride solution is the condition of 50mmol/L
Lower to cross Toyopeal DEAE-650M chromatographic columns, gel adsorption 4h is collected and is flowed through liquid；
（3）It takes and flows through liquid in mass ratio 75：1：0.4 adds in Tween-80, tributyl phosphate, and 5.5h is stirred in 25 DEG C,
Liquid must be stirred, takes and is stirred liquid in mass ratio 10：2：3 add in glycine, the mixing of a concentration of 2mol/L sodium chloride solutions,
3h is stood in 13 DEG C, centrifugation takes precipitation, repeats to add in glycine, sodium chloride solution, stands centrifugal process, take final precipitation；
（4）Take final precipitation in mass ratio 1：6 add in citrate buffer, 35min are stirred in 28 DEG C, ultrafiltration takes super
Filtrate takes ultrafiltrate in mass ratio 100：0.25：1 adds in R-gene mixing, adjusts pH to 7.0, is filtered through 0.22 μm of micropore
Membrane filtration takes filtrate to concentrate, and obtains concentrate；
（5）According to the mass fraction, take 25 parts of concentrates, 18 parts of triglycerides, 2 parts of lecithin, 140 parts of water, 14 parts it is a concentration of
50mmol/L aqueous trehaloses are ultrasonically treated 4min in 280W, ultrasonic liquid are taken to be freeze-dried, then at 99 DEG C of xeothermic inactivation 25min,
Obtain human fibrinogen.
Embodiment 3
CG-6 resins：Polymethyl acrylate synthetic resin, middle strong basicity are purchased from Shanghai vast of heaven honor Industrial Co., Ltd..
A kind of preparation method of human fibrinogen, includes the following steps：
（1）Take fresh human plasma in mass ratio 10：1 adds in the polyethylene glycol-1000 of a concentration of 0.1mol/L, mixed in 37 DEG C of stirrings
30min is closed, 20min is centrifuged in 4 DEG C, takes precipitation in mass ratio 1：8 add in citrate buffers extract 3 times, take extract by
Mass ratio 10：1 adds in lysine, is stirred 3h in 4 DEG C, obtains stirring mixture；
（2）Take stirring mixture in mass ratio 1：10 add in the ethyl alcohol of a concentration of 1.2mol/L, and 20min is centrifuged in 1000r/min,
Precipitate A is taken, according to the mass fraction, takes 15 parts of precipitate As, 5 parts of sodium citrates, 6 parts of R-genes, 0.9 part of sodium chloride, 90 parts of notes
It penetrates and is mixed with water, be stirred 2h in 30 DEG C, obtain mixed liquor, take mixed liquor prior to pH7.5,15 DEG C are crossed CG-6 resins, and oscillation is inhaled
Attached 3h collects primary and flows through liquid, then primary is taken to flow through liquid in pH8, and concentration of sodium chloride solution is mistake under conditions of 60mmol/L
Toyopeal DEAE-650M chromatographic columns, gel adsorption 5h, collection flow through liquid；
（3）It takes and flows through liquid in mass ratio 80：1：0.5 adds in Tween-80, tributyl phosphate, is stirred 6h in 26 DEG C, obtains
Liquid is stirred, takes and is stirred liquid in mass ratio 10：2：3 add in glycine, the mixing of a concentration of 2mol/L sodium chloride solutions, in
15 DEG C of standing 4h, centrifugation take precipitation, repeat to add in glycine, sodium chloride solution, stand centrifugal process, take final precipitation；
（4）Take final precipitation in mass ratio 1：6 add in citrate buffer, are stirred 30 ~ 40min in 30 DEG C, ultrafiltration takes
Ultrafiltrate takes ultrafiltrate in mass ratio 100：0.25：1 adds in R-gene mixing, pH to 7.5 is adjusted, through 0.22 μm of micropore
Membrane filtration takes filtrate to concentrate, and obtains concentrate；
（5）According to the mass fraction, take 30 parts of concentrates, 20 parts of triglycerides, 3 parts of lecithin, 150 parts of water, 15 parts it is a concentration of
50mmol/L aqueous trehaloses are ultrasonically treated 5min in 280W, ultrasonic liquid are taken to be freeze-dried, then at 100 DEG C of xeothermic inactivations
30min obtains human fibrinogen.
Comparative example：Certain domestic company human fibrinogen's commercialized product.
Product of the present invention is fully achieved or is more than after tested《Chinese Pharmacopoeia》The related request of version in 2015 and the country are
The similar product of listing, the comparative analysis such as table 1 of related data：
As can be seen from the above table, the present invention obtained by human fibrinogen, Key Quality Indicator such as purity, solidification vigor,
Redissolve the time, appearance is more than《Chinese Pharmacopoeia》The product of other companies listed with the country.
Claims (1)
1. a kind of preparation method of human fibrinogen, which is characterized in that the preparation method includes the following steps：
Take fresh human plasma in mass ratio 10：1 adds in the polyethylene glycol-1000 of a concentration of 0.1mol/L, mixed in 35 ~ 37 DEG C of stirrings
It closes, in 4 DEG C of centrifugations, takes precipitation in mass ratio 1：8 add in citrate buffer extracting, take extract in mass ratio 10：1 adds in
Lysine is stirred in 2 ~ 4 DEG C, obtains stirring mixture；
Take stirring mixture in mass ratio 1：7 ~ 10 add in the ethyl alcohol of a concentration of 1.2mol/L, are centrifuged in 1000r/min, take precipitation
A, according to the mass fraction, take 10 ~ 15 parts of precipitate As, 3 ~ 5 parts of sodium citrates, 4 ~ 6 parts of R-genes, 0.4 ~ 0.9 part of sodium chloride,
The mixing of 70 ~ 90 parts of waters for injection, is stirred 1 ~ 2h in 25 ~ 30 DEG C, obtains mixed liquor, takes mixed liquor prior to pH7.0 ~ 7.5,10 ~
15 DEG C are crossed CG-6 resins, and oscillation 2 ~ 3h of absorption collects primary and flows through liquid, then primary is taken to flow through liquid in pH7 ~ 8, sodium chloride solution is dense
It spends to cross Toyopeal DEAE-650M chromatographic columns, 3 ~ 5h of gel adsorption under conditions of 40 ~ 60mmol/L, collection flows through liquid；
It takes and flows through liquid in mass ratio 70 ~ 80：1：0.3 ~ 0.5 adds in Tween-80, tributyl phosphate, mixed in 23 ~ 26 DEG C of stirrings
It closes, liquid must be stirred, take and be stirred liquid in mass ratio 10：2：3 addition glycine, a concentration of 2mol/L sodium chloride solutions mix
It closes, in 10 ~ 15 DEG C of standings, centrifugation takes precipitation, repeats to add in glycine, sodium chloride solution, stands centrifugal process, and it is final heavy to take
It forms sediment；
Take final precipitation in mass ratio 1：6 add in citrate buffer, are stirred in 25 ~ 30 DEG C, and ultrafiltration takes ultrafiltrate,
Take ultrafiltrate in mass ratio 100：0.25：1 adds in R-gene mixing, pH to 6.5 ~ 7.5 is adjusted, through 0.22 μm of miillpore filter
Filtering, takes filtrate to concentrate, obtains concentrate；
According to the mass fraction, take 20 ~ 30 parts of concentrates, 15 ~ 20 parts of triglycerides, 1 ~ 3 part of lecithin, 120 ~ 150 parts of water, 12 ~
15 parts of a concentration of 50mmol/L aqueous trehaloses in 280W ultrasounds, take ultrasonic liquid to be freeze-dried, then at 98 ~ 100 DEG C of xeothermic inactivations
20 ~ 30min obtains human fibrinogen.
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