CN104231072B - 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
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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 extract the preparation work for extracting human fibrinogen in the waste material of platelet cofactor Ⅰ from cryoprecipitate
Skill, belongs to field of biological pharmacy.
Background technology
Fibrinogen（Fg）, synthesized and secreted by hepatocyte, be important thrombin in body hemostasis physiology.Storage
Stability is preferable, but heat stability is poor, can form irreversible precipitation at 56 DEG C, and in human normal plasma, Fg concentration is 2.4～4.0g/
L。
Used as a kind of Acute reaction protein, its blood content is the danger of the diseases such as ischemic cerebrocardiac disease to Fg extremely
The dangerous factor.Fg increases a kind of non-specific responding of often body, is common in the infection such as toxemia, pneumonia, cholecystitis, pulmonary tuberculosis
And the aseptic inflammation such as nephrotic syndrome, rheumatic fever, malignant tumor, cerebral thrombosiss, myocardial infarction；In addition such as surgical operation, radiation
Treatment, trimester of pregnancy also show Fg and slightly increase.Fg reduces more rare, but when which is less than 1.0g/L, body may occur in which that bleeding is levied
As.A kind of congenital afibrinogenemia is extremely rare hereditary, by autosomal recessive gene heredity,
This patient's liver can not synthesize Fg；The fibrinogenopenic reason of Secondary cases is due to fibrinolytic enzymes fiber egg
Caused by white, such as placental abruption, during childbirth, amniotic fluid intravasation forms thrombosis, causes disseminated inravascular coagulation, and activation is fine
Fibrillarin dissolves proenzyme, and in making blood, fibrinoclase vigor increases, solution fibrin, consumes original Fg in vivo, makes
Its content is reduced；Hepatic necrosis that serious liver parenchyma lesion, such as a variety of causes cause, chronic hepatopathy late period etc. also may occur in which Fg's
Reduce.
Clinically Fg is mainly used in congenital treatment, acquired Fibrinogen reduction or deficiency disease, and major Liver is damaged
Wound, liver cirrhosis, disseminated inravascular coagulation, postpartum hemorrhage and the blood coagulation disorderss caused because of major operation, wound or internal hemorrhage etc..
Research shows the blood clotting factories material such as fibre rich proteinogen, the cryoprecipitate that 400ml whole bloods are obtained in cryoprecipitate
Middle Fibrinogen >=150mg.In today that blood plasma raw material is increasingly in short supply, human fibrinogen is prepared by cryoprecipitate, it is right
The comprehensive utilization of cryoprecipitate, saves rare blood plasma resource, improves the market competitiveness and is significant.
In prior art, traditional fibre proteinogen preparation technology in S/D inactivation it is previous as do not carry out the gel adsorption removal of impurity,
Such as patent of invention《The production method of human fibrinogen》（Application publication number：101703763A）Deng, or use DEAE
Sephadex A50 gel adsorptions, such as patent of invention《Platelet cofactor Ⅰ, Fibrinogen and fiber are prepared by cryoprecipitate and combines egg
White method》（Application publication number：102295696A）.Patent of invention is found only at present《People is extracted from plasma component precipitation to coagulate
Blood factor VIII and the technique of human fibrinogen》（Application publication number：103351432 A）Using gel aluminum hydroxide, and hydrogen-oxygen
It is 3g/Kg to change aluminum adding proportion.Mechanical strength of the DEAE Sephadex A50 gels in swelling state is poor, in column chromatography
Middle volume and flow velocity can change.
Secondly, conventional preparation techniques are typically only with fine after the glycine sedimentation method or chilled alcohol precipitation method purification chromatography
Fibrillarin stock solution, such as《The technique for extracting human blood coagulation factors VIII and human fibrinogen from plasma component precipitation》（Shen Qing Publication
Number：103351432 A）、《The method that platelet cofactor Ⅰ, Fibrinogen and Fn Fiberonectin are prepared by cryoprecipitate》（Application
Publication No.：102295696A）Fibrinogen is obtained using two step glycine sedimentation method purification；《The preparation method of Fibrinogen》
（Application publication number：102286095A）Using plasma supernatant of the two step chilled alcohol precipitation method purification Jing after inactivation.
Moreover, the problems such as should further be appreciated that dissolubility in preparation technology and redissolve the time.Dissolving is difficult even to dissolve
After have a large amount of Denatured proteins to separate out, caused by Fibrinogen activation.Fibrinogen is easy to activation, especially exists
Ca2+In the presence of thrombin, thrombin will activate fibroblast cells monomer, the latter by water soluble Fibrinogen originally
It is aggregated, and in Ca2+In the presence of form water-fast fibrin, fibrin twists together agglomerating again, formed Fibrin Glue,
Therefore, filtration difficulty is easily caused in technique preparation process and the product redissolution time is long.
It can be seen that, existing human fibrinogen still suffers from some problems：（1）Purity is not high, and product impurity content is on the high side,
Easily there is the untoward reaction such as disseminated inravascular coagulation, erythra, tachycardia, heating in Clinical practice；（2）Dissolubility is low, makes
Used time redissolves overlong time, is not convenient to use, particularly in emergency situations.A large amount of Denatured proteins are had even after some dissolvings
Separate out.Most of product redissolves the time at 20 minutes or so, and some even up to 30 minutes or so；（3）Yield is not high, particularly
Human fibrinogen is extracted from cryoprecipitate.
At present, people's fiber of new high-purity and highly dissoluble is developed still in short situation in Fibrinogen market
Proteinogen extraction process seems particularly necessary.
The content of the invention
It is an object of the invention to provide a kind of high-purity, redissolve the time it is short extract the useless of platelet cofactor Ⅰ from cryoprecipitate
The preparation technology of human fibrinogen is extracted in material.
The major technique design of the present invention is as follows：
The present invention 2% gel aluminum hydroxide of optimization addition before S/D inactivation of virus is adsorbed；
The present invention is to the mono- step glycine sedimentation method of protein liquid Jing Jing after chromatography except fibrin monomer, fibrin are combined
After thing and fibrin lysate, then bis- step cold ethanol method purification of Jing, remove impurity protein, it is ensured that product purity is further carried
It is high；
The newly-increased addition AT- III inactivations thrombin of the present invention, EDTA remove Ca2+, centrifugation remove fibrin monomer impurity etc.
Step, to obtain high-purity, redissolution time short human fibrinogen.
The preparation technology of the present invention, it includes successively：Cryoprecipitate dissolving, the absorption of 2% gel aluminum hydroxide, regulation ion are strong
Degree, cascade filtration, S/D inactivation of virus, ion-exchange chromatography；Ion-exchange chromatography must collect eluent, eluent Jing ultrafiltration, thoroughly
Analysis, filtration, for the production of blood coagulation factor VIII, then the waste material for extracting platelet cofactor Ⅰ from cryoprecipitate collects Jing chromatographic column streams
Human fibrinogen is extracted in liquid out；Include successively：EDTA removes Ca2+, glycine precipitation, first time cold ethanol sink
Form sediment, AT- III inactivates thrombin, second chilled alcohol precipitation, nano-film filtration and xeothermic inactivation.
The present invention is achieved like this, and its concrete technology scheme is as follows：
（1）, quarantine after qualified human plasma gets quanrantine, 75% ethanol plasma bags surface is rushed with water for injection
Wash, be merged into and melt in slurry tank, melted with less than 30～35 DEG C recirculated waters, not higher than 4 DEG C of blood plasma temperature control；After melting, from
The heart, goes out liquid temp and controls at 0 ~ 4 DEG C, collect cryoprecipitate；
（2）, by step（1）Prepared thing add 3IU/ml heparin sodium aquas, stir to cryoprecipitate and be completely dissolved, circulate
The temperature control of water is at 20～28 DEG C；Start centrifugation, collect supernatant, weigh；
（3）, by step（2）Prepared thing supernatant with 0.5mol/L HCL adjust PH to 6.6～7.2；Add 2% hydrogen-oxygen
Change alumina gel, stirring；Start centrifugation, collect supernatant, weigh；
（4）, by " W（Adjust ionic strength buffer liquid）=supernatant weight/19 " calculate, and weigh the regulation ion of amount of calculation
Strength buffer is to step（3）Prepared thing supernatant in；Adjust supernatant PH to 6.8～7.5；Use regulatory protein concentration buffer
Liquid regulatory protein concentration is not more than 15.0g/L；Filter after filter element series connection with the filter element and 0.45um of 1.0um, collect filtrate,
Weigh；Adjust the compound method of ionic strength buffer liquid：48.5g trishydroxymethylaminomethane, 1.7g calcium chloride, 69.0g chlorinations
Sodium, 28.1g glycine, plus appropriate water for injection fully dissolve, and benefit injects water to 1L, and it is 6.8～7.2 to adjust PH；Adjust
The compound method of protein concentration buffer：Measure 0.05L and adjust ionic strength buffer liquid, inject water to 1L, adjusting PH is
6.4～6.8；
（5）, by step（4）Prepared thing filtrate volume 1/10 addition S/D solution, stir, temperature control exists
24～26 DEG C, continuous insulation 6 hours；0.45um filter element filterings；Filtrate is concentrated into protein concentration 1.5% with 100KD ultrafilter membranes,
Then with more than 2 times lavation buffer solution constant volume ultrafiltration of protein liquid weight, ultrafiltrate is obtained, is weighed；The preparation of lavation buffer solution
Method：2.5g trishydroxymethylaminomethane, 1.5g calcium chloride, 14.5g Sodium Chloride, plus appropriate water for injection fully dissolve, and add
To 1L, it is 6.4～6.8 to adjust PH to water for injection；
（6）, by step（5）Prepared thing ultrafiltrate carry out chromatography purification with ion exchange column, wash pillar with cleaning mixture
Until into baseline, with elution, collecting eluent（Eluent Jing ultrafiltration, dialysis, filtration, for blood coagulation factor VIII
Production）；The liquid that Jing chromatographic column effluents come is collected, is weighed；Add appropriate 0.01mol/L ethylenediaminetetraacetic acid（EDTA）Liquid
Dissolving, gentle agitation 10 hours, at 2～4 DEG C, PH is controlled 7.1～7.3 temperature control；Centrifugation, collects supernatant；Eluting delays
Rush the compound method of liquid：2.4g trishydroxymethylaminomethane, 0.09g calcium chloride, 40.9g Sodium Chloride, plus appropriate water for injection fill
Dissolving, benefit is divided to inject water to 1L, it is 6.4～6.8 to adjust PH；
（7）, in step（6）In the liquid of collection, add appropriate glycine, make whole glycine concentration be 6%, temperature control
At -1～-3 DEG C, precipitation is collected in stirring centrifugation；During precipitation adds 10 times of amount sodium citrate, sodium chloride buffer, stirring and dissolving is about
30min is compressed filtration, collects filtrate, by filtrate greenhouse cooling to 0～1 DEG C；Plus less than -15 DEG C of 95% ethanol, make ethanol
Final concentration of 8%（V/V）, at -1～-3 DEG C, after adding ethanol, pH value is 6.95～7.15 to temperature control；Stirring 30 minutes；From
The heart, goes out liquid temp and controls at -1～-3 DEG C, collect centrifuged deposit, weigh in centrifugal process；Sodium citrate, sodium chloride buffer
Compound method：14g sodium citrate, 9g Sodium Chloride, plus water for injection fully dissolves in right amount, benefit injects water to 1L, adjusts
PH is 7.00～7.10；
（8）, measure 10 times amount steps（7）Described sodium citrate, sodium chloride buffer, add AT- III to reach 1mU/ml, will
Step（7）Prepared thing precipitation add lysate, stirring and dissolving about 30min is compressed filtration；Filtrate is collected, by filtrate temperature
Degree is cooled to 0～1 DEG C；Plus less than -15 DEG C of 95% ethanol, make ethanol final concentration of 8%（V/V）, temperature control is -1～-3
DEG C, after adding ethanol, pH value is 6.95～7.15；Stirring 30 minutes；Centrifugation, goes out liquid temp control -1～-3 in centrifugal process
DEG C, centrifuged deposit is collected, is weighed；
（9）, by step（8）Prepared thing precipitation add 5 times of amount sodium citrate, Sodium Chloride, arginine hydrochloride buffer,
Stirring and dissolving is compressed filtration；Collect filtrate, metering；Dilute to match somebody with somebody, adjustment protein content is 2.0～3.0, and adjustment pH is 7.0
±0.1；Nano-film filtration（35nm）, collect filtrate；Compound method in sodium citrate, Sodium Chloride, arginine hydrochloride buffer：
15.5 ± 0.5g sodium citrate, 8.5g Sodium Chloride, 45g arginine hydrochloride, plus water for injection fully dissolves in right amount, adds injection
To 1L, it is 6.90～7.10 to adjust PH to water；
（10）, by step（9）0.2 μm of degerming filter element filtering subpackage of prepared thing filtrate Jing；The incoming jelly of the good product of subpackage
Dry cabinet carries out lyophilization；Offer for sale Zha Gai；99～100 DEG C, 30 minutes xeothermic inactivation of virus；Censorship, packs after the assay was approved, enters
Storehouse；
In addition to having and limiting, remaining is mass percent to the percent.
The positive effect of the present invention：
1st, in today that blood plasma raw material is increasingly in short supply, human fibrinogen is prepared by cryoprecipitate, to the comprehensive of cryoprecipitate
Close and utilize, improve the market competitiveness and be significant, on the other hand also can the rare blood plasma resource of indirect saving.
2nd, during ion-exchange chromatography purification Fibrinogen, use appropriate eluent（2.4g trihydroxy methyl amino
Methane, 0.09g calcium chloride, 40.9g Sodium Chloride, plus appropriate water for injection fully dissolve, and benefit injects water to 1L, adjust PH and are
6.4～6.8）The solution for eluting, then the operation such as Jing ultrafiltration, dialysis, filtration, can be used to produce platelet cofactor Ⅰ.
Preparation technology of the present invention compared to traditional fibre proteinogen, innovative point is：
1、Ca2+In the case of presence, thrombinogen can be catalyzed and become thrombin, Fibrinogen is activated into by thrombinFiber Albumen, cause filtration difficulty and redissolution time long.For this purpose, it is solidifying to increase the inactivations of AT- III before second chilled alcohol precipitation of the invention newly
Hemase and ion exchange loading flow through liquid addition EDTA and remove Ca2+Technique, and Jing centrifugations remove the impurity such as fibrin monomer, with
Obtain high-purity, redissolve time short human fibrinogen, quickly can dissolve at room temperature, meet the need of clinically emergency.This
Invented technology, human fibrinogen's redissolve 30min or so of the time by traditional handicraft, shorten within 15min.
2nd, in order to ensure Product Safety, before S/D inactivations, 2% gel aluminum hydroxide of optimization addition is adsorbed；Except S/
Outside D and xeothermic inactivation, in it is dilute with rear, degerming subpackage before increase nanometer film DV35 newly and cross and filter virus.
3rd, to Jing chromatography after the mono- step glycine sedimentation method of protein liquid Jing except fibrin monomer, fibrin complex and
After fibrin lysate, then bis- step cold ethanol method purification of Jing, remove impurity protein, it is ensured that product purity is further carried
Height, reduces adverse reaction rate.
4th, the present invention by test cold ethanol precipitation purifying fibrinogen below -15 DEG C of two step when, ethanol is optimum eventually
Concentration is 8%（V/V）.
The inventive method prepare product with《Chinese Pharmacopoeia》（Version in 2010, three）Described in human fibrinogen exist
The contrast of Key Quality Indicator such as table 1 below.
Table 1
| Project | The present invention | 《Chinese Pharmacopoeia》（Version in 2010, three） |
| Purity | Answer >=85.0% | It is not less than 70.0% |
| The redissolution time | Should be completely dissolved in 15 minutes | 30～37 DEG C of sterilizeds water for injection are added, is shaken gently for, should be completely dissolved in 30 minutes |
| Heat stability | 57 ± 0.5 DEG C 4 hours, perusal should be without gelation or floccule | 60 minutes are incubated in redissolving rearmounted 30～37 DEG C of water-baths, should be without grumeleuse or fibrin deposition. |
| Solidification vigor | Should be less than 55 seconds | Secondary measurement result meansigma methodss should be less than 60 seconds. |
| Osmotic pressure molar density | 240～1000mOsmol/kg | 240mOsmol/kg should be not less than |
In a word, the present invention increases nano-film filtration newly except virus in order to ensure safety in addition to S/D and xeothermic inactivation；Newly
Increase the inactivation thrombins of AT- III and EDTA removes Ca2+Technique, effectively prevents Fibrinogen in process of production from activating into fiber egg
In vain；The fibrin monomer and polymer removed in product is precipitated with glycine, to obtain highly purified human fibrinogen；Institute
Obtain formulation products safe and reliable, the redissolution time is short, meet the need of clinically emergency.For the comprehensive utilization of cryoprecipitate, save indirectly
About rare blood plasma resource is significant.
Description of the drawings
Fig. 1 is present invention process flow chart.
Specific embodiment
The present invention can be so that the invention will be further described, however, the scope of the present invention is simultaneously by the following examples
It is not limited to following embodiments.
Embodiment 1：By taking 20000 liters of blood plasma as an example, concrete preparation technology is as follows：
（1）, quarantine after qualified human plasma gets quanrantine, 75% ethanol plasma bags surface is rushed with water for injection
Wash, be merged into and melt in slurry tank, melted with less than 30～35 DEG C recirculated waters, not higher than 4 DEG C of blood plasma temperature control；After melting, from
The heart, goes out liquid temp and controls at 0～4 DEG C, collect to obtain cryoprecipitate 136.9kg；
（2）, by step（1）Prepared thing cryoprecipitate add 3IU/ml heparin sodium aquas, stir completely molten to cryoprecipitate
Solution, the temperature control of recirculated water is at 20～28 DEG C；Start centrifugation, collect supernatant, weigh to obtain 507.8kg；
（3）, by step（2）Prepared thing supernatant with 0.5mol/L HCL adjust PH to 6.6～7.2；Plus 2% hydroxide
Alumina gel, stirring；Start centrifugation, collect supernatant, weigh to obtain 540.2kg；
（4）, by " W（Adjust ionic strength buffer liquid）=supernatant weight/19 " calculate, and weigh the regulation ion of amount of calculation
Strength buffer is to step（3）Prepared thing supernatant in；Adjust supernatant PH to 6.8～7.5；Use regulatory protein concentration buffer
Liquid regulatory protein concentration is not more than 15.0g/L；Filter after filter element series connection with the filter element and 0.45um of 1.0um, collect filtrate,
Weigh to obtain 568.7kg；
（5）, by step（4）Prepared thing filtrate volume 1/10 addition S/D solution, stir, temperature control exists
24～26 DEG C, continuous insulation 6 hours；0.45um filter element filterings；Filtrate is concentrated into protein concentration about with 100KD ultrafilter membranes
1.5%, then with more than 2 times lavation buffer solution constant volume ultrafiltration of protein liquid weight, i.e. ultrafiltrate, weigh to obtain 611.6L；
（6）, by step（5）Prepared thing ultrafiltrate carry out chromatography purification with ion exchange column, wash pillar with cleaning mixture
Until into baseline, with elution, collecting eluent（Eluent Jing ultrafiltration, dialysis, filtration, for blood coagulation factor VIII
Production）；The liquid that Jing chromatographic column effluents come is collected, is weighed；Add appropriate 0.01mol/L ethylenediaminetetraacetic acid（EDTA）Liquid
Dissolving, gentle agitation 10 hours, at 2～4 DEG C, PH is controlled 7.1～7.3 temperature control；Centrifugation, collects supernatant, weighs
614.4L；
（7）, in step（6）In the liquid of collection, add appropriate glycine, make whole glycine concentration be 6%, temperature control
At -1～-3 DEG C, precipitation is collected in stirring centrifugation；During precipitation adds 10 times of amount sodium citrate, sodium chloride buffer, stirring and dissolving is about
30min is compressed filtration, collects filtrate, by filtrate greenhouse cooling to 0～1 DEG C；Plus less than -15 DEG C of 95% ethanol, make ethanol
Final concentration of 8%（V/V）, at -1～-3 DEG C, after adding ethanol, pH value is 7.00～7.10 to temperature control；Stirring 30 minutes；From
The heart, goes out liquid temp and controls at -1～-3 DEG C, collect centrifuged deposit, and weigh to obtain 31.5kg；
（8）, measure 10 times amount steps（8）Described sodium citrate, sodium chloride buffer, add AT- III to reach 1mU/ml, will
Step（7）Prepared thing precipitation add lysate, stirring and dissolving about 30min is compressed filtration；Filtrate is collected, by filtrate temperature
Degree is cooled to 0～1 DEG C；Plus less than -15 DEG C of 95% ethanol, make ethanol final concentration of 8%（V/V）, temperature control is -1～-3
DEG C, after adding ethanol, pH value is 7.00～7.10；Stirring 30 minutes；Centrifugation, goes out liquid temp and controls at -1～-3 DEG C, collect centrifugation
Postprecipitation, weigh to obtain 23.1kg；
（9）, by step（8）Prepared thing precipitation add 5 times of amount sodium citrate, Sodium Chloride, arginine hydrochloride buffer,
Stirring and dissolving is compressed filtration；Filtrate is collected, 135L is measured to obtain；Dilute to match somebody with somebody, adjustment protein content is 2.0～3.0, adjusts pH
For 7.0 ± 0.1；Nano-film filtration（35nm）, filtrate is collected, 173.2L is weighed to obtain；
（10）, by step（9）0.2 μm of degerming filter element filtering subpackage of prepared thing filtrate Jing, subpackage loading amount is every bottle of 25mL,
Subpackage quantity is 6765 bottles；The incoming lyophilizing cabinet of the good product of subpackage carries out lyophilization；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；
In addition to having and limiting, remaining is mass percent to the percent.
The verification result of the product that Jing the present embodiment is prepared is referring to table 2 below～table 6.
Table 2：Redissolve timing
Table 3, osmotic pressure molar density is determined
Table 4：Stability test
Table 5, solidification vitality test
Table 6：Purity and Fibrinogen total amount are determined
1st, sample protein matter content is determined：
2nd, sample solidifiable protein content determination：Take sample after redissolution10.0 Ml, plus physiological sodium chloride is diluted to50
Ml, takes sample after dilution5.0 Ml, plus physiological sodium chloride5.0 Ml, adds per ml thrombin solutions containing 3IU（Containing 0.05mmol/
L calcium chloride）10.0 ml.Mix.Put 37 DEG C of water-baths 20 Minute, separating coagulated protein, then by coagulated protein access digestive tube.
3rd, result is calculated：
3.1 Fibrinogen purity（%）=solidifiable protein content/protein content × 100
=2.16% /2.54 ×100=85.1%
3.2 Fibrinogen total amounts（G/ bottles）=solidifiable protein content × sample marker volume of dissolution
=2.16% × 25=0.5g/ bottles.
Claims (6)
1. a kind of to extract the preparation technology for extracting human fibrinogen in the waste material of platelet cofactor Ⅰ from cryoprecipitate, its feature exists
In preparation technology is as follows：
（1）, collect cryoprecipitate；
（2）, by step（1）Cryoprecipitate add 3IU/ml heparin sodium aquas in, stir to cryoprecipitate and be completely dissolved, recirculated water
Temperature control is at 20～28 DEG C；Start centrifugation, collect supernatant, weigh；
（3）, by step（2）Prepared thing supernatant with 0.5mol/L HCL adjust PH to 6.6～7.2；Add 2% aluminium hydroxide
Gel, stirring；Start centrifugation, collect supernatant, weigh；
（4）, calculate by " adjust ionic strength buffer liquid W=supernatant weight/19 ", the regulation ionic strength for weighing amount of calculation is delayed
Liquid is rushed to step（3）Prepared thing supernatant in；Adjust supernatant PH to 6.8～7.5；Adjusted with regulatory protein concentration buffer liquid
Protein concentration, protein concentration are finally adjusted to concentration value for no more than 15.0g/L；With the filter of the filter element and 0.45um of 1.0um
Core cascade filtration, collects filtrate, weighs；
（5）, by step（4）Prepared thing filtrate volume 1/10 addition S/D solution, stir, temperature control 24～
26 DEG C, continuous insulation 6 hours；0.45um filter element filterings；Filtrate is concentrated into protein concentration 1.5% with 100KD ultrafilter membranes, then
With more than 2 times lavation buffer solution constant volume ultrafiltration of protein liquid weight, ultrafiltrate is obtained, is weighed；
（6）, by step（5）Prepared thing ultrafiltrate carry out chromatography purification with ion exchange column, washed with cleaning mixture pillar until
Into baseline, with elution, eluent is collected, eluent Jing ultrafiltration, dialysis, filtration, for the production of blood coagulation factor VIII；
The waste material that platelet cofactor Ⅰ is extracted from cryoprecipitate again extracts human fibrin in collecting the liquid that Jing chromatographic column effluents come
It is former；The liquid that Jing chromatographic column effluents come is weighed；Add appropriate 0.01mol/L ethylenediaminetetraacetic acid（EDTA）Liquid dissolves, slightly
Stirring 10 hours, at 2～4 DEG C, PH is controlled 7.1～7.3 temperature control；Centrifugation, collects supernatant；
（7）, in step（6）In the liquid of collection, adding appropriate glycine, whole glycine concentration being made for 6%, temperature control is -1
～-3 DEG C, precipitation is collected in stirring centrifugation；During precipitation adds the sodium citrate of 10 times of weight portions, sodium chloride buffer, stirring and dissolving
30min is compressed filtration, collects filtrate, by filtrate greenhouse cooling to 0～1 DEG C；Plus less than -15 DEG C of 95% ethanol, make ethanol
Final concentration V/V is 8%, and at -1～-3 DEG C, after adding ethanol, pH value is 6.95～7.15 to temperature control；Stirring 30 minutes；Centrifugation,
Go out liquid temp to control at -1～-3 DEG C in centrifugal process, collect centrifuged deposit, weigh；
（8）, measure 10 times of weight portion steps（7）Described sodium citrate, sodium chloride buffer, add the inactivation thrombins of AT- III
So that final III valence values of AT- of solution are 1mU/ml, by step（7）Prepared thing precipitation add lysate, stirring and dissolving
30min is compressed filtration, collects filtrate, by filtrate greenhouse cooling to 0～1 DEG C；Plus less than -15 DEG C of 95% ethanol, make ethanol
Final concentration V/V is 8%, and at -1～-3 DEG C, after adding ethanol, pH value is 6.95～7.15 to temperature control；Stirring 30 minutes；Centrifugation,
Go out liquid temp to control at -1～-3 DEG C in centrifugal process, collect centrifuged deposit, weigh；
（9）, by step（8）Prepared thing precipitation add the sodium citrate of 5 times of weight portions, Sodium Chloride, arginine hydrochloride buffer
In, stirring and dissolving is compressed filtration；Collect filtrate, metering；Dilute to match somebody with somebody, adjustment protein content is 2.0～3.0, and adjustment pH is
7.0±0.1；35nm nano-film filtrations, collect filtrate；
（10）, by step（9）0.2 μm of degerming filter element filtering subpackage of prepared thing filtrate Jing；The incoming lyophilizing cabinet of the good product of subpackage
Carry out lyophilization；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；
In addition to having and limiting, remaining is mass percent to the percent.
2. a kind of extraction from cryoprecipitate according to claim 1 extract in the waste material of platelet cofactor Ⅰ human fibrinogen's
Preparation technology, it is characterised in that step（4）The compound method of described regulation ionic strength buffer liquid：48.5g trihydroxy methyl ammonia
Methylmethane, 1.7g calcium chloride, 69.0g Sodium Chloride, 28.1g glycine, plus appropriate water for injection fully dissolve, and add water for injection
To 1L, it is 6.8～7.2 to adjust PH；The compound method of the regulatory protein concentration buffer liquid：Measure 0.05L and adjust ionic strength
Buffer, injects water to 1L, and it is 6.4～6.8 to adjust PH.
3. a kind of extraction from cryoprecipitate according to claim 1 extract in the waste material of platelet cofactor Ⅰ human fibrinogen's
Preparation technology, it is characterised in that step（5）The compound method of described lavation buffer solution：2.5g trishydroxymethylaminomethane,
1.5g calcium chloride, 14.5g Sodium Chloride, plus appropriate water for injection fully dissolve, and benefit injects water to 1L, adjust PH be 6.4～
6.8。
4. according to a kind of system for extracting extraction human fibrinogen in the waste material of platelet cofactor Ⅰ from cryoprecipitate described in claim 1
Standby technique, it is characterised in that step（6）The compound method of described eluent：2.4g trishydroxymethylaminomethane, 0.09g chlorine
Change calcium, 40.9g Sodium Chloride, plus appropriate water for injection fully to dissolve, benefit injects water to 1L, it is 6.4～6.8 to adjust PH.
5. a kind of extraction from cryoprecipitate according to claim 1 extract in the waste material of platelet cofactor Ⅰ human fibrinogen's
Preparation technology, it is characterised in that step（7）Described sodium citrate, the compound method of sodium chloride buffer：14g sodium citrate,
9g Sodium Chloride, plus water for injection fully dissolves in right amount, benefit injects water to 1L, and it is 7.00～7.10 to adjust PH.
6. a kind of extraction from cryoprecipitate according to claim 1 extract in the waste material of platelet cofactor Ⅰ human fibrinogen's
Preparation technology, it is characterised in that step（9）Described sodium citrate, Sodium Chloride, the compound method of arginine hydrochloride buffer：
15.5 ± 0.5g sodium citrate, 8.5g Sodium Chloride, 45g arginine hydrochloride, plus water for injection fully dissolves in right amount, adds injection
To 1L, it is 6.90～7.10 to adjust PH to water.
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| 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 |
| KR101841587B1 (en) * | 2016-01-12 | 2018-05-14 | 주식회사 녹십자홀딩스 | Purification of 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 |
| 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 |
| 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 |
| 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 |
| CN113754757B (en) * | 2021-07-01 | 2024-06-14 | 华兰生物工程股份有限公司 | Preparation method of human fibrinogen |
| 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 |
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