CN102212129B - Method for extracting human fibrinogen from component I through column chromatography - Google Patents
Method for extracting human fibrinogen from component I through column chromatography Download PDFInfo
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Abstract
The invention relates to a method for extracting human fibrinogen from a component I through column chromatography. The problem of low purity, high stability and high yield of the human fibrinogen can be effectively solved. The method comprises the following steps of: (1) dissolving and filtering FI precipitate; (2) inactivating S/D ; (3) precipitating glycine for the first time; (4) dissolving and filtering the precipitate obtained in the precipitation for the first time; (5) performing column chromatography; (6) precipitating glycine for the second time; (7) dissolving and filtering the precipitate obtained in the precipitation for the second time; (8) precipitating; (9) packing the product in bags and freeze-drying the product; and (10) inactivating the product through hot air. In the method, glycine serves as a precipitator, so the finally obtained product contains about 0.5 percent of glycine; and the glycine and arginine hydrochloride are combined to serve as a stabilizer of the product, so the stability of freeze-dried product can be improved and the quality, the purity and the yield of the final product are improved. Therefore, the method is an innovation for preparing the human fibrinogen.
Description
One, technical field
The present invention relates to field of biological pharmacy, particularly a kind of method of extracting human fibrinogen from component I through column chromatography.
Two, background technology
Human fibrinogen (Human Fibrinogen) is human blood coagulation I, is one of major ingredient of plasma proteins, and molecular weight is about 340,000, and iso-electric point 5.5 is heated to 57 ℃ of very fast sex change.Its content in blood plasma is high, is about 2-4g/L, is the main determining factor of blood plasma viscosity, and liver is synthetic fine former main place.
Human fibrinogen (Fg) can improve rapidly fibrinogenic content in blood, by the effect of zymoplasm, makes Fibrinogen be converted into insoluble scleroproein, and the formed elements such as blood cell clot that congeals in network blood reaches the purpose of hemostasis.The blood coagulation disorders that the fibrinogenopenia that is used for the treatment of postpartum hemorrhage and causes because of major operation, wound or internal hemorrhage etc. causes, and thorax, belly and urinary tract etc. are large-scale operating hemorrhage etc., also effective to treating haemophiliachemophiliac complication.
Because fibrinogen content in blood plasma is very abundant, and the fibrinogen molecule amount is very large, so Fibrinogen is separating-purifying from blood plasma more easily, and this product clinical demand amount is also larger, so separation and purification human fibrinogen, not only improve the comprehensive utilization of valuable blood plasma resource, greater significance is also arranged simultaneously aspect medical and health.
The human fibrinogen be existing national standard blood products, be embodied in version in 2010 " in three ones of Chinese pharmacopoeia, clinical use is for many years at home for this medicine, its production technique is comparative maturity, it is 200810046747.5 and the number of patent application human fibrinogen's that is 200910237204.6 production method that number of patent application is also arranged in recent years, but at present domestic and international most of blood products producer all adopts cold ethanol method to take blood plasma as raw material basically, extract the human fibrinogen from the component I of Cohn6 method, the purity that this method major defect is goods is lower, general 70～80%, outward appearance is poor, quality stability is poor.Also there is report to extract the human fibrinogen from cryoprecipitate, because the fibrinogenic content in cryoprecipitate is lower, the product ultimate yield is not high from FI, and cryoprecipitate is the starting raw material as human blood coagulation factor VII I, if for the production of the human fibrinogen, certainly will reduce the turnout of human blood coagulation factor VII I, yield is low.Therefore, human fibrinogen's preparation method improvement and bring new ideas urgently.
Three, summary of the invention
For above-mentioned situation, for overcoming the defect of prior art, purpose of the present invention just is to provide a kind of method of extracting human fibrinogen from component I through column chromatography, can effectively solve human fibrinogen's purity high, good stability, the problem that yield is high.
The technical scheme that the present invention solves is to be realized by following steps:
(1) component I resolution of precipitate and filtration: the first lysate of 5～15 times of volumes for precipitation, 20-26 ℃ of stirring and dissolving 1 hour, the goods clarification filtration after dissolving, measure product volume;
(2) S/D deactivation: products temperature is controlled to 24～26 ℃, adds the ratio of 100ml in every liter of filtrate, slowly add the 11%S/D solution of mass concentration under whipped state, keep 24～26 ℃ of products temperatures 6 hours;
(3) glycine precipitates for the first time: in metrology steps 2, deactivation liquid is long-pending, add the glycine powder by 1.5～2.2mol/L concentration, after fully dissolving, be cooled to 2～8 ℃, carry out continuously centrifuged with the speed of 4200rpm and separate 40 minutes, 0 ℃ of design temperature, collecting precipitation;
(4) dissolving of primary sedimentation and filtration: the second lysate of 5～10 times of volumes for precipitation, 20-26 ℃ of stirring and dissolving 1 hour, filter collection filtrate after dissolving;
(5) column chromatography: the DEAE-650M gel is first used the citrate buffer balance, then the filtrate upper prop in step (4) is carried out to chromatography, collects effluent liquid, after chromatography finishes, by the citrate buffer detergent gel of 3 times of column volumes, is incorporated in effluent liquid;
(6) glycine precipitates for the second time: the chromatography effluent liquid adds the glycine powder by 1.5～2.2mol/L concentration, after fully dissolving, is cooled to 2～8 ℃, carries out continuously centrifuged with whizzer with the speed of 4200rpm and separates 40 minutes ,-5 ℃ of collecting precipitations;
(7) dissolving of secondary sedimentation and filtration: the 3rd lysate of 3～5 times of volumes for precipitation, room temperature is dissolved 1 hour, after dissolving, filters;
(8) preparation: detect and filter rear goods protein content, prepared with the 3rd lysate according to protein content, making protein content in end article is 25g/L;
(9) packing and freeze-drying: after preparation, the goods degerming is sub-packed in the 50ml vial, 25ml/ bottle, freeze-drying after packing;
(10) xeothermic deactivation: in step 9 after freeze-drying goods be placed in water-bath, keep 100 ℃ of bath temperatures, 30 minutes.
The method is adopt column chromatography technology and extract the human fibrinogen in conjunction with the glycine precipitator method from component I (FI), the glycine precipitator method that adopt in this production technique are than traditional cold ethanol precipitator method mild condition, can guarantee the activity of goods and the stability of goods, added DEAE-650M gel adsorption process, other thrombin of carrying secretly in can adsorption production process, further ensured the stable of goods, made raw material with component I and can improve every liter of blood plasma human fibrinogen yield.This method adopts glycine as precipitation agent, makes in end article to be entrained with the glycine of 0.5% left and right, and glycine is combined with arginine hydrochloride as the goods stablizer, can strengthen the stability after the goods freeze-drying.So this production technique can improve the quality of goods, the yield of purity, stability and the finished product, be preparation human fibrinogen's innovation.
Four, accompanying drawing explanation
Fig. 1 is preparation technology's schema of the present invention.
Fig. 2 is the Inactivation Dynamics graphic representation of S/D method of the present invention to PRV.
The Inactivation Dynamics graphic representation that Fig. 3 is S/D method treatment S indbis of the present invention.
Fig. 4 is the Inactivation Dynamics graphic representation of dry heating method inactivation of virus of the present invention to EMC.
Five, embodiment
Below in conjunction with process flow sheet, the specific embodiment of the present invention is elaborated.
As shown in Figure 1, the present invention is realized by following steps in concrete enforcement:
(1) component I resolution of precipitate and filtration: the first lysate of 5～15 times of volumes for precipitation, 20-26 ℃ of stirring and dissolving 1 hour, the goods clarification filtration after dissolving, measure product volume; Described the first lysate is: in every 1000ml aqueous solution for injection, contain: 10～14g trisodium citrate, 9gNaCl, 8～12g sucrose, 2～4gTris (Tutofusin tris), 3～5g lysine hydrochloride, adjust pH value: 6.90-7.10 with HCl;
(2) S/D deactivation: products temperature is controlled to 24～26 ℃, adds the ratio of 100ml in every liter of filtrate, slowly add the 11%S/D inactivator of mass concentration under whipped state, keep 24～26 ℃ of products temperatures 6 hours; Described 11%S/D solution is to contain in the 1000ml aqueous solution for injection: 110g tween-80,33g tributyl phosphate; Described S/D inactivator, use organic solvent/stain remover mixture (S/D) to destroy the adipose membrane of enveloped virus, once destroy the virus of adipose membrane, can not with cells infected, be combined, this method is the deactivation lipid-coated virus effectively again, but can not the non-enveloped virus of deactivation;
(3) glycine precipitates for the first time: in metrology steps 2, deactivation liquid is long-pending, add the glycine powder by 1.5～2.2mol/L concentration, after fully dissolving, be cooled to 2～8 ℃, carry out continuously centrifuged with the speed of 4200rpm and separate 40 minutes, 0 ℃ of design temperature, collecting precipitation;
(4) dissolving of primary sedimentation and filtration: the second lysate of 5～10 times of volumes for precipitation, 20-26 ℃ of stirring and dissolving 1 hour, filter collection filtrate after dissolving; Described the second lysate is to contain in the 1000ml aqueous solution for injection: 5～10g trisodium citrate adds HCl and adjusts pH value: 6.90-7.10;
(5) column chromatography: the DEAE-650M gel is first used the citrate buffer balance, again the filtrate upper prop in step (4) is carried out to chromatography, collect effluent liquid, chromatography finishes the rear citrate buffer detergent gel with 3 times of column volumes, be incorporated in effluent liquid, silica gel column chromatography moving phase is water;
(6) glycine precipitates for the second time: the chromatography effluent liquid adds the glycine powder by 1.5～2.2mol/L concentration, after fully dissolving, is cooled to 2～8 ℃, carries out continuously centrifuged with whizzer with the speed of 4200rpm and separates 40 minutes ,-5 ℃ of collecting precipitations;
(7) dissolving of secondary sedimentation and filtration: the 3rd lysate of 3～5 times of volumes for precipitation, room temperature is dissolved 1 hour, after dissolving, filters; Described the 3rd lysate is to contain in the 1000ml aqueous solution for injection: the Sodium Citrate of 14～16.5g, and 30～55g arginine hydrochloride, the sodium-chlor of 7～9g, adjust pH value of solution 6.9～7.1 with HCl or NaOH;
(8) preparation: detect and filter rear goods protein content, prepared with the 3rd lysate according to protein content, making protein content in end article is 25g/L;
(9) packing and freeze-drying: after preparation, the goods degerming is sub-packed in the 50ml vial, 25ml/ bottle, freeze-drying after packing;
(10) xeothermic deactivation: in step 9 after freeze-drying goods be placed in water-bath, keep 100 ℃ of bath temperatures, 30 minutes.
Clarification filtration alleged in described step (1) adopts the filter plate clarification filtration, and its filter plate aperture is 0.7 μ m～1.0 μ m;
In described step (3) and step (6), the concentration of glycine is 1.5～2.2mol/L.
The present invention, in concrete enforcement, also can be provided by following examples:
Embodiment 1:
The present invention is realized by following steps in concrete enforcement:
(1) component I resolution of precipitate and filtration: the first lysate of 5 times of volumes for precipitation, 20 ℃ of stirring and dissolving 1 hour, the goods clarification filtration after dissolving, measure product volume; Described the first lysate is: in every 1000ml aqueous solution for injection, contain: the 10g trisodium citrate, and 9g NaCl, 8g sucrose, 2g Tris, the 3g lysine hydrochloride, adjusting the pH value with HCl is 6.90;
(2) S/D deactivation: products temperature is controlled to 24 ℃, adds the ratio of 100ml in every liter of filtrate, slowly add the 11%S/D inactivator of mass concentration under whipped state, keep 24 ℃ of products temperatures 6 hours;
(3) glycine precipitates for the first time: in metrology steps 2, deactivation liquid is long-pending, by 1.5mol/L concentration, adds the glycine powder, after fully dissolving, is cooled to 2 ℃, carries out continuously centrifuged with the speed of 4200rpm and separates 40 minutes, 0 ℃ of collecting precipitation;
(4) dissolving of primary sedimentation and filtration: the second lysate of 5 times of volumes for precipitation, 20 ℃ of stirring and dissolving 1 hour, filter collection filtrate after dissolving; Described the second lysate is to contain in the 1000ml aqueous solution for injection: the 5g trisodium citrate, and adding HCl adjustment pH value is 6.90;
(5) column chromatography: the DEAE-650M gel is first used the citrate buffer balance, then the filtrate upper prop in step (4) is carried out to chromatography, collects effluent liquid, after chromatography finishes, by the citrate buffer detergent gel of 3 times of column volumes, is incorporated in effluent liquid;
(6) glycine precipitates for the second time: the chromatography effluent liquid adds the glycine powder by 1.5mol/L concentration, after fully dissolving, is cooled to 2 ℃, carries out continuously centrifuged with whizzer with the speed of 4200rpm and separates 40 minutes ,-5 ℃ of collecting precipitations;
(7) dissolving of secondary sedimentation and filtration: the 3rd lysate of 3 times of volumes for precipitation, room temperature is dissolved 1 hour, after dissolving, filters; Described the 3rd lysate is to contain in the 1000ml aqueous solution for injection: the Sodium Citrate of 14g, and the 30g arginine hydrochloride, the sodium-chlor of 7g, adjusting pH value of solution with HCl or NaOH is 6.9;
(8) preparation: detect and filter rear goods protein content, prepared with the 3rd lysate according to protein content, making protein content in end article is 25g/L;
(9) packing and freeze-drying: after preparation, the goods degerming is sub-packed in the 50ml vial, 25ml/ bottle, freeze-drying after packing;
(10) xeothermic deactivation: in step 9 after freeze-drying goods be placed in water-bath, keep 100 ℃ of bath temperatures, 30 minutes.
Clarification filtration alleged in described step (1) adopts the filter plate clarification filtration, and its filter plate aperture is 0.7 μ m.
Embodiment 2:
(1) component I resolution of precipitate and filtration: the first lysate of 8 times of volumes for precipitation, 23 ℃ of stirring and dissolving 1 hour, the goods clarification filtration after dissolving, measure product volume; Described the first lysate is: in every 1000ml aqueous solution for injection, contain: the 12g trisodium citrate, and 9gNaCl, 10g sucrose, 3g Tris, the 4g lysine hydrochloride, adjusting the pH value with HCl is 7;
(2) S/D deactivation: products temperature is controlled to 25 ℃, adds the ratio of 100ml in every liter of filtrate, slowly add the 11%S/D inactivator of mass concentration under whipped state, keep 25 ℃ of products temperatures 6 hours;
(3) glycine precipitates for the first time: in metrology steps 2, deactivation liquid is long-pending, by 1.8mol/L concentration, adds the glycine powder, after fully dissolving, is cooled to 5 ℃, carries out continuously centrifuged with the speed of 4200rpm and separates 40 minutes, 0 ℃ of collecting precipitation;
(4) dissolving of primary sedimentation and filtration: the second lysate of 7 times of volumes for precipitation, 23 ℃ of stirring and dissolving 1 hour, filter collection filtrate after dissolving; Described the second lysate is to contain in the 1000ml aqueous solution for injection: the 7.5g trisodium citrate, and adding HCl adjustment pH value is 7;
(5) column chromatography: the DEAE-650M gel is first used the citrate buffer balance, then the filtrate upper prop in step (4) is carried out to chromatography, collects effluent liquid, after chromatography finishes, by the citrate buffer detergent gel of 3 times of column volumes, is incorporated in effluent liquid;
(6) glycine precipitates for the second time: the chromatography effluent liquid adds the glycine powder by 1.8mol/L concentration, after fully dissolving, is cooled to 5 ℃, carries out continuously centrifuged with whizzer with the speed of 4200rpm and separates 40 minutes ,-5 ℃ of collecting precipitations;
(7) dissolving of secondary sedimentation and filtration: the 3rd lysate of 4 times of volumes for precipitation, room temperature is dissolved 1 hour, after dissolving, filters; Described the 3rd lysate is to contain in the 1000ml aqueous solution for injection: the Sodium Citrate of 15.2g, and the 43g arginine hydrochloride, the sodium-chlor of 8g, adjusting pH value of solution with HCl or NaOH is 7;
(8) preparation: detect and filter rear goods protein content, prepared with the 3rd lysate according to protein content, making protein content in end article is 25g/L;
(9) packing and freeze-drying: after preparation, the goods degerming is sub-packed in the 50ml vial, 25ml/ bottle, freeze-drying after packing;
(10) xeothermic deactivation: in step 9 after freeze-drying goods be placed in water-bath, keep 100 ℃ of bath temperatures, 30 minutes.
Clarification filtration alleged in described step (1) adopts the filter plate clarification filtration, and its filter plate aperture is 0.85 μ m.
Embodiment 3:
(1) component I resolution of precipitate and filtration: the first lysate of 15 times of volumes for precipitation, 26 ℃ of stirring and dissolving 1 hour, the goods clarification filtration after dissolving, measure product volume; Described the first lysate is: in every 1000ml aqueous solution for injection, contain: the 14g trisodium citrate, and 9g NaCl, 12g sucrose, 4g Tris, the 5g lysine hydrochloride, adjusting the pH value with HCl is 7.10;
(2) S/D deactivation: products temperature is controlled to 26 ℃, adds the ratio of 100ml in every liter of filtrate, slowly add the 11%S/D inactivator of mass concentration under whipped state, keep 26 ℃ of products temperatures 6 hours;
(3) glycine precipitates for the first time: in metrology steps 2, deactivation liquid is long-pending, by 2.2mol/L concentration, adds the glycine powder, after fully dissolving, is cooled to 8 ℃, carries out continuously centrifuged with the speed of 4200rpm and separates 40 minutes, 0 ℃ of collecting precipitation;
(4) dissolving of primary sedimentation and filtration: the second lysate of 10 times of volumes for precipitation, 26 ℃ of stirring and dissolving 1 hour, filter collection filtrate after dissolving; Described the second lysate is to contain in the 1000ml aqueous solution for injection: the 10g trisodium citrate, and adding HCl adjustment pH value is 7.10;
(5) column chromatography: the DEAE-650M gel is first used the citrate buffer balance, then the filtrate upper prop in step (4) is carried out to chromatography, collects effluent liquid, after chromatography finishes, by the citrate buffer detergent gel of 3 times of column volumes, is incorporated in effluent liquid;
(6) glycine precipitates for the second time: the chromatography effluent liquid adds the glycine powder by 2.2mol/L concentration, after fully dissolving, is cooled to 8 ℃, carries out continuously centrifuged with whizzer with the speed of 4200rpm and separates 40 minutes ,-5 ℃ of collecting precipitations;
(7) dissolving of secondary sedimentation and filtration: the 3rd lysate of 5 times of volumes for precipitation, room temperature is dissolved 1 hour, after dissolving, filters; Described the 3rd lysate is to contain in the 1000ml aqueous solution for injection: the Sodium Citrate of 16.5g, and the 55g arginine hydrochloride, the sodium-chlor of 9g, adjusting pH value of solution with HCl or NaOH is 7.1;
(8) preparation: detect and filter rear goods protein content, prepared with the 3rd lysate according to protein content, making protein content in end article is 25g/L;
(9) packing and freeze-drying: after preparation, the goods degerming is sub-packed in the 50ml vial, 25ml/ bottle, freeze-drying after packing;
(10) xeothermic deactivation: in step 9 after freeze-drying goods be placed in water-bath, keep 100 ℃ of bath temperatures, 30 minutes;
Clarification filtration alleged in described step (1) adopts the filter plate clarification filtration, and its filter plate aperture is 1.0 μ m.
Product of the present invention met or exceeded fully after tested " related request and the domestic like product gone on the market of Chinese pharmacopoeia 2010 version (three ones), the comparative analysis of related data sees the following form:
Product of the present invention and domestic list marketing quality product be data (specification: the 0.5g/ bottle) relatively
As can be seen from the above table, the prepared human fibrinogen of the present invention, its Key Quality Indicator as purity, solidify vigor, redissolution time, outward appearance all over the " product of Chinese pharmacopoeia and domestic other company of having gone on the market.
S/D method inactivation of virus compliance test result:
S/D inactivation of virus of the present invention and xeothermic inactivation of virus all verified, the result is as follows:
After the S/D method that the present invention adopts is processed, the decline titre of indicator virus is as follows:
The inactivating efficacy of S/D method of the present invention to PRV:
S/D method of the present invention to the Inactivation Dynamics curve of PRV as shown in Figure 2.
After the present invention adopts the S/D method to process to the inactivating efficacy of Sindbis:
The Inactivation Dynamics curve of S/D method treatment S indbis of the present invention as shown in Figure 3.
Result shows, S/D method inactivation of virus of the present invention is through 25 ± 1 ℃ of water bath processing after 1 hour, the decline titre of indicator virus PRV and Sindbis has been greater than 4lg, therefore think effectively these two kinds of indicator viruses of deactivation of this method (25 ± 1 ℃ water bath processing 6 hours).
Dry heating method inactivation of virus compliance test result:
Xeothermic inactivation of virus of the present invention verifies, the result is as follows:
The inactivating efficacy of the dry heating method inactivation of virus that the present invention adopts to EMC:
The dry heating method inactivation of virus that the present invention adopts to the Inactivation Dynamics curve of EMC as shown in Figure 4.
Result shows, dry heating method of the present invention (100 ℃ 30 minutes) viral inactivation treatment effective deactivation EMC virus just after 20 minutes, the validity of this method inactivation of virus effect of sufficient proof.
By above-mentioned situation, shown, the present invention adopts the absorption of DEAE-650M gel, and effectively assorted thrombin in absorbent articles, make goods more stable.
This inhale to adopt the glycine precipitator method to carry out the separation of protein, and the separation condition gentleness can better keep activity and the purity of protein.
The present invention be take component I as starting raw material, can keep the yield of the finished product.
The present invention be take the waste products component I of producing human serum albumin and is carried out human fibrinogen's production as raw material, its preparation process is: add 8% (V/V) ethanol in blood plasma, the pH value is adjusted into: 7.10 ± 0.10, outlet temperature :-2.5 ± 0.5 ℃, by the centrifuging and taking precipitation, both obtained, supernatant is for producer's seralbumin.So both improved the comprehensive utilization of raw blood plasma, and prevented the wasting of resources and, to the pollution of environment, can provide again the medicine of more high-qualitys, alleviate the situation that domestic raw blood plasma is in short supply, economic benefit of the present invention and social benefit are remarkable.
Claims (3)
1. the method for an extracting human fibrinogen from component I through column chromatography, is characterized in that, is to be realized by following steps:
(1) component I resolution of precipitate and filtration: the first lysate of 5～8 times of volumes for precipitation, 20-26 ℃ of stirring and dissolving 1 hour, the goods clarification filtration after dissolving, measure product volume; Described the first lysate is: in every 1000ml aqueous solution for injection, contain: 10～14g trisodium citrate, and 9g NaCl, 8～12g sucrose, 2～4gTris, 3～5g lysine hydrochloride, adjust pH value: 6.90-7.10 with HCl;
(2) S/D deactivation: products temperature is controlled to 24～26 ℃, adds the ratio of 100ml in every liter of filtrate, slowly add the 11%S/D inactivator of mass concentration under whipped state, keep 24～26 ℃ of products temperatures 6 hours; Described 11%S/D inactivator is to contain in the 1000ml aqueous solution for injection: 110g tween-80,33g tributyl phosphate;
(3) glycine precipitates for the first time: in metrology steps (2), deactivation liquid is long-pending, add the glycine powder by 1.5～2.2mol/L concentration, after fully dissolving, be cooled to 2～8 ℃, carry out continuously centrifuged with the speed of 4200rpm and separate 40 minutes, 0 ℃ of design temperature, collecting precipitation;
(4) dissolving of primary sedimentation and filtration: the second lysate of 5～7 times of volumes for precipitation, 20-26 ℃ of stirring and dissolving 1 hour, filter collection filtrate after dissolving; Described the second lysate is to contain in the 1000ml aqueous solution for injection: 5～10g trisodium citrate adds HCl and adjusts pH value: 6.90-7.10;
(5) column chromatography: the DEAE-650M gel is first used the citrate buffer balance, then the filtrate upper prop in step (4) is carried out to chromatography, collects effluent liquid, after chromatography finishes, by the citrate buffer detergent gel of 3 times of column volumes, is incorporated in effluent liquid;
(6) glycine precipitates for the second time: the chromatography effluent liquid adds the glycine powder by 1.5～2.2mol/L concentration, after fully dissolving, is cooled to 2～8 ℃, carries out continuously centrifuged with whizzer with the speed of 4200rpm and separates 40 minutes ,-5 ℃ of collecting precipitations;
(7) dissolving of secondary sedimentation and filtration: the 3rd lysate of 3～5 times of volumes for precipitation, room temperature is dissolved 1 hour, after dissolving, filters; Described the 3rd lysate is to contain in the 1000ml aqueous solution for injection: the Sodium Citrate of 14～16.5g, and 30～55g arginine hydrochloride, the sodium-chlor of 7～9g, adjust pH value of solution 6.9～7.1 with HCl or NaOH;
(8) preparation: detect and filter rear goods protein content, prepared with the 3rd lysate according to protein content, making protein content in end article is 25g/L;
(9) packing and freeze-drying: after preparation, the goods degerming is sub-packed in the 50ml vial, 25ml/ bottle, freeze-drying after packing;
(10) xeothermic deactivation: after the middle freeze-drying of step (9), goods are placed in water-bath, keep 100 ℃ of bath temperatures, 30 minutes;
Clarification filtration alleged in described step (1) adopts the filter plate clarification filtration, and its filter plate aperture is 0.7 μ m～1.0 μ m.
2. the method for extracting human fibrinogen from component I through column chromatography according to claim 1, is characterized in that, by described following steps, realized:
(1) component I resolution of precipitate and filtration: the first lysate of 5 times of volumes for precipitation, 20 ℃ of stirring and dissolving 1 hour, the goods clarification filtration after dissolving, measure product volume; Described the first lysate is: in every 1000ml aqueous solution for injection, contain: the 10g trisodium citrate, and 9g NaCl, 8g sucrose, 2g Tris, the 3g lysine hydrochloride, adjusting the pH value with HCl is 6.90;
(2) S/D deactivation: products temperature is controlled to 24 ℃, adds the ratio of 100ml in every liter of filtrate, slowly add the 11%S/D inactivator of mass concentration under whipped state, keep 24 ℃ of products temperatures 6 hours;
(3) glycine precipitates for the first time: in metrology steps (2), deactivation liquid is long-pending, by 1.5mol/L concentration, adds the glycine powder, after fully dissolving, is cooled to 2 ℃, with the speed of 4200rpm, carries out continuously centrifuged separation 40 minutes, 0 ℃ of collecting precipitation;
(4) dissolving of primary sedimentation and filtration: the second lysate of 5 times of volumes for precipitation, 20 ℃ of stirring and dissolving 1 hour, filter collection filtrate after dissolving; Described the second lysate is to contain in the 1000ml aqueous solution for injection: the 5g trisodium citrate, and adding HCl adjustment pH value is 6.90;
(5) column chromatography: the DEAE-650M gel is first used the citrate buffer balance, then the filtrate upper prop in step (4) is carried out to chromatography, collects effluent liquid, after chromatography finishes, by the citrate buffer detergent gel of 3 times of column volumes, is incorporated in effluent liquid;
(6) glycine precipitates for the second time: the chromatography effluent liquid adds the glycine powder by 1.5mol/L concentration, after fully dissolving, is cooled to 2 ℃, carries out continuously centrifuged with whizzer with the speed of 4200rpm and separates 40 minutes ,-5 ℃ of collecting precipitations;
(7) dissolving of secondary sedimentation and filtration: the 3rd lysate of 3 times of volumes for precipitation, room temperature is dissolved 1 hour, after dissolving, filters; Described the 3rd lysate is to contain in the 1000ml aqueous solution for injection: the Sodium Citrate of 14g, and the 30g arginine hydrochloride, the sodium-chlor of 7g, adjusting pH value of solution with HCl or NaOH is 6.9;
(8) preparation: detect and filter rear goods protein content, prepared with the 3rd lysate according to protein content, making protein content in end article is 25g/L;
(9) packing and freeze-drying: after preparation, the goods degerming is sub-packed in the 50ml vial, 25ml/ bottle, freeze-drying after packing;
(10) xeothermic deactivation: after the middle freeze-drying of step (9), goods are placed in water-bath, keep 100 ℃ of bath temperatures, 30 minutes;
Clarification filtration alleged in described step (1) adopts the filter plate clarification filtration, and its filter plate aperture is 0.7 μ m.
3. the method for extracting human fibrinogen from component I through column chromatography according to claim 1, is characterized in that, by described following steps, realized:
(1) component I resolution of precipitate and filtration: the first lysate of 8 times of volumes for precipitation, 23 ℃ of stirring and dissolving 1 hour, the goods clarification filtration after dissolving, measure product volume; Described the first lysate is: in every 1000ml aqueous solution for injection, contain: the 12g trisodium citrate, and 9gNaCl, 10g sucrose, 3g Tris, the 4g lysine hydrochloride, adjusting the pH value with HCl is 7;
(2) S/D deactivation: products temperature is controlled to 25 ℃, adds the ratio of 100ml in every liter of filtrate, slowly add the 11%S/D inactivator of mass concentration under whipped state, keep 25 ℃ of products temperatures 6 hours;
(3) glycine precipitates for the first time: in metrology steps (2), deactivation liquid is long-pending, by 1.8mol/L concentration, adds the glycine powder, after fully dissolving, is cooled to 5 ℃, with the speed of 4200rpm, carries out continuously centrifuged separation 40 minutes, 0 ℃ of collecting precipitation;
(4) dissolving of primary sedimentation and filtration: the second lysate of 7 times of volumes for precipitation, 23 ℃ of stirring and dissolving 1 hour, filter collection filtrate after dissolving; Described the second lysate is to contain in the 1000ml aqueous solution for injection: the 7.5g trisodium citrate, and adding HCl adjustment pH value is 7;
(5) column chromatography: the DEAE-650M gel is first used the citrate buffer balance, then the filtrate upper prop in step (4) is carried out to chromatography, collects effluent liquid, after chromatography finishes, by the citrate buffer detergent gel of 3 times of column volumes, is incorporated in effluent liquid;
(6) glycine precipitates for the second time: the chromatography effluent liquid adds the glycine powder by 1.8mol/L concentration, after fully dissolving, is cooled to 5 ℃, carries out continuously centrifuged with whizzer with the speed of 4200rpm and separates 40 minutes ,-5 ℃ of collecting precipitations;
(7) dissolving of secondary sedimentation and filtration: the 3rd lysate of 4 times of volumes for precipitation, room temperature is dissolved 1 hour, after dissolving, filters; Described the 3rd lysate is to contain in the 1000ml aqueous solution for injection: the Sodium Citrate of 15.2g, and the 43g arginine hydrochloride, the sodium-chlor of 8g, adjusting pH value of solution with HCl or NaOH is 7;
(8) preparation: detect and filter rear goods protein content, prepared with the 3rd lysate according to protein content, making protein content in end article is 25g/L;
(9) packing and freeze-drying: after preparation, the goods degerming is sub-packed in the 50ml vial, 25ml/ bottle, freeze-drying after packing;
(10) xeothermic deactivation: after the middle freeze-drying of step (9), goods are placed in water-bath, keep 100 ℃ of bath temperatures, 30 minutes;
Clarification filtration alleged in described step (1) adopts the filter plate clarification filtration, and its filter plate aperture is 0.85 μ m.
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| 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 |
| 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 |
| CN112354002A (en) * | 2020-11-12 | 2021-02-12 | 广东深蓝生物科技有限公司 | Hemostatic sealant and preparation method thereof |
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