CN102212129A - 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 column chromatography extracts human fibrinogen's method from component I.
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 height in blood plasma 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 fibrinogenic content in the blood rapidly, by the effect of zymoplasm, makes Fibrinogen be converted into insoluble scleroproein, and formed elements such as blood cell clot that congeals into reaches the hematostatic purpose in the network blood.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 is very abundant in the blood plasma, and the fibrinogen molecule amount is very big, so more easily separating from blood plasma, Fibrinogen purifies, and this product clinical demand amount is also bigger, so separation and purification human fibrinogen, not only improve the comprehensive utilization of valuable blood plasma resource, big meaning is also arranged aspect medical and health simultaneously.
The human fibrinogen be existing national standard blood products, be embodied in version in 2010 " in three ones of the Chinese pharmacopoeia, the clinical at home use of this medicine for many years, its production technique is comparative maturity, in recent years also have number of patent application be 200810046747.5 and number of patent application be 200910237204.6 human fibrinogen's production method, but it is raw material with blood plasma that at present domestic and international most of blood products producer all adopts cold ethanol method basically, from the component I of Cohn6 method, extract the human fibrinogen, the purity that this method major defect is goods is lower, general 70～80%, outward appearance is relatively poor, and quality stability is poor.Also there is report from cryoprecipitate, to extract the human fibrinogen, because the fibrinogenic content in the cryoprecipitate is lower, the product ultimate yield is not from the FI height, and cryoprecipitate is the starting raw material as human blood coagulation factor VII I, if be used to produce the human fibrinogen, certainly will reduce the turnout of human blood coagulation factor VII I, yield is low.Therefore, human fibrinogen's preparation method demands urgently improving and innovation.
Three, summary of the invention
At above-mentioned situation, for overcoming the defective of prior art, purpose of the present invention just provides a kind of column chromatography extracts the human fibrinogen from component I method, can effectively solve human fibrinogen's purity height, 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: precipitation is with first lysate of 5～15 times of volumes, 20-26 ℃ of stirring and dissolving 1 hour, and the goods clarification filtration after the dissolving measures product volume;
(2) S/D deactivation: products temperature is controlled at 24～26 ℃, adds the ratio of 100ml, under whipped state, slowly add the 11%S/D solution of mass concentration, kept 24～26 ℃ of products temperatures 6 hours in every liter of filtrate;
(3) glycine precipitates for the first time: deactivation liquid is long-pending in the metrology steps 2, add the glycine powder by 1.5～2.2mol/L concentration, fully dissolve postcooling to 2～8 ℃, carry out continuously centrifuged with the speed of 4200rpm and separated 40 minutes, 0 ℃ of design temperature, collecting precipitation;
(4) dissolving of primary sedimentation and filtration: precipitation is with second lysate of 5～10 times of volumes, 20-26 ℃ of stirring and dissolving 1 hour, and dissolving after-filtration, collection filtrate;
(5) column chromatography: the DEAE-650M gel is used the citrate buffer balance earlier, again the filtrate upper prop in the step (4) is carried out chromatography, collects effluent liquid, and chromatography finishes the citrate buffer detergent gel of back with 3 times of column volumes, incorporates in the effluent liquid;
(6) glycine precipitation for the second time: the chromatography effluent liquid adds the glycine powder by 1.5～2.2mol/L concentration, fully dissolves postcooling to 2～8 ℃, carries out continuously centrifuged separation 40 minutes ,-5 ℃ of collecting precipitations with whizzer with the speed of 4200rpm;
(7) dissolving of secondary sedimentation and filtration: precipitation was dissolved 1 hour with the 3rd lysate of 3～5 times of volumes, room temperature, after the dissolving, filtered;
(8) preparation: detect to filter back goods protein content, prepare with the 3rd lysate, make that protein content is 25g/L in the end article according to protein content;
(9) packing and freeze-drying: the goods degerming of preparation back is sub-packed in the 50ml vial 25ml/ bottle, freeze-drying after the packing;
(10) xeothermic deactivation: in the step 9 after the freeze-drying goods place water-bath, keep 100 ℃ of bath temperatures, 30 minutes.
This method is to 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 the glycine that is entrained with in the end article about 0.5%, and glycine and arginine hydrochloride are united as the goods stablizer, can strengthen the stability after the goods freeze-drying.So this production technique can improve Products Quality, the yield of purity, stability and the finished product is innovations of preparation human fibrinogen.
Four, description of drawings
Fig. 1 is preparation technology's schema of the present invention.
Fig. 2 is the deactivation kinetic curve figure of S/D method of the present invention to PRV.
Fig. 3 is the deactivation kinetic curve figure of S/D method treatment S indbis of the present invention.
Fig. 4 is the deactivation kinetic curve figure 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.
By shown in Figure 1, the present invention is realized by following steps in concrete enforcement:
(1) component I resolution of precipitate and filtration: precipitation is with first lysate of 5～15 times of volumes, 20-26 ℃ of stirring and dissolving 1 hour, and the goods clarification filtration after the dissolving measures product volume; Described first lysate is: contain in every 1000ml aqueous solution for injection: 10～14g trisodium citrate, and 9gNaCl, 8～12g sucrose, 2～4gTris (Tutofusin tris), 3～5g lysine hydrochloride is adjusted pH value: 6.90-7.10 with HCl;
(2) S/D deactivation: products temperature is controlled at 24～26 ℃, adds the ratio of 100ml, under whipped state, slowly add the 11%S/D inactivator of mass concentration, kept 24～26 ℃ of products temperatures 6 hours in every liter of filtrate; 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 promptly uses organic solvent/stain remover mixture (S/D) to destroy the adipose membrane of enveloped virus, can not combine with cells infected in case destroy the virus of adipose membrane again, and this method is the deactivation lipid-coated virus effectively, but can not the non-enveloped virus of deactivation;
(3) glycine precipitates for the first time: deactivation liquid is long-pending in the metrology steps 2, add the glycine powder by 1.5～2.2mol/L concentration, fully dissolve postcooling to 2～8 ℃, carry out continuously centrifuged with the speed of 4200rpm and separated 40 minutes, 0 ℃ of design temperature, collecting precipitation;
(4) dissolving of primary sedimentation and filtration: precipitation is with second lysate of 5～10 times of volumes, 20-26 ℃ of stirring and dissolving 1 hour, and dissolving after-filtration, collection filtrate; Described 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 used earlier the citrate buffer balance, again the filtrate upper prop in the step (4) is carried out chromatography, collect effluent liquid, chromatography finishes the citrate buffer detergent gel of back with 3 times of column volumes, incorporate in the effluent liquid, silica gel column chromatography moving phase is water;
(6) glycine precipitation for the second time: the chromatography effluent liquid adds the glycine powder by 1.5～2.2mol/L concentration, fully dissolves postcooling to 2～8 ℃, carries out continuously centrifuged separation 40 minutes ,-5 ℃ of collecting precipitations with whizzer with the speed of 4200rpm;
(7) dissolving of secondary sedimentation and filtration: precipitation was dissolved 1 hour with the 3rd lysate of 3～5 times of volumes, room temperature, after the dissolving, filtered; 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 is adjusted pH value of solution 6.9～7.1 with HCl or NaOH;
(8) preparation: detect to filter back goods protein content, prepare with the 3rd lysate, make that protein content is 25g/L in the end article according to protein content;
(9) packing and freeze-drying: the goods degerming of preparation back is sub-packed in the 50ml vial 25ml/ bottle, freeze-drying after the packing;
(10) xeothermic deactivation: in the step 9 after the freeze-drying goods place water-bath, keep 100 ℃ of bath temperatures, 30 minutes.
Clarification filtration alleged in the described step (1) adopts the filter plate clarification filtration, and its filter plate aperture is 0.7 μ m～1.0 μ m;
The concentration of glycine is 1.5～2.2mol/L in described step (3) and the step (6).
The present invention also can be provided by following examples in concrete enforcement:
Embodiment 1:
The present invention is realized by following steps in concrete enforcement:
(1) component I resolution of precipitate and filtration: precipitation is with first lysate of 5 times of volumes, 20 ℃ of stirring and dissolving 1 hour, and the goods clarification filtration after the dissolving measures product volume; Described first lysate is: contain in every 1000ml aqueous solution for injection: 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 at 24 ℃, adds the ratio of 100ml, under whipped state, slowly add the 11%S/D inactivator of mass concentration, kept 24 ℃ of products temperatures 6 hours in every liter of filtrate;
(3) glycine precipitation for the first time: deactivation liquid is long-pending in the metrology steps 2, press 1.5mol/L concentration and adds the glycine powder, fully dissolves postcooling to 2 ℃, carries out continuously centrifuged separation 40 minutes, 0 ℃ of collecting precipitation with the speed of 4200rpm;
(4) dissolving of primary sedimentation and filtration: precipitation is with second lysate of 5 times of volumes, 20 ℃ of stirring and dissolving 1 hour, and dissolving after-filtration, collection filtrate; Described 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 used the citrate buffer balance earlier, again the filtrate upper prop in the step (4) is carried out chromatography, collects effluent liquid, and chromatography finishes the citrate buffer detergent gel of back with 3 times of column volumes, incorporates in the effluent liquid;
(6) glycine precipitation for the second time: the chromatography effluent liquid press 1.5mol/L concentration and is added the glycine powder, fully dissolves postcooling to 2 ℃, carries out continuously centrifuged separation 40 minutes ,-5 ℃ of collecting precipitations with whizzer with the speed of 4200rpm;
(7) dissolving of secondary sedimentation and filtration: precipitation was dissolved 1 hour with the 3rd lysate of 3 times of volumes, room temperature, after the dissolving, filtered; 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 to filter back goods protein content, prepare with the 3rd lysate, make that protein content is 25g/L in the end article according to protein content;
(9) packing and freeze-drying: the goods degerming of preparation back is sub-packed in the 50ml vial 25ml/ bottle, freeze-drying after the packing;
(10) xeothermic deactivation: in the step 9 after the freeze-drying goods place water-bath, keep 100 ℃ of bath temperatures, 30 minutes.
Clarification filtration alleged in the 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: precipitation is with first lysate of 8 times of volumes, 23 ℃ of stirring and dissolving 1 hour, and the goods clarification filtration after the dissolving measures product volume; Described first lysate is: contain in every 1000ml aqueous solution for injection: 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 at 25 ℃, adds the ratio of 100ml, under whipped state, slowly add the 11%S/D inactivator of mass concentration, kept 25 ℃ of products temperatures 6 hours in every liter of filtrate;
(3) glycine precipitation for the first time: deactivation liquid is long-pending in the metrology steps 2, press 1.8mol/L concentration and adds the glycine powder, fully dissolves postcooling to 5 ℃, carries out continuously centrifuged separation 40 minutes, 0 ℃ of collecting precipitation with the speed of 4200rpm;
(4) dissolving of primary sedimentation and filtration: precipitation is with second lysate of 7 times of volumes, 23 ℃ of stirring and dissolving 1 hour, and dissolving after-filtration, collection filtrate; Described 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 used the citrate buffer balance earlier, again the filtrate upper prop in the step (4) is carried out chromatography, collects effluent liquid, and chromatography finishes the citrate buffer detergent gel of back with 3 times of column volumes, incorporates in the effluent liquid;
(6) glycine precipitation for the second time: the chromatography effluent liquid press 1.8mol/L concentration and is added the glycine powder, fully dissolves postcooling to 5 ℃, carries out continuously centrifuged separation 40 minutes ,-5 ℃ of collecting precipitations with whizzer with the speed of 4200rpm;
(7) dissolving of secondary sedimentation and filtration: precipitation was dissolved 1 hour with the 3rd lysate of 4 times of volumes, room temperature, after the dissolving, filtered; 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 to filter back goods protein content, prepare with the 3rd lysate, make that protein content is 25g/L in the end article according to protein content;
(9) packing and freeze-drying: the goods degerming of preparation back is sub-packed in the 50ml vial 25ml/ bottle, freeze-drying after the packing;
(10) xeothermic deactivation: in the step 9 after the freeze-drying goods place water-bath, keep 100 ℃ of bath temperatures, 30 minutes.
Clarification filtration alleged in the 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: precipitation is with first lysate of 15 times of volumes, 26 ℃ of stirring and dissolving 1 hour, and the goods clarification filtration after the dissolving measures product volume; Described first lysate is: contain in every 1000ml aqueous solution for injection: 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 at 26 ℃, adds the ratio of 100ml, under whipped state, slowly add the 11%S/D inactivator of mass concentration, kept 26 ℃ of products temperatures 6 hours in every liter of filtrate;
(3) glycine precipitation for the first time: deactivation liquid is long-pending in the metrology steps 2, press 2.2mol/L concentration and adds the glycine powder, fully dissolves postcooling to 8 ℃, carries out continuously centrifuged separation 40 minutes, 0 ℃ of collecting precipitation with the speed of 4200rpm;
(4) dissolving of primary sedimentation and filtration: precipitation is with second lysate of 10 times of volumes, 26 ℃ of stirring and dissolving 1 hour, and dissolving after-filtration, collection filtrate; Described 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 used the citrate buffer balance earlier, again the filtrate upper prop in the step (4) is carried out chromatography, collects effluent liquid, and chromatography finishes the citrate buffer detergent gel of back with 3 times of column volumes, incorporates in the effluent liquid;
(6) glycine precipitation for the second time: the chromatography effluent liquid press 2.2mol/L concentration and is added the glycine powder, fully dissolves postcooling to 8 ℃, carries out continuously centrifuged separation 40 minutes ,-5 ℃ of collecting precipitations with whizzer with the speed of 4200rpm;
(7) dissolving of secondary sedimentation and filtration: precipitation was dissolved 1 hour with the 3rd lysate of 5 times of volumes, room temperature, after the dissolving, filtered; 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 to filter back goods protein content, prepare with the 3rd lysate, make that protein content is 25g/L in the end article according to protein content;
(9) packing and freeze-drying: the goods degerming of preparation back is sub-packed in the 50ml vial 25ml/ bottle, freeze-drying after the packing;
(10) xeothermic deactivation: in the step 9 after the freeze-drying goods place water-bath, keep 100 ℃ of bath temperatures, 30 minutes;
Clarification filtration alleged in the 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 that has 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, is solidified vigor, redissolution time, outward appearance all above the " product of Chinese pharmacopoeia and domestic other company of having gone on the market at its Key Quality Indicator such as purity.
S/D method inactivation of virus compliance test result:
S/D inactivation of virus of the present invention and xeothermic inactivation of virus verify that all the result is as follows in checking:
After the S/D method that the present invention adopts was handled, the decline titre of indicator virus was as follows:
S/D method of the present invention is to the inactivating efficacy of PRV:
S/D method of the present invention is seen shown in Figure 2 to the deactivation kinetic curve of PRV.
The present invention adopts the S/D method to handle the inactivating efficacy of back to Sindbis:
The deactivation kinetic curve of S/D method treatment S indbis of the present invention is seen shown in Figure 3.
The 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 is greater than 4lg, so 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 that the result is as follows in checking:
The dry heating method inactivation of virus that the present invention adopts is to the inactivating efficacy of EMC:
The dry heating method inactivation of virus that the present invention adopts is seen shown in Figure 4 to the deactivation kinetic curve of EMC.
The result shows, dry heating method of the present invention (100 ℃ 30 minutes) viral inactivation treatment effective deactivation EMC virus just after 20 minutes fully proves the validity of this method inactivation of virus effect.
Shown that by above-mentioned situation the present invention adopts the absorption of DEAE-650M gel, effectively assorted thrombin in the absorbent articles makes goods more stable.
Inhale for this and adopt the glycine precipitator method to carry out proteinic separation, the separation condition gentleness can better keep activity of proteins and purity.
The present invention is starting raw material with the component I, can keep the yield of the finished product.
The present invention is the production that raw material carries out the human fibrinogen with the waste products component I of producing human serum albumin, its preparation process is: add 8% (V/V) ethanol in the blood plasma, the pH value is adjusted into: 7.10 ± 0.10, outlet temperature :-2.5 ± 0.5 ℃, after the centrifuging and taking precipitation both got, supernatant is used for producer's seralbumin.So both improved the comprehensive utilization of raw blood plasma, and prevented the wasting of resources and, can provide more fine medicines again, alleviated domestic raw blood plasma situation in short supply, economic benefit of the present invention and obvious social benefit the pollution of environment.
Claims (4)
1. a column chromatography extracts human fibrinogen's method from component I, it is characterized in that, be to realize by following steps:
(1) component I resolution of precipitate and filtration: precipitation is with first lysate of 5～15 times of volumes, 20-26 ℃ of stirring and dissolving 1 hour, and the goods clarification filtration after the dissolving measures product volume; Described first lysate is: contain in every 1000ml aqueous solution for injection: 10～14g trisodium citrate, and 9g NaCl, 8～12g sucrose, 2～4gTris, 3～5g lysine hydrochloride is adjusted pH value: 6.90-7.10 with HCl;
(2) S/D deactivation: products temperature is controlled at 24～26 ℃, adds the ratio of 100ml, under whipped state, slowly add the 11%S/D inactivator of mass concentration, kept 24～26 ℃ of products temperatures 6 hours in every liter of filtrate; 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: deactivation liquid is long-pending in the metrology steps 2, add the glycine powder by 1.5～2.2mol/L concentration, fully dissolve postcooling to 2～8 ℃, carry out continuously centrifuged with the speed of 4200rpm and separated 40 minutes, 0 ℃ of design temperature, collecting precipitation;
(4) dissolving of primary sedimentation and filtration: precipitation is with second lysate of 5～10 times of volumes, 20-26 ℃ of stirring and dissolving 1 hour, and dissolving after-filtration, collection filtrate; Described 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 used the citrate buffer balance earlier, again the filtrate upper prop in the step (4) is carried out chromatography, collects effluent liquid, and chromatography finishes the citrate buffer detergent gel of back with 3 times of column volumes, incorporates in the effluent liquid;
(6) glycine precipitation for the second time: the chromatography effluent liquid adds the glycine powder by 1.5～2.2mol/L concentration, fully dissolves postcooling to 2～8 ℃, carries out continuously centrifuged separation 40 minutes ,-5 ℃ of collecting precipitations with whizzer with the speed of 4200rpm;
(7) dissolving of secondary sedimentation and filtration: precipitation was dissolved 1 hour with the 3rd lysate of 3～5 times of volumes, room temperature, after the dissolving, filtered; 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 is adjusted pH value of solution 6.9～7.1 with HCl or NaOH;
(8) preparation: detect to filter back goods protein content, prepare with the 3rd lysate, make that protein content is 25g/L in the end article according to protein content;
(9) packing and freeze-drying: the goods degerming of preparation back is sub-packed in the 50ml vial 25ml/ bottle, freeze-drying after the packing;
(10) xeothermic deactivation: in the step 9 after the freeze-drying goods place water-bath, keep 100 ℃ of bath temperatures, 30 minutes.
Clarification filtration alleged in the described step (1) adopts the filter plate clarification filtration, and its filter plate aperture is 0.7 μ m～1.0 μ m;
The concentration of glycine is 1.5～2.2mol/L in described step (3) and the step (6).
2. column chromatography according to claim 1 extracts human fibrinogen's method from component I, it is characterized in that, is realized by described following steps:
(1) component I resolution of precipitate and filtration: precipitation is with first lysate of 5 times of volumes, 20 ℃ of stirring and dissolving 1 hour, and the goods clarification filtration after the dissolving measures product volume; Described first lysate is: contain in every 1000ml aqueous solution for injection: 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 at 24 ℃, adds the ratio of 100ml, under whipped state, slowly add the 11%S/D inactivator of mass concentration, kept 24 ℃ of products temperatures 6 hours in every liter of filtrate;
(3) glycine precipitation for the first time: deactivation liquid is long-pending in the metrology steps 2, press 1.5mol/L concentration and adds the glycine powder, fully dissolves postcooling to 2 ℃, carries out continuously centrifuged separation 40 minutes, 0 ℃ of collecting precipitation with the speed of 4200rpm;
(4) dissolving of primary sedimentation and filtration: precipitation is with second lysate of 5 times of volumes, 20 ℃ of stirring and dissolving 1 hour, and dissolving after-filtration, collection filtrate; Described 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 used the citrate buffer balance earlier, again the filtrate upper prop in the step (4) is carried out chromatography, collects effluent liquid, and chromatography finishes the citrate buffer detergent gel of back with 3 times of column volumes, incorporates in the effluent liquid;
(6) glycine precipitation for the second time: the chromatography effluent liquid press 1.5mol/L concentration and is added the glycine powder, fully dissolves postcooling to 2 ℃, carries out continuously centrifuged separation 40 minutes ,-5 ℃ of collecting precipitations with whizzer with the speed of 4200rpm;
(7) dissolving of secondary sedimentation and filtration: precipitation was dissolved 1 hour with the 3rd lysate of 3 times of volumes, room temperature, after the dissolving, filtered; 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 to filter back goods protein content, prepare with the 3rd lysate, make that protein content is 25g/L in the end article according to protein content;
(9) packing and freeze-drying: the goods degerming of preparation back is sub-packed in the 50ml vial 25ml/ bottle, freeze-drying after the packing;
(10) xeothermic deactivation: in the step 9 after the freeze-drying goods place water-bath, keep 100 ℃ of bath temperatures, 30 minutes.
Clarification filtration alleged in the described step (1) adopts the filter plate clarification filtration, and its filter plate aperture is 0.7 μ m.
3. column chromatography according to claim 1 extracts human fibrinogen's method from component I, it is characterized in that, is realized by described following steps:
(1) component I resolution of precipitate and filtration: precipitation is with first lysate of 8 times of volumes, 23 ℃ of stirring and dissolving 1 hour, and the goods clarification filtration after the dissolving measures product volume; Described first lysate is: contain in every 1000ml aqueous solution for injection: the 12g trisodium citrate, and 9g NaCl, 10g sucrose, 3g Tris, the 4g lysine hydrochloride, adjusting the pH value with HCl is 7;
(2) S/D deactivation: products temperature is controlled at 25 ℃, adds the ratio of 100ml, under whipped state, slowly add the 11%S/D inactivator of mass concentration, kept 25 ℃ of products temperatures 6 hours in every liter of filtrate;
(3) glycine precipitation for the first time: deactivation liquid is long-pending in the metrology steps 2, press 1.8mol/L concentration and adds the glycine powder, fully dissolves postcooling to 5 ℃, carries out continuously centrifuged separation 40 minutes, 0 ℃ of collecting precipitation with the speed of 4200rpm;
(4) dissolving of primary sedimentation and filtration: precipitation is with second lysate of 7 times of volumes, 23 ℃ of stirring and dissolving 1 hour, and dissolving after-filtration, collection filtrate; Described 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 used the citrate buffer balance earlier, again the filtrate upper prop in the step (4) is carried out chromatography, collects effluent liquid, and chromatography finishes the citrate buffer detergent gel of back with 3 times of column volumes, incorporates in the effluent liquid;
(6) glycine precipitation for the second time: the chromatography effluent liquid press 1.8mol/L concentration and is added the glycine powder, fully dissolves postcooling to 5 ℃, carries out continuously centrifuged separation 40 minutes ,-5 ℃ of collecting precipitations with whizzer with the speed of 4200rpm;
(7) dissolving of secondary sedimentation and filtration: precipitation was dissolved 1 hour with the 3rd lysate of 4 times of volumes, room temperature, after the dissolving, filtered; 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 to filter back goods protein content, prepare with the 3rd lysate, make that protein content is 25g/L in the end article according to protein content;
(9) packing and freeze-drying: the goods degerming of preparation back is sub-packed in the 50ml vial 25ml/ bottle, freeze-drying after the packing;
(10) xeothermic deactivation: in the step 9 after the freeze-drying goods place water-bath, keep 100 ℃ of bath temperatures, 30 minutes.
Clarification filtration alleged in the described step (1) adopts the filter plate clarification filtration, and its filter plate aperture is 0.85 μ m.
4. column chromatography according to claim 1 extracts human fibrinogen's method from component I, it is characterized in that, is realized by described following steps:
(1) component I resolution of precipitate and filtration: precipitation is with first lysate of 15 times of volumes, 26 ℃ of stirring and dissolving 1 hour, and the goods clarification filtration after the dissolving measures product volume; Described first lysate is: contain in every 1000ml aqueous solution for injection: 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 at 26 ℃, adds the ratio of 100ml, under whipped state, slowly add the 11%S/D inactivator of mass concentration, kept 26 ℃ of products temperatures 6 hours in every liter of filtrate;
(3) glycine precipitation for the first time: deactivation liquid is long-pending in the metrology steps 2, press 2.2mol/L concentration and adds the glycine powder, fully dissolves postcooling to 8 ℃, carries out continuously centrifuged separation 40 minutes, 0 ℃ of collecting precipitation with the speed of 4200rpm;
(4) dissolving of primary sedimentation and filtration: precipitation is with second lysate of 10 times of volumes, 26 ℃ of stirring and dissolving 1 hour, and dissolving after-filtration, collection filtrate; Described 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 used the citrate buffer balance earlier, again the filtrate upper prop in the step (4) is carried out chromatography, collects effluent liquid, and chromatography finishes the citrate buffer detergent gel of back with 3 times of column volumes, incorporates in the effluent liquid;
(6) glycine precipitation for the second time: the chromatography effluent liquid press 2.2mol/L concentration and is added the glycine powder, fully dissolves postcooling to 8 ℃, carries out continuously centrifuged separation 40 minutes ,-5 ℃ of collecting precipitations with whizzer with the speed of 4200rpm;
(7) dissolving of secondary sedimentation and filtration: precipitation was dissolved 1 hour with the 3rd lysate of 5 times of volumes, room temperature, after the dissolving, filtered; 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 to filter back goods protein content, prepare with the 3rd lysate, make that protein content is 25g/L in the end article according to protein content;
(9) packing and freeze-drying: the goods degerming of preparation back is sub-packed in the 50ml vial 25ml/ bottle, freeze-drying after the packing;
(10) xeothermic deactivation: in the step 9 after the freeze-drying goods place water-bath, keep 100 ℃ of bath temperatures, 30 minutes;
Clarification filtration alleged in the described step (1) adopts the filter plate clarification filtration, and its filter plate aperture is 1.0 μ m.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106800583A (en) * | 2015-11-26 | 2017-06-06 | 上海洲跃生物科技有限公司 | It is a kind of instant without the cryodesiccant human fibrinogen preparation technology for separating out |
| 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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| CN101229367A (en) * | 2008-01-21 | 2008-07-30 | 江西博雅生物制药股份有限公司 | Process of preparing human fibrinogen preparation |
| CN101703763A (en) * | 2009-11-05 | 2010-05-12 | 绿十字(中国)生物制品有限公司 | Production method of human fibrinogen |
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| CN101229367A (en) * | 2008-01-21 | 2008-07-30 | 江西博雅生物制药股份有限公司 | Process of preparing human fibrinogen preparation |
| CN101703763A (en) * | 2009-11-05 | 2010-05-12 | 绿十字(中国)生物制品有限公司 | Production method of human fibrinogen |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106800583A (en) * | 2015-11-26 | 2017-06-06 | 上海洲跃生物科技有限公司 | It is a kind of instant without the cryodesiccant human fibrinogen preparation technology for separating out |
| 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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| CN102212129B (en) | 2013-12-18 |
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