CN113214384B - Preparation method of canine fibrinogen and product thereof - Google Patents
Preparation method of canine fibrinogen and product thereof Download PDFInfo
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Abstract
The method for producing the canine fibrinogen adopts the method of extracting and separating the canine fibrinogen from the low-temperature ethanol component I, and the S/D method and the water bath heat treatment method adopted in the production are used for double virus inactivation, so that the lipid-coated viruses and the non-lipid-coated viruses can be effectively inactivated, and the safety of the product is ensured; the purity of the extracted product is up to more than 90%, the content of impurity protein is low, and the clinical side effect of the product is small. The invention has the following advantages: 1. the manufacturing method provided by the invention has high extraction efficiency. 2. The freeze-drying stabilizer used in the invention ensures that the temperature change of the product is stable in the freeze-drying process, the structure of the freeze-dried product is uniform, the stability of the fibrinogen of dogs is ensured, and the biological activity of the fibrinogen is protected; 3. in the technological process of the invention, the technological re-dissolution time of fibrinogen is shortened to about 10 minutes, thereby gaining precious time for rescuing the life of pets clinically.
Description
Technical Field
The invention relates to the field of canine blood products, in particular to a preparation method of canine fibrinogen.
Background
Fibrinogen (Fg) is a protein component in plasma, the content of which is up to 2-4 g/L, plays a key role in the canine blood coagulation system, and the final stage of blood coagulation is that Fg is converted into fibrin under the action of thrombin, and fibrin and other blood cell components are aggregated into insoluble aggregates to achieve the purpose of hemostasis. Fg plays an important role in platelet aggregation and blood viscosity besides directly participating in the coagulation process, and is an important cause of cardiovascular and cerebrovascular diseases. The Fg deficiency commonly associated diseases comprise congenital fibrinogen reduction or deficiency, and acquired fibrinogen deficiency caused by severe liver injury, liver cirrhosis, disseminated intravascular coagulation, trauma, postpartum hemorrhage, major surgery, internal hemorrhage and the like. Supplemental Fg is currently the only reliable treatment due to congenital factor induced reduction or deficiency, and for acquired reduction or deficiency due to different factors, infusion of different doses of Fg formulation is also required depending on the etiology.
At present, the raw materials for extracting the fibrinogen mainly comprise two components, namely, a component (FI) precipitate and a plasma cryoprecipitate, which are generated by a low-temperature ethanol process. Most blood product manufacturers at home and abroad basically use FI precipitation as a raw material for extracting fibrinogen. The F1 sediment mainly contains fibrinogen, FVIII, fibronectin Fn and other components. Cryoprecipitation is a white flocculent precipitate of fresh frozen plasma (Fresh frozen plasma, FFP) after thawing at low temperature, which is rich in fviii, von willebrand factor, fibrinogen, fibronectin and factor XIII. Simultaneously, cold precipitation is also a main starting material for the preparation of FVIII, and at present, processes for simultaneously extracting FVIII and fibrinogen from cold precipitation have been reported.
In the field of human medicine, fibrinogen and a composite preparation thereof are widely used, but at present, no fibrinogen preparation is applied to the field of pets, and canine fibrinogen produced by me company is prepared by taking low-temperature ethanol F1 precipitate as a raw material, purifying the fibrinogen by adopting the manufacturing processes of extraction, adsorption, centrifugation and the like and adopting the international advanced ion exchange chromatography technology, so that the purity of the fibrinogen product is greatly improved, and the fibrinogen is a pet disease such as: 1. congenital fibrinogen reduction or deficiency. 2. Reduced acquired fibrinogen, severe liver injury; cirrhosis of the liver; disseminated intravascular coagulation; the method provides an effective diagnosis and treatment means for postpartum hemorrhage and coagulation disorders caused by fibrinogen deficiency due to major surgery, trauma or internal hemorrhage and the like.
Disclosure of Invention
The invention aims to provide a preparation method of fibrinogen with high dissolution speed, short production period, quick redissolution during use, good biological activity and high yield in the production process.
In order to solve the technical problems, the invention provides a preparation method of fibrinogen, which sequentially comprises the following steps:
a method for preparing canine fibrinogen, which is characterized by comprising the following steps:
(1) Thawing qualified canine plasma, stirring to dissolve the plasma completely, adjusting the plasma temperature to-4-0 ℃, adjusting the pH value to 6.4-7.4, adding pre-cooled ethanol to a final concentration of 8% -10% (g/100 ml), and keeping the temperature at-4-0 ℃ and centrifuging at 13000-18000rpm to retain precipitate;
(2) Dissolving the precipitate with 8-10 times of dissolving solution, centrifuging with continuous centrifuge (kept at-4-0deg.C, 13000-18000 rpm) to obtain supernatant;
(3) Adding Tween-80 and TNBP into the obtained supernatant to make the final concentration of Tween-80 in the solution be 1% and the final concentration of TNBP be 0.3%, stirring the obtained liquid in a special virus inactivation tank at a low speed of 40-100 rmp, and preserving the temperature for 6-8 hours at 24-26 ℃;
(4) Chromatography: adsorbing the solution obtained in the step (3) by using a DEAE-650M gel chromatographic column, collecting filtrate passing through the chromatographic column, washing with citrate buffer solution with the volume of 3 times of gel volume after adsorption, and washing out the residual canine fibrinogen in the chromatographic column and collecting the residual canine fibrinogen;
(5) Adding glycine/sodium chloride into the liquid collected in the step (4), wherein the final concentration glycine is 0.9-1.5 mol/L; the sodium chloride is 1.5-2.5 mol/L. After glycine/sodium chloride is fully dissolved, cooling the solution to 0-10 ℃, and continuously centrifuging the solution at a speed of 2000-5000 rmp by using a centrifuge;
(6) Dissolving the precipitate obtained in the step (5) by using a dissolving solution, stirring for 2-6 hours to completely dissolve the precipitate, then adjusting the protein concentration to 2.0-3.0%, and adjusting the pH value of the product to 6.4-7.4 by using a 0.5M hydrochloric acid solution or a 0.5M sodium hydroxide solution;
(7) And (3) sterilizing and filtering: sterilizing and filtering the liquid obtained in the step (6) by using a sterilized filter;
(8) And (3) adding a freeze-drying protective agent into the secondary dissolved clear liquid obtained in the step (7), and freeze-drying to obtain a fibrinogen freeze-dried product.
(9) And (3) heat treatment: and (3) carrying out water bath heat treatment at 100+/-2 ℃ for 30 minutes on the product obtained in the step (8).
The method for producing the canine fibrinogen is characterized in that the step (1) is performed with centrifugation at 0-4 ℃.
The method for producing the canine fibrinogen is characterized in that in the step (1), the pH value of a dissolving solution is regulated to be 6.4-7.4 by using 0.1M acetic acid-sodium acetate buffer solution.
The preparation method of fibrinogen is characterized in that the concentration of cold ethanol is 50% -95% v/v.
The preparation method of fibrinogen is characterized in that the freeze-drying protective agent is composed of mannitol and dextran-40 glucose injection according to a specific dosage proportion, wherein the concentration of mannitol is 4% -15% w/v, and the concentration of dextran-40 glucose injection is 3% -8% w/v. The preparation method of fibrinogen is characterized in that the dissolving buffer solution contains 1.5-2.0 g/L sodium citrate, 6.0-8.0 g/L sodium chloride and 8.0-10.0 g/L glycine.
Definition of terms in connection with the present invention
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The invention has the advantages that:
1. the method for producing the canine fibrinogen adopts the method for extracting and separating the canine fibrinogen from the component I, and the S/D method and the water bath heat treatment method adopted in the production are used for double virus inactivation, so that the lipid-coated virus and the non-lipid-coated virus can be effectively inactivated, and the safety of the product is ensured; the purity of the extracted product is up to more than 90%, the content of impurity protein is low, and the clinical side effect of the product is small.
2. According to the canine fibrinogen, mannitol and dextran-40 glucose injection are adopted as the stabilizing agents in the production process, so that the temperature change of the product is stable in the freeze-drying process, the structure of the freeze-dried product is uniform, and the stability of the canine fibrinogen is ensured.
3. In the process of the invention, the process redissolution time of fibrinogen is shortened to about 10 minutes (about 37 ℃ water bath) from 2 hours (about 37 ℃ water bath) of the general process. The process redissolution time is shortened, and the probability of bacterial reproduction in the process preparation process is reduced, so that the pyrogen in the product is reduced, and valuable time is gained for rescuing the life of pets clinically;
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the invention without departing from the spirit and scope of the invention, but these modifications and substitutions are intended to be within the scope of the invention.
EXAMPLE 1 preparation of canine fibrinogen preparation
1. Preparation method
(1) 100L of canine plasma which is qualified in inspection is dissolved, the plasma is completely dissolved by stirring, the temperature of the plasma is regulated to 0 ℃, the pH value of the plasma is regulated to 6.4, precooled ethanol is added to a final concentration of 10% (g/100 ml), and the precipitate is reserved by centrifugation;
(2) Adding 8 times of dissolving solution into the precipitate for dissolving, and centrifuging by using a continuous centrifuge to obtain a supernatant;
(3) Adding Tween-80 and TNBP into the obtained supernatant to make the final concentration of Tween-80 in the solution be 1% and the final concentration of TNBP be 0.3%, stirring the obtained liquid in a special virus inactivation tank at a low speed of 40rmp, and preserving the temperature for 6 hours at 24-26 ℃;
(4) Chromatography: adsorbing the solution obtained in the step (3) by using a DEAE-650M gel chromatographic column, collecting filtrate passing through the chromatographic column, washing with citrate buffer solution with the volume of 3 times of gel volume after adsorption, and washing out the residual canine fibrinogen in the chromatographic column and collecting the residual canine fibrinogen;
(5) Adding glycine/sodium chloride into the liquid collected in the step (4), wherein the final concentration glycine is 0.9mol/L; sodium chloride is 1.5mol/L. After glycine/sodium chloride is fully dissolved, cooling the solution to 0 ℃, and continuously centrifuging the solution at a speed of 2000rmp by using a centrifuge;
(6) Dissolving the precipitate obtained in the step (5) by using a dissolving solution, stirring for 2 hours to completely dissolve the precipitate, then adjusting the protein concentration to 2.0-3.0%, and adjusting the pH value of the product to 6.4 by using a 0.5M hydrochloric acid solution or a 0.5M sodium hydroxide solution;
(7) And (3) sterilizing and filtering: sterilizing and filtering the liquid obtained in the step (6) by using a sterilized filter;
(8) And (3) adding a freeze-drying protective agent into the secondary dissolved clear liquid obtained in the step (7), and freeze-drying to obtain a fibrinogen freeze-dried product.
(9) And (3) heat treatment: and (3) carrying out water bath heat treatment at 100+/-2 ℃ for 30 minutes on the product obtained in the step (8).
The protein concentration, purity, clotting activity and the like of the prepared canine fibrinogen are measured according to the method specified in Chinese pharmacopoeia.
EXAMPLE 2 preparation of canine fibrinogen preparation
1. Preparation method
(1) 100L of canine plasma which is qualified in inspection is dissolved, the plasma is completely dissolved by stirring, the temperature of the plasma is regulated to-2 ℃, the pH value of the plasma is regulated to 7.0, precooled ethanol is added to a final concentration of 9% (g/100 ml), and the precipitate is reserved by centrifugation;
(2) Adding 9 times of dissolving solution into the precipitate for dissolving, and centrifuging by using a continuous centrifuge to obtain a supernatant;
(3) Adding Tween-80 and TNBP into the obtained supernatant to make the final concentration of Tween-80 in the solution be 1% and the final concentration of TNBP be 0.3%, stirring the obtained liquid in a special virus inactivation tank at a low speed of 80rmp, and preserving the temperature for 7 hours at 24-26 ℃;
(4) Chromatography: adsorbing the solution obtained in the step (3) by using a DEAE-650M gel chromatographic column, collecting filtrate passing through the chromatographic column, washing with citrate buffer solution with the volume of 3 times of gel volume after adsorption, and washing out the residual canine fibrinogen in the chromatographic column and collecting the residual canine fibrinogen;
(5) Adding glycine/sodium chloride into the liquid collected in the step (4), wherein the final concentration glycine is 1.2mol/L; sodium chloride is 2.0mol/L. After glycine/sodium chloride is fully dissolved, cooling the solution to 5 ℃, and continuously centrifuging the solution at a speed of 3000rmp by using a centrifuge;
(6) Dissolving the precipitate obtained in the step (5) by using a dissolving solution, stirring for 4 hours to completely dissolve the precipitate, then adjusting the protein concentration to 2.0-3.0%, and adjusting the pH value of the product to 7.0 by using a 0.5M hydrochloric acid solution or a 0.5M sodium hydroxide solution;
(7) And (3) sterilizing and filtering: sterilizing and filtering the liquid obtained in the step (6) by using a sterilized filter;
(8) And (3) adding a freeze-drying protective agent into the secondary dissolved clear liquid obtained in the step (7), and freeze-drying to obtain a fibrinogen freeze-dried product.
(9) And (3) heat treatment: and (3) carrying out water bath heat treatment at 100+/-2 ℃ for 30 minutes on the product obtained in the step (8).
The protein concentration, purity, clotting activity and the like of the prepared canine fibrinogen are measured according to the method specified in Chinese pharmacopoeia.
EXAMPLE 3 preparation of canine fibrinogen preparation
1. Preparation method
(1) Dissolving qualified canine plasma, stirring to dissolve completely, adjusting plasma temperature to-4deg.C, adjusting pH to 7.4, adding pre-cooled ethanol to final concentration of 8% (g/100 ml), centrifuging, and retaining precipitate;
(2) Adding 10 times of dissolving solution into the precipitate for dissolving, and centrifuging by using a continuous centrifuge to obtain a supernatant;
(3) Adding Tween-80 and TNBP into the obtained supernatant to make the final concentration of Tween-80 in the solution be 1% and the final concentration of TNBP be 0.3%, stirring the obtained liquid in a special virus inactivation tank at a low speed of 100rmp, and preserving the temperature for 8 hours at 24-26 ℃;
(4) Chromatography: adsorbing the solution obtained in the step (3) by using a DEAE-650M gel chromatographic column, collecting filtrate passing through the chromatographic column, washing with citrate buffer solution with the volume of 3 times of gel volume after adsorption, and washing out the residual canine fibrinogen in the chromatographic column and collecting the residual canine fibrinogen;
(5) Adding glycine/sodium chloride into the liquid collected in the step (4), wherein the final concentration glycine is 1.5mol/L; sodium chloride is 2.5mol/L. After glycine/sodium chloride is fully dissolved, cooling the solution to 10 ℃, and continuously centrifuging the solution at a speed of 5000rmp by using a centrifuge;
(6) Dissolving the precipitate obtained in the step (5) by using a dissolving solution, stirring for 6 hours to completely dissolve the precipitate, then adjusting the protein concentration to 2.0-3.0%, and adjusting the pH value of the product to 7.4 by using a 0.5M hydrochloric acid solution or a 0.5M sodium hydroxide solution;
(7) And (3) sterilizing and filtering: sterilizing and filtering the liquid obtained in the step (6) by using a sterilized filter;
(8) And (3) adding a freeze-drying protective agent into the secondary dissolved clear liquid obtained in the step (7), and freeze-drying to obtain a fibrinogen freeze-dried product.
(9) And (3) heat treatment: and (3) carrying out water bath heat treatment at 100+/-2 ℃ for 30 minutes on the product obtained in the step (8).
The protein concentration, purity, clotting activity and the like of the prepared canine fibrinogen are measured according to the method specified in Chinese pharmacopoeia.
The comparison result of the preparation process and the quality standard of fibrinogen products in Chinese pharmacopoeia is shown in Table 1.
TABLE 1
The specific detection method and standard adopted are as follows:
[ PREPARATION ] it should be an off-white or pale yellow loose body. After reconstitution, clear solution should be used, with slight opalescence.
The protein content of the sample is diluted to 10g/L by physiological sodium chloride solution, and the protein content is 6.5-7.5.
[ vacuum ] the flask should be subjected to blue-violet glow by detection with a high-frequency spark vacuum tester.
The test sample was equilibrated to 30-37℃and sterilized 5ml of water for injection at 30-37℃was added thereto, and the mixture was gently shaken to dissolve the sample completely within 30 minutes.
[ PREPARATION ] A sample is diluted with a physiological sodium chloride solution to 2 to 3 mg/lml fibrinogen, and the protein content (P, g/L) is measured (Kjeldahl method).
10ml of the above sample solution was further taken, an equivalent amount of thrombin solution (containing 0.05mmol/L calcium chloride) containing 3 ml of U/ml was added, the mixture was left at 37℃for 20 minutes, the precipitate was separated by centrifugation or filtration at 2500 rpm, and after washing 3 times with a physiological sodium chloride solution, the content of coagulable protein (F, g/L) was determined (Kjeldahl method), and the purity of the sample was calculated as below, and should be not lower than 70.0%.
Fibrinogen purity (%) = (F/P) ×100%
Based on the measured content of the coagulable protein and the labeled amount, the total amount of fibrinogen per bottle is calculated to be not less than 0.1g.
[ coagulability ] A thrombin solution (3 IU/ml) preheated to 37 ℃ was added to 0.5ml, and then a sample solution diluted to 3mg/ml with a physiological sodium chloride solution was added to 0.5ml, and the mixture was shaken well. The setting time was recorded at 37 ℃. The average of the results of the two determinations should not exceed 60 seconds.
The foreign virus test is carried out according to the annex of the current Chinese animal pharmacopoeia, and no foreign virus pollution is needed.
The test is carried out according to the annex of the current Chinese animal pharmacopoeia, and the bacteria-free growth is needed.
[ safety test ] 2 healthy beagle dogs (body weight: about 5 kg) of 16 to 22 weeks old were used, and the recovered fibrinogen was intravenously injected into the forelimbs at a dose of 40mg/kg, and the temperature of the rectum of the dogs was measured once daily for 14 days after the injection. During the observation period, the body temperature rise value of each test dog should not exceed 0.8 ℃, such as exceeding 0.8 ℃ and the duration should not exceed two times, and the test dogs should be healthy and alive, which can be judged to be in accordance with the regulations.
[ authentication test ]
Reagent(s)
Rabbit anti-canine plasma serum, rabbit anti-equine plasma serum, rabbit anti-bovine plasma serum, rabbit anti-porcine plasma serum, agar, amino black, methanol, glacial acetic acid, decolorized solution and the like.
Operating procedure
Preparation of
1% agarose gel plate: 1.0g of agarose is weighed, added into 99ml of physiological saline solution, placed into boiling water, heated in a water bath until the agarose is fully swelled, poured into a flat plate with the thickness of about 4-5 mm, and punched by a puncher after solidification, wherein the diameter of the hole is 3mm, and the distance between the holes is 3mm.
0.5% amino black staining solution: 0.5g of amino black 10B is weighed and dissolved in a mixed solution of 50ml of methanol, 10ml of glacial acetic acid and 40ml of water.
Decolorization liquid: 45ml of ethanol, 5ml of glacial acetic acid and 50ml of water are measured and mixed uniformly.
Sample addition
1 canine fibrinogen was dissolved in 5ml of water for injection, a perforated agarose gel plate was taken, rabbit anti-canine plasma serum was added to the central well, and positive control (canine plasma), negative control (saline), and different dilutions (1:4 and 1:8) of canine fibrinogen, 5 μl/well, were added to the surrounding wells, respectively.
The method for adding rabbit anti-horse plasma serum, rabbit anti-pig plasma serum and rabbit anti-cow plasma serum is the same as that described above.
Incubation
The agarose gel plates after loading were placed in a horizontal wet box and left to stand horizontally at 37℃for 24 hours.
Dyeing
The agarose gel was thoroughly soaked with physiological saline solution to remove unbound protein. And (3) putting the soaked agarose gel into 0.5% amino black staining solution for staining for 5-30 seconds.
Decoloring (decoloring)
And placing the dyed agarose gel plate into a decoloring solution until the gel plate is decolored until the background is colorless and the precipitation line is clear blue.
Result determination
The test is established if the corresponding precipitation lines should appear between the rabbit anti-canine plasma serum, the rabbit anti-equine plasma serum, the rabbit anti-bovine plasma serum and the rabbit anti-porcine plasma serum and the respective positive control plasma and no precipitation lines appear between the positive control plasma and the negative control plasma.
A corresponding precipitation line should appear between the test sample and the rabbit anti-canine plasma serum, and no precipitation line should appear between the test sample and the rabbit anti-equine plasma serum, the rabbit anti-porcine plasma serum and the rabbit anti-bovine plasma serum.
[ pyrogen test ]
Experimental animal
The 3 healthy applicable rabbits with the same source and strain, the weight of 1.7-3.0 kg, the body temperature of 38.0-39.6 ℃ and the highest and lowest body temperature difference of not more than 0.4 ℃ are selected.
Inspection method
After measuring the normal body temperature, within 15 minutes, the canine fibrinogen is warmed to about 38 ℃ according to the dosage of 40mg/kg, then slowly injected from the auricular vein, then the body temperature is measured for 1 time every 30 minutes, 6 times are measured, and the normal body temperature is subtracted from the highest one of the 6 times of body temperature, thus obtaining the elevated temperature of the rabbit body temperature. If 1 rabbit body temperature is raised by 0.6 ℃ or higher than 0.6 ℃ or the total body temperature of 3 rabbits reaches 1.3 ℃ or higher than 1.3 ℃,5 rabbits should be taken for retesting, and the detection method is the same as that described above.
Result determination
In the first 3 rabbits, the body temperature rise is lower than 0.6 ℃, and the total body temperature rise of the 3 rabbits is lower than 1.3 ℃; or in the retested 5 rabbits, the body temperature of the rabbits with the temperature rise of 0.6 ℃ or higher than 0.6 ℃ is not more than 1, and the total body temperature rise of 8 rabbits in the initial test and the retested is 3.5 ℃ or lower than 3.5 ℃, and the pyrogen inspection is judged to be qualified.
Of the 3 rabbits in the initial test, more than 1 rabbit with a temperature rise of 0.6 ℃ or higher than 0.6 ℃; or in the retest 5 rabbits, the body temperature rises by 0.6 ℃ or more than 0.6 ℃ to more than 1 rabbit; or the total temperature rise of 8 rabbits in initial and repeated tests exceeds 3.5 ℃, and the pyrogen examination is judged to be unqualified.
As can be seen from Table 1, the quality and yield of the canine fibrinogen prepared by the method are superior to the quality standard of fibrinogen specified in Chinese pharmacopoeia, the purity of the fibrinogen reaches more than 90%, and the redissolution time is shortened to about 10 minutes.
Example 4 control test
The preparation method of the dog fibrinogen by using the common fibrinogen in the field of human medicine is described in CN101703763, and specifically comprises the following steps:
1. plasma collection: the method comprises the steps of naturally thawing frozen raw material plasma 100L meeting national quarantine period regulations at room temperature, sterilizing the surface of a plasma bag by using 75% alcohol, flushing the alcohol on the surface of the plasma bag by using water for injection at 20 ℃, breaking the bag, collecting the plasma in a special plasma collecting tank with an interlayer, circularly heating the plasma by using circulating water at 25 ℃, timely transferring the thawed plasma to a special centrifugal tank, controlling the temperature of the plasma at 0 ℃, and injecting the plasma into a centrifugal machine for continuous centrifugal separation at 22000rmp speed to obtain sediment.
2. Cold precipitation, dissolution and centrifugation:
taking out the cryoprecipitate stored in a freezer below-30deg.C, crushing into small pieces, adding 16L injectable water, and stirring for 300 min to dissolve the cryoprecipitate completely. The temperature of the solution was adjusted to 20℃and the pH was adjusted to 6.4 using a 0.1M acetic acid solution, followed by continuous centrifugation at 22000rmp, and the supernatant obtained by centrifugation was subjected to clean filtration with a clean filter to obtain 17L of filtrate.
3. Virus inactivation:
170ml of calcium chloride solution was added to the collected centrifugal supernatant to make the concentration of calcium chloride in the solution to 1mmol/L, the temperature of the product in the collection tank was adjusted to 30 ℃, and then Tween-80178.5 g and TNBP53.7ml were added to make the final concentrations of both in the solution to 1% and 0.3%, respectively. In a special virus inactivation tank, stirring at a low speed of 40rmp, heating at 24 ℃ for 7 hours, and inactivating the lipid-enveloped virus possibly remained in the product or brought in during production. The workplace, air conditioner and equipment after virus inactivation are completely separated from those before inactivation.
4. And (3) chromatographic refining:
the virus-inactivated solution was adsorbed by a DEAE-650M gel column, and the filtrate passing through the column was collected at this time. After the adsorption, the gel is washed by citrate buffer solution with the volume of 3 times of gel volume, and the gel is collected into a special collecting tank.
5. And (3) precipitation and centrifugal separation:
adding glycine into the collected filtrate, cooling to 0 ℃ after full dissolution, and carrying out continuous centrifugal separation at the speed of 22000rmp by using a centrifugal machine, wherein the fibrinogen precipitate is obtained. This step also removes Tween-80 and TNBP for virus inactivation.
6. Preparing:
dissolving secondary precipitation by using a buffer solution of sodium citrate and sucrose to ensure that the concentration of the sodium citrate in the solution after dissolution reaches 39mmol/L and the concentration of the sucrose reaches 40g/L, stirring for 2 hours to completely dissolve the precipitate, then regulating the concentration of protein to 2.0%, and regulating the pH value of the product to 6.4 by using 2.0ml of a 0.5M hydrochloric acid solution.
7. And (3) sterilizing and filtering:
the prepared liquid was sterilized and filtered by a sterilized filter having a pore size of 0.45. Mu.m. After the filtration is finished, the integrity of the degerming filter is checked, and the degerming filter is rapidly filtered once again when unqualified, so that the product is ensured to meet the sterile requirement.
8. Split charging and freeze-drying:
subpackaging according to the marked loading amount in the area with the cleanliness of 100 levels, rapidly freezing the freeze dryer to below-40 ℃ before freeze drying, wherein the temperature of the product in the freeze drying process cannot exceed 35 ℃, and the freeze drying time is 4 hours. The freeze-dried product is completely sealed with a rubber plug in a vacuum state, and sterile air is injected into the freeze dryer to balance the pressure.
9. And (3) heat treatment:
and carrying out water bath heat treatment at 100+/-2 ℃ for 30 minutes on the freeze-dried product, inactivating non-lipid-coated viruses possibly remained or carried in production, and ensuring the safety of the product.
The properties of the obtained product are as follows: the obtained product is yellow powdery solid, the sample is balanced to 30-37 ℃, 5ml of sterilized water for injection is added at 30-37 ℃ and is gently shaken, and the precipitate is always suspended and the complete redissolution is not realized. The fibrinogen content in the sample is 11.5mg/ml, and the purity is only 65%. The analysis was mainly due to: human and canine blood differ in major components and constitution, blood rheological index, and specific shape of human fibrinogen and canine fibrinogen, for example: panJ.J., chenQ.S., huangW., chinaOncology,2011, 21 (9), 708-712 (Pan Jianji, chen Jisong, huang Wei. Journal of chinese cancer, 2011, 21 (9), 708-712), woodB.R., caspersP., puppelsG.J., anal & bioanal. Chem., 2007, 387 (5), 1691-703; premasiriW.R., leeJ.C., zieglerL.D., J.Phys.Chem.B,2012, 116 (31), 9376-86. Therefore, the technical proposal that can not simply utilize fibrinogen in human blood can not be applied to the treatment process of the canine fibrinogen, and the steps and specific parameters in the technical proposal can not be realized by creative labor.
These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the invention without departing from the spirit and scope of the invention, but these modifications and substitutions are intended to be within the scope of the invention.
Claims (6)
1. A method for preparing canine fibrinogen, which is characterized by comprising the following steps:
(1) Thawing qualified canine plasma, stirring to dissolve the plasma completely, adjusting the plasma temperature to-4-0 ℃, adjusting the pH value to 6.4-7.4, adding pre-cooled ethanol to a final concentration of 8% -10% (g/100 ml), and centrifuging to retain precipitate;
(2) Adding 8-10 times of dissolving solution into the precipitate for dissolving, and centrifuging by using a continuous centrifuge to obtain a supernatant;
(3) Adding Tween-80 and TNBP into the obtained supernatant to make the final concentration of Tween-80 in the solution be 1% and the final concentration of TNBP be 0.3%, stirring the obtained liquid in a special virus inactivation tank at a low speed of 40-100 rmp, and preserving the temperature for 6-8 hours at 24-26 ℃;
(4) Chromatography: adsorbing the solution obtained in the step (3) by using a DEAE-650M gel chromatographic column, collecting filtrate passing through the chromatographic column, washing with citrate buffer solution with the volume of 3 times of gel volume after adsorption, and washing out the residual canine fibrinogen in the chromatographic column and collecting the residual canine fibrinogen;
(5) Adding glycine/sodium chloride into the liquid collected in the step (4), wherein the final concentration glycine is 0.9-1.5 mol/L; the sodium chloride is 1.5-2.5 mol/L, after glycine/sodium chloride is fully dissolved, the solution is cooled to 0-10 ℃, and continuous centrifugal separation is carried out on the solution at the speed of 2000-5000 rmp by a centrifugal machine;
(6) Dissolving the precipitate obtained in the step (5) by using a dissolving solution, stirring for 2-6 hours to completely dissolve the precipitate, then adjusting the protein concentration to 2.0-3.0%, and adjusting the pH value of the product to 6.4-7.4 by using a 0.5M hydrochloric acid solution or a 0.5M sodium hydroxide solution;
(7) And (3) sterilizing and filtering: sterilizing and filtering the liquid obtained in the step (6) by using a sterilized filter;
(8) Adding a freeze-drying protective agent into the secondary dissolved clear liquid obtained in the step (7), and freeze-drying to obtain a fibrinogen freeze-dried product;
(9) And (3) heat treatment: and (3) carrying out water bath heat treatment at 100+/-2 ℃ for 30 minutes on the product obtained in the step (8).
2. The method for producing canine fibrinogen according to claim 1, wherein the centrifugation is performed at 0 to 4 ℃ in step (1).
3. The method of producing canine fibrinogen according to claim 1, wherein the PH of the solution in step (1) is adjusted to 6.4 to 7.4 with 0.1M acetic acid-sodium acetate buffer.
4. The method for producing fibrinogen according to claim 1, wherein the concentration of cold ethanol is 50% to 95% v/v.
5. The preparation method of fibrinogen according to claim 1, wherein the freeze-drying protective agent is prepared from mannitol and dextran-40 glucose injection according to a specific dosage proportion, wherein the concentration of mannitol is 4% -15% w/v, and the concentration of dextran-40 glucose injection is 3% -8% w/v.
6. The method for preparing fibrinogen according to claim 1, wherein the dissolving buffer comprises 1.5-2.0 g/L sodium citrate, 6.0-8.0 g/L sodium chloride and 8.0-10.0 g/L glycine.
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