CN112972755A

CN112972755A - Preparation method of biological hemostatic material based on porcine fibrinogen and thrombin

Info

Publication number: CN112972755A
Application number: CN202110322473.3A
Authority: CN
Inventors: 余美伦; 赵丽; 朱佳伟; 秦香芹; 范宝祥; 宫佳欣; 孟庆雪; 耿东涛; 李常禄; 傅文上; 白莹莹; 东立国; 吴艺婕
Original assignee: Harbin Hanbang Medical Science And Technology Co ltd
Current assignee: Harbin Hanbang Biopharmaceutical Co Ltd
Priority date: 2021-03-25
Filing date: 2021-03-25
Publication date: 2021-06-18
Anticipated expiration: 2041-03-25
Also published as: CN112972755B

Abstract

本发明提供了一种基于猪源纤维蛋白原和凝血酶的生物止血材料的制备方法，涉及止血材料技术领域。所述方法包括：(a)分别配制纤维蛋白原溶液和凝血酶溶液；(b)将所述纤维蛋白原溶液和所述凝血酶溶液分别制成纤维蛋白粉和凝血酶粉；以及(c)将所述纤维蛋白原粉和凝血酶粉与氯化钙混合均匀。本发明提供了简单高效的止血材料的制备方法，所制备得到的止血材料每克止血粉含有纤维蛋白原80mg，凝血酶400IU，以止血粉为基础制备的止血贴每平方厘米含有纤维蛋白原8mg，凝血酶40IU；上述止血材料能快速形成稳定胶体，不易断裂，能够起到有效的止血防护效果。The invention provides a preparation method of a biological hemostatic material based on pig-derived fibrinogen and thrombin, and relates to the technical field of hemostatic materials. The method comprises: (a) preparing a fibrinogen solution and a thrombin solution respectively; (b) preparing the fibrinogen solution and the thrombin solution into fibrin powder and thrombin powder, respectively; and (c) Mix the fibrinogen powder and thrombin powder with calcium chloride uniformly. The invention provides a simple and efficient preparation method of hemostatic material. The prepared hemostatic material contains 80 mg of fibrinogen and 400 IU of thrombin per gram of hemostatic powder, and the hemostatic patch prepared based on the hemostatic powder contains 8 mg of fibrinogen per square centimeter. , thrombin 40IU; the hemostatic material can quickly form a stable colloid, not easy to break, and can play an effective hemostatic protection effect.

Description

Preparation method of biological hemostatic material based on porcine fibrinogen and thrombin

Technical Field

The invention relates to the technical field of hemostatic materials, in particular to a preparation method of a biological hemostatic material based on porcine fibrinogen and thrombin.

Background

The fibrin adhesive is a surgical hemostatic, mainly contains fibrinogen and thrombin, has unique hemostatic effects on bleeding and fine venous bleeding, has the effects of tissue sealing, wound healing promotion and the like, and is widely used in the field of global surgery since the approval of European Union in 1972. The porcine fibrin adhesive is a new hemostatic material in recent years, adopts porcine blood as a raw material, extracts active ingredients in the porcine blood, is applied to the surgical hemostasis field in the form of a freeze-dried powder injection preparation, can effectively replace human or bovine fibrin adhesive, does not have human and animal diseases and human infectious diseases which are easily obtained by cattle, and is widely applied.

The fibrin adhesive is applied by respectively dissolving fibrinogen and thrombin lyophilized powder, mixing the two solutions by using a special instrument and spraying the two solutions on an affected part immediately, so that complete hemostasis effect can be achieved within 30 seconds.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a biological hemostatic material based on porcine fibrinogen and thrombin and a preparation method thereof. The invention aims to solve the problem that the use process of the pig-derived hemostatic material in the prior art is complicated (fibrinogen and thrombin are required to be dissolved respectively for use), and provides a simple and efficient preparation method of the hemostatic material.

The technical scheme provided by the invention is as follows:

in one aspect, the present invention provides a method for preparing a biological hemostatic material based on porcine fibrinogen and thrombin, the method comprising:

(a) preparing a fibrinogen solution and a thrombin solution respectively; the fibrinogen solution comprises 14-18 g of fibrinogen, 3-5 g of 20% human serum albumin solution, 1.0-1.4 g of sodium acetate, 2.5-3.5 g of glycine, 0.7-1.1 g of sodium citrate, 0.3-0.5 g of polysorbate 80, 1.0-1.4 g of arginine hydrochloride, 0.3-0.9g of sodium chloride, 1-3 g of trehalose and 64-96 g of mannitol per 1000g of the solution;

the thrombin solution contains 70000-90000 IU of thrombin, 3-5 g of 20% human serum albumin solution, 2-3 g of glycine, 3-9 g of sodium chloride and 74-107 g of mannitol per 1000g of solution;

(b) preparing the fibrinogen solution and the thrombin solution into fibrinogen powder and thrombin powder respectively; and

(c) and uniformly mixing the fibrinogen powder, the thrombin powder and calcium chloride.

The 20% human serum albumin solution is a solution with the concentration of human serum albumin being 20%, and is purchased from commercial sources.

In a particular embodiment, the invention begins with the extraction of fibrinogen and thrombin from porcine plasma. In a preferred embodiment, the present invention employs the extraction method of the previously applied patent technology (CN101371921B) to prepare and extract fibrinogen and thrombin stock.

Specifically, the preparation method of the fibrinogen comprises the following steps: adding 3.8mg of anticoagulant sodium citrate into 1mL of pig whole blood, centrifuging at 3500r/min at the low temperature of 2-5 ℃ for 30 minutes, separating plasma, sterilizing by using a 0.2-micron microporous filter membrane, and filtering to obtain filtrate; when the temperature of the solution is reduced to-2 ℃, ethanol which is pre-cooled to-20 ℃ is added until the final concentration is 8%, and protein precipitate is obtained by low-temperature precipitation (the amount of the fibrinogen added in the invention is converted according to the amount of the added protein precipitate).

The preparation method of the thrombin freeze-dried powder comprises the following steps: adding 13.4g of sodium oxalate into each ml of pig whole blood, and centrifugally separating to obtain anticoagulated pig plasma; adding 0.1g of barium sulfate into 1mL of blood plasma to adsorb pig prothrombin, and washing the precipitate; eluting prothrombin with 1ml of 0.3mg/ml sodium citrate solution per 1g of precipitate, adding 5mg of tissue thromboplastin and 3mg of calcium chloride per ml of precipitate for activation, and centrifuging to remove precipitate; adding polysorbate-8010 mg and tributyl phosphate 3mg into each ml; stirring for 8 hours at 24-28 ℃ to inactivate S/D virus, carrying out ultrafiltration washing purification, and concentrating to obtain a thrombin solution.

In one embodiment, the fibrinogen solution has a protein content of 14-18 mg/mL; the titer of the thrombin is 70-90 IU/mL.

In one embodiment, the mixing mass ratio of the fibrinogen powder and the thrombin powder to the calcium chloride is (0.8-1): (0.8-1): (0.003-0.005). The particle size of the calcium chloride powder is 50-75 μm.

In one embodiment, the step (a) further comprises: adjusting the pH of the fibrinogen solution to 6.7-7.2; and adjusting the pH value of the thrombin solution to be 6.3-6.8.

In a specific embodiment, the pH of the fibrinogen solution is adjusted to between 6.7 and 7.2 using 0.1% acetic acid and 0.1% sodium hydroxide.

In a specific embodiment, the pH of the thrombin solution is adjusted to between 6.7 and 7.2 using 0.1% sodium hydroxide.

In one embodiment, a color-labeling substrate is further included in the fibrinogen solution; preferably, the color-coded substrate is VB 2;

more preferably, the VB2 is added in an amount of 0.10 to 0.15g per 1000g of the fibrinogen solution.

In one embodiment, the method further comprises the steps of preparing a hemostatic patch by mixing the fibrinogen powder containing the color-labeled substrate, the thrombin powder and calcium chloride to form a mixed powder and the porcine collagen;

preferably, the mixed powder is prepared into a paste by using an organic solvent, and then the paste is spread on the pre-frozen porcine collagen.

In a specific embodiment, the extracted fibrinogen and thrombin and the auxiliary materials are respectively freeze-dried or spray-dried, and then the freeze-dried or spray-dried products are crushed and mixed to form uniform powder, namely the hemostatic powder.

In one embodiment, the preparation of the hemostatic powder further comprises the step of performing Co-60 radiation sterilization on the hemostatic powder. Preferably, the radiation dose is greater than 20 KGy.

In a specific embodiment, VB2 is used as a color marking substrate during the freeze-drying or spray-drying of the fibrinogen, is prepared into yellow powder, is mixed with thrombin powder and is fixed on a substrate material such as collagen in a co-freeze-drying or coating mode to form the hemostatic plaster with certain shape and mechanical strength.

In one embodiment, the method uses freeze drying or low temperature spray drying to produce a powder.

In one embodiment, the freeze drying comprises pre-freezing the front box for 4-5 hours at-50 ℃ to-55 ℃, adjusting the back box to-60 ℃ to-65 ℃, the primary sublimation time to be 20-26 hours, the primary sublimation end temperature to be 10-16 ℃, the secondary drying time to be 5-7 hours, and the secondary drying end temperature to be 31-39 ℃;

drying and then crushing; preferably, crushing by using a jet mill, wherein the air pressure of the jet mill is adjusted to 0.7-0.8 MPa; the particle size after grinding is 25-50 μm.

In a specific embodiment, the fibrinogen solution is added to a square tray or the fibrinogen is lyophilized in an integral freeze dryer. Pre-freezing the front box at-55 ℃ for 5 hours, adjusting the rear box to-60 ℃, controlling the primary sublimation time to be 22 hours, controlling the primary sublimation end temperature to be 12 ℃, controlling the secondary drying time to be 5 hours, and controlling the secondary drying end temperature to be 39 ℃. And (3) extracting or taking out the freeze-dried blocks after freeze-drying, crushing by using a jet mill, adjusting the air pressure to 0.7MPa, collecting powder with the particle size of about 25-50 mu m, and taking out for later use.

In a specific embodiment, the thrombin solution is added to a square tray or the fibrinogen is lyophilized in an integral lyophilizer. Pre-freezing the front box at-55 ℃ for 5 hours, adjusting the rear box to-60 ℃, controlling the primary sublimation time to be 24 hours, controlling the primary sublimation end temperature to be 12 ℃, controlling the secondary drying time to be 7 hours, and controlling the secondary drying end temperature to be 32 ℃. And (3) extracting or taking out the freeze-dried blocks after freeze-drying, crushing by using a jet mill, adjusting the air pressure to 0.7MPa, collecting powder with the particle size of about 25-50 mu m, and taking out for later use.

In one embodiment, the air inlet temperature of the low-temperature spray drying is 40-45 ℃, the air outlet temperature is 30-32 ℃, and the particle size of the spray powder is 50-75 μm.

In another aspect, the invention provides a biological hemostatic material prepared by the preparation method. The hemostatic material of the present invention may be a hemostatic powder or a hemostatic patch.

Has the advantages that:

(1) the preparation method is simple and efficient, is convenient to use, and can be used directly by preparing the fibrinogen solution and the thrombin solution into powder respectively and then mixing the powder with the calcium chloride powder to prepare the hemostatic material (such as hemostatic powder).

(2) The hemostatic material (such as hemostatic powder) produced by the invention can form stable colloid in 15s after contacting with blood, water and other liquids, thereby greatly accelerating the clinical use process. The hemostatic powder produced by the invention has uniform particle fineness and good fluidity, and can be suitable for different using modes, including but not limited to external spray, powder or clinically used spraying equipment.

(3) The invention fully optimizes the content and proportion of fibrinogen and thrombin in the hemostatic powder, and when each gram of the hemostatic powder contains 80mg of fibrinogen and 400IU of thrombin, the hemostatic effect is optimal.

(4) The colloid formed by the hemostatic powder prepared by the invention does not have the fracture phenomenon after being folded for 20 times at 180 degrees, so that the product does not have the fracture phenomenon under the action of conventional external force when in use, has the hemostatic protection effect to the utmost extent, and has the adhesive force of more than 200g/cm²And after the product is sprayed, the product is directly covered on the surface of a wound and is not easy to fall off, the use performance of the product is prevented from being influenced by partial or total falling off, the elasticity of the colloid is greater than 8cm, the colloid can be better fitted with the wound, the contact with human tissues is more flexible and flexible, the discomfort of the wound caused by the over hardness of the colloid is avoided, and the use performance of the product is better exerted.

(5) The hemostatic plaster is obtained by further processing a preparation on the basis of hemostatic powder, the obtained plaster is similar to the hemostatic powder in performance, each square centimeter of plaster contains 8mg of fibrinogen and 40IU of thrombin, collagen is used as a substrate of the plaster, so that the plaster has certain ductility, mechanical deformation capacity and water absorption capacity, the water absorption capacity can reach 20 times of the self weight, the plaster can effectively adhere to wound properties after forming colloid when meeting water, the plaster can be trimmed into various shapes by using sterile surgical scissors before use so as to be convenient for surgical use, and the plaster can be further prepared into a portable plaster for acute traumatic hemorrhage hemostasis to replace external gauze or other blood absorption hemostatic materials to a certain extent.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

EXAMPLE 1 formulation screening of hemostatic powders

1. Screening of fibrinogen and thrombin ratio

Fibrinogen and thrombin are main components of the hemostatic product, and the content and proportion of the fibrinogen and the thrombin directly influence the hemostatic effect of the hemostatic powder. Taking gelling time and gel strength as judgment, setting the lowest fibrinogen concentration of 40mg/g and the highest fibrinogen concentration of 120mg/g, setting the lowest thrombin concentration of 200IU/g and the highest thrombin concentration of 800IU/g, and setting the design parameters of a response surface as follows:

a factor of 2; 1, imitation;

14, basic times; total number of tests 14;

the basic block group is 2; the total number of the groups is 2;

two level factors: a full factor;

cubic point 4;

the center point of the cube is 3;

the shaft point is 4;

the central point of the axis point is 3;

α:1.41421。

response surface method experiments are carried out on the optimal proportion and content of the fibrinogen and the thrombin, and specific experimental data are as follows:

the optimal proportion is calculated to be the fibrinogen content of 88.427mg/g and the thrombin content of 460.915IU/g, and the gel forming time is too long when the thrombin content is lower than 200IU/g through analysis; when the fibrinogen (fibrinogen) content is less than 40mg/g, the colloidal strength (adhesive force) is poor. The ratio of fibrinogen to thrombin in the process is determined to be 80 + -10 mg/400 + -50 IU.

2. Influence of trehalose content and Co-60 radiation dose on product quality and safety

Trehalose can protect proteins from being damaged by Co-60 radiation for inactivating bacterial viruses, and meanwhile Co-60 radiation inactivation is an important process step for inactivating solid preparations, so that the quality and safety of products are directly influenced by the trehalose content and the Co-60 radiation dose. In order to reduce the dosage of trehalose and the radiation dose while ensuring the safety, a single-factor multi-level experiment is designed, and the data and the results are as follows:

the result shows that when the content of the trehalose is more than 1mg/g, the product can be effectively prevented from being influenced by radiation, but the radiation dose is less than 20KGy, so that the virus can not be effectively removed, and the process adopts the addition of the trehalose of 1mg/g and carries out the radiation inactivation of more than 20 KGy.

EXAMPLE 2 hemostatic powder preparation (Freeze drying + jet milling)

1. Preparing a fibrinogen precipitation compound solution which contains 0.12 percent of sodium acetate, 0.093 percent of sodium citrate, 0.032 percent of sodium chloride, 0.3 percent of glycine and 0.12 percent of arginine hydrochloride.

2. Taking 0.46kg of alcohol precipitation in the fibrinogen extraction process, and redissolving with 22.24kg of the solution (70-90% of the mass of the alcohol precipitation is the mass of the fibrinogen).

3. 95.0g of albumin, 23.6g of trehalose and 9.5g of tween 80 and 1628.8g of mannitol are added into the solution in the step 2, stirred and dissolved completely, and the pH is adjusted to 6.83 by using 0.1% acetic acid and 0.1% sodium hydroxide.

4. Introducing the solution into a square metal tray with a liquid level of 1-2cm, and pre-freezing the square metal tray in a freeze dryer at-55 deg.C for 5 hr. After the freeze-drying is finished, the temperature of the freeze-drying machine is adjusted to be 12 ℃, the temperature rise time is 18 hours, the heat is preserved for 4 hours for freeze-drying, after the freeze-drying is finished, the temperature is adjusted to be 39 ℃, the temperature rise time is 5 hours, and the drying time is 2 hours.

5. The tray was taken out, and the cake was collected and put in a pneumatic dryer, and pulverized while adjusting the pressure to 0.7MPa, and about 2281.3g of powder was collected.

6. The extracted thrombin solution was diluted with 0.32% NaCl solution to obtain 13.27kg of thrombin stock solution with a titer of about 87 IU/ml.

7. Adding 53.08g of human serum albumin, 39.81g of glycine and 1191.6g of mannitol, stirring and mixing uniformly, and adjusting the pH value to 6.4 by using 0.1% sodium hydroxide.

8. Introducing the above solution into square tray, keeping liquid level at 1-2cm, and pre-freezing the square metal tray in a freeze dryer at-55 deg.C for 5 hr. The temperature of the freeze dryer is adjusted to be 12 ℃, the temperature rise time is 20 hours, the heat preservation is carried out for 4 hours, the temperature is adjusted to be 32 ℃, the temperature rise time is 5 hours, and the drying time is 2 hours.

9. The tray was taken out, the cake was collected and put in an air jet mill, and the pressure was adjusted to 0.7MPa to mill, and about 1297.3g of powder was collected.

10. And respectively taking 1000.0g of the dry powder, adding 5g of calcium chloride, and completely mixing all the powder in a four-dimensional mixer to obtain a finished product of the hemostatic powder.

EXAMPLE 3 hemostatic powder preparation (spray drying)

2. Taking 0.53kg of alcohol precipitation in the fibrinogen extraction process, and redissolving with 26.44kg of the solution.

3. To the above-mentioned reconstituted solution, 107.5g of albumin, 26.4g of trehalose, 10.7g of tween 80 and 1833.7g of mannitol were added, and the mixture was stirred until completely dissolved, and the pH was adjusted to 6.91 with 0.1% acetic acid and 0.1% sodium hydroxide.

4. The extracted thrombin solution was diluted with 0.32% NaCl solution to obtain 20.36kg of thrombin stock solution with a titer of about 81 IU/ml.

5. 81.44g of human serum albumin, 61.08g of glycine and 1828.3g of mannitol are added, stirred and mixed evenly, and then the pH value is adjusted to 6.6 by 0.1% of sodium hydroxide.

6. And (3) respectively carrying out low-temperature spray drying on the solutions, adjusting the air inlet temperature of the spray drying agent to be 45 ℃, the air outlet temperature to be 30 ℃ and the spraying speed to be 100ml/min, respectively drying, and respectively collecting 2403.2g of fibrinogen powder and 1763.9g of thrombin powder.

7. And (3) taking 1000.0g of the fibrinogen powder and the thrombin powder, adding 5g of calcium chloride, and mixing by using a four-dimensional mixer to obtain a finished product of the hemostatic powder.

Example 4 hemostatic patch preparation (spray drying)

2. Taking 0.19kg of alcohol precipitation in the fibrinogen extraction process, and redissolving with 10.0kg of the solution.

3. The above compound solution is added with albumin 40.0g, trehalose 10.1g, tween 80 4.2g, mannitol 689.3g, VB₂1.2g was added, stirred to dissolve completely, and the pH was adjusted to 6.88 with 0.1% acetic acid and 0.1% sodium hydroxide. (1001.3)

4. The extracted thrombin solution was diluted with 0.32% NaCl solution to obtain 10.21kg of thrombin stock solution with a titer of about 83 IU/ml.

5. Adding human serum albumin 40.9g, glycine 30.7g and mannitol 924.9g, stirring and mixing uniformly, and adjusting pH to 6.7 with 0.1% sodium hydroxide. (1000.5)

6. And (3) respectively carrying out low-temperature spray drying on the solutions, adjusting the air inlet temperature of the spray drying agent to be 45 ℃, the air outlet temperature to be 30 ℃ and the spraying speed to be 100ml/min, respectively drying, and respectively collecting 827.3g of fibrinogen powder and 816.8g of thrombin powder.

7. The fibrinogen powder and the thrombin powder (800.0 g) were each mixed with calcium chloride (4.0 g) in a four-dimensional mixer.

8. Dissolving type I collagen in 6% acetic acid solution, introducing medical carbon dioxide, and making into 0.5% collagen solution.

9. Adding N-hydroxy thiosuccinimide cross-linking agent (other cross-linking agent such as glutaraldehyde and ultraviolet rays can be used) into collagen solution, cross-linking at room temperature for 2 hr, and transferring to 2-8 deg.C refrigerator for further cross-linking for 24 hr.

10. Injecting the crosslinked collagen solution into a metal mold, pre-freezing for 6 hours at-50 ℃, then placing the mold into a freeze dryer, heating to the final temperature of 5 ℃ for 12 hours, preserving heat for 5 hours, then adjusting the temperature to 22 ℃, heating to 6 hours, preserving heat for 3 hours, and carrying out freeze-drying molding on the collagen.

11. And (3) putting the collagen in pure water for washing for 3 times, taking out the collagen, squeezing out water, putting the collagen in physiological saline for washing for 2 times again, squeezing out water, and putting the collagen in a pre-freezing condition at the temperature of 50 ℃ below zero for 5 hours.

12. And (3) mixing the mixed powder in the step (7) with about 450ml of absolute ethyl alcohol to form paste, placing the paste at the temperature of minus 50 ℃ for precooling for 3 hours, then flatly paving the paste on the pre-frozen collagen prepared in the step (11), shaking and flatly paving the paste, and then placing the paste in a freeze dryer for freeze drying.

13. And adjusting the temperature of the freeze dryer to 15 ℃, raising the temperature for 20 hours, keeping the temperature for 4 hours, and freeze-drying and forming to obtain the hemostatic patch.

Example 5

1. Preparing a fibrinogen precipitation compound solution which contains 0.10 percent of sodium acetate, 0.07 percent of sodium citrate, 0.03 percent of sodium chloride, 0.25 percent of glycine and 0.10 percent of arginine hydrochloride.

2. Taking 0.16kg of alcohol precipitation in the fibrinogen extraction process, and redissolving with 7.76kg of the solution.

3. The above-mentioned compound solution was added with albumin 24.1g, trehalose 7.9g, tween 80 2.4g, and mannitol 754.8g, stirred to dissolve completely, and adjusted to pH 6.93 with 0.1% acetic acid and 0.1% sodium hydroxide.

4. The extracted thrombin solution was diluted with 0.32% NaCl solution to obtain 9.8kg of thrombin stock solution with a titer of about 81 IU/ml.

5. Adding human serum albumin 29.5g, glycine 19.8g and mannitol 1048.8g, stirring and mixing uniformly, and adjusting pH to 6.62 with 0.1% sodium hydroxide.

6. And (3) respectively carrying out low-temperature spray drying on the solutions, adjusting the air inlet temperature of the spray drying agent to be 45 ℃, the air outlet temperature to be 30 ℃ and the spraying speed to be 100ml/min, respectively drying, and respectively collecting 869.4g of fibrinogen powder and 1028.2g of thrombin powder.

7. And mixing the fibrinogen powder and the thrombin powder of 800.0g with calcium chloride of 4g in a four-dimensional mixer to obtain the hemostatic powder finished product.

Example 6

1. Preparing a fibrinogen precipitation compound solution which contains 0.14 percent of sodium acetate, 0.11 percent of sodium citrate, 0.09 percent of sodium chloride, 0.35 percent of glycine and 0.14 percent of arginine hydrochloride.

2. Taking 0.21kg of alcohol precipitation in the fibrinogen extraction process, and redissolving with 10.20kg of the solution.

3. 51.7g of albumin, 31.1g of trehalose and 5.3g of tween 80 and 661.1g of mannitol are added to the above-mentioned compound solution, and stirred to be completely dissolved, and the pH value is adjusted to 6.95 with 0.1% acetic acid and 0.1% sodium hydroxide.

4. The extracted thrombin solution was diluted with 0.9% NaCl solution to obtain 9.4kg of thrombin stock solution with a titer of about 80 IU/ml.

5. Adding 51.6g of human serum albumin, 31.1g of glycine and 762.8g of mannitol, stirring and mixing uniformly, and adjusting the pH value to 6.64 by using 0.1% sodium hydroxide.

6. And (3) respectively carrying out low-temperature spray drying on the solutions, adjusting the air inlet temperature of the spray drying agent to be 45 ℃, the air outlet temperature to be 30 ℃ and the spraying speed to be 100ml/min, respectively drying, and respectively collecting 912.0g of fibrinogen powder and 814.3g of thrombin powder.

Examples of effects

The mechanical ability and the adhesive ability of the hemostatic powder produced in the above practical examples were measured as follows:

finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. a preparation method based on the biological hemostatic material of pig-derived fibrinogen and thrombin, is characterized in that, described method comprises:

(a) respectively preparing fibrinogen solution and thrombin solution; wherein the fibrinogen solution contains 14-18g fibrinogen, 3-5g 20% human albumin solution, 1.0-1.4g per 1000g solution Sodium acetate, 2.5-3.5g glycine, 0.7-1.1g sodium citrate, 0.3-0.5g polysorbate 80, 1.0-1.4g arginine hydrochloride, 0.3-0.9g sodium chloride, 1-3g trehalose, 64～96g mannitol;

The thrombin solution contains 70000～90000IU thrombin, 3～5g 20% human albumin solution, 2～3g glycine, 3～9g sodium chloride, 74～107g mannitol per 1000g solution;

(b) preparing the fibrinogen solution and the thrombin solution into fibrinogen powder and thrombin powder, respectively; and,

(c) Mixing the fibrinogen powder and the thrombin powder with calcium chloride uniformly.

2 . The preparation method according to claim 1 , wherein the protein content in the fibrinogen solution is 14-18 mg/g; the titer of the thrombin is 70-90 IU/mL. 3 .

3. preparation method according to claim 1 and 2 is characterized in that, the mixing mass ratio of described fibrinogen powder and thrombin powder and calcium chloride is (0.8-1): (0.8-1): ( 0.003-0.005).

The preparation method according to claim 1, wherein the step (a) further comprises: adjusting the pH of the fibrinogen solution to be between 6.7 and 7.2; adjusting the pH of the thrombin solution Adjust to between 6.3 and 6.8.

5. The preparation method according to claim 1, wherein the fibrinogen solution further comprises a color-labeled substrate; preferably, the color-labeled substrate is VB2;

More preferably, the amount of VB2 added is 0.10-0.15 g per 1000 g of fibrinogen solution.

6. The preparation method according to claim 5, wherein the method further comprises mixing the fibrinogen powder containing the color-labeled substrate, the thrombin powder and the calcium chloride mixed powder with the mixed powder. Hemostatic patch is made with pig collagen;

Preferably, the mixed powder is prepared into a paste with an organic solvent, and then spread on the pre-frozen porcine-derived collagen.

7. The preparation method according to claim 1 or 4, characterized in that, in the method, in step (b), freeze-drying or low-temperature spray-drying is used to prepare powder.

8. The preparation method according to claim 7, wherein the freeze-drying comprises pre-freezing the front box for 4 to 5 hours at -50°C to -55°C, and the rear box is adjusted to -60 to -65°C, The primary sublimation time is 20 to 26 hours, the primary sublimation end point temperature is 10°C to 16°C, the secondary drying time is 5 to 7 hours, and the secondary drying end point temperature is 31°C to 39°C;

After drying, pulverize; preferably, pulverize with a jet mill, and the air pressure of the jet mill is adjusted to 0.7-0.8 MPa; the particle size after pulverization is 25-50 μm.

9 . The preparation method according to claim 7 , wherein the air inlet temperature of the low-temperature spray drying is 40° C. to 45° C., the air outlet temperature is 30° C. to 32° C., and the particle size of the spray-dried powder is 50° C. 10 . -75μm.

10. The biological hemostatic material prepared by the preparation method according to any one of claims 1-9.