CN112354002A - Hemostatic sealant and preparation method thereof - Google Patents
Hemostatic sealant and preparation method thereof Download PDFInfo
- Publication number
- CN112354002A CN112354002A CN202011264246.1A CN202011264246A CN112354002A CN 112354002 A CN112354002 A CN 112354002A CN 202011264246 A CN202011264246 A CN 202011264246A CN 112354002 A CN112354002 A CN 112354002A
- Authority
- CN
- China
- Prior art keywords
- solution
- fibrinogen
- supernatant
- thrombin
- centrifuging
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/10—Polypeptides; Proteins
- A61L24/106—Fibrin; Fibrinogen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/001—Use of materials characterised by their function or physical properties
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/10—Polypeptides; Proteins
- A61L24/108—Specific proteins or polypeptides not covered by groups A61L24/102 - A61L24/106
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/04—Materials for stopping bleeding
Abstract
The invention discloses a hemostatic sealant and a preparation method thereof, wherein the hemostatic sealant comprises a fibrinogen solution and a thrombin solution; the fibrin solution comprises the following components per kilogram: 2-8 g of fibrinogen, 6-9 g of sodium chloride, 6-10 g of sodium citrate, 5-8 g of sucrose, 1-3 g of Tris and 5-8 g of lysine hydrochloride; the thrombin solution comprises the following components per kilogram: 2-8 g of thrombin, 5-10 g of sodium chloride, 3-6 g of Tris, 4-8 g of sodium acetate trihydrate and 0.2-0.6 ml of glacial acetic acid; the sealant consists of a fibrinogen solution and a thrombin solution, and has excellent hemostatic effect; in addition, the fibrinogen and the thrombin can be stored in specific solutions, so that the storage stability of the fibrinogen and the thrombin can be improved, the storage time is long, and the excellent biological activity is maintained.
Description
Technical Field
The invention relates to the technical field of hemostasis, in particular to a hemostatic sealant and a preparation method thereof.
Background
Fibrinogen is an active protein component in animal and human blood plasma, human fibrinogen is produced in large scale in developed countries such as the United states, Austria and Germany before the 70 th 20 th century, the curative effect is confirmed, and the demand is increased year by year, but human fibrinogen lacks an effective virus inactivation step in the preparation process and becomes a pollution source for virus transmission, and then production and sale are gradually prohibited in various countries. After 70S, with the maturation of the S/D virus inactivation method, the production of blood coagulation factor products such as fibrinogen, blood coagulation VIII factor, prothrombin complex and the like was resumed. The fibrinogen derived from pig plasma has the advantages of wide source, low cost and high virus safety.
Thrombin is a specific external hemostatic biological product and can directly act on fibrinogen in blood. At present, prothrombin is mainly prepared from animal plasma and human plasma in China, and then becomes thrombin through activation of an activator. It catalyzes the cleavage of the fibrinous atoms fibrinopeptides A and B to convert them into insoluble fibrin clots. Thrombin has effects of accelerating blood coagulation and promoting hemostasis.
However, fibrinogen and thrombin are prone to fire after leaving the body, and have short shelf life and poor stability.
Disclosure of Invention
The invention aims to provide a hemostatic sealant, which comprises a fibrinogen solution and a thrombin solution and has excellent hemostatic effect; in addition, the stability of the fibrinogen and the thrombin can be improved and the preservation time can be long by storing the fibrinogen and the thrombin in a specific solution.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
in a first aspect, the invention provides a hemostatic sealant comprising a fibrinogen solution and a thrombin solution;
the fibrin solution comprises the following components per kilogram:
2-8 g of fibrinogen, 6-9 g of sodium chloride, 6-10 g of sodium citrate, 5-8 g of sucrose, 1-3 g of Tris and 5-8 g of lysine hydrochloride;
the thrombin solution comprises the following components per kilogram:
2-8 g of thrombin, 5-10 g of sodium chloride, 3-6 g of Tris, 4-8 g of sodium acetate trihydrate and 0.2-0.6 ml of glacial acetic acid.
The sealant consists of a fibrinogen solution and a thrombin solution, and has excellent hemostatic effect; in addition, the fibrinogen and the thrombin can be stored in specific solutions, so that the storage stability of the fibrinogen and the thrombin can be improved, the storage time is long, and the excellent biological activity is maintained. In the use process of the hemostatic sealing agent, the fibrinogen solution and the thrombin solution are directly mixed according to the mass ratio of 1: 0.8-1: 1.2, and the hemostatic sealing agent is used for wound surfaces within 3 minutes, so that the hemostatic sealing agent can play roles in stopping bleeding and adhering wounds.
Preferably, the pH value of the first suspension buffer is 7.1-7.3.
Preferably, the pH value of the second suspension buffer is 4.9-5.9.
The stability and storage time of the fibrinogen and thrombin can be better improved by controlling the pH of the first suspension buffer and the pH of the second suspension buffer.
The second aspect of the present invention provides a preparation method of the hemostatic sealing agent, wherein the preparation method comprises the following steps:
(a) diluting porcine plasma by using 0.05M acetic acid-sodium acetate buffer solution containing 0.1M sodium chloride and having a pH value of 5.8, cooling, adding ethanol with a volume of 0.5 times that of the diluted solution, stirring and standing for 6-10 h, centrifuging and collecting supernate;
(b) precooling the supernatant obtained in the step (a), adjusting the pH value to 7.4-7.8, adding ethanol with the volume 0.5 times that of the precooled supernatant, stirring and standing for 6-10 h, and centrifuging to obtain a fibrinogen crude product and a supernatant;
(c) dissolving a fibrinogen crude product in a first suspension buffer solution, centrifuging to collect supernatant, performing virus inactivation treatment on the supernatant, then cooling the virus-inactivated supernatant, adding low-temperature ethanol, centrifuging to collect precipitate, and obtaining crude precipitate;
(d) dissolving the coarse precipitate in a first suspension buffer solution, filtering to remove the precipitate, cooling the filtrate, adding low-temperature ethanol, centrifuging to obtain fibrinogen, and suspending the fibrinogen in the first buffer solution to obtain a fibrinogen solution;
(e) cooling the supernatant obtained in the step (b), adding low-temperature ethanol, centrifuging and collecting precipitates to obtain a prothrombin crude product;
(f) placing the prothrombin crude product into a second suspension buffer solution for dissolving, performing filter pressing to collect precipitates, then placing the precipitates into the second suspension buffer solution, then adding a calcium chloride solution into the second suspension buffer solution, diluting, then adding polyethylene glycol, performing filter pressing to collect supernate;
(g) and (f) performing virus inactivation treatment on the supernatant collected in the step (f), then, adsorbing the supernatant by using an adsorption resin, and then, eluting and ultrafiltering by using a second suspension to obtain a thrombin solution.
The preparation method can fully extract the fibrinogen and the thrombin in the pig plasma, has higher yield and ensures the activity of the extracted fibrinogen and the thrombin; in addition, the safety of the product can be guaranteed to the maximum extent through virus inactivation treatment.
Preferably, the virus inactivation treatment mode is an S/D virus inactivation method.
Preferably, the low-temperature ethanol temperature is-5 to-25 ℃;
the final concentration of the low-temperature ethanol added in the step (c), (d) or (e) is 7-9 percent respectively. The yield of the fibrinogen and the thrombin can be better improved by limiting the temperature of the low-temperature ethanol and the addition amount of the low-temperature ethanol.
Preferably, in the step (f), the calcium chloride solution is added to a final concentration of 0.01-0.03 mol/L calcium chloride. The addition of the calcium chloride solution can activate prothrombin to obtain thrombin, so that the prepared thrombin has excellent biological activity.
Preferably, the final concentration of the polyethylene glycol after addition is 10% to 15%. The impurity protein can be better precipitated by adding the polyethylene glycol so as to be removed, and the purity of the prepared thrombin is improved.
Preferably, the ultrafiltration is for the retention of macromolecular proteins with a molecular weight of 100 Kda.
Compared with the prior art, the invention has the beneficial effects that at least:
the sealant consists of a fibrinogen solution and a thrombin solution, and has excellent hemostatic effect; in addition, the fibrinogen and the thrombin can be stored in specific solutions, so that the storage stability of the fibrinogen and the thrombin can be improved, the storage time is long, and the excellent biological activity is maintained.
The preparation method can fully extract the fibrinogen and the thrombin in the pig plasma, has higher yield and ensures the activity of the extracted fibrinogen and the thrombin; in addition, the safety of the product can be guaranteed to the maximum extent through virus inactivation treatment.
Detailed Description
The following describes embodiments of the present invention in detail with reference to the following embodiments. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.
It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.
Example 1
First, hemostatic sealant
The hemostatic sealing agent comprises fibrinogen solution and thrombin solution;
the fibrin solution per kilogram comprises the following components:
2g of fibrinogen, 6g of sodium chloride, 10g of sodium citrate, 5g of sucrose, 3g of Tris and 5g of lysine hydrochloride;
the thrombin solution per kg comprises the following components:
6.1g of thrombin, 5g of sodium chloride, 6g of Tris, 4g of sodium acetate trihydrate and 0.6ml of glacial acetic acid;
the pH of the first suspension buffer was 7.1; the pH of the second suspension buffer was 5.9.
Second, preparation method
The preparation method of the hemostatic sealant comprises the following steps:
(a) diluting pig plasma with 0.1M sodium chloride, pH5.8 0.05M acetic acid-sodium acetate buffer solution by equal times (equal volume), cooling, adding 0.5 volume times of ethanol, stirring, standing for 8 hr, centrifuging, and collecting supernatant;
(b) precooling the supernatant obtained in the step (a), adjusting the pH value to 7.5, adding ethanol with the volume 0.5 times that of the precooled supernatant, stirring and standing for 8 hours, and centrifuging to obtain a fibrinogen crude product and a supernatant;
(c) dissolving a fibrinogen crude product in a first suspension buffer solution at 25 ℃, centrifuging to collect a supernatant, adding Tween-80 and tributyl phosphate into the supernatant to enable the final concentration of the Tween-80 to be 1% and the final concentration of the tributyl phosphate to be 0.3%, then performing virus inactivation treatment at 25 ℃ for 6h, subsequently cooling the supernatant to 4 ℃, adding-20 ℃ low-temperature ethanol to enable the final concentration of the ethanol to be 8%, centrifuging to collect precipitates, and obtaining a crude precipitate;
(d) dissolving the coarse precipitate in a first suspension buffer solution at 25 ℃, filtering to remove the precipitate, cooling the filtrate to 4 ℃, adding ethanol at the low temperature of-20 ℃ until the final concentration of the ethanol is 8%, and centrifuging to obtain fibrinogen; suspending fibrinogen in a first buffer solution to obtain a fibrinogen solution;
(e) cooling the supernatant obtained in the step (b) to 4 ℃, then adding low-temperature ethanol with the temperature of minus 20 ℃ until the final concentration of the ethanol is 8%, centrifuging and collecting precipitates to obtain a prothrombin crude product;
(f) dissolving the prothrombin crude product in a second suspension buffer solution at 25 ℃, performing filter pressing to collect precipitates, placing the precipitates in the second suspension buffer solution, adding a calcium chloride solution into the second suspension buffer solution until the final concentration of calcium chloride is 0.02mol/L for activation, adding an equal volume of the second suspension buffer solution for dilution, then adding polyethylene glycol according to the mass ratio of 12% to precipitate impure proteins, and performing filter pressing to collect supernate;
(g) adding Tween-80 and tributyl phosphate into the supernatant collected in the step (f) to enable the final concentration of Tween-80 to be 1% and the final concentration of tributyl phosphate to be 0.3%, then performing virus inactivation treatment at 25 ℃ for 6h, subsequently, adsorbing the supernatant by using SP-C50 adsorption resin, eluting by using a second suspension, and ultrafiltering to obtain a thrombin solution with molecular weight cutoff of 100 Kda.
Example 2
First, hemostatic sealant
The hemostatic sealing agent comprises fibrinogen solution and thrombin solution;
the fibrin solution per kilogram comprises the following components:
8g of fibrinogen, 9g of sodium chloride, 6g of sodium citrate, 8g of sucrose, 1g of Tris and 8g of lysine hydrochloride;
the thrombin solution per kg comprises the following components:
2.7 g of thrombin, 10g of sodium chloride, 3g of Tris, 8g of sodium acetate trihydrate and 0.2ml of glacial acetic acid;
the pH of the first suspension buffer was 7.3; the pH of the second suspension buffer was 4.9.
Second, preparation method
The preparation method of the hemostatic sealant comprises the following steps:
(a) diluting pig plasma with 0.1M sodium chloride, pH5.8 0.05M acetic acid-sodium acetate buffer solution by equal times (equal volume), cooling, adding 0.5 volume times of ethanol, stirring, standing for 8 hr, centrifuging, and collecting supernatant;
(b) precooling the supernatant obtained in the step (a), adjusting the pH value to 7.5, adding ethanol with the volume 0.5 times that of the precooled supernatant, stirring and standing for 8 hours, and centrifuging to obtain a fibrinogen crude product and a supernatant;
(c) dissolving a fibrinogen crude product in a first suspension buffer solution at 25 ℃, centrifuging to collect a supernatant, adding Tween-80 and tributyl phosphate into the supernatant to enable the final concentration of the Tween-80 to be 1% and the final concentration of the tributyl phosphate to be 0.3%, then performing virus inactivation treatment at 25 ℃ for 6h, subsequently cooling the supernatant to 4 ℃, adding-20 ℃ low-temperature ethanol to enable the final concentration of the ethanol to be 8%, centrifuging to collect precipitates, and obtaining a crude precipitate;
(d) dissolving the coarse precipitate in a first suspension buffer solution at 25 ℃, filtering to remove the precipitate, cooling the filtrate to 4 ℃, adding ethanol at the low temperature of-20 ℃ until the final concentration of the ethanol is 8%, and centrifuging to obtain fibrinogen; suspending fibrinogen in a first buffer solution to obtain a fibrinogen solution;
(e) cooling the supernatant obtained in the step (b) to 4 ℃, then adding low-temperature ethanol with the temperature of minus 20 ℃ until the final concentration of the ethanol is 8%, centrifuging and collecting precipitates to obtain a prothrombin crude product;
(f) dissolving the prothrombin crude product in a second suspension buffer solution at 25 ℃, performing filter pressing to collect precipitates, placing the precipitates in the second suspension buffer solution, adding a calcium chloride solution into the second suspension buffer solution until the final concentration of calcium chloride is 0.02mol/L for activation, adding an equal volume of the second suspension buffer solution for dilution, then adding polyethylene glycol according to the mass ratio of 12% to precipitate impure proteins, and performing filter pressing to collect supernate;
(g) adding Tween-80 and tributyl phosphate into the supernatant collected in the step (f) to enable the final concentration of Tween-80 to be 1% and the final concentration of tributyl phosphate to be 0.3%, then performing virus inactivation treatment at 25 ℃ for 6h, subsequently, adsorbing the supernatant by using SP-C50 adsorption resin, eluting by using a second suspension, and ultrafiltering to obtain a thrombin solution with molecular weight cutoff of 100 Kda.
Example 3
First, hemostatic sealant
The hemostatic sealing agent comprises fibrinogen solution and thrombin solution;
the fibrin solution per kilogram comprises the following components:
6g of fibrinogen, 8g of sodium chloride, 8g of sodium citrate, 6g of sucrose, 2g of Tris and 7g of lysine hydrochloride;
the thrombin solution per kg comprises the following components:
5.7g of thrombin, 8g of sodium chloride, 5g of Tris, 6g of sodium acetate trihydrate and 0.4ml of glacial acetic acid;
the pH of the first suspension buffer was 7.2; the pH of the second suspension buffer was 5.4.
Second, preparation method
The preparation method of the hemostatic sealant comprises the following steps:
(a) diluting pig plasma with 0.1M sodium chloride, pH5.8 0.05M acetic acid-sodium acetate buffer solution by equal times (equal volume), cooling, adding 0.5 volume times of ethanol, stirring, standing for 8 hr, centrifuging, and collecting supernatant;
(b) precooling the supernatant obtained in the step (a), adjusting the pH value to 7.5, adding ethanol with the volume 0.5 times that of the precooled supernatant, stirring and standing for 8 hours, and centrifuging to obtain a fibrinogen crude product and a supernatant;
(c) dissolving a fibrinogen crude product in a first suspension buffer solution at 25 ℃, centrifuging to collect a supernatant, adding Tween-80 and tributyl phosphate into the supernatant to enable the final concentration of the Tween-80 to be 1% and the final concentration of the tributyl phosphate to be 0.3%, then performing virus inactivation treatment at 25 ℃ for 6h, subsequently cooling the supernatant to 4 ℃, adding-20 ℃ low-temperature ethanol to enable the final concentration of the ethanol to be 8%, centrifuging to collect precipitates, and obtaining a crude precipitate;
(d) dissolving the coarse precipitate in a first suspension buffer solution at 25 ℃, filtering to remove the precipitate, cooling the filtrate to 4 ℃, adding ethanol at the low temperature of-20 ℃ until the final concentration of the ethanol is 8%, and centrifuging to obtain fibrinogen; suspending fibrinogen in a first buffer solution to obtain a fibrinogen solution;
(e) cooling the supernatant obtained in the step (b) to 4 ℃, then adding low-temperature ethanol with the temperature of minus 20 ℃ until the final concentration of the ethanol is 8%, centrifuging and collecting precipitates to obtain a prothrombin crude product;
(f) dissolving the prothrombin crude product in a second suspension buffer solution at 25 ℃, performing filter pressing to collect precipitates, placing the precipitates in the second suspension buffer solution, adding a calcium chloride solution into the second suspension buffer solution until the final concentration of calcium chloride is 0.02mol/L for activation, adding an equal volume of the second suspension buffer solution for dilution, then adding polyethylene glycol according to the mass ratio of 12% to precipitate impure proteins, and performing filter pressing to collect supernate;
(g) adding Tween-80 and tributyl phosphate into the supernatant collected in the step (f) to enable the final concentration of Tween-80 to be 1% and the final concentration of tributyl phosphate to be 0.3%, then performing virus inactivation treatment at 25 ℃ for 6h, subsequently, adsorbing the supernatant by using SP-C50 adsorption resin, eluting by using a second suspension, and ultrafiltering to obtain a thrombin solution with molecular weight cutoff of 100 Kda.
Comparative example 1
The comparative example discloses a hemostatic sealant which is substantially the same as the hemostatic sealant in example 3, except that the sodium citrate in the fibrinogen solution in example 3 is replaced with an equal fraction of sodium citrate;
the preparation method of the hemostatic sealant is the same as that in example 3.
Comparative example 2
This comparative example discloses a hemostatic sealant that is essentially the same as the hemostatic sealant of example 3, except that the pH of the fibrinogen solution of example 3 is adjusted to 7.5;
the preparation method of the hemostatic sealant is the same as that in example 3.
Comparative example 3
This comparative example discloses a hemostatic sealant that is the same as the hemostatic sealant in example 3;
the preparation method of the hemostatic sealant is basically the same as that of example 3, except that the final concentration of the hemostatic sealant is 10% after adding low-temperature ethanol in steps (c) and (d).
Experimental example 1
The fibrinogen solutions prepared in examples 1 to 3 and comparative examples 1 to 2 were selected, respectively; the fibrinogen solution was stored at 4 ℃ for 80 days and observed as shown in Table 1:
TABLE 1 storage of different fibrinogen solutions
| Group of | Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 |
| In the state of solution | Slight turbidity | Slight turbidity | Clarification | Flocculent precipitate | Flocculent precipitate |
As can be seen from table 1, it is,
the fibrinogen solution prepared by the embodiment of the invention has better stability and longer storage time.
Experimental example 2
The fibrinogen solutions of examples 1 to 3 and comparative examples 1 to 3 were prepared according to the methods described above, and the amounts of fibrinogen obtained in step (c) were measured to calculate the amounts of fibrinogen extracted per liter of pig plasma in each of the examples and comparative examples, and the calculation results are shown in table 2:
TABLE 2
| Group of | Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 | Comparative example 3 |
| Yield of the product | 2.36g/L | 2.24g/L | 2.43g/L | 2.37g/L | 2.25g/L | 1.97g/L |
As can be seen from Table 2:
the embodiment of the application claims that the technical scheme has the advantages of better extracting fibrinogen and better extracting fibrinogen in pig plasma by selecting the low-temperature ethanol content.
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; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.
Claims (9)
1. A hemostatic sealant, comprising a fibrinogen solution and a thrombin solution;
the fibrin solution comprises the following components per kilogram:
2-8 g of fibrinogen, 6-9 g of sodium chloride, 6-10 g of sodium citrate, 5-8 g of sucrose, 1-3 g of Tris and 5-8 g of lysine hydrochloride;
the thrombin solution comprises the following components per kilogram:
2-8 g of thrombin, 5-10 g of sodium chloride, 3-6 g of Tris, 4-8 g of sodium acetate trihydrate and 0.2-0.6 ml of glacial acetic acid.
2. The hemostatic sealant according to claim 1, wherein the pH value of the first suspension buffer is 7.1-7.3.
3. The hemostatic sealant according to claim 1, wherein the pH value of the second suspension buffer is 4.9-5.9.
4. A method of preparing a hemostatic sealant according to any one of claims 1 to 3, comprising the steps of:
(a) diluting porcine plasma by using 0.05M acetic acid-sodium acetate buffer solution containing 0.1M sodium chloride and having a pH value of 5.8, cooling, adding ethanol with a volume of 0.5 times that of the diluted solution, stirring and standing for 6-10 h, centrifuging and collecting supernate;
(b) precooling the supernatant obtained in the step (a), adjusting the pH value to 7.4-7.8, adding ethanol with the volume 0.5 times that of the precooled supernatant, stirring and standing for 6-10 h, and centrifuging to obtain a fibrinogen crude product and a supernatant;
(c) dissolving a fibrinogen crude product in a first suspension buffer solution, centrifuging to collect supernatant, performing virus inactivation treatment on the supernatant, then cooling the virus-inactivated supernatant, adding low-temperature ethanol, centrifuging to collect precipitate, and obtaining crude precipitate;
(d) dissolving the coarse precipitate in a first suspension buffer solution, filtering to remove the precipitate, cooling the filtrate, adding low-temperature ethanol, centrifuging to obtain fibrinogen, and suspending the fibrinogen in the first buffer solution to obtain a fibrinogen solution;
(e) cooling the supernatant obtained in the step (b), adding low-temperature ethanol, centrifuging and collecting precipitates to obtain a prothrombin crude product;
(f) placing the prothrombin crude product into a second suspension buffer solution for dissolving, performing filter pressing to collect precipitates, then placing the precipitates into the second suspension buffer solution, then adding a calcium chloride solution into the second suspension buffer solution, diluting, then adding polyethylene glycol, performing filter pressing to collect supernate;
(g) and (f) performing virus inactivation treatment on the supernatant collected in the step (f), then, adsorbing the supernatant by using an adsorption resin, and then, eluting and ultrafiltering by using a second suspension to obtain a thrombin solution.
5. The method according to claim 4, wherein the virus inactivation is performed by S/D virus inactivation.
6. The method according to claim 4, wherein the low-temperature ethanol temperature is-5 to-25 ℃;
the final concentration of the low-temperature ethanol added in the step (c), (d) or (e) is 7-9 percent respectively.
7. The method according to claim 4, wherein the calcium chloride solution is added to the calcium chloride solution in a final concentration of 0.01 to 0.03mol/L in the step (f).
8. The method according to claim 4, wherein the final concentration of the polyethylene glycol after the addition is 10 to 15%.
9. The method of claim 4, wherein the ultrafiltration is performed to retain a macromolecular protein having a molecular weight of 100 Kda.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011264246.1A CN112354002A (en) | 2020-11-12 | 2020-11-12 | Hemostatic sealant and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011264246.1A CN112354002A (en) | 2020-11-12 | 2020-11-12 | Hemostatic sealant and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112354002A true CN112354002A (en) | 2021-02-12 |
Family
ID=74514575
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011264246.1A Pending CN112354002A (en) | 2020-11-12 | 2020-11-12 | Hemostatic sealant and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112354002A (en) |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101371921A (en) * | 2008-10-08 | 2009-02-25 | 余美伦 | Instant lyophilized fibrinogen and fibrin ferment formulation composition, preparation method and use thereof |
| CN102178975A (en) * | 2011-04-25 | 2011-09-14 | 福建南生科技有限公司 | Fibrous protein hemostatic patch and making method thereof |
| CN102212129A (en) * | 2011-05-05 | 2011-10-12 | 邦和药业股份有限公司 | Method for extracting human fibrinogen from component I through column chromatography |
| CN103160486A (en) * | 2013-04-02 | 2013-06-19 | 黑龙江迪龙制药有限公司 | Preparation method of porcine thrombin |
| CN104387466A (en) * | 2014-11-11 | 2015-03-04 | 贵州中泰生物科技有限公司 | Method for separating fibrinogen from Cohn component I sediment |
| CN104436171A (en) * | 2014-12-25 | 2015-03-25 | 华兰生物工程股份有限公司 | Method for preparing human fibrinogen preparation and preparation prepared by method |
| CN105039295A (en) * | 2015-09-15 | 2015-11-11 | 上海洲跃生物科技有限公司 | Method for preparing human thrombin from cold-removing glue plasma |
| CN105326859A (en) * | 2015-11-09 | 2016-02-17 | 上海洲跃生物科技有限公司 | Method for preparing human prothrombin complex from Cohn blood plasma component III |
| US20160067371A1 (en) * | 2013-04-22 | 2016-03-10 | Sealantium Medical Ltd | Fibrinogen-based tissue adhesive patches |
| 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 |
| CN107699554A (en) * | 2017-11-17 | 2018-02-16 | 浙江丰安生物制药有限公司 | A kind of preparation method of pig blood fibrin ferment |
-
2020
- 2020-11-12 CN CN202011264246.1A patent/CN112354002A/en active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101371921A (en) * | 2008-10-08 | 2009-02-25 | 余美伦 | Instant lyophilized fibrinogen and fibrin ferment formulation composition, preparation method and use thereof |
| CN102178975A (en) * | 2011-04-25 | 2011-09-14 | 福建南生科技有限公司 | Fibrous protein hemostatic patch and making method thereof |
| CN102212129A (en) * | 2011-05-05 | 2011-10-12 | 邦和药业股份有限公司 | Method for extracting human fibrinogen from component I through column chromatography |
| CN103160486A (en) * | 2013-04-02 | 2013-06-19 | 黑龙江迪龙制药有限公司 | Preparation method of porcine thrombin |
| US20160067371A1 (en) * | 2013-04-22 | 2016-03-10 | Sealantium Medical Ltd | Fibrinogen-based tissue adhesive patches |
| CN104387466A (en) * | 2014-11-11 | 2015-03-04 | 贵州中泰生物科技有限公司 | Method for separating fibrinogen from Cohn component I sediment |
| CN104436171A (en) * | 2014-12-25 | 2015-03-25 | 华兰生物工程股份有限公司 | Method for preparing human fibrinogen preparation and preparation prepared by method |
| CN105039295A (en) * | 2015-09-15 | 2015-11-11 | 上海洲跃生物科技有限公司 | Method for preparing human thrombin from cold-removing glue plasma |
| CN105326859A (en) * | 2015-11-09 | 2016-02-17 | 上海洲跃生物科技有限公司 | Method for preparing human prothrombin complex from Cohn blood plasma component III |
| 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 |
| CN107699554A (en) * | 2017-11-17 | 2018-02-16 | 浙江丰安生物制药有限公司 | A kind of preparation method of pig blood fibrin ferment |
Non-Patent Citations (2)
| Title |
|---|
| 温辉梁: "《生物化工产品生产技术》", 31 December 2004, 江西科学技术出版社 * |
| 程文康: "《人凝血酶及纤维蛋白原的制备》", 《中国优秀硕士学位论文全文数据库 (工程科技Ⅰ辑)》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0317376B1 (en) | Preparation of a concentrate of high-purity human factor IX and of other plasma proteins | |
| CA1126652A (en) | Antithrombin preparation and process for the production thereof | |
| CA1074698A (en) | Method of collecting anti-hemophilic factor viii from blood and blood plasma | |
| AT407115B (en) | METHOD FOR PRODUCING A CONCENTRATE OF STANDARDIZED, HUMAN FROM WILLEBRAND FACTOR | |
| EP0782616B1 (en) | Therapeutic grade thrombin production and products | |
| RU2603103C2 (en) | Method of producing fibrinogen using strong anion exchange resin and fibrinogen-containing product | |
| US4470969A (en) | Process for producing a concentrate of coagulation factors VII and VIIa | |
| CA1243950A (en) | Process for producing a high purity antihemophilic factor concentrate | |
| US7816495B2 (en) | Processes for the preparation of fibrinogen | |
| CN102614219A (en) | Method for preparing human prothrombin complex with high yield | |
| JPH0424360B2 (en) | ||
| JPH06505494A (en) | Preparation of factor IX | |
| CN109071596B (en) | Method for purifying fibrinogen | |
| CN112354002A (en) | Hemostatic sealant and preparation method thereof | |
| CN113652414B (en) | Preparation method of high-purity human thrombin | |
| JP3944267B2 (en) | Method for activating prothrombin and method for producing thrombin based on the method | |
| EP0813598B1 (en) | Thrombin preparation | |
| US4406886A (en) | Purification of antihemophilia factor VIII by precipitation with zinc ions | |
| US5286849A (en) | Purification of factor IX | |
| CN113577295B (en) | Human fibrinogen dry heat treatment stabilizer and application thereof | |
| JP3195439B2 (en) | Method for purifying factor XIII | |
| CN114106145B (en) | Process for producing blood-source human blood coagulation factor VIII/von Willebrand factor complex | |
| JPH0733799A (en) | Thrombin having high specific activity and its production | |
| CN115197315A (en) | Method for preparing human fibrinogen by using cryoprecipitate as raw material and using chromatographic method | |
| CN115386564A (en) | Method for purifying urokinase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |