CN112029015A - Production and purification process of high-purity low-molecular-weight heparin sodium - Google Patents
Production and purification process of high-purity low-molecular-weight heparin sodium Download PDFInfo
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- CN112029015A CN112029015A CN202011126351.9A CN202011126351A CN112029015A CN 112029015 A CN112029015 A CN 112029015A CN 202011126351 A CN202011126351 A CN 202011126351A CN 112029015 A CN112029015 A CN 112029015A
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
- C08B37/0078—Degradation products
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Abstract
The invention discloses a production and purification process of high-purity low-molecular-weight heparin sodium, which comprises the following steps: s1 weighing 80-120 g of crude heparin sodium, dissolving with purified water, controlling the temperature of the solution to be below 30 ℃, stirring and dissolving to prepare 10-20% (w/v) of crude heparin sodium solution; s2, adding the crude heparin sodium solution into a NaCl solution with the concentration of 2% to prepare a crude heparin sodium solution with the concentration of 5% -10% (w/v), adding alkaline protease, and performing heat preservation and enzymolysis at 35-50 ℃ for 3-6 h to obtain an enzymolysis solution; s3 centrifuging the enzymolysis liquid obtained in step S2 to obtain supernatant and precipitate, and discarding insoluble impurities. The invention also enables macromolecular substances such as residual protein and the like in the produced low molecular weight heparin to be intercepted together by using an ultrafiltration method, improves the safety of the low molecular weight heparin, and can ensure that the low molecular weight heparin sodium has low protein content, high purity and high yield.
Description
Technical Field
The invention relates to the technical field of biochemical engineering, in particular to a production and purification process of high-purity low-molecular-weight heparin sodium.
Background
Heparin has been the first choice of drugs for preventing and treating thrombosis since the last 80 s. The low molecular heparin is prepared by specific chemical cutting and purification separation of common heparin, and gradually replaces the market position of the traditional heparin due to the advantages of definite curative effect, small side effect, predictability and the like.
The existing purification methods are mainly divided into the following methods: (1) the enoxaparin sodium is decolorized by activated carbon, filtered by macroporous resin, and the filtrate is lyophilized to obtain purified enoxaparin sodium, the decolorization effect of the method is good, but the method has the defects that a large amount of products are adsorbed in the activated carbon and the resin, and the yield can only reach about 60%; (2) the pH value of enoxaparin sodium is adjusted by sodium hydroxide, hydrogen peroxide is used for decolorization, and the enoxaparin sodium is subjected to sodium chloride and ethanol washing and freeze-drying to obtain purified enoxaparin sodium; (3) sequentially passing the enoxaparin sodium through a hydrophobic chromatographic column and anion exchange resin, performing nanofiltration and desalination, precipitating, and drying to obtain purified enoxaparin sodium, wherein the method needs multiple chromatographic columns, and the product loss is large; (4) the enoxaparin sodium is sequentially filtered, micro-filtered and ultrafiltered by a device, and freeze-dried to obtain purified enoxaparin sodium, the loss of products is small in the method, but the operation pressure of the hierarchical ultrafiltration is large, so that the method is not suitable for industrial application; (5) the enoxaparin sodium is repeatedly treated by sodium chloride solution and methanol to finally obtain enoxaparin sodium with the standard clarity.
Disclosure of Invention
Based on the technical problems in the background art, the invention provides a production and purification process of high-purity low-molecular-weight heparin sodium.
The invention provides a production and purification process of high-purity low-molecular-weight heparin sodium, which comprises the following steps:
s1 weighing 80-120 g of crude heparin sodium, dissolving with purified water, controlling the temperature of the solution to be below 30 ℃, stirring and dissolving to prepare 10-20% (w/v) of crude heparin sodium solution;
s2, adding the crude heparin sodium solution into a NaCl solution with the concentration of 2% to prepare a crude heparin sodium solution with the concentration of 5% -10% (w/v), adding alkaline protease, and performing heat preservation and enzymolysis at 35-50 ℃ for 3-6 h to obtain an enzymolysis solution;
s3, centrifugally separating the enzymolysis liquid obtained in the step S2 to obtain supernatant and precipitate, and discarding insoluble impurities;
s4, adding ethanol or methanol into the supernatant obtained in the step S2, stirring and standing to obtain a precipitate;
s5, dissolving the precipitate in distilled water, slowly adding sodium nitrite to degrade heparin sodium under stirring until the degradation reaction is finished, adding sodium hydroxide, and adding sodium borohydride to reduce the heparin sodium;
s6 dissolving the precipitate with purified water to 10-15 wt% solution, filtering with 0.1 μm filter membrane, and spray drying to obtain heparin sodium product.
Preferably, in the step S4, the temperature is adjusted to 0 to 4 ℃, the mixture is left for 5 to 12 hours, the precipitate is collected, the precipitate is dissolved in distilled water with the volume of 4 to 8 times that of the solution, after the precipitate is completely dissolved, hydrogen peroxide with the volume of 0.5 to 1.2 percent of the solution is added for oxidation for 8 to 12 hours, the mixture is filtered by a filter membrane, ethanol with the concentration of 95 percent and the volume of 2 times that of the solution is added for precipitation, and the precipitate is left for 6 to 15 hours, so that the precipitate is obtained.
Preferably, the rotation speed of the centrifugal separation in the step S3 is 1000-3000 r/min, and the centrifugal time is 10-60 min.
Preferably, the volume ratio of the ethanol or the methanol to the supernatant in the step S4 is 1-5:1, the stirring time is 1-3 hours, and the standing time is 3-10 hours.
Preferably, the dosage of the alkaline protease is 0.5-1% of the crude heparin sodium solution.
Preferably, after the reduction in step S5 is finished, a 1-ten-thousand-molecular-weight ultrafilter is used to intercept macromolecules, the pH of the ultrafiltrate is adjusted to 6.2-7.0, ethanol with the volume 1.5 times that of the ultrafiltrate is added for precipitation for 3-12 hours, and finally ethanol with the concentration of 95% is used for dehydration and drying, so as to obtain the low-molecular-weight heparin sodium.
Preferably, the preparation method of the crude heparin sodium in step S1 comprises the following steps:
s1 raw material treatment: carefully cleaning the pork intestines with clear water to remove internal and external dirt, and mincing the pork intestines into meat paste;
s2, performing ultrasonic enzymolysis extraction, adding 4-6 times of water and 20-25% of NaOH into the obtained meat paste-shaped pork sausage, adding 0.5-1.5% of protease, stirring for 15-20 min in an ultrasonic oscillator, filtering to obtain an enzymolysis solution, and performing ion exchange adsorption to obtain resin;
s3 heparin elution: filtering the resin to obtain an eluent;
s4 ethanol precipitation: filtering the eluent to obtain a precipitate, and drying the precipitate to obtain the crude product of heparin sodium.
According to the production and purification process of the high-purity low-molecular-weight heparin sodium, the ultrafiltration method is used, so that residual proteins and other macromolecular substances in the produced low-molecular-weight heparin are intercepted together, the safety of the low-molecular-weight heparin is improved, the low-molecular-weight heparin sodium can be low in protein content and high in purity, and the yield is high.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.
A production and purification process of high-purity low-molecular-weight heparin sodium comprises the following steps:
s1 weighing 80-120 g of crude heparin sodium, dissolving with purified water, controlling the temperature of the solution to be below 30 ℃, stirring and dissolving to prepare 10-20% (w/v) of crude heparin sodium solution;
s2, adding the crude heparin sodium solution into a NaCl solution with the concentration of 2% to prepare a crude heparin sodium solution with the concentration of 5% -10% (w/v), adding alkaline protease, and performing heat preservation and enzymolysis at 35-50 ℃ for 3-6 h to obtain an enzymolysis solution;
s3, centrifugally separating the enzymolysis liquid obtained in the step S2 to obtain supernatant and precipitate, and discarding insoluble impurities;
s4, adding ethanol or methanol into the supernatant obtained in the step S2, stirring and standing to obtain a precipitate;
s5, dissolving the precipitate in distilled water, slowly adding sodium nitrite to degrade heparin sodium under stirring until the degradation reaction is finished, adding sodium hydroxide, and adding sodium borohydride to reduce the heparin sodium;
s6 dissolving the precipitate with purified water to 10-15 wt% solution, filtering with 0.1 μm filter membrane, and spray drying to obtain heparin sodium product.
In the invention, the temperature is adjusted to 0-4 ℃ in the step S4, the mixture is placed for 5-12 h, the precipitate is collected and dissolved in distilled water with the volume of 4-8 times that of the precipitate, after the precipitate is completely dissolved, hydrogen peroxide with the volume of 0.5-1.2% of the solution is added for oxidation for 8-12 h, the mixture is filtered by a filter membrane, ethanol with the concentration of 95% and the volume of 2 times that of the solution is added for precipitation, and the precipitate is placed for 6-15 h to obtain the precipitate.
In the invention, the rotation speed of centrifugal separation in the step S3 is 1000-3000 r/min, and the centrifugal time is 10-60 min.
In the invention, the volume ratio of the ethanol or methanol to the supernatant in the step S4 is 1-5:1, the stirring time is 1-3 h, and the standing time is 3-10 h.
In the invention, the dosage of the alkaline protease is 0.5-1% of the crude heparin sodium solution.
In the invention, after the reduction in the step S5 is finished, an ultrafilter with the molecular weight of 1 ten thousand is adopted to intercept macromolecules, the pH value of ultrafiltrate is adjusted to 6.2-7.0, ethanol with the volume of 1.5 times of that of the ultrafiltrate is added for precipitation for 3-12 hours, and finally ethanol with the concentration of 95% is used for dehydration and drying, so that the low molecular weight heparin sodium is obtained.
In the invention, the preparation method of the crude heparin sodium in the step S1 comprises the following steps:
s1 raw material treatment: carefully cleaning the pork intestines with clear water to remove internal and external dirt, and mincing the pork intestines into meat paste;
s2, performing ultrasonic enzymolysis extraction, adding 4-6 times of water and 20-25% of NaOH into the obtained meat paste-shaped pork sausage, adding 0.5-1.5% of protease, stirring for 15-20 min in an ultrasonic oscillator, filtering to obtain an enzymolysis solution, and performing ion exchange adsorption to obtain resin;
s3 heparin elution: filtering the resin to obtain an eluent;
s4 ethanol precipitation: filtering the eluent to obtain a precipitate, and drying the precipitate to obtain the crude product of heparin sodium.
The invention comprises the following steps: raw material treatment: carefully cleaning the pork intestines with clear water to remove internal and external dirt, and mincing the pork intestines into meat paste; performing ultrasonic enzymolysis extraction, adding 4-6 times of water and 20-25% of NaOH into the obtained meat paste-shaped pork sausage, adding 0.5-1.5% of protease, stirring for 15-20 min in an ultrasonic oscillator, filtering to obtain an enzymolysis solution, and performing ion exchange adsorption to obtain resin; heparin elution: filtering the resin to obtain an eluent; ethanol precipitation: filtering the eluent to obtain a precipitate, drying the precipitate to obtain crude heparin sodium, weighing 80-120 g of crude heparin sodium, dissolving the crude heparin sodium by using purified water, controlling the temperature of the solution to be below 30 ℃, stirring and dissolving to prepare 10-20% (w/v) of crude heparin sodium solution; adding a 2% NaCl solution into the crude heparin sodium solution to prepare a 5% -10% (w/v) crude heparin sodium solution, adding alkaline protease, and performing heat preservation and enzymolysis at 35-50 ℃ for 3-6 hours to obtain an enzymolysis solution; centrifuging the obtained enzymolysis solution to obtain supernatant and precipitate, and discarding insoluble impurities; adding ethanol or methanol into the supernatant obtained in the step S2, stirring and standing to obtain a precipitate; dissolving the precipitate in distilled water, slowly adding sodium nitrite to degrade heparin sodium under stirring until the degradation reaction is finished, adding sodium hydroxide, and adding sodium borohydride to reduce the heparin sodium; dissolving the precipitate with purified water to obtain 10-15 wt% solution, filtering with 0.1 micron filter membrane, and spray drying to obtain heparin sodium product.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (7)
1. A production and purification process of high-purity low-molecular-weight heparin sodium is characterized by comprising the following steps:
s1 weighing 80-120 g of crude heparin sodium, dissolving with purified water, controlling the temperature of the solution to be below 30 ℃, stirring and dissolving to prepare 10-20% (w/v) of crude heparin sodium solution;
s2, adding the crude heparin sodium solution into a NaCl solution with the concentration of 2% to prepare a crude heparin sodium solution with the concentration of 5% -10% (w/v), adding alkaline protease, and performing heat preservation and enzymolysis at 35-50 ℃ for 3-6 h to obtain an enzymolysis solution;
s3, centrifugally separating the enzymolysis liquid obtained in the step S2 to obtain supernatant and precipitate, and discarding insoluble impurities;
s4, adding ethanol or methanol into the supernatant obtained in the step S2, stirring and standing to obtain a precipitate;
s5, dissolving the precipitate in distilled water, slowly adding sodium nitrite to degrade heparin sodium under stirring until the degradation reaction is finished, adding sodium hydroxide, and adding sodium borohydride to reduce the heparin sodium;
s6 dissolving the precipitate with purified water to 10-15 wt% solution, filtering with 0.1 μm filter membrane, and spray drying to obtain heparin sodium product.
2. The process for producing and purifying high-purity low-molecular-weight heparin sodium according to claim 1, wherein the temperature of step S4 is adjusted to 0-4 ℃, the mixture is placed for 5-12 hours, the precipitate is collected and dissolved in distilled water with the volume 4-8 times that of the solution, after the precipitate is completely dissolved, hydrogen peroxide with the volume 0.5% -1.2% that of the solution is added for 8-12 hours, the solution is filtered by a filter membrane, ethanol with the concentration of 95% and the volume 2 times that of the solution is added for precipitation, and the precipitate is placed for 6-15 hours to obtain the precipitate.
3. The process for producing and purifying high-purity low-molecular-weight heparin sodium according to claim 1, wherein the rotation speed of the centrifugal separation in the step S3 is 1000-3000 r/min, and the centrifugal time is 10-60 min.
4. The production and purification process of high-purity low-molecular-weight heparin sodium according to claim 1, wherein the volume ratio of ethanol or methanol to the supernatant in step S4 is 1-5:1, the stirring time is 1-3 h, and the standing time is 3-10 h.
5. The process for producing and purifying high-purity low-molecular-weight heparin sodium according to claim 1, wherein the dosage of the alkaline protease is 0.5-1% of the crude heparin sodium solution.
6. The production and purification process of high-purity low-molecular-weight heparin sodium according to claim 1, wherein after the reduction in step S5 is finished, a 1 ten thousand molecular-weight ultrafilter is used to intercept macromolecules, the pH of the ultrafiltrate is adjusted to 6.2-7.0, ethanol with the volume 1.5 times that of the ultrafiltrate is added for precipitation for 3-12 hours, and finally, ethanol with the concentration of 95% is used for dehydration and drying to obtain the low-molecular-weight heparin sodium.
7. The process for producing and purifying high-purity low-molecular-weight heparin sodium according to claim 1, wherein the preparation method of the crude heparin sodium in step S1 comprises the following steps:
s1 raw material treatment: carefully cleaning the pork intestines with clear water to remove internal and external dirt, and mincing the pork intestines into meat paste;
s2, performing ultrasonic enzymolysis extraction, adding 4-6 times of water and 20-25% of NaOH into the obtained meat paste-shaped pork sausage, adding 0.5-1.5% of protease, stirring for 15-20 min in an ultrasonic oscillator, filtering to obtain an enzymolysis solution, and performing ion exchange adsorption to obtain resin;
s3 heparin elution: filtering the resin to obtain an eluent;
s4 ethanol precipitation: filtering the eluent to obtain a precipitate, and drying the precipitate to obtain the crude product of heparin sodium.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113004436A (en) * | 2021-04-30 | 2021-06-22 | 山东万邦赛诺康生化制药股份有限公司 | Preparation method of dalteparin sodium and application of method in preparation of low-molecular-weight heparin sodium |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107011463A (en) * | 2017-05-31 | 2017-08-04 | 广元市海天实业有限责任公司 | A kind of production method of low molecular weight heparin sodium |
| CN107011464A (en) * | 2017-05-31 | 2017-08-04 | 广元市海天实业有限责任公司 | A kind of efficient crude heparin sodium production technology |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107011463A (en) * | 2017-05-31 | 2017-08-04 | 广元市海天实业有限责任公司 | A kind of production method of low molecular weight heparin sodium |
| CN107011464A (en) * | 2017-05-31 | 2017-08-04 | 广元市海天实业有限责任公司 | A kind of efficient crude heparin sodium production technology |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113004436A (en) * | 2021-04-30 | 2021-06-22 | 山东万邦赛诺康生化制药股份有限公司 | Preparation method of dalteparin sodium and application of method in preparation of low-molecular-weight heparin sodium |
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Application publication date: 20201204 |