CN110922506A - High-clarity heparin sodium - Google Patents
High-clarity heparin sodium Download PDFInfo
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- CN110922506A CN110922506A CN201911266359.2A CN201911266359A CN110922506A CN 110922506 A CN110922506 A CN 110922506A CN 201911266359 A CN201911266359 A CN 201911266359A CN 110922506 A CN110922506 A CN 110922506A
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
Abstract
The invention relates to high-clarity heparin sodium, which comprises the following components in percentage by weight: carrying out enzymolysis, oxidation and ethanol precipitation on the heparin sodium crude product, filtering, and collecting feed liquid I; adding a sodium carbonate solution and dichloromethane into the feed liquid I, standing, filtering for the second time, collecting filtrate, precipitating with ethanol again, and collecting feed liquid II; and diluting and acidifying the feed liquid II, and filtering again to obtain the high-purity heparin sodium. Solves the problem that the existing heparin sodium is relatively rich in residual protein and nucleic acid during refining, and greatly reduces insoluble particles during refining. Thereby obtaining heparin sodium with higher purity.
Description
Technical Field
The invention relates to a method for refining crude heparin sodium, in particular to a method for producing raw material heparin sodium suitable for producing low molecular weight heparin.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.
Heparin sodium is a clinical commonly used anticoagulant drug, widely exists in internal organs and intestinal mucosa of mammals, is mainly mucopolysaccharide sulfate consisting of D-glucosamine, L-iduronic acid, M-acetylglucosamine and D-glucuronic acid in an alternating manner, has good activity in aspects of anticoagulation, promotion of release of plasmepsin, body cytolytic system supplement and the like as a natural anticoagulant substance, and is widely used for treatment of epidemic encephalitis, septicemia, thromboembolism, acute myocardial infarction, arteriosclerosis and the like. In recent years, the low molecular weight heparin sodium product is emerging, so that the application of the heparin sodium is more and more extensive.
The low molecular weight heparin sodium (calcium) is prepared by chemically degrading heparin sodium, has better antithrombotic and anticoagulant properties, is more convenient to use clinically, and also has strong anti-inflammatory and anti-tumor effects, so that the low molecular weight heparin sodium (calcium) is more and more favored clinically.
The main raw material of the low molecular weight heparin sodium (calcium) is heparin sodium, and the quality of the heparin sodium directly influences the quality of the low molecular weight heparin sodium (calcium). If impurities present in heparin sodium are carried over into the low molecular weight heparin, their quality will inevitably be adversely affected. Because heparin mainly exists in the form of heparin sodium-protein complex in animals, a certain amount of impurities such as protein and the like are remained in crude heparin sodium extracted from the animals at present, and certain influence is generated on the activity of the heparin sodium. It also has certain influence on the production of low molecular weight heparin sodium (calcium), especially the absorbance and clarity of low molecular weight heparin sodium (calcium).
Disclosure of Invention
In order to overcome the problems, the invention provides a preparation process of high-purity heparin sodium, which solves the problem that the existing heparin sodium is relatively rich in residual protein and nucleic acid during refining and greatly reduces insoluble particles during refining. Thereby obtaining heparin sodium with higher purity.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:
a preparation process of high-purity heparin sodium comprises the following steps:
carrying out enzymolysis, oxidation and ethanol precipitation on the heparin sodium crude product, filtering, and collecting feed liquid I;
adding a sodium carbonate solution and dichloromethane into the feed liquid I, standing, filtering for the second time, collecting filtrate, precipitating with ethanol again, and collecting feed liquid II;
and diluting and acidifying the feed liquid II, filtering again, and freeze-drying to obtain the high-purity heparin sodium.
For the purification problem of heparin sodium, because the content of impurities is trace and the composition is difficult to determine, no suitable method for removing trace impurities and improving the purity of heparin sodium is found all the time at present. In the research of the application, it is occasionally found that: in the refining process of heparin sodium, dichloromethane has outstanding effect on removing trace impurities, and the quality of refined heparin sodium is improved, so that raw material heparin sodium which is more favorable for preparing low-molecular heparin sodium can be provided.
The research finds that: the purity of the heparin sodium is gradually improved along with the increase of the adding amount of the dichloromethane, but when the adding amount of the dichloromethane reaches a certain amount, the purity of the heparin sodium is not greatly improved by continuously increasing the adding amount of the dichloromethane, so that in some embodiments, the volume ratio of the dichloromethane to the feed liquid I is 0.2-5% to improve the treatment efficiency.
The research of the application finds that: in a weakly alkaline environment, a better heparin sodium purification effect can be obtained. Therefore, in some embodiments, the mass-to-volume ratio of the sodium carbonate solution to the feed liquid I is 0.3-0.5% to effectively purify heparin sodium.
In some embodiments, the resting time is 0.5-6h, residual impurities are effectively ethanol precipitated by resting, and removed by subsequent filtration.
In some embodiments, the absorbance of the oxidized feed solution is less than 0.2; if the concentration is more than 0.2, the enzymolysis and oxidation treatment are repeated to improve the efficiency of subsequent purification.
In some embodiments, the absorbance of the filtrate after ethanol precipitation is less than 0.2; if the concentration is more than 0.2, the oxidation and ethanol precipitation treatment are repeated, so that the early treatment effect is ensured, and the subsequent purification treatment is facilitated.
The specific purification method of heparin sodium is not particularly limited in this application, and in some embodiments, the specific steps of the enzymatic hydrolysis are as follows: diluting the heparin sodium crude product into a solution with the concentration of 5-15 w/v%, heating to 35.0-55.0 ℃, adjusting the pH value to 7-9, adding dissolved protease, keeping the temperature and the pH value for 6 hours under stirring, adding a calcium chloride solution, continuously stirring, and filtering; the covalent bond between heparin and protein is cut off by protease, so that heparin is dissociated.
In some embodiments, the specific steps of the oxidation are: adjusting the temperature of the filtrate to room temperature, adjusting the pH value of the feed liquid to 10-11, adding hydrogen peroxide for oxidation under the condition of keeping 10-11, keeping the pH value to 10-11, and keeping the oxidation time to 6-20 hours, wherein the whole hydrogen peroxide addition amount is not more than 4% so as to oxidize protein and nucleic acid.
In some embodiments, the specific steps of the re-ethanol precipitation are: adding 40.0-50.0 deg.C ethanol into the feed liquid, controlling the ethanol concentration of the feed liquid at 46 + -2% and the temperature at 35.0-40.0 deg.C, standing at the constant temperature, precipitating with ethanol for 4 + -1 hr, and repeating the ethanol precipitation twice; impurities such as crude protein in the system are removed by an alcohol precipitation mode, and the purity is improved.
In some embodiments, the specific steps of lyophilization are: diluting to 8-16% solution, adjusting pH to 6.0 + -0.5, filtering, and freeze drying to obtain high purity heparin sodium, wherein freeze drying process can avoid deterioration of heat sensitive substance, so that heparin sodium has good activity.
The invention also provides the high-purity heparin sodium prepared by any one of the methods.
The invention also provides application of the high-purity heparin sodium in preparing antithrombotic, anticoagulant, anti-inflammatory or antitumor drugs.
The invention has the beneficial effects that:
(1) the invention provides a preparation process of high-purity heparin sodium, which solves the problem that the existing heparin sodium is relatively rich in residual protein and nucleic acid during refining and greatly reduces insoluble particles during refining. Thereby obtaining the heparin sodium with higher purity.
(2) The operation method is simple, low in cost, universal and easy for large-scale production.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. 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 application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular is intended to include the plural unless the context clearly dictates otherwise, and it should be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of features, steps, operations, devices, components, and/or combinations thereof.
As introduced in the background art, the problem that a certain amount of impurities such as protein and the like remain in the existing heparin sodium, which affects the activity of the heparin sodium and the quality of the low molecular weight heparin sodium (calcium) prepared by taking the heparin sodium as a raw material subsequently is solved. Therefore, the invention provides a preparation process of high-purity heparin sodium, which specifically comprises the following steps:
1. and (3) enzymolysis protein removal: diluting the heparin sodium crude product into a solution with the concentration of 5-15% (w/v) by using purified water, heating to 35.0-55.0 ℃, adjusting the pH value to 7-9, adding dissolved 2709 protease, keeping the temperature and the pH value for 6 hours under stirring, adding a calcium chloride solution, continuously stirring, and filtering by using a plate-and-frame filter;
2. and (3) oxidation: adjusting the temperature of the filtrate to room temperature, adjusting the pH value of the feed liquid to 10-11 by using 4mol/L sodium hydroxide solution, adding 30% hydrogen peroxide of 1% of the volume of the feed liquid under the condition of keeping the pH value of 10-11 for oxidation, detecting the pH change of the feed liquid every half hour, if the pH is lower than 10, timely adjusting the pH value to 10-11, and supplementing 0.5% of hydrogen peroxide, wherein the adding amount of the whole hydrogen peroxide is not more than 4%. The oxidation time is 6-20 hours. And (4) taking the feed liquid, detecting that the absorbance of 280 nanometers is less than 0.2 after filtering the filtrate. If the absorbance is greater than 0.2, the process of previous 2 is repeated.
3. And (3) carrying out alcohol precipitation after oxidation, and filtering the feed liquid according to the volume of the feed liquid: ethanol volume 1: 1.0-1: 1.2 adding ethanol heated to 40.0-50.0 deg.C, controlling the ethanol concentration of the feed liquid to 40 + -2%, the temperature to 35.0-40.0 deg.C, and standing for precipitation for 4 + -1 h.
4. And (5) filtering, taking the filtrate for detection, and performing step 5 if the absorbance of the filtrate is less than 0.2. If the absorbance is greater than 0.2, the steps of 2 and 3 are repeated.
5. Impurity removal: filtering with a plate-frame filter, collecting filtrate, adding anhydrous sodium carbonate with a volume of 0.3% of that of sodium carbonate solution, adding dichloromethane with a volume of 0.2-5% (V/V) of the solution under stirring, standing for 0.5-6 hr, and filtering with a plate-frame filter;
6. ethanol precipitation again: according to the volume ratio of the feed liquid to the ethanol, namely 1: 1.1-1: 1.3 adding ethanol heated to 40.0-50.0 deg.C, controlling ethanol concentration of the feed liquid at 46 + -2% and temperature at 35.0-40.0 deg.C, standing for 4 + -1 h, and repeating precipitation twice.
7. And (3) freeze drying: preparing the precipitate obtained in the step 6 into a solution with the concentration of 8-16% by using water for injection, adjusting the pH value to 6.0 +/-0.5 by using 4mol/L hydrochloric acid, filtering, and freeze-drying to obtain the high-purity heparin sodium.
The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.
Example 1:
a preparation process of high-purity heparin sodium specifically comprises the following steps:
1. and (3) enzymolysis protein removal: diluting the heparin sodium crude product into a solution with the concentration of 10% (w/v) by using purified water, heating to 45.0 ℃, adjusting the pH value to 7-9, adding dissolved 2709 protease, keeping the temperature and the pH value under stirring for 6 hours, adding a calcium chloride solution, continuously stirring, and filtering by using a plate-and-frame filter;
2. and (3) oxidation: adjusting the temperature of the filtrate to room temperature, adjusting the pH value of the feed liquid to 10-11 by using 4mol/L sodium hydroxide solution, adding 30% hydrogen peroxide of 1% of the volume of the feed liquid under the condition of keeping the pH value of 10-11, detecting the pH change of the feed liquid every half hour, adjusting the pH value to 10-11 in time if the pH value is lower than 10, and supplementing 0.5% of hydrogen peroxide, wherein the adding amount of the whole hydrogen peroxide is not more than 4%. The oxidation time is 6-20 hours. And (4) taking the feed liquid, detecting that the absorbance of 280 nanometers is less than 0.2 after filtering the filtrate. If the absorbance is greater than 0.2, the process of previous 2 is repeated.
3. And (3) carrying out alcohol precipitation after oxidation, and filtering the feed liquid according to the volume of the feed liquid: adding ethanol with the volume of 1:1.1 into the ethanol, heating to 45.0 ℃, controlling the concentration of ethanol in the feed liquid to be 40 +/-2% and the temperature to be 37.0 ℃, and standing and precipitating for 4 hours under the condition of heat preservation.
4. And (5) filtering, taking the filtrate for detection, and performing step 5 if the absorbance of the filtrate is less than 0.2. If the absorbance is greater than 0.2, the steps of 2 and 3 are repeated.
5. Impurity removal: filtering with a plate-frame filter, collecting filtrate, adding anhydrous sodium carbonate with a volume of 0.3% of that of sodium carbonate solution, adding dichloromethane with a volume of 2.5% (V/V) of the solution under stirring, standing for 3 hr, and filtering with the plate-frame filter;
6. ethanol precipitation again: adding ethanol heated to 45 ℃ according to the volume ratio of the feed liquid to the ethanol of 1:1.2, controlling the concentration of the ethanol in the feed liquid to be 46 +/-2% and the temperature to be 37 ℃, keeping the temperature, standing and precipitating for 4 hours, and repeating precipitation twice.
7. And (3) freeze drying: preparing the precipitate obtained in the step 6 into a solution with the concentration of 12% by using water for injection, adjusting the pH value to 6.0 +/-0.5 by using 4mol/L hydrochloric acid, filtering, and freeze-drying to obtain the high-purity heparin sodium.
Example 2:
a preparation process of high-purity heparin sodium specifically comprises the following steps:
1. and (3) enzymolysis protein removal: diluting the heparin sodium crude product into a solution with the concentration of 5% (w/v) by using purified water, heating to 55.0 ℃, adjusting the pH value to 7-9, adding dissolved 2709 protease, keeping the temperature and the pH value under stirring for 6 hours, adding a calcium chloride solution, continuously stirring, and filtering by using a plate-and-frame filter;
2. and (3) oxidation: adjusting the temperature of the filtrate to room temperature, adjusting the pH value of the feed liquid to 10-11 by using 4mol/L sodium hydroxide solution, adding 30% hydrogen peroxide of 1% of the volume of the feed liquid under the condition of keeping the pH value of 10-11, detecting the pH change of the feed liquid every half hour, adjusting the pH value to 10-11 in time if the pH value is lower than 10, and supplementing 0.5% of hydrogen peroxide, wherein the adding amount of the whole hydrogen peroxide is not more than 4%. The oxidation time was 20 hours. And (4) taking the feed liquid, detecting that the absorbance of 280 nanometers is less than 0.2 after filtering the filtrate. If the absorbance is greater than 0.2, the process of previous 2 is repeated.
3. And (3) carrying out alcohol precipitation after oxidation, and filtering the feed liquid according to the volume of the feed liquid: adding ethanol heated to 50.0 ℃ into ethanol with the volume of 1:1.0, controlling the concentration of ethanol in the feed liquid to be 40 +/-2% and the temperature to be 40.0 ℃, and standing and precipitating for 5 hours under the condition of heat preservation.
4. And (5) filtering, taking the filtrate for detection, and performing step 5 if the absorbance of the filtrate is less than 0.2. If the absorbance is greater than 0.2, the steps of 2 and 3 are repeated.
5. Impurity removal: filtering with a plate-frame filter, collecting filtrate, adding anhydrous sodium carbonate with a volume of 0.3% of that of sodium carbonate solution, adding dichloromethane with a volume of 0.2% (V/V) of the solution under stirring, standing for 6 hours, and filtering with the plate-frame filter;
6. ethanol precipitation again: adding ethanol heated to 50.0 ℃ according to the volume ratio of the ethanol to the feed liquid of 1:1.1-1, controlling the concentration of the ethanol in the feed liquid to be 46 +/-2% and the temperature to be 40.0 ℃, preserving heat, standing and precipitating for 5h, and repeating the precipitation twice.
7. And (3) freeze drying: preparing the precipitate obtained in the step 6 into a solution with the concentration of 8% by using water for injection, adjusting the pH value to 6.0 +/-0.5 by using 4mol/L hydrochloric acid, filtering, and freeze-drying to obtain the high-purity heparin sodium.
Example 3:
a preparation process of high-purity heparin sodium specifically comprises the following steps:
1. and (3) enzymolysis protein removal: diluting the heparin sodium crude product into a solution with the concentration of 15% (w/v) by using purified water, heating to 35.0 ℃, adjusting the pH value to 7-9, adding dissolved 2709 protease, keeping the temperature and the pH value under stirring for 6 hours, adding a calcium chloride solution, continuously stirring, and filtering by using a plate-and-frame filter;
2. and (3) oxidation: adjusting the temperature of the filtrate to room temperature, adjusting the pH value of the feed liquid to 10-11 by using 4mol/L sodium hydroxide solution, adding 30% hydrogen peroxide of 1% of the volume of the feed liquid under the condition of keeping the pH value of 10-11, detecting the pH change of the feed liquid every half hour, adjusting the pH value to 10-11 in time if the pH value is lower than 10, and supplementing 0.5% of hydrogen peroxide, wherein the adding amount of the whole hydrogen peroxide is not more than 4%. The oxidation time was 6 hours. And (4) taking the feed liquid, detecting that the absorbance of 280 nanometers is less than 0.2 after filtering the filtrate. If the absorbance is greater than 0.2, the previous step 2 is repeated.
3. And (3) carrying out alcohol precipitation after oxidation, and filtering the feed liquid according to the volume of the feed liquid: adding ethanol with the volume of 1:1.2 into the ethanol which is heated to 40.0 ℃, controlling the concentration of the ethanol in the feed liquid to be 40 +/-2% and the temperature to be 35.0 ℃, and standing and precipitating for 3 hours under the condition of heat preservation.
4. And (5) filtering, taking the filtrate for detection, and performing step 5 if the absorbance of the filtrate is less than 0.2. If the absorbance is greater than 0.2, the steps of 2 and 3 are repeated.
5. Impurity removal: filtering with a plate-frame filter, collecting filtrate, adding anhydrous sodium carbonate with a volume of 0.3% of that of sodium carbonate solution, adding dichloromethane with a volume of 5% (V/V) of the solution under stirring, standing for 0.5 hr, and filtering with the plate-frame filter;
6. ethanol precipitation again: according to the volume of the feed liquid, the volume of ethanol is 1: 1.3 adding ethanol heated to 40.0 ℃, controlling the concentration of ethanol in the feed liquid to be 46 +/-2 percent and the temperature to be 35.0 ℃, preserving heat, standing and precipitating for 3 hours, and repeating precipitation twice.
7. And (3) freeze drying: preparing the precipitate obtained in the step 6 into a solution with the concentration of 16% by using water for injection, adjusting the pH value to 6.0 +/-0.5 by using 4mol/L hydrochloric acid, filtering, and freeze-drying to obtain the high-purity heparin sodium.
Comparative example 1
The difference from example 1 is that: in step 5, no dichloromethane was added.
Comparative example 2
The difference from example 1 is that: in step 5, trichloroacetic acid was added instead of dichloromethane.
Comparative example 3
The difference from example 1 is that: in step 5, trifluorotrichloroethane was added in place of dichloromethane.
Comparative example 4
The difference from example 1 is that: in step 5, vinyl acetate was added instead of dichloromethane.
Two methods are adopted in the experimental process to judge whether the residues of protein and nucleic acid in the heparin sodium meet the specification: one is clarity, which is less than 0.5 specified by the quality standard of heparin sodium recorded in Chinese pharmacopoeia; one is to detect ultraviolet absorption at 260nm and 280nm, and the quality standard stated in Chinese pharmacopoeia specifies that the ultraviolet absorption of heparin sodium at 260nm and 280nm should be less than 0.1, and the specific detection method is as follows: taking the product, adding water to make into solution containing 4mg per 1ml, and measuring by ultraviolet spectrophotometry (appendix IV A), wherein the absorbance at 260nm wavelength is not more than 0.10; at the wavelength of 280nm, the absorbance of the solution is not more than 0.10;
TABLE 1 heparin sodium detection results
From the above experimental data, it can be seen that, in the experiment in which dichloromethane is added in step 5, compared with the experiment in which dichloromethane, trichloroacetic acid, trifluorotrichloroethane and vinyl acetate are not added, the clarity of the product meets the standard requirement, and the ultraviolet absorption value at 260nm/280nm is lower than that of other comparative experiments, which indicates that the content of impurities in the protein in the product is very low, so that the quality of the heparin sodium product is better and better by adding dichloromethane.
In order to further prove that the product quality obtained by the step 5 is better in the process, 5 batches of raw material heparin sodium prepared by a dichloromethane adding process and 5 batches of heparin sodium prepared by a dichloromethane adding process are respectively used as starting materials to respectively prepare 5 batches of low molecular heparin sodium by the same process, and the clarity and ultraviolet absorption values at 260nm and 280nm of the low molecular heparin sodium are detected.
Table 2 preparation of low molecular weight heparin sodium detection results
According to experimental data, the low-molecular heparin sodium is further prepared by using the heparin sodium prepared by the dichloromethane adding process, the content of protein and nucleic acid impurities in the obtained low-molecular heparin sodium is lower, and the clarity is better, so that the heparin sodium prepared by the dichloromethane adding process is more suitable for preparing high-quality low-molecular heparin sodium.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or some of them can be substituted. Any modification, equivalent replacement, or improvement made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood that the scope of the present invention is not limited thereto, and those skilled in the art will appreciate that various modifications and variations can be made without inventive changes by those skilled in the art based on the technical solutions of the present invention.
Claims (10)
1. A preparation process of high-purity heparin sodium is characterized by comprising the following steps:
carrying out enzymolysis, oxidation and ethanol precipitation on the heparin sodium crude product, filtering, and collecting feed liquid I;
adding a sodium carbonate solution and dichloromethane into the feed liquid I, standing, filtering for the second time, collecting filtrate, precipitating with ethanol again, and collecting feed liquid II;
and diluting and acidifying the feed liquid II, and filtering again to obtain the high-purity heparin sodium.
2. The process for preparing high-purity heparin sodium according to claim 1, wherein the volume ratio of the dichloromethane to the feed liquid I is 0.2-5%.
3. The process for preparing high-purity heparin sodium according to claim 1, wherein the mass volume ratio of the sodium carbonate solution to the feed liquid I is 0.3-0.5%.
4. The process for preparing high purity heparin sodium according to claim 1, wherein the resting time is 0.5-6 h.
5. The process for preparing high-purity heparin sodium according to claim 1, wherein the absorbance of the oxidized feed liquid is less than 0.2; if the content is more than 0.2, the enzymolysis and oxidation treatment are repeated.
6. The process for preparing high-purity heparin sodium according to claim 1, wherein the absorbance of the filtrate after ethanol precipitation is less than 0.2; if the concentration is more than 0.2, the oxidation and ethanol precipitation treatment is repeated.
7. The process for preparing high-purity heparin sodium according to claim 1, wherein the enzymolysis specifically comprises the following steps: diluting the heparin sodium crude product into a solution with the concentration of 5-15 w/v%, heating to 35.0-55.0 ℃, adjusting the pH value to 7-9, adding dissolved protease, keeping the temperature and the pH value for 6 hours under stirring, adding a calcium chloride solution, continuously stirring, and filtering;
or the oxidation comprises the following specific steps: adjusting the temperature of the filtrate to room temperature, adjusting the pH value of the feed liquid to 10-11, adding hydrogen peroxide for oxidation under the condition of keeping 10-11, keeping the pH value to 10-11, and keeping the oxidation time to 6-20 hours, wherein the whole addition amount of the hydrogen peroxide is not more than 4%.
8. The process for preparing high-purity heparin sodium according to claim 1, wherein the specific steps of ethanol precipitation again are as follows: adding 40.0-50.0 deg.C ethanol into the feed liquid, controlling the ethanol concentration of the feed liquid at 46 + -2% and the temperature at 35.0-40.0 deg.C, standing for 4 + -1 hr, and repeating ethanol precipitation twice;
the freeze-drying comprises the following specific steps: diluting to 8-16% solution, adjusting pH to 6.0 + -0.5, filtering, and freeze drying to obtain high purity heparin sodium.
9. High purity heparin sodium prepared by the method of any one of claims 1-8.
10. Use of the high purity heparin sodium of claim 9 in the preparation of an antithrombotic, anticoagulant, anti-inflammatory or antitumor medicament.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113061199A (en) * | 2021-04-13 | 2021-07-02 | 重庆博万生物制药有限公司 | Process for concentrating and extracting crude heparin sodium by using nanofiltration membrane |
| CN115028757A (en) * | 2022-06-29 | 2022-09-09 | 江苏麦德森制药有限公司 | Decolorizing method of heparin sodium |
-
2019
- 2019-12-11 CN CN201911266359.2A patent/CN110922506A/en not_active Withdrawn
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113061199A (en) * | 2021-04-13 | 2021-07-02 | 重庆博万生物制药有限公司 | Process for concentrating and extracting crude heparin sodium by using nanofiltration membrane |
| CN115028757A (en) * | 2022-06-29 | 2022-09-09 | 江苏麦德森制药有限公司 | Decolorizing method of heparin sodium |
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Application publication date: 20200327 |