CN115057950A - Method for preparing ultra-low molecular heparin from nadroparin calcium waste - Google Patents
Method for preparing ultra-low molecular heparin from nadroparin calcium waste Download PDFInfo
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- 239000002699 waste material Substances 0.000 title claims abstract description 61
- 239000003055 low molecular weight heparin Substances 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 35
- 229950003543 nadroparin calcium Drugs 0.000 title claims abstract description 33
- 238000001556 precipitation Methods 0.000 claims abstract description 60
- 239000007788 liquid Substances 0.000 claims abstract description 33
- 229940127215 low-molecular weight heparin Drugs 0.000 claims abstract description 23
- 238000004519 manufacturing process Methods 0.000 claims abstract description 18
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 14
- 238000004108 freeze drying Methods 0.000 claims abstract description 13
- 238000004090 dissolution Methods 0.000 claims abstract description 9
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 8
- 230000003647 oxidation Effects 0.000 claims abstract description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 68
- 239000006228 supernatant Substances 0.000 claims description 34
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 28
- 239000002244 precipitate Substances 0.000 claims description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 22
- 239000003480 eluent Substances 0.000 claims description 14
- 239000011780 sodium chloride Substances 0.000 claims description 14
- 150000003138 primary alcohols Chemical class 0.000 claims description 13
- 150000003333 secondary alcohols Chemical class 0.000 claims description 13
- 230000001376 precipitating effect Effects 0.000 claims description 12
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 10
- 239000012535 impurity Substances 0.000 claims description 5
- 238000011068 loading method Methods 0.000 claims description 5
- 230000002378 acidificating effect Effects 0.000 claims description 3
- 229920000669 heparin Polymers 0.000 abstract description 10
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 abstract description 7
- 229960001008 heparin sodium Drugs 0.000 abstract description 7
- 239000002994 raw material Substances 0.000 abstract description 5
- 230000010100 anticoagulation Effects 0.000 abstract description 4
- 230000002265 prevention Effects 0.000 abstract description 3
- 208000007536 Thrombosis Diseases 0.000 abstract description 2
- 230000001590 oxidative effect Effects 0.000 abstract 1
- 230000000052 comparative effect Effects 0.000 description 5
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 3
- 239000003146 anticoagulant agent Substances 0.000 description 3
- 229960002897 heparin Drugs 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 2
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 description 2
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 2
- 229940127219 anticoagulant drug Drugs 0.000 description 2
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 2
- 238000002144 chemical decomposition reaction Methods 0.000 description 2
- 229960002442 glucosamine Drugs 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- AEMOLEFTQBMNLQ-HNFCZKTMSA-N L-idopyranuronic acid Chemical compound OC1O[C@@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-HNFCZKTMSA-N 0.000 description 1
- 230000002785 anti-thrombosis Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 229940097043 glucuronic acid Drugs 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
Classifications
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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
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
- C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
-
- 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
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
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- Polymers & Plastics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Sustainable Development (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Diabetes (AREA)
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- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention discloses a method for preparing ultra-low molecular heparin from nadroparin calcium waste, which comprises the following steps: s1, collecting waste liquid, S2, oxidizing, S3, performing primary precipitation, S4, performing secondary dissolution, S5, performing chromatography, S6, and freeze-drying; the method takes the waste in the process of producing nadroparin calcium by using common heparin sodium as a raw material, and prepares the ultra-low molecular weight heparin through the steps of oxidation, primary precipitation, secondary dissolution, chromatography, freeze-drying and the like, thereby solving the waste problem, changing waste into valuable, improving the utilization rate of the waste, reducing the production cost, saving a large amount of resources, and ensuring that the quality of the prepared ultra-low molecular weight heparin is within the specified standard, so the method has wide application prospect in the prevention and treatment of anticoagulation and thrombosis resistance.
Description
Technical Field
The invention belongs to the technical field of biological medicine preparation, and particularly relates to a method for preparing ultra-low molecular heparin from nadroparin calcium waste.
Background
Ultra-low molecular weight heparin (ultra low molecular weight heparin) is prepared by taking heparin from porcine intestinal mucosa as a raw material and carrying out chemical degradation. The structure of the heparin-based anticoagulant mainly comprises 4-12 monosaccharides which are mainly glucuronic acid, glucosamine, iduronic acid and derivatives of glucuronic acid and glucosamine, the molecular weight of the ultra-low molecular heparin is 2000-3600, the ultra-low molecular heparin-based anticoagulant mainly is clinically applied to prevention and treatment of anticoagulation and antithrombotic, and in addition, the anticoagulation activity of the ultra-low molecular heparin is very low and is about 5IU/mg, so that the safety of clinical application is higher than that of heparin and low molecular weight heparin.
At present, the ultra-low molecular heparin is directly produced by using common heparin sodium as a raw material through a chemical degradation method, an enzymolysis method or a hydrogen peroxide method, and the ultra-low molecular heparin can be obtained through the methods, but the problems that the utilization rate of the raw material of the heparin sodium is low, partial heparin sodium is lost in the production process, the production cost is increased and the like exist.
In the production and preparation process of nadroparin calcium, the main process is that ordinary heparin sodium is subjected to over-cracking under an acidic condition through sodium nitrite, partial small molecules are removed through twice alcohol precipitation, and finally the molecular weight of the product is controlled to meet the quality standard. However, in the process of two times of alcohol precipitation, small molecules are lost and wasted.
In view of the above, there is a need to provide a method for preparing ultra-low molecular weight heparin from nadroparin calcium waste to solve the above technical problems.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for preparing ultra-low molecular heparin from nadroparin calcium waste. The method collects the waste materials in the process of producing nadroparin calcium by using common heparin sodium, and reprocesses the waste materials, so that the waste materials are changed into valuable, the ultra-low molecular weight heparin is produced, the production cost is reduced, and the resources are saved.
In order to solve the problems in the prior art, the invention is realized by the following technical scheme:
a method for preparing ultra-low molecular heparin from nadroparin calcium waste comprises the following steps:
s1, collecting waste liquid: collecting waste liquid in the production process of nadroparin calcium, adding ethanol, precipitating, discarding supernatant after precipitation, and adding water to dissolve precipitate to obtain a dissolved solution;
s2, oxidation: adjusting the pH value of the dissolving solution obtained in the step S1 to be alkaline, then adding a hydrogen peroxide solution, and carrying out an oxidation reaction to obtain a first solution;
s3, primary precipitation: adjusting the pH value of the first solution obtained in the step S2 to be acidic, adding ethanol, and performing primary precipitation;
s4, secondary dissolution: discarding the supernatant obtained in the step S3 after the primary precipitation, and adding water to dissolve the precipitate to obtain a second solution;
s5, chromatography: loading the second solution obtained in the step S4 into a chromatographic column, and then eluting by using a first sodium chloride solution to remove small molecular impurities; then eluting by using a second sodium chloride solution, and collecting eluent;
s6, freeze-drying: and (4) adding ethanol into the eluent obtained in the step (S5), standing, collecting supernatant, adding ethanol into the supernatant, standing, discarding the supernatant, adding water into the precipitate for dissolving, and freeze-drying to obtain the ultra-low molecular heparin.
Further, in step S1, the collecting of the waste liquid specifically includes the following steps:
s11, collecting 5000-15000 ml of primary alcohol precipitation waste liquid in the production process of nadroparin calcium, adding 95% ethanol with the volume 0.5 times that of the primary alcohol precipitation waste liquid, precipitating, discarding supernatant after precipitation, and adding water with the volume 0.2 times that of the primary alcohol precipitation waste liquid to dissolve precipitate so as to obtain a first dissolved solution;
s12, collecting 5000-15000 ml of secondary alcohol precipitation waste liquid in the nadroparin calcium production process, adding 95% ethanol with the volume 0.5 times that of the secondary alcohol precipitation waste liquid, precipitating, discarding supernatant after precipitation, and adding water with the volume 0.2 times that of the secondary alcohol precipitation waste liquid to dissolve precipitate so as to obtain a second dissolved solution;
and S13, combining the first solution and the second solution to obtain the solution.
Further, in step S11, the average molecular weight of the first solution is 650 to 1200, the ratio of components having a molecular weight of less than 2000 is 92 to 99.95%, the ratio of components having a molecular weight of 2000 to 4000 is 0 to 8%, and the ratio of components having a molecular weight of more than 8000 is 0 to 0.1%.
Furthermore, in step S12, the average molecular weight of the second solution is 1400-1900, the component ratio with the molecular weight less than 2000 is 64-84%, the component ratio with the molecular weight between 2000-4000 is 15-33%, and the component ratio with the molecular weight more than 8000 is 0-3%.
In step S2, the pH is 9.5 to 11.0, the mass concentration of the hydrogen peroxide solution is 30%, the addition amount is 2% of the volume of the solution, the temperature of the oxidation reaction is room temperature, and the time is 4 to 8 hours.
Further, in step S3, the pH value is 6.0 to 7.0, the concentration of ethanol is 95%, the addition amount is 3.5 times of the volume of the solution, the temperature of the primary precipitation is room temperature, and the time is 16 to 24 hours.
Further, in step S4, the mass concentration of the second solution is 1 to 9%.
Further, in step S5, the mass concentration of the first sodium chloride solution is 0.2 to 0.4%; the mass concentration of the second sodium chloride solution is 15-20%.
Further, in step S6, the amount of 95% ethanol added to the eluent is 0.5-0.8 times of the volume of the eluent; the amount of 95% ethanol added to the supernatant was 2.5 times the volume of the eluate.
Compared with the prior art, the invention has the following advantages:
the method takes the waste in the process of producing nadroparin calcium by using common heparin sodium as a raw material, and prepares the ultra-low molecular weight heparin through the steps of oxidation, primary precipitation, secondary dissolution, chromatography, freeze-drying and the like, thereby solving the waste problem, changing waste into valuable, improving the utilization rate of the waste, reducing the production cost, saving a large amount of resources, and ensuring that the quality of the prepared ultra-low molecular weight heparin is within the specified standard, so the method has wide application prospect in the prevention and treatment of anticoagulation and thrombosis resistance.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and all other embodiments obtained by those skilled in the art without any inventive work are within the scope of the present invention.
The conventional reagents and equipment used in the present invention are commercially available unless otherwise specified.
Example 1
A method for preparing ultra-low molecular heparin from nadroparin calcium waste comprises the following steps:
s11, collecting 5000ml of primary alcohol precipitation waste liquid in the production process of nadroparin calcium, adding 2500ml of 95% ethanol, precipitating, discarding supernatant after precipitation, adding 100ml of water to dissolve precipitate to obtain a first dissolved solution, wherein the average molecular weight of the first dissolved solution is 673, the component proportion of the molecular weight of less than 2000 is 99.95%, the component proportion of the molecular weight of 2000-4000 is 0.05%, and the component proportion of the molecular weight of more than 8000 is 0%;
s12, collecting 5000ml of secondary alcohol precipitation waste liquid in the production process of nadroparin calcium, adding 2500ml of 95% ethanol, precipitating, discarding supernatant after precipitation, adding 100ml of water to dissolve precipitate to obtain a second dissolved solution, wherein the average molecular weight of the second dissolved solution is 1428, the component proportion with the molecular weight of less than 2000 is 82.7%, the component proportion with the molecular weight of 2000-4000 is 16.3%, and the component proportion with the molecular weight of more than 8000 is 1%;
s13, mixing the first solution and the second solution to obtain 200ml of solution;
s2, oxidation: adjusting the pH value of the solution obtained in the step S1 to 10.5, then adding 4ml of 30% hydrogen peroxide solution, and carrying out oxidation reaction for 4h at room temperature to obtain a first solution;
s3, primary precipitation: adjusting the pH value of the first solution obtained in the step S2 to 6.5, adding 700ml of 95% ethanol, and performing primary precipitation at room temperature for 16 h;
s4, secondary dissolution: discarding the supernatant obtained in the step S3 after the primary precipitation, adding 250ml of water to dissolve the precipitate to obtain a second solution;
s5, chromatography: loading the second solution obtained in the step S4 into a chromatographic column, and then eluting by using a first sodium chloride solution with the mass concentration of 0.4% to remove small molecular impurities; then, eluting by using a second sodium chloride solution with the mass concentration of 20%, and collecting 300ml of eluent;
s6, freeze-drying: and (4) adding 180ml of 95% ethanol into the eluent obtained in the step (S5), standing for 4 hours, collecting supernatant, adding 750ml of 95% ethanol into the supernatant, standing for 8 hours, discarding the supernatant, adding water into the precipitate for dissolving, and freeze-drying to obtain 12.33g of the ultra-low molecular weight heparin.
Example 2
A method for preparing ultra-low molecular heparin from nadroparin calcium waste comprises the following steps:
s11, collecting 10000ml of primary alcohol precipitation waste liquid in the production process of nadroparin calcium, adding 5000ml of 95% ethanol, precipitating, discarding supernatant after precipitation, adding 200ml of water to dissolve precipitate to obtain a first dissolved solution, wherein the average molecular weight of the first dissolved solution is 716, the component proportion of the molecular weight of the first dissolved solution is 99.75%, the component proportion of the molecular weight of the first dissolved solution is 0.25% in the range of 2000-4000, and the component proportion of the molecular weight of the first dissolved solution is 0% in the range of 8000;
s12, collecting 10000ml of secondary alcohol precipitation waste liquid in the production process of nadroparin calcium, adding 5000ml of 95% ethanol, precipitating, discarding supernatant after precipitation, adding 200ml of water to dissolve precipitate to obtain a second dissolved solution, wherein the average molecular weight of the second dissolved solution is 1415, the component proportion of the molecular weight of less than 2000 is 83.4%, the component proportion of the molecular weight of 2000-4000 is 15.8%, and the component proportion of the molecular weight of more than 8000 is 0.8%;
s13, mixing the first solution and the second solution to obtain 400ml of solution;
s2, oxidation: adjusting the pH value of the solution obtained in the step S1 to 9.5, then adding 8ml of 30% hydrogen peroxide solution, and carrying out oxidation reaction for 6h at room temperature to obtain a first solution;
s3, primary precipitation: adjusting the pH value of the first solution obtained in the step S2 to 6.0, adding 1400ml of 95% ethanol, and performing primary precipitation at room temperature for 20 h;
s4, secondary dissolution: discarding the supernatant obtained in the step S3 after the primary precipitation, adding 500ml of water to dissolve the precipitate to obtain a second solution;
s5, chromatography: loading the second solution obtained in the step S4 into a chromatographic column, and then eluting by using a first sodium chloride solution with the mass concentration of 0.2% to remove small molecular impurities; then, eluting by using a second sodium chloride solution with the mass concentration of 15%, and collecting 600ml of eluent;
s6, freeze-drying: adding 300ml of 95% ethanol into the eluent obtained in the step S5, standing for 4h, collecting supernatant, adding 1500ml of 95% ethanol into the supernatant, standing for 8h, discarding the supernatant, adding water to dissolve the precipitate, and freeze-drying to obtain 33.50g of the ultra-low molecular heparin.
Example 3
A method for preparing ultra-low molecular heparin from nadroparin calcium waste comprises the following steps:
s11, collecting 15000ml of primary alcohol precipitation waste liquid in the production process of nadroparin calcium, adding 7500ml of 95% ethanol, precipitating, discarding supernatant after precipitation, adding 300ml of water to dissolve precipitate to obtain a first dissolved solution, wherein the average molecular weight of the first dissolved solution is 1188, the component proportion of the molecular weight of less than 2000 is 92.1%, the component proportion of the molecular weight of 2000-4000 is 7.8%, and the component proportion of the molecular weight of more than 8000 is 0.1%;
s12, collecting 15000ml of secondary alcohol precipitation waste liquid in the production process of nadroparin calcium, adding 7500ml of 95% ethanol, precipitating, discarding supernatant after precipitation, adding 300ml of water to dissolve precipitate to obtain a second dissolved solution, wherein the average molecular weight of the second dissolved solution is 1804, the component proportion with the molecular weight of less than 2000 is 64%, the component proportion with the molecular weight of 2000-4000 is 33%, and the component proportion with the molecular weight of more than 8000 is 3%;
s13, mixing the first solution and the second solution to obtain 600ml of solution;
s2, oxidation: adjusting the pH value of the solution obtained in the step S1 to 11.0, then adding 12ml of 30% hydrogen peroxide solution, and carrying out oxidation reaction for 8h at room temperature to obtain a first solution;
s3, primary precipitation: adjusting the pH value of the first solution obtained in the step S2 to 7.0, adding 2100ml of 95% ethanol, and performing primary precipitation at room temperature for 24 h;
s4, secondary dissolution: discarding the supernatant obtained in the step S3 after the primary precipitation, adding 750ml of water to dissolve the precipitate to obtain a second solution;
s5, chromatography: loading the second solution obtained in the step S4 into a chromatographic column, and then eluting by using a first sodium chloride solution with the mass concentration of 0.3% to remove small molecular impurities; then, a second sodium chloride solution with the mass concentration of 17% is used for elution, and 900ml of eluent is collected;
s6, freeze-drying: and (4) adding 540ml of 95% ethanol into the eluent obtained in the step (S5), standing for 4h, collecting supernatant, adding 2250ml of 95% ethanol into the supernatant, standing for 8h, discarding the supernatant, adding water into the precipitate for dissolving, and freeze-drying to obtain 40.21g of the ultra-low molecular weight heparin.
Comparative example 1
The method for preparing ultra-low molecular weight heparin from nadroparin calcium waste is basically the same as that in example 2, except that the secondary alcohol precipitation waste liquid in step S12 is changed into the primary alcohol precipitation waste liquid in step S11.
Comparative example 2
The method for preparing ultra-low molecular weight heparin from nadroparin calcium waste is basically the same as that in example 2, except that the primary alcohol precipitation waste liquid in step S11 is changed into the secondary alcohol precipitation waste liquid in step S12.
Example 4
The performance indexes of the ultra-low molecular weight heparin prepared in the examples 1 to 3 and the comparative examples 1 to 2 are tested, and the results are shown in the following table 1:
TABLE 1 ultra-low molecular heparin performance index test results
The results in the table show that the performance indexes of the ultra-low molecular weight heparin prepared in the examples 1 to 3 are within the specified standards, the ultra-low molecular weight heparin prepared in the comparative example 1 is prepared by using the primary alcohol precipitation waste liquid, the molecular weight of the primary alcohol precipitation waste liquid is low, the average molecular weight of the finally prepared ultra-low molecular weight heparin is small, the 500-8000 fractions are outside the indexes, the ultra-low molecular weight heparin prepared in the comparative example 2 is prepared by using the secondary alcohol precipitation waste liquid, the molecular weight of the secondary alcohol precipitation waste liquid is high, the average molecular weight of the finally prepared ultra-low molecular weight heparin is high, and the 500-8000 fractions are outside the indexes.
Although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that the following descriptions are only illustrative and not restrictive, and that the scope of the present invention is not limited to the above embodiments: any person skilled in the art can modify or easily conceive the technical solutions described in the foregoing embodiments or equivalent substitutes for some technical features within the technical scope of the present disclosure; such modifications, changes or substitutions do not depart from the spirit and scope of the embodiments of the present invention, and they should be construed as being included therein.
Claims (9)
1. A method for preparing ultra-low molecular heparin from nadroparin calcium waste is characterized by comprising the following steps:
s1, collecting waste liquid: collecting waste liquid in the production process of nadroparin calcium, adding ethanol, precipitating, discarding supernatant after precipitation, and adding water to dissolve precipitate to obtain a dissolved solution;
s2, oxidation: adjusting the pH value of the dissolving solution obtained in the step S1 to be alkaline, then adding a hydrogen peroxide solution, and carrying out oxidation reaction to obtain a first solution;
s3, primary precipitation: adjusting the pH value of the first solution obtained in the step S2 to be acidic, adding ethanol, and performing primary precipitation;
s4, secondary dissolution: discarding the supernatant obtained in the step S3 after the primary precipitation, and adding water to dissolve the precipitate to obtain a second solution;
s5, chromatography: loading the second solution obtained in the step S4 into a chromatographic column, and then eluting by using a first sodium chloride solution to remove small molecular impurities; then eluting by using a second sodium chloride solution, and collecting eluent;
s6, freeze-drying: and (4) adding ethanol into the eluent obtained in the step (S5), standing, collecting supernatant, adding ethanol into the supernatant, standing, discarding the supernatant, adding water into the precipitate for dissolving, and freeze-drying to obtain the ultra-low molecular heparin.
2. The method for preparing ultra-low molecular weight heparin from nadroparin calcium waste material as claimed in claim 1, wherein the step of collecting the waste liquid in step S1 specifically comprises the following steps:
s11, collecting 5000-15000 ml of primary alcohol precipitation waste liquid in the nadroparin calcium production process, adding 95% ethanol with the volume 0.5 times of that of the primary alcohol precipitation waste liquid, precipitating, discarding supernatant after precipitation, adding water with the volume 0.2 times of that of the primary alcohol precipitation waste liquid to dissolve precipitate, and obtaining a first dissolved solution;
s12, collecting 5000-15000 ml of secondary alcohol precipitation waste liquid in the nadroparin calcium production process, adding 95% ethanol with the volume 0.5 times that of the secondary alcohol precipitation waste liquid, precipitating, discarding supernatant after precipitation, and adding water with the volume 0.2 times that of the secondary alcohol precipitation waste liquid to dissolve precipitate so as to obtain a second dissolved solution;
and S13, combining the first solution and the second solution to obtain a solution.
3. The method of claim 2, wherein in step S11, the first solution has an average molecular weight of 650 to 1200, a component ratio of 92 to 99.95% with a molecular weight of less than 2000, a component ratio of 0 to 8% with a molecular weight of 2000 to 4000, and a component ratio of 0 to 0.1% with a molecular weight of more than 8000.
4. The method of claim 2, wherein in step S12, the average molecular weight of the second solution is 1400-1900, the component ratio with molecular weight less than 2000 is 64-84%, the component ratio with molecular weight between 2000 and 4000 is 15-33%, and the component ratio with molecular weight more than 8000 is 0-3%.
5. The method of claim 1, wherein in step S2, the pH value is 9.5-11.0, the hydrogen peroxide solution has a mass concentration of 30%, the amount of the hydrogen peroxide solution is 2% of the volume of the solution, and the temperature of the oxidation reaction is room temperature and the time is 4-8 h.
6. The method of claim 1, wherein in step S3, the pH value is 6.0-7.0, the concentration of ethanol is 95%, the addition amount is 3.5 times of the volume of the dissolution solution, the temperature of the primary precipitation is room temperature, and the time is 16-24 h.
7. The method for preparing ultra-low molecular weight heparin from nadroparin calcium waste as claimed in claim 1, wherein the mass concentration of the second solution in step S4 is 1-9%.
8. The method for preparing ultra-low molecular weight heparin from nadroparin calcium waste according to claim 1, wherein in step S5, the mass concentration of the first sodium chloride solution is 0.2-0.4%; the mass concentration of the second sodium chloride solution is 15-20%.
9. The method for preparing ultra-low molecular weight heparin from nadroparin calcium waste as claimed in claim 1, wherein in step S6, 95% ethanol is added to the eluent in an amount of 0.5-0.8 times of the volume of the eluent; the amount of 95% ethanol added to the supernatant was 2.5 times the volume of the eluate.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1749284A (en) * | 2005-09-19 | 2006-03-22 | 南京健友生物化学制药有限公司 | Purifying method for low molecule heparin |
| CN102731683A (en) * | 2012-07-17 | 2012-10-17 | 湖北亿诺瑞生物制药有限公司 | Method of separating natural low molecular heparin from heparin waste liquor |
| CN104558250A (en) * | 2015-02-03 | 2015-04-29 | 华北制药华坤河北生物技术有限公司 | Method for producing nadroparin calcium by using crude sodium heparin products |
| CN110885386A (en) * | 2019-12-23 | 2020-03-17 | 天津生物化学制药有限公司 | Method for extracting ultra-low molecular weight heparin sodium by using nadroparin calcium waste |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1749284A (en) * | 2005-09-19 | 2006-03-22 | 南京健友生物化学制药有限公司 | Purifying method for low molecule heparin |
| CN102731683A (en) * | 2012-07-17 | 2012-10-17 | 湖北亿诺瑞生物制药有限公司 | Method of separating natural low molecular heparin from heparin waste liquor |
| CN104558250A (en) * | 2015-02-03 | 2015-04-29 | 华北制药华坤河北生物技术有限公司 | Method for producing nadroparin calcium by using crude sodium heparin products |
| CN110885386A (en) * | 2019-12-23 | 2020-03-17 | 天津生物化学制药有限公司 | Method for extracting ultra-low molecular weight heparin sodium by using nadroparin calcium waste |
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