A method for preparing ultra-low molecular weight heparin from nadroparin calcium waste

Abstract

The invention discloses a method for preparing ultra-low molecular weight heparin from nadroparin calcium waste, comprising the following steps: S1, collecting waste liquid, S2, oxidation, S3, primary precipitation, S4, secondary dissolution, S5, chromatography, S6, freeze drying. The invention uses waste in the process of producing nadroparin calcium from ordinary heparin sodium as raw material, and prepares ultra-low molecular weight heparin through oxidation, primary precipitation, secondary dissolution, chromatography, freeze drying and other steps, thereby solving the problem of waste waste and turning waste into treasure, improving the utilization rate of waste, reducing production cost, saving a large amount of resources, and the quality of the prepared ultra-low molecular weight heparin is within the specified standard. Therefore, the invention has a wide application prospect in the prevention and treatment of anticoagulation and antithrombosis.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine preparation, and in particular relates to a method for preparing ultra-low molecular weight heparin from nadroparin calcium waste. Background Art

[0002] Ultra low molecular weight heparin is made from heparin from porcine intestinal mucosa through chemical degradation. Its structure is mainly composed of 4 to 12 monosaccharides, which are mainly glucuronic acid, glucosamine and iduronic acid and their derivatives. The molecular weight of ultra low molecular weight heparin is 2000 to 3600. It is mainly used in the prevention and treatment of anticoagulation and antithrombosis in clinical practice. In addition, the anticoagulant activity of ultra low molecular weight heparin is very low, about 5IU / mg. Therefore, its safety in clinical application is higher than that of heparin and low molecular weight heparin.

[0003] At present, ultra-low molecular weight heparin is mainly produced directly using ordinary heparin sodium as raw material through chemical degradation, enzymatic hydrolysis or hydrogen peroxide method. Although ultra-low molecular weight heparin can be obtained through these methods, there are problems such as low utilization rate of heparin sodium raw material and partial loss of heparin sodium during the production process, thereby increasing production costs.

[0004] In the production and preparation of nadroparin calcium, the main process is to over-crack ordinary heparin sodium with sodium nitrite under acidic conditions, remove some small molecules through two alcohol precipitations, and finally control the product molecular weight to meet the quality standards. However, during the two alcohol precipitations, there is loss and waste of small molecules.

[0005] In view of this, it is necessary to provide a method for preparing ultra-low molecular weight heparin from nadroparin calcium waste to solve the above technical problems. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for preparing ultra-low molecular weight heparin from nadroparin calcium waste. The present invention collects waste from the process of producing nadroparin calcium from ordinary heparin sodium, reprocesses the waste, turns waste into treasure, produces ultra-low molecular weight heparin, reduces production costs, and saves resources.

[0007] In order to solve the above problems existing in the prior art, the present invention is achieved through the following technical solutions:

[0008] A method for preparing ultra-low molecular weight heparin from nadroparin calcium waste comprises the following steps:

[0009] S1. Collection of waste liquid: collecting waste liquid from the production process of nadroparin calcium, then adding ethanol to precipitate, discarding the supernatant after the precipitation, adding water to dissolve the precipitate to obtain a dissolved liquid;

[0010] S2, oxidation: adjusting the pH value of the solution obtained in step S1 to be alkaline, then adding a hydrogen peroxide solution to perform an oxidation reaction to obtain a first solution;

[0011] S3, primary precipitation: adjusting the pH value of the first solution obtained in step S2 to be acidic, adding ethanol, and performing primary precipitation;

[0012] S4, secondary dissolution: discard the supernatant after the primary precipitation obtained in step S3, add water to dissolve the precipitate, and obtain a second solution;

[0013] S5, chromatography: loading the second solution obtained in step S4 into a chromatography column, and then eluting with the first sodium chloride solution to remove small molecule impurities; then eluting with the second sodium chloride solution, and collecting the eluate;

[0014] S6, freeze-drying: adding ethanol to the eluate obtained in step S5, collecting the supernatant after standing, adding ethanol to the supernatant after standing, discarding the supernatant, dissolving the precipitate in water, and freeze-drying to obtain ultra-low molecular weight heparin.

[0015] Furthermore, in step S1, the collection of waste liquid specifically includes the following steps:

[0016] S11, collecting 5000-15000 ml of the first alcohol precipitation waste liquid in the production process of nadroparin calcium, adding 95% ethanol with a volume of 0.5 times the volume of the first alcohol precipitation waste liquid, precipitating, discarding the supernatant after the precipitation, adding water with a volume of 0.2 times the volume of the first alcohol precipitation waste liquid to dissolve the precipitate, and obtaining a first dissolved solution;

[0017] S12, collecting 5000-15000 ml of secondary alcohol precipitation waste liquid in the production process of nadroparin calcium, adding 95% ethanol with a volume of 0.5 times the volume of the secondary alcohol precipitation waste liquid, precipitating, discarding the supernatant after the precipitation, adding water with a volume of 0.2 times the volume of the secondary alcohol precipitation waste liquid to dissolve the precipitate, and obtaining a second dissolved solution;

[0018] S13, combining the first dissolving solution and the second dissolving solution to obtain a dissolving solution.

[0019] Furthermore, in step S11, the average molecular weight of the first dissolving solution is 650-1200, the proportion of components with a molecular weight less than 2000 is 92-99.95%, the proportion of components with a molecular weight between 2000 and 4000 is 0-8%, and the proportion of components with a molecular weight greater than 8000 is 0-0.1%.

[0020] Furthermore, in step S12, the average molecular weight of the second dissolving solution is 1400-1900, the proportion of components with a molecular weight less than 2000 is 64-84%, the proportion of components with a molecular weight between 2000 and 4000 is 15-33%, and the proportion of components with a molecular weight greater than 8000 is 0-3%.

[0021] Furthermore, in step S2, the pH value is 9.5-11.0, the mass concentration of the hydrogen peroxide solution is 30%, the added amount is 2% of the volume of the dissolved solution, the temperature of the oxidation reaction is room temperature, and the time is 4-8 hours.

[0022] Furthermore, in step S3, the pH value is 6.0-7.0, the concentration of ethanol is 95%, the amount added is 3.5 times the volume of the dissolved solution, the temperature of the first precipitation is room temperature, and the time is 16-24 hours.

[0023] Furthermore, in step S4, the mass concentration of the second solution is 1-9%.

[0024] Furthermore, 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%.

[0025] Furthermore, in step S6, the amount of 95% ethanol added to the eluent is 0.5 to 0.8 times the volume of the eluent; and the amount of 95% ethanol added to the supernatant is 2.5 times the volume of the eluent.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] The invention uses waste materials in the process of producing nadroparin calcium from ordinary heparin sodium as raw materials, and prepares ultra-low molecular weight heparin through the steps of oxidation, primary precipitation, secondary dissolution, chromatography and freeze drying, thereby solving the problem of waste material waste and turning waste into treasure, thereby improving the utilization rate of waste materials, reducing production costs, and saving a large amount of resources. Moreover, the quality of the prepared ultra-low molecular weight heparin is within the prescribed standards, and therefore, the ultra-low molecular weight heparin has broad application prospects in the prevention and treatment of anticoagulation and antithrombosis. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field without creative work based on the embodiments of the present invention are within the scope of protection of the present invention.

[0029] The conventional reagents and equipment used in the present invention, unless otherwise specified, are commercially available.

[0030] Example 1

[0031] A method for preparing ultra-low molecular weight heparin from nadroparin calcium waste comprises the following steps:

[0032] S11, collecting 5000 ml of the primary alcohol precipitation waste liquid in the production process of nadroparin calcium, adding 2500 ml of 95% ethanol, precipitating, discarding the supernatant after the precipitation, adding 100 ml of water to dissolve the precipitate, and obtaining a first dissolving solution, wherein the average molecular weight of the first dissolving solution is 673, the proportion of components with a molecular weight less than 2000 is 99.95%, the proportion of components with a molecular weight of 2000 to 4000 is 0.05%, and the proportion of components with a molecular weight greater than 8000 is 0%;

[0033] S12, collecting 5000ml of secondary alcohol precipitation waste liquid in the production process of nadroparin calcium, adding 2500ml of 95% ethanol, precipitating, discarding the supernatant after the precipitation, adding 100ml of water to dissolve the precipitate, and obtaining a second dissolving solution, wherein the average molecular weight of the second dissolving solution is 1428, the proportion of components with a molecular weight less than 2000 is 82.7%, the proportion of components with a molecular weight of 2000 to 4000 is 16.3%, and the proportion of components with a molecular weight greater than 8000 is 1%;

[0034] S13, combining the first dissolved solution and the second dissolved solution to obtain 200 ml of dissolved solution;

[0035] S2, oxidation: adjusting the pH value of the solution obtained in step S1 to 10.5, then adding 4 ml of 30% hydrogen peroxide solution, and performing oxidation reaction at room temperature for 4 hours to obtain a first solution;

[0036] S3, primary precipitation: adjusting the pH value of the first solution obtained in step S2 to 6.5, adding 700 ml of 95% ethanol, and performing primary precipitation at room temperature for 16 hours;

[0037] S4, secondary dissolution: discard the supernatant after the primary precipitation obtained in step S3, add 250 ml of water to dissolve the precipitate to obtain a second solution;

[0038] S5, chromatography: the second solution obtained in step S4 is loaded into a chromatography column, and then eluted with a first sodium chloride solution having a mass concentration of 0.4% to remove small molecule impurities; then eluted with a second sodium chloride solution having a mass concentration of 20%, and 300 ml of the eluate is collected;

[0039] S6. Lyophilization: add 180 ml of 95% ethanol to the eluate obtained in step S5, let it stand for 4 hours, collect the supernatant, add 750 ml of 95% ethanol to the supernatant, let it stand for 8 hours, discard the supernatant, dissolve the precipitate in water, and after lyophilization, 12.33 g of ultra-low molecular weight heparin can be obtained.

[0040] Example 2

[0041] A method for preparing ultra-low molecular weight heparin from nadroparin calcium waste comprises the following steps:

[0042] S11, collecting 10000ml of the primary alcohol precipitation waste liquid in the production process of nadroparin calcium, adding 5000ml of 95% ethanol to precipitate, discarding the supernatant after the precipitation, adding 200ml of water to dissolve the precipitate, and obtaining a first dissolving solution, wherein the average molecular weight of the first dissolving solution is 716, the proportion of components with a molecular weight less than 2000 is 99.75%, the proportion of components with a molecular weight of 2000 to 4000 is 0.25%, and the proportion of components with a molecular weight greater than 8000 is 0%;

[0043] S12, collecting 10000ml of secondary alcohol precipitation waste liquid in the production process of nadroparin calcium, adding 5000ml of 95% ethanol, precipitating, discarding the supernatant after the precipitation, adding 200ml of water to dissolve the precipitate, and obtaining a second dissolving solution, wherein the average molecular weight of the second dissolving solution is 1415, the proportion of components with a molecular weight less than 2000 is 83.4%, the proportion of components with a molecular weight of 2000 to 4000 is 15.8%, and the proportion of components with a molecular weight greater than 8000 is 0.8%;

[0044] S13, combining the first dissolved solution and the second dissolved solution to obtain 400 ml of dissolved solution;

[0045] S2, oxidation: adjusting the pH value of the solution obtained in step S1 to 9.5, then adding 8 ml of 30% hydrogen peroxide solution, and performing oxidation reaction at room temperature for 6 hours to obtain a first solution;

[0046] S3, primary precipitation: adjusting the pH value of the first solution obtained in step S2 to 6.0, adding 1400 ml of 95% ethanol, and performing primary precipitation at room temperature for 20 hours;

[0047] S4, secondary dissolution: discard the supernatant after the primary precipitation obtained in step S3, add 500 ml of water to dissolve the precipitate to obtain a second solution;

[0048] S5, chromatography: the second solution obtained in step S4 is loaded into a chromatography column, and then eluted with a first sodium chloride solution having a mass concentration of 0.2% to remove small molecule impurities; then eluted with a second sodium chloride solution having a mass concentration of 15%, and 600 ml of the eluate is collected;

[0049] S6. Lyophilization: add 300 ml of 95% ethanol to the eluate obtained in step S5, let it stand for 4 hours, collect the supernatant, add 1500 ml of 95% ethanol to the supernatant, let it stand for 8 hours, discard the supernatant, dissolve the precipitate in water, and after lyophilization, 33.50 g of ultra-low molecular weight heparin can be obtained.

[0050] Example 3

[0051] A method for preparing ultra-low molecular weight heparin from nadroparin calcium waste comprises the following steps:

[0052] S11, collecting 15000ml of the primary alcohol precipitation waste liquid in the production process of nadroparin calcium, adding 7500ml of 95% ethanol, precipitating, discarding the supernatant after the precipitation, adding 300ml of water to dissolve the precipitate, and obtaining a first dissolving solution, wherein the average molecular weight of the first dissolving solution is 1188, the proportion of components with a molecular weight less than 2000 is 92.1%, the proportion of components with a molecular weight of 2000 to 4000 is 7.8%, and the proportion of components with a molecular weight greater than 8000 is 0.1%;

[0053] S12, collecting 15000ml of secondary alcohol precipitation waste liquid in the production process of nadroparin calcium, adding 7500ml of 95% ethanol, precipitating, discarding the supernatant after the precipitation, adding 300ml of water to dissolve the precipitate, and obtaining a second dissolving solution, wherein the average molecular weight of the second dissolving solution is 1804, the proportion of components with a molecular weight less than 2000 is 64%, the proportion of components with a molecular weight of 2000 to 4000 is 33%, and the proportion of components with a molecular weight greater than 8000 is 3%;

[0054] S13, combining the first dissolved solution and the second dissolved solution to obtain 600 ml of dissolved solution;

[0055] S2, oxidation: adjusting the pH value of the solution obtained in step S1 to 11.0, then adding 12 ml of 30% hydrogen peroxide solution, and performing oxidation reaction at room temperature for 8 hours to obtain a first solution;

[0056] S3, primary precipitation: adjusting the pH value of the first solution obtained in step S2 to 7.0, adding 2100 ml of 95% ethanol, and performing primary precipitation at room temperature for 24 hours;

[0057] S4, secondary dissolution: discard the supernatant after the primary precipitation obtained in step S3, add 750 ml of water to dissolve the precipitate to obtain a second solution;

[0058] S5, chromatography: the second solution obtained in step S4 is loaded into a chromatography column, and then eluted with a first sodium chloride solution having a mass concentration of 0.3% to remove small molecule impurities; then eluted with a second sodium chloride solution having a mass concentration of 17%, and 900 ml of the eluate is collected;

[0059] S6. Lyophilization: add 540 ml of 95% ethanol to the eluate obtained in step S5, let it stand for 4 hours, collect the supernatant, add 2250 ml of 95% ethanol to the supernatant, let it stand for 8 hours, discard the supernatant, dissolve the precipitate in water, and after lyophilization, 40.21 g of ultra-low molecular weight heparin can be obtained.

[0060] Comparative Example 1

[0061] 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 replaced by the primary alcohol precipitation waste liquid in step S11.

[0062] Comparative Example 2

[0063] 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 replaced by the secondary alcohol precipitation waste liquid in step S12.

[0064] Example 4

[0065] The performance indicators of the ultra-low molecular weight heparins prepared in Examples 1 to 3 and Comparative Examples 1 to 2 were tested, and the results are shown in Table 1 below:

[0066] Table 1 Performance index test results of ultra-low molecular weight heparin

[0067]

[0068] As can be seen from the results in the table, the performance indicators of the ultra-low molecular weight heparins obtained in Examples 1 to 3 are all within the specified standards. In Comparative Example 1, only the primary alcohol precipitation waste liquid was used for preparation. Since the molecular weight of the primary alcohol precipitation waste liquid is relatively low, the average molecular weight of the ultra-low molecular weight heparin finally obtained is relatively small, and 500 to 8000 fractions are outside the index. In Comparative Example 2, only the secondary alcohol precipitation waste liquid was used for preparation. Since the molecular weight of the secondary alcohol precipitation waste liquid is relatively high, the average molecular weight of the ultra-low molecular weight heparin finally obtained is relatively high, and 500 to 8000 fractions are outside the index. The results show that after the primary alcohol precipitation waste liquid and the secondary alcohol precipitation waste liquid are mixed, they complement each other, so that the performance indicators of the ultra-low molecular weight heparin obtained are all within the specified standards.

[0069] The above embodiments are only specific implementation methods of the present invention, which are used to illustrate the technical solutions of the present invention rather than to limit them. The protection scope of the present invention is not limited thereto. Although the present invention has been described in detail with reference to the aforementioned embodiments, ordinary technicians in the field should understand that any technician familiar with the technical field can still modify the technical solutions recorded in the aforementioned embodiments within the technical scope disclosed by the present invention, or can easily think of changes, or make equivalent replacements for some of the technical features therein; and these modifications, changes or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be covered within the protection scope of the present invention.

Claims

1. A method for preparing ultra-low molecular weight heparin from nadroparin calcium waste, characterized in that: The steps include: S1. Collection of waste liquid: collecting waste liquid from the production process of nadroparin calcium, then adding ethanol to precipitate, discarding the supernatant after the precipitation, adding water to dissolve the precipitate to obtain a dissolved liquid; The collection of waste liquid specifically includes the following steps: S11, collecting 5000-15000 ml of the first alcohol precipitation waste liquid in the production process of nadroparin calcium, adding 95% ethanol with a volume of 0.5 times the volume of the first alcohol precipitation waste liquid, performing precipitation, discarding the supernatant after the precipitation, adding water with a volume of 0.2 times the volume of the first alcohol precipitation waste liquid to dissolve the precipitate, and obtaining a first dissolved solution; S12, collecting 5000-15000 ml of secondary alcohol precipitation waste liquid in the production process of nadroparin calcium, adding 95% ethanol with a volume of 0.5 times the volume of the secondary alcohol precipitation waste liquid, precipitating, discarding the supernatant after the precipitation, adding water with a volume of 0.2 times the volume of the secondary alcohol precipitation waste liquid to dissolve the precipitate, and obtaining a second dissolved solution; S13, combining the first dissolved solution and the second dissolved solution to obtain a dissolved solution; S2, oxidation: adjusting the pH value of the solution obtained in step S1 to be alkaline, then adding a hydrogen peroxide solution to perform an oxidation reaction to obtain a first solution; S3, primary precipitation: adjusting the pH value of the first solution obtained in step S2 to be acidic, adding ethanol, and performing primary precipitation; S4, secondary dissolution: discard the supernatant after the primary precipitation obtained in step S3, add water to dissolve the precipitate, and obtain a second solution; S5, chromatography: loading the second solution obtained in step S4 into a chromatography column, and then eluting with the first sodium chloride solution to remove small molecule impurities; then eluting with the second sodium chloride solution, and collecting the eluate; S6, freeze-drying: adding ethanol to the eluate obtained in step S5, allowing the mixture to stand, collecting the supernatant, adding ethanol to the supernatant, allowing the mixture to stand, discarding the supernatant, dissolving the precipitate in water, and freeze-drying to obtain ultra-low molecular weight heparin; In step S11, the average molecular weight of the first dissolving solution is 650-1200, the proportion of components with a molecular weight less than 2000 is 92-99.95%, the proportion of components with a molecular weight of 2000-4000 is 0-8%, and the proportion of components with a molecular weight greater than 8000 is 0-0.1%; In step S12, the average molecular weight of the second dissolving solution is 1400-1900, the proportion of components with a molecular weight less than 2000 is 64-84%, the proportion of components with a molecular weight between 2000 and 4000 is 15-33%, and the proportion of components with a molecular weight greater than 8000 is 0-3%.

2. The method for preparing ultra-low molecular weight heparin from nadroparin calcium waste according to claim 1, characterized in that: In step S2, the pH value is 9.5-11.0, the mass concentration of the hydrogen peroxide solution is 30%, the added amount is 2% of the volume of the dissolved solution, the temperature of the oxidation reaction is room temperature, and the time is 4-8 hours.

3. The method for preparing ultra-low molecular weight heparin from nadroparin calcium waste according to claim 1, characterized in that: In step S3, the pH value is 6.0-7.0, the concentration of ethanol is 95%, the amount added is 3.5 times the volume of the dissolved solution, the temperature of the first precipitation is room temperature, and the time is 16-24 hours.

4. The method for preparing ultra-low molecular weight heparin from nadroparin calcium waste according to claim 1, characterized in that: In step S4, the mass concentration of the second solution is 1-9%.

5. The method for preparing ultra-low molecular weight heparin from nadroparin calcium waste according to claim 1, characterized in that: 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%.

6. The method for preparing ultra-low molecular weight heparin from nadroparin calcium waste according to claim 1, characterized in that: In step S6, the amount of 95% ethanol added to the eluent is 0.5 to 0.8 times the volume of the eluent; the amount of 95% ethanol added to the supernatant is 2.5 times the volume of the eluent.

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