A method for preparing calcium nafamostat

Abstract

The application discloses a preparation method of calcium nartograstim, and belongs to the technical field of biological medicines. By optimizing key steps such as degradation, reduction, ion exchange and oxidation of heparin sodium, and combining with specific anion exchange resin, the yield and purity of the product are significantly improved. The method comprises the following steps: mixing heparin sodium with purified water, degrading by adjusting pH value and adding sodium nitrite; continuing to react with sodium hydroxide and sodium borohydride, and then collecting precipitate through sterilization treatment and ethanol precipitation. Subsequently, the precipitate is reacted with anion exchange resin in purified water, and after washing and precipitation, oxidation reaction is carried out. Finally, calcium nartograstim is obtained through filtration, dehydration and drying. Compared with the prior art, the preparation method has higher yield and purity, the molecular weight distribution is more concentrated, the proportion of effective components is higher, and the proportions of small molecule and large molecule impurities are significantly reduced, so that the quality and curative effect of the product are improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for preparing Nagqu heparin calcium. Background Technology

[0002] Nadroparin calcium is an important anticoagulant widely used in the prevention and treatment of thrombosis. Its preparation process typically involves complex chemical reactions and purification steps, including the degradation, reduction, ion exchange, and oxidation of heparin sodium. Traditional methods for preparing nadroparin calcium have some drawbacks, such as low yield, uneven molecular weight distribution, and high impurity content, which limit its efficacy and safety in clinical applications.

[0003] Among existing preparation methods, Chinese invention patent CN104072639A discloses a method for producing nagqu heparin calcium. This method achieves a "one-pot" process through specific process parameters, integrating steps such as degradation, reduction, ethanol precipitation, calcium conversion via ion exchange resin, oxidation, filtration, precipitation, and dehydration. This simplifies the operation process and improves production efficiency. However, this method still has some problems, such as low yield and uneven molecular weight distribution, which affect the quality and efficacy of the product.

[0004] Furthermore, traditional anion exchange resins suffer from low adsorption efficiency, poor selectivity, and insufficient stability in the preparation of nadroparin calcium, making it difficult to further improve product purity and yield. Therefore, developing a new method for preparing nadroparin calcium, optimizing process parameters, improving product yield and purity, and ensuring the concentration of molecular weight distribution are important directions for current research. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to improve the yield and purity of nagqu heparin calcium by optimizing the preparation process, particularly by improving the preparation method of anion exchange resin, while ensuring the concentration of molecular weight distribution, thereby enhancing the quality and efficacy of the product.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0007] A method for preparing Nagqu heparin calcium is as follows:

[0008] Step 1: First, mix heparin sodium with purified water, with 9-10 L of purified water for every 1.0 kg of heparin sodium. Adjust the pH of the solution to 2.5-3.0 using 3-5 mol / L hydrochloric acid. Then, add sodium nitrite, with a mass ratio of heparin sodium to sodium nitrite of 100:2-3. Stir at 20-30°C for 1-5 hours to obtain the degradation solution. Next, adjust the pH of the degradation solution to 9-10 using 3-6 mol / L sodium hydroxide aqueous solution, and then add boron hydrochloride. Sodium nitrite and sodium borohydride were mixed in a mass ratio of 1.0:0.3-0.4. The mixture was stirred for 15-25 hours. The pH of the solution was then adjusted to 3-4 with 3-5 mol / L hydrochloric acid and stirred for 10-30 minutes. The pH was then adjusted to 7-8 with 4-6 mol / L sodium hydroxide aqueous solution. The mixture was then sterilized. Ethanol was then added to precipitate the precipitate at a volume ratio of 1:0.5-2. After standing for 5-15 hours, the supernatant was removed and the precipitate was collected.

[0009] Step 2: Dissolve the precipitate obtained in Step 1 in purified water at a mass-to-volume ratio of precipitate to purified water of 1:8-10. Then add anion exchange resin at a mass-to-volume ratio of heparin sodium to anion exchange resin of 1:4-6. Stir for 5-10 hours to allow the resin to fully adsorb the precipitate. Next, prepare a 0.05-0.2 mol / L calcium chloride solution equal in mass to the resin and add it to the resin for washing. Stir for 0.5-2 hours and then drain. Repeat the above washing process once, draining after each stirring. Combine the two washing solutions and stir at 15-20°C for 5-10 minutes. Then add ethanol at a volume ratio of washing solution to ethanol of 1:1-3 and stir for 5-10 minutes. After the precipitate has settled for 5-15 hours, remove the supernatant and collect the precipitate.

[0010] Step 3: Dissolve the precipitate obtained in Step 2 in purified water, with a mass-to-volume ratio of precipitate to purified water of 1:4-5; add solid calcium chloride, with a solution volume to calcium chloride feed ratio of 1.5-2 kg calcium chloride per 100 L of solution; adjust the pH value to 10-12 with saturated calcium hydroxide solution; add hydrogen peroxide at a temperature of 20-40℃ for oxidation, with a total solution volume to hydrogen peroxide feed volume ratio of 100:1-1.5, while maintaining the pH value at 10.5-11; the oxidation reaction continues for 2-4 hours; finally, adjust the pH value to 6-6.5 with 3-5 mol / L hydrochloric acid to obtain the reaction solution.

[0011] Step 4: Filter the reaction solution from Step 3 through a filter membrane. Precipitate the filtrate with 1-3 times its volume of ethanol. After standing for 5-10 hours, remove the supernatant by vacuum extraction. Add ethanol again for dehydration. Stir to loosen the precipitate into fine particles. After standing for 0.5-2 hours, remove the supernatant again by vacuum extraction. Repeat the above dehydration operation 1-3 times. Finally, centrifuge the precipitate to remove water and place it in a vacuum drying oven. Dry it at 50-70°C for 24-72 hours to obtain nagqu heparin calcium.

[0012] The sterilization process in step 1 involves sterilizing the solution with an ultraviolet lamp for 20 to 30 minutes.

[0013] The pore size of the filter membrane in step 4 is 0.15–0.25 μm.

[0014] The preparation method of the anion exchange resin is as follows, in parts by weight:

[0015] S1. Weigh 0.5–2 parts of azobisisobutyronitrile, 50–70 parts of n-hexane, 100–150 parts of monomer compound, and 15–25 parts of dried nano-silica. Stir until homogeneous to obtain a mixture. Simultaneously, weigh 5–10 parts of sodium carboxymethyl cellulose and 70–90 parts of potassium chloride. Dissolve them in 400–500 parts of water at 80–95°C and mix until homogeneous to obtain a mixed solvent. Cool to 30–50°C. Mix the mixture with the mixed solvent. Heat to 60–80°C. Add 0.3–0.8 parts of 0.5–2 wt% colorimetric solution. Add 4–6 parts of crosslinking agent. Heat to 75–85°C. Stir and react at this temperature for 5–20 hours. After the reaction is complete, extract with anhydrous ethanol. Dry and sieve the product to obtain a pretreated product.

[0016] S2. Weigh 15-25 parts of the pretreated material prepared in step S1 and add it to 150-250 parts of dimethyl sulfoxide. Swell for 3-8 hours, add 40-60 parts of amidoyl-cysteine ​​and 100-150 parts of dimethyl sulfoxide, and add 180-220 parts of 10-18 wt% sodium ethoxide ethanol solution. Heat to 70-90°C and react at this temperature for 5-15 hours. After the reaction is complete, filter to remove the reaction solution, wash the product with water, and vacuum dry the washed product at 50-70°C for 5-15 hours to obtain the anion exchange resin.

[0017] The particle size range of the nano-silica is 10–100 nm.

[0018] The monomeric compound is at least one of 1,2-dichloro-4-isopropenylbenzene, 1-chloro-4-[1-(4-chlorophenyl)vinyl]benzene, and 1,1-bis-(4-chlorophenyl)-1,3-butadiene.

[0019] The crosslinking agent is at least one of divinylbenzene, 1,2,3-trivinylbenzene, 1,4-diamino-2,5-divinylbenzene, and 1,4-dialdehyde-2,5-divinylbenzene.

[0020] The colorimetric solution is at least one of methyl orange ethanol solution, methylene blue ethanol solution, and bromophenol blue ethanol solution.

[0021] Azobisisobutyronitrile (AIBN) is used as an initiator to initiate the polymerization reaction of monomer compounds and form the main structure of the resin.

[0022] Hexane is used as a solvent to dissolve monomeric compounds and nano-silica, ensuring their uniform distribution in the reaction system.

[0023] Monomer compounds, as the main monomers in polymerization, form the main structure of the resin and affect its chemical properties and performance.

[0024] The dried nano-silica serves as the core material, providing a core-shell structure that enhances the resin's mechanical strength and thermal stability, increases specific surface area, and improves adsorption capacity and selectivity.

[0025] Sodium carboxymethyl cellulose is used as a dispersant to form a uniform dispersion system, ensuring the uniform mixing of nano-silica and other substances, and improving the stability and flowability of the mixture.

[0026] Potassium chloride, as an electrolyte, forms a mixed solvent with sodium carboxymethyl cellulose, providing an ionic environment and promoting the reaction.

[0027] Colorimetric solutions, as indicators, use color changes to indicate the progress of a reaction and help control reaction conditions.

[0028] Crosslinking agents react with monomer compounds to form crosslinking networks, which enhance the mechanical strength and chemical stability of the resin, and improve adsorption efficiency and selective separation ability.

[0029] Dimethyl sulfoxide is used as a swelling agent to dissolve the pretreated material, allowing it to swell fully and improving the efficiency of the reaction.

[0030] As a functionalizing agent, amidocysteine ​​reacts with resin to introduce specific functional groups and improve the resin's performance.

[0031] Sodium ethoxide in ethanol acts as a catalyst, promoting the reaction and increasing the reaction rate.

[0032] Anhydrous ethanol is used as an extractant to extract unreacted monomers and impurities, thereby improving the purity of the product.

[0033] Compared with existing technologies, it has the following advantages:

[0034] 1) This invention significantly improves the yield and purity of nagqu heparin calcium by optimizing its preparation process. Simultaneously, the molecular weight distribution is more concentrated, the proportion of active ingredients is higher, and the proportion of small and large molecular impurities is significantly reduced, thereby improving the product's quality and efficacy.

[0035] 2) This invention prepares a high-performance anion exchange resin by selecting specific monomer compounds and crosslinking agents, resulting in a resin with a more uniform molecular structure and higher chemical stability. This resin exhibits higher adsorption efficiency and selective separation capability during the preparation of heparin calcium nadroprolide, reducing residual impurities and improving the concentration of the product's molecular weight distribution and yield.

[0036] 3) This invention optimizes and simplifies multiple steps in the preparation process. By precisely controlling the reaction conditions and using a specific filter membrane, the number of operation steps is reduced, production costs are lowered, and production efficiency is improved. Detailed Implementation

[0037] Main source of materials:

[0038] Nano-silica, particle size: 20nm, specific surface area: 200m² 2 / g, Product Model: PST-Q02, Nanjing Baoket New Materials Co., Ltd.

[0039] Commercially available anion exchange resin, model: D301, particle size: 0.315-1.25mm, from Langfang Tengjing Environmental Protection Technology Co., Ltd.

[0040] The design concept of this invention is to improve the yield and purity of nadroparin calcium by optimizing the preparation process, while ensuring a concentrated molecular weight distribution. Specifically, by precisely controlling the parameters of key steps such as the degradation, reduction, ion exchange, and oxidation of heparin sodium, combined with a specific anion exchange resin preparation method, efficient ion exchange and impurity removal are achieved. Furthermore, by selecting suitable crosslinking agents and monomer compounds, the performance of the resin is further optimized, thereby achieving a higher yield and a more concentrated molecular weight distribution in the preparation of nadroparin calcium.

[0041] Example 1

[0042] A method for preparing Nagqu heparin calcium is as follows:

[0043] Step 1: First, mix heparin sodium with purified water, with 9.3L of purified water for every 1kg of heparin sodium. Adjust the pH of the solution to 2.6 using 4mol / L hydrochloric acid. Then add sodium nitrite, with a mass ratio of heparin sodium to sodium nitrite of 100:2.8. Stir at 25℃ for 3 hours to obtain the degradation solution. Next, adjust the pH of the degradation solution to 9.5 using 5mol / L sodium hydroxide aqueous solution. Then add sodium borohydride, with a mass ratio of sodium nitrite to sodium borohydride of 1:0.38. Continue stirring and reacting for 20 hours. Then adjust the pH of the solution to 3.5 using 4mol / L hydrochloric acid and stir for 20 minutes. Then adjust the pH to 7.4 using 5mol / L sodium hydroxide aqueous solution. After that, sterilize the solution with a 254nm wavelength ultraviolet lamp for 25 minutes. Then add ethanol for precipitation, with a solution volume to ethanol volume ratio of 1:1. After standing for 10 hours, remove the supernatant and collect the precipitate.

[0044] Step 2: Dissolve the precipitate obtained in Step 1 in purified water at a mass-to-volume ratio of precipitate to purified water of 1:9.5. Then add anion exchange resin at a mass-to-volume ratio of heparin sodium to anion exchange resin of 1:5. Stir for 8 hours to allow the resin to fully adsorb the precipitate. Next, prepare a 0.1 mol / L calcium chloride solution equal in mass to the resin and add it to the resin for washing. Stir for 1 hour and then drain. Repeat the above washing process once, draining after each stirring. Combine the two washing solutions and stir at 18°C ​​for 8 minutes. Then add ethanol at a volume ratio of washing solution to ethanol of 1.0:2 and stir for 8 minutes. After the precipitate has settled for 10 hours, remove the supernatant and collect the precipitate.

[0045] Step 3: Dissolve the precipitate obtained in Step 2 in purified water at a mass-to-volume ratio of precipitate to purified water of 1:4.5; add calcium chloride solid at a ratio of 1.6 kg calcium chloride per 100 L of solution; adjust the pH to 11 with a saturated calcium hydroxide solution; add hydrogen peroxide at 30 °C for oxidation at a ratio of 100:1.3 between the total solution volume and the hydrogen peroxide volume, while maintaining the pH at 10.8; continue the oxidation reaction for 4 hours; finally, adjust the pH to 6.2 with 4 mol / L hydrochloric acid to obtain the reaction solution.

[0046] Step 4: Filter the reaction solution from Step 3 through a 0.2 μm filter membrane. Precipitate the filtrate with 2.0 times its volume of ethanol. After standing for 8 hours, remove the supernatant by vacuum extraction. Add ethanol again for dehydration. Stir to loosen the precipitate into fine particles. After standing for 1 hour, remove the supernatant again by vacuum extraction. Repeat the above dehydration operation 3 times. Finally, centrifuge the precipitate to remove water and place it in a vacuum drying oven. Dry it at 60°C for 48 hours to obtain Nagqu heparin calcium.

[0047] The preparation method of the anion exchange resin is as follows:

[0048] S1. Weigh 1g of azobisisobutyronitrile, 60g of n-hexane, 130g of 1,1-bis-(4-chlorophenyl)-1,3-butadiene, and 20g of dried nano-silica. Stir until homogeneous to obtain a mixture. Simultaneously, weigh 8g of sodium carboxymethyl cellulose and 80g of potassium chloride, dissolve them in 450g of water at 90℃, mix them until homogeneous to obtain a mixed solvent. Cool to 40℃, mix the mixture with the mixed solvent, heat to 70℃, add 0.5g of 1wt% methyl orange ethanol solution, add 5g of 1,4-diamino-2,5-divinylbenzene, heat to 80℃, and stir the reaction at this temperature for 12 hours. After the reaction is complete, extract with anhydrous ethanol, dry and sieve the product to obtain the pretreated product.

[0049] S2. Weigh 20g of the pretreated material prepared in step S1 and add it to 200g of dimethyl sulfoxide. Swell for 5 hours, add 50g of amidoyl-cysteine ​​and 120g of dimethyl sulfoxide, add 200g of 15wt% sodium ethoxide ethanol solution, heat to 80℃, and react at this temperature for 10 hours. After the reaction is complete, filter to remove the reaction solution, wash the product with water, and vacuum dry the washed product at 60℃ for 10 hours to obtain anion exchange resin.

[0050] Example 2

[0051] The preparation method of Nagqu heparin calcium is basically the same as that in Example 1, except that the preparation method of the anion exchange resin is different.

[0052] The preparation method of the anion exchange resin is as follows:

[0053] S1. Weigh 1g of azobisisobutyronitrile, 60g of n-hexane, 130g of 1-chloro-4-[1-(4-chlorophenyl)vinyl]benzene and 20g of dried nano-silica, stir evenly to obtain a mixture. At the same time, weigh 8g of sodium carboxymethyl cellulose and 80g of potassium chloride, dissolve them in 450g of water at 90℃, mix evenly to obtain a mixed solvent, cool to 40℃, mix the mixture with the mixed solvent, heat to 70℃, add 0.5g of 1wt% methyl orange ethanol solution dropwise, add 5g of 1,4-diamino-2,5-divinylbenzene, heat to 80℃, stir the reaction at this temperature for 12 hours. After the reaction is complete, extract with anhydrous ethanol, dry and sieve the product to obtain the pretreated product.

[0054] S2. Weigh 20g of the pretreated material prepared in step S1 and add it to 200g of dimethyl sulfoxide. Swell for 5 hours, add 50g of amidoyl-cysteine ​​and 120g of dimethyl sulfoxide, add 200g of 15wt% sodium ethoxide ethanol solution, heat to 80℃, and react at this temperature for 10 hours. After the reaction is complete, filter to remove the reaction solution, wash the product with water, and vacuum dry the washed product at 60℃ for 10 hours to obtain anion exchange resin.

[0055] Example 3

[0056] The preparation method of Nagqu heparin calcium is basically the same as that in Example 1, except that the preparation method of the anion exchange resin is different.

[0057] The preparation method of the anion exchange resin is as follows:

[0058] S1. Weigh 1g of azobisisobutyronitrile, 60g of n-hexane, 130g of 1,2-dichloro-4-isopropenylbenzene and 20g of dried nano-silica, stir evenly to obtain a mixture. At the same time, weigh 8g of sodium carboxymethyl cellulose and 80g of potassium chloride, dissolve them in 450g of water at 90℃, mix evenly to obtain a mixed solvent, cool to 40℃, mix the mixture with the mixed solvent, heat to 70℃, add 0.5g of 1wt% methyl orange ethanol solution dropwise, add 5g of 1,4-diamino-2,5-divinylbenzene, heat to 80℃, stir and react at this temperature for 12 hours. After the reaction is complete, extract with anhydrous ethanol, dry and sieve the product to obtain the pretreated product.

[0059] S2. Weigh 20g of the pretreated material prepared in step S1 and add it to 200g of dimethyl sulfoxide. Swell for 5 hours, add 50g of amidoyl-cysteine ​​and 120g of dimethyl sulfoxide, add 200g of 15wt% sodium ethoxide ethanol solution, heat to 80℃, and react at this temperature for 10 hours. After the reaction is complete, filter to remove the reaction solution, wash the product with water, and vacuum dry the washed product at 60℃ for 10 hours to obtain anion exchange resin.

[0060] Example 4

[0061] The preparation method of Nagqu heparin calcium is basically the same as that in Example 1, except that the preparation method of the anion exchange resin is different.

[0062] The preparation method of the anion exchange resin is as follows:

[0063] S1. Weigh 1g of azobisisobutyronitrile, 60g of n-hexane, 130g of 1,1-bis-(4-chlorophenyl)-1,3-butadiene, and 20g of dried nano-silica. Stir until homogeneous to obtain a mixture. Simultaneously, weigh 8g of sodium carboxymethyl cellulose and 80g of potassium chloride, dissolve them in 450g of water at 90℃, mix them until homogeneous to obtain a mixed solvent. Cool to 40℃, mix the mixture with the mixed solvent, heat to 70℃, add 0.5g of 1wt% methyl orange ethanol solution, add 5g of 1,2,3-trivinylbenzene, heat to 80℃, and stir the reaction at this temperature for 12 hours. After the reaction is complete, extract with anhydrous ethanol, dry and sieve the product to obtain the pretreated product.

[0064] S2. Weigh 20g of the pretreated material prepared in step S1 and add it to 200g of dimethyl sulfoxide. Swell for 5 hours, add 50g of amidoyl-cysteine ​​and 120g of dimethyl sulfoxide, add 200g of 15wt% sodium ethoxide ethanol solution, heat to 80℃, and react at this temperature for 10 hours. After the reaction is complete, filter to remove the reaction solution, wash the product with water, and vacuum dry the washed product at 60℃ for 10 hours to obtain anion exchange resin.

[0065] Example 5

[0066] The preparation method of Nagqu heparin calcium is basically the same as that in Example 1, except that the preparation method of the anion exchange resin is different.

[0067] The preparation method of the anion exchange resin is as follows:

[0068] S1. Weigh 1g of azobisisobutyronitrile, 60g of n-hexane, 130g of 1,1-bis-(4-chlorophenyl)-1,3-butadiene and 20g of dried nano-silica, stir evenly to obtain a mixture. At the same time, weigh 8g of sodium carboxymethyl cellulose and 80g of potassium chloride, dissolve them in 450g of water at 90℃, mix evenly to obtain a mixed solvent, cool to 40℃, mix the mixture with the mixed solvent, heat to 70℃, add 0.5g of 1wt% methyl orange ethanol solution dropwise, add 5g of p-divinylbenzene, heat to 80℃, stir the reaction at this temperature for 12 hours. After the reaction is complete, extract with anhydrous ethanol, dry and sieve the product to obtain the pretreated product.

[0069] S2. Weigh 20g of the pretreated material prepared in step S1 and add it to 200g of dimethyl sulfoxide. Swell for 5 hours, add 50g of amidoyl-cysteine ​​and 120g of dimethyl sulfoxide, add 200g of 15wt% sodium ethoxide ethanol solution, heat to 80℃, and react at this temperature for 10 hours. After the reaction is complete, filter to remove the reaction solution, wash the product with water, and vacuum dry the washed product at 60℃ for 10 hours to obtain anion exchange resin.

[0070] Example 6

[0071] The preparation method of Nagqu heparin calcium is basically the same as that in Example 1, except that the preparation method of the anion exchange resin is different.

[0072] The preparation method of the anion exchange resin is as follows:

[0073] S1. Weigh 1g of azobisisobutyronitrile, 60g of n-hexane, 130g of 1,1-bis-(4-chlorophenyl)-1,3-butadiene, and 20g of dried nano-silica. Stir until homogeneous to obtain a mixture. Simultaneously, weigh 8g of sodium carboxymethyl cellulose and 80g of potassium chloride, dissolve them in 450g of water at 90℃, mix them until homogeneous to obtain a mixed solvent. Cool to 40℃, mix the mixture with the mixed solvent, heat to 70℃, add 0.5g of 1wt% methyl orange ethanol solution, add 5g of 1,4-dialdehyde-2,5-divinylbenzene, heat to 80℃, and stir the reaction at this temperature for 12 hours. After the reaction is complete, extract with anhydrous ethanol, dry and sieve the product to obtain the pretreated product.

[0074] S2. Weigh 20g of the pretreated material prepared in step S1 and add it to 200g of dimethyl sulfoxide. Swell for 5 hours, add 50g of amidoyl-cysteine ​​and 120g of dimethyl sulfoxide, add 200g of 15wt% sodium ethoxide ethanol solution, heat to 80℃, and react at this temperature for 10 hours. After the reaction is complete, filter to remove the reaction solution, wash the product with water, and vacuum dry the washed product at 60℃ for 10 hours to obtain anion exchange resin.

[0075] Comparative Example 1

[0076] The preparation method of Nagqu heparin calcium is basically the same as that in Example 1, except that the preparation method of the anion exchange resin is different.

[0077] The preparation method of the anion exchange resin is as follows:

[0078] S1. Weigh 1g of azobisisobutyronitrile, 60g of n-hexane, 130g of p-chloromethylstyrene, and 20g of dried nano-silica. Stir until homogeneous to obtain a mixture. Simultaneously, weigh 8g of sodium carboxymethyl cellulose and 80g of potassium chloride, dissolve them in 450g of water at 90℃, and mix until homogeneous to obtain a mixed solvent. Cool to 40℃, mix the mixture with the mixed solvent, heat to 70℃, add 0.5g of 1wt% methyl orange ethanol solution, add 5g of 1,4-diamino-2,5-divinylbenzene, heat to 80℃, and stir the reaction at this temperature for 12 hours. After the reaction is complete, extract with anhydrous ethanol, dry and sieve the product to obtain the pretreated product.

[0079] S2. Weigh 20g of the pretreated material prepared in step S1 and add it to 200g of dimethyl sulfoxide. Swell for 5 hours, add 50g of amidoyl-cysteine ​​and 120g of dimethyl sulfoxide, add 200g of 15wt% sodium ethoxide ethanol solution, heat to 80℃, and react at this temperature for 10 hours. After the reaction is complete, filter to remove the reaction solution, wash the product with water, and vacuum dry the washed product at 60℃ for 10 hours to obtain anion exchange resin.

[0080] Comparative Example 2

[0081] The preparation method of Nagqu heparin calcium is basically the same as that in Example 1, except that the preparation method of the anion exchange resin is different.

[0082] The preparation method of the anion exchange resin is as follows:

[0083] S1. Weigh 1g of azobisisobutyronitrile, 60g of n-hexane, 130g of 1,1-bis-(4-chlorophenyl)-1,3-butadiene and 20g of dried nano-silica, stir evenly to obtain a mixture. At the same time, weigh 8g of sodium carboxymethyl cellulose and 80g of potassium chloride, dissolve them in 450g of water at 90℃, mix evenly to obtain a mixed solvent, cool to 40℃, mix the mixture with the mixed solvent, heat to 70℃, add 0.5g of 1wt% methyl orange ethanol solution dropwise, add 5g of divinylbenzene, heat to 80℃, stir the reaction at this temperature for 12 hours. After the reaction is complete, extract with anhydrous ethanol, dry and sieve the product to obtain the pretreated product.

[0084] S2. Weigh 20g of the pretreated material prepared in step S1 and add it to 200g of dimethyl sulfoxide. Swell for 5 hours, add 50g of amidoyl-cysteine ​​and 120g of dimethyl sulfoxide, add 200g of 15wt% sodium ethoxide ethanol solution, heat to 80℃, and react at this temperature for 10 hours. After the reaction is complete, filter to remove the reaction solution, wash the product with water, and vacuum dry the washed product at 60℃ for 10 hours to obtain anion exchange resin.

[0085] Comparative Example 3

[0086] The preparation method of Nagqu heparin calcium is basically the same as that in Example 1, except that the anion exchange resin is replaced with an equal amount of commercially available anion exchange resin.

[0087] Test Example 1

[0088] Weight yield test

[0089] The test method for the weight yield of nagqu heparin calcium is as follows: First, accurately weigh the mass (M) of the heparin sodium raw material used to prepare nagqu heparin calcium. 原料 Then, all steps are completed according to the preparation process of Nagqu heparin calcium to finally obtain the finished Nagqu heparin calcium product, and the mass of the finished product is accurately weighed (M). 成品 The formula for calculating weight yield is: Weight yield (%) = (M) / (M) 成品 ) / (M 原料 ×100. During the test, a high-precision balance must be used for weighing to ensure the accuracy of the weighing results; before weighing the finished product, it should be ensured that the Nagqu heparin calcium has been fully dried and all moisture has been removed; to ensure the reliability of the results, three experiments were conducted and the average yield was calculated. The specific test data are shown in Table 1.

[0090] Table 1

[0091] Experimental protocol Weight yield (%) Example 1 71.4 Example 2 68.9 Example 3 68.3 Example 4 69.5 Example 5 69.1 Example 6 73.1 Comparative Example 1 67.9 Comparative Example 2 66.8 Comparative Example 3 64.8

[0092] Test Example 2

[0093] Yield test

[0094] The molecular weight distribution of the nadroparin calcium prepared in this invention was determined according to the European Pharmacopoeia EP7.0, and the detailed determination results are shown in Table 2.

[0095] Table 2

[0096]

[0097] The more concentrated the molecular weight of heparin calcium in naqué, the lower the proportion of small and large molecules, and the higher the proportion of the effective active ingredient.

[0098] As can be seen from test examples 1 and 2, the nagqu heparin calcium obtained by the preparation method of Example 1 of the present invention has a good molecular weight distribution and yield.

[0099] The 1,1-bis-(4-chlorophenyl)-1,3-butadiene used in Example 1 of this invention, compared to the 1-chloro-4-[1-(4-chlorophenyl)vinyl]benzene in Example 2, the 1,2-dichloro-4-isopropenylbenzene in Example 3, and the p-chloromethylstyrene in Comparative Example 1, provides a more uniform molecular structure and more stable chemical properties in the preparation of heparin calcium nadroprolene. This structural characteristic allows the resin to more effectively perform ion exchange and impurity removal during the preparation of heparin calcium nadroprolene, thereby improving the concentration of the molecular weight distribution and the yield of the product. Specifically, the bischlorophenyl structure of 1,1-bis-(4-chlorophenyl)-1,3-butadiene may enhance the hydrophobicity and selectivity of the resin, which is beneficial to improving the adsorption efficiency and selective separation of the resin, thus resulting in a higher yield and better molecular weight distribution of the final product. Furthermore, the stability of this structure may also reduce the degradation or deactivation of the resin during the reaction process, thereby improving the efficiency and yield of the entire preparation process. Therefore, the preparation method of Example 1 exhibits superior performance in the production of heparin calcium nadroprolene.

[0100] In this invention, Example 1 uses 1,4-diamino-2,5-divinylbenzene as a crosslinking agent to prepare an anion exchange resin, which exhibits superior performance in the preparation of nadroparin calcium. The structural characteristics of this crosslinking agent enable it to form a more stable three-dimensional network structure during resin synthesis, thereby improving the resin's mechanical strength and chemical stability. In the preparation of nadroparin calcium, this resin can more effectively adsorb and exchange target ions, reducing residual impurities and thus improving the purity and uniformity of the molecular weight distribution of the product. Furthermore, the introduction of 1,4-diamino-2,5-divinylbenzene may also enhance the resin's ion exchange capacity, allowing for a more efficient completion of the ion exchange process under the same process conditions, thereby increasing the yield of nadroparin calcium. In contrast, although the 1,2,3-trivinylbenzene of Example 4, the p-divinylbenzene of Example 5, and the divinylbenzene of Comparative Example 2 can also be used for resin preparation, their crosslinking efficiency and resin properties may not be as good as those of 1,4-diamino-2,5-divinylbenzene. Therefore, they exhibited lower yields and wider molecular weight distributions in the preparation of heparin calcium in nadroprolide.

[0101] In this invention, Example 6 uses 1,4-dialdehyde-2,5-divinylbenzene as a crosslinking agent to prepare an anion exchange resin, which exhibits a higher yield in the preparation of heparin calcium in nadroprolide. From a molecular structure perspective, the aldehyde group (-CHO) of 1,4-dialdehyde-2,5-divinylbenzene has high reactivity and can undergo more effective crosslinking reactions with other groups in the resin, forming a more stable three-dimensional network structure. This structural stability helps to improve the resin's adsorption capacity and ion exchange efficiency, thereby more effectively adsorbing and exchanging target ions in the preparation of heparin calcium in nadroprolide, reducing impurity residues, and improving product purity and yield.

[0102] The above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing Nagqu heparin calcium, characterized in that, The method is as follows: Step 1: Mix sodium heparin with purified water, adjust the pH of the solution with hydrochloric acid, add sodium nitrite and stir to react, and obtain the degradation solution; adjust the pH of the degradation solution with sodium hydroxide aqueous solution, add sodium borohydride and continue stirring to react; then adjust the pH of the solution with hydrochloric acid and sodium hydroxide aqueous solution, sterilize, add ethanol to precipitate, and collect the precipitate after standing. Step 2: Dissolve the precipitate obtained in Step 1 in purified water, add it to anion exchange resin and stir to allow the resin to fully adsorb; wash the resin with calcium chloride solution, stir and drain; combine the washing liquids, add ethanol, let stand and collect the precipitate. Step 3: Dissolve the precipitate obtained in Step 2 in purified water, add calcium chloride solid, adjust the pH value with calcium hydroxide saturated solution, add hydrogen peroxide at a certain temperature to carry out the oxidation reaction, maintain the pH value, and finally adjust the pH value with hydrochloric acid to obtain the reaction solution. Step 4: Filter the reaction solution from Step 3 through a filter membrane, precipitate the filtrate with ethanol, let it stand, remove the supernatant under vacuum, add ethanol for dehydration, stir to loosen the precipitate, let it stand, remove the supernatant under vacuum again, and repeat the dehydration operation; after centrifugation to dehydrate the precipitate, put it in a vacuum drying oven to dry, and obtain nagqu heparin calcium. The preparation method of the anion exchange resin is as follows: S1. Weigh azobisisobutyronitrile, n-hexane, monomer compound and dried nano-silica, and stir evenly to obtain a mixture; weigh sodium carboxymethyl cellulose and potassium chloride, dissolve in hot water to obtain a mixed solvent, cool and mix the mixture with the mixed solvent, heat and add colorimetric solution and crosslinking agent dropwise, continue to heat and stir the reaction, after the reaction is completed, extract with anhydrous ethanol, dry and sieve to obtain the pretreated product. S2. Weigh the pretreated material prepared in step S1, add it to dimethyl sulfoxide to swell, add amidoyl-cysteine ​​and dimethyl sulfoxide, then add sodium ethoxide ethanol solution, heat and react, filter after the reaction is complete, wash the product with water, and vacuum dry to obtain anion exchange resin. The monomeric compound is 1,1-bis-(4-chlorophenyl)-1,3-butadiene; the crosslinking agent is one of 1,4-diamino-2,5-divinylbenzene and 1,4-dialdehyde-2,5-divinylbenzene.

2. The method for preparing nagqu heparin calcium as described in claim 1, characterized in that, The method is as follows: Step 1: First, mix heparin sodium with purified water, with 9-10 L of purified water for every 1.0 kg of heparin sodium. Adjust the pH of the solution to 2.5-3.0 using 3-5 mol / L hydrochloric acid. Then, add sodium nitrite, with a mass ratio of heparin sodium to sodium nitrite of 100:2-3. Stir at 20-30°C for 1-5 hours to obtain the degradation solution. Next, adjust the pH of the degradation solution to 9-10 using 3-6 mol / L sodium hydroxide aqueous solution, and then add boron hydrochloride. Sodium nitrite and sodium borohydride were mixed in a mass ratio of 1.0:0.3~0.

4. The mixture was stirred and reacted for 15~25 hours. Then, the pH of the solution was adjusted to 3~4 with 3~5 mol / L hydrochloric acid and stirred for 10~30 minutes. The pH was then adjusted to 7~8 with 4~6 mol / L sodium hydroxide aqueous solution. After sterilization, ethanol was added for precipitation. The volume ratio of the solution to the ethanol was 1:0.5~2. After standing for 5~15 hours, the supernatant was removed and the precipitate was collected. Step 2: Dissolve the precipitate obtained in Step 1 in purified water at a mass-to-volume ratio of precipitate to purified water of 1:8-10. Then add anion exchange resin at a mass-to-volume ratio of heparin sodium to anion exchange resin of 1:4-6. Stir for 5-10 hours to allow the resin to fully adsorb the precipitate. Next, prepare a 0.05-0.2 mol / L calcium chloride solution equal in mass to the resin, add it to the resin for washing, and drain after stirring for 0.5-2 hours. Repeat the washing process once, draining the solution after each stirring. Combine the two washing solutions and stir at 15-20°C for 5-10 minutes. Then add ethanol at a volume ratio of 1:1-3 and stir for 5-10 minutes. After the precipitate has settled for 5-15 hours, remove the supernatant and collect the precipitate. Step 3: Dissolve the precipitate obtained in Step 2 in purified water, with a mass-to-volume ratio of precipitate to purified water of 1:4~5; add calcium chloride solid, with a solution volume to calcium chloride feed ratio of 1.5~2 kg calcium chloride per 100 L of solution; adjust the pH value to 10~12 with saturated calcium hydroxide solution; add hydrogen peroxide at a temperature of 20~40℃ for oxidation, with a total solution volume to hydrogen peroxide feed volume ratio of 100:1~1.5, while maintaining the pH value at 10.5~11; the oxidation reaction continues for 2~4 hours; finally, adjust the pH value to 6~6.5 with 3~5 mol / L hydrochloric acid to obtain the reaction solution. Step 4: Filter the reaction solution from Step 3 through a filter membrane. Precipitate the filtrate with 1-3 times its volume of ethanol. After standing for 5-10 hours, remove the supernatant by vacuum extraction. Add ethanol again for dehydration. Stir to loosen the precipitate into fine particles. After standing for 0.5-2 hours, remove the supernatant again by vacuum extraction. Repeat the above dehydration operation 1-3 times. Finally, centrifuge the precipitate to remove water and place it in a vacuum drying oven. Dry it at 50-70°C for 24-72 hours to obtain Nagqu heparin calcium.

3. The method for preparing nagqu heparin calcium as described in claim 1 or 2, characterized in that, The sterilization process in step 1 involves sterilizing the solution with an ultraviolet lamp for 20 to 30 minutes.

4. The method for preparing nagqu heparin calcium as described in claim 1 or 2, characterized in that, The pore size of the filter membrane in step 4 is 0.15~0.25μm.

5. The method for preparing nagqu heparin calcium as described in claim 1 or 2, characterized in that, The preparation method of the anion exchange resin is as follows, in parts by weight: S1. Weigh 0.5-2 parts of azobisisobutyronitrile, 50-70 parts of n-hexane, 100-150 parts of monomer compound and 15-25 parts of dried nano-silica, stir evenly to obtain a mixture. At the same time, weigh 5-10 parts of sodium carboxymethyl cellulose and 70-90 parts of potassium chloride, dissolve them in 400-500 parts of water at 80-95℃, mix evenly to obtain a mixed solvent, cool to 30-50℃, mix the mixture with the mixed solvent, heat to 60-80℃, add 0.3-0.8 parts of 0.5-2wt% colorimetric solution dropwise, add 4-6 parts of crosslinking agent, heat to 75-85℃, stir and react at this temperature for 5-20 hours. After the reaction is complete, extract with anhydrous ethanol, dry and sieve the product to obtain the pretreated product. S2. Weigh 15-25 parts of the pretreated material prepared in step S1 and add it to 150-250 parts of dimethyl sulfoxide. Swell for 3-8 hours, add 40-60 parts of amidoyl-cysteine ​​and 100-150 parts of dimethyl sulfoxide, and add 180-220 parts of 10-18 wt% sodium ethoxide ethanol solution. Heat to 70-90℃ and react at this temperature for 5-15 hours. After the reaction is complete, filter to remove the reaction solution, wash the product with water, and vacuum dry the washed product at 50-70℃ for 5-15 hours to obtain the anion exchange resin.

6. The method for preparing nagqu heparin calcium as described in claim 5, characterized in that, The particle size range of the nano-silica is 10~100nm.

7. The method for preparing nagqu heparin calcium as described in claim 5, characterized in that, The colorimetric solution is at least one of methyl orange ethanol solution, methylene blue ethanol solution, and bromophenol blue ethanol solution.

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