Efficient extraction method for reducing content of impurity residues in heparin sodium

Abstract

The invention relates to the technical field of heparin sodium purification, and discloses an efficient extraction method for reducing the content of impurity residues in heparin sodium, which comprises the following preparation steps: S1, mixing small intestine mucosa with deionized water, adding alkaline protease, filtering after enzymolysis is completed, and collecting filtrate; s2, modified adsorption resin is added, alkaline protease is added again after stirring adsorption is completed, modified adsorption resin is added again after enzymolysis is completed, elution is conducted after filtering and washing are conducted, and an eluent and a washing solution are combined to obtain a mixed solution; and S3, adding an ethanol water solution, filtering and collecting heparin sodium precipitate, and purifying and drying to obtain the heparin sodium. According to the method, secondary enzymolysis and process adsorption are adopted, so that the operation time can be effectively shortened, the adsorption efficiency is improved, non-enzymolysis impurities doped in the adsorption process can be effectively avoided, resin adopted for adsorption is modified, the service life of the resin is prolonged, the use efficiency of the resin is improved, and the resin can still keep good adsorption capacity at high temperature.

Description

Technical Field

[0001] The invention relates to the technical field of sodium heparin purification, in particular to a high-efficiency extraction method for reducing the residual impurity content in sodium heparin. Background Art

[0002] Heparin sodium has an irreplaceable position as an important anticoagulant drug in the medical field. It is mainly extracted from animal tissues such as the pig small intestinal mucosa. As clinical demand continues to grow, the traditional extraction process that relies on long-term salting out or acid hydrolysis and insufficient separation and purification has been difficult to meet the needs of large-scale production due to its low efficiency, poor purity, large consumption of chemical reagents and water resources, and wastewater pollution. Although the enzymatic hydrolysis method has received attention in recent years and provided new possibilities for the application of adsorption resins, it still faces the dilemma of low extraction rate and purity. Further research and optimization are urgently needed to promote the innovation of heparin sodium extraction process to meet medical needs and reduce environmental pressure;

[0003] China's invention patent CN103848929B discloses an efficient extraction process for sodium heparin, which specifically includes enzymolysis of a mixture of intestinal mucosa and water, wherein the ratio of intestinal mucosa to water is 1:4, and then conventional adsorption, incremental adsorption, elution, precipitation purification, and drying to obtain sodium heparin. Since the extraction process of the invention adds secondary enzymolysis and incremental adsorption, the yield of sodium heparin is improved, and the purity of the obtained sodium heparin is also improved. Specifically, the yield of sodium heparin can reach 100 million IU / 1200-1400 pig intestines, which is 30-45% higher than the conventional process; the purity of the produced sodium heparin can reach 110-130 IU / mg; the efficient extraction process of the invention saves about 40% of water and 20-40% of salt, uses less water and salt, and has high economic benefits. It is an efficient sodium heparin extraction process. However, the adsorption resin in this preparation method is often exposed to an environment above 60°C, and due to the use of secondary enzymatic hydrolysis, which is accompanied by the inactivation of proteases and the denaturation and precipitation of proteins after enzymatic hydrolysis, the temperature of the process is generally between 70 and 80°C. The working temperature of traditional adsorption resins is 40 to 60°C, which cannot adapt to this process well. Summary of the invention

[0004] The object of the present invention is to provide a highly efficient extraction method for reducing the residual impurity content in heparin sodium, so as to solve the problems existing in the prior art.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] An efficient extraction method for reducing the residual impurity content in heparin sodium comprises the following preparation steps:

[0007] S1. Mix the small intestinal mucosa and deionized water in a mass ratio of 1:4-5, then stir and heat to 50-55°C, add 5%-8% alkaline protease of the mass of the small intestinal mucosa after heating, stir evenly, adjust the pH to 8.5-9 with 1.5-2mol / L sodium hydroxide solution, and then adjust the salt concentration to 1.5%-3% with 1-2mol / L sodium chloride solution, stir and hydrolyze for 2-3h, heat to 80-85°C and keep warm for 8-10min after the hydrolysis is completed, and naturally precipitate after the insulation is completed, select 100-mesh filter cloth for filtration, and collect the filtrate;

[0008] S2, cooling the filtrate prepared in S1 to 65-70°C, adding a modified adsorption resin of 0.25-0.3 times the mass of the small intestinal mucosa, stirring and adsorbing for 2-3 hours, adding alkaline protease of 1%-2% of the mass of the small intestinal mucosa again after the stirring and adsorption is completed, and enzymolysis is performed for 2-3 hours. After the enzymolysis is completed, the temperature is raised to 80-85°C and kept warm for 8-10 minutes. After the insulation is completed, adding a modified adsorption resin of 0.1-0.2 times the mass of the small intestinal mucosa again, stirring and adsorbing again for 5-8 hours, filtering out the modified adsorption resin, washing and eluting, and combining the eluate and the washing liquid to obtain a mixed solution;

[0009] S3. Add 90% to 95% ethanol aqueous solution to the mixed solution in S2 until the ethanol mass fraction of the mixed solution is 35% to 40%. After mixing and stirring, let stand for 8 to 10 hours, filter and collect the heparin sodium precipitate, and then purify and dry to obtain heparin sodium.

[0010] Preferably, the washing in S2 comprises the following steps: washing the modified adsorption resin with a 1-1.25 mol / L sodium chloride aqueous solution for 20-30 min, and collecting the washing liquid.

[0011] Preferably, the elution in S2 comprises the following steps: statically eluting the modified adsorption resin with a 4 mol / L sodium chloride aqueous solution for 3 to 3.5 hours, and collecting the eluate.

[0012] Preferably, the small intestinal mucosa is one of porcine small intestinal mucosa and sheep small intestinal mucosa.

[0013] Preferably, the alkaline protease is composed of 2709 alkaline protease and trypsin in a mass ratio of 2:1 to 1.2.

[0014] Preferably, the modified adsorption resin comprises the following preparation steps: adding the post-crosslinked resin to diethylenetriamine with a mass of 4.8 to 5 times that of the post-crosslinked resin, swelling for 4 to 4.5 hours, then heating to 110 to 120°C, reacting for 11 to 12 hours, filtering after the reaction, adding the filtered product to toluene with a mass of 40 to 50 times that of the filtered product, then adding 0.4 to 0.5 times the mass of propylene glycol triglycidyl ether, stirring evenly, reacting for 10 to 12 hours at a temperature of 100 to 105°C, washing with 1 to 2 mol / L sodium chloride solution for 5 to 6 times after the reaction, then washing with deionized water for 5 to 6 times, and then extracting with anhydrous ethanol for 44 to 48 hours to obtain the modified adsorption resin.

[0015] Preferably, the post-crosslinking resin comprises the following preparation steps: adding a polystyrene precursor to a chloromethyl methyl ether solution with a mass of 6.2 to 6.4 times that of the polystyrene precursor, swelling for 4 to 4.5 hours at a temperature of 25 to 35° C., adding anhydrous tin tetrachloride with a mass of 4 to 4.2 times that of the polystyrene precursor after swelling, reacting for 15 to 17 hours, adding a 1 mol / L hydrochloric acid solution after the reaction is completed to stop the reaction, filtering and drying after the reaction is completed; adding the dried product to a 7 to 10 mol / L ... The mixture was swelled in 7.2 times of 1,2-dichloroethane for 4 to 4.5 hours, and then 0.16 to 0.18 times of the mass of the dry product was added with anhydrous ferric chloride, stirred for 30 to 35 minutes at a temperature of 40 to 45°C, and then heated to 80 to 85°C for reaction for 10 to 11 hours. After the reaction was completed, the mixture was cooled to room temperature and filtered, and the filtered product was washed alternately with 1% hydrochloric acid solution and anhydrous ethanol for 5 to 6 times, and then extracted with anhydrous ethanol for 10 to 12 hours to obtain a post-crosslinked resin.

[0016] Preferably, the polystyrene precursor comprises the following preparation steps: adding the oil phase to the water phase whose mass is 2.8 to 3.2 times of the oil phase at a temperature of 40 to 45°C, stirring for 30 to 35 minutes, then heating to 75 to 80°C and keeping warm for 16 to 18 hours, washing the reaction product with deionized water after the insulation, then extracting with acetone for 10 to 12 hours, and then screening the resin of 40 to 60 mesh and drying to obtain the polystyrene precursor.

[0017] Preferably, the aqueous phase comprises the following preparation steps: gelatin, sodium chloride, anhydrous sodium carbonate and magnesium sulfate heptahydrate are weighed in a mass ratio of 1:35-38:2.5-3.5:6.5-7.5, added to deionized water 230-250 times the mass of gelatin, stirred evenly, and prepared into an aqueous phase for use.

[0018] Preferably, the oil phase comprises the following preparation steps: styrene, divinylbenzene, porogen and benzoyl peroxide are weighed in a mass ratio of 1:1.1-1.2:2.8-3.2:0.04-0.05, stirred evenly, and prepared into an oil phase for use.

[0019] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0020] The present invention adopts secondary enzymolysis and process adsorption, which can effectively reduce the operation time and improve the adsorption efficiency. The secondary enzymolysis can effectively avoid the impurities that are not enzymolyzed during the adsorption process, and the resin used for adsorption is modified to improve its service life and use efficiency, so that it can still maintain good adsorption capacity at high temperature.

[0021] The modified adsorption resin has a high specific surface area and an adjustable pore structure. The preparation process is to first use a suspension polymerization method to synthesize a polystyrene precursor, which has a rich mesoporous and macroporous structure. Then, after chloromethylation, post-crosslinking and grafting modification, the target resin is obtained. During the post-crosslinking reaction, the chloromethyl groups suspended on the resin skeleton will cross-link with the benzene rings to form a network structure. The methylene cross-linking bridges generated in this process can divide the pores inside the resin, thereby promoting the formation of a new microporous structure. However, the skeleton of the post-crosslinked resin exhibits strong hydrophobicity and a low functional group loading, which leads to poor selectivity for the adsorbate. In view of this, the present invention uses diethylenetriamine to modify it. After modification with diethylenetriamine, the resin has a higher selectivity. After the diethylenetriamine grafting is completed, intermolecular crosslinking will further occur. Since diethylenetriamine itself has the characteristics of strong alkalinity and structural conjugation, the reaction of strong base degradation to weak base is effectively avoided, so that the heat resistance of the resin is improved. Subsequently, propylene glycol triglycidyl ether was used to fix some of the active amino groups still remaining on the resin, successfully enhancing its anti-migration and anti-aging capabilities, and ultimately further improving the overall stability of the resin. In addition, by using propylene glycol triglycidyl ether for modification and adding hydroxyl groups to it, the hydrophilic properties of the resin can be improved. DETAILED DESCRIPTION

[0022] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0023] Example 1

[0024] S1. Mix the small intestinal mucosa and deionized water in a mass ratio of 1:4, then stir and heat to 50°C, add 5% alkaline protease of the mass of the small intestinal mucosa after heating, stir evenly, adjust the pH to 8.5 with 1.5 mol / L sodium hydroxide solution, and then adjust the salt concentration to 1.5% with 1 mol / L sodium chloride solution, stir and hydrolyze for 2 hours, heat to 80°C and keep warm for 8 minutes after the hydrolysis is completed, and naturally precipitate after the insulation is completed, select 100 mesh filter cloth for filtration, and collect the filtrate; the alkaline protease is composed of 2709 alkaline protease and trypsin in a mass ratio of 2:1;

[0025] S2, the modified adsorption resin includes the following preparation steps: adding the post-crosslinked resin to diethylenetriamine with a mass of 4.8 times that of the post-crosslinked resin, swelling for 4 hours, then heating to 110°C, reacting for 11 hours, filtering after the reaction is completed, adding the filtered product to toluene with a mass of 40 times that of the filtered product, and then adding 0.4 times the mass of propylene glycol triglycidyl ether of the filtered product, stirring evenly, reacting for 10 hours at a temperature of 100°C, washing with 1 mol / L sodium chloride solution 5 times after the reaction is completed, and then washing with deionized water 5 times, and then extracting with anhydrous ethanol for 44 hours to obtain a modified adsorption resin;

[0026] S3, the post-crosslinking resin comprises the following preparation steps: adding a polystyrene precursor to a chloromethyl methyl ether solution of 6.2 times the mass of the polystyrene precursor, swelling for 4 hours at a temperature of 25°C, adding anhydrous tin tetrachloride of 4 times the mass of the polystyrene precursor after the swelling is completed, reacting for 15 hours, adding a 1 mol / L hydrochloric acid solution after the reaction is completed to stop the reaction, filtering and drying after the reaction is completed; adding the dried product to 1,2-dichloroethane of 7 times the mass of the dried product, swelling for 4 hours, then adding anhydrous ferric chloride of 0.16 times the mass of the dried product, stirring for 30 minutes at a temperature of 40°C, then heating to 80°C to react for 10 hours, cooling to room temperature after the reaction is completed, filtering, and washing the filtered product with a 1% hydrochloric acid solution and anhydrous ethanol alternately for 5 times, and extracting with anhydrous ethanol after washing. 10h, to obtain a post-crosslinked resin; the polystyrene precursor includes the following preparation steps: under the condition of a temperature of 40°C, adding the oil phase to a water phase 2.8 times the mass of the oil phase, stirring for 30min, then heating to 75°C and keeping warm for 16h, washing the reaction product with deionized water after the insulation, then extracting with acetone for 10h and then screening a 40-mesh resin to dry to obtain a polystyrene precursor; the water phase includes the following preparation steps: weighing gelatin, sodium chloride, anhydrous sodium carbonate and magnesium sulfate heptahydrate according to a mass ratio of 1:35:2.5:6.5, adding them to deionized water 230 times the mass of gelatin, stirring evenly, and preparing a water phase for standby use; the oil phase includes the following preparation steps: weighing styrene, divinylbenzene, porogen and benzoyl peroxide according to a mass ratio of 1:1.1:2.8:0.04, stirring evenly, and preparing an oil phase for standby use;

[0027] S4, cooling the filtrate prepared in S1 to 65°C, adding a modified adsorption resin of 0.25 times the mass of the small intestinal mucosa, stirring and adsorbing for 2 hours, adding alkaline protease of 1% of the mass of the small intestinal mucosa again after the stirring and adsorption is completed, enzymolysis for 2 hours, heating to 80°C and keeping warm for 8 minutes after the enzymolysis is completed, adding a modified adsorption resin of 0.1 times the mass of the small intestinal mucosa again after the insulation is completed, stirring and adsorbing for 5 hours again, filtering out the modified adsorption resin, washing and eluting, and combining the eluate and the washing liquid to obtain a mixed solution; the washing in S4 includes the following steps: washing the modified adsorption resin with a 1 mol / L sodium chloride aqueous solution for 20 minutes, collecting the washing liquid; the elution in S4 includes the following steps: statically eluting the modified adsorption resin with a 4 mol / L sodium chloride aqueous solution for 3 hours, and collecting the eluate;

[0028] S5. Add 90% ethanol aqueous solution to the mixed solution in S4 until the ethanol mass fraction of the mixed solution is 35%. Mix and stir, let stand for 8 hours, filter and collect the heparin sodium precipitate, and then purify and dry to obtain heparin sodium.

[0029] Example 2

[0030] S1. Mix the small intestinal mucosa and deionized water in a mass ratio of 1:4.5, then stir and heat to 52°C, add 6.5% alkaline protease of the mass of the small intestinal mucosa after heating, stir evenly, adjust the pH to 8.8 with 1.8 mol / L sodium hydroxide solution, and then adjust the salt concentration to 2.2% with 1.5 mol / L sodium chloride solution, stir and hydrolyze for 2.5 hours, heat to 82°C and keep warm for 9 minutes after the hydrolysis is completed, and naturally precipitate after the insulation is completed, select 100 mesh filter cloth for filtration, and collect the filtrate; the alkaline protease is composed of 2709 alkaline protease and trypsin in a mass ratio of 2:1.1;

[0031] S2, the modified adsorption resin includes the following preparation steps: adding the post-crosslinked resin to diethylenetriamine with a mass of 4.9 times that of the post-crosslinked resin, swelling for 4.2 hours, then heating to 115°C, reacting for 11.5 hours, filtering after the reaction, adding the filtered product to toluene with a mass of 45 times that of the filtered product, and then adding 0.45 times the mass of propylene glycol triglycidyl ether of the filtered product, stirring evenly, reacting for 11 hours at a temperature of 102°C, washing with 1.5 mol / L sodium chloride solution 5 times after the reaction, and then washing with deionized water 5 times, and then extracting with anhydrous ethanol for 46 hours to obtain a modified adsorption resin;

[0032] S3, the post-crosslinking resin comprises the following preparation steps: adding a polystyrene precursor to a chloromethyl methyl ether solution with a mass of 6.3 times that of the polystyrene precursor, swelling for 4.2 hours at a temperature of 30°C, adding anhydrous tin tetrachloride with a mass of 4.1 times that of the polystyrene precursor after swelling, reacting for 16 hours, adding a 1 mol / L hydrochloric acid solution to stop the reaction after the reaction is completed, filtering and drying after the reaction is completed; adding the dried product to 1,2-dichloroethane with a mass of 7.1 times that of the dried product, swelling for 4.2 hours, then adding anhydrous ferric chloride with a mass of 0.17 times that of the dried product, stirring for 32 minutes at a temperature of 42°C, then heating to 82°C to react for 10.5 hours, cooling to room temperature after the reaction is completed, filtering, and washing the filtered product with a 1% hydrochloric acid solution and anhydrous ethanol alternately for 5 times, and washing is completed. The polystyrene precursor comprises the following preparation steps: at a temperature of 42°C, the oil phase is added to a water phase 3 times the mass of the oil phase, stirred for 32 minutes, then heated to 78°C and kept warm for 17 hours, after the insulation, the reaction product is washed with deionized water, then extracted with acetone for 11 hours, and then 50 mesh resin is screened and dried to obtain a polystyrene precursor; the water phase comprises the following preparation steps: gelatin, sodium chloride, anhydrous sodium carbonate and magnesium sulfate heptahydrate are weighed in a mass ratio of 1:36:3:7, added to deionized water 240 times the mass of gelatin, stirred evenly, and prepared into a water phase for standby use; the oil phase comprises the following preparation steps: styrene, divinylbenzene, porogen and benzoyl peroxide are weighed in a mass ratio of 1:1.15:3:0.045, stirred evenly, and prepared into an oil phase for standby use;

[0033] S4, cooling the filtrate prepared in S1 to 68°C, adding a modified adsorption resin of 0.28 times the mass of the small intestinal mucosa, stirring and adsorbing for 2.5 hours, adding alkaline protease of 1.5% of the mass of the small intestinal mucosa again after the stirring and adsorption is completed, enzymolysis for 2.5 hours, heating to 82°C and keeping warm for 9 minutes after the enzymolysis is completed, adding a modified adsorption resin of 0.15 times the mass of the small intestinal mucosa again after the insulation is completed, stirring and adsorbing for 6 hours again, filtering out the modified adsorption resin, washing and eluting, and combining the eluate and the washing liquid to obtain a mixed solution; the washing in S4 includes the following steps: washing the modified adsorption resin with a 1.1 mol / L sodium chloride aqueous solution for 25 minutes, collecting the washing liquid; the elution in S4 includes the following steps: statically eluting the modified adsorption resin with a 4 mol / L sodium chloride aqueous solution for 3.2 hours, and collecting the eluate;

[0034] S5. Add 92% ethanol aqueous solution to the mixed solution in S4 until the ethanol mass fraction of the mixed solution is 38%. Mix and stir, let stand for 9 hours, filter and collect the heparin sodium precipitate, and then purify and dry to obtain heparin sodium.

[0035] Example 3

[0036] S1. Mix the small intestinal mucosa and deionized water in a mass ratio of 1:5, then stir and heat to 55°C, add 8% alkaline protease of the mass of the small intestinal mucosa after heating, stir evenly, adjust the pH to 9 with 2mol / L sodium hydroxide solution, and then adjust the salt concentration to 3% with 2mol / L sodium chloride solution, stir and hydrolyze for 3h, heat to 85°C and keep warm for 10min after the hydrolysis, precipitate naturally after the insulation, select 100-mesh filter cloth for filtration, and collect the filtrate; the alkaline protease is composed of 2709 alkaline protease and trypsin in a mass ratio of 2:1.2;

[0037] S2, the modified adsorption resin includes the following preparation steps: adding the post-crosslinked resin to diethylenetriamine with a mass of 5 times that of the post-crosslinked resin, swelling for 4.5 hours, then heating to 120°C, reacting for 12 hours, filtering after the reaction is completed, adding the filtered product to toluene with a mass of 50 times that of the filtered product, and then adding 0.5 times the mass of propylene glycol triglycidyl ether of the filtered product, stirring evenly, reacting for 12 hours at a temperature of 105°C, washing with 2 mol / L sodium chloride solution 6 times after the reaction is completed, and then washing with deionized water 6 times, and then extracting with anhydrous ethanol for 48 hours to obtain a modified adsorption resin;

[0038] S3, the post-crosslinking resin comprises the following preparation steps: adding a polystyrene precursor to a chloromethyl methyl ether solution with a mass of 6.4 times that of the polystyrene precursor, swelling for 4.5 hours at a temperature of 35°C, adding anhydrous tin tetrachloride with a mass of 4.2 times that of the polystyrene precursor after swelling, reacting for 17 hours, adding a 1 mol / L hydrochloric acid solution to stop the reaction after the reaction is completed, filtering and drying after the reaction is completed; adding the dried product to 1,2-dichloroethane with a mass of 7.2 times that of the dried product, swelling for 4.5 hours, then adding anhydrous ferric chloride with a mass of 0.18 times that of the dried product, stirring for 35 minutes at a temperature of 45°C, then heating to 85°C to react for 11 hours, cooling to room temperature after the reaction is completed, filtering, and washing the filtered product with a 1% hydrochloric acid solution and anhydrous ethanol alternately for 6 times, and washing with anhydrous ethanol after washing. The polystyrene precursor comprises the following preparation steps: at a temperature of 45°C, the oil phase is added to a water phase 3.2 times the mass of the oil phase, stirred for 35 minutes, then heated to 80°C and kept warm for 18 hours, after the insulation, the reaction product is washed with deionized water, then extracted with acetone for 12 hours, and then a 60-mesh resin is screened and dried to obtain a polystyrene precursor; the water phase comprises the following preparation steps: gelatin, sodium chloride, anhydrous sodium carbonate and magnesium sulfate heptahydrate are weighed in a mass ratio of 1:38:3.5:7.5, added to deionized water 250 times the mass of gelatin, stirred evenly, and prepared into a water phase for standby use; the oil phase comprises the following preparation steps: styrene, divinylbenzene, porogen and benzoyl peroxide are weighed in a mass ratio of 1:1.2:3.2:0.05, stirred evenly, and prepared into an oil phase for standby use;

[0039] S4, cooling the filtrate prepared in S1 to 70°C, adding a modified adsorption resin of 0.3 times the mass of the small intestinal mucosa, stirring and adsorbing for 3 hours, adding 2% of the mass of the small intestinal mucosa alkaline protease again after the stirring and adsorption is completed, enzymolysis for 3 hours, heating to 85°C and keeping warm for 10 minutes after the enzymolysis is completed, adding 0.2 times the mass of the small intestinal mucosa modified adsorption resin again after the insulation is completed, stirring and adsorbing for 8 hours, filtering out the modified adsorption resin, washing and eluting, and combining the eluate and the washing liquid to obtain a mixed solution; the washing in S4 includes the following steps: washing the modified adsorption resin with a 1.25 mol / L sodium chloride aqueous solution for 30 minutes, collecting the washing liquid; the elution in S4 includes the following steps: statically eluting the modified adsorption resin with a 4 mol / L sodium chloride aqueous solution for 3.5 hours, and collecting the eluate;

[0040] S5. Add 95% ethanol aqueous solution to the mixed solution in S4 until the ethanol mass fraction of the mixed solution is 40%. After mixing and stirring, let stand for 10 hours, filter and collect the heparin sodium precipitate, and then purify and dry to obtain heparin sodium.

[0041] Comparative Example 1

[0042] The only difference from Example 2 is step S2: the modified adsorption resin includes the following preparation steps: adding the post-cross-linked resin to diethylenetriamine (4.9 times the mass of the post-cross-linked resin), swelling for 4.2 hours, then heating to 115°C, reacting for 11.5 hours, washing with 1.5 mol / L sodium chloride solution for 5 times after the reaction, then washing with deionized water for 5 times, and then extracting with anhydrous ethanol for 46 hours to obtain the modified adsorption resin.

[0043] Comparative Example 2

[0044] The only difference from Example 2 is step S4: the modified adsorption resin in step S4 is replaced by the post-crosslinking resin prepared in step S3.

[0045] Comparative Example 3

[0046] The only difference from Example 2 is step S4: the modified adsorption resin in step S4 is replaced by commercially available D208 resin.

[0047] Heparin sodium extraction rate and potency test

[0048] Heparin sodium was prepared according to the steps in Examples 1 to 3 of the present invention and Comparative Examples 1 to 3.

[0049] Extraction rate calculation:

[0050] The heparin sodium content in the extract was quantitatively determined by measuring the absorbance of heparin sodium at a specific wavelength. Subsequently, the extraction rate was calculated according to a specific formula based on the measured heparin sodium content and the amount of raw materials input. The calculation formula is: extraction rate (%) = (measured heparin sodium amount / theoretical heparin sodium content) × 100%. In order to ensure the accuracy and repeatability of the test results, at least three repeated experiments were carried out for each experimental condition, and the average value was taken. The specific test results are presented in Table 1.

[0051] Potency test:

[0052] The titer test of sodium heparin mainly focuses on determining its anticoagulant activity. After all, its titer is closely related to the effectiveness of clinical application. First, a series of sodium heparin standard solutions of known concentrations are prepared to construct a standard curve. Then, the sodium heparin prepared by the present invention is dissolved in a specific solvent and prepared into a test solution. Then, its titer is evaluated by measuring the change in plasma clotting time after adding sodium heparin. During the specific operation, the sample solution and the standard solution are respectively added to the test system containing the coagulation system, and the reaction time of each sample and standard is recorded in detail, such as clotting time or other related indicators. Finally, a standard curve is drawn according to the reaction time of the standard, and the titer of sodium heparin is calculated according to the reaction time of the sample and the standard curve. To ensure the accuracy and repeatability of the results, each sample must be tested independently at least three times, and the corresponding test results are listed in Table 1.

[0053] Table 1. Experimental data of heparin sodium extraction rate and potency test

[0054]

[0055]

[0056] From the comparison of the experimental data in Table 1, it can be found that Examples 1 to 3 all maintain good performance and are much higher than Comparative Examples 1 to 3. It can be expected that the performance will decrease in Comparative Examples 1 to 3, mainly because the use of traditional or unmodified adsorption resins will accelerate decomposition during the heating process, resulting in a decrease in the final adsorption effect. In Comparative Example 1, since it is not modified by propylene glycol triglycidyl ether, it will still partially decompose during the heating process, resulting in a decrease in the final adsorption effect. In Comparative Example 2, since it uses a post-crosslinking resin and does not use diethylenetriamine for grafting, its selectivity decreases, and it also decomposes the fastest during the heating process, so its final decrease is the fastest.

[0057] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations within the meaning and scope of the equivalent elements of the claims be included in the invention. Any marking in a claim should not be considered as limiting the claim to which it relates.

Claims

1. An efficient extraction method for reducing the residual impurity content in heparin sodium, characterized in that: The method comprises the following preparation steps: S1. Mix the small intestinal mucosa and deionized water in a mass ratio of 1:4-5, then stir and heat to 50-55°C, add 5%-8% alkaline protease of the mass of the small intestinal mucosa after heating, stir evenly, adjust the pH to 8.5-9 with 1.5-2mol / L sodium hydroxide solution, and then adjust the salt concentration to 1.5%-3% with 1-2mol / L sodium chloride solution, stir and hydrolyze for 2-3h, heat to 80-85°C and keep warm for 8-10min after the hydrolysis is completed, and naturally precipitate after the insulation is completed, select 100-mesh filter cloth for filtration, and collect the filtrate; S2, cooling the filtrate prepared in S1 to 65-70°C, adding a modified adsorption resin of 0.25-0.3 times the mass of the small intestinal mucosa, stirring and adsorbing for 2-3 hours, adding alkaline protease of 1%-2% of the mass of the small intestinal mucosa again after the stirring and adsorption is completed, and enzymolysis is performed for 2-3 hours. After the enzymolysis is completed, the temperature is raised to 80-85°C and kept warm for 8-10 minutes. After the insulation is completed, adding a modified adsorption resin of 0.1-0.2 times the mass of the small intestinal mucosa again, stirring and adsorbing again for 5-8 hours, filtering out the modified adsorption resin, washing and eluting, and combining the eluate and the washing liquid to obtain a mixed solution; S3. Add 90% to 95% ethanol aqueous solution to the mixed solution in S2 until the ethanol mass fraction of the mixed solution is 35% to 40%. After mixing and stirring, let stand for 8 to 10 hours, filter and collect the heparin sodium precipitate, and then purify and dry to obtain heparin sodium.

2. The efficient extraction method for reducing the residual impurity content in heparin sodium according to claim 1, characterized in that: The washing in S2 comprises the following steps: washing the modified adsorption resin with a 1-1.25 mol / L sodium chloride aqueous solution for 20-30 min, and collecting the washing liquid.

3. The efficient extraction method for reducing the residual impurity content in heparin sodium according to claim 1, characterized in that: The elution in S2 comprises the following steps: statically eluting the modified adsorption resin with a 4 mol / L sodium chloride aqueous solution for 3 to 3.5 hours, and collecting the eluate.

4. The efficient extraction method for reducing the residual impurity content in heparin sodium according to claim 1, characterized in that: The small intestinal mucosa is one of pig small intestinal mucosa and sheep small intestinal mucosa.

5. The efficient extraction method for reducing the residual impurity content in heparin sodium according to claim 1, characterized in that: The alkaline protease is composed of 2709 alkaline protease and trypsin in a mass ratio of 2:1 to 1.

2.

6. The efficient extraction method for reducing the residual impurity content in heparin sodium according to claim 1, characterized in that: The modified adsorption resin comprises the following preparation steps: adding the post-crosslinked resin to diethylenetriamine with a mass of 4.8 to 5 times that of the post-crosslinked resin, swelling for 4 to 4.5 hours, then heating to 110 to 120° C., reacting for 11 to 12 hours, filtering after the reaction is completed, adding the filtered product to toluene with a mass of 40 to 50 times that of the filtered product, then adding 0.4 to 0.5 times that of propylene glycol triglycidyl ether with a mass of 0.4 to 0.5 times that of the filtered product, stirring evenly, reacting for 10 to 12 hours at a temperature of 100 to 105° C., washing with 1 to 2 mol / L sodium chloride solution for 5 to 6 times after the reaction is completed, then washing with deionized water for 5 to 6 times, and then extracting with anhydrous ethanol for 44 to 48 hours to obtain the modified adsorption resin.

7. The efficient extraction method for reducing the residual impurity content in heparin sodium according to claim 6, characterized in that: The post-crosslinking resin comprises the following preparation steps: adding a polystyrene precursor to a chloromethyl methyl ether solution with a mass of 6.2 to 6.4 times the mass of the polystyrene precursor, swelling for 4 to 4.5 hours at a temperature of 25 to 35° C., adding anhydrous tin tetrachloride with a mass of 4 to 4.2 times the mass of the polystyrene precursor after the swelling is completed, reacting for 15 to 17 hours, adding a 1 mol / L hydrochloric acid solution after the reaction is completed to stop the reaction, filtering and drying after the reaction is completed; adding the dried product to a 7 to 7.2 mass of the dried product times the mass of 1,2-dichloroethane, swell for 4-4.5h, then add anhydrous ferric chloride in an amount of 0.16-0.18 times the mass of the dry product, stir for 30-35min at a temperature of 40-45°C, then heat to 80-85°C for reaction for 10-11h, cool to room temperature and filter after the reaction is completed, and wash the filtered product alternately with 1% hydrochloric acid solution and anhydrous ethanol for 5-6 times, and extract with anhydrous ethanol for 10-12h after washing to obtain a post-crosslinking resin.

8. The efficient extraction method for reducing the residual impurity content in heparin sodium according to claim 7, characterized in that: The polystyrene precursor comprises the following preparation steps: adding the oil phase to the water phase whose mass is 2.8 to 3.2 times of the oil phase at a temperature of 40 to 45° C., stirring for 30 to 35 minutes, then heating to 75 to 80° C. and keeping the temperature for 16 to 18 hours, washing the reaction product with deionized water after the heat preservation, then extracting with acetone for 10 to 12 hours, and then screening the resin of 40 to 60 meshes to dry to obtain the polystyrene precursor.

9. The efficient extraction method for reducing the residual impurity content in heparin sodium according to claim 8, characterized in that: The aqueous phase comprises the following preparation steps: gelatin, sodium chloride, anhydrous sodium carbonate and magnesium sulfate heptahydrate are weighed according to a mass ratio of 1:35-38:2.5-3.5:6.5-7.5, added into deionized water with a mass of 230-250 times that of gelatin, stirred evenly, and prepared into an aqueous phase for standby use.

10. The efficient extraction method for reducing the residual impurity content in heparin sodium according to claim 8, characterized in that: The oil phase comprises the following preparation steps: styrene, divinylbenzene, porogen and benzoyl peroxide are weighed in a mass ratio of 1:1.1-1.2:2.8-3.2:0.04-0.05, and stirred evenly to prepare an oil phase for standby use.

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