A preparation method of heparin sodium

Abstract

The present invention provides a method for preparing sodium heparin, belonging to the technical field of heparin. The method comprises the following steps: 1) mixing heparin with water, and adjusting the temperature of the resulting aqueous heparin solution to 0-5°C and acidic; 2) centrifuging the acidic heparin solution at 0-5°C to obtain a supernatant; 3) adjusting the supernatant to alkaline, controlling the temperature of the alkaline supernatant at 0-5°C, adding calcium chloride and hydrogen peroxide, and oxidizing at 5-15°C; 4) adding sodium carbonate to the oxidation product, stirring and centrifuging in sequence, and detecting the optical absorption of the centrifuge at 260nm, 280nm, and 400nm using a spectrophotometer; 5) adding sodium chloride to the centrifuge, adjusting the temperature to neutral, adding ethanol, and allowing the centrifuge to stand to obtain sodium heparin. The sodium heparin prepared by the method provided by the present invention has high activity and high yield, and the process is simple, making it suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of heparin, and in particular relates to a method for preparing heparin sodium. Background Art

[0002] Heparin is the preferred anticoagulant drug, exhibiting anticoagulant, antithrombotic, and lipid-lowering effects. Its active content determines its anticoagulant effect. Heparin sodium is also the starting material for all low-molecular-weight heparins, and its active content and disaccharide structure are closely related to the activity and structure of low-molecular-weight heparins.

[0003] The starting material for heparin sodium, crude heparin, is primarily derived from porcine intestinal mucosa. The production process for heparin sodium involves the removal of nucleic acids, proteins, and related substances from crude heparin. Currently, enzymatic hydrolysis, salt hydrolysis, and high-temperature treatment combined with oxidation are commonly used to remove nucleic acids and proteins from crude heparin. These impurity removal processes are performed under alkaline conditions. Under alkaline conditions, the 2-O-sulfate group on iduronic acid can be hydrolyzed, thereby inactivating the heparin and causing some damage to its structure.

[0004] Patent application number 201910035877.7 discloses a process for preparing high-purity heparin sodium. This process employs an integrated enzymatic and salt hydrolysis method, with a protective agent added to protect the heparin from damage during the high-temperature dissociation and oxidation process. However, this method is complex and costly. Summary of the Invention

[0005] The invention provides a method for preparing heparin sodium. The prepared heparin sodium has high activity, high yield and simple process.

[0006] In order to achieve the above object, the present invention provides a method for preparing heparin sodium, comprising the following steps:

[0007] 1) mixing heparin with water, adjusting the temperature of the obtained heparin aqueous solution to 0-5° C. and adjusting it to acidity to obtain an acidic heparin solution;

[0008] 2) centrifuging the acidic heparin solution at 0-5° C. to obtain a supernatant;

[0009] 3) adjusting the supernatant to alkaline, controlling the temperature of the alkaline supernatant at 0-5° C., adding calcium chloride and hydrogen peroxide, and oxidizing at 5-15° C. to obtain an oxidation product;

[0010] 4) adding sodium carbonate to the oxidation product, stirring and centrifuging in sequence, and detecting the absorbance of the centrifuge at 260 nm, 280 nm, and 400 nm using a spectrophotometer; when the absorbance at 260 nm is <0.1, the absorbance at 280 nm is <0.1, and the absorbance at 400 nm is <0.040, proceeding to the next step; otherwise, repeating steps 3) and 4) to continue oxidation until the absorbance at 260 nm, the absorbance at 280 nm is <0.1, and the absorbance at 400 nm is <0.040;

[0011] 5) Sodium chloride is added to the centrifuge solution, and after adjusting to neutrality, ethanol is added and the solution is allowed to stand to obtain heparin sodium.

[0012] Preferably, the concentration of the heparin aqueous solution in step 1) is 10 wt% to 15 wt%; and hydrochloric acid is used to adjust the pH value to 3 to 4 in step 1).

[0013] Preferably, the centrifugal speed in step 2) is 3000-5000 r / min, and the time is 5-10 min.

[0014] Preferably, in step 3), sodium hydroxide solution is used to adjust the pH value to 9-10.

[0015] Preferably, in step 3), the amount of calcium chloride added is 3% of the volume of the supernatant, and the amount of hydrogen peroxide added is 1% to 2% of the volume of the supernatant.

[0016] Preferably, the oxidation time in step 3) is 6 to 12 hours.

[0017] Preferably, the amount of sodium carbonate added in step 4) is 3% of the volume of the oxidation product.

[0018] Preferably, in step 4), the stirring speed is 100-300 r / min, and the time is 50-70 min; the centrifugal speed is 3000-5000 r / min, and the time is 5-10 min.

[0019] Preferably, in step 5), the amount of sodium chloride added is 1% of the volume of the centrifuge; and the amount of ethanol added is 70% to 100% of the volume of the centrifuge.

[0020] Preferably, in step 5), when the impurity content in the obtained heparin sodium is ≤2%, the standing is completed.

[0021] Compared with the prior art, the advantages and positive effects of the present invention are:

[0022] 1. This invention utilizes the chemical properties of crude heparin to replace the complex processes of enzymatic hydrolysis, salt hydrolysis, and high-temperature treatment. Under a low-temperature environment, it maximizes the preservation of heparin's structure while rapidly removing nucleic acids and proteins from the crude heparin. Compared to existing technologies, the operation is simpler, faster, and more time-saving.

[0023] 2. The crude heparin is not treated with high temperature, which not only reduces the degree of damage to the heparin structure, but also does not cause the color of the crude product to darken due to high temperature heating, reduces the difficulty of oxidation, and reduces the number of oxidation times.

[0024] 3. Oxidation under low temperature and alkaline environment can not only effectively remove colored substances, nucleic acids, and proteins, but also retain the heparin structure to the greatest extent.

[0025] 4. Adding an appropriate amount of calcium chloride during oxidation can greatly increase the effect of removing colored substances, nucleic acids, and proteins, reduce production processes, shorten production cycles, and improve yields. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the disaccharide spectrum of the crude heparin in Example 1;

[0027] Figure 2 This is the disaccharide spectrum of the crude heparin after oxidation in Example 1;

[0028] Figure 3 This is a detection spectrum of relevant impurities in the heparin sodium finished product prepared in Example 1;

[0029] Figure 4 This is the disaccharide spectrum of the crude heparin after oxidation in Comparative Example 1;

[0030] Figure 5 This is a detection spectrum of relevant impurities in the heparin sodium finished product prepared in Comparative Example 1;

[0031] Figure 6 This is the disaccharide spectrum of the crude heparin after oxidation in Comparative Example 2;

[0032] Figure 7 This is a detection spectrum of relevant impurities in the heparin sodium finished product prepared in Comparative Example 2;

[0033] Figure 8 This is the disaccharide spectrum of the crude heparin after oxidation in Comparative Example 3;

[0034] Figure 9 This is a detection spectrum of relevant impurities in the heparin sodium finished product prepared in Comparative Example 3;

[0035] Figure 10 This is the disaccharide spectrum of the crude heparin after oxidation in Comparative Example 4;

[0036] Figure 11This is a detection spectrum of relevant impurities in the heparin sodium finished product prepared in Comparative Example 4. DETAILED DESCRIPTION

[0037] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0038] The present invention provides a method for preparing heparin sodium, comprising the following steps:

[0039] 1) mixing heparin with water, adjusting the temperature of the obtained heparin aqueous solution to 0-5° C. and adjusting it to acidity to obtain an acidic heparin solution;

[0040] 2) centrifuging the acidic heparin solution at 0-5° C. to obtain a supernatant;

[0041] 3) adjusting the supernatant to alkaline, controlling the temperature of the alkaline supernatant at 0-5° C., adding calcium chloride and hydrogen peroxide, and oxidizing at 5-15° C. to obtain an oxidation product;

[0042] 4) adding sodium carbonate to the oxidation product, stirring and centrifuging in sequence, and detecting the absorbance of the centrifuge at 260 nm, 280 nm, and 400 nm using a spectrophotometer; when the absorbance at 260 nm is <0.1, the absorbance at 280 nm is <0.1, and the absorbance at 400 nm is <0.040, proceeding to the next step; otherwise, repeating steps 3) and 4) to continue oxidation until the absorbance at 260 nm, the absorbance at 280 nm is <0.1, and the absorbance at 400 nm is <0.040;

[0043] 5) Sodium chloride is added to the centrifuge solution, and after adjusting to neutrality, ethanol is added and the solution is allowed to stand to obtain heparin sodium.

[0044] The present invention mixes heparin with water, adjusts the temperature of the resulting heparin aqueous solution to 0-5°C, and adjusts the solution to acidity to obtain an acidic heparin solution. In the present invention, the concentration of the heparin aqueous solution is preferably 10 wt% to 15 wt%. In the present invention, hydrochloric acid is preferably used to adjust the pH to 3-4.

[0045] After obtaining the acidic heparin solution, the present invention centrifuges the acidic heparin solution at 0-5°C to obtain a supernatant. In the present invention, the centrifugal speed is preferably 3000-5000 r / min, more preferably 4000 r / min; and the centrifugal time is preferably 5-10 min.

[0046] In the present invention, centrifugation is performed under acidic conditions, which can quickly, effectively and to the greatest extent remove protein and nucleic acid impurities in crude heparin, thereby reducing the number of subsequent process steps.

[0047] After obtaining the supernatant, the present invention adjusts the supernatant to alkaline, controls the temperature of the alkaline supernatant to 0-5°C, adds calcium chloride and hydrogen peroxide, and performs oxidation at 5-15°C to obtain an oxidized product. In the present invention, sodium hydroxide solution is preferably used to adjust the pH to 9-10. In the present invention, the amount of calcium chloride added is preferably 3% of the volume of the supernatant, and the amount of hydrogen peroxide added is preferably 1%-2% of the volume of the supernatant. In the present invention, the oxidation time is preferably 6-12 hours.

[0048] After obtaining the oxidation product, the present invention adds sodium carbonate to the oxidation product and stirs and centrifuges in sequence. The centrifuge is tested for light absorption at 260 nm, 280 nm, and 400 nm using a spectrophotometer. When the light absorption at 260 nm is <0.1, the light absorption at 280 nm is <0.1, and the light absorption at 400 nm is <0.040, the process proceeds to the next step. Otherwise, steps 3) and 4) are repeated to continue oxidation until the light absorption at 260 nm, 280 nm, and 400 nm is <0.1. In the present invention, the amount of sodium carbonate added is preferably 3% of the volume of the oxidation product. In the present invention, the stirring speed is preferably 100 to 300 r / min, and the time is preferably 50 to 70 min. The centrifuge speed is preferably 3000 to 5000 r / min, and the time is preferably 5 to 10 min.

[0049] According to the present invention, an appropriate amount of calcium chloride is added before oxidation, and the addition of calcium chloride before oxidation can enhance the oxidation effect of hydrogen peroxide and more effectively remove nucleic acids, proteins and colored substances in crude heparin. An appropriate amount of sodium carbonate is added after oxidation to form calcium carbonate precipitate with the calcium chloride, which is removed by centrifugation, thereby improving the purity of the product.

[0050] After obtaining the centrifuge, the present invention preferably uses a spectrophotometer to detect the optical absorption of the centrifuge at 260 nm, 280 nm, and 400 nm. In the present invention, using a spectrophotometer to detect the optical absorption of the centrifuge at 260 nm, 280 nm, and 400 nm can monitor and control the removal effect of nucleic acids, proteins, and colored substances.

[0051] After obtaining the centrifuge, the present invention adds sodium chloride to the centrifuge, adjusts it to neutrality, then adds ethanol and allows the centrifuge to stand to obtain heparin sodium. In the present invention, the amount of sodium chloride added is preferably 1% of the volume of the centrifuge; the amount of ethanol added is preferably 70% to 100% of the volume of the centrifuge. In the present invention, the standing is preferably terminated when the impurity content of the obtained heparin sodium is ≤2%. In the present invention, to save time, it is preferably allowed to stand for 3 to 4 hours for an impurity test. If the impurity content is ≤2wt%, the standing is stopped. If the impurity content is >2wt%, the standing is continued.

[0052] Under alkaline conditions, the 2-O-sulfate group on iduronic acid can be hydrolyzed, thereby making heparin lose its activity and causing a certain degree of damage to the heparin structure. Currently, enzymatic hydrolysis, salting, and high-temperature treatment are used to remove nucleic acids and proteins in crude heparin, and the processes of these impurity removals are all carried out under alkaline conditions. The present invention takes into account the chemical properties of crude heparin itself, and the heparin solution is centrifuged under acidic conditions to quickly, effectively, and to the greatest extent remove protein and nucleic acid impurities in crude heparin, reduce, without the need for enzymatic hydrolysis and salting, and carry out under low temperature conditions, while rapidly removing nucleic acids and proteins in crude heparin, protecting the structure of heparin to the greatest extent.

[0053] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0054] Example 1

[0055] 1. Weigh 60.05 g crude heparin (the disaccharide spectrum of crude heparin is as follows Figure 1 ), add 600 mL of purified water, dissolve, and place in a freezer to cool to 5°C. Adjust the pH of the solution to 3.5 with hydrochloric acid and stir at 100 rpm until the solution is uniform. Centrifuge the solution at 4000 rpm for 5 minutes at 5°C to obtain a supernatant.

[0056] 2. Transfer the supernatant to another container and adjust the pH to 9.5 with sodium hydroxide solution (the temperature is controlled at 5-10°C during the entire process). After adjustment, add 18.10 g of calcium chloride, dissolve it, and then add 2% hydrogen peroxide by volume of the supernatant. Oxidize at 10°C for 8 h to obtain the oxidation product.

[0057] 3. Add 18.00 g of sodium carbonate to the oxidation product and stir at 100 r / min for 1 hour. Centrifuge at 4000 r / min for 5 minutes and collect the centrifuge liquid.

[0058] 4. The centrifuge was tested for absorbance at 260 nm, 280 nm, and 400 nm. At this point, the absorbance at 400 nm was 0.036, while the absorbance at 260 nm and 280 nm was >0.1. Therefore, the centrifuge was adjusted to a pH of 9.5 using sodium hydroxide solution (the temperature was maintained at 5-10°C throughout the process). After adjustment, 18.10 g of calcium chloride was added and dissolved. Then, 2% hydrogen peroxide (total volume) was added to the supernatant and oxidized at 10°C for 8 h. 18.00 g of sodium carbonate was added to the oxidized product, stirred at 100 rpm for 1 hour, centrifuged at 4000 rpm for 5 minutes, and the centrifuge was collected. The centrifuge was again tested for absorbance at 260 nm and 280 nm. The absorbance at 260 nm was 0.088, and the absorbance at 280 nm was 0.058. The absorbance at 260 nm, 280 nm, and 400 nm all met the requirements. The disaccharide spectrum of crude heparin after oxidation is as follows Figure 2 shown.

[0059] 5. Add sodium chloride to the centrifuge (the amount added is 1% of the volume of the centrifuge), then adjust the pH to 7.0 with hydrochloric acid, add 0.8 times the volume of ethanol to the centrifuge, and after standing for 4 hours, the impurity content is 1.04% (the impurity detection spectrum in the finished product is as shown in the figure). Figure 3 The activity and yield of the obtained heparin sodium were tested, and the specific results are shown in Table 1. The disaccharide composition of the obtained heparin sodium is shown in Table 2.

[0060] Table 1 Activity and yield

[0061] FXa FIIa Weight yield 223 IU / mg 227 IU / mg 45％

[0062] Table 2 Disaccharide composition (%)

[0063]

[0064]

[0065] Example 2

[0066] 1. Weigh 60.15 g of crude heparin and add 500 mL of purified water. Dissolve the mixture and place in a freezer to cool to 0°C. Adjust the pH of the solution to 4.0 with hydrochloric acid and stir at 100 rpm until the solution is homogeneous. Centrifuge the mixture at 4000 rpm for 10 minutes at 4°C.

[0067] 2. Transfer the supernatant to another container and quickly adjust the pH to 10.0 with sodium hydroxide solution (the temperature is controlled at 15°C during the entire process). After adjustment, add 18.05 g of calcium chloride, dissolve it, and then add 2% hydrogen peroxide by volume of the supernatant. Oxidize at 15°C for 12 hours.

[0068] 3. Add 18.12 g of sodium carbonate to the oxidation product and stir at 100 r / min for 1 hour. Centrifuge at 4000 r / min for 5 minutes and collect the centrifuge liquid.

[0069] 4. The centrifuge was tested for absorbance at 260 nm, 280 nm, and 400 nm. At this point, the absorbance at 400 nm was 0.036, while the absorbance at 260 nm and 280 nm was >0.1. Therefore, the centrifuge was rapidly adjusted to a pH of 10.0 using sodium hydroxide solution (the temperature was maintained at 15°C throughout the entire process). After adjustment, 18.05 g of calcium chloride was added and dissolved. Then, hydrogen peroxide (2% by volume of the feed solution) was added and oxidation was carried out at 15°C for 12 h. 18.12 g of sodium carbonate was added to the oxidation product, stirred at 100 rpm for 1 hour, centrifuged at 4000 rpm for 5 minutes, and the centrifuge was collected. The centrifuge was again tested for absorbance at 260 nm and 280 nm. At this point, the absorbance at 260 nm was 0.086, and the absorbance at 280 nm was 0.056. The absorbance at 260 nm, 280 nm, and 400 nm all met the requirements.

[0070] 5. Sodium chloride was added to the centrifuge (1% of the centrifuge volume), and then the pH was adjusted to 6.5 with hydrochloric acid. Ethanol (0.8 times the volume of the centrifuge volume) was added and the mixture was allowed to stand for 4 hours. The impurity content was determined to be 1.12%. The mixture was then allowed to stand for 4 hours to obtain heparin sodium. The activity and yield of the obtained heparin sodium were tested, and the specific results are shown in Table 3. The disaccharide composition of the obtained heparin sodium is shown in Table 4.

[0071] Table 3 Activity and yield

[0072] FXa FIIa Weight yield 221 IU / mg 222 IU / mg 46％

[0073] Table 4 Disaccharide composition (%)

[0074] Disaccharide components Crude heparin After oxidation is completed Linkage 2.88 1.81 ΔIVA 10.10 4.27 Linkageox 3.73 0.38 ΔIVSgal 0.14 0.16 ΔIVS 5.96 3.04 ΔIIA 4.31 3.38 ΔIIIA 2.04 1.61 ΔIISgal 0.73 1.20 ΔIIS 10.08 11.45 ΔIIIS 7.33 7.27 ΔIA 1.19 1.51 ΔIIA-IVSglu 0.34 0.59 ΔIS 47.16 58.72 ΔIIA-IISglu 2.02 2.48

[0075] Comparative Example 1

[0076] 1. Heat 600 mL of purified water to 50°C, add 2% sodium chloride by volume of purified water while stirring, dissolve it, and add 60.05 g of crude heparin (the disaccharide spectrum of crude heparin is as follows Figure 1As shown), stirring and dissolving was continued for 1 hour, the temperature was lowered to 35°C, 1.2 times the weight of crude heparin sodium to potency ratio of dissolved pancreatic enzyme was added, the mixture was stirred evenly, the temperature was controlled at 35°C, 4 mol / L sodium hydroxide solution was added dropwise, the pH was adjusted to 7.0, and the mixture was heated for 3 hours. Anhydrous sodium carbonate at a concentration of 0.3% by volume of the feed solution and 0.25% by volume of sodium bisulfite were added, the temperature was raised to 50°C, 2.0% by volume of the solution of anhydrous calcium chloride was added, the temperature was raised to 80°C, the mixture was heated for 30 minutes, the temperature was cooled by water and filtered, anhydrous sodium carbonate at 2% by volume of the solution was added, the mixture was heated and stirred for 30 minutes, and then filtered.

[0077] 2. The pH of the solution was adjusted to 10 with a 4 mol / L sodium hydroxide solution. Anhydrous sodium carbonate (0.3% by volume), anhydrous sodium sulfate (0.25%), and sodium chloride (2%) were added. The solution temperature was controlled at 30°C. Hydrogen peroxide (2% by volume) was added and oxidized for 12 hours. The absorbance at 260 nm was 0.144, at 280 nm was 0.099, and at 400 nm was 0.052.

[0078] 3. After the oxidation in step 2 is completed, the solution is filtered and ethanol heated to 40.0°C is added according to the ratio of feed liquid volume to ethanol volume = 1:1.0. The alcohol content of the feed liquid is controlled to 38% and the temperature is 35.0°C. The mixture is kept warm and allowed to settle for 3.0 hours.

[0079] 4. The precipitate obtained after the precipitation in step 3 was dissolved in 6.9 times the volume of purified water. The temperature of the solution was adjusted to 25.0°C. The pH of the solution was adjusted to 11 with 4 mol / L sodium hydroxide solution. 0.3% of anhydrous sodium carbonate and 0.25% of anhydrous sodium sulfate were added and stirred to dissolve. Sodium chloride was then added at 2.0% of the solution volume. Hydrogen peroxide (original concentration: 30%) was added at 2.0% of the solution volume and oxidized for 12 hours. At this point, the optical absorption at 260 nm was 0.108, the optical absorption at 280 nm was 0.083, and the optical absorption at 400 nm was 0.047.

[0080] 5. After the oxidation in step 4 is completed, filter the solution, add ethanol that has been heated to 40.0°C according to the ratio of feed liquid volume to ethanol volume = 1:1.1, control the alcohol content of the feed liquid to 44%, the temperature to 35.0°C, and keep it warm and let it settle for 3.0 hours. 6. Freeze drying: Use 3 times the amount of purified water as the crude sodium heparin to fully dissolve the precipitate obtained in step 5, adjust the pH value to 5.5 with 4mol / L hydrochloric acid, filter, freeze-dry, and obtain the finished sodium heparin. The impurity content was detected to be 1.76%. The activity and yield of the obtained sodium heparin were tested, and the specific results are shown in Table 5. The disaccharide composition of the obtained sodium heparin is shown in Table 6. The disaccharide spectrum of the crude heparin after oxidation is shown in Table 6. Figure 4 As shown, the impurity detection spectrum in the finished product is as follows Figure 5 shown.

[0081] Table 5 Activity and yield

[0082] FXa FIIa Weight yield 215 IU / mg 210 IU / mg 41％

[0083] Table 6 Disaccharide composition (%)

[0084]

[0085]

[0086] Comparative Example 2

[0087] The difference from Example 1 is that calcium chloride was not added before oxidation, and the other operations were exactly the same as in Example 1. The impurity content of the obtained heparin sodium was 1.97%. The activity and yield of the obtained heparin sodium were tested, and the specific results are shown in Table 7. The disaccharide composition of the obtained heparin sodium is shown in Table 8. The disaccharide spectrum of the crude heparin after oxidation is shown in Table 8. Figure 6 As shown, the impurity detection spectrum in the finished product is as follows Figure 7 shown.

[0088] Table 7 Activity and yield

[0089] FXa FIIa Weight yield 220 IU / mg 224 IU / mg 40％

[0090] Table 8 Disaccharide composition (%)

[0091] Disaccharide components Crude heparin After oxidation is completed Linkage 2.18 1.37 ΔIVA 9.73 3.99 Linkageox 5.58 0.54 ΔIVSgal 0.00 0.22 ΔIVS 5.57 2.87 ΔIIA 4.50 3.43 ΔIIIA 1.87 1.68 ΔIISgal 0.38 1.14 ΔIIS 9.31 11.43 ΔIIIS 7.17 7.33 ΔIA 1.19 1.55 ΔIIA-IVSglu 0.00 0.38 ΔIS 46.47 59.40 ΔIIA-IISglu 2.12 2.48

[0092] Comparative Example 3

[0093] The difference from Example 1 is that in step 1, the pH of the heparin solution is not adjusted to acidic before centrifugation, and the other operations are exactly the same as in Example 1. The specific operations of step 1 are:

[0094] Weigh 60.05 g of crude heparin (the disaccharide spectrum of crude heparin is as follows Figure 1 ), add 600 mL of purified water, dissolve, place in a freezer and cool to 5°C, stir at 100 r / min until the liquid is uniform. Centrifuge the material at 4000 r / min at 5°C for 5 minutes to obtain a supernatant.

[0095] The impurity content of the obtained heparin sodium was 1.34%. The activity and yield of the obtained heparin sodium were tested, and the specific results are shown in Table 9. The disaccharide composition of the obtained heparin sodium is shown in Table 10. The disaccharide spectrum of the crude heparin after oxidation is shown in Table 10. Figure 8 As shown, the impurity detection spectrum in the finished product is as follows Figure 9 shown.

[0096] Table 9 Activity and yield

[0097] FXa FIIa Weight yield 216 IU / mg 211 IU / mg 40％

[0098] Table 10 Disaccharide composition (%)

[0099] Disaccharide components Crude heparin After oxidation is completed Linkage 2.18 1.62 ΔIVA 9.73 4.57 Linkageox 5.58 0.49 ΔIVSgal 0.00 0.19 ΔIVS 5.57 3.10 ΔIIA 4.50 3.46 ΔIIIA 1.87 1.55 ΔIISgal 0.38 1.35 ΔIIS 9.31 10.77 ΔIIIS 7.17 7.04 ΔIA 1.19 1.36 ΔIIA-IVSglu 0.00 0.45 ΔIS 46.47 59.50 ΔIIA-IISglu 2.12 2.46

[0100] Comparative Example 4

[0101] The difference from Example 1 is that the entire process is carried out at room temperature (20-25°C), and the other operations are exactly the same as in Example 1. The impurity content in the obtained heparin sodium is 1.86%. The activity and yield of the obtained heparin sodium were tested, and the specific results are shown in Table 11. The disaccharide composition of the obtained heparin sodium is shown in Table 12. The disaccharide spectrum of the crude heparin after oxidation is shown in Table 12. Figure 10 As shown, the impurity detection spectrum in the finished product is as follows Figure 11 shown.

[0102] Table 11 Activity and yield

[0103]

[0104]

[0105] Table 12 Disaccharide composition (%)

[0106] Disaccharide components Crude heparin After oxidation is completed Linkage 2.18 1.63 ΔIVA 9.73 5.28 Linkageox 5.58 0.44 ΔIVSgal 0.00 0.25 ΔIVS 5.57 3.64 ΔIIA 4.50 3.46 ΔIIIA 1.87 1.56 ΔIISgal 0.38 1.89 ΔIIS 9.31 10.70 ΔIIIS 7.17 7.40 ΔIA 1.19 1.44 ΔIIA-IVSglu 0.00 0.50 ΔIS 46.47 57.46 ΔIIA-IISglu 2.12 2.30

[0107] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing heparin sodium, characterized in that: The steps include: 1) mixing heparin with water, adjusting the temperature of the obtained heparin aqueous solution to 0-5° C. and adjusting it to acidity to obtain an acidic heparin solution; 2) centrifuging the acidic heparin solution at 0-5° C. to obtain a supernatant; 3) adjusting the supernatant to alkaline, controlling the temperature of the alkaline supernatant at 0-5° C., adding calcium chloride and hydrogen peroxide, and oxidizing at 5-15° C. to obtain an oxidation product; 4) adding sodium carbonate to the oxidation product, stirring and centrifuging in sequence, and detecting the absorbance of the centrifuge at 260 nm, 280 nm, and 400 nm using a spectrophotometer; when the absorbance at 260 nm is <0.1, the absorbance at 280 nm is <0.1, and the absorbance at 400 nm is <0.040, proceeding to the next step; otherwise, repeating steps 3) and 4) to continue oxidation until the absorbance at 260 nm, the absorbance at 280 nm is <0.1, and the absorbance at 400 nm is <0.040; 5) adding sodium chloride to the centrifuge solution, adjusting the solution to neutrality, adding ethanol, and allowing the solution to stand to obtain heparin sodium; The concentration of the heparin aqueous solution in step 1) is 10 wt% to 15 wt%; in step 1), the pH value is adjusted to 3 to 4 using hydrochloric acid; In step 3), the pH value is adjusted to 9-10 using sodium hydroxide solution; In step 3), the amount of calcium chloride added is 3% of the volume of the supernatant, and the amount of hydrogen peroxide added is 1% to 2% of the volume of the supernatant.

2. The preparation method according to claim 1, characterized in that The centrifugal speed in step 2) is 3000-5000 r / min, and the time is 5-10 min.

3. The preparation method according to claim 1, characterized in that The oxidation time in step 3) is 6 to 12 hours.

4. The preparation method according to claim 1, characterized in that In step 4), the amount of sodium carbonate added is 3% of the volume of the oxidation product.

5. The preparation method according to claim 1, characterized in that In step 4), the stirring speed is 100-300 r / min and the time is 50-70 min; the centrifugal speed is 3000-5000 r / min and the time is 5-10 min.

6. The preparation method according to claim 1, characterized in that In step 5), the amount of sodium chloride added is 1% of the volume of the centrifuge; the amount of ethanol added is 70% to 100% of the volume of the centrifuge.

7. The preparation method according to claim 1, characterized in that In step 5), when the impurity content of the obtained heparin sodium is ≤2%, the standing is completed.

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