Method for purifying and refining heparin sodium crude product

Abstract

The invention provides a heparin sodium crude product purifying and refining method, which comprises: adding a heparin sodium crude product into a sodium chloride solution, and completely dissolving to obtain a heparin sodium crude product solution; adding trypsin into the heparin sodium crude product solution, performing enzymolysis, and inactivating to obtain an enzymolysis solution; adding hydrogen peroxide into the enzymolysis solution, carrying out first oxidation, and carrying out alcohol precipitation to obtain a first precipitate; adding the first precipitate into a sodium chloride solution, adding anion exchange resin, adsorbing, filtering, eluting, and carrying out alcohol precipitation to obtain a second precipitate; and adding the second precipitate into a sodium chloride solution, adding hydrogen peroxide, carrying out second oxidation, filtering, freeze-drying, and crushing to obtain a pure heparin sodium product. According to the method, separation, purification and efficient extraction of the heparin sodium are realized, the recovery rate and titer of the pure heparin sodium product are improved, and the heparin sodium bulk drug meeting the pharmacopoeia standard is produced.

Description

Technical Field

[0001] The present invention relates to the technical field of biological purification, and particularly to a method for purifying and refining crude heparin sodium. Background Art

[0002] As an important anticoagulant drug, heparin sodium plays an irreplaceable role in the medical field. It is mainly extracted from animal tissues, especially porcine intestinal mucosa, and is widely used in anticoagulant therapy and the prevention of thrombotic diseases. Due to the increasing clinical demand for heparin sodium, it has become particularly important to develop an efficient, economical, and scalable purification and refining process.

[0003] The crude heparin sodium directly extracted from porcine intestinal mucosa contains impurities such as proteins, nucleic acids, and inorganic salts, with a titer of 10 - 150 IU / mg. It cannot be used as the raw material of heparin sodium for medical use and must be purified to have medical use. Many production enterprises are unable to further produce high-titer and structurally intact heparin sodium due to backward production processes, low yields, and high production costs when purifying crude heparin sodium. Therefore, the production of high-quality heparin sodium with a unit titer greater than 180 IU / mg and a complete structure from crude heparin sodium has become the research focus in the industry.

[0004] Currently, the methods for purifying crude heparin sodium at home and abroad all have certain limitations. On the one hand, traditional methods usually rely on long-term salting out or acid hydrolysis processes, and these methods have low extraction efficiency and cannot meet the requirements of large-scale production. To overcome the above problems, the hydrogen peroxide oxidation method has been used to refine heparin sodium in recent years, with simple process operation and high recovery rate. However, the most difficult problem in the process is to filter out acidic heteroproteins, and how to ensure the yield and titer is the key. Therefore, studying a more reasonable process method to improve the quality of refined heparin sodium, especially on the basis of increasing the unit titer to greater than 180 IU / mg while maintaining the complete molecular structure of heparin sodium, is an important factor in improving the quality of heparin sodium raw material products. Summary of the Invention

[0005] Based on the technical problems existing in the background art, the present invention proposes a method for purifying and refining crude heparin sodium, which realizes the separation and purification of heparin sodium and efficient extraction, improves the recovery rate and titer of pure heparin sodium, and produces heparin sodium raw materials that meet the pharmacopoeia standards.

[0006] The method for purifying and refining crude heparin sodium proposed by the present invention includes the following steps:

[0007] S1. Add the crude heparin sodium to a sodium chloride solution, and after complete dissolution, obtain a crude heparin sodium solution;

[0008] S2. Add trypsin to the crude heparin sodium solution, inactivate it after enzymatic hydrolysis to obtain an enzymatically hydrolyzed solution;

[0009] S3. Add hydrogen peroxide to the enzymatically hydrolyzed solution, after the first oxidation, perform alcohol precipitation to obtain the first precipitate;

[0010] S4. Add the first precipitate to a sodium chloride solution, add an anion exchange resin, after adsorption, filter, elute, and perform alcohol precipitation to obtain the second precipitate;

[0011] S5. Add the second precipitate to a sodium chloride solution, add hydrogen peroxide, after the second oxidation, filter, lyophilize, and pulverize to obtain pure heparin sodium.

[0012] In the present invention, aiming at the other mucopolysaccharides, a large amount of undigested proteins and nucleic acid substances contained in heparin sodium, heparin sodium is refined by the two - step hydrogen peroxide oxidation method to maximize the removal of impurities such as proteins and nucleic acids that are tightly bound to heparin, improve the extraction efficiency, and maintain the activity of heparin sodium, and finally obtain a heparin sodium product with high purity and high quality.

[0013] Preferably, in step S1, add the crude heparin sodium to a 2 - 3 wt% sodium chloride solution, raise the temperature to 55 - 60 °C and stir for 4 - 5 h to obtain a crude heparin sodium solution.

[0014] Preferably, in step S2, add trypsin to the crude heparin sodium solution, the addition amount of trypsin is 0.5 - 2% of the mass of the crude heparin sodium, adjust the pH to 8.0 - 8.5, raise the temperature to 55 - 60 °C, after enzymatic hydrolysis for 2 - 3 h, raise the temperature to 80 - 85 °C, inactivate and stir for 20 - 30 min to obtain an enzymatically hydrolyzed solution.

[0015] Preferably, in step S3, lower the temperature of the enzymatically hydrolyzed solution below 30 °C, adjust the pH to 10.0 - 10.5, add a hydrogen peroxide solution, the addition amount of the hydrogen peroxide solution is 2 - 3% of the volume of the enzymatically hydrolyzed solution, oxidize for 12 - 18 h to obtain the first oxidized solution.

[0016] Preferably, in step S3, it further includes performing heat denaturation on the first oxidized solution;

[0017] Preferably, the heat denaturation includes: adding calcium chloride and sodium sulfite to the first oxidized solution, the addition amount of calcium chloride is 5 - 10% of the mass of the crude heparin sodium, the addition amount of sodium sulfite is 0.5 - 1% of the mass of the crude heparin sodium, adjust the pH to 8.0 - 8.5, raise the temperature to 85 - 95 °C, perform heat denaturation for 15 - 20 min, after filtration, add anhydrous sodium carbonate to the obtained filtrate, the addition amount of anhydrous sodium carbonate is the same as that of calcium chloride, adjust the pH to 9.0 - 10.0, filter to obtain a filtrate;

[0018] Preferably, the ethanol precipitation includes: adding 95% ethanol to the filtrate, where the volume of ethanol is 0.7 - 1 times that of the filtrate, and standing at room temperature for 4 - 6 h.

[0019] Preferably, in step S4, the first precipitate is added to a 2 - 3 wt% sodium chloride solution, and then an anion exchange resin is added. The addition amount of the anion exchange resin is 1 - 3% of the mass of the crude heparin sodium. The pH is adjusted to 7.0 - 8.0, and the temperature is raised to 55 - 60 °C, followed by stirring and adsorbing for 4 - 6 h. Then, filtration is carried out to collect the resin. A 5 - 7 wt% sodium chloride solution that completely submerges the resin amount is added to the resin, and the temperature is raised to 55 - 60 °C, followed by stirring for 0.5 - 1 h. After filtering and drying, the washed resin is obtained. Then, a 20 - 25 wt% sodium chloride solution that completely submerges the amount of the washed resin is added, and the temperature is raised to 55 - 60 °C, followed by stirring for 3 - 4 h. Then, the resin is separated, and the eluate is collected. The addition of a 20 - 25 wt% sodium chloride solution that completely submerges the amount of the washed resin is repeated once. The two eluates are combined, and 95% ethanol is added. The volume of ethanol is 1 - 1.5 times that of the eluate to obtain the second precipitate.

[0020] Preferably, the anion exchange resin is a polyvinylpyrrolidone - modified macroporous basic anion exchange resin;

[0021] The polyvinylpyrrolidone - modified macroporous basic anion exchange resin is obtained by swelling macroporous chloromethylated polystyrene - divinylbenzene copolymer beads with a solvent, adding a tertiary aminoacrylate monomer for quaternization reaction, and then carrying out a copolymerization reaction with N - vinylpyrrolidone;

[0022] Preferably, the macroporous chloromethylated polystyrene - divinylbenzene copolymer beads are macroporous chloromethylated polystyrene - divinylbenzene copolymer beads; the tertiary aminoacrylate monomer is dimethylaminoethyl methacrylate.

[0023] In the present invention, on the one hand, the anion exchange resin belongs to a quaternary ammonium salt - type macroporous basic anion exchange resin, which can adsorb heparin sodium with strong - charge polyanions well. On the other hand, the grafted polyvinylpyrrolidone has good hydrophilicity, further enhancing the adsorption of polysaccharide - type heparin sodium. When this anion exchange resin is used as an adsorption resin, the absorption effect is good, and mucopolysaccharides can be effectively removed by washing with a low - concentration salt solution. Then, heparin sodium is obtained through replacement with a high - concentration salt solution and the resin, further realizing the efficient purification and separation of heparin sodium.

[0024] Preferably, in step S5, the second precipitate is added to a 2 - 3 wt% sodium chloride solution, the pH is adjusted to 10.0 - 10.5, and a hydrogen peroxide solution is added. The addition amount of the hydrogen peroxide solution is 1 - 2% of the solution volume, and oxidation is carried out for 6 - 12 h to obtain the second oxidation solution.

[0025] Preferably, in step S5, after filtering the second oxidation and decolorization solution, it is added to a freeze dryer for freeze-drying treatment. After pulverization, pure heparin sodium is obtained.

[0026] The present invention also provides a heparin sodium raw material drug, which includes the pure heparin sodium obtained by the above purification and refinement method.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention uses crude heparin sodium as a raw material to prepare pure heparin sodium: while realizing the separation and purification of heparin sodium based on the hydrogen peroxide secondary oxidation method, trypsin enzymolysis and resin adsorption are used to further remove the impurities contained in the crude heparin sodium, so that the heparin sodium titer has been significantly increased. In addition, calcium chloride is used to accelerate the adsorption and precipitation of alkaline proteins, and sodium sulfite has a protective effect on heparin sodium; furthermore, heparin is adsorbed by anion exchange resin, with a high titer recovery rate, good dynamic separation effect, and high degree of continuity. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a process flow diagram of the purification and refinement method of the crude heparin sodium described in Example 1 of the present invention;

[0030] Figure 2 It is a structural schematic diagram of the anion exchange resin described in Example 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] Hereinafter, the technical solutions of the present invention will be described in detail through specific examples. However, it should be clearly stated that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

[0032] Example 1

[0033] Referring to Figure 1 , this example provides a purification and refinement method for crude heparin sodium, including:

[0034] (1) Dissolution: 1 kg of crude heparin sodium (titer: 105.1 IU / mg) is added to a 2 wt% sodium chloride solution. After heating to 55 °C and stirring for 5 h, the crude heparin sodium is completely dissolved to obtain a 10% (w / v) crude heparin sodium solution;

[0035] (2) Enzymolysis: The pH of the crude heparin sodium solution is adjusted to 8.0. After heating to 55 °C, 0.01 kg of trypsin is added. After enzymolysis for 3 h, the pH is adjusted to 6.5, heated to 80 °C, inactivated for 30 min, and then filtered through a 100-mesh filter bag to obtain an enzymolysis solution;

[0036] (3) First oxidation: Cool the enzymolysis solution to below 30 °C, adjust the pH to 10.0, add 30 v% hydrogen peroxide accounting for 2% of the volume of the enzymolysis solution, stir and oxidize for 16 h, then filter to obtain the first oxidation solution;

[0037] (4) Thermal denaturation: Add 0.1 kg of calcium chloride and 0.01 kg of sodium sulfite to the first oxidation solution, adjust the pH to 8.0, heat up to 90 °C, keep warm for thermal denaturation for 15 min, filter through a 0.22 μm microporous membrane, then add 0.1 kg of anhydrous sodium carbonate to the obtained filtrate, adjust the pH to 9.5, filter through a 0.22 μm microporous membrane, cool the obtained filtrate to below 30 °C, add 95% ethanol accounting for 0.7 times the volume of the oxidation solution, stir for 30 min, then precipitate for 6 h to obtain the first precipitate;

[0038] (5) Adsorption: Add the first precipitate to a 2 wt% sodium chloride solution, adjust the pH to 8.0 to obtain a 10% (w / v) solution, add 0.02 kg of anion exchange resin, heat up to 55 °C and stir for adsorption for 6 h, filter, collect the resin, repeatedly wash the obtained resin with water until the water becomes clear, filter dry, then add a 5 wt% sodium chloride solution that completely submerges the resin amount, heat up to 55 °C and stir for 1 h, filter dry, add the washed resin to a 20 wt% sodium chloride solution that completely submerges the washed resin amount, heat up to 55 °C, stir for 4 h, then separate the resin, collect the eluate, repeat adding a 20 wt% sodium chloride solution that completely submerges the washed resin amount once, combine the two eluates to obtain the eluate, add 95% ethanol accounting for 1 time the volume of the eluate, stir for 30 min, precipitate for 6 h to obtain the second precipitate;

[0039] The anion exchange resin is a polyvinylpyrrolidone-modified macroporous basic anion exchange resin, which is prepared by the following method:

[0040] Add macroporous chloromethylated polystyrene-divinylbenzene copolymer beads (chloromethylated chlorinated beads, chlorine content is 20.8%) to N,N-dimethylformamide and swell for 2 h, then add dimethylaminoethyl methacrylate accounting for 60% of the mass of the chloromethylated chlorinated beads, heat up to 50 °C and stir for reaction for 24 h, cool to room temperature, add N-vinylpyrrolidone monomer accounting for 40% of the mass of the chloromethylated chlorinated beads and azobisisobutyronitrile accounting for 1% of the mass of the chloromethylated chlorinated beads, heat up to 80 °C and stir for reaction for 12 h, cool to room temperature, then filter out the obtained resin, add it to distilled water, fully soak and swell, then filter dry, add a sodium hydroxide solution with a volume 2 times that of 2wr% and stir for 2 h, wash with distilled water until neutral, filter dry to obtain the polyvinylpyrrolidone-modified macroporous basic anion exchange resin;

[0041] (6) Second oxidation: Add the second precipitate into a 2 wt% sodium chloride solution to obtain a 10% (w / v) solution. Adjust the pH to 10.0, add 1% (by volume) of 30 v% hydrogen peroxide to the solution, and stir and oxidize for 12 h to obtain the second oxidation solution;

[0042] (7) Freeze-drying and pulverization: Adjust the pH of the second oxidation solution to 6.9, filter it through a 0.22 μm microporous membrane into a clean area freeze-drying tray, perform vacuum freeze-drying for 75 h, and then pulverize it to obtain pure heparin sodium, which can be used as the raw material drug of heparin sodium. The yield of pure heparin sodium is 93.3%.

[0043] The indicators of the pure heparin sodium obtained in this example are shown in the following table:

[0044] Test Items Specified by USP Test Results Appearance White or off-white powder White powder pH value (1% aqueous solution) 5.0-7.5 7.2 Nucleic Acid Absorbance at 260 nm ≤ 0.20 0.083 Heavy Metal Content ≤ 30 ppm 12 ppm Potency (USP) ≥ 180 IU / mg 205.4 Protein ≤ 1% (w / w) 0.52％ Bacterial Endotoxin < 0.030 EU / IU 0.007

[0045] The pure heparin sodium prepared by the method described in Example 1 has a relatively high unit potency and other aspects.

[0046] Example 2

[0047] This example presents a method for purification and refinement of crude heparin sodium, including:

[0048] (1) Dissolution: Add 1 kg of crude heparin sodium (potency: 105.1 IU / mg) into a 3 wt% sodium chloride solution, heat to 60 °C and stir for 4 h until the crude heparin sodium is completely dissolved to obtain a 10% (w / v) crude heparin sodium solution;

[0049] (2) Enzymatic hydrolysis: Adjust the pH of the crude heparin sodium solution to 8.5, heat to 60 °C, add 0.01 kg of trypsin, after enzymatic hydrolysis for 2 h, adjust the pH to 7.0, heat to 85 °C, inactivate for 20 min, and filter through a 100-mesh filter bag to obtain the enzymatic hydrolysis solution;

[0050] (3) First oxidation: Cool the enzymatic hydrolysis solution to below 30 °C, adjust the pH to 10.5, add 3% (by volume) of 30 v% hydrogen peroxide to the enzymatic hydrolysis solution, stir and oxidize for 12 h, and then filter to obtain the first oxidation solution;

[0051] (4) Thermal denaturation: Add 0.05 kg of calcium chloride and 0.005 kg of sodium sulfite to the first oxidation solution, adjust the pH to 8.5, heat to 85 °C, keep warm for thermal denaturation for 20 min, filter through a 0.22 μm microporous membrane, add 0.05 kg of anhydrous sodium carbonate to the obtained filtrate, adjust the pH to 10.0, filter through a 0.22 μm microporous membrane, cool the obtained filtrate to below 30 °C, add 1 time (by volume) of 95% ethanol to the oxidation solution, stir for 30 min, and precipitate for 4 h to obtain the first precipitate;

[0052] (5) Adsorption: Add the first precipitate into a 3 wt% sodium chloride solution, adjust the pH to 7.0 to obtain a 10% (w / v) solution, add 0.03 kg of anion exchange resin, raise the temperature to 60 °C, stir and adsorb for 4 h, filter, collect the resin, repeatedly rinse the obtained resin with water until the water becomes clear, filter and dry, then add a 7 wt% sodium chloride solution that completely submerges the resin amount to the obtained resin, raise the temperature to 60 °C, stir for 0.5 h, filter and dry, add a 25 wt% sodium chloride solution that completely submerges the amount of the washed resin to the obtained washed resin, raise the temperature to 60 °C, stir for 3 h, separate the resin, collect the eluate, repeat adding a 25 wt% sodium chloride solution that completely submerges the amount of the washed resin once, combine the two eluates to obtain an eluate, add 95% ethanol with a volume 1.5 times that of the eluate, stir for 30 min, and precipitate for 6 h to obtain a second precipitate;

[0053] The anion exchange resin is a polyvinylpyrrolidone-modified macroporous basic anion exchange resin, which is prepared by the method described in Example 1;

[0054] (6) Second oxidation: Add the second precipitate into a 3 wt% sodium chloride solution to obtain a 10% (w / v) solution, adjust the pH to 10.5, add 30 v% hydrogen peroxide with a volume of 2% of the solution volume, stir and oxidize for 6 h to obtain a second oxidation solution;

[0055] (7) Freeze-drying and pulverization: Adjust the pH of the second oxidation solution to 6.5, filter through a 0.22 μm microporous filter membrane into a clean area freeze-drying tray, vacuum freeze-dry for 75 h, and then pulverize to obtain pure heparin sodium, which can be used as a heparin sodium raw material drug. The yield of pure heparin sodium is 92.6%.

[0056] The indicators of the pure heparin sodium obtained in this example are shown in the following table:

[0057]

[0058]

[0059] The pure heparin sodium prepared by the method described in Example 2 also has a relatively high unit potency and other aspects.

[0060] Comparative Example 1

[0061] This comparative example presents a method for the purification and refinement of crude heparin sodium, including:

[0062] (1) Dissolution: Add 1 kg of crude heparin sodium (potency: 105.1 IU / mg) into a 2 wt% sodium chloride solution, raise the temperature to 55 °C, stir for 5 h until the crude heparin sodium is completely dissolved to obtain a 10% (w / v) crude heparin sodium solution;

[0063] (2) Enzymatic hydrolysis: Adjust the pH of the crude heparin sodium solution to 8.0. After heating to 55°C, add 0.01 kg of trypsin. After enzymatic hydrolysis for 3 h, adjust the pH to 6.5, heat to 80°C, inactivate for 30 min, and filter through a 100-mesh filter bag to obtain the enzymatic hydrolysate;

[0064] (3) Oxidation: Cool the enzymatic hydrolysate to below 30°C, adjust the pH to 10.0, add 30 v% hydrogen peroxide accounting for 2% of the volume of the enzymatic hydrolysate, stir and oxidize for 16 h, and filter to obtain the oxidized solution;

[0065] (4) Thermal denaturation: Add 0.1 kg of calcium chloride and 0.01 kg of sodium sulfite to the oxidized solution, adjust the pH to 8.0, heat to 90°C, keep warm for thermal denaturation for 15 min, filter through a 0.22-μm microporous membrane filter. Then add 0.1 kg of anhydrous sodium carbonate to the obtained filtrate, adjust the pH to 9.5, filter through a 0.22-μm microporous membrane filter. Cool the obtained filtrate to below 30°C, add 95% ethanol accounting for 70% of the volume of the oxidized solution, stir for 30 min, and precipitate for 6 h to obtain the first precipitate;

[0066] (5) Adsorption: Add the first precipitate to a 2 wt% sodium chloride solution, adjust the pH to 8.0 to obtain a 10% (w / v) solution, add 0.02 kg of anion exchange resin, heat to 55°C and stir for adsorption for 6 h, filter, collect the resin, repeatedly wash the obtained resin with water until the water becomes clear, filter and dry. Then add a 5 wt% sodium chloride solution that completely submerges the resin amount to the obtained resin, heat to 55°C and stir for 1 h, filter and dry. Add a 20 wt% sodium chloride solution that completely submerges the amount of the washed resin to the obtained washed resin, heat to 55°C, stir for 4 h, separate the resin, collect the eluate, repeat adding a 20 wt% sodium chloride solution that completely submerges the amount of the washed resin once, combine the two eluates to obtain the eluate, add 95% ethanol with the same volume as the eluate, stir for 30 min, and precipitate for 6 h to obtain the second precipitate;

[0067] The anion exchange resin is a macroporous alkaline anion exchange resin modified with polyvinylpyrrolidone, which is prepared by the method described in Example 1;

[0068] (6) Freeze-drying and pulverization: Add the second precipitate to a 2 wt% sodium chloride solution to obtain a 10% (w / v) solution, adjust the pH to 6.9, filter through a 0.22-μm microporous membrane filter to a clean area freeze-drying tray, vacuum freeze-dry for 75 h, and pulverize to obtain pure heparin sodium, which can be used as heparin sodium raw material medicine. The yield of pure heparin sodium is 90.4%.

[0069] The indicators of the pure heparin sodium obtained in this comparative example are shown in the following table:

[0070]

[0071]

[0072] The pure heparin sodium prepared by the method described in Comparative Example 1 has relatively low unit potency and other aspects.

[0073] Comparative Example 2

[0074] This comparative example presents a method for purifying and refining crude heparin sodium, including:

[0075] (1) Dissolution: 1 kg of crude heparin sodium (potency: 105.1 IU / mg) was added to a 2 wt% sodium chloride solution, and the temperature was raised to 55 °C, followed by stirring for 5 h until the crude heparin sodium was completely dissolved, obtaining a 10% (w / v) crude heparin sodium solution;

[0076] (2) Enzymatic hydrolysis: The pH of the crude heparin sodium solution was adjusted to 8.0, and after raising the temperature to 55 °C, 0.01 kg of trypsin was added. After enzymatic hydrolysis for 3 h, the pH was adjusted to 6.5, and the temperature was raised to 80 °C. After inactivating for 30 min, it was filtered through a 100-mesh filter bag to obtain an enzymatic hydrolysate;

[0077] (3) First oxidation: The enzymatic hydrolysate was cooled to below 30 °C, the pH was adjusted to 10.0, and 2% of the volume of the enzymatic hydrolysate of 30 v% hydrogen peroxide was added. After stirring and oxidizing for 16 h, it was filtered to obtain a first oxidation solution;

[0078] (4) Thermal denaturation: 0.1 kg of calcium chloride and 0.01 kg of sodium sulfite were added to the first oxidation solution, the pH was adjusted to 8.0, the temperature was raised to 90 °C, and it was kept for thermal denaturation for 15 min. After filtering through a 0.22-μm microporous filter membrane, 0.1 kg of anhydrous sodium carbonate was added to the obtained filtrate, the pH was adjusted to 9.5, and after filtering through a 0.22-μm microporous filter membrane, the obtained filtrate was cooled to below 30 °C, and 70% of the volume of the oxidation solution of 95% ethanol was added. After stirring for 30 min, it was precipitated for 6 h to obtain a first precipitate;

[0079] (5) Adsorption: Add the first precipitate into a 2 wt% sodium chloride solution, adjust the pH to 8.0 to obtain a 10% (w / v) solution, add 0.02 kg of anion exchange resin (D204 resin), raise the temperature to 55 °C, stir and adsorb for 6 h, filter, collect the resin, repeatedly rinse the obtained resin with water until the water becomes clear, filter dry, then add a 5 wt% sodium chloride solution that completely submerges the resin amount to the obtained resin, raise the temperature to 55 °C, stir for 1 h, filter dry, add a 20 wt% sodium chloride solution that completely submerges the amount of the washed resin to the obtained washed resin, raise the temperature to 55 °C, stir for 4 h, separate the resin, collect the eluate, repeat adding a 20 wt% sodium chloride solution that completely submerges the amount of the washed resin once, combine the two eluates to obtain an eluate, add 95% ethanol in an amount equal to 1 times the volume of the eluate, stir for 30 min, precipitate for 6 h to obtain the second precipitate;

[0080] (6) Second oxidation: Add the second precipitate into a 2 wt% sodium chloride solution to obtain a 10% (w / v) solution, adjust the pH to 10.0, add 30 v% hydrogen peroxide in an amount equal to 1% of the solution volume, stir and oxidize for 12 h to obtain the second oxidation solution;

[0081] (7) Freeze-drying and pulverization: Adjust the pH of the second oxidation solution to 6.9, filter through a 0.22 μm microporous membrane into a clean area freeze-drying tray, vacuum freeze-dry for 75 h, and then pulverize to obtain pure heparin sodium, which can be used as heparin sodium raw material medicine. The yield of pure heparin sodium is 89.3%.

[0082] The indicators of the pure heparin sodium obtained in this example are shown in the following table:

[0083] Test Items Specified by USP Test Results Appearance White or off-white powder White powder pH value (1% aqueous solution) 5.0-7.5 7.3 Nucleic Acid Absorbance at 260 nm ≤ 0.20 0.134 Heavy Metal Content ≤ 30 ppm 19 ppm Potency (USP) ≥ 180 IU / mg 171.3 Protein ≤ 1% (w / w) 1.32％ Bacterial Endotoxin < 0.030 EU / IU 0.015

[0084] The pure heparin sodium prepared by the method described in Comparative Example 2 has a lower unit potency and other aspects.

[0085] Comparative Example 3

[0086] This comparative example presents a method for purification and refinement of crude heparin sodium, including:

[0087] (1) Dissolution: Add 1 kg of crude heparin sodium (potency 105.1 IU / mg) into a 2 wt% sodium chloride solution, raise the temperature to 55 °C, stir for 5 h until the crude heparin sodium is completely dissolved to obtain a 10% (w / v) crude heparin sodium solution;

[0088] (2) Enzymatic hydrolysis: Adjust the pH of the crude heparin sodium solution to 8.0, raise the temperature to 55 °C, add 0.01 kg of trypsin, after enzymatic hydrolysis for 3 h, adjust the pH to 6.5, raise the temperature to 80 °C, inactivate for 30 min, and then filter through a 100-mesh filter bag to obtain the enzymatic hydrolysis solution;

[0089] (3) First oxidation: Cool the enzymatic hydrolysate to below 30 °C, adjust the pH to 10.0, add 30v% hydrogen peroxide accounting for 2% of the volume of the enzymatic hydrolysate, stir and oxidize for 16 h, then filter to obtain the first oxidation solution;

[0090] (4) Thermal denaturation: Add 0.1 kg of calcium chloride and 0.01 kg of sodium sulfite to the first oxidation solution, adjust the pH to 8.0, heat up to 90 °C, keep warm for thermal denaturation for 15 min, filter through a 0.22 μm microporous membrane, then add 0.1 kg of anhydrous sodium carbonate to the obtained filtrate, adjust the pH to 9.5, filter through a 0.22 μm microporous membrane, cool the obtained filtrate to below 30 °C, add 95% ethanol accounting for 0.7 times the volume of the oxidation solution, stir for 30 min, then precipitate for 6 h to obtain the first precipitate;

[0091] (5) Adsorption: Add the first precipitate to a 2 wt% sodium chloride solution, adjust the pH to 8.0 to obtain a 10% (w / v) solution, add 0.02 kg of anion exchange resin, heat up to 55 °C and stir for adsorption for 6 h, filter, collect the resin, repeatedly wash the obtained resin with water until the water becomes clear, filter dry, then add a 5 wt% sodium chloride solution that completely submerges the resin amount, heat up to 55 °C and stir for 1 h, filter dry, add the washed resin to a 20 wt% sodium chloride solution that completely submerges the washed resin amount, heat up to 55 °C, stir for 4 h, then separate the resin, collect the eluate, repeat adding a 20 wt% sodium chloride solution that completely submerges the washed resin amount once, combine the two eluates to obtain the eluate, add 95% ethanol accounting for 1 time the volume of the eluate, stir for 30 min, precipitate for 6 h to obtain the second precipitate;

[0092] The anion exchange resin is a modified macroporous basic anion exchange resin, which is prepared by the following method:

[0093] Add macroporous chloromethylated polystyrene - divinylbenzene copolymer beads (chloromethylated chlorinated beads, chlorine content is 20.8%) to N, N - dimethylformamide and swell for 2 h, then add dimethylaminoethyl methacrylate accounting for 60% of the mass of the chloromethylated chlorinated beads, heat up to 50 °C and stir for reaction for 24 h, filter out the obtained resin after cooling to room temperature, add it to distilled water for full immersion and swelling, then filter dry, add a 2wr% sodium hydroxide solution with a volume 2 times that of the resin and stir for 2 h, wash with distilled water until neutral, filter dry to obtain the modified macroporous basic anion exchange resin;

[0094] (6) Second oxidation: Add the second precipitate to a 2 wt% sodium chloride solution to obtain a 10% (w / v) solution, adjust the pH to 10.0, add 30v% hydrogen peroxide accounting for 1% of the volume of the solution, stir and oxidize for 12 h to obtain the second oxidation solution;

[0095] (7) Lyophilization and pulverization: Adjust the pH of the secondary oxidation solution to 6.9, filter it through a 0.22 μm microporous membrane into a lyophilization tray in the clean area, conduct vacuum freeze-drying for 75 h, and then pulverize it to obtain pure heparin sodium, which can be used as the raw material for heparin sodium. The yield of pure heparin sodium is 90.9%.

[0096] The indicators of the pure heparin sodium obtained in this comparative example are shown in the following table:

[0097]

[0098]

[0099] The pure heparin sodium prepared by the method described in Comparative Example 3 has very low unit potency and other aspects.

[0100] Comparative Example 4

[0101] This comparative example presents a method for purifying and refining crude heparin sodium, including:

[0102] (1) Dissolution: Add 1 kg of crude heparin sodium (potency: 105.1 IU / mg) to a 2 wt% sodium chloride solution, raise the temperature to 55 °C, and stir for 5 h until the crude heparin sodium is completely dissolved to obtain a 10% (w / v) crude heparin sodium solution;

[0103] (2) Enzymatic hydrolysis: Adjust the pH of the crude heparin sodium solution to 8.0, raise the temperature to 55 °C, add 0.01 kg of trypsin, after enzymatic hydrolysis for 3 h, adjust the pH to 6.5, raise the temperature to 80 °C, inactivate for 30 min, and then filter through a 100-mesh filter bag to obtain an enzymatic hydrolysate;

[0104] (3) First oxidation: Cool the enzymatic hydrolysate to below 30 °C, adjust the pH to 10.0, add 2% of the volume of the enzymatic hydrolysate of 30 v% hydrogen peroxide, stir and oxidize for 16 h, and then filter to obtain the first oxidation solution;

[0105] (4) Thermal denaturation: Add 0.1 kg of calcium chloride and 0.01 kg of sodium sulfite to the first oxidation solution, adjust the pH to 8.0, raise the temperature to 90 °C, keep it warm for thermal denaturation for 15 min, filter through a 0.22 μm microporous membrane, add 0.1 kg of anhydrous sodium carbonate to the obtained filtrate, adjust the pH to 9.5, filter through a 0.22 μm microporous membrane, cool the obtained filtrate to below 30 °C, add 0.7 times the volume of the oxidation solution of 95% ethanol, stir for 30 min, and then precipitate for 6 h to obtain the first precipitate;

[0106] (5) Adsorption: Add the first precipitate into a 2 wt% sodium chloride solution, adjust the pH to 8.0 to obtain a 10% (w / v) solution, add 0.02 kg of anion exchange resin, raise the temperature to 55 °C, stir and adsorb for 6 h, filter, collect the resin, repeatedly rinse the obtained resin with water until the water becomes clear, filter and dry, then add a 5 wt% sodium chloride solution that completely submerges the resin amount to the obtained resin, raise the temperature to 55 °C, stir for 1 h, filter and dry, add a 20 wt% sodium chloride solution that completely submerges the amount of the washed resin to the obtained washed resin, raise the temperature to 55 °C, stir for 4 h, separate the resin, collect the eluate, repeat adding a 20 wt% sodium chloride solution that completely submerges the amount of the washed resin once, combine the two eluates to obtain an eluate, add 95% ethanol with a volume 1 time that of the eluate, stir for 30 min, precipitate for 6 h to obtain the second precipitate;

[0107] The anion exchange resin is a modified macroporous basic anion exchange resin, which is prepared by the following method:

[0108] Add macroporous chloromethylated polystyrene-divinylbenzene copolymer beads (chloromethylated chlorinated beads, chlorine content is 20.8%) into N,N-dimethylformamide to swell for 2 h, then add triethylamine with a mass 60% of the chloromethylated chlorinated beads, raise the temperature to 50 °C and stir and react for 24 h, after cooling to room temperature, add N-vinylpyrrolidone monomer with a mass 40% of the chloromethylated chlorinated beads and azobisisobutyronitrile with a mass 1% of the chloromethylated chlorinated beads, raise the temperature to 80 °C and stir and react for 12 h, after cooling to room temperature, filter out the obtained resin, filter out the obtained resin after cooling to room temperature, add it into distilled water, fully soak and swell, then filter and dry, add a 2wr% sodium hydroxide solution with a volume 2 times that of it and stir for 2 h, wash with distilled water until neutral, filter and dry to obtain the modified macroporous basic anion exchange resin;

[0109] (6) Second oxidation: Add the second precipitate into a 2 wt% sodium chloride solution to obtain a 10% (w / v) solution, adjust the pH to 10.0, add 30 v% hydrogen peroxide with a volume 1% of the solution volume, stir and oxidize for 12 h to obtain a second oxidation solution;

[0110] (7) Freeze-drying and pulverization: Adjust the pH of the second oxidation solution to 6.9, filter it through a 0.22 μm microporous membrane into a clean area freeze-drying tray, vacuum freeze-dry for 75 h, and then pulverize to obtain pure heparin sodium, which can be used as heparin sodium raw material medicine, and the yield of pure heparin sodium is 91.5%.

[0111] The indicators of the pure heparin sodium obtained in this comparative example are shown in the following table:

[0112]

[0113]

[0114] The pure heparin sodium prepared by the method described in Comparative Example 4 has relatively low unit potency and other aspects.

[0115] As mentioned above, only the preferred specific embodiments of the present invention are described. However, the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, should be covered within the protection scope of the present invention.

Claims

1. A method for purifying crude heparin sodium, characterized in that: The steps include: S1. Adding crude heparin sodium into sodium chloride solution and dissolving completely to obtain crude heparin sodium solution; S2, adding trypsin to the crude heparin sodium solution, enzymolyzing and inactivating it to obtain an enzymolysis solution; S3, adding hydrogen peroxide to the enzymatic solution, after the first oxidation, ethanol precipitation, to obtain a first precipitate; S4, adding the first precipitate to a sodium chloride solution, adding an anion exchange resin, after adsorption, filtering, eluting, and alcohol precipitation to obtain a second precipitate; S5. Add the second precipitate into a sodium chloride solution, add hydrogen peroxide, and after the second oxidation, filter, freeze-dry, and grind to obtain pure heparin sodium.

2. The method for purifying crude heparin sodium according to claim 1, characterized in that: In step S1, crude heparin sodium is added to a 2-3 wt % sodium chloride solution, heated to 55-60° C. and stirred for 4-5 hours to obtain a crude heparin sodium solution.

3. The method for purifying crude heparin sodium according to claim 1 or 2, characterized in that: In step S2, trypsin is added to the crude heparin sodium solution, the amount of trypsin added is 0.5-2% of the crude heparin sodium quality, the pH is adjusted to 8.0-8.5, the temperature is raised to 55-60° C., after enzymolysis for 2-3 hours, the temperature is raised to 80-85° C., and the solution is inactivated and stirred for 20-30 minutes to obtain an enzymolysis solution.

4. The method for purifying crude heparin sodium according to any one of claims 1 to 3, characterized in that: In step S3, the temperature of the enzymatic hydrolysate is lowered to below 30° C., the pH is adjusted to 10.0-10.5, and a hydrogen peroxide solution is added in an amount of 2-3% of the volume of the enzymatic hydrolysate. The solution is oxidized for 12-18 hours to obtain a first oxidation solution.

5. The method for purifying crude heparin sodium according to claim 4, characterized in that: Step S3 also includes thermally denaturing the first oxidation solution; Preferably, the thermal denaturation comprises: adding calcium chloride and sodium sulfite to the first oxidation solution, wherein the amount of calcium chloride added is 5-10% of the crude heparin sodium quality, and the amount of sodium sulfite added is 0.5-1% of the crude heparin sodium quality, adjusting the pH to 8.0-8.5, heating to 85-95° C., thermal denaturation for 15-20 min, filtering, adding anhydrous sodium carbonate to the obtained filtrate, wherein the amount of anhydrous sodium carbonate added is the same as that of calcium chloride, adjusting the pH to 9.0-10.0, filtering, and obtaining a filtrate; Preferably, the alcohol precipitation comprises: adding 95% ethanol to the filtrate, wherein the volume of the ethanol is 0.7-1 times the volume of the filtrate, and standing at room temperature for 4-6 hours.

6. The method for purifying crude heparin sodium according to any one of claims 1 to 5, characterized in that: In step S4, the first precipitate is added to a 2-3wt% sodium chloride solution, and then an anion exchange resin is added, the amount of the anion exchange resin added is 1-3% of the crude heparin sodium quality, the pH is adjusted to 7.0-8.0, the temperature is raised to 55-60°C and stirred for adsorption for 4-6h, filtered, the resin is collected, and a 5-7wt% sodium chloride solution is added to the resin to completely immerse the resin, the temperature is raised to 55-60°C and stirred for 0.5-1h, filtered to obtain washed resin, and then a 20-25wt% sodium chloride solution is added to completely immerse the washed resin, the temperature is raised to 55-60°C and stirred for 3-4h, the resin is separated, the eluent is collected, and the 20-25wt% sodium chloride solution is added to completely immerse the washed resin. The two eluents are combined, and 95% ethanol is added, and the volume of ethanol is 1-1.5 times the volume of the eluent to obtain a second precipitate.

7. The method for purifying crude heparin sodium according to claim 6, characterized in that: The anion exchange resin is a polyvinyl pyrrolidone modified macroporous alkaline anion exchange resin; The polyvinyl pyrrolidone modified macroporous alkaline anion exchange resin is obtained by swelling macroporous chloroalkylated polystyrene-divinylbenzene copolymer beads with a solvent, adding tertiary amino acrylate monomers to carry out quaternary ammonium salt reaction, and then copolymerizing with N-vinyl pyrrolidone; Preferably, the macroporous chloroalkylated polystyrene-divinylbenzene copolymer beads are macroporous chloromethylated polystyrene-divinylbenzene copolymer beads; and the tertiary amino acrylate monomer is dimethylaminoethyl methacrylate.

8. The method for purifying crude heparin sodium according to any one of claims 1 to 7, characterized in that: In step S5, the second precipitate is added to a 2-3 wt % sodium chloride solution, the pH is adjusted to 10.0-10.5, and a hydrogen peroxide solution is added, the amount of hydrogen peroxide solution added is 1-2% of the solution volume, and oxidation is performed for 6-12 hours to obtain a second oxidation solution.

9. The method for purifying crude heparin sodium according to any one of claims 1 to 8, characterized in that: In step S5, the second oxidation decolorization solution is filtered, added to a freeze dryer for freeze drying, and crushed to obtain pure heparin sodium.

10. A heparin sodium bulk drug, characterized in that: The invention comprises a pure heparin sodium product obtained by the purification method according to any one of claims 1 to 9.

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