Heparin sodium bulk drug and preparation method thereof

Abstract

The invention provides a heparin sodium bulk drug and a preparation method thereof, and the preparation method comprises the following steps: dissolving a heparin sodium crude product in a sodium chloride solution to obtain a crude product solution; carrying out enzymolysis on the crude product solution, and carrying out alcohol precipitation to obtain a precipitate; after dissolving the precipitate, purifying the precipitate by a strongly basic anion exchange column to obtain an eluent; and purifying the eluent by using a weakly alkaline anion exchange column to obtain the heparin sodium bulk drug, the strongly basic anion exchange column is filled with strongly basic quaternary ammonium type anion resin, and the strongly basic quaternary ammonium type anion resin is obtained by carrying out quaternary ammonium salinization reaction on tertiary amine anion exchange resin and halogenated quaternary ammonium salt; the alkalescent anion exchange column is filled with alkalescent imidazole type anion resin, and the alkalescent imidazole type anion resin is obtained by carrying out condensation reaction on primary amine anion exchange resin and alkenyl acyl chloride and then carrying out copolymerization reaction with vinyl imidazole. The heparin sodium obtained in the invention has high purity and high activity, and can be used as an injection-grade heparin sodium bulk drug.

Description

Technical Field

[0001] The present invention relates to the technical field of heparin sodium bulk drugs, and particularly relates to a heparin sodium bulk drug and a preparation method thereof. Background Art

[0002] Heparin sodium is an acidic mucopolysaccharide with anticoagulant effects. As a natural anticoagulant substance, heparin sodium has attracted the attention of countries around the world. Although heparin sodium has been used clinically for more than 60 years, there is still no product that can completely replace it so far. Therefore, it remains one of the most important biochemical drugs for anticoagulation and antithrombosis. Heparin has a strong anticoagulant effect and is the first choice drug for preventing and treating thromboembolic diseases such as deep vein thrombosis. With the in-depth research, it is found that heparin not only has anticoagulant, antithrombotic and lipid-regulating effects, but also has a variety of biological functions such as anti-inflammatory, anti-allergic, antiviral and anti-cancer.

[0003] The heparin sodium bulk drug is derived from crude heparin. Crude heparin is sourced from the small intestinal mucosa of healthy animals, which contains a large amount of impurity proteins, impurity nucleic acids, microorganisms, etc. It is necessary to go through physical and chemical extraction and separation processes to obtain heparin with a complete natural structural group in a targeted manner, so as to produce the heparin sodium bulk drug. However, the heparin bulk drugs produced in China have problems of low titer and low extraction rate, and most of them are exported to foreign countries at a low price in the form of crude heparin sodium. In view of the problems existing in the current production of heparin sodium, it is of great significance to study the technology for efficient extraction, separation and purification of heparin.

[0004] However, most of the existing heparin sodium extraction and purification methods use acid-base treatment to remove impurity proteins, nucleic acids and other substances, use multiple oxidation methods to remove pigment substances, and use multiple ethanol fractionation methods to remove other heteropolysaccharides. The above acid-base treatment method is prone to high residual amounts of nucleic acids and pigments, resulting in an increase in the number of subsequent oxidations; acid treatment and multiple oxidations are prone to partial inactivation of heparin sodium, making it difficult to obtain heparin sodium with a high titer; multiple ethanol fractionations are prone to reduce the yield and cause waste of materials.

[0005] The advantages of the enzymatic extraction of heparin polysaccharide are mild reaction conditions, and the yield can be significantly improved by removing protein impurities through enzymatic methods. However, this technology also has certain limitations: (1) The optimal temperature and optimal pH value of the enzyme are often within a very small range. To increase the enzyme activity to the maximum value, the enzyme reaction temperature and pH must be strictly controlled. A slight fluctuation in the reaction conditions may greatly reduce the enzyme activity, so the requirements for test equipment are relatively high. (2) During the enzymatic extraction process, the enzyme may react with the polysaccharide, causing polysaccharide degradation. (3) The enzyme itself is also a protein, so the enzyme needs to be inactivated and the protein impurities need to be removed after extracting heparin.

[0006] Zhou Xianwan from Peking University used the enzymatic hydrolysis method to refine heparin sodium and achieved good results (Zhou Xianwan, He Wei. Refinement of Heparin Sodium by Enzymatic Degradation. Chinese Journal of Biochemistry and Molecular Biology, 1999, 6(18): 488-490). However, in the preparation process, the heparin content in the enzymatic hydrolysate is generally above 100,000 U / L, but the heparin sodium adsorbed from the enzymatic hydrolysate by ion exchange is incomplete, and the residual solution often contains a relatively high amount of heparin sodium, thus affecting the recovery rate of heparin sodium. This requires first selecting a resin with good adsorption effect, strong adsorption capacity, alkali resistance, and heat resistance; and determining the optimal adsorption conditions of the resin. Summary of the Invention

[0007] Based on the technical problems existing in the background art, the present invention provides a heparin sodium raw material drug and its preparation method, which has the characteristics of simple operation, short process time, and high product recovery rate. The obtained heparin sodium not only has high purity and high activity, but also is used as an injection-grade heparin sodium raw material drug.

[0008] The preparation method of a heparin sodium raw material drug provided by the present invention includes the following steps:

[0009] S1. Dissolve the crude heparin sodium in a sodium chloride solution to obtain a crude product solution;

[0010] S2. After enzymatic hydrolysis of the crude product solution, perform alcohol precipitation to obtain a precipitate;

[0011] S3. After dissolving the precipitate, purify it on a strongly basic anion exchange column to obtain an eluate;

[0012] S4. After purifying the eluate on a weakly basic anion exchange column again, the heparin sodium raw material drug is obtained;

[0013] In step S3, the strongly basic anion exchange column is filled with a strongly basic quaternary ammonium type anion resin, which is obtained by performing a quaternary ammonium saltification reaction on a tertiary amine anion exchange resin and a quaternary ammonium halide;

[0014] In step S4, the weakly basic anion exchange column is filled with a weakly basic imidazole type anion resin, which is obtained by performing a condensation reaction on a primary amine anion exchange resin and an alkenyl acyl chloride, and then performing a copolymerization reaction with vinyl imidazole.

[0015] In the present invention, after enzymatic hydrolysis of the crude heparin sodium, heparin sodium is fully released from the protein complex; heparin sodium is first purified on a strongly basic anion exchange column to remove impurities such as heteroproteins and heteropolysaccharides, and then decolorized on a weakly basic anion exchange column to remove impurities such as pigments; through two-step purification of the strongly basic anion exchange column and the weakly basic anion exchange column, the present invention avoids the introduction of foreign substances, improves the activity of heparin sodium, and also ensures the purity of the product.

[0016] In the present invention, a strongly basic anion exchange column is filled with a strongly basic quaternary ammonium type anion resin, which uses a tertiary amine anion exchange resin as the matrix and utilizes the tertiary amine groups it has to carry out a quaternary ammonium salification reaction with a quaternary ammonium halide. In this way, a large number of quaternary ammonium cation structures are introduced onto the backbone of the exchange resin, obtaining an anion exchange resin with a higher amount of quaternary ammonium cations, achieving a greater retention effect for heparin sodium, heteroproteins, heteropolysaccharides, etc., thereby realizing the effective separation and purification of heparin sodium and obtaining a higher recovery efficiency.

[0017] In the present invention, a weakly basic anion exchange column is filled with a weakly basic imidazole type anion resin, which uses a primary amine anion exchange resin as the matrix and utilizes the primary amine groups it has to carry out a condensation reaction with an alkenyl acyl chloride. In this way, a large number of alkenyl structures are introduced onto the backbone of the exchange resin, which can carry out a copolymerization reaction with vinyl imidazole, thereby obtaining an anion exchange resin with a polyvinyl imidazole side chain. Utilizing the affinity between the imidazole group and heparin sodium, heparin sodium is further purified and decolorized.

[0018] Preferably, step S1 specifically includes: adding the crude heparin sodium product to a 2-3 wt% sodium chloride solution, heating to 45-55 °C and stirring for 4-6 h to obtain a crude product solution;

[0019] Preferably, the mass-volume ratio of the crude heparin sodium product to the sodium chloride solution is 1 g:5-10 mL.

[0020] Preferably, in step S2, the enzymatic hydrolysis specifically includes: adjusting the pH of the crude product solution to 7.5-8.5, heating to 45-55 °C, adding trypsin, incubating for 3-5 h, and then heating to 85-95 °C to inactivate, obtaining an enzymatic hydrolysis solution;

[0021] Preferably, the addition amount of the trypsin is 1-3% of the mass of the crude heparin sodium product;

[0022] Preferably, the alcohol precipitation specifically includes: adding 95% ethanol with a volume 1-2 times that of the enzymatic hydrolysis solution, stirring evenly, standing for precipitation for 4-6 h, and filtering to obtain a precipitate.

[0023] Preferably, step S3 specifically includes: dissolving the precipitate in purified water, adjusting the pH to 8.0-9.0, heating to 40-50 °C and then loading onto the column at a loading rate of 1.0-1.5 BV / h. After completion, impurities and heparin sodium are eluted successively to obtain an eluate;

[0024] Preferably, the elution of impurities includes: first washing with 2-3 BV of distilled water at 40-50 °C at a flow rate of 2-3 BV / h, and then washing with 3-4 BV of 5-8 wt% sodium chloride solution at a flow rate of 1-2 BV / h;

[0025] Preferably, the elution of sodium heparin includes: eluting with 2 - 3 BV of sodium chloride solution at a temperature of 40 - 50°C and a concentration of 20 - 25 wt%, with a flow rate of 0.5 - 1 BV / h.

[0026] Preferably, in the strongly basic quaternary ammonium type anion resin, the tertiary amine anion exchange resin is D301 resin, and the halogenated quaternary ammonium salt is at least one of 2 - chloroethyltrimethylammonium chloride, 3 - chloropropyltrimethylammonium chloride, or 4 - chlorobutyltrimethylammonium chloride;

[0027] Preferably, the mass ratio of the tertiary amine anion exchange resin to the halogenated quaternary ammonium salt is 1:0.3 - 0.5.

[0028] Preferably, before the strongly basic quaternary ammonium type anion resin is used in the strongly basic anion exchange column, it further includes adding the strongly basic quaternary ammonium type anion resin to 95% ethanol with a volume 1 - 2 times that of the resin and stirring for 1 - 2 h, then adding it to 1 - 3 mo1 / L hydrochloric acid with a volume 4 - 6 times that of the resin and stirring for 1 - 2 h, and then adding it to a 1 - 3 mo1 / L sodium hydroxide solution with a volume 2 - 4 times that of the resin and stirring for 2 - 3 h.

[0029] Preferably, step S4 specifically includes: loading the eluate onto the column at a loading speed of 1.0 - 1.5 BV / h. After completion, impurities and sodium heparin are eluted successively to obtain a pure sodium heparin solution, which is freeze - dried and pulverized to obtain the sodium heparin raw material drug;

[0030] Preferably, washing the impurities includes: washing with 2 - 3 BV of distilled water at a temperature of 40 - 50°C, with a flow rate of 2 - 3 BV / h;

[0031] Preferably, the elution of sodium heparin includes: eluting with 2 - 3 BV of sodium chloride solution at a temperature of 40 - 50°C and a concentration of 5 - 10 wt%, with a flow rate of 0.5 - 1 BV / h.

[0032] Preferably, in the weakly basic imidazole type anion resin, the primary amine anion exchange resin is D318 resin, the alkenyl acyl chloride is at least one of 2 - butenoyl chloride, acryloyl chloride, or methacryloyl chloride, and the vinyl imidazole is N - vinyl imidazole;

[0033] Preferably, the mass ratio of the primary amine anion exchange resin, the alkenyl acyl chloride, and the vinyl imidazole is 1:0.05 - 0.2:0.3 - 0.5.

[0034] Preferably, before the weakly basic imidazole type anion resin is used in the weakly basic anion exchange column, it further includes soaking the weakly basic imidazole type anion resin in 95% ethanol with a volume 2 - 4 times that of the resin for 1 - 2 h.

[0035] The present invention also provides a sodium heparin raw material drug, which is prepared by the above - mentioned preparation method.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] In the present invention, after the crude heparin sodium solution is treated by enzymatic degreasing, heparin sodium is fully freed from the protein complex, avoiding the interference of excessive grease on the resin in ion exchange, and greatly improving the adsorption capacity and utilization rate of the resin; in the process of purifying with ion exchange resin, a combination of two-step purification with a strongly basic anion exchange column and a weakly basic anion exchange column is adopted to maximize the dissociation of heparin sodium from proteins, and impurities such as proteins, chondroitin sulfate, and dermatan sulfate are removed more thoroughly, resulting in a significant increase in the purity, activity, and yield of the heparin sodium finished product, and obtaining an ideal extraction effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is a schematic flow chart of the preparation method of the heparin sodium raw material drug described in Example 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0039] Next, the technical solutions of the present invention will be described in detail through specific examples. 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.

[0040] Example 1

[0041] Refer to Figure 1 , this example proposes a preparation method of a heparin sodium raw material drug, including:

[0042] (1) Dissolution: Dissolve the crude heparin sodium in a 2.5 wt% sodium chloride solution at a ratio of 1 g:8 mL, heat to 50 °C, and keep stirring for 5 h to fully dissolve the crude heparin sodium to obtain a crude product solution;

[0043] (2) Enzymatic hydrolysis: Adjust the pH of the crude product solution to 8.0 with 2 mo1 / L sodium hydroxide solution, raise the temperature to 50 °C, then add trypsin, and the addition amount of trypsin is 2.0% of the mass of the crude heparin sodium. Keep the solution at pH 8.0 and temperature 50 °C for enzymatic hydrolysis for 4 h, then raise the temperature to 90 °C and keep warm for inactivation for 0.5 h to obtain an enzymatic hydrolysate;

[0044] (3) Filtration: Cool the enzymatic hydrolysate to below 60 °C, centrifuge at high speed for 2 h, and after reducing the pressure of the centrifugate, filter until clear to obtain a filtrate;

[0045] (4) Precipitation: Add 1.5 times the volume of 95% ethanol to the filtrate, stir evenly, then let it stand for precipitation for 5 h, and after vacuum filtration, obtain a precipitate;

[0046] (5) First column loading: Add a strongly basic quaternary ammonium anion resin to distilled water and soak it fully. After swelling, filter it dry. Add 1.5 times the volume of 95% ethanol and stir for 1.5 hours. Wash it with distilled water and filter it dry. Then add 5 times the volume of 2 mol / L hydrochloric acid and stir it for 2 hours. Wash it with distilled water until it is neutral. Filter it dry. Then add 3 times the volume of 2 mol / L sodium hydroxide solution and stir it for 2 hours. Wash it with distilled water until it is neutral. Filter it dry. Finally, load the column with 2.5wt% sodium chloride solution to obtain a strongly basic anion exchange column.

[0047] The strong alkaline quaternary ammonium anion resin is prepared by the following method: D301 resin is added to N-methylpyrrolidone to swell for 2 hours, 3-chloropropyltrimethylammonium chloride is added at 40% of the mass of D301 resin, the temperature is raised to 60° C. and stirred for reaction for 24 hours, after the reaction is completed, the resin is poured into acetone for precipitation, filtered, and vacuum dried to obtain a strong alkaline quaternary ammonium anion resin;

[0048] (6) First purification: The precipitate was added into purified water at a ratio of 1 g:5 mL, stirred for 2 h and dissolved completely, the pH was adjusted to 8.5, the temperature was raised to 45° C., and the ion exchange was carried out in the first ion exchange column by an infusion pump at a rate of 1.0 BV / h until the effluent was found to be free of heparin, then the ion exchange was stopped and balanced for 1 h; the balanced first ion exchange column was first washed with 2 BV of 45° C. distilled water at a flow rate of 2.5 BV / h, then washed with 3 BV of 6 wt % sodium chloride solution at a flow rate of 1.5 BV / h, the pH of the sodium chloride solution was adjusted to 8.5, and finally eluted three times with 3 BV of 45° C. 20 wt %, 25 wt %, and 25 wt % sodium chloride solutions at a flow rate of 0.5 BV / h, and the eluate was collected to obtain the first eluate;

[0049] (7) Second column loading: Add the weakly basic imidazole anion resin into distilled water and soak it fully, swell it, and then filter it dry. Then add it into 3 times the volume of 95% ethanol and stir it for 2 hours. Then wash it with distilled water, filter it dry, and finally load it with 2.5wt% sodium chloride solution to obtain a weakly basic anion exchange column.

[0050] The weakly basic imidazole anion resin is prepared by the following method: D318 resin is added to a 5wt% sodium hydroxide solution for immersion, washed with distilled water, filtered, added to dichloromethane for swelling for 2h, and then 10% of acryloyl chloride and 35% of triethylamine by weight of the D318 resin are added, stirred for reaction for 12h, filtered, and dried to obtain olefinic D318 resin; the olefinic D318 resin is added to ethanol, and then 40% of N-vinyl imidazole and 1% of azobisisobutyronitrile by weight of the D318 resin are added, the temperature is raised to 60°C under nitrogen protection, stirred for reaction for 24h, filtered, washed, and vacuum dried to obtain the weakly basic imidazole anion resin;

[0051] (8) Second purification: The first eluate is input into the second ion exchange column by a peristaltic pump for ion exchange. The loading rate is 1.5 BV / h. After the loading is completed, it is equilibrated for 1 h. First, it is washed with 2 BV of distilled water at a temperature of 45 °C with a flow rate of 2 BV / h, and then eluted with 3 BV of a 7.5 wt% sodium chloride solution at a temperature of 45 °C with a flow rate of 0.5 BV / h. The eluate is collected to obtain the second eluate;

[0052] (9) Microfiltration: 95% ethanol with a volume 0.7 times that of the second eluate is added to the second eluate for alcohol precipitation. After stirring evenly, it is allowed to stand for precipitation for 4 h. After vacuum filtration, the obtained precipitate is dissolved in distilled water to obtain a 20 wt% solution, which is filtered through a 0.45-μm microfiltration membrane to obtain a filtrate;

[0053] (10) Freeze-drying and pulverization: The above-mentioned filtrate is placed in a freeze dryer for freeze-drying, pulverized and passed through a 60-mesh sieve to obtain high-quality heparin sodium, which is the heparin sodium raw material drug.

[0054] Example 2

[0055] This example provides a preparation method of heparin sodium raw material drug, including:

[0056] (1) Dissolution: The crude heparin sodium is dissolved in a 3 wt% sodium chloride solution at a ratio of 1 g:5 mL, heated to 45 °C and kept stirring for 6 h to fully dissolve the crude heparin sodium to obtain a crude product solution;

[0057] (2) Enzymatic hydrolysis: The pH of the crude product solution is adjusted to 7.5 with 2 mo1 / L sodium hydroxide solution, and after heating to 55 °C, trypsin is added. The addition amount of trypsin is 3.0% of the mass of the crude heparin sodium. The enzymatic hydrolysis is carried out for 3 h under the conditions of keeping the solution pH at 7.5 and the temperature at 55 °C, and then heated to 85 °C and kept warm for inactivation for 0.5 h to obtain an enzymatic hydrolysate;

[0058] (3) Filtration: The enzymatic hydrolysate is cooled to below 60 °C, centrifuged at high speed for 2 h, and after the centrifugate is depressurized, it is filtered until clear to obtain a filtrate;

[0059] (4) Precipitation: 95% ethanol with a volume 1 time that of the filtrate is added to the filtrate, stirred evenly, allowed to stand for precipitation for 6 h, and after vacuum filtration, a precipitate is obtained;

[0060] (5) First column packing: Add strongly basic quaternary ammonium type anion resin into distilled water, soak it fully, filter it after swelling, add it into 95% ethanol with a volume 1 time that of the resin and stir for 2 h, wash it with distilled water until clean, filter it, then add it into 3 mol / L hydrochloric acid with a volume 4 times that of the resin and stir for 1 h, wash it with distilled water until neutral, filter it, then add it into 1 mol / L sodium hydroxide solution with a volume 4 times that of the resin and stir for 3 h, wash it with distilled water until neutral, filter it, and finally pack the column with 2.5 wt% sodium chloride solution to obtain a strongly basic anion exchange column;

[0061] The strongly basic quaternary ammonium type anion resin is prepared by the method described in Example 1;

[0062] (6) First purification: Add the precipitate into purified water according to the ratio of 1 g:5 mL, stir for 2 h until completely dissolved, adjust the pH to 8.0, heat up to 50 °C, input it into the first ion exchange column with an infusion pump for ion exchange, and the loading rate is 1.5 BV / h. Stop the ion exchange until no heparin is detected in the effluent, and balance for 1 h; First, wash the balanced first ion exchange column with 3 BV of distilled water at 50 °C with a flow rate of 3 BV / h, then wash it with 4 BV of 5 wt% sodium chloride solution with a flow rate of 2 BV / h, adjust the pH of the sodium chloride solution to 8.0, and finally elute it twice with 2 BV of 20 wt% and 25 wt% sodium chloride solution at 50 °C with a flow rate of 0.5 BV / h, collect the eluate to obtain the first eluate;

[0063] (7) Second column packing: Add weakly basic imidazole type anion resin into distilled water, soak it fully and swell, then filter it, add it into 95% ethanol with a volume 2 times that of the resin and stir for 2 h, wash it with distilled water until clean, filter it, and finally pack the column with 2.5 wt% sodium chloride solution to obtain a weakly basic anion exchange column;

[0064] The weakly basic imidazole type anion resin is prepared by the method described in Example 1;

[0065] (8) Second purification: Input the first eluate into the second ion exchange column with an infusion pump for ion exchange, and the loading rate is 1.0 BV / h. After the loading is completed, balance for 1 h. First, wash it with 3 BV of distilled water at 50 °C with a flow rate of 3 BV / h, then elute it with 2 BV of 5 wt% sodium chloride solution at 50 °C with a flow rate of 0.5 BV / h, collect the eluate to obtain the second eluate;

[0066] (9) Microfiltration: Add 95% ethanol with a volume 1 time that of the second eluate for alcohol precipitation, stir evenly, let it stand for precipitation for 4 h, after vacuum filtration, add the obtained precipitate into distilled water to dissolve to obtain a 20 wt% solution, and filter it with a 0.45 μm microfiltration membrane to obtain a filtrate;

[0067] (10) Freeze-drying and pulverizing: The filtrate is placed in a freeze dryer and freeze-dried, pulverized and passed through a 60-mesh sieve to obtain fine heparin sodium, which is the heparin sodium bulk drug.

[0068] Example 3

[0069] This embodiment provides a method for preparing a heparin sodium bulk drug, comprising:

[0070] (1) Dissolution: Dissolve the crude heparin sodium in a 2 wt % sodium chloride solution at a ratio of 1 g:10 mL, heat to 55° C., and stir for 4 h to fully dissolve the crude heparin sodium to obtain a crude solution;

[0071] (2) Enzymatic hydrolysis: The crude solution was adjusted to pH 8.5 with 2 mol / L sodium hydroxide solution, and the temperature was raised to 45°C. Trypsin was added, and the amount of trypsin added was 1.0% of the quality of the crude heparin sodium. The solution was kept at pH 8.5 and the temperature was 45°C for enzymatic hydrolysis for 5 h. The solution was then heated to 95°C and kept incubated for 0.5 h to obtain an enzymatic hydrolyzate.

[0072] (3) Filtration: The enzymatic hydrolyzate is cooled to below 60°C, centrifuged at high speed for 2 h, the centrifuged liquid is decompressed, and filtered until clear to obtain a filtrate;

[0073] (4) Precipitation: Add 2 times the volume of 95% ethanol to the filtrate, stir evenly, let stand for 4 hours, and obtain the precipitate after vacuum filtration;

[0074] (5) First column loading: Add a strongly basic quaternary ammonium anion resin to distilled water and soak it fully. After swelling, filter it dry. Add it to 95% ethanol twice its volume and stir it for 1 hour. Wash it with distilled water and filter it dry. Then add 1 mol / L hydrochloric acid six times its volume and stir it for 2 hours. Wash it with distilled water until it is neutral. Filter it dry. Then add 3 mol / L sodium hydroxide solution twice its volume and stir it for 2 hours. Wash it with distilled water until it is neutral. Filter it dry. Finally, load the column with 2.5 wt% sodium chloride solution to obtain a strongly basic anion exchange column.

[0075] The strong alkaline quaternary ammonium anion resin is prepared by the method described in Example 1;

[0076] (6) First purification: the precipitate was added into purified water at a ratio of 1 g:5 mL, stirred for 2 h and dissolved completely, the pH was adjusted to 9.0, the temperature was raised to 40° C., and the ion exchange was carried out in the first ion exchange column by an infusion pump at a rate of 1.0 BV / h until the effluent was found to be free of heparin, then the ion exchange was stopped and balanced for 1 h; the balanced first ion exchange column was first washed with 2 BV of 40° C. distilled water at a flow rate of 2 BV / h, then washed with 3 BV of 8 wt % sodium chloride solution at a flow rate of 1 BV / h, the pH of the sodium chloride solution was adjusted to 9.0, and finally eluted three times with 3 BV of 40° C. 20 wt %, 25 wt %, and 25 wt % sodium chloride solutions at a flow rate of 1 BV / h, and the eluate was collected to obtain the first eluate;

[0077] (7) Second column loading: Add the weakly basic imidazole anion resin into distilled water and soak it fully, swell it, and then filter it dry. Then add it into 4 times the volume of 95% ethanol and stir it for 1 hour. Then wash it with distilled water, filter it dry, and finally load it with 2.5wt% sodium chloride solution to obtain a weakly basic anion exchange column.

[0078] The weakly basic imidazole anion resin is prepared by the method described in Example 1;

[0079] (8) Second purification: The first eluate was introduced into the second ion exchange column by an infusion pump at a rate of 1.5 BV / h. After the column was loaded, it was balanced for 1 hour. It was first washed with 2 BV of distilled water at a temperature of 40° C. at a flow rate of 2 BV / h, and then eluted with 3 BV of 10 wt % sodium chloride solution at a temperature of 40° C. at a flow rate of 1 BV / h. The eluate was collected to obtain the second eluate.

[0080] (9) Microporous filtration: Add 1 volume of 95% ethanol to the second eluate, stir evenly, let stand for 4 hours, vacuum filter, add the resulting precipitate into distilled water to dissolve, and obtain a 20 wt % solution. Filter with a 0.45 μm microporous membrane to obtain a filtrate.

[0081] (10) Freeze-drying and pulverizing: The filtrate is placed in a freeze dryer and freeze-dried, pulverized and passed through a 60-mesh sieve to obtain fine heparin sodium, which is the heparin sodium bulk drug.

[0082] Comparative Example 1

[0083] This comparative example proposes a method for preparing a heparin sodium bulk drug, comprising:

[0084] (1) Dissolution: Dissolve the crude heparin sodium in a 2.5 wt % sodium chloride solution at a ratio of 1 g:8 mL, heat to 50° C., and stir for 5 h to fully dissolve the crude heparin sodium to obtain a crude solution;

[0085] (2) Enzymatic hydrolysis: The crude solution was adjusted to pH 8.0 with 2 mol / L sodium hydroxide solution, and the temperature was raised to 50°C. Trypsin was added, and the amount of trypsin added was 2.0% of the quality of the crude heparin sodium. The solution was hydrolyzed for 4 h under the conditions of maintaining the pH of the solution at 8.0 and the temperature at 50°C. The solution was then heated to 90°C and kept incubated for 0.5 h to obtain an enzymatic hydrolyzate.

[0086] (3) Filtration: The enzymatic hydrolyzate is cooled to below 60°C, centrifuged at high speed for 2 h, the centrifuged liquid is decompressed, and filtered until clear to obtain a filtrate;

[0087] (4) Precipitation: Add 1.5 times the volume of 95% ethanol to the filtrate, stir evenly, let stand for 5 hours, and obtain the precipitate after vacuum filtration;

[0088] (5) First column loading: Add a strongly basic quaternary ammonium anion resin to distilled water and soak it fully. After swelling, filter it dry. Add 1.5 times the volume of 95% ethanol and stir for 1.5 hours. Wash it with distilled water and filter it dry. Then add 5 times the volume of 2 mol / L hydrochloric acid and stir it for 2 hours. Wash it with distilled water until it is neutral. Filter it dry. Then add 3 times the volume of 2 mol / L sodium hydroxide solution and stir it for 2 hours. Wash it with distilled water until it is neutral. Filter it dry. Finally, load the column with 2.5wt% sodium chloride solution to obtain a strongly basic anion exchange column.

[0089] The strong alkaline quaternary ammonium anion resin is prepared by the method described in Example 1;

[0090] (6) First purification: The precipitate was added into purified water at a ratio of 1 g:5 mL, stirred for 2 h and dissolved completely, the pH was adjusted to 8.5, the temperature was raised to 45° C., and the ion exchange was carried out in the first ion exchange column by an infusion pump at a rate of 1.0 BV / h until the effluent was found to be free of heparin, then the ion exchange was stopped and balanced for 1 h; the balanced first ion exchange column was first washed with 2 BV of 45° C. distilled water at a flow rate of 2.5 BV / h, then washed with 3 BV of 6 wt % sodium chloride solution at a flow rate of 1.5 BV / h, the pH of the sodium chloride solution was adjusted to 8.5, and finally eluted three times with 3 BV of 45° C. 20 wt %, 25 wt %, and 25 wt % sodium chloride solutions at a flow rate of 0.5 BV / h, and the eluate was collected to obtain the first eluate;

[0091] (7) Microporous filtration: add 0.7 times the volume of 95% ethanol to the first eluate, stir evenly, let stand for 4 hours, vacuum filter, add the resulting precipitate into distilled water to dissolve, and obtain a 20 wt% solution. Filter with a 0.45 μm microporous membrane to obtain a filtrate.

[0092] (8) Freeze-drying and pulverizing: The filtrate is placed in a freeze dryer and freeze-dried, pulverized and passed through a 60-mesh sieve to obtain fine heparin sodium, which is the heparin sodium bulk drug.

[0093] Comparative Example 2

[0094] This comparative example proposes a method for preparing a heparin sodium bulk drug, comprising:

[0095] (1) Dissolution: Dissolve the crude heparin sodium in a 2.5 wt % sodium chloride solution at a ratio of 1 g:8 mL, heat to 50° C., and stir for 5 h to fully dissolve the crude heparin sodium to obtain a crude solution;

[0096] (2) Enzymatic hydrolysis: The crude solution was adjusted to pH 8.0 with 2 mol / L sodium hydroxide solution, and the temperature was raised to 50°C. Trypsin was added, and the amount of trypsin added was 2.0% of the quality of the crude heparin sodium. The solution was hydrolyzed for 4 h under the conditions of maintaining the pH of the solution at 8.0 and the temperature at 50°C. The solution was then heated to 90°C and kept incubated for 0.5 h to obtain an enzymatic hydrolyzate.

[0097] (3) Filtration: The enzymatic hydrolyzate is cooled to below 60°C, centrifuged at high speed for 2 h, the centrifuged liquid is decompressed, and filtered until clear to obtain a filtrate;

[0098] (4) Precipitation: Add 1.5 times the volume of 95% ethanol to the filtrate, stir evenly, let stand for 5 hours, and obtain the precipitate after vacuum filtration;

[0099] (5) First column loading: Add D254 resin (strong alkaline quaternary ammonium anion resin) to distilled water for soaking, filter dry after swelling, add 1.5 times the volume of 95% ethanol and stir for 1.5 hours, wash with distilled water, filter dry, then add 5 times the volume of 2 mol / L hydrochloric acid and stir for 2 hours, wash with distilled water until neutral, filter dry, then add 3 times the volume of 2 mol / L sodium hydroxide solution and stir for 2 hours, wash with distilled water until neutral, filter dry, and finally load the column with 2.5wt% sodium chloride solution to obtain a strong alkaline anion exchange column;

[0100] (6) First purification: The precipitate was added into purified water at a ratio of 1 g:5 mL, stirred for 2 h and dissolved completely, the pH was adjusted to 8.5, the temperature was raised to 45° C., and the ion exchange was carried out in the first ion exchange column by an infusion pump at a rate of 1.0 BV / h until the effluent was found to be free of heparin, then the ion exchange was stopped and balanced for 1 h; the balanced first ion exchange column was first washed with 2 BV of 45° C. distilled water at a flow rate of 2.5 BV / h, then washed with 3 BV of 6 wt % sodium chloride solution at a flow rate of 1.5 BV / h, the pH of the sodium chloride solution was adjusted to 8.5, and finally eluted three times with 3 BV of 45° C. 20 wt %, 25 wt %, and 25 wt % sodium chloride solutions at a flow rate of 0.5 BV / h, and the eluate was collected to obtain the first eluate;

[0101] (7) Second column loading: Add the weakly basic imidazole anion resin into distilled water and soak it fully, swell it, and then filter it dry. Then add it into 3 times the volume of 95% ethanol and stir it for 2 hours. Then wash it with distilled water, filter it dry, and finally load it with 2.5wt% sodium chloride solution to obtain a weakly basic anion exchange column.

[0102] The weakly basic imidazole anion resin is prepared by the method described in Example 1;

[0103] (8) Second purification: The first eluate was transferred into the second ion exchange column by an infusion pump at a rate of 1.5 BV / h. After loading, the column was balanced for 1 hour. The column was first washed with 2 BV of distilled water at a temperature of 45° C. at a flow rate of 2 BV / h. The column was then eluted with 3 BV of a 7.5 wt % sodium chloride solution at a temperature of 45° C. at a flow rate of 0.5 BV / h. The eluate was collected to obtain a second eluate.

[0104] (9) Microporous filtration: add 0.7 times the volume of 95% ethanol to the second eluate, stir evenly, let stand for 4 hours, vacuum filter, add the obtained precipitate into distilled water to dissolve, and obtain a 20 wt % solution. Filter with a 0.45 μm microporous membrane to obtain a filtrate;

[0105] (10) Freeze-drying and pulverizing: The filtrate is placed in a freeze dryer and freeze-dried, pulverized and passed through a 60-mesh sieve to obtain fine heparin sodium, which is the heparin sodium bulk drug.

[0106] Comparative Example 3

[0107] This comparative example proposes a method for preparing a heparin sodium bulk drug, comprising:

[0108] (1) Dissolution: Dissolve the crude heparin sodium in a 2.5 wt % sodium chloride solution at a ratio of 1 g:8 mL, heat to 50° C., and stir for 5 h to fully dissolve the crude heparin sodium to obtain a crude solution;

[0109] (2) Enzymatic hydrolysis: The crude solution was adjusted to pH 8.0 with 2 mol / L sodium hydroxide solution, and the temperature was raised to 50°C. Trypsin was added, and the amount of trypsin added was 2.0% of the quality of the crude heparin sodium. The solution was hydrolyzed for 4 h under the conditions of maintaining the pH of the solution at 8.0 and the temperature at 50°C. The solution was then heated to 90°C and kept incubated for 0.5 h to obtain an enzymatic hydrolyzate.

[0110] (3) Filtration: The enzymatic hydrolyzate is cooled to below 60°C, centrifuged at high speed for 2 h, the centrifuged liquid is decompressed, and filtered until clear to obtain a filtrate;

[0111] (4) Precipitation: Add 1.5 times the volume of 95% ethanol to the filtrate, stir evenly, let stand for 5 hours, and obtain the precipitate after vacuum filtration;

[0112] (5) First column loading: Add a strongly basic quaternary ammonium anion resin to distilled water and soak it fully. After swelling, filter it dry. Add 1.5 times the volume of 95% ethanol and stir for 1.5 hours. Wash it with distilled water and filter it dry. Then add 5 times the volume of 2 mol / L hydrochloric acid and stir it for 2 hours. Wash it with distilled water until it is neutral. Filter it dry. Then add 3 times the volume of 2 mol / L sodium hydroxide solution and stir it for 2 hours. Wash it with distilled water until it is neutral. Filter it dry. Finally, load the column with 2.5wt% sodium chloride solution to obtain a strongly basic anion exchange column.

[0113] The strong alkaline quaternary ammonium anion resin is prepared by the method described in Example 1;

[0114] (6) First purification: The precipitate was added into purified water at a ratio of 1 g:5 mL, stirred for 2 h and dissolved completely, the pH was adjusted to 8.5, the temperature was raised to 45° C., and the ion exchange was carried out in the first ion exchange column by an infusion pump at a rate of 1.0 BV / h until the effluent was found to be free of heparin, then the ion exchange was stopped and balanced for 1 h; the balanced first ion exchange column was first washed with 2 BV of 45° C. distilled water at a flow rate of 2.5 BV / h, then washed with 3 BV of 6 wt % sodium chloride solution at a flow rate of 1.5 BV / h, the pH of the sodium chloride solution was adjusted to 8.5, and finally eluted three times with 3 BV of 45° C. 20 wt %, 25 wt %, and 25 wt % sodium chloride solutions at a flow rate of 0.5 BV / h, and the eluate was collected to obtain the first eluate;

[0115] (7) Second column loading: D301 resin (weakly basic tertiary amine type anion resin) was added to distilled water to soak and swell, then filtered and dried. Then, it was added to 3 times the volume of 95% ethanol and stirred for 2 h. The mixture was washed with distilled water, filtered and dried. Finally, a 2.5 wt % sodium chloride solution was used to load the column to obtain a weakly basic anion exchange column.

[0116] (8) Second purification: The first eluate is input into the second ion exchange column for ion exchange using an infusion pump at a loading rate of 1.5 BV / h. After loading, it is equilibrated for 1 h. First, it is washed with 2 BV of distilled water at a temperature of 45 °C at a flow rate of 2 BV / h, and then eluted with 3 BV of a 7.5 wt% sodium chloride solution at a temperature of 45 °C at a flow rate of 0.5 BV / h. The eluate is collected to obtain the second eluate;

[0117] (9) Microfiltration: 0.7 times the volume of 95% ethanol is added to the second eluate for alcohol precipitation. After stirring evenly, it is allowed to stand for precipitation for 4 h. After vacuum filtration, the obtained precipitate is dissolved in distilled water to obtain a 20 wt% solution, which is filtered through a 0.45 μm microfiltration membrane to obtain a filtrate;

[0118] (10) Freeze-drying and pulverization: The above-mentioned filtrate is placed in a freeze-dryer for freeze-drying, pulverized, and sieved through a 60-mesh sieve to obtain high-quality heparin sodium, which is the heparin sodium raw material drug.

[0119] Under the same conditions, the yield, potency yield, and unit potency of the high-quality heparin sodium obtained in the examples and comparative examples were tested, and the results are shown in Table 1 below:

[0120] Yield (%) = (weight of high-quality heparin sodium / weight of crude heparin sodium) × 100%; Potency yield (%) = (total potency of high-quality heparin sodium / total potency of crude heparin sodium) × 100%;

[0121] Table 1 Results of yield verification of the heparin sodium refining method

[0122] Unit potency IU / mg Potency yield % Yield % Example 1 202.3 96.5 90.3 Example 2 196.7 95.9 91.4 Example 3 195.5 93.5 89.7 Comparative Example 1 171.2 82.4 91.1 Comparative Example 2 174.6 79.0 85.7 Comparative Example 3 180.9 84.5 88.9

[0123] As can be seen from Table 1, the high-quality heparin sodium obtained by the preparation methods provided in Examples 1-3 has a relatively high potency yield and productivity.

[0124] The above is only a preferred specific embodiment of the present invention, but 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 preparing a heparin sodium bulk drug, characterized in that: The steps include: S1, dissolving crude heparin sodium in sodium chloride solution to obtain a crude solution; S2, enzymatically hydrolyzing the crude product solution, and then precipitating with alcohol to obtain a precipitate; S3, after dissolving the precipitate, purifying it on a strong alkaline anion exchange column to obtain an eluent; S4, purifying the eluate by a weakly alkaline anion exchange column to obtain the heparin sodium bulk drug; In step S3, the strong basic anion exchange column is filled with a strong basic quaternary ammonium anion resin, which is obtained by quaternizing a tertiary amine anion exchange resin and a halogenated quaternary ammonium salt; In step S4, the weakly basic anion exchange column is filled with a weakly basic imidazole type anion resin, which is obtained by condensing a primary amine anion exchange resin with olefinic acid chloride and then copolymerizing it with vinyl imidazole.

2. The method for preparing the heparin sodium bulk drug according to claim 1, characterized in that: Step S1 specifically comprises: adding crude heparin sodium to a 2-3 wt % sodium chloride solution, heating to 45-55° C. and stirring for 4-6 hours to obtain a crude solution; Preferably, the mass volume ratio of the crude heparin sodium to the sodium chloride solution is 1 g:5-10 mL.

3. The method for preparing the heparin sodium bulk drug according to claim 1 or 2, characterized in that: In step S2, the enzymatic hydrolysis specifically includes: adjusting the pH of the crude product solution to 7.5-8.5, heating to 45-55° C., adding trypsin, keeping the temperature for 3-5 hours, and then heating to 85-95° C. to inactivate the solution, thereby obtaining an enzymatic hydrolyzate; Preferably, the amount of trypsin added is 1-3% of the quality of crude heparin sodium; Preferably, the alcohol precipitation specifically comprises: adding 1-2 times the volume of 95% ethanol to the enzymatic hydrolysate, stirring evenly, standing and settling for 4-6 hours, and filtering to obtain a precipitate.

4. The method for preparing the heparin sodium bulk drug according to any one of claims 1 to 3, characterized in that: Step S3 specifically comprises: adding purified water to dissolve the precipitate, adjusting the pH to 8.0-9.0, heating to 40-50°C and then loading onto a column at a loading rate of 1.0-1.5 BV / h, and after completion, eluting impurities and sodium heparin in turn to obtain an eluate; Preferably, eluting impurities comprises: first washing with 2-3 BV of 40-50° C. distilled water at a flow rate of 2-3 BV / h, and then washing with 3-4 BV of 5-8 wt % sodium chloride solution at a flow rate of 1-2 BV / h; Preferably, eluting the sodium heparin comprises: eluting with 2-3 BV of a 20-25 wt % sodium chloride solution at a temperature of 40-50° C. and a flow rate of 0.5-1 BV / h.

5. The method for preparing the heparin sodium bulk drug according to any one of claims 1 to 4, characterized in that: In the strongly basic quaternary ammonium anion resin, the tertiary amine anion exchange resin is D301 resin, and the halogenated quaternary ammonium salt is at least one of 2-chloroethyltrimethylammonium chloride, 3-chloropropyltrimethylammonium chloride or 4-chlorobutyltrimethylammonium chloride; Preferably, the mass ratio of the tertiary amine anion exchange resin to the halogenated quaternary ammonium salt is 1:0.3-0.

5.

6. The method for preparing the heparin sodium bulk drug according to any one of claims 1 to 5, characterized in that: Before the strong alkaline quaternary ammonium anion resin is used in a strong alkaline anion exchange column, the strong alkaline quaternary ammonium anion resin is added to 1-2 times the volume of 95% ethanol and stirred for 1-2 hours, then added to 4-6 times the volume of 1-3 mol / L hydrochloric acid and stirred for 1-2 hours, and then added to 2-4 times the volume of 1-3 mol / L sodium hydroxide solution and stirred for 2-3 hours.

7. The method for preparing the heparin sodium bulk drug according to any one of claims 1 to 6, characterized in that: Step S4 specifically comprises: loading the eluate onto a column at a loading rate of 1.0-1.5 BV / h, and after completion, eluting impurities and heparin sodium in turn to obtain a pure heparin sodium solution, freeze-drying, and crushing to obtain the heparin sodium bulk drug; Preferably, washing the impurities comprises: washing with 2-3 BV of distilled water at a temperature of 40-50° C. at a flow rate of 2-3 BV / h; Preferably, eluting the sodium heparin comprises: eluting with 2-3 BV of a 5-10 wt % sodium chloride solution at a temperature of 40-50° C., at a flow rate of 0.5-1 BV / h.

8. The method for preparing the heparin sodium bulk drug according to any one of claims 1 to 7, characterized in that: In the weakly basic imidazole type anion resin, the primary amine anion exchange resin is D318 resin, the olefinic acid chloride is at least one of 2-butylene acid chloride, acryloyl chloride or methacryloyl chloride, and the vinyl imidazole is N-vinyl imidazole; Preferably, the mass ratio of primary amine anion exchange resin, olefinic acid chloride and vinyl imidazole is 1:0.05-0.2:0.3-0.

5.

9. The method for preparing the heparin sodium bulk drug according to any one of claims 1 to 8, characterized in that: Before the weakly basic imidazole type anion resin is used in the weakly basic anion exchange column, the weakly basic imidazole type anion resin is added into 95% ethanol with a volume of 2-4 times and soaked for 1-2 hours.

10. A heparin sodium bulk drug, characterized in that: The compound is prepared by the preparation method according to any one of claims 1 to 9.

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