Preparation method and application of heparin sodium composition

Abstract

The invention relates to the technical field of biological pharmacy, and discloses a preparation method and application of a heparin sodium composition, medical-grade yeast cell wall polysaccharide and aloe polysaccharide are selected as main raw materials, medical-grade sodium chloride and medical-grade ethanol are selected as auxiliary materials, the medical-grade yeast cell wall polysaccharide and the aloe polysaccharide are pretreated, and the pretreated medical-grade yeast cell wall polysaccharide and the pretreated medical-grade aloe polysaccharide are added into the pretreated medical-grade yeast cell wall polysaccharide and the pretreated medical-grade ethanol; the method comprises the following steps: reacting by adopting a continuous reaction container of immobilized heparinase, introducing nitrogen to protect the structure of heparin sodium, performing combined purification on the reacted solution by adopting anion exchange chromatography and gel permeation chromatography to obtain a heparin sodium solution, and performing crystallization optimization on the heparin sodium solution by adopting a step-by-step alcohol precipitation method to obtain the heparin sodium. According to the present invention, the temperature reduction rate is controlled to achieve the dynamic crystallization control so as to obtain the high-purity heparin sodium crystal, the pharmaceutical grade sodium chloride is added in the vaporization manner to obtain the heparin sodium composition, and the devices used in the processes have the automatic monitoring and regulation functions; the prepared heparin sodium composition has the advantages of high efficiency, low cost, easiness in operation and good structure protection.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and specifically to a preparation method and application of a heparin sodium composition. Background Art

[0002] In the medical field, heparin sodium, as a key anticoagulant drug, its preparation process has always been the focus of research and development. However, there are some defects in the existing preparation methods. For example, in the traditional preparation technology, the chemical cleavage method will damage the structure of heparin sodium during the operation process, which will not only affect the purity of the product, but also lead to low yield and reduced activity, unable to meet the requirements of high-quality production. In addition, some special preparation processes, such as preparing with heated vegetable oil and ultrasonic extraction and separation technology, require specific equipment and conditions to achieve. These specific equipment include precise temperature control systems, ultrasonic generators, as well as specific solvents and reaction media. These requirements are obviously not conducive to large-scale industrial production and are difficult to meet the large market demand for heparin sodium. Therefore, developing a new preparation method for heparin sodium composition, which can achieve efficient production while ensuring product quality, has become an urgent problem to be solved in this field. Summary of the Invention

[0003] (I) Technical Problems to be Solved

[0004] Aiming at the deficiencies of the prior art, the present invention provides a preparation method and application of a heparin sodium composition, which has the advantages of high efficiency, low cost, easy operation and good structure protection, and solves the problems of easy structure damage, low purity, low yield, high cost and unfavorable industrial production in the traditional preparation process.

[0005] (II) Technical Solutions

[0006] To achieve the above object, the present invention provides the following technical solutions: A preparation method of a heparin sodium composition, comprising the following steps: Step 1. Raw material selection: The main raw materials are medical-grade yeast cell wall polysaccharide and aloe polysaccharide, and the auxiliary materials are pharmaceutical-grade sodium chloride and pharmaceutical-grade ethanol; Step 2. Raw material pretreatment: The medical-grade yeast cell wall polysaccharide and aloe polysaccharide are respectively pulverized, centrifuged and purified; Step 3. Enzymatic hydrolysis optimization process: React using a continuous reaction vessel with immobilized heparinase. After setting the enzymatic hydrolysis parameters, add a stabilizer, and at the same time introduce nitrogen to protect the structure of heparin sodium; Step 4. Purification improvement process: Purify the reacted solution by combining anion exchange chromatography and gel permeation chromatography to obtain a heparin sodium solution; Step 5. Crystallization optimization: The heparin sodium solution is subjected to crystallization optimization by the stepwise alcohol precipitation method, and dynamic crystallization control is achieved by controlling the cooling rate to obtain high-purity heparin sodium crystals. The heparin sodium composition is obtained by adding pharmaceutical-grade sodium chloride in a vaporization manner. Step 6. Equipment optimization: All the equipment used in the preparation process has automatic monitoring and regulation functions, and various process parameters are fed back in real time.

[0007] Preferably, in the pretreatment process of the pharmaceutical-grade yeast cell wall polysaccharide in Step 2: The pharmaceutical-grade yeast cell wall polysaccharide is put into a pulverizer and pulverized to 100 - 120 mesh. The pulverized yeast cell wall polysaccharide is transferred to a centrifuge tube, and deionized water with a volume of 80% of the yeast cell wall polysaccharide is added. The rotation speed is set to 8000 - 9000 r / min, and the centrifugation time is 10 - 12 min. Column chromatography is used for purification, with deionized water as the eluent, and the elution flow rate is controlled between 1 - 2 ml / min. The centrifuged yeast cell wall polysaccharide solution is passed through a Sephadex G-50 column, and the eluent containing high-purity yeast cell wall polysaccharide is collected.

[0008] Preferably, in the pretreatment process of the pharmaceutical-grade aloe polysaccharide: The pharmaceutical-grade aloe polysaccharide is put into a low-temperature pulverizer, and the temperature is controlled below 0 °C and pulverized to 80 - 100 mesh. The pulverized aloe polysaccharide is put into a centrifuge tube, and deionized water with a volume of 65% of the aloe polysaccharide is added. The centrifuge tube is placed in a high-speed centrifuge, and the rotation speed is set to 9000 - 10000 r / min, and the centrifugation time is 12 - 15 min. Finally, ultrafiltration technology is used for purification. Under a pressure of 0.1 - 0.15 MPa, the centrifuged aloe polysaccharide solution is ultrafiltered to obtain a high-purity aloe polysaccharide solution.

[0009] Preferably, after the pretreatment of the pharmaceutical-grade yeast cell wall polysaccharide and aloe polysaccharide, they are mixed in a ratio of 6:5 to obtain a crude heparin extract.

[0010] Preferably, in the enzymatic hydrolysis optimization process in Step 3: S3.1. Set the enzymatic hydrolysis parameters: Add the crude heparin extract to a continuous reaction vessel with immobilized heparinase, set the pH of the enzymatic hydrolysis parameters to 6.9 ± 0.1, the temperature to 7 - 9 °C, and the flow rate to be controlled between 2.5 - 3 L / min; S3.2. Real-time monitoring: The molecular weight distribution is monitored in real time by on-line HPLC in the reaction vessel, and the target range is 14 - 15 kDa; S3.3. Protection structure: Add a composite stabilizer of 0.1 mM EDTA + 0.05% (mass percentage) of sodium thiosulfate to the reaction vessel, and nitrogen with a purity greater than 99.99% is introduced during the reaction to protect the internal environment of the reaction vessel.

[0011] Preferably, in the anion exchange chromatography in Step 4, DEAE cellulose is selected for the preliminary purification of sodium heparin, and elution is performed with a linear gradient of 0.5→2.0 M NaCl.

[0012] Preferably, in the gel permeation chromatography in Step 4, the reaction solution of the anion exchange chromatography is further passed through the gel permeation chromatography.

[0013] Preferably, in the crystallization optimization process in Step 5: S5.1, Reagent preparation: Prepare ethanol reagents with concentrations of 38% - 40% and 72% - 75%. S5.2, Reactor preparation: Perform staged precipitation on the sodium heparin solution, and prepare two groups of reaction vessels, A and B. Place the sodium heparin solution in Reaction Vessel A. S5.3, First stage: Gradually add dropwise the ethanol reagent with a concentration of 38% - 40% to Reaction Vessel A, and at the same time, turn on the low-speed stirring device, set the stirring speed at 60 - 80 r / min. When the initial temperature is 45 - 50 °C, use a temperature control device to gradually lower the solution temperature at a rate of 0.4 - 0.5 °C / min, with a cooling duration of 10 - 15 min. Finally, preliminary precipitate the miscellaneous proteins at the bottom of Reaction Vessel A. S5.4, Second stage: Transfer the sodium heparin solution on the upper layer of Reaction Vessel A to Reaction Vessel B, add the ethanol reagent with a concentration of 72% - 75%, turn on the low-speed stirring, maintain the rotation speed between 50 - 80 r / min, and gradually lower the temperature at a rate of 0.4 - 0.5 °C / min. S5.5, Centrifugation: Centrifuge the mixture after precipitation in the second stage, select a high-speed centrifuge, set the rotation speed at 7000 - 8000 r / min, and the centrifugation duration at 15 - 20 min, and collect the precipitated part. S5.6, Washing: Wash the collected precipitate 2 - 4 times with anhydrous ethanol, and stir with a glass rod each time during washing. S5.7, Drying: After washing, transfer the precipitate to a vacuum drying oven, set the temperature between 40 - 50 °C, maintain the vacuum degree between -0.08 and -0.5 MPa, and the drying duration at 5 - 6 h to finally obtain high-purity sodium heparin crystals.

[0014] Preferably, in the equipment optimization in Step 6, sensors are installed on both the reaction vessel and the chromatography column to monitor the temperature, pressure, and flow rate parameters in real time. When abnormal fluctuations in the parameters are detected, the equipment can automatically make adjustments or issue an alarm.

[0015] An application of a composition of sodium heparin, wherein the composition of sodium heparin obtained according to the above preparation method is used for the prevention and treatment of thrombosis.

[0016] Compared with the prior art, the present invention provides a method for preparing a heparin sodium composition and its application, having the following beneficial effects: 1. Through low-temperature pulverization, multi-stage centrifugation, immobilized enzymatic hydrolysis, dual-column chromatography purification, and stepwise alcohol precipitation crystallization, the present invention helps to improve the purity (≥98%) and yield (≥80%) of heparin sodium. Among them, dynamic crystallization control can ensure the high quality and production stability of the product, resulting in high anticoagulant activity (≥178 IU / mg), low impurity content (≤0.3%), and high crystallization purity (≥98.8%) of the finished product.

[0017] 2. All the equipment used in the entire preparation process of the present invention has automatic monitoring and regulation functions, which can real-time feedback various process parameters, ensure the stability and repeatability of the preparation process, reduce the impact of human operation errors on product quality, and thus improve the consistency of production efficiency and product quality. High-precision sensors are installed at key parts of the equipment, such as reaction vessels and chromatography columns, which helps to accurately monitor temperature, pressure, and flow rate parameters. When the parameters show abnormal fluctuations, the equipment can automatically adjust or issue an alarm, thus ensuring the smooth progress of the preparation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a flowchart of the method of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0020] Please refer to Figure 1 , a method for preparing a heparin sodium composition, comprising the following steps: Step 1. Raw material selection: The main raw materials are medical-grade yeast cell wall polysaccharide and aloe polysaccharide to ensure reducing the interference of impurities on the subsequent preparation process from the source. The auxiliary materials are pharmaceutical-grade sodium chloride (USP standard) and pharmaceutical-grade ethanol (≥99.8%) to ensure the high quality of the reagents and avoid introducing additional impurities; Step 2. Raw material pretreatment: The medical-grade yeast cell wall polysaccharide and aloe polysaccharide are respectively pulverized, centrifuged, and purified; Step 3. Enzymatic hydrolysis optimization process: React in a continuous reaction vessel with immobilized heparinase. After setting the enzymatic hydrolysis parameters, add a stabilizer and simultaneously introduce nitrogen to protect the structure of heparin sodium; Step 4. Purification and enhancement process: The reacted solution is purified by a combination of anion exchange chromatography and gel permeation chromatography to obtain a heparin sodium solution. By combining the advantages of the two techniques, it can not only improve the purity but also retain the activity of heparin sodium, meeting the requirements of high-quality production. It can efficiently and stably prepare high-purity heparin sodium, while also avoiding the problems of structural damage and impurity residues in traditional processes, and is suitable for large-scale industrial production; Step 5. Crystallization optimization: The heparin sodium solution is subjected to crystallization optimization by the stepwise alcohol precipitation method. Dynamic crystallization control is achieved by controlling the cooling rate to obtain high-purity heparin sodium crystals. A heparin sodium composition is obtained by adding pharmaceutical-grade sodium chloride in a vaporization manner; The specific operation of vaporization is as follows: Place the pharmaceutical-grade sodium chloride in a vaporization device, set the heating temperature of the device to 300 - 400 °C to vaporize the sodium chloride, and then pass the generated sodium chloride vapor through a high-temperature glass tube and slowly introduce it into the reaction vessel containing heparin sodium crystals. Control the sodium chloride vapor flow rate at 0.5 - 1.0 kg / m³ per hour and the introduction time at 2 - 4 h to ensure that the sodium chloride and heparin sodium are fully and evenly mixed; Step 6. Equipment optimization: All the equipment used in the preparation process has automated monitoring and regulation functions, which can real-time feedback various process parameters, ensuring the stability and repeatability of the preparation process and reducing the impact of human operation errors on product quality; Advantages: All the equipment used in the entire preparation process has automated monitoring and regulation functions, which can real-time feedback various process parameters, ensuring the stability and repeatability of the preparation process and reducing the impact of human operation errors on product quality, improving the consistency of production efficiency and product quality. High-precision sensors are installed at key parts of the equipment, such as the reaction vessel and chromatography column, which helps to accurately monitor temperature, pressure, and flow rate parameters. When the parameters show abnormal fluctuations, the equipment can automatically adjust or issue an alarm, thus ensuring the smooth progress of the preparation process.

[0021] Specifically, the pretreatment process of medical-grade yeast cell wall polysaccharide in Step 2: Put the medical-grade yeast cell wall polysaccharide into a crusher and crush it to 100 - 120 mesh to increase the specific surface area and create favorable conditions for subsequent reactions. Transfer the crushed yeast cell wall polysaccharide to a centrifuge tube, add deionized water with a volume 80% of that of the yeast cell wall polysaccharide, and gently shake to preliminarily disperse the polysaccharide. Set the rotation speed to 8000 - 9000 r / min and the centrifugation duration to 10 - 12 min to remove incompletely crushed large particle impurities and insoluble substances with a relatively high density. Use column chromatography for purification, with deionized water as the eluent, control the elution flow rate between 1 - 2 ml / min, pass the centrifuged yeast cell wall polysaccharide solution through a Sephadex G-50 column, load the sample slowly, and finally collect the eluent containing high-purity yeast cell wall polysaccharide based on the difference in retention time of the polysaccharide and impurities in the gel column.

[0022] Specifically, the pretreatment process of medical-grade aloe polysaccharide: Since aloe polysaccharide is soft in texture and may contain moisture, a low-temperature crusher is selected for processing. During the crushing process, with the help of liquid nitrogen refrigeration, put the medical-grade aloe polysaccharide into the low-temperature crusher, control the temperature below 0 °C to prevent the polysaccharide from degrading due to temperature rise, crush the aloe polysaccharide to 80 - 100 mesh, put the crushed aloe polysaccharide into a centrifuge tube, add deionized water with a volume 65% of that of the aloe polysaccharide to make it disperse evenly, place the centrifuge tube in a high-speed centrifuge, set the rotation speed to 9000 - 10000 r / min, and set the centrifugation duration to 12 - 15 min to remove insoluble impurities. Finally, use ultrafiltration technology for purification. Under a pressure of 0.1 - 0.15 MPa, ultrafilter the centrifuged aloe polysaccharide solution to remove impurities and salts with a relatively small molecular weight, and finally obtain a high-purity aloe polysaccharide solution.

[0023] Specifically, after the pretreatment of medical-grade yeast cell wall polysaccharide and aloe polysaccharide, mix them in a ratio of 6:5 to obtain a crude heparin extract.

[0024] Specifically, the enzymatic hydrolysis optimization process in Step 3: S3.1. Set the enzymatic hydrolysis parameters: Add the crude heparin extract to a continuous reaction vessel with immobilized heparinase, set the pH of the enzymatic hydrolysis parameters to 6.9 ± 0.1, the temperature to 7 - 9 °C, and control the flow rate between 2.5 - 3 L / min; S3.2. Real-time monitoring: Real-time monitor the molecular weight distribution through on-line HPLC in the reaction vessel, with the target range being 14 - 15 kDa, so as to ensure that the reaction proceeds in the expected direction and precisely control the molecular structure of heparin sodium; S3.3. Protection Structure: Add a composite stabilizer of 0.1 mM EDTA + 0.05% sodium thiosulfate by mass percentage in the reaction vessel. During the reaction process, introduce nitrogen with a purity greater than 99.99% to protect the internal environment of the reaction vessel. The composite stabilizer of EDTA and sodium thiosulfate and nitrogen can protect the structural integrity of heparin sodium to the greatest extent and avoid structural damage caused by oxidation and other factors during the enzymatic hydrolysis process.

[0025] Specifically, for anion exchange chromatography in Step 4: Select DEAE cellulose to preliminarily purify heparin sodium. By adjusting the pH value and ionic strength of the solution, negatively charged heparin sodium binds to the exchanger and is eluted with a linear gradient of 0.5→2.0 M NaCl. Based on the difference in charges carried by heparin sodium and other impurities, heparin sodium is adsorbed through electrostatic interaction. Under the elution of sodium chloride solutions with different concentrations, the effective separation of heparin sodium from impurities is achieved, improving the product purity.

[0026] Specifically, for gel permeation chromatography in Step 4: Further pass the reaction solution of anion exchange chromatography through gel permeation chromatography to remove small molecule impurities and residual buffer components, and improve the purity of heparin sodium.

[0027] Specifically, for the crystallization optimization process in Step 5: S5.1. Reagent Preparation: Prepare ethanol reagents with concentrations of 38% - 40% and 72% - 75%, ensuring that the purity and concentration of the ethanol reagents meet the experimental requirements, providing a suitable precipitant for the subsequent precipitation step; S5.2. Reactor Preparation: Perform a staged precipitation operation on the heparin sodium solution, and prepare two groups of reaction vessels, A and B. Place the heparin sodium solution in reaction vessel A. This method facilitates subsequent operations for different components, namely the preliminary precipitation of miscellaneous proteins and the precipitation of heparin sodium, greatly improving the accuracy of separation; S5.3. The First Stage: Gradually add the ethanol reagent with a concentration of 38% - 40% dropwise to reaction vessel A while turning on the low-speed stirring device, set the stirring speed at 60 - 80 r / min. When the initial temperature is 45 - 50 °C, ensure that the ethanol can be evenly dispersed in the solution. Use a temperature control device to gradually reduce the solution temperature at a rate of 0.4 - 0.5 °C / min for a cooling duration of 10 - 15 min. Finally, preliminarily precipitate the miscellaneous proteins at the bottom of reaction vessel A. During this process, the miscellaneous proteins will gradually aggregate due to changes in solubility, and slow cooling promotes the orderly aggregation and precipitation of the miscellaneous proteins to improve the effect and purity of the miscellaneous protein precipitation; S5.4, Second Stage: Transfer the heparin sodium solution on the upper layer of the A reaction vessel to the B reaction vessel, add ethanol reagent with a concentration of 72% - 75%, start low-speed stirring, maintain the rotation speed between 50 - 80 r / min to ensure that the ethanol can be evenly dispersed in the solution, and gradually cool down at a rate of 0.4 - 0.5 °C / min. The ethanol at this concentration and the slow cooling condition can promote the orderly arrangement of heparin sodium molecules, crystallize out, and finally form high-quality crystals; S5.5, Centrifugation: Centrifuge the mixed solution after precipitation in the second stage. Select a high-speed centrifuge, set the rotation speed at 7000 - 8000 r / min, and the centrifugation duration at 15 - 20 min. With the help of centrifugal force, quickly separate the precipitate from the supernatant and collect the precipitate part; S5.6, Washing: Wash the collected precipitate with absolute ethanol 2 - 4 times. Stir with a glass rod each time during washing to ensure that the impurities and solvents remaining on the surface of the precipitate are fully removed; S5.7, Drying: After washing, transfer the precipitate to a vacuum drying oven, set the temperature between 40 - 50 °C, maintain the vacuum degree between -0.08 to -0.5 MPa, and the drying duration at 5 - 6 h. After vacuum drying, thoroughly remove the residual moisture and finally obtain high-purity heparin sodium crystals; Advantages: Through this fractional precipitation method, it helps to achieve dynamic crystallization control, effectively separate impurity proteins and purified heparin sodium, improve the crystallization purity and yield. Throughout the process, by precisely controlling the ethanol concentration, stirring speed, cooling rate, and subsequent centrifugation, washing, and drying conditions, ensure the high quality and stability of the heparin sodium product.

[0028] Specifically, equipment optimization in step six: Install sensors on both the reaction vessel and the chromatography column to monitor temperature, pressure, and flow rate parameters in real time. When abnormal fluctuations in the parameters are detected, the equipment can automatically adjust or issue an alarm to ensure the smooth progress of the preparation process. The specific abnormal situations and handling measures are as follows: (1) When abnormal temperature fluctuations occur, the control system of the equipment will automatically start the heating or cooling device for adjustment. If the temperature is lower than the set value, the heater can be automatically turned on to gradually increase the temperature at the set power until it reaches the preset temperature range; if the temperature is too high, start the cooling circulation system to lower the temperature through cooling water or refrigerant; (2) When the pressure exceeds the set upper limit, the control system of the equipment will automatically open the pressure relief valve to reduce the pressure; if the pressure is lower than the lower limit, restore the pressure by adjusting the intake air volume or closing some exhaust ports; (3) When the flow rate deviates from the set value, the control system of the equipment will automatically adjust the rotation speed of the pump or the opening of the valve to ensure the stability of the flow rate, and precisely adjust the flow rate by controlling the rotation speed of the delivery pump through a frequency converter; When the device detects any of the above abnormal fluctuations, the control system of the device will immediately issue an alarm to timely remind the operator to handle it. Through the above automated detection and regulation measures, the stability of the heparin sodium preparation process can be ensured, thereby reducing the impact of human operation errors on product quality and improving production quality.

[0029] Specifically, for the application of a composition of heparin sodium, the composition of heparin sodium obtained according to the above preparation method is used for the prevention and treatment of thrombosis.

[0030] The following are the finished product processes of Examples 1-2 prepared by the method of the present invention, and the finished product processes of Comparative Examples 1-2 prepared by the traditional method, specifically: Example 1: Heparin sodium prepared by the method of the present invention S1. Raw material selection: medical-grade yeast cell wall polysaccharide and aloe polysaccharide (purity ≥ 99.5%), pharmaceutical-grade sodium chloride, pharmaceutical-grade ethanol (≥ 99.8%); S2. Preparation steps: cryogenic pulverization (-18 °C), centrifugation and purification treatment; S3. Enzymatic hydrolysis optimization (pH 6.9, 8 °C, flow rate 3 L / min, on-line HPLC monitoring); S4. Anion exchange chromatography (DEAE cellulose, 0.5 - 2.0 M NaCl gradient elution) + gel permeation chromatography; S5. Stepwise alcohol precipitation crystallization (38% ethanol precipitates miscellaneous proteins, 75% ethanol crystallizes heparin sodium, cooling rate 0.5 °C / min); S6. Automated equipment monitoring and regulation.

[0031] Example 2: Heparin sodium prepared by the method of the present invention (slightly adjusted) T1. Raw material selection: the same as in Example 1; T2. Preparation steps: cryogenic pulverization (-20 °C), centrifugation and purification treatment; T3. Enzymatic hydrolysis optimization (pH 7.0, 7 °C, flow rate 2.5 L / min, on-line HPLC monitoring); T4. Anion exchange chromatography (DEAE cellulose, 0.5 - 2.0 M NaCl gradient elution) + gel permeation chromatography; T5. Stepwise alcohol precipitation crystallization (38% ethanol precipitates miscellaneous proteins, 72% ethanol crystallizes heparin sodium, cooling rate 0.4 °C / min); T6. Automated equipment monitoring and regulation.

[0032] Comparative Example 1: Traditional chemical cleavage method F1. Raw material selection: sheep intestinal mucosa element (purity about 90%), industrial-grade ethanol; F2. Preparation steps: Crushing at room temperature, single-stage extraction (pH 7.0, room temperature); F3. Chemical lysis (high-temperature acid-base treatment); F4. Purification by precipitation method; F5. Direct ethanol precipitation (70% ethanol, rapid cooling); F6. Manual operation, without automated equipment.

[0033] Comparative Example 2: Traditional enzymatic hydrolysis method G1. Raw material selection: Intestinal mucosa element of sheep (purity about 90%), industrial-grade ethanol; G2. Crushing at room temperature, single-stage extraction (pH 7.0, room temperature); G3. Enzymatic hydrolysis (unimmobilized enzyme, room temperature, without real-time monitoring); G4. Purification by precipitation method; G5. Direct ethanol precipitation (70% ethanol, rapid cooling) G6. Manual operation, without automated equipment.

[0034] The following Table 1 shows the comparison of quality inspection data between the examples and the comparative examples: Table 1

[0035] The following information is obtained from the analysis of Table 1: The method of the present invention (Examples 1 and 2): Through low-temperature crushing, immobilized enzymatic hydrolysis, double-column chromatography purification and stepwise alcohol precipitation crystallization, it helps to improve the purity (≥98%) and yield (≥80%) of heparin sodium. Dynamic crystallization control and optimization of automated equipment can ensure the high quality and production stability of the product, making the finished product have high anticoagulant activity (≥178 IU / mg), low impurity content (≤0.3%) and high crystallization purity (≥98.8%).

[0036] Traditional methods (Comparative Examples 1 and 2): The chemical lysis method (Comparative Example 1) and the traditional enzymatic hydrolysis method (Comparative Example 2) have lower purity and yield, higher impurity content, poorer structural integrity, lower anticoagulant activity, and lower crystallization purity and yield. Manual operation results in poor production stability and large fluctuations in product quality.

[0037] Summary: Therefore, by optimizing each step, the present invention overall improves the preparation efficiency and product quality of heparin sodium, thus being suitable for large-scale industrial production Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A preparation method of a heparin sodium composition, characterized in that, It includes the following steps: Step 1, raw material selection: The main raw materials are medical-grade yeast cell wall polysaccharide and aloe polysaccharide, and the auxiliary materials are pharmaceutical-grade sodium chloride and pharmaceutical-grade ethanol; Step 2, raw material pretreatment: The medical-grade yeast cell wall polysaccharide and aloe polysaccharide are respectively pulverized, centrifuged and purified; Step 3, enzymatic hydrolysis optimization process: React in a continuous reaction vessel with immobilized heparinase. After setting the enzymatic hydrolysis parameters, add a stabilizer and simultaneously introduce nitrogen to protect the structure of heparin sodium; Step 4, purification improvement process: Purify the reacted solution by a combination of anion exchange chromatography and gel permeation chromatography to obtain a heparin sodium solution; Step 5, crystallization optimization: Crystallize and optimize the heparin sodium solution by the stepwise alcohol precipitation method. Achieve dynamic crystallization control by controlling the cooling rate to obtain high-purity heparin sodium crystals. Add pharmaceutical-grade sodium chloride by vaporization to obtain a composition of heparin sodium; Step 6, equipment optimization: All the equipment used in the preparation process has the functions of automatic monitoring and regulation, and can provide real-time feedback on various process parameters.

2. The preparation method of a heparin sodium composition according to claim 1, characterized in that: The pretreatment process of medical-grade yeast cell wall polysaccharide in Step 2: Put the medical-grade yeast cell wall polysaccharide into a pulverizer and pulverize it to 100-120 mesh. Transfer the pulverized yeast cell wall polysaccharide to a centrifuge tube, add deionized water with a volume 80% of the yeast cell wall polysaccharide, set the rotation speed to 8000-9000 r / min, and the centrifugation time to 10-12 min. Use column chromatography for purification, use deionized water as the eluent, control the elution flow rate between 1-2 ml / min, pass the centrifuged yeast cell wall polysaccharide solution through a Sephadex G-50 column, and collect the eluent containing high-purity yeast cell wall polysaccharide.

3. The preparation method of a heparin sodium composition according to claim 1, characterized in that: The pretreatment process of medical-grade aloe polysaccharide: Put the medical-grade aloe polysaccharide into a low-temperature pulverizer, control the temperature below 0 °C, and pulverize it to 80-100 mesh. Put the pulverized aloe polysaccharide into a centrifuge tube, add deionized water with a volume 65% of the aloe polysaccharide, place the centrifuge tube in a high-speed centrifuge, set the rotation speed to 9000-10000 r / min, and the centrifugation time to 12-15 min. Finally, use ultrafiltration technology for purification. Under a pressure of 0.1-0.15 MPa, ultrafilter the centrifuged aloe polysaccharide solution to obtain a high-purity aloe polysaccharide solution.

4. The preparation method of a heparin sodium composition according to claim 1, characterized in that: After the medical-grade yeast cell wall polysaccharide and aloe polysaccharide are pretreated, they are mixed in a ratio of 6:5 to obtain a crude heparin extract.

5. The preparation method of a heparin sodium composition according to claim 1, characterized in that: The enzymatic hydrolysis optimization process in Step 3: S3.1, set the enzymatic hydrolysis parameters: Add the crude heparin extract to a continuous reaction vessel with immobilized heparinase. Set the pH of the enzymatic hydrolysis parameters to 6.9±0.1, the temperature to 7-9 °C, and the flow rate to be controlled between 2.5-3 L / min; S3.2, real-time monitoring: Real-time monitor the molecular weight distribution by on-line HPLC in the reaction vessel, and the target range is 14-15 kDa; S3.

3. Protection Structure: Add a composite stabilizer of 0.1 mM EDTA and 0.05% sodium thiosulfate by mass percentage in the reaction vessel, and introduce nitrogen with a purity greater than 99.99% during the reaction process to protect the internal environment of the reaction vessel.

6. A method for preparing a heparin sodium composition according to claim 1, characterized in that: In the anion exchange chromatography in Step 4, DEAE cellulose is selected for the preliminary purification of heparin sodium, and eluted with a linear gradient of 0.5→2.0 M NaCl.

7. The preparation method of a heparin sodium composition according to claim 1, wherein: In the gel permeation chromatography in Step 4, the reaction solution of the anion exchange chromatography is further passed through the gel permeation chromatography.

8. A method for preparing a heparin sodium composition according to claim 1, wherein: The crystallization optimization process in Step 5: S5.

1. Reagent Preparation: Prepare ethanol reagents with concentrations of 38% - 40% and 72% - 75%. S5.

2. Reactor Preparation: Perform staged precipitation operations on the heparin sodium solution, and prepare two groups of reaction vessels, A and B. Place the heparin sodium solution in reaction vessel A. S5.

3. First Stage: Gradually add the ethanol reagent with a concentration of 38% - 40% dropwise to reaction vessel A, and at the same time turn on the low-speed stirring device, set the stirring speed at 60 - 80 r / min. When the initial temperature is 45 - 50 °C, use a temperature control device to gradually lower the solution temperature at a rate of 0.4 - 0.5 °C / min, and the cooling duration is 10 - 15 min. Finally, preliminary precipitate the impurity proteins at the bottom of reaction vessel A. S5.

4. Second Stage: Transfer the heparin sodium solution on the upper layer of reaction vessel A to reaction vessel B, add the ethanol reagent with a concentration of 72% - 75%, turn on the low-speed stirring, maintain the rotation speed between 50 - 80 r / min, and gradually lower the temperature at a rate of 0.4 - 0.5 °C / min. S5.

5. Centrifugation: Centrifuge the mixture after precipitation in the second stage. Select a high-speed centrifuge, set the rotation speed at 7000 - 8000 r / min, and the centrifugation duration is 15 - 20 min. Collect the precipitated part. S5.

6. Washing: Wash the collected precipitate 2 - 4 times with anhydrous ethanol, and stir with a glass rod each time during washing. S5.

7. Drying: After washing, transfer the precipitate to a vacuum drying oven, set the temperature between 40 - 50 °C, maintain the vacuum degree between -0.08 to -0.5 MPa, and the drying duration is 5 - 6 h to finally obtain high-purity heparin sodium crystals.

9. A method for preparing a heparin sodium composition according to claim 1, characterized in that: In the equipment optimization in Step 6, sensors are installed on both the reaction vessel and the chromatography column to monitor the temperature, pressure, and flow rate parameters in real time. When abnormal fluctuations in the parameters are detected, the equipment can automatically adjust or issue an alarm.

10. Use of a composition of heparin sodium, characterized in that, The composition of heparin sodium obtained by the preparation method according to Claim 1 is used for the prevention and treatment of thrombosis.

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