阅读说明：本技术 一种降低肝素钠中硫酸皮肤素含量的方法 (Method for reducing content of dermatan sulfate in heparin sodium ) 是由 李成斌 田丹 宋威 于 2021-10-20 设计创作，主要内容包括：本发明涉及肝素钠的精制工艺,尤其涉及一种降低肝素钠中硫酸皮肤素含量的方法。所述方法包括：将待处理的肝素钠溶解、并将得到的肝素钠溶解液进行酶解,离心后取上清液进行碱化处理,再次离心后取上清液进行梯度层析；其中,在进行梯度层析时,采用阴离子层析柱,依次使用3±0.1％的氯化钠溶液、5±0.1％的氯化钠溶液、7±0.1％的氯化钠溶液、18±0.1％的氯化钠溶液进行洗脱,收集18±0.1％的氯化钠溶液的洗脱液。本发明通过特定的处理方法、尤其是控制梯度层析,最终达到最大限度去除硫酸皮肤素的目的,使得硫酸皮肤素含量由原来的1.2～1.8％,降低至0.22～0.83％。(The invention relates to a refining process of heparin sodium, in particular to a method for reducing the content of dermatan sulfate in heparin sodium. The method comprises the following steps: dissolving heparin sodium to be treated, carrying out enzymolysis on the obtained heparin sodium dissolving solution, centrifuging, taking supernate, carrying out alkalization treatment, centrifuging again, taking supernate, and carrying out gradient chromatography; wherein, when the gradient chromatography is carried out, an anion chromatographic column is adopted, 3 plus or minus 0.1 percent sodium chloride solution, 5 plus or minus 0.1 percent sodium chloride solution, 7 plus or minus 0.1 percent sodium chloride solution and 18 plus or minus 0.1 percent sodium chloride solution are sequentially used for elution, and the eluent of the 18 plus or minus 0.1 percent sodium chloride solution is collected. The method finally achieves the aim of removing the dermatan sulfate to the maximum extent by a specific treatment method, particularly by controlling gradient chromatography, so that the content of the dermatan sulfate is reduced to 0.22-0.83% from the original 1.2-1.8%.)

1. A method for reducing the content of dermatan sulfate in heparin sodium is characterized by comprising the following steps: dissolving heparin sodium to be treated, carrying out enzymolysis on the obtained heparin sodium dissolving solution, centrifuging, taking supernate, carrying out alkalization treatment, centrifuging again, taking supernate, and carrying out gradient chromatography;

wherein, when the gradient chromatography is carried out, an anion chromatographic column is adopted, 3 plus or minus 0.1 percent sodium chloride solution, 5 plus or minus 0.1 percent sodium chloride solution, 7 plus or minus 0.1 percent sodium chloride solution and 18 plus or minus 0.1 percent sodium chloride solution are sequentially used for elution, and the eluent of the 18 plus or minus 0.1 percent sodium chloride solution is collected.

2. The method according to claim 1, wherein the volume of the anion chromatography column is 1.5-2.5 times of the volume of the heparin sodium dissolving solution.

3. The method according to claim 1 or 2, characterized in that 3 ± 0.1% sodium chloride solution is used for elution, and the elution volume is 1-3 times of the volume of the anion chromatographic column;

and/or eluting with 5 + -0.1% sodium chloride solution until the effluent is clarified by alcohol detection;

and/or eluting with 7 +/-0.1% sodium chloride solution, wherein the elution volume is 1-2 times of the volume of the anion chromatographic column;

and/or eluting with 18 + -0.1% sodium chloride solution until the effluent is clarified by alcohol detection.

4. The method according to any one of claims 1 to 3, further comprising: carrying out oxidation decoloration on the eluent;

preferably, the oxidative decoloration is specifically: adjusting the pH value of the eluent to 9.5-10.5, then adding hydrogen peroxide, and stirring for 4-8 hours at room temperature;

more preferably, the concentration of the hydrogen peroxide is 25-35%; and taking the volume of the eluent as a reference, and adding 2-4% of hydrogen peroxide.

5. The method of claim 4, further comprising: carrying out alcohol precipitation on the eluent after oxidation and decoloration treatment;

preferably, the alcohol precipitation is specifically: adjusting the pH value of the eluent after oxidation and decoloration treatment to 6.0-8.0, adding ethanol, standing for 23-25 h at 3-5 ℃, and then centrifuging for 10-20 min at 3-5 ℃ and 3500-4500 rpm;

more preferably, the concentration of ethanol is not less than 90%; the addition amount of the ethanol is 1-2 times of that of the eluent after oxidation and decoloration treatment.

6. The method according to any one of claims 1 to 5, wherein the heparin sodium to be treated is dissolved by using a 3 ± 0.1% sodium chloride solution; in g/ml, the ratio of heparin sodium to be treated: 3 ± 0.1% sodium chloride solution ═ 1: 14 to 16.

7. The method according to any one of claims 1 to 6, wherein the enzyme for enzymatic hydrolysis is trypsin; trypsin in g/ml: 1, heparin sodium dissolving solution: 8000-12000;

preferably, the enzymolysis is carried out under the condition that the pH value is 7.0-9.0;

more preferably, the enzymolysis time is 5-8 h.

8. The method of claim 1, wherein both centrifugations are carried out at 3500-4500 rpm at 3-5 ℃ for 10-20 min;

and/or the alkalization treatment specifically comprises the following steps: adjusting the pH value of the supernatant after the first centrifugation to 10.0-13.0, then heating for 10-20 min at the temperature of not lower than 95 ℃, and cooling to room temperature; preferably, 5m sodium hydroxide solution is used as the pH regulator.

9. The method of claim 1, comprising the steps of:

(1) dissolving heparin sodium to be treated by adopting a 3 +/-0.1% sodium chloride solution to obtain a heparin sodium dissolving solution;

wherein, in g/ml, the ratio of heparin sodium to be treated: 3 ± 0.1% sodium chloride solution ═ 1: 14-16;

(2) adding trypsin into the heparin sodium dissolving solution, and carrying out enzymolysis for 5-8 h under the condition that the pH value is 7.0-9.0;

wherein, in g/ml, trypsin: 1, heparin sodium dissolving solution: 8000-12000;

(3) centrifuging the hydrolyzed heparin sodium solution at the temperature of 3-5 ℃ and the rpm of 3500-4500 for 10-20 min to obtain supernatant I;

(4) adjusting the pH value of the supernatant I to 10.0-13.0 by using a 5m sodium hydroxide solution, heating for 10-20 min at the temperature of not less than 95 ℃, and cooling to room temperature; centrifuging at 3-5 ℃ and 3500-4500 rpm for 10-20 min to obtain supernatant II;

(5) subjecting the supernatant II to gradient chromatography;

when gradient chromatography is carried out, an anion chromatographic column with the volume 1.5-2.5 times that of the heparin sodium dissolving solution is adopted, 3 +/-0.1% of sodium chloride solution is used for elution, and the elution volume is 1-3 times that of the anion chromatographic column; eluting with 5 + -0.1% sodium chloride solution until the effluent is clarified by alcohol detection; then eluting by using 7 +/-0.1% sodium chloride solution, wherein the elution volume is 1-2 times of the volume of the anion chromatographic column; finally, eluting with 18 +/-0.1% sodium chloride solution until effluent is clarified by alcohol detection, and collecting the eluate of the 18 +/-0.1% sodium chloride solution;

(6) adjusting the pH value of the eluent to 9.5-10.5, then adding hydrogen peroxide with the concentration of 25-35%, and stirring, oxidizing and decolorizing for 4-8 h at room temperature;

wherein the volume of the eluent is taken as a reference, and the addition amount of the hydrogen peroxide is 2-4%;

(7) adjusting the pH value of the eluent after oxidation and decoloration treatment to 6.0-8.0, adding ethanol with the concentration not lower than 90%, standing for 23-25 h at the temperature of 3-5 ℃, and then centrifuging for 10-20 min at the temperature of 3-5 ℃ and the rpm of 3500-4500;

wherein the addition amount of the ethanol is 1-2 times of that of the eluent after oxidation and decoloration treatment.

10. Heparin sodium obtained by the method according to any one of claims 1 to 9.

Technical Field

The invention relates to a refining process of heparin sodium, in particular to a method for reducing the content of dermatan sulfate in heparin sodium.

Background

Heparin sodium (heparin sodium) is an anticoagulant, a mucopolysaccharide substance, and is a sodium glucosamine sulfate salt extracted from intestinal mucosa of pig, cattle and sheep, secreted by mast cells in human body and naturally present in blood. Heparin sodium has the effects of preventing platelet aggregation and destruction, inhibiting the conversion of fibrinogen into fibrin monomers, inhibiting the formation of thromboxane and antagonizing thromboxane which has been formed, preventing the conversion of prothrombin into thrombin and antagonizing thrombin. The heparin sodium can delay or prevent blood coagulation in vitro and in vivo. At present, the most common raw material source of heparin sodium is pig small intestine, and crude heparin sodium is prepared by steps of small intestine mucosa scraping, enzymolysis, adsorption, chromatography and the like; however, crude heparin sodium requires further refinement before it can be used in clinical patients.

Currently, the refining process of heparin sodium is mainly divided into two types: salt decomposition-resin adsorption method and enzymolysis-resin adsorption method, wherein dermatan sulfate is the main impurity. CN110713558A discloses an extraction process of heparin sodium, which comprises the following steps: treating raw materials, heating for enzymolysis, cooling for adsorption, eluting, precipitating, dehydrating and drying; however, the heparin sodium extracted by the above process has a problem of excessively high dermatan sulfate content. On the other hand, the production of heparin sodium was carried out by a method of purifying heparin sodium according to other patent documents and literatures, and as a result, it was found that the content of dermatan sulfate was close to 2.0% of the qualified line although it was acceptable. CN201410396942.6 discloses a method for controlling the total content of dermatan sulfate and chondroitin sulfate in a heparin sodium raw material, which adopts sodium hypochlorite hydrolysis 2, tert-butyl peroxide oxidation and isopropanol precipitation to effectively remove dermatan sulfate and chondroitin sulfate in the heparin sodium raw material and reduce the total content of dermatan sulfate and chondroitin sulfate to be less than 0.1%; however, the above method does not take into account the need for removing nucleic acids, so that the problem of nucleic acid failure may occur. On the basis of ensuring that the content of various impurities in the heparin sodium raw material meets the pharmacopoeia standard, the content of dermatan sulfate is reduced as much as possible, and the method has practical production significance.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a method for reducing the content of dermatan sulfate in heparin sodium, which can obviously reduce the content of dermatan sulfate in heparin sodium; the invention also aims to provide the heparin sodium prepared by the method.

Specifically, the invention provides the following technical scheme:

the invention provides a method for reducing the content of dermatan sulfate in heparin sodium, which comprises the following steps: dissolving heparin sodium to be treated, carrying out enzymolysis on the obtained heparin sodium dissolving solution, centrifuging, taking supernate, carrying out alkalization treatment, centrifuging again, taking supernate, and carrying out gradient chromatography;

wherein, when the gradient chromatography is carried out, an anion chromatographic column is adopted, 3 plus or minus 0.1 percent sodium chloride solution, 5 plus or minus 0.1 percent sodium chloride solution, 7 plus or minus 0.1 percent sodium chloride solution and 18 plus or minus 0.1 percent sodium chloride solution are sequentially used for elution, and the eluent of the 18 plus or minus 0.1 percent sodium chloride solution is collected.

In the prior art, related substances mainly comprising dermatan sulfate are eluted from heparin sodium by a 5-9% sodium chloride solution by adopting a chromatography method; however, if the sodium chloride concentration is too low, the dermatan sulfate is not completely removed, and the related content is not qualified; if the concentration of sodium chloride is too high, heparin sodium will be eluted.

The invention aims to reduce the content of dermatan sulfate in heparin sodium, tries a large number of chromatographic processes, and finds that the content of dermatan sulfate in heparin sodium is hopefully reduced by adopting a gradient chromatography mode in the research and development process.

Further, in the process of gradient chromatography, an anion chromatographic column is adopted, and 3 +/-0.1% sodium chloride solution, 5 +/-0.1% sodium chloride solution, 7 +/-0.1% sodium chloride solution and 18 +/-0.1% sodium chloride solution are sequentially used for elution, so that the content of dermatan sulfate in heparin sodium can be obviously reduced.

Specifically, 3 + -0.1% NaCl solution is eluted for the purpose of column equilibration and removal of nucleic acid proteins remaining after partial basification centrifugation; dermatan sulfate is mainly removed in the elution process of 5 +/-0.1% sodium chloride solution, but according to the traditional process and the experimental methods of other patent documents, the content of related substances in the produced refined heparin sodium product is lower than 2% but higher than 1% (even close to 2%), and once the process is amplified, the content of related substances is easy to fail; on the basis of the elution step, residual dermatan sulfate can be removed and heparin sodium can be prevented from being eluted by further eluting with 7 +/-0.1% sodium chloride solution; finally, the sodium chloride solution with the concentration of 18 +/-0.1% is used for elution, and the eluent of the sodium chloride solution with the concentration of 18 +/-0.1% is collected, so that the content of the dermatan sulfate in the heparin sodium can be obviously reduced.

In order to further reduce the content of dermatan sulfate in heparin sodium, the invention optimizes the steps of gradient chromatography, and the steps are as follows:

preferably, the volume of the anion chromatographic column is 1.5-2.5 times of the volume of the heparin sodium dissolving solution;

further, when 3 +/-0.1% sodium chloride solution is used for elution, the elution volume is 1-3 times of the volume of the anion chromatographic column;

further, when 5 +/-0.1% sodium chloride solution is used for elution, the elution is carried out until the effluent is clarified by alcohol detection;

further, when 7 +/-0.1% sodium chloride solution is used for elution, the elution volume is 1-2 times of the volume of the anion chromatographic column;

further, when the elution is carried out by using 18 +/-0.1% sodium chloride solution, the elution is carried out until the effluent is clarified by alcohol detection.

In the above technical scheme, "alcohol detection clarification" specifically means: the chromatography immediate effluent is detected by 95% ethanol solution until no turbidity is detected.

Preferably, the method further comprises: carrying out oxidation decoloration on the eluent;

further, the oxidation decoloring specifically comprises: adjusting the pH value of the eluent to 9.5-10.5, then adding hydrogen peroxide, and stirring for 4-8 hours at room temperature;

furthermore, the concentration of the hydrogen peroxide is 25-35%; and taking the volume of the eluent as a reference, and adding 2-4% of hydrogen peroxide.

Preferably, the method further comprises: carrying out alcohol precipitation on the eluent after oxidation and decoloration treatment;

further, the alcohol precipitation specifically comprises: adjusting the pH value of the eluent after oxidation and decoloration treatment to 6.0-8.0, adding ethanol, standing for 23-25 h at 3-5 ℃, and then centrifuging for 10-20 min at 3-5 ℃ and 3500-4500 rpm;

further, the concentration of the ethanol is not lower than 90%; the addition amount of the ethanol is 1-2 times of that of the eluent after oxidation and decoloration treatment.

Preferably, 3 +/-0.1% sodium chloride solution is adopted to dissolve heparin sodium to be treated; in g/ml, the ratio of heparin sodium to be treated: 3 ± 0.1% sodium chloride solution ═ 1: 14 to 16.

Further, the dissolution is carried out at 40-50 ℃.

Preferably, the enzyme for enzymolysis is trypsin; trypsin in g/ml: 1, heparin sodium dissolving solution: 8000-12000;

further, the enzymolysis is carried out under the condition that the pH value is 7.0-9.0;

furthermore, the enzymolysis time is 5-8 h.

Preferably, the centrifugation is carried out for 10-20 min at 3500-4500 rpm at 3-5 ℃ for two times.

Preferably, the alkalization treatment is specifically as follows: adjusting the pH value of the supernatant after the first centrifugation to 10.0-13.0, then heating for 10-20 min at the temperature of not lower than 95 ℃, and cooling to room temperature; preferably, 5m sodium hydroxide solution is used as the pH regulator.

As a better technical scheme of the invention, the method comprises the following steps:

(1) dissolving heparin sodium to be treated by adopting a 3 +/-0.1% sodium chloride solution to obtain a heparin sodium dissolving solution;

wherein, in g/ml, the ratio of heparin sodium to be treated: 3 ± 0.1% sodium chloride solution ═ 1: 14-16;

(2) adding trypsin into the heparin sodium dissolving solution, and carrying out enzymolysis for 5-8 h under the condition that the pH value is 7.0-9.0;

wherein, in g/ml, trypsin: 1, heparin sodium dissolving solution: 8000-12000;

(3) centrifuging the hydrolyzed heparin sodium solution at the temperature of 3-5 ℃ and the rpm of 3500-4500 for 10-20 min to obtain supernatant I;

(4) adjusting the pH value of the supernatant I to 10.0-13.0 by using a 5m sodium hydroxide solution, heating for 10-20 min at the temperature of not less than 95 ℃, and cooling to room temperature; centrifuging at 3-5 ℃ and 3500-4500 rpm for 10-20 min to obtain supernatant II;

(5) subjecting the supernatant II to gradient chromatography;

when gradient chromatography is carried out, an anion chromatographic column with the volume 1.5-2.5 times that of the heparin sodium dissolving solution is adopted, 3 +/-0.1% of sodium chloride solution is used for elution, and the elution volume is 1-3 times that of the anion chromatographic column; eluting with 5 + -0.1% sodium chloride solution until the effluent is clarified by alcohol detection; then eluting by using 7 +/-0.1% sodium chloride solution, wherein the elution volume is 1-2 times of the volume of the anion chromatographic column; finally, eluting with 18 +/-0.1% sodium chloride solution until effluent is clarified by alcohol detection, and collecting the eluate of the 18 +/-0.1% sodium chloride solution;

(6) adjusting the pH value of the eluent to 9.5-10.5, then adding hydrogen peroxide with the concentration of 25-35%, and stirring, oxidizing and decolorizing for 4-8 h at room temperature;

wherein the volume of the eluent is taken as a reference, and the addition amount of the hydrogen peroxide is 2-4%;

(7) adjusting the pH value of the eluent after oxidation and decoloration treatment to 6.0-8.0, adding ethanol with the concentration not lower than 90%, standing for 23-25 h at the temperature of 3-5 ℃, and then centrifuging for 10-20 min at the temperature of 3-5 ℃ and the rpm of 3500-4500;

wherein the addition amount of the ethanol is 1-2 times of that of the eluent after oxidation and decoloration treatment.

Therefore, the method effectively reduces the content of dermatan sulfate in heparin sodium through specific steps of dissolution, enzymolysis, primary centrifugation, alkalization, secondary centrifugation, gradient chromatography, oxidation decoloration, alcohol precipitation and the like.

The invention also provides heparin sodium prepared by the method.

The invention has the beneficial effects that:

the method provided by the invention solves the problem that the skin sulfate content in the traditional refined heparin sodium sold on the market is higher; specifically, the purpose of removing the dermatan sulfate to the maximum extent is finally achieved through a specific treatment method, particularly control of gradient chromatography, so that the content of the dermatan sulfate is reduced to 0.22-0.83% from 1.2-1.8% of the original content.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.

Example 1

The embodiment provides a method for reducing the content of dermatan sulfate in heparin sodium, which comprises the following steps:

(1) weighing 40.08g of crude heparin sodium, dissolving with 600ml of 3% sodium chloride solution (weighing 18.16g of sodium chloride solid, dissolving with purified water and fixing the volume to 600ml), and heating and stirring in a water bath at 40 ℃ to promote dissolution to obtain a heparin sodium solution;

(2) weighing 60.03mg of trypsin, slowly adding the trypsin into the heparin sodium dissolving solution under the stirring condition, and stirring for enzymolysis for 8 hours under the condition that the pH value is 8.63;

(3) loading the enzymolyzed heparin sodium solution into a 1L centrifugal barrel, centrifuging at 4 deg.C and 4000rpm for 10min, and collecting supernatant I;

(4) adjusting the pH value of the supernatant I to 11.82 by using a 5m sodium hydroxide solution, heating the supernatant I in a water bath kettle at 100 ℃ for 10min, and cooling the supernatant I to room temperature;

(5) the alkalized heparin sodium solution is respectively filled into a 1L centrifugal barrel and is centrifuged for 10min at the temperature of 4 ℃ and the rpm of 4000 to obtain supernatant liquid II;

(6) measuring the volume of the supernatant II to be 566ml, loading the supernatant II into an anion chromatographic column with the volume of 1L of the column, then reducing the flow rate, eluting by using a 3% sodium chloride solution, eluting by using a 5% sodium chloride solution after 1.5L, detecting the chromatography instant effluent by using a 95% ethanol solution after 2.0L of elution, detecting the chromatography instant effluent by using a 95% ethanol solution, clarifying the chromatography instant effluent after 400ml of elution, eluting by using a 7% sodium chloride solution, eluting by 1.2L, finally eluting by using an 18% sodium chloride solution, detecting the chromatography instant effluent by using a 95% ethanol solution, stopping elution after the detection result is clear, and collecting the eluent (2400ml) of the 18% sodium chloride solution;

(7) adjusting the pH value of the eluent to 9.80, then adding 72ml of 30% hydrogen peroxide, and stirring at room temperature (25 ℃) for oxidation and decoloration for 4 hours;

(8) adjusting the pH of the eluent after oxidation and decoloration to 7.02, adding 2500ml of 95% ethanol, performing overnight ethanol precipitation for 24h at 4 ℃, then centrifuging for 15min at 4 ℃ and 4000rpm, removing the supernatant, and keeping the precipitate;

(9) drying the precipitate in a constant-temperature oven at 40 ℃ for 16h, dissolving with purified water, placing into suitable freeze-drying boxes, and freeze-drying in a freeze-drying machine for 47 h; obtaining the refined heparin sodium.

The refined heparin sodium prepared by the embodiment is 24.65g, the titer is about 210IU, and the dermatan sulfate content is 0.22%.

Example 2

The embodiment provides a method for reducing the content of dermatan sulfate in heparin sodium, which comprises the following steps:

(1) weighing 40.23g of crude heparin sodium, dissolving with 600ml of 3% sodium chloride solution (weighing 18.09g of sodium chloride solid, dissolving with purified water and fixing the volume to 600ml), and heating and stirring in a water bath at 40 ℃ to promote dissolution to obtain a heparin sodium solution;

(2) weighing 60.04mg of trypsin, slowly adding into the heparin sodium solution under stirring, and stirring for enzymolysis for 8h under the condition that the pH value is 8.80;

(3) loading the enzymolyzed heparin sodium solution into a 1L centrifugal barrel, centrifuging at 4 deg.C and 4000rpm for 10min, and collecting supernatant I;

(4) adjusting the pH value of the supernatant I to 12.61 by using a 5m sodium hydroxide solution, heating the supernatant I in a water bath kettle at 100 ℃ for 10min, and cooling the supernatant I to room temperature;

(5) the alkalized heparin sodium solution is respectively filled into a 1L centrifugal barrel and is centrifuged for 10min at the temperature of 4 ℃ and the rpm of 4000 to obtain supernatant liquid II;

(6) measuring the volume of the supernatant II to be 593ml, loading the supernatant II into an anion chromatographic column with the volume of 1L of column, then reducing the flow rate, eluting by using a 3% sodium chloride solution, eluting by using a 5% sodium chloride solution after 1.5L of elution, detecting the chromatographic instant effluent by using a 95% ethanol solution after 2.0L of elution, wherein the detection result is clear, then eluting by using a 7% sodium chloride solution, eluting by 1.2L, and finally eluting by using an 18% sodium chloride solution, detecting the chromatographic instant effluent by using a 95% ethanol solution, wherein the detection result is clear, stopping elution, and collecting the eluent (2400ml) of the 18% sodium chloride solution;

(7) adjusting the pH value of the eluent to 10.20, then adding 72ml of 30% hydrogen peroxide, and stirring at room temperature (25 ℃) for oxidation and decoloration for 4 hours;

(8) adjusting the pH of the eluent after oxidation and decoloration to 6.94, adding 2500ml of 95% ethanol, performing overnight ethanol precipitation for 24h at 4 ℃, then centrifuging for 15min at 4 ℃ and 4000rpm, removing the supernatant, and keeping the precipitate;

(9) drying the precipitate in a constant-temperature oven at 40 ℃ for 16h, dissolving with purified water, placing into suitable freeze-drying boxes, and freeze-drying in a freeze-drying machine for 47 h; obtaining the refined heparin sodium.

The refined heparin sodium prepared by the embodiment is 25.12g, the titer is about 230IU, and the dermatan sulfate content is 0.83 percent.

Comparative example 1

The comparative example provides a method for reducing the content of dermatan sulfate in heparin sodium, which comprises the following steps:

(1) weighing 40.14g of crude heparin sodium, dissolving with 600ml of 3% sodium chloride solution (weighing 18.07g of sodium chloride solid, dissolving with purified water and fixing the volume to 600ml), and heating and stirring in a water bath at 40 ℃ to promote dissolution to obtain a heparin sodium solution;

(2) weighing 60.22mg of trypsin, slowly adding the trypsin into the heparin sodium dissolving solution under the stirring condition, and stirring and carrying out enzymolysis for 8 hours under the condition that the pH value is 7.92;

(3) loading the enzymolyzed heparin sodium solution into a 1L centrifugal barrel, centrifuging at 4 deg.C and 4000rpm for 10min, and collecting supernatant I;

(4) adjusting the pH value of the supernatant I to 11.47 by using a 5m sodium hydroxide solution, heating the supernatant I in a water bath kettle at 100 ℃ for 10min, and cooling the supernatant I to room temperature;

(5) the alkalized heparin sodium solution is respectively filled into a 1L centrifugal barrel and is centrifuged for 10min at the temperature of 4 ℃ and the rpm of 4000 to obtain supernatant liquid II;

(6) measuring the volume of the supernatant II to be 575ml, loading the supernatant II into an anion chromatographic column with the volume of 1L of the column, then reducing the flow rate, eluting by using a 3% sodium chloride solution, eluting by using a 5% sodium chloride solution after 1.5L of the elution, detecting the chromatography instant effluent by using a 95% ethanol solution after 2.3L of the elution, wherein the detection result is clear, finally eluting by using an 18% sodium chloride solution, detecting the chromatography instant effluent by using a 95% ethanol solution, stopping the elution when the detection result is clear, and collecting the eluate (2400ml) of the 18% sodium chloride solution;

(7) adjusting the pH value of the eluent to 10.32, then adding 72ml of 30% hydrogen peroxide, and stirring at room temperature (25 ℃) for oxidation and decoloration for 4 hours;

(8) adjusting the pH of the eluent after oxidation and decoloration to 7.10, adding 2500ml of 95% ethanol, performing overnight ethanol precipitation for 24h at 4 ℃, then centrifuging for 15min at 4 ℃ and 4000rpm, removing the supernatant, and leaving the precipitate;

(9) drying the precipitate in a constant-temperature oven at 40 ℃ for 16h, dissolving with purified water, placing into suitable freeze-drying boxes, and freeze-drying in a freeze-drying machine for 47 h; obtaining the refined heparin sodium.

The refined heparin sodium prepared by the comparative example has 28.35g, the titer is about 230IU, and the dermatan sulfate content is 1.80 percent.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.