WO2021114941A1 - Low molecular weight dermatan sulfate, refining process therefor and application thereof - Google Patents

Abstract

The present application provides a low molecular weight dermatan sulfate. The number-average molecular weight of the low molecular weight dermatan sulfate is 2,000 to 5,000 Da, and the weight-average molecular weight of the low molecular weight dermatan sulfate is 2,500 to 6,500 Da. Also provided is a refining process for the low molecular weight dermatan sulfate, comprising: 1) mixing a dermatan sulfate solution and chondroitin sulfate B enzyme for enzymolysis to obtain feed liquid; 2) adding a strong oxidant to the feed liquid obtained in step 1) for oxidation reaction, and filtering to obtain filtered feed liquid; and 3) performing circulating ultrafiltration on the filtered feed liquid obtained in step 2), to obtain the low molecular weight dermatan sulfate. The low molecular weight dermatan sulfate is more suitable for human body absorption, and has a wide application prospect in antithrombotic drugs. The refining process for the low molecular weight dermatan sulfate is a process for refining the low molecular weight dermatan sulfate using an enzyme-ultrafiltration method, the reaction is mild, stable and controllable, the process repeatability is high, certain economic benefits and environmental protection effects are achieved, and the refining process is suitable for industrial application and promotion.

Description

Low-molecular-weight dermatan sulfate and its refined technology and application

LOW-MOLECULAR WEIGHT DERMATAN SULFATE AND REFINING PROCESS AND APPLICATION THEREOF

This application requires the priority of a Chinese patent application submitted to the Chinese Patent Office on December 11, 2019, with the application number 201911267218.2. The invention title is "A process and application for the purification of low molecular weight dermatan sulfate by enzyme-ultrafiltration method". The entire content is incorporated into this application by reference.

Technical field

The application belongs to the technical field of dermatan sulfate, and relates to a low-molecular-weight dermatan sulfate and its refining process and application, and in particular to a low-molecular-weight dermatan sulfate and the process and application for refining low-molecular-weight dermatan sulfate by an enzyme-ultrafiltration method.

Background technique

Dermatansulfate (DS for short) is the most widely distributed extracellular matrix glycosaminoglycan in animals, and it is the main component of vascular wall proteoglycan. Dermatan sulfate, as a glycosaminoglycan, is widely distributed in animal tissues. As an important part of connective tissue, DS has a variety of pharmacological and physiological functions. It can be used as medicine and health food. It is mainly used for osteoarthritis (oa) and coronary atherosclerotic heart disease (coronary heart disease). The antithrombotic effect of DS is equivalent to or even stronger than that of heparin, and the antithrombotic effect of DS does not depend on plasma antithrombin III, but is catalyzed by heparin cofactor II to inhibit thrombin. The bleeding side effect is less than that of heparin. It has gradually become a very Promising new type of antithrombotic drugs.

As for the source of dermatan sulfate, natural dermatan sulfate can be used directly, which is mainly derived from animal tissue extraction. Because it is mixed with other glycosaminoglycans, proteins, nucleic acids and other components in the animal body, extraction and purification are difficult. In addition, the relative molecular mass of natural DS is generally between 15,000 and 45,000 Da. The molecular weight is large, which is not conducive to human absorption. If it is directly used in the human body, there are two main pharmacokinetic problems: one is short half-life, and the other is subcutaneous and muscle When injected internally, its bioavailability is low; and the higher the relative molecular mass of DS, the lower its bioavailability.

Therefore, it is necessary to prepare Low Molecular Weight Dermatan Sulfate (LMWDS) with small molecular weight and narrow molecular weight distribution to increase the bioavailability of DS and improve its pharmacokinetic characteristics. LMWDS is a small molecule polymer prepared by degrading natural dermatan sulfate through chemical, biological and other means. It is easily absorbed by the human body, has high antithrombotic activity and high human availability.

At present, there are researches on the degradation of DS by chemical and physical methods to prepare LMWDS, but there are problems in the preparation process such as low yield and difficult control of molecular weight distribution. For example, the chemical method to degrade DS is to degrade dermatan sulfate using chemical reagents such as hydrochloric acid and hydrogen peroxide. Both of these methods use strong acid or strong oxidizing chemical reagents to hydrolyze dermatan sulfate chain scission. Dermatan sulfate is hydrolyzed in hydrochloric acid solution, but the amount of hydrochloric acid used is large, the hydrolysis reaction progresses slowly, and the hydrolysis reaction is not easy to control, which easily causes excessive hydrolysis or insufficient hydrolysis. The use of hydrogen peroxide to degrade dermatan sulfate also has problems such as difficult to control degradation parameters, poor reaction stability and reproducibility, resulting in low yield, uneven molecular weight distribution of degradation products, and uncontrollable curative effects.

Therefore, how to find a suitable method for the degradation of dermatan sulfate, that is, a refining process of low molecular weight dermatan sulfate, to solve the above-mentioned problems in the existing dermatan sulfate degradation process, has become a research and development manufacturer in the industry. One of the problems that many front-line researchers need to solve urgently.

Summary of the invention

In view of this, based on the technical problems to be solved above, this application provides a low-molecular-weight dermatan sulfate and its refining process and application, especially a process for refining low-molecular-weight dermatan sulfate by an enzyme-ultrafiltration method.

This application provides a low-molecular-weight dermatan sulfate, the number-average molecular weight of the low-molecular-weight dermatan sulfate is 2000-5000 Da;

The weight average molecular weight of the low molecular weight dermatan sulfate is 2500-6500 Da.

Preferably, the molecular weight distribution of the weight average molecular weight of the low molecular weight dermatan sulfate is:

Dermatan sulfate with a molecular weight of less than 2000 Da accounts for less than 10 wt% of the total low molecular weight dermatan sulfate;

Dermatan sulfate with a molecular weight greater than 8000 Da accounts for less than 12 wt% of the total low molecular weight dermatan sulfate;

Dermatan sulfate with a molecular weight of 2000-8000 Da accounts for 78 wt% to 90 wt% of the total low molecular weight dermatan sulfate.

Preferably, the weight average molecular weight of the low molecular weight dermatan sulfate is 3500-6000 Da;

The number average molecular weight of the low molecular weight dermatan sulfate is 3000 to 4500 Da;

The specific rotation of the low molecular weight dermatan sulfate is less than or equal to -40°;

The purity of the low molecular weight dermatan sulfate is more than 99%.

This application provides a refining process for low molecular weight dermatan sulfate, which includes the following steps:

1) After mixing the dermatan sulfate solution and chondroitin sulfate B enzyme, after enzymatic hydrolysis, the material liquid is obtained;

2) After adding a strong oxidant to the material liquid obtained in the above step to carry out the oxidation reaction, it is filtered to obtain the filtered material liquid;

3) After the filtered material liquid obtained in the above steps is circulated ultrafiltration, low molecular weight dermatan sulfate is obtained.

Preferably, the dermatan sulfate solution includes an aqueous solution of dermatan sulfate;

The pH of the dermatan sulfate solution is 6.5 to 8.5;

The mass concentration of the dermatan sulfate solution is 8%-12%;

The weight average molecular weight of the dermatan sulfate is 15000-45000 Da;

The volume ratio of the chondroitin sulfate B enzyme to the dermatan sulfate solution is 0.001% to 0.003%;

The concentration of the chondroitin sulfate B enzyme is 8000-12000 IU/mL;

The temperature of the enzymatic hydrolysis is 35-37°C;

The enzymolysis time is 3 to 5 hours.

Preferably, the strong oxidant includes one or more of potassium permanganate, sodium permanganate, hydrogen peroxide and sodium hypochlorite;

The mass ratio of the strong oxidant to the material liquid is 2% to 5%;

The temperature of the oxidation reaction is 80-90°C;

The oxidation reaction time is 30 to 45 minutes;

The oxidation reaction further includes a step of standing and cooling and/or a step of removing impurities;

The filtration is microfiltration membrane filtration;

The pore size of the microfiltration membrane is 0.1-0.45 μm.

Preferably, before the circulating ultrafiltration, a dilution step is further included;

The mass concentration of the diluted filtered feed liquid is 3% to 5%;

Preferably, the ultrafiltration is tangential flow ultrafiltration;

The specific steps of the cyclic ultrafiltration are:

After the first ultrafiltration membrane is used for the first cycle of ultrafiltration, the permeate is collected, and then the second ultrafiltration membrane is used for the second cycle of ultrafiltration, and then the retentate is collected;

The filtration accuracy of the first ultrafiltration membrane is 8000 to 12000 Da;

The number of times of the first cycle of ultrafiltration is 100 to 500 times;

The filtration accuracy of the second ultrafiltration membrane is 800-1200 Da;

The number of times of the second cycle ultrafiltration is 100 to 500 times.

Preferably, after the cyclic ultrafiltration, a post-treatment step is further included;

The post-treatment step includes one or more steps of dissolution, precipitation, re-dissolution, filtration, freeze-drying, grinding and mixing;

The post-processing steps are specifically:

After dissolving the retentate obtained after circulating ultrafiltration and sodium chloride, add ethanol and let stand, then discard the supernatant to obtain a precipitate;

After re-dissolving the precipitate obtained in the above steps, freeze-drying and grinding, the finished product low molecular weight dermatan sulfate is obtained;

The volume ratio of the sodium chloride to the retentate is 2% to 4%;

The volume ratio of the ethanol to the dissolved material is (2～3):1;

The standing time is 6-8h.

Preferably, the dermatan sulfate is obtained from the by-product of heparin sodium rich in dermatan sulfate;

The preparation process is:

A) Dissolve the by-product of heparin sodium rich in dermatan sulfate and water to obtain the original solution;

B) After the stock solution obtained in the above steps is adsorbed by resin, it is eluted with sodium chloride solution to obtain the eluted stock solution;

C) After mixing the eluted stock solution obtained in the above steps with ethanol and standing, then drying to obtain dermatan sulfate;

Preferably, the mass concentration of the stock solution is 8%-12%;

The temperature of the resin adsorption is 50-70°C;

The resin column adsorbed by the resin is an anion resin column;

The mass concentration of the sodium chloride solution is preferably 2%-9%;

The elution includes eluting impurities first and then eluting the resin.

Preferably, the volume ratio of the ethanol to the eluted stock solution is preferably (1.8-2.4):1;

The mixing and standing time is preferably 7-9 hours;

The mixing and standing is preferably two mixing and standing;

The drying temperature is preferably 50-60°C.

Preferably, the refining process specifically includes the following steps:

(1) Add water to dissolve the by-product of heparin sodium rich in dermatan sulfate to obtain a dissolved material liquid;

(2) Pass the feed solution described in (1) through an anion resin column, and control the temperature of the feed solution to about 50-70°C for resin adsorption; firstly, elution is carried out with a sodium chloride solution of the first mass concentration to remove Impurities; then eluted with a second mass concentration of sodium chloride solution to collect the salinity eluted material liquid;

(3) Add ethanol to the material liquid collected in (2) for precipitation and place, transfer the precipitation supernatant to another material tank, add ethanol for precipitation and place, discard the supernatant, and collect the precipitate;

(4) Dry the sediment collected in (3) to remove the ethanol; then add water to dissolve it and dissolve it into a material liquid;

(5) Adjust the pH of the material liquid obtained in (4) to 6.5-8.5, control the temperature to 35-37°C, and add chondroitin sulfate B enzyme for enzymatic hydrolysis;

(6) Raise the temperature of the material liquid described in (5), add potassium permanganate, continue to stir, and oxidize; leave the material liquid to stand still after oxidation to cool down, filter to remove impurity products after oxidation of potassium permanganate, and then filter Membrane filtration and impurity removal;

(7) Dilute the filtered material in (6), use the first filter membrane for circulating ultrafiltration, and collect the permeate; then use the second filter membrane for the collected permeate for circulating ultrafiltration to collect the retentate, Control the molecular weight during sampling and control the weight-average molecular weight between 3000 Da and 5000 Da;

(8) Add sodium chloride to dissolve the retentate collected in (7), add ethanol, and stir well, then set aside.

(9) Discard the supernatant precipitated in (8), dissolve the precipitate, dissolve the precipitate, filter, freeze-dry, grind, mix, and package.

This application provides the application of the low-molecular-weight dermatan sulfate described in any one of the above or the low-molecular-weight dermatan sulfate refined by the purification process of any one of the above in the preparation of antithrombotic drugs.

This application provides a low molecular weight dermatan sulfate, the number average molecular weight of the low molecular weight dermatan sulfate is 2000-5000 Da; the weight average molecular weight of the low molecular weight dermatan sulfate is 2500-6500 Da. Compared with the prior art, this application aims at natural dermatan sulfate, which is more difficult to extract and purify. In addition, the relative molecular mass of natural DS is generally between 15,000 and 45,000 Da. The molecular weight is large, which is not conducive to human absorption. There are problems with short half-life and low bioavailability when injected subcutaneously and intramuscularly.

This application creatively obtains a low molecular weight dermatan sulfate. The refined low molecular weight dermatan sulfate has a narrower weight average molecular weight distribution and data molecular weight distribution, is more suitable for human body absorption, and has a wide range of applications in antithrombotic drugs prospect.

This application is more aimed at the process of extracting heparin sodium from pig small intestinal mucosa, a large amount of dermatan sulfate will be collected together with heparin, and my country is a big producer of heparin sodium, in the production process of heparin sodium, a large amount of DS rich will be produced. By-products, this part of by-products are generally treated as waste, causing resource waste, environmental pollution and other problems. This application provides a refining process for low molecular weight dermatan sulfate, which is a process for refining low molecular weight dermatan sulfate by an enzyme-ultrafiltration method, especially using heparin sodium by-product rich in dermatan sulfate as a raw material, which can be degraded by use The biological enzyme of dermatan sulfate--chondroitin sulfate B enzyme, combined with ultrafiltration method, has obtained an "enzyme-ultrafiltration method" comprehensive refining process, which has a small molecular weight, uniform molecular weight distribution, high purity, and is beneficial to human absorption. The low molecular weight dermatan sulfate effectively solves the problem of using hydrochloric acid, hydrogen peroxide and other chemical reagents to degrade dermatan sulfate. The amount of hydrochloric acid is large, the hydrolysis reaction progresses slowly, and the hydrolysis reaction is not easy to control, which is likely to cause excessive hydrolysis. Or the degree of hydrolysis is not enough, and the degradation parameters are not easy to control, the reaction stability and reproducibility are poor, resulting in low yield, uneven molecular weight distribution of degradation products, and uncontrollable curative effects.

In the overall process of this application, the combination of resin elution and ethanol precipitation process can effectively remove impurities such as nucleic acid, protein, heparin, etc., and ensure product purity; the enzymatic hydrolysis process through the treatment of chondroitin sulfate B enzyme can effectively reduce the molecular weight of the product; through ultrafiltration The process can effectively control the molecular weight of the product. The low-molecular-weight dermatan sulfate refining process provided in this application has a mild, stable, and controllable reaction, high process repeatability, certain economic benefits and environmental protection effects, and is suitable for industrial promotion and application.

The experimental results show that the low molecular weight dermatan sulfate refining process provided in this application can control the 260nm and 280nm light absorption of the reaction nucleic acid and protein concentration in the product below 0.06, and can control the impurity content of heparin impurities below 0.5% ( Can reflect the high purity from the side). After the product is hydrolyzed and ultrafiltered, the weight average molecular weight is controlled within the range of 4000～4700Da, the molecular weight distribution is narrow, the proportion of components with weight average molecular weight greater than 8000Da does not exceed 6.5%, and the proportion of components with molecular weight fragments less than 2000Da does not exceed 8.5%. In addition, the process can effectively retain the chondroitin sulfate components in the raw materials, and the enzymatic hydrolysis conditions can ensure that the molecular weight of the intermediate product is close to the finished product after groping and verification, the ultrafiltration loss is small, and the total product yield exceeds 40%.

Detailed ways

In order to further understand the present application, the preferred embodiments of the present application are described below in conjunction with examples, but it should be understood that these descriptions are only for further illustrating the features and advantages of the present application rather than limiting the patent requirements of the present application.

All the raw materials in this application have no special restrictions on their sources, and they can be purchased on the market or prepared according to conventional methods well known to those skilled in the art.

There is no particular limitation on the purity of all the raw materials in this application. This application preferably adopts the purity that is conventional in analytical purity, the medical field or its application field.

All noun expressions and abbreviations in this application belong to conventional noun expressions and abbreviations in the field. Each noun expression and abbreviation is clear and definite in its related application field. Those skilled in the art can clearly, accurately and uniquely based on the noun expressions and abbreviations Understand.

This application provides a low-molecular-weight dermatan sulfate, the number-average molecular weight of the low-molecular-weight dermatan sulfate is 2000-5000 Da;

The weight average molecular weight of the low molecular weight dermatan sulfate is 2500-6500 Da.

This application has no special restrictions on the definition of the weight average molecular weight, and the definition of the weight average molecular weight well known to those skilled in the art, namely Mw, can be selected by those skilled in the art according to the application situation, application requirements or product performance requirements. Adjusted, the weight average molecular weight of the low molecular weight dermatan sulfate described in the present application is 2500-6500 Da, preferably 3000-6000 Da, more preferably 3500-5500 Da, more preferably 4000-5000 Da, and specifically may be 3500-6000 Da.

This application has no special restrictions on the definition of the number average molecular weight, as long as the number average molecular weight is well known to those skilled in the art, that is, Mn. Those skilled in the art can make selections according to application conditions, application requirements or product performance requirements. Adjusted, the number average molecular weight of the low molecular weight dermatan sulfate described in this application is 2000-5000 Da, preferably 2200-4700 Da, more preferably 2500-4500 Da, more preferably 2700-4200 Da, more preferably 3000-4000 Da, more preferably 3200 to 3800 Da, specifically 3000 to 4500 Da.

In principle, this application does not specifically limit the specific molecular weight distribution of the low-molecular-weight dermatan sulfate. Those skilled in the art can make selections and adjustments according to production conditions, application requirements or quality requirements. This application is to ensure the high molecular weight of low-molecular-weight dermatan sulfate. Quality, narrow the width of the molecular weight distribution, which is more conducive to human body absorption. The specific molecular weight distribution of the weight average molecular weight of the low molecular weight dermatan sulfate is preferably:

Dermatan sulfate with a molecular weight of less than 2000 Da accounts for less than 10 wt% of the total low molecular weight dermatan sulfate;

Among them, the molecular weight limit may be preferably: dermatan sulfate with a molecular weight of less than 2100 Da, more preferably less than 2200 Da, accounts for less than 10 wt% of the total low molecular weight dermatan sulfate. Among them, the percentage limit may be preferably 9 wt% or less, more preferably 8 wt% or less, more preferably 7 wt% or less, more preferably 6 wt% or less, and more preferably 5 wt% or less.

Dermatan sulfate with a molecular weight greater than 8000 Da accounts for less than 12 wt% of the total low molecular weight dermatan sulfate;

Among them, the molecular weight limit may preferably be: dermatan sulfate with a molecular weight greater than 7800 Da, more preferably greater than 7600 Da accounts for 12wt% or less of the total low molecular weight dermatan sulfate; wherein the percentage limit may preferably be 11wt% or less, more preferably 10wt% % Or less, more preferably 9 wt% or less, more preferably 8 wt% or less, more preferably 7 wt% or less.

Dermatan sulfate with a molecular weight of 2000 to 8000 Da accounts for 78 wt% to 90 wt% of the total low molecular weight dermatan sulfate;

Among them, the molecular weight limit may preferably be: 2200-7600Da, more preferably 2100-7800Da, the dermatan sulfate accounts for 78wt%-90wt% of the total low molecular weight dermatan sulfate; wherein the percentage limit may preferably be 80wt%-88wt %, more preferably 82wt% to 86wt%.

In principle, this application has no special restrictions on other parameters of the low-molecular-weight dermatan sulfate. Those skilled in the art can make selections and adjustments according to production conditions, application requirements or quality requirements. This application is to ensure the high-quality of low-molecular-weight dermatan sulfate , Narrowing the width of molecular weight distribution, is more conducive to human body absorption, the specific rotation of the low molecular weight dermatan sulfate is preferably less than or equal to -35°, more preferably less than or equal to -40°, more preferably less than or equal to -42°, more preferably Less than or equal to -45°. The purity of the low molecular weight dermatan sulfate is 99% or more, more preferably 99.3% or more, and more preferably 99.5% or more. In this application, the lower the total content of heparin impurities, the higher the purity, and the purity of low molecular weight dermatan sulfate can be reflected by its numerical side.

This application also provides a refining process for the low-molecular-weight dermatan sulfate described in any one of the above, including the following steps:

1) After mixing the dermatan sulfate solution and chondroitin sulfate B enzyme, after enzymatic hydrolysis, the material liquid is obtained;

2) After adding a strong oxidant to the material liquid obtained in the above step to carry out the oxidation reaction, it is filtered to obtain the filtered material liquid;

3) After the filtered material liquid obtained in the above steps is circulated ultrafiltration, low molecular weight dermatan sulfate is obtained.

The preferred principles or specific preferred schemes such as the properties, structure and ratio of the materials in the preparation method described in this application and the aforementioned preferred principles or specific preferred schemes such as the properties, structure and ratio of low molecular weight dermatan sulfate, preferably one-to-one correspondence, in This will not be repeated one by one.

In this application, the dermatan sulfate solution and the chondroitin sulfate B enzyme are mixed first, and then subjected to enzymatic hydrolysis to obtain a feed solution.

In principle, this application has no special restrictions on the specific parameters of the dermatan sulfate. Those skilled in the art can select and adjust according to the production situation, application requirements or quality requirements. This application is to ensure the high quality of low molecular weight dermatan sulfate. The narrow molecular weight distribution width is more conducive to human absorption and ensures the stability and repeatability of the refining process. The weight average molecular weight of the dermatan sulfate can be 15000-45000Da, or 20000-43000Da, or 25000-40000Da, or 30,000 ~37000Da.

In principle, this application has no special restrictions on the specific solvent of the dermatan sulfate solution. Those skilled in the art can select and adjust according to the production situation, application requirements or quality requirements. This application is to ensure the high quality of low molecular weight dermatan sulfate. The narrowing of the molecular weight distribution width is more conducive to human body absorption and ensures the stability and repeatability of the refining process. The dermatan sulfate solution preferably includes a dermatan sulfate aqueous solution.

In principle, this application has no special restrictions on the specific parameters of the dermatan sulfate solution. Those skilled in the art can select and adjust according to the production situation, application requirements or quality requirements. This application is to ensure the high quality of low molecular weight dermatan sulfate. Narrowing the width of molecular weight distribution is more conducive to human absorption, ensuring the stability and repeatability of the refining process. The pH value of the dermatan sulfate solution is preferably 6.5-8.5, more preferably 6.7-8.3, more preferably 7.0-8.0, More preferably, it is 7.2 to 7.8. The mass concentration of the dermatan sulfate solution is preferably 8% to 12%, more preferably 8.5% to 11.5%, more preferably 9% to 11%, more preferably 9.5% to 10.5%.

In principle, this application does not specifically limit the source of the dermatan sulfate. Those skilled in the art can make selections and adjustments according to production conditions, application requirements or quality requirements. This application is capable of ensuring the high quality of low molecular weight dermatan sulfate. Narrowing the width of molecular weight distribution is more conducive to human body absorption, and on the basis of ensuring the stability and repeatability of the refining process, it also has greater environmental protection and economic benefits. The dermatan sulfate is particularly preferably heparin rich in dermatan sulfate The sodium by-product is obtained after preparation.

More preferably, the preparation process may specifically include the following steps:

A) Dissolve the DS-rich heparin sodium by-product and water to obtain the original solution;

B) After the stock solution obtained in the above steps is adsorbed by resin, it is eluted with sodium chloride solution to obtain the eluted stock solution;

C) After mixing the eluted stock solution obtained in the above steps with ethanol and standing, then drying to obtain dermatan sulfate;

This application first dissolves the DS-rich heparin sodium by-product and water to obtain the original solution.

In principle, this application has no special restrictions on the specific parameters of the stock solution. Those skilled in the art can select and adjust according to the production situation, application requirements or quality requirements. This application is to ensure the high quality of low molecular weight dermatan sulfate and narrow the molecular weight. The distribution width is more conducive to human body absorption. The mass concentration of the stock solution is preferably 8%-12%, more preferably 8.5%-11.5%, more preferably 9%-11%, and more preferably 9.5%-10.5%.

In this application, the stock solution obtained in the above steps is adsorbed by resin, and then eluted with sodium chloride solution to obtain the eluted stock solution.

In principle, this application does not specifically limit the specific parameters of the resin adsorption. Those skilled in the art can select and adjust according to the production situation, application requirements or quality requirements. This application is to ensure the high quality of low molecular weight dermatan sulfate. The width of the molecular weight distribution is more conducive to human body absorption. The resin adsorption temperature is preferably 50-70°C, more preferably 52-68°C, more preferably 55-65°C, and more preferably 58-63°C. The resin column used for resin adsorption preferably includes an anion resin column, more preferably a large pore anion resin column, and specifically may be an A98 resin column.

In principle, this application has no special restrictions on the specific parameters of the sodium chloride solution. Those skilled in the art can make selections and adjustments according to production conditions, application requirements or quality requirements. This application is to ensure the high quality of low molecular weight dermatan sulfate. Narrowing the width of the molecular weight distribution is more conducive to human absorption. The mass concentration of the sodium chloride solution is preferably 2%-9%, more preferably 3%-8%, and more preferably 4%-7%.

In principle, this application does not specifically limit the specific elution process. Those skilled in the art can make selections and adjustments according to production conditions, application requirements or quality requirements. This application is to ensure the high quality of low molecular weight dermatan sulfate. The molecular weight distribution width is more conducive to human body absorption. The elution includes eluting impurities first and then eluting resin (eluting dermatan sulfate adsorbed on resin). More specifically, the specific process of elution may be:

After the stock solution obtained in the above steps is adsorbed on the resin, the resin is eluted with a sodium chloride solution with a mass concentration of 2% to 3% (preferably 2.2% to 2.8%, more preferably 2.4% to 2.6%). 2 to 3 resin volumes) to remove impurities. Then use 7%-9% (preferably 7.2%-8.8%, more preferably 7.5%-85%, more preferably 7.8%-8.3%) mass concentration of sodium chloride solution to elute the resin (elution 3~ 4 resin volumes) to elute the adsorbed dermatan sulfate to obtain the eluted stock solution.

At the end of the application, the eluted stock solution obtained in the above steps and ethanol are mixed and allowed to stand, and then dried to obtain dermatan sulfate.

In principle, this application does not specifically limit the specific amount of ethanol. Those skilled in the art can make selections and adjustments according to production conditions, application requirements or quality requirements. This application ensures the high quality of low molecular weight dermatan sulfate and narrows the molecular weight. The distribution width is more conducive to human body absorption. The volume ratio of the ethanol to the eluted stock solution is preferably (1.8～2.4):1, more preferably (1.9～2.3):1, more preferably (2.0～2.2) ):1.

In principle, this application does not specifically limit the specific parameters of the mixing and standing. Those skilled in the art can select and adjust according to the production situation, application requirements or quality requirements. This application is to ensure the high quality of low molecular weight dermatan sulfate. Narrow molecular weight distribution width is more conducive to human absorption. The mixing and standing time is preferably 7-9 hours, more preferably 7.2-8.8 hours, more preferably 7.4-8.6 hours, more preferably 7.6-8.4 hours, more preferably It is 7.8 to 8.2 hours.

In principle, this application does not specifically limit the specific process of mixing and standing. Those skilled in the art can select and adjust according to the production situation, application requirements or quality requirements. This application is to ensure the high quality of low molecular weight dermatan sulfate. The narrow molecular weight distribution width is more conducive to human body absorption. The mixing and standing is preferably multiple times of mixing and standing, more preferably two times of mixing and standing, which may specifically include the following steps:

Mix the eluted stock solution obtained in the above steps and 0.3 to 0.4 volume multiple (more preferably 0.32 to 0.38, more preferably 0.34 to 0.36) ethanol and let stand for 2 to 3 hours (more preferably 2.2 to 2.8 hours, and more (Preferably 2.4 to 2.6 hours), extract this part of the supernatant into another material tank, add this part of the supernatant again to 1.5-2.0 volume multiples (more preferably 1.6-1.9, more preferably 1.7-1.8) The ethanol is mixed, and the precipitate is left standing for 5-6 hours again (more preferably 5.2-5.8 hours, more preferably 5.4-5.6 hours), and the part of the precipitate is collected.

In principle, this application has no special restrictions on the specific parameters of the drying. Those skilled in the art can select and adjust according to the production situation, application requirements or quality requirements. This application is to ensure the high quality of low molecular weight dermatan sulfate and narrow the molecular weight. The distribution width is more conducive to human body absorption, and the drying temperature is preferably 50-60°C, more preferably 52-58°C, and more preferably 54-56°C.

In principle, this application does not specifically limit the specific drying process. Those skilled in the art can make selections and adjustments according to production conditions, application requirements or quality requirements. This application ensures the high quality of low molecular weight dermatan sulfate and reduces the molecular weight. The distribution width is more conducive to human body absorption. The specific drying process can be:

After mixing and standing, the precipitate is collected and dried to remove ethanol to obtain dermatan sulfate.

This application does not have any special restrictions on the source of the chondroitin sulfate B enzyme, as long as the conventional source of chondroitin sulfate B enzyme is well known to those skilled in the art, and those skilled in the art can make selections according to production conditions, application requirements or quality requirements. It can be adjusted, either commercially available or produced according to conventional methods.

In principle, this application has no special restrictions on the specific parameters of the chondroitin sulfate B enzyme. Those skilled in the art can select and adjust according to the production situation, application requirements or quality requirements. This application is to ensure the high quality of low molecular weight dermatan sulfate. , Narrowing the width of molecular weight distribution, more conducive to human absorption, the acid chondroitin B enzyme concentration is preferably 8000-12000IU/mL, more preferably 8500-11500IU/mL, more preferably 9000-11000IU/mL, more preferably 9500～10500IU/mL.

In principle, this application does not specifically limit the specific dosage of the chondroitin sulfate B enzyme. Those skilled in the art can select and adjust according to the production situation, application requirements or quality requirements. This application is to ensure the high quality of low molecular weight dermatan sulfate. , Narrowing the width of molecular weight distribution, is more conducive to human absorption, the volume ratio of the chondroitin sulfate B enzyme to the dermatan sulfate solution is preferably 0.001% to 0.003%, more preferably 0.0012% to 0.0028%, more preferably 0.0015 % To 0.0025%, more preferably 0.0018% to 0.0022%.

In principle, this application has no special restrictions on the specific parameters of the enzymatic hydrolysis. Those skilled in the art can select and adjust according to the production situation, application requirements or quality requirements. This application is to ensure the high quality of low molecular weight dermatan sulfate. The molecular weight distribution width is more conducive to human absorption. The pH value of the enzymatic hydrolysis (the pH value of the dermatan sulfate solution) is preferably 6.5 to 8.5, more preferably 6.7 to 8.3, more preferably 7.0 to 8.0, and more preferably 7.2 to 7.8. The temperature of the enzymatic hydrolysis (the control temperature of the dermatan sulfate solution) is preferably 35 to 37°C, more preferably 35.2 to 36.8°C, more preferably 35.5 to 36.5°C, and more preferably 35.7 to 36.3°C. The enzymatic hydrolysis time is preferably 3 to 5 hours, more preferably 3.2 to 4.8 hours, more preferably 3.5 to 4.5 hours, and more preferably 3.7 to 4.3 hours.

The application subsequently adds a strong oxidant to the material liquid obtained in the above steps to carry out the oxidation reaction, and then filters the material liquid to obtain the filtered material liquid.

In principle, this application does not specifically limit the specific selection and dosage of the strong oxidant. Those skilled in the art can select and adjust according to the production situation, application requirements or quality requirements. This application is to ensure the high quality of low molecular weight dermatan sulfate. Narrowing the molecular weight distribution width is more conducive to human body absorption. The strong oxidant preferably includes one or more of potassium permanganate, sodium permanganate, hydrogen peroxide and sodium hypochlorite, more preferably potassium permanganate and permanganese Sodium, hydrogen peroxide or sodium hypochlorite. The mass ratio of the strong oxidant in the feed liquid is preferably 2% to 5%, more preferably 2.5% to 4.5%, and more preferably 3% to 4%.

In principle, this application does not specifically limit the specific conditions of the oxidation reaction. Those skilled in the art can select and adjust according to the production situation, application requirements or quality requirements. This application is to ensure the high quality of low molecular weight dermatan sulfate. The width of the molecular weight distribution is more conducive to human absorption. The temperature of the oxidation reaction is preferably 80-90°C, more preferably 82-88°C, and more preferably 84-86°C. The time of the oxidation reaction is preferably 30 to 45 minutes, more preferably 32 to 43 minutes, and more preferably 35 to 40 minutes.

This application is to complete and refine the entire refining process, better ensure the high quality of low molecular weight dermatan sulfate, narrow the molecular weight distribution width, and be more conducive to human absorption. After the oxidation reaction, it also preferably includes a standing and cooling step and/or The impurity removal step is more preferably a standing cooling step and an impurity removal step. More specifically, it is more preferable to: stand the oxidized material liquid to stand and lower the temperature to below 50°C, filter and remove the impurity products after the oxidation of potassium permanganate, and then proceed to the subsequent steps. Use a filter membrane to filter and remove impurities.

In principle, this application has no special restrictions on the specific conditions of the filtration. Those skilled in the art can select and adjust according to the production situation, application requirements or quality requirements. This application is to ensure the high quality of low molecular weight dermatan sulfate and narrow the molecular weight. The distribution width is more conducive to human body absorption, and the filtration is preferably microfiltration membrane filtration. Further, the pore size of the microfiltration membrane is preferably 0.1 to 0.45 μm, more preferably 0.15 to 0.4 μm, more preferably 0.2 to 0.35 μm, more preferably 0.25 to 0.3 μm.

At the end of this application, the filtered material liquid obtained in the above steps is subjected to circulating ultrafiltration to obtain low molecular weight dermatan sulfate.

This application is to complete and refine the entire refining process, to better ensure the high quality of the low molecular weight dermatan sulfate, narrow the molecular weight distribution width, and is more conducive to human absorption. The cyclic ultrafiltration preferably further includes a dilution step. Specifically, the mass concentration of the diluted filtered feed liquid is preferably 3% to 5%, more preferably 3.2% to 4.8%, more preferably 3.5% to 4.5%, more preferably 3.7% to 4.2%.

In principle, this application has no special restrictions on the specific method of ultrafiltration. Those skilled in the art can select and adjust according to the production situation, application requirements or quality requirements. This application is to ensure the high quality of low molecular weight dermatan sulfate. The width of molecular weight distribution is more conducive to human body absorption, and the ultrafiltration is preferably tangential flow ultrafiltration.

In principle, this application does not specifically limit the specific steps of the cyclic ultrafiltration. Those skilled in the art can select and adjust according to the production situation, application requirements or quality requirements. This application is to ensure the high quality of low molecular weight dermatan sulfate. The narrow molecular weight distribution width is more conducive to human body absorption. The specific steps of the cyclic ultrafiltration are preferably:

After the first ultrafiltration membrane is used for the first cycle of ultrafiltration, the permeate is collected, and then the second ultrafiltration membrane is used for the second cycle of ultrafiltration, and then the retentate is collected.

In principle, this application does not specifically limit the specific parameters of the above-mentioned cyclic ultrafiltration. Those skilled in the art can select and adjust according to the production situation, application requirements or quality requirements. This application is to ensure the high quality of low molecular weight dermatan sulfate. The width of the molecular weight distribution is more conducive to human absorption. The filtration accuracy of the first ultrafiltration membrane is preferably 8000-12000 Da, more preferably 8500-11500 Da, more preferably 9000-11000 Da, and more preferably 9500-10500 Da. The number of times of the first cycle ultrafiltration is preferably 100 to 500 times, more preferably 150 to 450 times, more preferably 200 to 400 times, and more preferably 250 to 350 times. The filtration accuracy of the second ultrafiltration membrane is preferably 800 to 1200 Da, more preferably 850 to 1150 Da, more preferably 900 to 1100 Da, and more preferably 950 to 1050 Da. The number of times of the second cycle ultrafiltration is preferably 100 to 500 times, more preferably 150 to 450 times, more preferably 200 to 400 times, and more preferably 250 to 350 times.

This application specifically selects the membrane separation technology of tangential flow ultrafiltration (TFF), which is a kind of molecular weight level tangential flow filtration. The tangential flow filtration membrane can wash the membrane surface while filtering to keep the membrane surface Clean and maintain a stable filtration rate. This application uses tangential flow ultrafiltration technology, and more critically, uses specific parameters and combinations, which can separate and purify dermatan sulfate with uneven molecular weight distribution more quickly and efficiently.

This application is to complete and refine the entire refining process, to better ensure the high quality of the low molecular weight dermatan sulfate, narrow the molecular weight distribution width, and is more conducive to human absorption. The cyclic ultrafiltration preferably further includes a post-processing step.

In principle, this application does not specifically limit the specific steps of the above-mentioned post-processing. Those skilled in the art can select and adjust according to the production situation, application requirements or quality requirements. This application is to ensure the high quality of low molecular weight dermatan sulfate and narrow the molecular weight. The distribution width is more conducive to human body absorption. The post-processing steps preferably include one or more steps of dissolution, precipitation, re-dissolution, filtration, freeze-drying, grinding and mixing, and more preferably dissolution, precipitation, re-dissolution, filtration, and freezing. There are multiple steps in drying, grinding and mixing, more specifically, the following steps:

After dissolving the retentate and sodium chloride, add ethanol and let stand, discard the supernatant to obtain a precipitate;

After re-dissolving the precipitate obtained in the above steps, freeze-drying and grinding, the finished product low molecular weight dermatan sulfate is obtained.

In principle, this application does not specifically limit the specific parameters of the above-mentioned post-processing. Those skilled in the art can select and adjust according to the production situation, application requirements or quality requirements. This application ensures the high quality of low molecular weight dermatan sulfate and narrows the molecular weight. The distribution width is more conducive to human body absorption. The volume ratio of the sodium chloride to the retentate is preferably 2% to 4%, more preferably 2.2% to 3.8%, more preferably 2.5% to 3.5%, more preferably 2.7 %～3.2%. The volume ratio of the ethanol to the dissolved material is preferably (2 to 3):1, more preferably (2.2 to 2.8):1, and more preferably (2.4 to 2.6):1. The standing time is preferably 6-8h, more preferably 6.2-7.8h, more preferably 6.5-7.5h, more preferably 6.8-7.2h.

This application does not have any special restrictions on the specific steps and parameters of the above-mentioned re-dissolution, freeze-drying and milling, and the conventional steps and parameters corresponding to the above-mentioned process in the preparation process of this type of product are well known to those skilled in the art. It can be selected and adjusted according to production conditions, application requirements or quality requirements.

This application is to complete and refine the entire refining process, to better ensure the high quality of low molecular weight dermatan sulfate, narrow the molecular weight distribution width, and is more conducive to human absorption. The above refining process can specifically include the following steps:

A. Dissolve the DS-rich heparin sodium by-product with water to obtain the dissolved material liquid;

B. Pass the material liquid described in A through a large pore anion resin column, and control the temperature of the material liquid to about 50-70°C for resin adsorption. First eluting with the first mass concentration of sodium chloride solution (2 to 3 resin volumes) to remove impurities; then eluting with the second mass concentration of sodium chloride solution (3 to 4 resin volumes) , Collect the salinity eluted feed solution.

C. Add ethanol to the material liquid collected in B for precipitation, transfer the precipitation supernatant to another material tank, add ethanol for precipitation, discard the supernatant, and collect the precipitate;

D. Dry the precipitate collected in C to remove the ethanol. Then add water to dissolve, and dissolve into material liquid;

E. Adjust the pH of the material liquid obtained in D to 6.5-8.5, control the temperature to 35-37°C, and add chondroitin sulfate B enzyme for enzymatic hydrolysis.

F. Raise the temperature of the material liquid described in E, add potassium permanganate, continue to stir, and oxidize; leave the material liquid to stand still after oxidation to cool down, filter to remove impurity products after oxidation of potassium permanganate, and then filter with a filter membrane In addition to miscellaneous.

G. Dilute the filtered material in F, use the first membrane to perform cyclic ultrafiltration, and collect the permeate. The collected permeate is then subjected to cyclic ultrafiltration using the second filter membrane, the retentate is collected, and the molecular weight is controlled during sampling, and the weight average molecular weight is controlled to be 3000-5000 Da;

H. Add sodium chloride to the retentate collected in G to dissolve it, add ethanol, stir evenly, and then set it aside.

I. Discard the supernatant precipitated in H, dissolve the precipitate, dissolve the precipitate, filter, freeze-dry, grind, mix, and package.

The process for purifying low-molecular-weight dermatan sulfate provided by the enzyme-ultrafiltration method uses a large-pore anion resin column to purify and treat dermatan sulfate, chondroitin sulfate B enzyme to degrade dermatan sulfate, and uses tangential flow ultrafiltration technology The special combination of adjusting the molecular weight of low-molecular-weight dermatan sulfate resulted in an overall refining process. Among them, resin elution is used to purify dermatan sulfate, and dermatan sulfate is purified by adsorption and elution using a large-pore anion resin column (especially A98 resin). It is possible to remove a large amount of impurities in dermatan sulfate before enzymatic hydrolysis treatment; and use ethanol precipitation to purify dermatan sulfate. By adding a certain volume of ethanol, the dermatan sulfate is further purified; then it is degraded by chondroitin sulfate B enzyme, and the purified chondroitin sulfate is degraded into small molecules through the degradation of chondroitin sulfate B enzyme. Traditional degradation methods such as hydrochloric acid and hydrogen peroxide are used. The patented method has high degradation efficiency, stable and controllable treatment method, good repeatability, and high yield of low-molecular-weight dermatan sulfate. In addition, the degradation conditions of the patented method are mild to protect the integrity of the low-molecular sulfate dermatan sulfate structure as much as possible; at the same time, potassium permanganate oxidation is used to further purify the low-molecular-weight dermatan sulfate. Perform oxidation removal and modify the end structure of the prepared low-molecular-weight dermatan sulfate to stabilize the structure of the prepared low-molecular-weight dermatan sulfate; and use tangential flow ultrafiltration technology to control the molecular weight of low-molecular-weight dermatan sulfate. Parametric tangential flow ultrafiltration technology can control and adjust the molecular weight of low-molecular-weight dermatan sulfate to a level of 4KD suitable for human absorption.

Compared with the traditional preparation process of low-molecular-weight dermatan sulfate, the low-molecular-weight dermatan sulfate refining process provided in this application has mild, stable, and controllable process reaction conditions, high process repeatability, and high purity of the prepared low-molecular-weight dermatan sulfate product , Narrow molecular weight distribution, more suitable for human body absorption.

The application also provides the application of the low-molecular-weight dermatan sulfate described in any one of the above technical solutions or the low-molecular-weight dermatan sulfate refined by the refined process described in any one of the above technical solutions in the preparation of antithrombotic drugs.

This application does not specifically limit the specific application mode of the above-mentioned low molecular weight dermatan sulfate in the preparation of antithrombotic drugs, as long as the conventional application mode of such products well known to those skilled in the art can be used according to the production situation and application. Selection and adjustment of demand or quality requirements.

The above steps of the present application provide a process and application of low-molecular-weight dermatan sulfate and enzyme-ultrafiltration method for refining low-molecular-weight dermatan sulfate. The refined low-molecular-weight dermatan sulfate obtained in this application has a narrower weight average molecular weight distribution and data molecular weight distribution, is more suitable for human body absorption, and has broad application prospects in antithrombotic drugs.

The refining process of low molecular weight dermatan sulfate provided in this application is a process for refining low molecular weight dermatan sulfate by an enzyme-ultrafiltration method, especially using heparin sodium by-products rich in dermatan sulfate as raw materials, which can degrade skin sulfate The bio-enzyme-chondroitin sulfate B enzyme, combined with the ultrafiltration method, obtains the "enzyme-ultrafiltration method" comprehensive purification process, and prepares low molecular weights with small molecular weight, uniform molecular weight distribution, high purity, and good absorption by the human body. Dermatan sulfate effectively solves the problem of using hydrochloric acid, hydrogen peroxide and other chemical reagents to degrade dermatan sulfate. The amount of hydrochloric acid is large, the hydrolysis reaction progresses slowly, and the hydrolysis reaction is not easy to control, which is likely to cause excessive hydrolysis or degree of hydrolysis. Insufficient, and the degradation parameters are not easy to control, the reaction stability and reproducibility are poor, resulting in low yield, uneven molecular weight distribution of degradation products, and uncontrollable curative effects.

In the overall process of this application, the combination of resin elution and ethanol precipitation process can effectively remove impurities such as nucleic acid, protein, heparin, etc., and ensure product purity; the enzymatic hydrolysis process through the treatment of chondroitin sulfate B enzyme can effectively reduce the molecular weight of the product; through ultrafiltration The process can effectively control the molecular weight of the product. The low-molecular-weight dermatan sulfate refining process provided in this application has a mild, stable, and controllable reaction, high process repeatability, certain economic benefits and environmental protection effects, and is suitable for industrial promotion and application.

The experimental results show that the low molecular weight dermatan sulfate refining process provided in this application can control the 260nm and 280nm light absorption of the reaction nucleic acid and protein concentration in the product below 0.06, and can control the impurity content of heparin impurities below 0.5% ( Can reflect the high purity from the side). After the product is hydrolyzed and ultrafiltered, the weight average molecular weight is controlled within the range of 4000～4700Da, the molecular weight distribution is narrow, the proportion of components with weight average molecular weight greater than 8000Da does not exceed 6.5%, and the proportion of components with molecular weight fragments less than 2000Da does not exceed 8.5%. In addition, the process can effectively retain the chondroitin sulfate components in the raw materials, and the enzymatic hydrolysis conditions can ensure that the molecular weight of the intermediate product is close to the finished product after groping and verification, the ultrafiltration loss is small, and the total product yield exceeds 40%.

In order to further illustrate the present application, a low molecular weight dermatan sulfate provided by the present application and its purification process and application will be described in detail below in conjunction with examples. However, it should be understood that these examples are implemented on the premise of the technical solution of the present application. The detailed implementation and specific operation procedures are given to further illustrate the features and advantages of the application, rather than to limit the claims of the application, and the scope of protection of the application is not limited to the following embodiments.

Example 1

Weigh 1Kg of heparin sodium by-product and add 10L of water to dissolve it into a material solution with a concentration of about 10%. The material solution is heated to 60°C and slowly added to a resin column of about 10L resin for resin adsorption; after the adsorption is complete, use 25L of sodium chloride solution with a concentration of 3.5%, adjust the temperature to 60°C for elution, discard the eluate; then use 30L of a sodium chloride solution with a concentration of 8% (adjust the temperature to 60°C) for elution, Collect this part of the eluate to tank 1#; after the elution is completed, accurately measure the volume of the collected eluate to be 32L, add 12.8L ethanol, stir evenly, let stand for 3h, and transfer the supernatant suspension to tank 2# Medium (1# tank bottom sediment is heparinoid), add again 64L ethanol to 2# tank, stir well, let stand for 6 hours, discard the supernatant, collect the sediment on the bottom of tank 2#, and settle this part The product was dried at 55°C to obtain a pure DS product with a weighed weight of 712g.

Dissolve the pure DS with 7.0L of water and dissolve it into a material solution with a concentration of about 10%, adjust the pH to 8.0, control the temperature to 36℃, and add 0.07mL chondroitin sulfate B enzyme (enzyme activity of 10000IU/mL). ), the enzymatic hydrolysis is carried out for 4h. Raise the temperature of the material liquid to 80°C, add 21.3g of potassium permanganate, continue stirring, and oxidize for 45 minutes; after oxidation, the material liquid is allowed to stand and cool to below 50°C, and the potassium permanganate is removed with a plate and frame filter with filter paper. The impurity products after oxidation are filtered and removed with a 0.22um filter membrane. The volume of the collected material liquid is about 8L, 6L of purified water is added again, and the 10KD ultrafiltration membrane package is used for circulating ultrafiltration, and the permeate is collected (maintaining the outlet pressure of 0.1Mpa). Then the collected permeate is circulated ultrafiltration using a 1KD filter membrane, the retentate is collected, and the molecular weight is controlled in the sampling to control the molecular weight distribution, and the molecular weight distribution of the weight average molecular weight is controlled to be 3000-5000 Da.

The volume of the tested material is 10L, add 300g of sodium chloride to dissolve, then add 25L of ethanol, stir well, let it stand for 8h, discard the supernatant, dissolve the precipitate, filter, freeze-dry, grind, mix, and collect after freeze-drying 447g of low-molecular-weight dermatan sulfate boutique.

The low-molecular-weight dermatan sulfate product (LMWDS) prepared in Example 1 of the present application was tested.

See Table 1. Table 1 is the relevant detection data of LMWDS prepared in the examples of this application.

Table 1

The test results in Table 1 show that the low-molecular-weight heparins prepared by the refining scheme provided in this application have reliable quality and high purity.

Example 2

Weigh 50Kg of heparin sodium by-product and add 500L of water to dissolve it into a material solution with a concentration of about 10%. The material solution is heated to 60°C and slowly added to a resin column of about 500L resin for resin adsorption; after the adsorption is complete, use 1250L of sodium chloride solution with a concentration of 3.5%, adjust the temperature to 60°C for elution, discard the eluent; then use 1500L of a sodium chloride solution with a concentration of 8% (adjust the temperature to 60°C) for elution, Collect this part of the eluate to tank 5#; after the elution is completed, accurately measure the volume of the collected eluate to be 1600L, add 640L of ethanol, stir well, let stand for 3h, and transfer the supernatant suspension to tank 6# (5# tank bottom sediment is heparinoid), add 3000L ethanol to 6# tank again, stir well, let stand for 8 hours, discard the supernatant, collect the sediment at the bottom of 6# tank, and this part of the sediment Dry at 55°C to obtain a pure DS product with a weighed weight of 37.5Kg.

Dissolve the pure DS product with 370L of water and dissolve it into a material solution with a concentration of about 10%, adjust the pH to 8.0, control the temperature to 36°C, and add 3.7mL chondroitin sulfate B enzyme (enzyme activity of 10000IU/mL) to the material solution. Carry out enzymatic hydrolysis for 4h. Raise the temperature of the material liquid to 80°C, add 1.125Kg potassium permanganate, continue to stir, and oxidize for 45 minutes; after oxidation, the material liquid is allowed to stand and cool to below 50°C, and the potassium permanganate is removed with a plate and frame filter with filter paper. The impurity products after oxidation are filtered and removed with a 0.22um filter membrane. The volume of the collected material liquid is about 400L, 400L of purified water is added again, and the 10KD ultrafiltration membrane package is used for circulating ultrafiltration, and the permeate is collected (maintaining the outlet pressure of 0.1Mpa). Then the collected permeate is circulated ultrafiltration using a 1KD filter membrane, the retentate is collected, and the molecular weight is controlled during sampling to control the molecular weight distribution to control the molecular weight distribution to 3000-5000 Da. The volume of the tested material is 700L, add 21Kg of sodium chloride to dissolve, then add 1750L of ethanol, stir evenly, let it stand for 8h, discard the supernatant, dissolve the precipitate, filter, freeze-dry, grind, mix, and collect after freeze-drying The low-molecular-weight dermatan sulfate boutique is 24.2Kg.

The low-molecular-weight dermatan sulfate product (LMWDS) prepared in Example 2 of the present application was tested.

See Table 1. Table 1 is related detection data of LMWDS prepared in the examples of this application.

The test results in Table 1 show that the low-molecular-weight heparins prepared by the refining scheme provided in this application have reliable quality and high purity.

Example 3

Weigh 100Kg of heparin sodium by-product and add 1000L of water to dissolve it into a material solution with a concentration of about 10%. The material solution is heated to 60°C and slowly added to a resin column of about 1000L resin for resin adsorption; after the adsorption is completed, use 2500L of 3.5% sodium chloride solution, adjust the temperature to 60°C for elution, discard the eluate; then use 3000L of 8% sodium chloride solution (adjust the temperature to 60°C) for elution, Collect this part of the eluate to tank 5#; after the elution is completed, accurately measure the volume of the collected eluate to be 3200L, add 1280L ethanol, stir well, let stand for 3h, and transfer the supernatant suspension to tank 6# (5# tank bottom sediment is heparinoid), add 6000L ethanol to 6# tank again, stir well, let stand for 8 hours, discard the supernatant, collect the sediment at the bottom of tank 6#, and this part of the sediment Dry at 55°C to obtain a pure DS product, weighing 75.3Kg.

Dissolve the pure product of DS with 753L of water and dissolve it into a material solution with a concentration of about 10%, adjust the pH to 8.0, control the temperature to 36°C, and add 7.5mL chondroitin sulfate B enzyme (enzyme activity of 10000IU/mL) to the material solution. Carry out enzymatic hydrolysis for 4h. Raise the temperature of the material liquid to 80°C, add 2.25Kg potassium permanganate, continue to stir, and oxidize for 45 minutes; after oxidation, the material liquid is allowed to stand and cool to below 50°C, and the potassium permanganate is removed with a plate and frame filter with filter paper. The impurity products after oxidation are filtered and removed with a 0.22um filter membrane. The volume of the collected material liquid is about 800L, 600L of purified water is added again, and the 10KD ultrafiltration membrane package is used for circulating ultrafiltration, and the permeate is collected (maintaining the outlet pressure of 0.1Mpa). Then the collected permeate is circulated ultrafiltration using a 1KD filter membrane, the retentate is collected, and the molecular weight is controlled during sampling to control the molecular weight distribution to control the molecular weight distribution to 3000-5000 Da. The volume of the tested material is 1500L, add 45Kg of sodium chloride to dissolve, then add 3500L of ethanol, stir well, let it stand for 8h, discard the supernatant, dissolve the precipitate, filter, freeze-dry, grind, mix, and collect after freeze-drying The low-molecular-weight dermatan sulfate boutique 46.4Kg.

The low-molecular-weight dermatan sulfate product (LMWDS) prepared in Example 2 of the present application was tested.

See Table 1. Table 1 is the relevant detection data of LMWDS prepared in the examples of this application.

The test results in Table 1 show that the low-molecular-weight heparins prepared by the refining scheme provided in this application have reliable quality and high purity.

The above provides a detailed introduction to the low-molecular-weight dermatan sulfate provided by the present application and the process and application of the enzyme-ultrafiltration method for refining low-molecular-weight dermatan sulfate. In this article, specific examples are used to describe the principles and implementations of the present application. To elaborate, the descriptions of the above embodiments are only used to help understand the method and core ideas of the application, including the best way, and also enable any person skilled in the art to practice the application, including manufacturing and using any device or system, And implement any combination of methods. It should be pointed out that for those of ordinary skill in the art, without departing from the principles of this application, several improvements and modifications can be made to this application, and these improvements and modifications also fall within the protection scope of the claims of this application. The scope of patent protection in this application is defined by the claims, and may include other embodiments that can be thought of by those skilled in the art. If these other embodiments have structural elements that are not different from the literal expression of the claims, or if they include equivalent structural elements that are not substantially different from the literal expression of the claims, then these other embodiments should also be included in the scope of the claims.

Claims (14)

1. A low molecular weight dermatan sulfate, characterized in that the number average molecular weight of the low molecular weight dermatan sulfate is 2000-5000 Da;

   The weight average molecular weight of the low molecular weight dermatan sulfate is 2500-6500 Da.
2. The low molecular weight dermatan sulfate according to claim 1, wherein the molecular weight distribution of the weight average molecular weight of the low molecular weight dermatan sulfate is:

   Dermatan sulfate with a molecular weight of less than 2000 Da accounts for less than 10 wt% of the total low molecular weight dermatan sulfate;

   Dermatan sulfate with a molecular weight greater than 8000 Da accounts for less than 12 wt% of the total low molecular weight dermatan sulfate;

   Dermatan sulfate with a molecular weight of 2000-8000 Da accounts for 78 wt% to 90 wt% of the total low molecular weight dermatan sulfate.
3. The low-molecular-weight dermatan sulfate of claim 1, wherein the weight-average molecular weight of the low-molecular-weight dermatan sulfate is 3500-6000 Da;

   The number average molecular weight of the low molecular weight dermatan sulfate is 3000 to 4500 Da;

   The specific rotation of the low molecular weight dermatan sulfate is less than or equal to -40°;

   The purity of the low molecular weight dermatan sulfate is more than 99%.
4. A refining process for low molecular weight dermatan sulfate, which is characterized in that it comprises the following steps:

   1) After mixing the dermatan sulfate solution and chondroitin sulfate B enzyme, after enzymatic hydrolysis, the material liquid is obtained;

   2) After adding a strong oxidant to the material liquid obtained in the above step to carry out the oxidation reaction, it is filtered to obtain the filtered material liquid;

   3) After the filtered material liquid obtained in the above steps is circulated ultrafiltration, low molecular weight dermatan sulfate is obtained.
5. The refining process according to claim 4, wherein the dermatan sulfate solution comprises an aqueous solution of dermatan sulfate;

   The pH of the dermatan sulfate solution is 6.5 to 8.5;

   The mass concentration of the dermatan sulfate solution is 8%-12%;

   The weight average molecular weight of the dermatan sulfate is 15000-45000 Da;

   The volume ratio of the chondroitin sulfate B enzyme to the dermatan sulfate solution is 0.001% to 0.003%;

   The concentration of the chondroitin sulfate B enzyme is 8000-12000 IU/mL;

   The temperature of the enzymatic hydrolysis is 35-37°C;

   The enzymolysis time is 3 to 5 hours.
6. The refining process according to claim 4, wherein the strong oxidant comprises one or more of potassium permanganate, sodium permanganate, hydrogen peroxide and sodium hypochlorite;

   The mass ratio of the strong oxidant to the material liquid is 2% to 5%;

   The temperature of the oxidation reaction is 80-90°C;

   The oxidation reaction time is 30 to 45 minutes;

   The oxidation reaction further includes a step of standing and cooling and/or a step of removing impurities;

   The filtration is microfiltration membrane filtration;

   The pore size of the microfiltration membrane is 0.1-0.45 μm.
7. The refining process according to claim 4, characterized in that, before the circulating ultrafiltration, a dilution step is further included; the mass concentration of the diluted filtered feed liquid is 3% to 5%;
8. The refining process according to claim 4, wherein the ultrafiltration is tangential flow ultrafiltration;

   The specific steps of the cyclic ultrafiltration are:

   After the first ultrafiltration membrane is used for the first cycle of ultrafiltration, the permeate is collected, and then the second ultrafiltration membrane is used for the second cycle of ultrafiltration, and then the retentate is collected;

   The filtration accuracy of the first ultrafiltration membrane is 8000 to 12000 Da;

   The number of times of the first cycle of ultrafiltration is 100 to 500 times;

   The filtration accuracy of the second ultrafiltration membrane is 800-1200 Da;

   The number of times of the second cycle ultrafiltration is 100 to 500 times.
9. The refining process according to claim 4, characterized in that, after the cyclic ultrafiltration, a post-treatment step is further included;

   The post-treatment step includes one or more steps of dissolution, precipitation, re-dissolution, filtration, freeze-drying, grinding and mixing;

   The post-processing steps are specifically:

   After dissolving the retentate obtained after circulating ultrafiltration and sodium chloride, add ethanol and let stand, then discard the supernatant to obtain a precipitate;

   After re-dissolving the precipitate obtained in the above steps, freeze-drying and grinding, the finished product low molecular weight dermatan sulfate is obtained;

   The volume ratio of the sodium chloride to the retentate is 2% to 4%;

   The volume ratio of the ethanol to the dissolved material is (2～3):1;

   The standing time is 6-8h.
10. The refining process according to any one of claims 4-9, wherein the dermatan sulfate is obtained from the by-product of heparin sodium rich in dermatan sulfate;

    The preparation process is:

    A) Dissolve the by-product of heparin sodium rich in dermatan sulfate and water to obtain the original solution;

    B) After the stock solution obtained in the above steps is adsorbed by resin, it is eluted with sodium chloride solution to obtain the eluted stock solution;

    C) After mixing the eluted stock solution obtained in the above steps with ethanol and standing, then drying to obtain dermatan sulfate.
11. The refining process according to claim 10, wherein the mass concentration of the stock solution is 8%-12%;

    The temperature of the resin adsorption is 50-70°C;

    The resin column adsorbed by the resin is an anion resin column;

    The mass concentration of the sodium chloride solution is preferably 2%-9%;

    The elution includes eluting impurities first and then eluting the resin.
12. The refining process according to claim 10, wherein the volume ratio of the ethanol to the eluted stock solution is preferably (1.8-2.4):1;

    The mixing and standing time is preferably 7-9 hours;

    The mixing and standing is preferably two mixing and standing;

    The drying temperature is preferably 50-60°C.
13. The refining process according to claim 4, wherein the refining process specifically includes the following steps:

    (1) Add water to dissolve the by-product of heparin sodium rich in dermatan sulfate to obtain a dissolved material liquid;

    (2) Pass the feed solution described in (1) through an anion resin column, and control the temperature of the feed solution to about 50-70°C for resin adsorption; firstly, elution is carried out with a sodium chloride solution of the first mass concentration to remove Impurities; then eluted with a second mass concentration of sodium chloride solution to collect the salinity eluted material liquid;

    (3) Add ethanol to the material liquid collected in (2) for precipitation and place, transfer the precipitation supernatant to another material tank, add ethanol for precipitation and place, discard the supernatant, and collect the precipitate;

    (4) Dry the sediment collected in (3) to remove the ethanol; then add water to dissolve it and dissolve it into a material liquid;

    (5) Adjust the pH of the material liquid obtained in (4) to 6.5-8.5, control the temperature to 35-37°C, and add chondroitin sulfate B enzyme for enzymatic hydrolysis;

    (6) Raise the temperature of the material liquid described in (5), add potassium permanganate, continue to stir, and oxidize; leave the material liquid to stand still after oxidation to cool down, filter to remove impurity products after oxidation of potassium permanganate, and then filter Membrane filtration and impurity removal;

    (7) Dilute the filtered material in (6), use the first filter membrane for circulating ultrafiltration, and collect the permeate; then use the second filter membrane for the collected permeate for circulating ultrafiltration to collect the retentate, Control the molecular weight during sampling and control the weight-average molecular weight between 3000 Da and 5000 Da;

    (8) Add sodium chloride to dissolve the retentate collected in (7), add ethanol, and stir well, then set aside.

    (9) Discard the supernatant precipitated in (8), dissolve the precipitate, dissolve the precipitate, filter, freeze-dry, grind, mix, and package.
14. The use of the low-molecular-weight dermatan sulfate according to any one of claims 1 to 3 or the low-molecular-weight dermatan sulfate refined by the refining process of any one of claims 4-13 in the preparation of antithrombotic drugs.