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

Low molecular weight dermatan sulfate, refining process therefor and application thereof Download PDF

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Publication number
WO2021114941A1
WO2021114941A1 PCT/CN2020/125013 CN2020125013W WO2021114941A1 WO 2021114941 A1 WO2021114941 A1 WO 2021114941A1 CN 2020125013 W CN2020125013 W CN 2020125013W WO 2021114941 A1 WO2021114941 A1 WO 2021114941A1
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Prior art keywords
molecular weight
dermatan sulfate
low molecular
ultrafiltration
application
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PCT/CN2020/125013
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French (fr)
Chinese (zh)
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董磊
李玮涛
艾自明
孙守政
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东营天东制药有限公司
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Publication of WO2021114941A1 publication Critical patent/WO2021114941A1/en

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0069—Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates

Definitions

  • 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.
  • 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).
  • oa osteoarthritis
  • coronary heart disease coronary atherosclerotic 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.
  • 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.
  • 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.
  • LMWDS Low Molecular Weight Dermatan Sulfate
  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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.
  • 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 ⁇ m.
  • a dilution step is further included;
  • the mass concentration of the diluted filtered feed liquid is 3% to 5%;
  • the ultrafiltration is tangential flow 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.
  • 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:
  • 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.
  • the dermatan sulfate is obtained from the by-product of heparin sodium rich in dermatan sulfate;
  • the preparation process is:
  • 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.
  • 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.
  • the refining process specifically includes the following steps:
  • 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.
  • this application aims at natural dermatan sulfate, which is more difficult to extract and purify.
  • 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.
  • 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).
  • 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%.
  • 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%.
  • This application preferably adopts the purity that is conventional in analytical purity, the medical field or its application field.
  • 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.
  • 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.
  • 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.
  • 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;
  • 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.
  • 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;
  • 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;
  • 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%.
  • 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.
  • 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:
  • the dermatan sulfate solution and the chondroitin sulfate B enzyme are mixed first, and then subjected to enzymatic hydrolysis to obtain a feed solution.
  • 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.
  • the dermatan sulfate solution preferably includes a dermatan sulfate aqueous solution.
  • 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%.
  • 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.
  • the preparation process may specifically include the following steps:
  • This application first dissolves the DS-rich heparin sodium by-product and water to obtain the original solution.
  • 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%.
  • 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.
  • 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.
  • 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%.
  • 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:
  • 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.
  • 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
  • the eluted stock solution obtained in the above steps and ethanol are mixed and allowed to stand, and then dried to obtain dermatan sulfate.
  • 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.
  • 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.
  • 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:
  • 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.
  • 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:
  • 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.
  • 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%.
  • 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.
  • 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%.
  • 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.
  • 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.
  • 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 ⁇ m, more preferably 0.15 to 0.4 ⁇ m, more preferably 0.2 to 0.35 ⁇ m, more preferably 0.25 to 0.3 ⁇ m.
  • the filtered material liquid obtained in the above steps is subjected to circulating ultrafiltration to obtain low molecular weight dermatan sulfate.
  • the cyclic ultrafiltration preferably further includes a dilution step.
  • 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%.
  • 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.
  • 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:
  • the permeate is collected, and then the second ultrafiltration membrane is used for the second cycle of ultrafiltration, and then the retentate is collected.
  • 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.
  • THF tangential flow ultrafiltration
  • 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.
  • the cyclic ultrafiltration preferably further includes a post-processing step.
  • 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:
  • the finished product low molecular weight dermatan sulfate is obtained.
  • 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 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:
  • 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;
  • 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.
  • 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).
  • 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.
  • 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.
  • the low-molecular-weight dermatan sulfate refining process 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 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.
  • 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).
  • 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%.
  • 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%.
  • 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.
  • LMWDS low-molecular-weight dermatan sulfate product
  • Table 1 is the relevant detection data of LMWDS prepared in the examples of this application.
  • 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.
  • LMWDS low-molecular-weight dermatan sulfate product
  • Table 1 is related detection data of LMWDS prepared in the examples of this application.
  • 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
  • LMWDS low-molecular-weight dermatan sulfate product
  • Table 1 is the relevant detection data of LMWDS prepared in the examples of this application.

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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

低分子硫酸皮肤素及其精制工艺与应用Low-molecular-weight dermatan sulfate and its refined technology and application
LOW-MOLECULAR WEIGHT DERMATAN SULFATE AND REFINING PROCESS AND APPLICATION THEREOFLOW-MOLECULAR WEIGHT DERMATAN SULFATE AND REFINING PROCESS AND APPLICATION THEREOF
本申请要求在2019年12月11日提交中国专利局、申请号为201911267218.2、发明名称为“一种酶-超滤法精制低分子硫酸皮肤素的工艺、应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。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.
技术领域Technical field
本申请属于硫酸皮肤素技术领域，涉及一种低分子量硫酸皮肤素及其精制工艺、应用，尤其涉及一种低分子量硫酸皮肤素以及酶-超滤法精制低分子硫酸皮肤素的工艺、应用。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.
背景技术Background technique
硫酸皮肤素(dermatansulfate，简称DS)，是动物体内分布最广泛的一种细胞外基质糖胺多糖，为血管壁蛋白多糖的主要成份。硫酸皮肤素作为一种糖胺多糖，广泛分布于动物组织中。DS作为***的重要组成部分，具有多种药理作用与生理功能，可用作药物和保健食品，主要用于骨关节炎(oa)和冠状动脉粥样硬化性心脏病(冠心病)，其抗血栓作用与肝素相当甚至更强，而且DS的抗血栓作用不依赖血浆抗凝血酶III，而是通过肝素辅因子II催化抑制凝血酶，出血副作用比肝素小，目前已逐渐成为一种很有前途的新型抗血栓药物。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.
在硫酸皮肤素的来源上，可以直接使用天然的硫酸皮肤素，主要来源于动物组织提取，由于和动物体内其它糖胺聚糖、蛋白、核酸等组分混合，因此提取纯化难度较高。另外，天然DS相对分子质量一般在15000～45000Da之间，分子量大，不利于人体吸收，若直接用于人体，存在着两个主要的药动学问题:一是半衰期短，二是皮下和肌内注射时，其生物利用度低；且DS相对分子质量越高，其生物利用度就越低。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.
因而这就需要制备出分子量小、分子量分布窄的低分子硫酸皮肤素(Low Molecular Weight Dermatan Sulfate，简称LMWDS)，以提高DS的生物利用度并改善其药动学特征。LMWDS是一种通过化学、生物等手段将天然硫酸皮肤素降解制备的小分子聚合物，易被人体吸收，抗血栓活性高，人体利用度高。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.
目前有研究通过化学法和物理法降解DS以制备LMWDS，但制备过程中存在收率低和分子量分布不易控制等问题。如化学法降解DS，就是利用盐酸、过氧化氢等化学试剂将硫酸皮肤素降解。这两种方式都是通过用强酸或强氧化性化学试剂将硫酸皮肤素断链水解。硫酸皮肤素在盐酸溶液中水解，但使用盐酸量大，水解反应进行缓慢，并且水解反应 不易控，易造成水解过剧或水解程度不够。使用过氧化氢氧化降解硫酸皮肤素同样存在降解参数不易控制，反应稳定性及重复性差等问题，造成收率低，降解产物分子量分布不均一，疗效不可控等问题。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.
因此，如何找到一种适宜的硫酸皮肤素的降解方法，即一种低分子量硫酸皮肤素的精制工艺，解决现有的硫酸皮肤素降解工艺中存在的上述问题，已成业内研发型生产厂家和诸多一线研究人员亟待解决的问题之一。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.
发明内容Summary of the invention
有鉴于此，本申请基于以上所要解决的技术问题，提供一种低分子量硫酸皮肤素及其精制工艺、应用，特别是一种酶-超滤法精制低分子硫酸皮肤素的工艺。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.
本申请提供了一种低分子量硫酸皮肤素，所述低分子量硫酸皮肤素的数均分子量为2000～5000Da；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;
所述低分子量硫酸皮肤素的重均分子量为2500～6500Da。The weight average molecular weight of the low molecular weight dermatan sulfate is 2500-6500 Da.
优选的，所述低分子量硫酸皮肤素的重均分子量的分子量分布为：Preferably, the molecular weight distribution of the weight average molecular weight of the low molecular weight dermatan sulfate is:
分子量小于2000Da的硫酸皮肤素占所述低分子量硫酸皮肤素全部的10wt％以下；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;
分子量大于8000Da的硫酸皮肤素占所述低分子量硫酸皮肤素全部的12wt％以下；Dermatan sulfate with a molecular weight greater than 8000 Da accounts for less than 12 wt% of the total low molecular weight dermatan sulfate;
分子量为2000～8000Da的硫酸皮肤素占所述低分子量硫酸皮肤素全部的78wt％～90wt％。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.
优选的，所述低分子量硫酸皮肤素的重均分子量为3500～6000Da；Preferably, the weight average molecular weight of the low molecular weight dermatan sulfate is 3500-6000 Da;
所述低分子量硫酸皮肤素的数均分子量为3000～4500Da；The number average molecular weight of the low molecular weight dermatan sulfate is 3000 to 4500 Da;
所述低分子量硫酸皮肤素的比旋度小于等于-40°；The specific rotation of the low molecular weight dermatan sulfate is less than or equal to -40°;
所述低分子量硫酸皮肤素的纯度为99％以上。The purity of the low molecular weight dermatan sulfate is more than 99%.
本申请提供了一种低分子量硫酸皮肤素的精制工艺，包括以下步骤：This application provides a refining process for low molecular weight dermatan sulfate, which includes the following steps:
1)将硫酸皮肤素溶液和硫酸软骨素B酶混合后，进行酶解后，得到料液；1) After mixing the dermatan sulfate solution and chondroitin sulfate B enzyme, after enzymatic hydrolysis, the material liquid is obtained;
2)向上述步骤得到的料液，加入强氧化剂进行氧化反应后，再经过过滤，得到滤后料液；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)将上述步骤得到的滤后料液经过循环超滤后，得到低分子量硫酸皮肤素。3) After the filtered material liquid obtained in the above steps is circulated ultrafiltration, low molecular weight dermatan sulfate is obtained.
优选的，所述硫酸皮肤素溶液包括硫酸皮肤素水溶液；Preferably, the dermatan sulfate solution includes an aqueous solution of dermatan sulfate;
所述硫酸皮肤素溶液的pH值为6.5～8.5；The pH of the dermatan sulfate solution is 6.5 to 8.5;
所述硫酸皮肤素溶液的质量浓度为8％～12％；The mass concentration of the dermatan sulfate solution is 8%-12%;
所述硫酸皮肤素的重均分子量为15000～45000Da；The weight average molecular weight of the dermatan sulfate is 15000-45000 Da;
所述硫酸软骨素B酶与所述硫酸皮肤素溶液的体积比为0.001％～0.003％；The volume ratio of the chondroitin sulfate B enzyme to the dermatan sulfate solution is 0.001% to 0.003%;
所述硫酸软骨素B酶的浓度为8000～12000IU/mL；The concentration of the chondroitin sulfate B enzyme is 8000-12000 IU/mL;
所述酶解的温度为35～37℃；The temperature of the enzymatic hydrolysis is 35-37°C;
所述酶解的时间为3～5h。The enzymolysis time is 3 to 5 hours.
优选的，所述强氧化剂包括高锰酸钾、高锰酸钠、过氧化氢和次氯酸钠中的一种或多种；Preferably, the strong oxidant includes one or more of potassium permanganate, sodium permanganate, hydrogen peroxide and sodium hypochlorite;
所述强氧化剂占所述料液的质量比为2％～5％；The mass ratio of the strong oxidant to the material liquid is 2% to 5%;
所述氧化反应的温度为80～90℃；The temperature of the oxidation reaction is 80-90°C;
所述氧化反应的时间为30～45分钟；The oxidation reaction time is 30 to 45 minutes;
所述氧化反应后还包括静置降温步骤和/或除杂步骤；The oxidation reaction further includes a step of standing and cooling and/or a step of removing impurities;
所述过滤为微滤膜过滤；The filtration is microfiltration membrane filtration;
所述微滤膜的孔径为0.1～0.45μm。The pore size of the microfiltration membrane is 0.1-0.45 μm.
优选的，所述循环超滤前还包括稀释步骤；Preferably, before the circulating ultrafiltration, a dilution step is further included;
所述稀释的滤后料液的质量浓度为3％～5％；The mass concentration of the diluted filtered feed liquid is 3% to 5%;
优选的，所述超滤为切向流超滤；Preferably, the ultrafiltration is tangential flow ultrafiltration;
所述循环超滤的具体步骤为：The specific steps of the cyclic ultrafiltration are:
先采用第一超滤膜进行第一循环超滤后，收集透过液，再采用第二超滤膜进行第二循环超滤后，收集截留液；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;
所述第一超滤膜的过滤精度为8000～12000Da；The filtration accuracy of the first ultrafiltration membrane is 8000 to 12000 Da;
所述第一循环超滤的次数为100～500次；The number of times of the first cycle of ultrafiltration is 100 to 500 times;
所述第二超滤膜的过滤精度为800～1200Da；The filtration accuracy of the second ultrafiltration membrane is 800-1200 Da;
所述第二循环超滤的次数为100～500次。The number of times of the second cycle ultrafiltration is 100 to 500 times.
优选的，所述循环超滤后还包括后处理步骤；Preferably, after the cyclic ultrafiltration, a post-treatment step is further included;
所述后处理步骤包括溶解、沉淀、再溶解、过滤、冷冻干燥、研磨和混合中的一步或多步；The post-treatment step includes one or more steps of dissolution, precipitation, re-dissolution, filtration, freeze-drying, grinding and mixing;
所述后处理步骤具体为：The post-processing steps are specifically:
将循环超滤后得到的截留液和氯化钠溶解后，再加入乙醇静置后，舍弃上清液，得到沉淀；After dissolving the retentate obtained after circulating ultrafiltration and sodium chloride, add ethanol and let stand, then discard the supernatant to obtain a precipitate;
将上述步骤得到的沉淀再次溶解、冷冻干燥和磨粉后，得到成品低分子量硫酸皮肤素；After re-dissolving the precipitate obtained in the above steps, freeze-drying and grinding, the finished product low molecular weight dermatan sulfate is obtained;
所述氯化钠与所述截留液体积比为2％～4％；The volume ratio of the sodium chloride to the retentate is 2% to 4%;
所述乙醇与溶解后的物料体积比值为(2～3)：1；The volume ratio of the ethanol to the dissolved material is (2～3):1;
所述静置的时间为6～8h。The standing time is 6-8h.
优选的，所述硫酸皮肤素由富含硫酸皮肤素的肝素钠副产物经制备后得到；Preferably, the dermatan sulfate is obtained from the by-product of heparin sodium rich in dermatan sulfate;
所述制备过程为：The preparation process is:
A)将富含硫酸皮肤素的肝素钠副产物和水进行溶解，得到原液；A) Dissolve the by-product of heparin sodium rich in dermatan sulfate and water to obtain the original solution;
B)将上述步骤得到的原液经过树脂吸附后，再用氯化钠溶液洗脱，得到洗脱后的原液；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)将上述步骤得到的洗脱后的原液和乙醇混合静置后，再经过干燥，得到硫酸皮肤素；C) After mixing the eluted stock solution obtained in the above steps with ethanol and standing, then drying to obtain dermatan sulfate;
优选的，所述原液的质量浓度为8％～12％；Preferably, the mass concentration of the stock solution is 8%-12%;
所述树脂吸附的温度为50～70℃；The temperature of the resin adsorption is 50-70°C;
所述树脂吸附的树脂柱为阴离子树脂柱；The resin column adsorbed by the resin is an anion resin column;
所述氯化钠溶液的质量浓度优选为2％～9％；The mass concentration of the sodium chloride solution is preferably 2%-9%;
所述洗脱包括先洗脱杂质和再洗脱树脂。The elution includes eluting impurities first and then eluting the resin.
优选的，所述乙醇与所述洗脱后的原液的体积比优选为(1.8～2.4)：1；Preferably, the volume ratio of the ethanol to the eluted stock solution is preferably (1.8-2.4):1;
所述混合静置的时间优选为7～9小时；The mixing and standing time is preferably 7-9 hours;
所述混合静置优选为两次混合静置；The mixing and standing is preferably two mixing and standing;
所述干燥的温度优选为50～60℃。The drying temperature is preferably 50-60°C.
优选的，所述精制过程具体包括以下步骤：Preferably, the refining process specifically includes the following steps:
(1)将富含硫酸皮肤素的肝素钠副产物加水进行溶解，得到溶解后的料液；(1) Add water to dissolve the by-product of heparin sodium rich in dermatan sulfate to obtain a dissolved material liquid;
(2)将(1)中所述料液通过阴离子树脂柱，控制料液温度约50～70℃，进行树脂吸附；首先用第一质量浓度的氯化钠溶液进行洗脱，以洗脱去除杂质；再用第二质量浓度的氯化钠溶液洗脱，收集该盐度洗脱的料液；(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)将(2)中所收集的料液加入乙醇进行沉淀放置，将沉淀上清液转移至另一物料罐中，补加乙醇进行沉淀放置，弃取上清，收集沉淀物；(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)将(3)中收集沉淀物进行烘干，去除乙醇；然后加水进行溶解，溶解为料液；(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)将(4)中所得的料液调整pH为6.5～8.5，温度控制至35～37℃，加入硫酸软骨素B酶，进行酶解；(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)将(5)中所述料液升温，加入高锰酸钾，持续搅拌，进行氧化；将氧化后料液静置降温，过滤去除高锰酸钾氧化后的杂质产物，再用滤膜进行过滤除杂；(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)将(6)中过滤后料液稀释，使用第一滤膜进行循环超滤，收集透过液；再将收集的透过液使用第二滤膜进行循环超滤，收集截留液，取样中控检测分子量，控制重均分子量在3000～5000Da；(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)将(7)中收集的截留液，加入氯化钠进行溶解，加入乙醇，搅拌均匀后，放置。(8) Add sodium chloride to dissolve the retentate collected in (7), add ethanol, and stir well, then set aside.
(9)弃取(8)中沉淀的上清，溶解沉淀物，将沉淀物溶解，过滤、冷冻干燥、磨粉、混合、包装。(9) Discard the supernatant precipitated in (8), dissolve the precipitate, dissolve the precipitate, filter, freeze-dry, grind, mix, and package.
本申请提供了上述任意一项所述的低分子量硫酸皮肤素或上述任意一项所述的精制工艺所精制的低分子量硫酸皮肤素在制备抗血栓药物中的应用。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.
本申请提供了一种低分子量硫酸皮肤素，所述低分子量硫酸皮肤素的数均分子量为2000～5000Da；所述低分子量硫酸皮肤素的重均分子量为2500～6500Da。与现有技术相比，本申请针对天然硫酸皮肤素，提取纯化难度较高。而且天然DS相对分子质量一般在15000～45000Da之间，分子量大，不利于人体吸收，存在半衰期短以及皮下和肌内注射时，生物利用度低的问题。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.
本申请创造性的得到了一种低分子量硫酸皮肤素，该精制的低分子量硫酸皮肤素，具有较窄的重均分子量分布和数据分子量分布，更加适合人体吸收，在抗血栓药物中具有广泛的应用前景。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.
本申请更针对由猪小肠黏膜提取肝素钠的过程中，大量的硫酸皮肤素会和肝素一起被收集，而我国是肝素钠生产大国，在肝素钠在生产过程中，会产生大量富含DS的副产物，这部分副产物一般作为废弃物处理，造成资源浪费、环境污染等问题的现状。本申请提供了一种低分子量硫酸皮肤素的精制工艺，是一种酶-超滤法精制低分子硫酸皮肤素的工艺，特别以富含硫酸皮肤素的肝素钠副产物为原料，利用可以降解硫酸皮肤素的生物酶--硫酸软骨素B酶，结合超滤法，得到了一种“酶-超滤法”综合精制工艺，制备出了分子量小、分子量分布均一、纯度高、利于人体吸收的低分子硫酸皮肤素，有效的解决了现有的利用盐酸、过氧化氢等化学试剂降解硫酸皮肤素，存在的盐酸用量大，水解反应进行缓慢，并且水解反应不易控，易造成水解过剧或水解程度不够，以及降解参数不易控制，反应稳定性及重复性差，造成收率低，降解产物分子量分布不均一，疗效不可控等问题。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.
本申请整体工艺中，结合树脂洗脱和乙醇沉淀工艺可有效去除核酸、蛋白、类肝素等杂质，保证产品纯度；通过硫酸软骨素B酶处理的酶解工艺可有效降低产品分子量；通过超滤工艺可有效控制产品分子量。本申请提供的低分子量硫酸皮肤素精制工艺，反应温和、稳定、可控，工艺重复性高，具有一定的经济效益和环境保护作用，适于工业化推广和应用。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.
实验结果表明，本申请提供的低分子量硫酸皮肤素精制工艺，可以将产品中反应核酸、蛋白浓度的260nm、280nm光吸收控制在0.06以下，可以将肝素类杂质的杂质含量控制在0.5％以下(可侧面反映纯度高)。产品经过酶解和超滤后，重均分子量控制在 4000～4700Da范围内，分子量分布窄，重均分子量大于8000Da的组分占比不超过6.5％，分子量片段小于2000Da的组分占比不超过8.5％。另外，该工艺可有效保留原料中硫酸软骨素组分，酶解条件经摸索验证后能够保证中间产物的分子量与成品较接近，超滤损失少，产品总收率超过40％。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%.
具体实施方式Detailed ways
为了进一步了解本申请，下面结合实施例对本申请的优选实施方案进行描述，但是应当理解，这些描述只是为进一步说明本申请的特征和优点而不是对本申请专利要求的限制。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.
本申请所有原料，对其来源没有特别限制，在市场上购买的或按照本领域技术人员熟知的常规方法制备的即可。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.
本申请所有原料，对其纯度没有特别限制，本申请优选采用分析纯、医药领域或其应用领域常规的纯度。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.
本申请所有名词表达和简称均属于本领域常规名词表达和简称，每个名词表达和简称在其相关应用领域内均是清楚明确的，本领域技术人员根据名词表达和简称，能够清楚准确唯一的进行理解。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.
本申请提供了一种低分子量硫酸皮肤素，所述低分子量硫酸皮肤素的数均分子量为2000～5000Da；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;
所述低分子量硫酸皮肤素的重均分子量为2500～6500Da。The weight average molecular weight of the low molecular weight dermatan sulfate is 2500-6500 Da.
本申请对所述重均分子量的定义没有特别限制，以本领域技术人员熟知的重均分子量的定义即可，即Mw，本领域技术人员可以根据应用情况、应用需求或产品性能要求进行选择和调整，本申请所述低分子量硫酸皮肤素的重均分子量为2500～6500Da，优选为3000～6000Da，更优选为3500～5500Da，更优选为4000～5000Da，具体可以为3500～6000Da。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.
本申请对所述数均分子量的定义没有特别限制，以本领域技术人员熟知的数均分子量的定义即可，即Mn，本领域技术人员可以根据应用情况、应用需求或产品性能要求进行选择和调整，本申请所述低分子量硫酸皮肤素的数均分子量为2000～5000Da，优选为2200～4700Da，更优选为2500～4500Da，更优选为2700～4200Da，更优选为3000～4000Da，更优选为3200～3800Da，具体可以为3000～4500Da。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.
本申请原则上对所述低分子量硫酸皮肤素具体的分子量分布没有特别限制，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，本申请为保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，所述低分子量硫酸皮肤素的重均分子量的具体分子量分布优选为：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:
分子量小于2000Da的硫酸皮肤素占所述低分子量硫酸皮肤素全部的10wt％以下；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;
其中，分子量界限可以优选为：分子量小于2100Da，更优选为小于2200Da的硫酸皮肤素占所述低分子量硫酸皮肤素全部的10wt％以下。其中，百分比界限可以优选为9wt％以下，更优选为8wt％以下，更优选为7wt％以下，更优选为6wt％以下，更优选为5wt％以下。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.
分子量大于8000Da的硫酸皮肤素占所述低分子量硫酸皮肤素全部的12wt％以下；Dermatan sulfate with a molecular weight greater than 8000 Da accounts for less than 12 wt% of the total low molecular weight dermatan sulfate;
其中，分子量界限可以优选为：分子量大于7800Da，更优选为大于7600Da的硫酸皮肤素占所述低分子量硫酸皮肤素全部的12wt％以下；其中，百分比界限可以优选为11wt％以下，更优选为10wt％以下，更优选为9wt％以下，更优选为8wt％以下，更优选为7wt％以下。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.
分子量为2000～8000Da的硫酸皮肤素占所述低分子量硫酸皮肤素全部的78wt％～90wt％；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;
其中，分子量界限可以优选为：2200～7600Da，更优选为2100～7800Da，的硫酸皮肤素占所述低分子量硫酸皮肤素全部的78wt％～90wt％；其中，百分比界限可以优选为80wt％～88wt％，更优选为82wt％～86wt％。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%.
本申请原则上对所述低分子量硫酸皮肤素的其他参数没有特别限制，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，本申请为保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，所述低分子量硫酸皮肤素的比旋度优选小于等于-35°，更优选小于等于-40°，更优选为小于等于-42°，更优选为小于等于-45°。所述低分子量硫酸皮肤素的纯度为99％以上，更优选为99.3％以上，更优选为99.5％以上。在本申请中，肝素类杂质总含量越低则纯度越高，低分子量硫酸皮肤素的纯度可以通过其数值侧面得到反映。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.
本申请还提供了上述任一项所述的低分子量硫酸皮肤素的精制工艺，包括以下步骤：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)将硫酸皮肤素溶液和硫酸软骨素B酶混合后，进行酶解后，得到料液；1) After mixing the dermatan sulfate solution and chondroitin sulfate B enzyme, after enzymatic hydrolysis, the material liquid is obtained;
2)向上述步骤得到的料液，加入强氧化剂进行氧化反应后，再经过过滤，得到滤后料液；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)将上述步骤得到的滤后料液经过循环超滤后，得到低分子量硫酸皮肤素。3) After the filtered material liquid obtained in the above steps is circulated ultrafiltration, low molecular weight dermatan sulfate is obtained.
本申请所述制备方法中所述材料的性质、结构以及比例等优选原则或具体优选方案与前述低分子量硫酸皮肤素的性质、结构以及比例等优选原则或具体优选方案，优选一一对应，在此不再一一赘述。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.
本申请首先将硫酸皮肤素溶液和硫酸软骨素B酶混合后，进行酶解后，得到料液。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.
本申请原则上对所述硫酸皮肤素的具体参数没有特别限制，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，本申请为保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，保证精制工艺的稳定性和重复性，所述硫酸皮肤素的重均分子量可以为15000～45000Da，或者为20000～43000Da，或者为25000～40000Da，或者为30000～37000Da。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.
本申请原则上对所述硫酸皮肤素溶液的具体溶剂没有特别限制，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，本申请为保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，保证精制工艺的稳定性和重复性，所述硫酸皮肤素溶液优选包括硫酸皮肤素水溶液。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.
本申请原则上对所述硫酸皮肤素溶液的具体参数没有特别限制，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，本申请为保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，保证精制工艺的稳定性和重复性，所述硫酸皮肤素溶液的pH值优选为6.5～8.5，更优选为6.7～8.3，更优选为7.0～8.0，更优选为7.2～7.8。所述硫酸皮肤素溶液的质量浓度优选为8％～12％，更优选为8.5％～11.5％，更优选为9％～11％，更优选为9.5％～10.5％。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%.
本申请原则上对所述硫酸皮肤素的来源没有特别限制，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，本申请为能够在保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，保证精制工艺的稳定性和重复性的基础上，同时具有更大的环保意义和经济效益，所述硫酸皮肤素特别优选为富含硫酸皮肤素的肝素钠副产物经制备后得到。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.
更优选的，所述制备过程具体可以为以下步骤：More preferably, the preparation process may specifically include the following steps:
A)将富含DS的肝素钠副产物和水进行溶解，得到原液；A) Dissolve the DS-rich heparin sodium by-product and water to obtain the original solution;
B)将上述步骤得到的原液经过树脂吸附后，再用氯化钠溶液洗脱，得到洗脱后的原液；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)将上述步骤得到的洗脱后的原液和乙醇混合静置后，再经过干燥，得到硫酸皮肤素；C) After mixing the eluted stock solution obtained in the above steps with ethanol and standing, then drying to obtain dermatan sulfate;
本申请首先将富含DS的肝素钠副产物和水进行溶解，得到原液。This application first dissolves the DS-rich heparin sodium by-product and water to obtain the original solution.
本申请原则上对所述原液的具体参数没有特别限制，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，本申请为保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，所述原液的质量浓度优选为8％～12％，更优选为8.5％～11.5％，更优选为9％～11％，更优选为9.5％～10.5％。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%.
本申请再将上述步骤得到的原液经过树脂吸附后，再用氯化钠溶液洗脱，得到洗脱后的原液。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.
本申请原则上对所述树脂吸附的具体参数没有特别限制，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，本申请为保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，所述树脂吸附的温度优选为50～70℃，更优选为52～68℃，更优选为55～65℃，更优选为58～63℃。所述用于树脂吸附的树脂柱优选包括阴离子树脂柱，更优选为大孔径阴离子树脂柱，具体可以为A98树脂柱。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.
本申请原则上对所述氯化钠溶液的具体参数没有特别限制，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，本申请为保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，所述氯化钠溶液的质量浓度优选为2％～9％，更优选为3％～8％，更优选为4％～7％。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%.
本申请原则上对所述洗脱的具体过程没有特别限制，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，本申请为保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，所述洗脱包括先洗脱杂质和再洗脱树脂(洗脱树脂上吸附的硫酸皮肤素)。更具体的，所述洗脱的具体过程可以为：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:
将上述步骤得到的原液经过树脂吸附后，先使用2％～3％(优选为2.2％～2.8％，更优选为2.4％～2.6％)质量浓度的氯化钠溶液进行洗脱树脂(洗脱2～3个树脂体积)，以去除杂质。然后使用7％～9％(优选为7.2％～8.8％，更优选为7.5％～8.5％，更优选为7.8％～8.3％)质量浓度的氯化钠溶液进行洗脱树脂(洗脱3～4个树脂体积)，将吸附的硫酸皮肤素洗脱下来，得到洗脱后的原液。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.
本申请最后将上述步骤得到的洗脱后的原液和乙醇混合静置后，再经过干燥，得到硫酸皮肤素。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.
本申请原则上对所述乙醇的具体用量没有特别限制，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，本申请为保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，所述乙醇与所述洗脱后的原液的体积比优选为(1.8～2.4)：1，更优选为(1.9～2.3)：1，更优选为(2.0～2.2)：1。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.
本申请原则上对所述混合静置的具体参数没有特别限制，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，本申请为保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，所述混合静置的时间优选为7～9小时，更优选为7.2～8.8小时，更优选为7.4～8.6小时，更优选为7.6～8.4小时，更优选为7.8～8.2小时。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.
本申请原则上对所述混合静置的具体过程没有特别限制，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，本申请为保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，所述混合静置优选为多次混合静置，更优选为两次混合静置，具体可以为以下步骤：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:
将上述步骤得到的洗脱后的原液和0.3～0.4体积倍数(更优选为0.32～0.38，更优选为0.34～0.36)的乙醇混合静置2～3小时(更优选为2.2～2.8小时，更优选为2.4～2.6小时)后，抽取该部分的上清至另一物料罐中，将该部分上清液再次加入1.5～2.0体积倍数(更优选为1.6～1.9，更优选为1.7～1.8)的乙醇混合，再次静置沉淀5～6小时(更优选为5.2～5.8小时，更优选为5.4～5.6小时)，收集该部分沉淀物。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.
本申请原则上对所述干燥的具体参数没有特别限制，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，本申请为保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，所述干燥的温度优选为50～60℃，更优选为52～58℃，更优选为54～56℃。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.
本申请原则上对所述干燥的具体过程没有特别限制，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，本申请为保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，所述干燥的具体过程可以为：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:
将混合静置后收集沉淀物进行烘干，去除乙醇，得到硫酸皮肤素。After mixing and standing, the precipitate is collected and dried to remove ethanol to obtain dermatan sulfate.
本申请对所述硫酸软骨素B酶的来源没有特别限制，以本领域技术人员熟知的硫酸软骨素B酶常规来源即可，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，选择市售或者按照常规的方法生产得到均可。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.
本申请原则上对所述硫酸软骨素B酶的具体参数没有特别限制，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，本申请为保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，所述酸软骨素B酶的浓度优选为8000～12000IU/mL，更优选为8500～11500IU/mL，更优选为9000～11000IU/mL，更优选为9500～10500IU/mL。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.
本申请原则上对所述硫酸软骨素B酶的具体用量没有特别限制，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，本申请为保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，所述硫酸软骨素B酶与所述硫酸皮肤素溶液的体积比优选为0.001％～0.003％，更优选为0.0012％～0.0028％，更优选为0.0015％～0.0025％，更优选为0.0018％～0.0022％。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%.
本申请原则上对所述酶解的具体参数没有特别限制，本领域技术人员可以根据生产情 况、应用需求或质量要求进行选择和调整，本申请为保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，所述酶解的pH值(硫酸皮肤素溶液的pH值)优选为6.5～8.5，更优选为6.7～8.3，更优选为7.0～8.0，更优选为7.2～7.8。所述酶解的温度(硫酸皮肤素溶液的控制温度)优选为35～37℃，更优选为35.2～36.8℃，更优选为35.5～36.5℃，更优选为35.7～36.3℃。所述酶解的时间优选为3～5h，更优选为3.2～4.8h，更优选为3.5～4.5h，更优选为3.7～4.3h。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.
本申请随后向上述步骤得到的料液，加入强氧化剂进行氧化反应后，再经过过滤，得到滤后料液。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.
本申请原则上对所述强氧化剂的具体选择和用量没有特别限制，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，本申请为保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，所述强氧化剂优选包括高锰酸钾、高锰酸钠、过氧化氢和次氯酸钠中的一种或多种，更优选为高锰酸钾、高锰酸钠、过氧化氢或次氯酸钠。所述强氧化剂占所述料液的质量比优选为2％～5％，更优选为2.5％～4.5％，更优选为3％～4％。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%.
本申请原则上对所述氧化反应的具体条件没有特别限制，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，本申请为保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，所述氧化反应的温度优选为80～90℃，更优选为82～88℃，更优选为84～86℃。所述氧化反应的时间优选为30～45分钟，更优选为32～43分钟，更优选为35～40分钟。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.
本申请为完整和细化整个精制工艺，更好的保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，所述氧化反应后还优选包括静置降温步骤和/或除杂步骤，更优选为静置降温步骤和除杂步骤，更具体优选为：将氧化后料液静置降温至50℃以下，过滤去除高锰酸钾氧化后的杂质产物，之后再进行后续用滤膜进行过滤除杂的步骤。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.
本申请原则上对所述过滤的具体条件没有特别限制，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，本申请为保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，所述过滤优选为微滤膜过滤。进一步的，所述微滤膜的孔径优选为0.1～0.45μm，更优选为0.15～0.4μm，更优选为0.2～0.35μm，更优选为0.25～0.3μm。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.
本申请最后将上述步骤得到的滤后料液经过循环超滤后，得到低分子量硫酸皮肤素。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.
本申请为完整和细化整个精制工艺，更好的保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，所述循环超滤前优选还包括稀释步骤。具体的，所 述稀释的滤后料液的质量浓度优选为3％～5％，更优选为3.2％～4.8％，更优选为3.5％～4.5％，更优选为3.7％～4.2％。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%.
本申请原则上对所述超滤的具体方式没有特别限制，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，本申请为保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，所述超滤优选为切向流超滤。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.
本申请原则上对所述循环超滤的具体步骤没有特别限制，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，本申请为保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，所述循环超滤的具体步骤优选为：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:
先采用第一超滤膜进行第一循环超滤后，收集透过液，再采用第二超滤膜进行第二循环超滤后，收集截留液。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.
本申请原则上对上述循环超滤的具体参数没有特别限制，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，本申请为保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，所述第一超滤膜的过滤精度优选为8000～12000Da，更优选为8500～11500Da，更优选为9000～11000Da，更优选为9500～10500Da。所述第一循环超滤的次数优选为100～500次，更优选为150～450次，更优选为200～400次，更优选为250～350次。所述第二超滤膜的过滤精度优选为800～1200Da，更优选为850～1150Da，更优选为900～1100Da，更优选为950～1050Da。所述第二循环超滤的次数优选为100～500次，更优选为150～450次，更优选为200～400次，更优选为250～350次。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.
本申请特别选择切向流超滤(TFF)这种膜分离技术，是一种分子量水平的切向流过滤，切向流过滤膜，在过滤的同时能够对膜表面进行冲刷，使膜表面保持干净，保持稳定的过滤速度。本申请采用切向流超滤技术，更关键的采用了特定的参数和组合，能够对分子量分布不均一的硫酸皮肤素进行更加快捷、高效的分离与纯化。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.
本申请为完整和细化整个精制工艺，更好的保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，所述循环超滤后优选还包括后处理步骤。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.
本申请原则上对上述后处理的具体步骤没有特别限制，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，本申请为保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，所述后处理步骤优选包括溶解、沉淀、再溶解、过滤、冷冻干燥、研磨和混合中的一步或多步，更优选为溶解、沉淀、再溶解、过滤、冷冻干燥、研磨和混合中的多步，更具体可以为以下步骤：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:
将截留液和氯化钠溶解后，再加入乙醇静置后，舍弃上清液，得到沉淀；After dissolving the retentate and sodium chloride, add ethanol and let stand, discard the supernatant to obtain a precipitate;
将上述步骤得到的沉淀再次溶解、冷冻干燥和磨粉后，得到成品低分子量硫酸皮肤素。After re-dissolving the precipitate obtained in the above steps, freeze-drying and grinding, the finished product low molecular weight dermatan sulfate is obtained.
本申请原则上对上述后处理的具体参数没有特别限制，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整，本申请为保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，所述氯化钠与所述截留液体积比优选为2％～4％，更优选为2.2％～3.8％，更优选为2.5％～3.5％，更优选为2.7％～3.2％。所述乙醇与溶解后的物料体积比值优选为(2～3)：1，更优选为(2.2～2.8)：1，更优选为(2.4～2.6)：1。所述静置的时间优选为6～8h，更优选为6.2～7.8h，更优选为6.5～7.5h，更优选为6.8～7.2h。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.
本申请对上述再次溶解、冷冻干燥和磨粉的具体步骤和参数没有特别限制，以本领域技术人员熟知的此类产品制备过程中的上述过程相应的常规步骤和参数即可，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整。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.
本申请为完整和细化整个精制工艺，更好的保证低分子量硫酸皮肤素的高质量，缩窄分子量分布宽度，更加利于人体吸收，上述精制过程具体可以为以下步骤：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.将富含DS的肝素钠副产物加水进行溶解，得到溶解后的料液；A. Dissolve the DS-rich heparin sodium by-product with water to obtain the dissolved material liquid;
B.将A中所述料液通过大孔径阴离子树脂柱，控制料液温度约50～70℃，进行树脂吸附。首先用第一质量浓度的氯化钠溶液进行洗脱(2～3个树脂体积)，以洗脱去除杂质；再用第二质量浓度的氯化钠溶液洗脱(3～4个树脂体积)，收集该盐度洗脱的料液。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.将B中所收集的料液加入乙醇进行沉淀放置，将沉淀上清液转移至另一物料罐中，补加乙醇进行沉淀放置，弃取上清，收集沉淀物；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.将C中收集沉淀物进行烘干，去除乙醇。然后加水进行溶解，溶解为料液；D. Dry the precipitate collected in C to remove the ethanol. Then add water to dissolve, and dissolve into material liquid;
E.将D中所得的料液调整pH为6.5～8.5，温度控制至35～37℃，加入硫酸软骨素B酶，进行酶解。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.将E中所述料液升温，加入高锰酸钾，持续搅拌，进行氧化；将氧化后料液静置降温，过滤去除高锰酸钾氧化后的杂质产物，再用滤膜进行过滤除杂。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.将F中过滤后料液稀释，使用第一滤膜进行循环超滤，收集透过液。再将收集的透过液使用第二滤膜进行循环超滤，收集截留液，取样中控检测分子量，控制重均分子量在3000～5000Da；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.将G中收集的截留液，加入氯化钠进行溶解，加入乙醇，搅拌均匀后，放置。H. Add sodium chloride to the retentate collected in G to dissolve it, add ethanol, stir evenly, and then set it aside.
I.弃取H中沉淀的上清，溶解沉淀物，将沉淀物溶解，过滤、冷冻干燥、磨粉、混合、包装。I. Discard the supernatant precipitated in H, dissolve the precipitate, dissolve the precipitate, filter, freeze-dry, grind, mix, and package.
本申请提供的酶-超滤法精制低分子硫酸皮肤素的工艺，采用了大孔径阴离子树脂柱提纯处理硫酸皮肤素、硫酸软骨素B酶对硫酸皮肤素进行降解以及使用切向流超滤技术调整 低分子硫酸皮肤素的分子量的特别组合，得到了整体的精制工艺。其中，树脂洗脱进行硫酸皮肤素的提纯，通过使用大孔径阴离子树脂柱(特别是A98树脂)吸附、洗脱，对硫酸皮肤素进行提纯。能够做到在进行酶解处理前，将硫酸皮肤素中的大量杂质进行去除；而采用乙醇沉淀提纯硫酸皮肤素。通过加入一定体积的乙醇，将硫酸皮肤素进一步提纯；再用硫酸软骨素B酶降解处理，通过用硫酸软骨素B酶的降解作用，将提纯后的硫酸软骨素降解成小分子，相比于传统的使用盐酸、过氧化氢等降解方法，本专利方法降解效率高，处理方法稳定可控、重复性好，生产低分子硫酸皮肤素收率高。另外，本专利方法降解条件温和，尽可能保护低分硫酸皮肤素结构的完整；同时再结合使用高锰酸钾氧化进一步提纯低分子硫酸皮肤素，高锰酸钾对加入的硫酸软骨素B酶进行氧化去除，并且对制备的低分子硫酸皮肤素末端结构进行修饰，使制备的低分子硫酸皮肤素结构稳定；以及使用切向流超滤技术控制低分子硫酸皮肤素的分子量，使用本申请特定参数的切向流超滤技术，可以控制、调整低分子硫酸皮肤素的分子量至适合人体吸收的4KD水平。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.
本申请提供的低分子硫酸皮肤素精制工艺，相比与传统的低分子硫酸皮肤素制备工艺，工艺反应条件温和、稳定、可控，工艺重复性高，制备的低分子硫酸皮肤素成品纯度高、分子量分布窄、更加适合人体吸收。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.
本申请还提供了上述技术方案任意一项所述的低分子量硫酸皮肤素或上述技术方案任意一项所述的精致工艺所精制的低分子量硫酸皮肤素在制备抗血栓药物中的应用。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.
本申请对上述低分子量硫酸皮肤素在制备抗血栓药物中的具体应用方式没有特别限制，以本领域技术人员熟知的此类产品常规的应用方式即可，本领域技术人员可以根据生产情况、应用需求或质量要求进行选择和调整。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.
本申请上述步骤提供了一种低分子量硫酸皮肤素以及酶-超滤法精制低分子硫酸皮肤素的工艺、应用。本申请得到的精制的低分子量硫酸皮肤素，具有较窄的重均分子量分布和数据分子量分布，更加适合人体吸收，在抗血栓药物中具有广泛的应用前景。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.
本申请提供的低分子量硫酸皮肤素的精制工艺，是一种酶-超滤法精制低分子硫酸皮肤素的工艺，特别以富含硫酸皮肤素的肝素钠副产物为原料，利用可以降解硫酸皮肤素的生物酶--硫酸软骨素B酶，结合超滤法，得到了该“酶-超滤法”综合精制工艺，制备出了分子量小、分子量分布均一、纯度高、利于人体吸收的低分子硫酸皮肤素，有效的解决了现有的利用盐酸、过氧化氢等化学试剂降解硫酸皮肤素，存在的盐酸用量大，水解反应进行缓慢，并且水解反应不易控，易造成水解过剧或水解程度不够，以及降解参数不易控制，反应稳定性及重复性差，造成收率低，降解产物分子量分布不均一，疗效不可控等问题。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.
本申请整体工艺中，结合树脂洗脱和乙醇沉淀工艺可有效去除核酸、蛋白、类肝素等 杂质，保证产品纯度；通过硫酸软骨素B酶处理的酶解工艺可有效降低产品分子量；通过超滤工艺可有效控制产品分子量。本申请提供的低分子量硫酸皮肤素精制工艺，反应温和、稳定、可控，工艺重复性高，具有一定的经济效益和环境保护作用，适于工业化推广和应用。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.
实验结果表明，本申请提供的低分子量硫酸皮肤素精制工艺，可以将产品中反应核酸、蛋白浓度的260nm、280nm光吸收控制在0.06以下，可以将肝素类杂质的杂质含量控制在0.5％以下(可侧面反映纯度高)。产品经过酶解和超滤后，重均分子量控制在4000～4700Da范围内，分子量分布窄，重均分子量大于8000Da的组分占比不超过6.5％，分子量片段小于2000Da的组分占比不超过8.5％。另外，该工艺可有效保留原料中硫酸软骨素组分，酶解条件经摸索验证后能够保证中间产物的分子量与成品较接近，超滤损失少，产品总收率超过40％。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%.
为了进一步说明本申请，以下结合实施例对本申请提供的一种低分子量硫酸皮肤素及其精制工艺、应用进行详细描述，但是应当理解，这些实施例是在以本申请技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，只是为进一步说明本申请的特征和优点，而不是对本申请权利要求的限制，本申请的保护范围也不限于下述的实施例。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.
实施例1Example 1
称取肝素钠副产物1Kg加10L水进行溶解，溶解为约10％浓度的料液，将料液升温至60℃，缓慢加入约10L树脂的树脂柱，进行树脂吸附；待吸附完毕后，用25L浓度为3.5％的氯化钠溶液，调温度至60℃，进行洗脱，弃取洗脱液；再用30L浓度为8％的氯化钠溶液(调温度至60℃)进行洗脱，收集该部分洗脱液至1#罐；洗脱完毕后，准确测量收集洗脱液体积为32L，加入12.8L乙醇，搅拌均匀后，静置3h，将上清悬浊液转移至2#罐中(1#罐底沉淀物为类肝素)，向2#罐中再次加入64L乙醇，搅拌均匀，静置6小时，弃取上清液，收集2#罐底的沉淀物，将该部分沉淀物进行55℃烘干，得到DS纯品，称量重量为712g。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.
将DS纯品加7.0L水进行溶解，溶解为约10％浓度的料液，调整pH为8.0，温度控制至36℃，加入料液体积0.07mL硫酸软骨素B酶(酶活性为10000IU/mL)，进行酶解4h。将料液升温至80℃，加入21.3g高锰酸钾，持续搅拌，进行氧化45分钟；将氧化后料液静置降温至50℃以下，用加滤纸的板框过滤器去除高锰酸钾氧化后的杂质产物，再用0.22um滤膜进行过滤除杂。收集料液体积约8L，再次补加6L纯化水，使用10KD超滤膜包进行循环超滤，收集透过液(保持0.1Mpa出口压力)。再将收集的透过液使用1KD的滤膜进行循环超滤，收集截留液，取样中控检测分子量，控制重均分子量的分子量分布 在3000～5000Da。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.
检测物料体积为10L，加入300g氯化钠溶解，再加入25L乙醇，搅拌均匀后，放置8h，弃去上清，将沉淀物溶解，过滤、冷冻干燥、磨粉、混合、共收集冻干后的低分子硫酸皮肤素精品447g。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.
对本申请实施例1制备的低分子硫酸皮肤素精品(LMWDS)进行检测。The low-molecular-weight dermatan sulfate product (LMWDS) prepared in Example 1 of the present application was tested.
参见表1，表1为本申请实施例制备的LMWDS相关检测数据。See Table 1. Table 1 is the relevant detection data of LMWDS prepared in the examples of this application.
表1Table 1
Figure PCTCN2020125013-appb-000001
Figure PCTCN2020125013-appb-000001
表1检测结果显示，本申请提供的精制方案实施制备的低分子量类肝素质量可靠，纯度高。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.
实施例2Example 2
称取肝素钠副产物50Kg加500L水进行溶解，溶解为约10％浓度的料液，将料液升温至60℃，缓慢加入约500L树脂的树脂柱，进行树脂吸附；待吸附完毕后，用1250L浓度为3.5％的氯化钠溶液，调温度至60℃，进行洗脱，弃取洗脱液；再用1500L浓度为8％的氯化钠溶液(调温度至60℃)进行洗脱，收集该部分洗脱液至5#罐；洗脱完毕后，准确测量收集洗脱液体积为1600L，加入640L乙醇，搅拌均匀后，静置3h，将上清悬浊液转移至6#罐中(5#罐底沉淀物为类肝素)，向6#罐中再次加入3000L乙醇，搅拌均匀，静置8小时，弃取上清液，收集6#罐底的沉淀物，将该部分沉淀物进行55℃烘干，得到DS纯品，称量重量为37.5Kg。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.
将DS纯品加370L水进行溶解，溶解为约10％浓度的料液，调整pH为8.0，温度控制至36℃，加入料液3.7mL硫酸软骨素B酶(酶活性为10000IU/mL)，进行酶解4h。将料液升温至80℃，加入1.125Kg高锰酸钾，持续搅拌，进行氧化45分钟；将氧化后料液静置降温至50℃以下，用加滤纸的板框过滤器去除高锰酸钾氧化后的杂质产物，再用0.22um滤膜进行过滤除杂。收集料液体积约400L，再次补加400L纯化水，使用10KD超滤膜包进行循环超滤，收集透过液(保持0.1Mpa出口压力)。再将收集的透过液使用1KD的滤膜进行循环超滤，收集截留液，取样中控检测分子量，控制分子量分布在3000～5000Da。检测物料体积为700L，加入21Kg氯化钠溶解，再加入1750L乙醇，搅拌均匀后，放置8h，弃去上清，将沉淀物溶解，过滤、冷冻干燥、磨粉、混合、共收集冻干后的低分子硫酸皮肤素精品24.2Kg。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.
对本申请实施例2制备的低分子硫酸皮肤素精品(LMWDS)进行检测。The low-molecular-weight dermatan sulfate product (LMWDS) prepared in Example 2 of the present application was tested.
参见表1，表1为本申请实施例制备的LMWDS相关检测数据。See Table 1. Table 1 is related detection data of LMWDS prepared in the examples of this application.
表1检测结果显示，本申请提供的精制方案实施制备的低分子量类肝素质量可靠，纯度高。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.
实施例3Example 3
称取肝素钠副产物100Kg加1000L水进行溶解，溶解为约10％浓度的料液，将料液升温至60℃，缓慢加入约1000L树脂的树脂柱，进行树脂吸附；待吸附完毕后，用2500L浓度为3.5％的氯化钠溶液，调温度至60℃，进行洗脱，弃取洗脱液；再用3000L浓度为8％的氯化钠溶液(调温度至60℃)进行洗脱，收集该部分洗脱液至5#罐；洗脱完毕后，准确测量收集洗脱液体积为3200L，加入1280L乙醇，搅拌均匀后，静置3h，将上清悬浊液转移至6#罐中(5#罐底沉淀物为类肝素)，向6#罐中再次加入6000L乙醇，搅拌均匀，静置8小时，弃取上清液，收集6#罐底的沉淀物，将该部分沉淀物进行55℃烘干，得到DS纯品，称量重量为75.3Kg。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.
将DS纯品加753L水进行溶解，溶解为约10％浓度的料液，调整pH为8.0，温度控制至36℃，加入料液7.5mL硫酸软骨素B酶(酶活性为10000IU/mL)，进行酶解4h。将料液升温至80℃，加入2.25Kg高锰酸钾，持续搅拌，进行氧化45分钟；将氧化后料液静置降温至50℃以下，用加滤纸的板框过滤器去除高锰酸钾氧化后的杂质产物，再用0.22um滤膜进行过滤除杂。收集料液体积约800L，再次补加600L纯化水，使用10KD超滤膜包进行循环超滤，收集透过液(保持0.1Mpa出口压力)。再将收集的透过液使用1KD的滤膜进行循环超滤，收集截留液，取样中控检测分子量，控制分子量分布在3000～5000Da。检测物料体积为1500L，加入45Kg氯化钠溶解，再加入3500L乙醇，搅 拌均匀后，放置8h，弃去上清，将沉淀物溶解，过滤、冷冻干燥、磨粉、混合、共收集冻干后的低分子硫酸皮肤素精品46.4Kg。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.
对本申请实施例2制备的低分子硫酸皮肤素精品(LMWDS)进行检测。The low-molecular-weight dermatan sulfate product (LMWDS) prepared in Example 2 of the present application was tested.
参见表1，表1为本申请实施例制备的LMWDS相关检测数据。See Table 1. Table 1 is the relevant detection data of LMWDS prepared in the examples of this application.
表1检测结果显示，本申请提供的精制方案实施制备的低分子量类肝素质量可靠，纯度高。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.
以上对本申请提供的一种低分子量硫酸皮肤素以及酶-超滤法精制低分子硫酸皮肤素的工艺、应用进行了详细的介绍，本文中应用了具体个例对本申请的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本申请的方法及其核心思想，包括最佳方式，并且也使得本领域的任何技术人员都能够实践本申请，包括制造和使用任何装置或***，和实施任何结合的方法。应当指出，对于本技术领域的普通技术人员来说，在不脱离本申请原理的前提下，还可以对本申请进行若干改进和修饰，这些改进和修饰也落入本申请权利要求的保护范围内。本申请专利保护的范围通过权利要求来限定，并可包括本领域技术人员能够想到的其他实施例。如果这些其他实施例具有不是不同于权利要求文字表述的结构要素，或者如果它们包括与权利要求的文字表述无实质差异的等同结构要素，那么这些其他实施例也应包含在权利要求的范围内。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. 一种低分子量硫酸皮肤素，其特征在于，所述低分子量硫酸皮肤素的数均分子量为2000～5000Da；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;
    所述低分子量硫酸皮肤素的重均分子量为2500～6500Da。The weight average molecular weight of the low molecular weight dermatan sulfate is 2500-6500 Da.
  2. 根据权利要求1所述的低分子量硫酸皮肤素，其特征在于，所述低分子量硫酸皮肤素的重均分子量的分子量分布为：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:
    分子量小于2000Da的硫酸皮肤素占所述低分子量硫酸皮肤素全部的10wt％以下；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;
    分子量大于8000Da的硫酸皮肤素占所述低分子量硫酸皮肤素全部的12wt％以下；Dermatan sulfate with a molecular weight greater than 8000 Da accounts for less than 12 wt% of the total low molecular weight dermatan sulfate;
    分子量为2000～8000Da的硫酸皮肤素占所述低分子量硫酸皮肤素全部的78wt％～90wt％。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. 根据权利要求1所述的低分子量硫酸皮肤素，其特征在于，所述低分子量硫酸皮肤素的重均分子量为3500～6000Da；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;
    所述低分子量硫酸皮肤素的数均分子量为3000～4500Da；The number average molecular weight of the low molecular weight dermatan sulfate is 3000 to 4500 Da;
    所述低分子量硫酸皮肤素的比旋度小于等于-40°；The specific rotation of the low molecular weight dermatan sulfate is less than or equal to -40°;
    所述低分子量硫酸皮肤素的纯度为99％以上。The purity of the low molecular weight dermatan sulfate is more than 99%.
  4. 一种低分子量硫酸皮肤素的精制工艺，其特征在于，包括以下步骤：A refining process for low molecular weight dermatan sulfate, which is characterized in that it comprises the following steps:
    1)将硫酸皮肤素溶液和硫酸软骨素B酶混合后，进行酶解后，得到料液；1) After mixing the dermatan sulfate solution and chondroitin sulfate B enzyme, after enzymatic hydrolysis, the material liquid is obtained;
    2)向上述步骤得到的料液，加入强氧化剂进行氧化反应后，再经过过滤，得到滤后料液；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)将上述步骤得到的滤后料液经过循环超滤后，得到低分子量硫酸皮肤素。3) After the filtered material liquid obtained in the above steps is circulated ultrafiltration, low molecular weight dermatan sulfate is obtained.
  5. 根据权利要求4所述的精制工艺，其特征在于，所述硫酸皮肤素溶液包括硫酸皮肤素水溶液；The refining process according to claim 4, wherein the dermatan sulfate solution comprises an aqueous solution of dermatan sulfate;
    所述硫酸皮肤素溶液的pH值为6.5～8.5；The pH of the dermatan sulfate solution is 6.5 to 8.5;
    所述硫酸皮肤素溶液的质量浓度为8％～12％；The mass concentration of the dermatan sulfate solution is 8%-12%;
    所述硫酸皮肤素的重均分子量为15000～45000Da；The weight average molecular weight of the dermatan sulfate is 15000-45000 Da;
    所述硫酸软骨素B酶与所述硫酸皮肤素溶液的体积比为0.001％～0.003％；The volume ratio of the chondroitin sulfate B enzyme to the dermatan sulfate solution is 0.001% to 0.003%;
    所述硫酸软骨素B酶的浓度为8000～12000IU/mL；The concentration of the chondroitin sulfate B enzyme is 8000-12000 IU/mL;
    所述酶解的温度为35～37℃；The temperature of the enzymatic hydrolysis is 35-37°C;
    所述酶解的时间为3～5h。The enzymolysis time is 3 to 5 hours.
  6. 根据权利要求4所述的精制工艺，其特征在于，所述强氧化剂包括高锰酸钾、高锰酸钠、过氧化氢和次氯酸钠中的一种或多种；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;
    所述强氧化剂占所述料液的质量比为2％～5％；The mass ratio of the strong oxidant to the material liquid is 2% to 5%;
    所述氧化反应的温度为80～90℃；The temperature of the oxidation reaction is 80-90°C;
    所述氧化反应的时间为30～45分钟；The oxidation reaction time is 30 to 45 minutes;
    所述氧化反应后还包括静置降温步骤和/或除杂步骤；The oxidation reaction further includes a step of standing and cooling and/or a step of removing impurities;
    所述过滤为微滤膜过滤；The filtration is microfiltration membrane filtration;
    所述微滤膜的孔径为0.1～0.45μm。The pore size of the microfiltration membrane is 0.1-0.45 μm.
  7. 根据权利要求4所述的精制工艺，其特征在于，所述循环超滤前还包括稀释步骤；所述稀释的滤后料液的质量浓度为3％～5％；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. 根据权利要求4所述的精制工艺，其特征在于，所述超滤为切向流超滤；The refining process according to claim 4, wherein the ultrafiltration is tangential flow ultrafiltration;
    所述循环超滤的具体步骤为：The specific steps of the cyclic ultrafiltration are:
    先采用第一超滤膜进行第一循环超滤后，收集透过液，再采用第二超滤膜进行第二循环超滤后，收集截留液；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;
    所述第一超滤膜的过滤精度为8000～12000Da；The filtration accuracy of the first ultrafiltration membrane is 8000 to 12000 Da;
    所述第一循环超滤的次数为100～500次；The number of times of the first cycle of ultrafiltration is 100 to 500 times;
    所述第二超滤膜的过滤精度为800～1200Da；The filtration accuracy of the second ultrafiltration membrane is 800-1200 Da;
    所述第二循环超滤的次数为100～500次。The number of times of the second cycle ultrafiltration is 100 to 500 times.
  9. 根据权利要求4所述的精制工艺，其特征在于，所述循环超滤后还包括后处理步骤；The refining process according to claim 4, characterized in that, after the cyclic ultrafiltration, a post-treatment step is further included;
    所述后处理步骤包括溶解、沉淀、再溶解、过滤、冷冻干燥、研磨和混合中的一步或多步；The post-treatment step includes one or more steps of dissolution, precipitation, re-dissolution, filtration, freeze-drying, grinding and mixing;
    所述后处理步骤具体为：The post-processing steps are specifically:
    将循环超滤后得到的截留液和氯化钠溶解后，再加入乙醇静置后，舍弃上清液，得到沉淀；After dissolving the retentate obtained after circulating ultrafiltration and sodium chloride, add ethanol and let stand, then discard the supernatant to obtain a precipitate;
    将上述步骤得到的沉淀再次溶解、冷冻干燥和磨粉后，得到成品低分子量硫酸皮肤素；After re-dissolving the precipitate obtained in the above steps, freeze-drying and grinding, the finished product low molecular weight dermatan sulfate is obtained;
    所述氯化钠与所述截留液体积比为2％～4％；The volume ratio of the sodium chloride to the retentate is 2% to 4%;
    所述乙醇与溶解后的物料体积比值为(2～3)：1；The volume ratio of the ethanol to the dissolved material is (2～3):1;
    所述静置的时间为6～8h。The standing time is 6-8h.
  10. 根据权利要求4～9任意一项所述的精制工艺，其特征在于，所述硫酸皮肤素由富含硫酸皮肤素的肝素钠副产物经制备后得到；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;
    所述制备过程为：The preparation process is:
    A)将富含硫酸皮肤素的肝素钠副产物和水进行溶解，得到原液；A) Dissolve the by-product of heparin sodium rich in dermatan sulfate and water to obtain the original solution;
    B)将上述步骤得到的原液经过树脂吸附后，再用氯化钠溶液洗脱，得到洗脱后的原液；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)将上述步骤得到的洗脱后的原液和乙醇混合静置后，再经过干燥，得到硫酸皮肤素。C) After mixing the eluted stock solution obtained in the above steps with ethanol and standing, then drying to obtain dermatan sulfate.
  11. 根据权利要求10所述的精制工艺，其特征在于，所述原液的质量浓度为8％～12％；The refining process according to claim 10, wherein the mass concentration of the stock solution is 8%-12%;
    所述树脂吸附的温度为50～70℃；The temperature of the resin adsorption is 50-70°C;
    所述树脂吸附的树脂柱为阴离子树脂柱；The resin column adsorbed by the resin is an anion resin column;
    所述氯化钠溶液的质量浓度优选为2％～9％；The mass concentration of the sodium chloride solution is preferably 2%-9%;
    所述洗脱包括先洗脱杂质和再洗脱树脂。The elution includes eluting impurities first and then eluting the resin.
  12. 根据权利要求10所述的精制工艺，其特征在于，所述乙醇与所述洗脱后的原液的体积比优选为(1.8～2.4)：1；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;
    所述混合静置的时间优选为7～9小时；The mixing and standing time is preferably 7-9 hours;
    所述混合静置优选为两次混合静置；The mixing and standing is preferably two mixing and standing;
    所述干燥的温度优选为50～60℃。The drying temperature is preferably 50-60°C.
  13. 根据权利要求4所述的精制工艺，其特征在于，所述精制过程具体包括以下步骤：The refining process according to claim 4, wherein the refining process specifically includes the following steps:
    (1)将富含硫酸皮肤素的肝素钠副产物加水进行溶解，得到溶解后的料液；(1) Add water to dissolve the by-product of heparin sodium rich in dermatan sulfate to obtain a dissolved material liquid;
    (2)将(1)中所述料液通过阴离子树脂柱，控制料液温度约50～70℃，进行树脂吸附；首先用第一质量浓度的氯化钠溶液进行洗脱，以洗脱去除杂质；再用第二质量浓度的氯化钠溶液洗脱，收集该盐度洗脱的料液；(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)将(2)中所收集的料液加入乙醇进行沉淀放置，将沉淀上清液转移至另一物料罐中，补加乙醇进行沉淀放置，弃取上清，收集沉淀物；(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)将(3)中收集沉淀物进行烘干，去除乙醇；然后加水进行溶解，溶解为料液；(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)将(4)中所得的料液调整pH为6.5～8.5，温度控制至35～37℃，加入硫酸软骨素B酶，进行酶解；(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)将(5)中所述料液升温，加入高锰酸钾，持续搅拌，进行氧化；将氧化后料液静置降温，过滤去除高锰酸钾氧化后的杂质产物，再用滤膜进行过滤除杂；(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)将(6)中过滤后料液稀释，使用第一滤膜进行循环超滤，收集透过液；再将收集的透过液使用第二滤膜进行循环超滤，收集截留液，取样中控检测分子量，控制重均分子量在3000～5000Da；(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)将(7)中收集的截留液，加入氯化钠进行溶解，加入乙醇，搅拌均匀后，放置。(8) Add sodium chloride to dissolve the retentate collected in (7), add ethanol, and stir well, then set aside.
    (9)弃取(8)中沉淀的上清，溶解沉淀物，将沉淀物溶解，过滤、冷冻干燥、磨粉、 混合、包装。(9) Discard the supernatant precipitated in (8), dissolve the precipitate, dissolve the precipitate, filter, freeze-dry, grind, mix, and package.
  14. 根据权利要求1～3任意一项所述的低分子量硫酸皮肤素或权利要求4～13任意一项所述的精制工艺所精制的低分子量硫酸皮肤素在制备抗血栓药物中的应用。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.
PCT/CN2020/125013 2019-12-11 2020-10-30 Low molecular weight dermatan sulfate, refining process therefor and application thereof WO2021114941A1 (en)

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CN113735994A (en) * 2020-05-29 2021-12-03 江苏唯高生物科技有限公司 Process for preparing sulodexide raw material

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