WO2021114941A1 - Sulfate de dermatan de bas poids moléculaire, son procédé de raffinage et son application - Google Patents

Sulfate de dermatan de bas poids moléculaire, son procédé de raffinage et son application 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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molecular weight
dermatan sulfate
low molecular
ultrafiltration
application
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PCT/CN2020/125013
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English (en)
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/fr

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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

La présente invention concerne un sulfate de dermatan de bas poids moléculaire. Le poids moléculaire moyen en nombre du sulfate de dermatan de bas poids moléculaire est de 2 000 à 5 000 Da, et le poids moléculaire moyen en poids du sulfate de dermatan de bas poids moléculaire est de 2 500 à 6 500 Da. L'invention concerne également un procédé de raffinage pour le sulfate de dermatan de bas poids moléculaire, comprenant : 1) le mélange d'une solution de sulfate de dermatan et d'enzyme de sulfate de chondroïtine B pour l'enzymolyse pour obtenir du liquide d'alimentation ; 2) l'addition d'un oxydant fort au liquide d'alimentation obtenu dans l'étape 1) pour la réaction d'oxydation, et le filtrage pour obtenir le liquide d'alimentation filtré ; et 3) l'exécution de l'ultrafiltration de circulation sur le liquide d'alimentation filtré obtenu dans l'étape 2), pour obtenir le sulfate de dermatan de bas poids moléculaire. Le sulfate de dermatan de bas poids moléculaire convient mieux à l'absorption par le corps humain, et présente de larges perspectives d'application dans les médicaments antithrombotiques. Le procédé de raffinage du sulfate de dermatan de bas poids moléculaire est un procédé de raffinage du sulfate de dermatan de bas poids moléculaire utilisant un procédé d'ultrafiltration par enzyme, la réaction est modérée, stable et contrôlable, la répétabilité du procédé est élevée, certains avantages économiques et effets de protection environnementale sont atteints, et le procédé de raffinage convient à l'application et à la promotion industrielles.
PCT/CN2020/125013 2019-12-11 2020-10-30 Sulfate de dermatan de bas poids moléculaire, son procédé de raffinage et son application WO2021114941A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0269937A2 (fr) * 1986-11-24 1988-06-08 MEDIOLANUM FARMACEUTICI s.r.l. Procédé de préparation contrôlée de glycosaminoglycanes de bas poids moléculaire
CN1445245A (zh) * 2003-04-18 2003-10-01 山东大学 一种低分子硫酸皮肤素及其制备方法
CN103602711A (zh) * 2013-11-08 2014-02-26 青岛贝尔特生物科技有限公司 一种治疗心肌炎的低分子硫酸软骨素的制备方法
CN109384861A (zh) * 2018-09-30 2019-02-26 临沂新程金锣肉制品集团有限公司 一种肝素钠废液提取硫酸皮肤素的方法
CN111040047A (zh) * 2019-12-11 2020-04-21 东营天东制药有限公司 一种酶-超滤法精制低分子硫酸皮肤素的工艺、应用

Family Cites Families (3)

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CN104530260A (zh) * 2014-12-10 2015-04-22 重庆都好生物科技有限公司 利用猪肺联产高纯度肝素钠和硫酸皮肤素的方法
CN106188341B (zh) * 2016-07-27 2019-09-17 河北常山生化药业股份有限公司 肝素钠下脚料制备高纯度硫酸皮肤素工艺
CN107936139A (zh) * 2016-10-12 2018-04-20 山东万邦赛诺康生化制药股份有限公司 一种硫酸皮肤素的制备工艺

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0269937A2 (fr) * 1986-11-24 1988-06-08 MEDIOLANUM FARMACEUTICI s.r.l. Procédé de préparation contrôlée de glycosaminoglycanes de bas poids moléculaire
CN1445245A (zh) * 2003-04-18 2003-10-01 山东大学 一种低分子硫酸皮肤素及其制备方法
CN103602711A (zh) * 2013-11-08 2014-02-26 青岛贝尔特生物科技有限公司 一种治疗心肌炎的低分子硫酸软骨素的制备方法
CN109384861A (zh) * 2018-09-30 2019-02-26 临沂新程金锣肉制品集团有限公司 一种肝素钠废液提取硫酸皮肤素的方法
CN111040047A (zh) * 2019-12-11 2020-04-21 东营天东制药有限公司 一种酶-超滤法精制低分子硫酸皮肤素的工艺、应用

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