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Preparation method of ultra-high purity heparan sulfate

A heparan sulfate, ultra-high-purity technology, applied in the field of biomedicine, can solve the problems of low anticoagulant activity, mixed glycosaminoglycans, difficult to enlarge process conditions, etc., and achieves simple operation, stable product quality, improved yield and high efficiency. The effect of purity

Inactive Publication Date: 2020-05-15
HUBEI YINUORUI BIOLOGICAL PHARMA
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  • Abstract
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  • Application Information

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Problems solved by technology

This patented method is to obtain heparan sulfate after removing oil, acidic protein, basic protein and nucleic acid in the duodenum in essence, but because other types of glycosaminoglycans (such as dermatan sulfate) will also be adsorbed by the resin, and Elutes at immediate sodium chloride concentrations, resulting in intermingling of glycosaminoglycans with heparan sulfate
Although the subsequent steps use the acid-adjusting centrifugation method to remove dermatan sulfate, but this method will leave dermatan sulfate in the supernatant, so heparan sulfate is always accompanied by dermatan sulfate, so the purity of heparan sulfate obtained is not high
[0005] Chinese patent ZL 201510394022.5 discloses a method for separating heparinoid from waste protein by-product of heparin. In this method, the waste protein by-product of heparin is dissolved, an adsorbent is added, alcohol precipitation is eluted, oxidative alcohol precipitation is dried, and dried heparinoid is obtained. However, this type of heparin dry product not only contains heparan sulfate, but also at least contains mucopolysaccharides such as fast heparin with high anticoagulant activity, indicating that the purity of heparan sulfate in heparin dry product is not high
The patented method obtains heparan sulfate through resin adsorption and elution, but also mixes glycosaminoglycans such as dermatan sulfate, so the purity of heparan sulfate is not high
[0011] Chinese patent application 201410819670.6 discloses a method for isolating heparan sulfate from animal lungs. In this method, hydrolytic enzymes are used to degrade the saline extract of animal lungs, and then oxidants and activated carbon are added to the enzymolysis solution for oxidative adsorption and decolorization. , and then use acetone to fractionally precipitate the oxidized solution, and dehydrate and dry to obtain the crude heparan sulfate. Then, the aqueous solution of the crude heparan sulfate is sequentially separated by membrane separation and anion exchange chromatography, and heparan sulfate is mixed with dermatan sulfate and sulfuric acid. Impurities such as chondroitin and heparin are separated, and then the eluate is concentrated by ultrafiltration using a 5000-7000Da ultrafiltration membrane, and finally desalted and freeze-dried by gel filtration chromatography to obtain heparan sulfate high-quality goods, but the process of the invention is lengthy , requires complex intermediate process control, and the process conditions are not easy to scale up, so the prospect of industrialization is worrying
However, the higher the purity of heparan sulfate, the lower the anticoagulant activity, so the purity of heparan sulfate obtained by this patent is not high
And this method also has the problem of source of raw material and product safety

Method used

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  • Preparation method of ultra-high purity heparan sulfate
  • Preparation method of ultra-high purity heparan sulfate
  • Preparation method of ultra-high purity heparan sulfate

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Effect test

Embodiment 1

[0040] The preparation of embodiment 1 ultra-high purity heparan sulfate

[0041] (1) Primary dissolution: add 2% (w / w) salt (sodium chloride) water to 50.23 g of heparin sodium by-product to dissolve into a 5% (w / w) solution;

[0042] (2) Primary precipitation: add 0.5 times the volume of 95% ethanol, and precipitate at 40°C for 3 hours;

[0043] (3) Secondary dissolution: dissolve the precipitate in (2) with 2% (w / w) salt (sodium chloride) water into a 5% (w / w) solution;

[0044] (4) Oxidation: Use 4M sodium hydroxide solution to adjust the pH of the solution in (3) to 11.0, add a total volume of 2% hydrogen peroxide solution (30% concentration), and oxidize at room temperature for 4 hours;

[0045] (5) Secondary precipitation: adjust the pH to 6.0 with hydrochloric acid, add 0.4 times the volume of 95% ethanol, precipitate at 5°C for 16 hours, and collect the supernatant;

[0046] (6) Three precipitations: add 1.0 times volume of 95% ethanol to the supernatant in (5) to p...

Embodiment 2

[0053] The preparation of embodiment 2 ultra-high purity heparan sulfate

[0054] (1) Primary dissolution: add 2% (w / w) salt (sodium chloride) water to 50.55 g of heparin sodium by-product to dissolve into a 10% (w / w) solution;

[0055] (2) Primary precipitation: add 0.8 times the volume of 95% ethanol, and precipitate at 40°C for 4 hours;

[0056] (3) Secondary dissolution: dissolve the precipitate in (2) with 2% (w / w) salt (sodium chloride) water into a 10% (w / w) solution;

[0057] (4) Oxidation: Use 4M sodium hydroxide solution to adjust the pH of the solution in (3) to 11.0, add a total volume of 2% hydrogen peroxide solution (30% concentration), and oxidize at room temperature for 6 hours;

[0058] (5) Secondary precipitation: adjust the pH to 6.5 with hydrochloric acid, add 0.5 times the volume of 95% ethanol, precipitate at 5°C for 20 hours, and collect the supernatant;

[0059] (6) Three precipitations: add 1.5 times volume of 95% ethanol to the supernatant in (5) to...

Embodiment 3

[0066] The preparation of embodiment 3 ultra-high purity heparan sulfate

[0067] (1) Primary dissolution: Add 2% (w / w) salt (sodium chloride) water to 50.41 g of heparin sodium by-product to dissolve into a 15% (w / w) solution;

[0068] (2) Primary precipitation: add 1.1 times the volume of 95% ethanol, and precipitate at 40°C for 5 hours;

[0069] (3) Secondary dissolution: dissolve the precipitate in (2) with 2% (w / w) salt (sodium chloride) water into a 15% (w / w) solution;

[0070] (4) Oxidation: Use 4M sodium hydroxide solution to adjust the pH of the solution in (3) to 11.0, add a total volume of 2% hydrogen peroxide solution (30% concentration), and oxidize at room temperature for 8 hours;

[0071] (5) Secondary precipitation: adjust the pH to 7.0 with hydrochloric acid, add 0.6 times the volume of 95% ethanol, precipitate at 5°C for 24 hours, and collect the supernatant;

[0072] (6) Three precipitations: add 2.0 times volume of 95% ethanol to the supernatant in (5) to...

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Abstract

The invention discloses a preparation method of ultrahigh-purity heparan sulfate. The preparation method comprises the following steps: dissolving and precipitating a heparin sodium byproduct to obtain a heparan sulfate crude product; and carrying out secondary dissolution, oxidation and cold precipitation on the heparan sulfate crude product to obtain supernatant, precipitating again, re-dissolving, adsorbing, eluting and precipitating to finally obtain the ultra-high purity heparan sulfate. According to the method, the problem that heparan sulfate is difficult to separate from dermatan sulfate, chondroitin sulfate and heparin sodium is solved, the yield and purity of heparan sulfate are improved, and in addition, the method is simple to operate, stable in product quality and suitable forlarge-scale production.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for preparing ultra-high-purity heparan sulfate. Background technique [0002] Heparan sulfate is a natural glycosaminoglycan, similar in structure to heparin sodium, but has a smaller molecular weight, higher degree of acetylation, less sulfate group content, and moderate anticoagulant activity. Heparan sulfate is used for antithrombotic, anti-inflammatory, hypolipidemic, and renal protection of diabetic patients. It is the most popular mucopolysaccharide biochemical drug studied this year. Heparan sulfate, heparin sodium, dermatan sulfate, and chondroitin sulfate have similar properties, and it is difficult to purify heparan sulfate by traditional ion exchange chromatography and fractional precipitation with organic solvents. [0003] Chinese patent ZL 201510127522.2 discloses a method for precisely quantitatively controlling the content of chondroitin sulfate (CS...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08B37/10
CPCC08B37/0075
Inventor 干浩董凯韩自江陈新伟付志豪周伟
Owner HUBEI YINUORUI BIOLOGICAL PHARMA
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