Purifying method for low molecule heparin

A technology of low molecular weight heparin and purification method, which is applied in the field of biomedicine and can solve the problems of difficulty in separating molecular weight distribution, narrowness, and reduced yield.

Inactive Publication Date: 2006-03-22
NANJING KING FRIEND BIOCHEM PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, fractional precipitation is mostly used for purification and separation, but fractional precipitation technology is difficult to separate fragments with narrow molecular weight distribution. For example, when the molecular weight distribution of dalteparin sodium is 5600-6400, fractional precipitation is difficult to achieve the separation effect
In order to achieve a reasonable fragment distribution, the yield is often reduced

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Dissolve 100g of heparin sodium in 1000ml of 1% NaCl solution, adjust the pH to 2.0-3.0, add 1.5-3.5% w / w sodium nitrite at room temperature, preferably 2.95g of sodium nitrite, react for 2 hours, add potassium iodide- The starch test paper does not turn blue, indicating that the reaction is complete. Adjust the pH to 9.5, add 1g of sodium borohydride, and let it stand overnight. Adjust the pH to 3.5, adjust the pH to 6.0 after one hour, add 0.40 times the volume of ethanol, the macromolecule precipitates out, suck out the supernatant, add 95% ethanol to 0.70-0.8 times the volume, overnight, precipitate, dehydrate, and dry to obtain the crude product 55g.

[0022] The results of molecular weight distribution of the sample determined by HPLC: 8000 part meets the regulations, the fluorescence chemical analysis measures -N-NO 8ppm, which is 0.25ppm higher than the pharmacopoeia, boron 6ppm, which is 1ppm higher than the pharmacopoeia, and needs to be purified as follows ...

Embodiment 2

[0028] Add 250-350 g of quaternary ammonium salt to 100 g of heparin sodium, and after the reaction is complete, dry to obtain about 350 g of heparin sodium quaternary ammonium salt.

[0029] Heparin quaternary ammonium salt was added with 400ml benzyl chloride and 2000ml dichloromethane, and reacted at room temperature for 24 hours.

[0030] Add 3500 ml of 10% sodium acetate methanol solution, dehydrate and dry to obtain 180 g of esterified heparin.

[0031] Dissolve 180 g of esterified heparin in 2000 ml of water, heat to 60°C, add 0.1M NaOH to react for one hour. After the reaction was completed, an equal volume of ethanol solution was added. Overnight, the precipitate was collected, dehydrated, and dried to obtain 58 g of crude product.

[0032] Molecular weight distribution was measured by HPLC, and both parts of molecular weight 8000 did not meet the requirements, and the content of benzyl alcohol was as high as 3% (less than 0.1% in Pharmacopoeia), which required furt...

Embodiment 3

[0036] Dissolve 100g of heparin sodium in a suitable solution, adjust the pH to about 2.4, and add 3ML of isoamyl nitrite for 1 to 2 hours. Potassium iodide-starch test paper does not turn blue, indicating that the reaction is complete. Adjust pH to about 9.5, add 0.8g sodium borohydride, overnight. Adjust the pH to 3.5 the next day, and adjust the pH to about 6.0 an hour later. Precipitate with one volume of ethanol, dehydrate and dry to obtain 62 g of crude product.

[0037] The molecular weight distribution measured by HPLC does not meet the requirements of the Pharmacopoeia, and further chromatographic purification is as follows. The crude product was dissolved in 350 ml of 1% sodium chloride solution, and 50 microliters of hydrobromic acid was added under airtight conditions to react for one hour. Pass the solution through the Sephadex G-50 chromatographic column according to the method of example 1, and the chromatographic conditions are the same as in example 1; meas...

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Abstract

The present invention relates to the extraction and purification method of low molecular weight heparin for treating blood coagulation and thrombus. The coarse low molecular weight heparin material is dissolved in NaCl solution and chromatographically separated with molecular sieve gel layer to obtain the chromatographic separated liquid, and the chromatographic separated liquid is alcohol precipitated, dewatered and dried to obtain low molecular weight heparin segment. Before chromatography, the coarse product is irradiated with ultraviolet lamp or reacted with oxidant sodium hypochlorite or hydrobromic acid to eliminate harmful ¿CN-NO- radical, and treated with strong anionic exchange resin to eliminate methanol and other chemical impurity. The present invention has the low molecular weight heparin product reaching relevant international standard, and the method is simple and high in yield.

Description

technical field [0001] The invention relates to a method for extracting and purifying low-molecular-weight heparin used for anticoagulant and antithrombotic drugs, belonging to the field of biomedicine. Background technique [0002] Heparin has been used in clinical anticoagulation therapy for more than 70 years. Since the 1980s, studies have found that as the molecular weight of heparin decreases, its anticoagulant activity decreases rapidly, but the anti-Xa activity, which is closely related to antithrombotic function, decreases relatively slowly. Based on this discovery, people have studied heparin fragments with higher antithrombotic activity-anti-Xa activity and lower bleeding tendency-low anticoagulant activity, that is, "low molecular weight heparin" that came into being later. Studies have shown that heparin fragments with a molecular weight below 2000 have no anti-Xa activity. Although fragments with a molecular weight greater than 8000 have high anti-Xa activity, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08B37/10
Inventor 唐明龙
Owner NANJING KING FRIEND BIOCHEM PHARMA CO LTD
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