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Production process for efficient separation of highly active hirudin based on anion exchange column

An anion exchange column and production process technology, applied in the directions of leech inhibitors, peptide preparation methods, protease inhibitors, etc., can solve the problems of unsuitability for injection, unsuitable for industrial production, low effective ingredients, etc. The effect of convenient use, low cost and low production cost

Active Publication Date: 2015-09-23
科康生物医药(深圳)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the above-mentioned method has many shortcomings, mainly because the active ingredients are not high after extraction, and the activity is generally about 150~6000ATU / g, and the activity varies according to different extracts, and the impurity content is high, so it is not suitable for injection medicine. The extraction methods are complicated, and some methods are slow and not suitable for industrial production. Therefore, there are still many people studying the extraction methods of hirudin, hoping to find an extraction method with high efficiency, low cost and high active ingredients.

Method used

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  • Production process for efficient separation of highly active hirudin based on anion exchange column
  • Production process for efficient separation of highly active hirudin based on anion exchange column
  • Production process for efficient separation of highly active hirudin based on anion exchange column

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Weigh 20g of leech powder (activity 300ATU / g) produced by Nanning Jinhai Kekang Pharmaceutical Technology Co., Ltd., add 20ml of ultrapure water, 20ml of 5%TCA (PH 1.15), centrifuge at 8000r / min for 20min, and supernatant for later use ;The precipitation continued to be extracted twice, all the supernatants were mixed, the pH was adjusted to 4.0, and three times of cold acetone was added to precipitate, and the HPLC test results were shown in figure 1 .

[0053] DEAE-silica gel column liquid chromatographic column (20×100mm) for separation and purification, the chromatographic column filler is macroporous silica gel coated with a layer of diethylaminoethyl methacrylate polymer on the surface, the pore diameter of the macroporous silica gel is 300 ?, and the particle size of the filler is is 30 μm.

[0054] The sample solution is detected by liquid chromatography with a UV detector, the detection wavelength is 210nm, the flow rate is 5mL / min, the gradient: 0%B (A: 10mM...

Embodiment 2

[0056] Weigh 20g of leech powder (activity 160ATU / g) produced by Nanning Jinhai Kekang Pharmaceutical Technology Co., Ltd., add 20ml of ultrapure water, 20ml of 5% TCA (PH 1.15), centrifuge at 8000r / min for 15min, and supernatant for later use ;The precipitation continued to be extracted twice, all the supernatants were mixed, the pH was adjusted to 4.0, and three times of cold acetone was added to precipitate, and the HPLC test results were shown in figure 1 .

[0057] DEAE-silica gel column liquid chromatographic column (20×100mm) for separation and purification, the chromatographic column filler is macroporous silica gel coated with a layer of diethylaminoethyl substituted cellulose on the surface, the pore diameter of macroporous silica gel is 400 ?, and the particle size of the filler is is 60 μm.

[0058] The sample solution is detected by liquid chromatography with a UV detector, the detection wavelength is 210nm, the flow rate is 5mL / min, the gradient: 0%B (A: 10mM T...

Embodiment 3

[0060] Weigh 20g of leech powder (activity 160ATU / g) produced by Nanning Jinhai Kekang Pharmaceutical Technology Co., Ltd., add 20ml of ultrapure water, 20ml of 5% TCA (PH 1.15), centrifuge at 8000r / min for 15min, and supernatant for later use ;The precipitation continued to be extracted twice, all the supernatants were mixed, the pH was adjusted to 4.0, and three times of cold acetone was added to precipitate, and the HPLC test results were shown in figure 1 .

[0061] DEAE-silica gel column liquid chromatographic column (20×100mm) for separation and purification, the chromatographic column filler is macroporous silica gel coated with a layer of diethylaminoethyl methacrylate polymer on the surface, the pore diameter of macroporous silica gel is 500 ?, and the packing particle size is 100 μm.

[0062] The sample solution is detected by liquid chromatography with a UV detector, the detection wavelength is 210nm, the flow rate is 5mL / min, the gradient: 0%B (A: 10mM Tris-HCl P...

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Abstract

The present invention is based on a novel anion exchange column (DEAE-silica gel separation column) to achieve efficient separation and purification of hirudin. The locust powder made from Hirudo phenatinae in Guangxi is used as a raw material, which belongs to the field of traditional Chinese medicine pharmaceuticals and involves the extraction of active ingredients from animals. The purpose is to solve the problems of complex extraction of hirudin, high cost and difficulty in industrial production. The present invention is characterized in that a new technique for separating and purifying hirudin is designed around the novel separation and purification system of the cheap DEAE-silica gel separation column. The process is simple and fast, and the cost is low; and the obtained hirudin freeze-dried powder has high activity, high recovery rate, high purity and long storage period, and can provide safe and high-quality raw materials for industries such as food, health food, medicine or cosmetics.

Description

technical field [0001] The process belongs to the fields of separation and purification of biomedicine, and refining of traditional Chinese medicine, and specifically relates to a production process for efficiently separating high-activity hirudin based on a novel anion exchange column. Background technique [0002] Hirudin is a protein that exists in the salivary glands of blood-sucking leeches (leeches). It has highly specific antithrombin activity and inhibits the binding of thrombin to substrates. It is the strongest natural thrombin found so far. specific inhibitors. In 1984, Haycraft found that leech extract had anticoagulant effect. In 1904, Jocoby isolated the active ingredient from medical leech for the first time and named it hirudin. In 1955, Markwardt successfully isolated and purified pure hirudin from the head of the medical leech, and began basic biochemical and pharmacological research. In 1970, he determined that hirudin was a specific inhibitor of thrombi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/815C07K1/18
CPCC07K14/815
Inventor 冯会藏赵堃李培培王辉赵平李璐璐黄明贤
Owner 科康生物医药(深圳)有限公司
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