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Method for removing endotoxin in recombinant hirudin protein solution

A technology of recombinant hirudin and protein solution, which is applied in the field of separation and purification of biomedicine, can solve the problems of little research on the method of removing endotoxin of recombinant hirudin, low recovery rate of recombinant hirudin protein, and inapplicability to industrial production, etc., reaching The method is simple, the service life is prolonged, and the recovery rate is guaranteed

Active Publication Date: 2021-07-06
宁波博睿瀚达生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006]Usually, pharmaceuticals and biological products have strict restrictions on endotoxins, but there is little research on the methods of recombinant hirudin to remove endotoxins
The patent application with the publication number CN106834395A uses the fermentation supernatant to adjust the pH and heat in the tank to improve the protein purity of recombinant hirudin, and uses low-temperature static filtration to remove impurity proteins and lipids, while endotoxin It belongs to the lipopolysaccharide component, and it is difficult to effectively remove it by static separation, and the separation process of this method is complicated, and the recovery rate of the obtained recombinant hirudin protein is low, which is not suitable for industrial production

Method used

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  • Method for removing endotoxin in recombinant hirudin protein solution

Examples

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Embodiment 1

[0039] This embodiment provides a method for removing endotoxin in recombinant hirudin protein solution by anion-exchange high-performance liquid chromatography, using diethylaminoethyl (DEAE) bonded silica gel matrix as a chromatographic column (particle size 5 μm, pore size 300 angstroms) , diameter 40mm × length 500mm) packing, the specific steps are as follows:

[0040] Sampling: Take the recombinant hirudin protein solution with a purity of 95%, inject it at a flow rate of 80ml / min, the injection volume is 30% of the maximum loading capacity of the chromatographic column, and use Tris with a concentration of 10mmol / L and a pH value of 7.0 - Rinse with HCl buffer balance solution for column balance, and the wash volume is 5 times the column volume;

[0041] Elution: The pH value is 7.0, the Tris-HCl buffer containing NaCl is used as the mobile phase for elution, wherein the concentration of NaCl is 120mmol / L, the concentration of Tris-HCl is 10mmol / L, and the effluent is c...

Embodiment 2

[0045] This example provides a method for removing endotoxin in recombinant hirudin protein solution by anion-exchange high-performance liquid chromatography, using DEAE-bonded silica gel matrix as a chromatographic column (5 μm in particle size, 300 angstrom in pore size, 40 mm in diameter x 500 mm in length) packing ,Specific steps are as follows:

[0046] Sampling: Take the recombinant hirudin protein solution with a purity of 95%, inject it at a flow rate of 80ml / min, the injection volume is 30% of the maximum loading capacity of the chromatographic column, and use Tris with a concentration of 10mmol / L and a pH value of 7.0 - Rinse with HCl buffer balance solution for column balance, and the wash volume is 5 times the column volume;

[0047] Elution: elution is carried out with Tris-HCl buffer containing NaCl at a pH of 7.0 as the mobile phase, wherein the concentration of NaCl is 200mmol / L and the concentration of Tris-HCl is 10mmol / L, and the effluent is collected to obt...

Embodiment 3

[0050] The difference between this example and Example 1 is that the sample injection volume is 25% of the maximum sample loading volume of the chromatographic column, and the rest of the operating methods and process conditions are the same as in Example 1.

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Abstract

The invention relates to the technical field of separation and purification of biological medicines, in particular to a method for removing endotoxin in a recombinant hirudin protein solution. The method performs separation by adopting anion exchange chromatography and comprises the following steps: injection: the recombinant hirudin protein solution is taken for injection, the injection volume is 25%-40% of the maximum load of a chromatographic column, and washing is performed with an equilibrium solution to perform column equilibrium; elution: isocratic elution is performed with an eluent, and an outflow part is collected to obtain a recombinant hirudin eluent; and column regeneration: isocratic elution is performed with regenerated liquid, and an outflow part is collected to obtain an endotoxin eluent. According to the method, according to the strength of adsorption of recombinant hirudin and endotoxin with an anion exchanger under neutral conditions, the isocratic elution is performed by increasing salt concentration in a mobile phase, anda good removal effect is achieved by adjusting the injection volume, thus guaranteeing a relatively high protein recovery rate, and meanwhile, regeneration of the chromatographic column is implemented. The method is simple, efficient, low in cost and suitable for industrial production.

Description

technical field [0001] The invention relates to the technical field of separation and purification of biomedicine, in particular to a method for removing endotoxin in a recombinant hirudin protein solution. Background technique [0002] Leech is a traditional Chinese medicine in my country. It was first recorded in "Shen Nong's Herbal Classic". It has the effects of breaking blood, removing blood stasis, and dysmenorrhea. Hirudin extracted from leech salivary glands is an anticoagulant protein, which has a strong inhibitory effect on thrombin, and can be used to inhibit the accumulation of fibrinogen and platelets in damaged blood vessels, prevent thrombosis, and treat disseminated blood vessels Diseases such as internal coagulation. Compared with traditional anticoagulant drugs such as heparin and aspirin, hirudin has the advantages of small dosage, high curative effect, less adverse reactions and high safety, and has good clinical application value. [0003] Natural hiru...

Claims

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Application Information

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IPC IPC(8): C07K14/815C07K1/18
CPCC07K14/815Y02A50/30
Inventor 朱文瑾李浛君陈平李浛民
Owner 宁波博睿瀚达生物科技有限公司
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