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Bionic affinity purifying process of interleukin I natural agonist

A purification method and antagonist technology, applied in the field of bionic affinity purification of interleukin I natural antagonists, can solve the problems of unstable properties, high cost of monoclonal antibody preparation, short service life, etc., and reduce the steps of large-scale purification , large-scale purification, and the effect of reducing production costs

Inactive Publication Date: 2008-04-02
上海荣君生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The monoclonal antibody used in this method is not suitable for large-scale production due to its high preparation cost, unstable properties and short service life

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] 1. Preparation of separation materials:

[0017] Take NH 2 -Sepharose (100ml), mixed with 100ml deionized water, stirred under ice bath conditions, when the temperature dropped to 5°C, slowly added triazoxide (4.9g dissolved in 100ml pre-cooled acetone), washed with saturated NaHCO 3 Keep the pH of the solution between 6 and 7, react at 5°C for 2 hours, take out, 3×10 times the volume of water / acetone (1:1, 1:3, 0:1, 1:1, 3:1, 1:0) were washed successively to remove unreacted triazoxide to obtain dichlorotriazoxide Sepharose (90ml).

[0018] Take dichlorotriazoxide Sepharose (49ml), weigh tryptophan (5g) and dissolve it with dimethyl sulfoxide (30ml) and deionized water (20ml), mix with dichlorotriazoxide Sepharose, stir at 50°C Under reaction 24h. During the reaction, saturated NaHCO 3 Keep the pH of the reaction system between 7 and 7.5. After the reaction, the reaction mixture was fully washed with 10×3 volumes of distilled water to obtain 1-tryptophan-2-chlorot...

Embodiment 2

[0023] 1. Preparation of separation materials:

[0024] Take NH 2 -Sepharose (100ml), mixed with 100ml deionized water, stirred under ice bath conditions, when the temperature dropped to 5°C, slowly added triazoxide (4.9g dissolved in 100ml pre-cooled acetone), washed with saturated NaHCO 3 Keep the pH of the solution between 6 and 7, react at 5°C for 2 hours, take out, 3×10 times the volume of water / acetone (1:1, 1:3, 0:1, 1:1, 3:1, 1:0) were washed successively to remove unreacted triazoxide to obtain dichlorotriazoxide Sepharose (90ml).

[0025] Take dichlorotriazoxide Sepharose (49ml), weigh tryptophan (5g) and dissolve it with dimethyl sulfoxide (30ml) and deionized water (20ml), mix with dichlorotriazoxide Sepharose, stir at 50°C Under reaction 24h. During the reaction, saturated NaHCO 3 Keep the pH of the reaction system between 7 and 7.5. After the reaction, the reaction mixture was fully washed with 10×3 volumes of distilled water to obtain 1-tryptophan-2-chlorot...

Embodiment 3

[0030] 1. Preparation of separation materials:

[0031] Take NH 2 -Sepharose (100ml), mixed with 100ml deionized water, stirred under ice bath conditions, when the temperature dropped to 5°C, slowly added triazoxide (4.9g dissolved in 100ml pre-cooled acetone), washed with saturated NaHCO 3 Keep the pH of the solution between 6 and 7, react at 5°C for 2 hours, take out, 3×10 times the volume of water / acetone (1:1, 1:3, 0:1, 1:1, 3:1, 1:0) were washed successively to remove unreacted triazoxide to obtain dichlorotriazoxide Sepharose (90ml).

[0032] Take dichlorotriazoxide Sepharose (49ml), weigh tryptophan (5g) and dissolve it with dimethyl sulfoxide (30ml) and deionized water (20ml), mix with dichlorotriazoxide Sepharose, stir at 50°C Under reaction 24h. During the reaction, saturated NaHCO 3 Keep the pH of the reaction system between 7 and 7.5. After the reaction, the reaction mixture was fully washed with 10×3 volumes of distilled water to obtain 1-tryptophan-2-chlorot...

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Abstract

The bionic affinity purifying process of natural interleukin I agonist belongs to the field of biotechnology. The present invention includes the following steps: 1. the separate reaction of basic chromatographic medium, after being activated with trichloro triazozine, to tyrosine and aminobenzo formamidine to synthesize bionic affinity separating material; and 2. preparing affinity chromatographic column with the synthesized bionic affinity separating material, making the protein sample containing human natural interleukin I agonist flow through the affinity chromatographic column for the protein containing human natural interleukin I agonist to be adsorbed onto the column, flushing the column to eliminate hetero protein, altering the buffering liquid for flushing the column so as to elute the protein containing human natural interleukin I agonist and to obtain purified protein containing human natural interleukin I agonist. The present invention has less purifying steps, long separating material life and low production cost.

Description

technical field [0001] The invention relates to a method in the field of biotechnology, in particular to a method for biomimetic affinity purification of interleukin I natural antagonist. Background technique [0002] Interleukin I natural antagonist (IL-1RA) can effectively block the activity of IL-1, make the body's excessively high TNF and IL-6 levels tend to normal without disturbing the internal environment balance, and treat certain cytokine disorders related inflammatory diseases. At present, recombinant human IL-1ra has been clinically used in the treatment of sepsis, rheumatoid arthritis, inflammatory bowel disease, asthma, myelogenous leukemia, and graft-versus-host reaction and other diseases. There are about 25 million asthma patients in my country, which has become the second largest respiratory disease, and about 20 million people suffer from allergic rhinitis. Therefore, there is an urgent need for large-scale purification techniques for the production of in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/54C07K1/22A61P29/00A61P19/02A61P11/06A61P1/00A61P35/00A61P37/06
Inventor 李荣秀贵艳丽程永刚
Owner 上海荣君生物医药科技有限公司
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