Bionic affinity purifying process of interleukin I natural agonist

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

Inactive Publication Date: 2006-05-17
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), mix with 100ml deionized water, stir under ice bath conditions, when the temperature drops to 5℃, slowly add triazoxide (4.9g 100ml pre-cooled acetone dissolved), use saturated NaHCO 3 Keep the pH of the solution between 6 and 7, react at 5°C for 2 hours, take out 3×10 volumes of water / acetone (1:1, 1:3, 0:1, 1:1, 3:1, 1:0) Wash in order to remove unreacted triazoxide to obtain dichlorotriazoxide Sepharose (90ml).

[0018] Take dichlorotriazoxide Sepharose (49ml), weigh tryptophan (5g), dissolve it with dimethyl sulfoxide (30ml) and deionized water (20ml), mix with dichlorotriazoxide Sepharose, stir at 50℃ Reaction for 24h. During the reaction, use saturated NaHCO 3 Keep the pH of the reaction system between 7 and 7.5. After the reaction, the reaction mixture was washed thoroughly with 10×3 times the volume of distilled water to obtain 1-tryptophan-2-chlorotriazide Sepharose (40 ml).

[0019] Tak...

Embodiment 2

[0023] 1. Preparation of separation materials:

[0024] Take NH 2 -Sepharose (100ml), mix with 100ml deionized water, stir under ice bath conditions, when the temperature drops to 5℃, slowly add triazoxide (4.9g 100ml pre-cooled acetone dissolved), use saturated NaHCO 3 Keep the pH of the solution between 6 and 7, react at 5°C for 2 hours, take out 3×10 volumes of water / acetone (1:1, 1:3, 0:1, 1:1, 3:1, 1:0) Wash in order to remove unreacted triazoxide to obtain dichlorotriazoxide Sepharose (90ml).

[0025] Take dichlorotriazoxide Sepharose (49ml), weigh tryptophan (5g), dissolve it with dimethyl sulfoxide (30ml) and deionized water (20ml), mix with dichlorotriazoxide Sepharose, stir at 50℃ Reaction for 24h. During the reaction, use saturated NaHCO 3 Keep the pH of the reaction system between 7 and 7.5. After the reaction, the reaction mixture was washed thoroughly with 10×3 times the volume of distilled water to obtain 1-tryptophan-2-chlorotriazide Sepharose (40 ml).

[0026] Tak...

Embodiment 3

[0030] 1. Preparation of separation materials:

[0031] Take NH 2 -Sepharose (100ml), mix with 100ml deionized water, stir under ice bath conditions, when the temperature drops to 5℃, slowly add triazoxide (4.9g 100ml pre-cooled acetone dissolved), use saturated NaHCO 3 Keep the pH of the solution between 6 and 7, react at 5°C for 2 hours, and take out 3×10 volumes of water / acetone (1:1, 1:3, 0:1, 1:1, 3:1, 1:0) Wash in order to remove unreacted triazoxide to obtain dichlorotriazoxide Sepharose (90ml).

[0032] Take dichlorotriazoxide Sepharose (49ml), weigh tryptophan (5g), dissolve it with dimethyl sulfoxide (30ml) and deionized water (20ml), mix with dichlorotriazide Sepharose, stir at 50℃ Reaction for 24h. During the reaction, use saturated NaHCO 3 Keep the pH of the reaction system between 7 and 7.5. After the completion of the reaction, the reaction mixture was thoroughly washed with 10×3 volumes of distilled water to obtain 1-tryptophan-2-chlorotriazine Sepharose (40 ml).

...

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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, specifically, a biomimetic affinity purification method of a natural interleukin I antagonist. Background technique [0002] Interleukin I natural antagonist (IL-1RA) can effectively block the activity of IL-1, make the body's excessive TNF and IL-6 levels normalize without disturbing the internal environment balance, and treat certain cytokine disorders Related inflammatory diseases. At present, recombinant human IL-1ra has been clinically tested for the treatment of sepsis, rheumatoid arthritis, inflammatory bowel disease, asthma, myeloid leukemia, and graft versus host response. There are about 25 million patients with asthma in my country, which has become the second largest respiratory disease, and about 20 million patients suffer from allergic rhinitis. Therefore, there is an urgent need for large-scale purification technology for the production of interleukin I natural antagonist (IL-1R...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/54A61P1/00A61P11/06A61P19/02A61P29/00A61P35/00A61P37/06C07K1/22
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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