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Refolding solution of recombinant human endostatin as well as preparation and application method thereof

A technology of endostatin and vascular endothelium, which is applied in the field of refolding solution of recombinant human endostatin, can solve the problems of matching and low refolding yield, and achieve the two goals of increasing refolding yield, promoting dissolution, maintaining and stabilizing The effect of hierarchical structure

Active Publication Date: 2014-08-20
DENOVO BIOPHARMA HANGZHOU LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention is that when refolding recombinant human vascular endostatin in the prior art, the refolding solution used cannot make the two pairs of disulfide bonds of endostatin properly paired, resulting in The problem of low yield, and then provide a refolding solution that can achieve high refolding yield of recombinant human endostatin, and also ensure the large-scale production of recombinant human endostatin

Method used

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  • Refolding solution of recombinant human endostatin as well as preparation and application method thereof
  • Refolding solution of recombinant human endostatin as well as preparation and application method thereof
  • Refolding solution of recombinant human endostatin as well as preparation and application method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] In this example, 1 L of the refolding solution of recombinant human endostatin is composed of 1 mol guanidine hydrochloride, 2 mmol reduced glutathione, 0.3 mmol oxidized glutathione, 2 mmol EDTA, 10 mmol HAc-NaAc , 10mmol DTT composition, pH value is 5.0.

[0035] When preparing the above-mentioned refolding liquid, it is first necessary to select the above-mentioned specific amounts of guanidine hydrochloride, reduced glutathione, oxidized glutathione, EDTA, DTT, and 1 L of HAc-NaAc solution, and then dissolve the above components in the HAc-NaAc solution.

[0036] When using the refolding solution prepared above to refold the denatured and purified recombinant human endostatin protein solution, first, 100 ml of the denatured and purified recombinant human endostatin protein solution with a protein concentration of 10 mg / ml Add it into 15L refolding solution at a rate of 8ml / min, mix evenly, let it stand at 4°C for 10 hours, and remove the refolding solution by ultra...

Embodiment 2

[0038] In this example, 1L of the refolding solution of recombinant human endostatin is composed of 2mol guanidine hydrochloride, 2.5mmol reduced glutathione, 0.2mmol oxidized glutathione, 8mmolEDTA, 20mmol HAc-NaAc Composition, pH 4.0.

[0039] When preparing the above-mentioned refolding liquid, it is first necessary to select the above-mentioned specific amounts of guanidine hydrochloride, reduced glutathione, oxidized glutathione, EDTA, and 1 L of HAc- NaAc solution, and then dissolve the above components in HAc-NaAc solution.

[0040] When using the refolding solution prepared above to refold the denatured and purified recombinant human endostatin protein solution, first, 125 ml of the denatured and purified recombinant human endostatin protein solution with a protein concentration of 8 mg / ml Add it into 12.5L refolding solution at a rate of 8ml / min, mix evenly, let it stand at 8°C for 24 hours, and remove the refolding solution by ultrafiltration equilibrium.

Embodiment 3

[0042] In this example, 1L of the refolding solution of recombinant human endostatin is composed of 1.2mol guanidine hydrochloride, 3mmol reduced glutathione, 0.25mmol oxidized glutathione, 5mmol EDTA, 15mmol HAc- Composition of NaAc, pH 4.5.

[0043]When preparing the above-mentioned refolding liquid, it is first necessary to select the above-mentioned specific amounts of guanidine hydrochloride, reduced glutathione, oxidized glutathione, EDTA, and 1 L of HAc- NaAc solution, and then dissolve the above components in HAc-NaAc solution.

[0044] When using the refolding solution prepared above to refold the denatured and purified recombinant human endostatin protein solution, first, 100 ml of the denatured and purified recombinant human endostatin protein solution with a protein concentration of 9 mg / ml Add it into 20L of refolding solution at a rate of 8ml / min, mix evenly, let stand at 2°C for 160h, remove the refolding solution by ultrafiltration equilibrium, and that’s it. ...

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Abstract

The invention relates to a refolding solution of recombinant human endostatin. The refolding solution comprises the following components: guanidine hydrochloride, reduced glutathione, oxidized glutathione and a complexing agent and the pH value of the refolding solution is 4.0-5.0. The invention also provides a preparation and application methods of the refolding solution of recombinant human endostatin. By virtue of the refolding solution of recombinant human endostatin disclosed by the invention, the high refolding yield of recombinant human endostatin can be realized, thereby ensuring the mass production of the recombinant human endostatin.

Description

technical field [0001] The invention belongs to the field of extraction and purification of recombinant human endostatin, and in particular relates to a refolding solution of recombinant human endostatin and its preparation and use methods. Background technique [0002] Human vascular endostatin is currently the most effective inhibitor of tumor angiogenesis with the best experimental effect. Experiments have shown that endostatin can inhibit vascular endothelial cells and growing blood vessels, and can better inhibit tumor growth and metastasis. In terms of its mechanism of action, human endostatin inhibits the blood supply of tumor tissue in the human body, causing the tumor to lack nutrients and oxygen and stop growing, and then gradually shrink until death. , has the advantage of no obvious toxic and side effects, and thus has received extensive attention from the medical community. [0003] At present, human endostatin is mainly prepared by obtaining engineering bacte...

Claims

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Application Information

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IPC IPC(8): C07K14/475C07K1/14
CPCC07K14/78
Inventor 荣志刚姚建林赵唯一黄佩华朱浩文陈卫崔志远徐霞
Owner DENOVO BIOPHARMA HANGZHOU LTD
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