A kind of renaturation method of recombinant protein

A technology of recombinant protein and denatured protein, which is applied in the field of fermentation industry, can solve the problems of insufficient uniformity of dissolved oxygen, uncontrollable process parameters, poor process repeatability and scalability, and achieve strong operability, easy promotion, and improved process reliability. controlling effect

Active Publication Date: 2022-01-18
BEIJING TIDE PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the traditional mixed renaturation method, the dissolved oxygen in the entire liquid environment is not uniform enough, the process parameters are uncontrollable, and the process repeatability and scalability are poor.

Method used

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  • A kind of renaturation method of recombinant protein
  • A kind of renaturation method of recombinant protein
  • A kind of renaturation method of recombinant protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1. Refolding of Recombinant Human Thrombopoietin Mimetic Peptide-Fc Fusion Protein (550ml)

[0060] (1) Preparation of denaturing solution

[0061] Prepare 50ml of denaturing solution, the components of which are: 8M guanidine hydrochloride, 20mM mercaptoethanol, 50mM Tris, and adjust the pH to 9.2 with hydrochloric acid. Add inclusion bodies until the final protein concentration of the refolding system is 0.5-1.5 mg / ml. Input amount of inclusion body: Weigh the inclusion body with a balance before and after drying, calculate the dry-wet weight ratio of the inclusion body, and make the dry weight 20g / L denaturing solution.

[0062] (2) Preparation of refolding solution

[0063] Prepare 500ml refolding liquid, its composition is: 2M urea, 50mM arginine, adjust with hydrochloric acid to make pH 9.2.

[0064] (3) mixed renaturation

[0065] Mix the denatured protein solution and the refolding solution at a ratio of 1:10 and carry out refolding in a 1L fermenter...

Embodiment 2

[0084] Example 2. Refolding of Recombinant Human Thrombopoietin Mimetic Peptide-Fc Fusion Protein (22L)

[0085] (1) Preparation of denaturing solution

[0086] Prepare 2L of denaturing solution, the components of which are: 8M guanidine hydrochloride, 10mM mercaptoethanol, 50mM Tris, and adjust the pH to 9.0 with hydrochloric acid. Input amount of inclusion bodies: Weigh the inclusion bodies with a balance before and after drying, and calculate the dry-wet weight ratio of the inclusion bodies so that the dry weight is 17.5g / L denaturing solution.

[0087] (2) Preparation of refolding solution

[0088] Prepare 20L of refolding liquid, its composition is: 2M urea, 50mM arginine, adjust the pH to 9.0 with hydrochloric acid.

[0089] (3) mixed renaturation

[0090] Denatured protein solution and refolding solution were mixed at a ratio of 1:10 and refolded in a 45L fermenter. Maintain the temperature at 5-15° C., ventilation oxygen partial pressure at 10%-60%, stirring speed ...

Embodiment 3

[0097] Example 3. Refolding of Recombinant Human Thrombopoietin Mimetic Peptide-Fc Fusion Protein (176L)

[0098] (1) Preparation of denaturing solution

[0099] Prepare 16L, its composition is: 8M guanidine hydrochloride, 20mM mercaptoethanol, 50mM Tris, adjust the pH to 9.0 with hydrochloric acid. Input amount of inclusion bodies: Weigh the inclusion bodies with a balance before and after drying, and calculate the dry-wet weight ratio of the inclusion bodies so that the dry weight is 17.5g / L denaturing solution.

[0100] (2) Preparation of refolding solution

[0101] Prepare 160L, its composition is: 2M urea, 50mM arginine, adjust the pH to 9.0 with hydrochloric acid.

[0102] (3) mixed renaturation

[0103] Denatured protein solution and refolding solution were mixed at a ratio of 1:10 and refolded in a 300L fermenter. Maintain the temperature at 5-15°C, ventilate oxygen partial pressure at 10%-60%, stirring speed at 60-80rpm, and pH 9.0 to maintain the renaturation con...

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Abstract

The invention belongs to the field of fermentation industry, and specifically relates to a refolding method of recombinant protein, which comprises fully dissolving the inclusion body of the recombinant protein with a denaturing solution, and taking the supernatant after centrifugation to obtain a denatured protein solution; mixing the denatured protein solution with the refolding solution Then add to the fermenter; control the temperature, pH and stirring speed of the fermenter, and at the same time inject gas under the liquid surface for renaturation. The present invention uses the fermenter fermented by bacteria to carry out renaturation, and utilizes the function of monitoring and recording parameters such as pH, stirring speed, and temperature in the fermenter, without the need for separate equipment and mixing devices, and at the same time adopts ventilation under the liquid surface, and uses Fermentation tank flowmeter and other devices effectively control the partial pressure to ensure the controllability of the entire renaturation process, which is conducive to the repeatability of the process. Compared with the traditional mixed renaturation method, the present invention improves the controllability, traceability and stability of the renaturation process, reduces disulfide bond mismatches, improves product quality, reduces operation difficulty, and successfully realizes Enhancing the scalability of the renaturation process.

Description

technical field [0001] The invention belongs to the field of fermentation industry, and in particular relates to a refolding method of recombinant protein, especially a large-scale refolding method of recombinant protein. Background technique [0002] Recombinant protein is a protein drug derived from living organisms produced by genetic engineering and cell engineering technologies. Recombinant proteins have become the most important products produced by biotechnology today. In the past two decades, protein drugs have developed rapidly. At present, there are nearly 100 recombinant protein drugs on the market, and thousands of them are under research and development. Europe and the United States have strong overall strength in protein drug research and have a large global market. Recombinant protein drugs can be divided into peptides and genetic engineering drugs, monoclonal antibodies and genetic engineering antibodies, and recombinant vaccines. Compared with previous sm...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/113C07K19/00
CPCC07K1/1136C07K14/524C07K2319/30
Inventor 齐连权李晓然崔颖王延山张琪姜玲郑博恺
Owner BEIJING TIDE PHARMA
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