Renaturation method of insulin glargine precursor

A technology of insulin glargine and precursor, applied in the preparation methods, chemical instruments and methods of insulin and peptides, etc., can solve problems such as low recovery rate, achieve the effects of less by-products, shorten reaction time, and increase protein content

Active Publication Date: 2014-04-02
ZHUHAI UNITED LAB
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  • Claims
  • Application Information

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Problems solved by technology

Wang Lili C07K14/62, Chinese patent CN103172727A discloses a method for high-efficiency size-exclusion chromatography renaturation and simultaneous purification of recombinant human proinsulin. This method was tried in the renatura

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  • Renaturation method of insulin glargine precursor
  • Renaturation method of insulin glargine precursor
  • Renaturation method of insulin glargine precursor

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Embodiment 1

[0056] Refolding of embodiment 1 insulin glargine precursor

[0057] At room temperature, dissolve 10 grams of insulin glargine precursor in 588 mL of 6M, pH9.5 urea solution to make the protein concentration of insulin glargine precursor about 17 g / L; then add 5 g of DTT to adjust the pH value to 9.5, control The temperature of the reaction system was 35° C., and the reaction was carried out for 30 minutes.

[0058] Add the denatured insulin glargine precursor solution to 10 mM, pH9.5 Tris-HCl buffer solution, and use the Tris-HCl buffer solution to set the volume to 10 L, so that the protein concentration of the insulin glargine precursor is 1.0 g / L, Then add 5g PEG4000, 500mL glycerol, 8mg CuSO 4 , adjust the pH value to 9.5, with 20cm 3 Air was continuously fed into the refolding solution at a rate of / H, the temperature of the reaction system was controlled at 4°C, and the reaction was stopped after 12 hours of reaction. Take 20 μL of the refolding solution and analyze...

Embodiment 2

[0059] Refolding of embodiment 2 insulin glargine precursor

[0060] At room temperature, dissolve 10 grams of insulin glargine precursor in 357 mL of 8M, pH 11.5 urea solution, so that the protein concentration of insulin glargine precursor is about 28 g / L, add 2.5 g of DTT, adjust the pH value to 11.5, control The temperature of the reaction system was 45° C., and the reaction was carried out for 60 minutes.

[0061] Add the denatured insulin glargine precursor solution to 50mM, pH11.5 Tris-HCl buffer solution, and use the Tris-HCl buffer solution to set the volume to 7.14L, so that the protein concentration of the insulin glargine precursor is 1.4g / L , then add 14.28g PEG4000, 1428mL glycerol, 22.85mg CuSO 4 , adjust the pH value to 11.5, to 714cm 3 Air was continuously fed into the refolding solution at a rate of / H, the temperature of the reaction system was controlled at 15°C, and the reaction was stopped after 25 hours of reaction. Take 20 μL of the refolding solutio...

Embodiment 3

[0062] Refolding of embodiment 3 insulin glargine precursor

[0063] At room temperature, dissolve 10 grams of insulin glargine precursor in 588 mL of 6M, pH 9.5 urea solution to make the protein concentration of insulin glargine precursor about 17 g / L, add 5 g of cysteine ​​hydrochloride to adjust the pH The value is 9.5, the temperature of the reaction system is controlled at 35° C., and the reaction is carried out for 30 minutes.

[0064] Add the denatured insulin glargine precursor solution to 10mM, pH9.5 glycine dilution buffer, and dilute to 10L with the glycine dilution buffer to make the protein concentration of the insulin glargine precursor 1.0g / L, then add 5g PEG4000, 500mL glycerol, 6.75mg CuCl 2 , adjust the pH value to 9.5, with 20cm 3 Air was continuously fed into the refolding solution at a rate of / H, and the temperature of the reaction system was controlled at 4 ° C. After 12 hours of reaction, the reaction was stopped. Take 20 μL of the refolding solution...

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Abstract

The invention discloses a renaturation method of an insulin glargine precursor, and belongs to the field of biomedical protein folding. The method comprises the following steps: dissolving the insulin glargine precursor in a modifier solution, adding a reducer for reduction, adjusting the pH value to be 9.5-11.5, controlling the temperature of a reaction system to be 35-45 DEG C, and reacting for 30-60 minutes to obtain a modified insulin glargine precursor solution; adding the modified insulin glargine precursor solution into a dilute buffer solution, adding a protein folding additive, adjusting the pH value to be 9.5-11.5, continuously inletting air into the solution, controlling the temperature of the reaction system to be 0-20 DEG C, and reacting for 2-40 hours to obtain an insulin glargine renaturation solution. According to the method, the renaturation reaction time is shortened, the correctly folded protein content is increased, the renaturation efficiency is improved to be 51%-62%, the production cost is reduced, and the large-scale industrialization production and application are facilitated.

Description

technical field [0001] The invention belongs to the field of biomedical protein folding, and in particular relates to a renaturation method of insulin glargine precursor, in particular to an air oxidation dilution renaturation method using polyethylene glycol, glycerin and metal ions as protein folding additives, Converting the misfolded insulin glargine precursor into a precursor with the correct spatial structure, the renaturation efficiency can exceed 50%, which reduces the generation of disulfide bond mismatch by-products and brings great convenience to the subsequent purification work . Background technique [0002] Glargine insulin (glycine arginine insulin) is a long-acting insulin biological product produced by recombinant DNA technology and used to treat type Ⅰ and Ⅱ diabetes. It adds two arginines at the carboxy-terminal of the B-chain of human insulin, and replaces the asparagine at the A21 position of the carboxyl-terminus of the A-chain with glycine, which prev...

Claims

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Application Information

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IPC IPC(8): C07K14/62C07K1/14
CPCC07K14/62
Inventor 黄晓泉肖拥军曹春来张伟
Owner ZHUHAI UNITED LAB
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