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Preparation method of insulin

A technology for insulin and proinsulin, which is applied in the field of insulin preparation, can solve the problems of uneasy control of the enzymatic digestion process, difficulty in industrial production, inconsistent action sites, etc., and achieve the effect of low cost, simple method and high recovery rate

Inactive Publication Date: 2013-12-04
GAN&LEE PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of adding two enzymes at the same time are 1. Since carboxypeptidase B can only enzymatically cut the basic amino acid at the carboxyl end of the peptide chain, carboxypeptidase B can only play a role after trypsin digestion, which will cause enzyme The cutting effect is incomplete and the efficiency is low; 2. The action sites of the two enzymes are inconsistent, which will lead to many by-products, and the enzyme cutting process is difficult to control
However, none of the preparation methods of the above-mentioned patents can meet this requirement. In fact, in the prior art, it is very difficult to prepare insulin with a purity of more than 99.7% with a high recovery rate, and to realize industrial production.

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1 prepares human insulin

[0041] Human proinsulin with the correct conformation was obtained according to steps A1 to C1 in the detailed description of the invention of European patent EP0055945, and prepared into a 1 mg / ml solution. Adjust the pH value of the above solution to 8 with concentrated ammonia water, then add trypsin so that the mass ratio of trypsin to human proinsulin is 1:200, react the mixture at 15°C for 2 hours, and adjust the pH value to 4 with hydrochloric acid To terminate the enzyme cleavage reaction to obtain Arg B31 - human insulin.

[0042] Purify the solution after the above enzyme digestion by hydrophobic chromatography, using phenyl sepharose as the chromatography medium, the eluent is ammonium sulfate solution dissolved in phosphate buffer, gradient 0M-2M, to obtain purified Arg B31 - a human insulin precursor.

[0043] The purified Arg B31 - Human insulin was made into a 1mg / ml solution with 1M Tris buffer at pH 7.5, and t...

Embodiment 2

[0046] Embodiment 2 prepares human insulin

[0047] According to Example 5-5.3 of Chinese patent CN98813941.3, the hGH-small proinsulin fusion protein with the correct conformation was obtained and prepared into a 10 mg / ml solution. Adjust the pH value of the above solution to 10.8 with NaOH, then add trypsin, make the mass ratio of trypsin and hGH-small proinsulin fusion protein 1:100, react the mixed solution at 4°C for 5 hours, adjust the pH value with phosphoric acid Adjusted to 3.5 to terminate the enzyme digestion reaction to obtain Arg B31 - human insulin.

[0048] The solution after the above enzyme digestion was subjected to cation exchange chromatography, using fast flow rate CM Sepharose TM As a chromatographic medium, the eluent is NaCl solution dissolved in 10mM citrate buffer, gradient 75mM-225mM, to obtain purified Arg B31 - a human insulin precursor.

[0049] The purified Arg B31 -Human insulin was prepared into a 10mg / ml solution with 50mM Tris-HCl buff...

Embodiment 3

[0052] Embodiment 3 prepares human insulin

[0053] Proinsulin in the correct conformation was obtained according to Example 1 of Chinese Patent CN200610117742.8 (paragraph 30-37 of the description), and prepared into a 15 mg / ml solution. Adjust the pH value of the above solution to 12 with NaOH, then add trypsin so that the mass ratio of trypsin to proinsulin is 1:400, react the mixture at 12°C for 3 hours, adjust the pH value to 4 with phosphoric acid to terminate Enzyme cleavage reaction to get Arg B31 - human insulin.

[0054] The solution after the above digestion was subjected to anion exchange chromatography, using DEAE Sepharose TM As a chromatographic medium, the eluent is NaCl solution dissolved in 0.1M Tris and 50% ethanol solution, gradient 20mM-50mM, to obtain purified Arg B31 - a human insulin precursor.

[0055] The purified Arg B31 -Human insulin is prepared as a 20mg / ml solution, and the pH value is adjusted to 10 with NaOH, and then carboxypeptidase B ...

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Abstract

The invention relates to a preparation method of insulin. The method which comprises two steps of chromatography purification can improve the enzymatic digestion efficiency, and the controllability in an enzymatic digestion process, so a complete reaction of trypsin and carboxypeptidase B is realized, and insulin precursors not reacted with the carboxypeptidase B can be thoroughly removed, so the purity and the quality of final insulin products are improved, the purity of the final insulin products can reach above 99.7%, and the recovery rate is high, thereby industrialized production can be carried out.

Description

technical field [0001] The invention relates to a preparation method of insulin, in particular to a preparation method comprising two steps of chromatographic purification of insulin. Background technique [0002] Diabetes is a common endocrine and metabolic disease. In recent years, the prevalence of diabetes has been increasing rapidly all over the world. In China, with the changes in people's lifestyles and the acceleration of the aging process, the prevalence of diabetes is on the rise. By the end of 2011, the number of diabetic patients in China had exceeded 92.4 million, becoming the second largest cardiovascular and cerebrovascular disease. Another important chronic non-communicable disease that seriously endangers people's health after disease and tumor. The acute and chronic complications of diabetes, especially the complications of chronic diseases, involve multiple organs, causing disability and high mortality, seriously affecting the physical and mental health ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/06C07K14/62C07K1/18
Inventor 王大梅李学勇刘晓宏甘忠如王冲
Owner GAN&LEE PHARMA
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