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Preparation method of human insulin esters

A technology of human insulin ester and human insulin, applied in the field of preparation of human insulin ester, can solve problems such as low insulin concentration, and achieve the effect of saving cost and improving use efficiency

Active Publication Date: 2015-05-06
ZHUHAI UNITED LAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The invention overcomes the defect of low insulin concentration in the existing insulin transpeptide reaction

Method used

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  • Preparation method of human insulin esters
  • Preparation method of human insulin esters
  • Preparation method of human insulin esters

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] (1) Take the human proinsulin solution expressed by Pichia pastoris (references: Gao Jiankun, Cai Shaoxi, Fan Kai, etc., containing short C-peptide human proinsulin analog desB 30 Expression and purification in Pichia pastoris, Advances in Biochemistry and Biophysics, 2008, 35 (1): 63-68 "prepared) after macroporous adsorption, reversed-phase chromatography was used to elute with isopropanol, wherein The eluate contains 25% isopropanol by volume, add trypsin according to the mass ratio of proinsulin to trypsin 300:1, stir slowly, and obtain desB-containing 30 The solution of human insulin was adjusted to pH 3.0 with 3M phosphoric acid, and the enzyme cleavage reaction was terminated. desB 30 desB in solution of human insulin 30 The concentration of human insulin is 1.0mM, add the equivalent of desB 30 Zinc chloride equivalent to the molar amount of human insulin, adjusted to pH 5.80 with 5M ammonia water, placed at 4-8°C for 4 hours, then suction-filtered with a 300-...

Embodiment 2

[0038] Dissolving o-tert-butyl ether-l-threonine tert-butyl acetate with desB using dimethyl sulfoxide 30 Human insulin water-containing powder (prepared according to step (1) of Example 1), followed by adding 1,4-butanediol and trypsin aqueous solution (prepared with pure water) to obtain a reaction system, stirred at 25±1°C, and transpeptide reaction 8h, to obtain human insulin ester; wherein, desB 30 The final concentration of human insulin in the reaction system is 17mM, desB 30 Human insulin and o-tert-butyl ether-l-threonine tert-butyl acetate in a molar ratio of 1:15, desB 30 The mass ratio of human insulin and trypsin is 40:1, water (including desB 30 The volume ratio of water contained in human insulin powder and water in trypsin aqueous solution), dimethyl sulfoxide and 1,4-butanediol is 20%:40%:40%.

[0039] HPLC detection and analysis detected the transpeptide efficiency at 2, 4, 6, and 8 hours, and the transpeptide efficiency at 2, 4, 6, and 8 hours were 85.52%...

Embodiment 3

[0041] (1) Take the aqueous solution of human insulin pro-insulin refolded after expression in Escherichia coli (references: Weng Liang, Liu Lijun, Wang Zhi, etc. Purification of recombinant human insulin and preliminary identification of its properties. Biotechnology Bulletin, 2003, 14 (5) : 387-389), add trypsin according to the mass ratio of proinsulin to trypsin 300:1, stir slowly, the solution after enzymatic digestion at 37℃, in which desB 30 The concentration of human insulin is 0.5mM according to desB 30 The molar ratio of human insulin: zinc chloride is 1:1.5, add zinc chloride, adjust the pH to 5.60 with 3M phosphoric acid, precipitate at 4-8°C for 4 hours, and obtain desB by suction filtration 30 Human insulin water-containing powder, its water content is 45% as measured by a moisture analyzer, and its content is detected by HPLC.

[0042] (2) Use dimethyl sulfoxide to dissolve o-tert-butyl ether-l-threonine tert-butyl ester hydrochloride (H-Thr(tBu)-OtBu.HCl) and ...

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Abstract

The invention discloses a preparation method of human insulin esters. The preparation method of the human insulin esters comprises the steps of: centrifuging or leaching a solution containing desB30 human insulin so as to obtain desB30 human insulin water-containing powder; and carrying out a transpeptidation reaction by using the desB30 human insulin water-containing powder to obtain human insulin esters, wherein the final concentration of the desB30 human insulin in the transpeptidation reaction system is 13 mM to 33 mM, the molar ratio of the desB30 human insulin to threonine esters or derivatives thereof is 1: 15-150, and the mass ratio of the desB30 human insulin to trypsin is 10-120: 1. The preparation method of the human insulin esters overcomes the disadvantage of relatively low concentration of insulin in the conventional insulin transpeptidation reaction, increases unit reaction efficiency as the concentration of the insulin in the peptide transferase reaction is up to 13 mM to 33 mM, saves cost as the transpeptidation efficiency can be up to 91.80% to the uppermost, and is suitable for industrial large-scale production.

Description

technical field [0001] The invention belongs to the field of human insulin preparation technology, and in particular relates to a preparation method of human insulin ester. Background technique [0002] As the population ages, eating disorders, physical inactivity and obesity continue to increase, the incidence of diabetes will increase in both developed and developing countries. There are currently more than 360 million diabetic patients worldwide, and diabetes has become the third leading cause of death after cancer and cardiovascular disease. At the same time, diabetes is a silent killer. Chronic complications of diabetes have become the main cause of disability or premature death of diabetic patients. Many early symptoms are easily ignored, while late complications bring people a lot of pain. [0003] At present, the number of diabetic patients in my country is close to 100 million. In current clinical application, insulin is the most effective drug for lowering blood ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/00
Inventor 曹春来谭叶林肖拥军
Owner ZHUHAI UNITED LAB
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