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Preparation method of recombinant human insulin

A technology of recombinant human insulin and human insulin, which is applied in fermentation and other directions, can solve the problems of low transpeptide rate, instability, high cost, etc., and achieve the effect of reducing dosage, reducing cost and reducing cost

Active Publication Date: 2013-09-18
ZHUHAI UNITED LAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The technical problem to be solved by the present invention is to improve the disadvantages of multi-step, low transpeptide rate, high cost and instability in the current preparation process of recombinant human insulin.

Method used

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  • Preparation method of recombinant human insulin
  • Preparation method of recombinant human insulin
  • Preparation method of recombinant human insulin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] (1) desB 30 Preparation of human insulin water-containing powder: recombinant human insulin precursor secreted and expressed by Pichia pastoris, purified by macroporous adsorption resin (references: Gao Jiankun, Cai Shaoxi, Fan Kai, etc., containing short C-peptide human proinsulin analog desB 30 Expression and purification in Pichia pastoris, Progress in Biochemistry and Biophysics, 2008, 35 (1): 63-68) The sample solution was diluted 2 times and then reversed-phase purified, gradient eluted with isopropanol, Obtain the recombinant human insulin precursor aqueous solution containing isopropanol (concentration is 24% by volume) (HPLC detection spectrum is as follows: figure 1 As shown in a), adjust the pH value to 8.5 with 5M ammonia water. Trypsin was added according to the mass ratio of trypsin to recombinant human insulin precursor 1:300, and digested at 25°C for 3 hours to obtain a digested solution. The recombinant digested rate was 97.55% by HPLC (the HPLC detect...

Embodiment 2

[0053] (1) desB 30 Preparation of aqueous human insulin powder: Dilute the recombinant human insulin precursor aqueous solution in Example 1 to a concentration of isopropanol of 10% by volume, and adjust the pH to 9 with 5M ammonia water. Add trypsin according to the mass ratio of trypsin to recombinant human insulin precursor 1:400, and digest at 25°C for 5 hours. The enzyme cleavage rate of recombinant human insulin precursor was detected to be 95.31%. Add zinc chloride according to the molar ratio of human insulin precursor to zinc chloride 1:1.5, adjust the pH value to 5.8 with 3M phosphoric acid solution, place it overnight at 4-8°C, and filter it with a 300-mesh filter membrane to obtain desB 30 Human insulin water-containing powder, and measure the water content and content.

[0054] (2) with desB 30 The reaction concentration of human insulin is 13.4mM for transpeptidation reaction to obtain recombinant human insulin ester: dissolve desB with dimethyl sulfoxide 30 ...

Embodiment 3

[0057] (1) desB 30 Preparation of human insulin water-containing powder: the recombinant human insulin precursor in Example 1 was eluted with ethanol during reverse-phase purification, the eluate was diluted to an ethanol concentration of 30% by volume, and the pH value was adjusted to 8.5 with 5M ammonia water. Add trypsin according to the mass ratio of trypsin to recombinant human insulin precursor 1:300, and digest at 25°C for 6 hours. The enzyme cleavage rate of recombinant human insulin precursor was detected to be 96.07%. Add zinc chloride according to the molar ratio of human insulin precursor to zinc chloride 1:0.5, adjust the pH to 5.80 with 3M phosphoric acid, place it overnight at 4-8°C, and centrifuge at 8000g to obtain desB 30 Human insulin aqueous powder. Determination of its moisture and content.

[0058] (2) with desB 30 The reaction concentration of human insulin is 12.1mM for transpeptidation reaction to obtain recombinant human insulin ester: dissolve de...

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Abstract

The invention discloses a preparation method of recombinant human insulin. The preparation method comprises the following steps of: performing an enzyme digestion reaction on a recombinant human insulin precursor in a mixed solution of an organic solution and water to obtain a human insulin solution containing desB30; centrifuging or suction filtrating the human insulin solution containing desB30 to obtain water-containing powder of desB30 human insulin; performing a transpeptidation reaction on the water-containing powder of desB30 human insulin to obtain human insulin ester, wherein the final concentration of the desB30 human insulin in a transpeptidation reaction system is 12.1mM-33mM, the mol ratio of the desB30 human insulin to threonine ester or a derivative thereof is 1:(15-150), and the mass ratio of the desB30 human insulin to trypsin is (10-80):1; degreasing the recombinant human insulin ester to obtain the recombinant human insulin. The method is suitable for industrial production of converting yeast-expressed proinsulin into the human insulin, high in yield, simple to operation and low in cost.

Description

technical field [0001] The invention belongs to the field of insulin preparation, in particular to a preparation method of recombinant human insulin. Background technique [0002] At present, according to the statistics of the International Diabetes Federation in 2011, the number of people with diabetes in the world is 366 million, and it is expected to reach 552 million in 2030, which is equivalent to an increase of one diabetes patient every ten seconds, or an increase of 10 million patients every year. In my country, the prevalence of diabetes has nearly doubled in the past 10 years. In 2010, the prevalence rate of adult diabetes in my country was 9.7%, and the total number of patients has exceeded 90 million, becoming the world's largest diabetes country; at the same time, the number of people with pre-diabetic impaired glucose tolerance reached 148 million The disease rate is 15.5%. So the market for treating diabetes with insulin is huge. [0003] In current clinical...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/06
Inventor 谭叶林肖拥军曹春来陈虹霖
Owner ZHUHAI UNITED LAB
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