Mono methoxy polyethylene glycol-insulin complex substance and its preparation method

A polyethylene glycol and monomethoxy technology, which is applied in the field of peptide or protein modification complexes and their preparation, can solve the problems of reduced bioavailability, small molecular weight, short half-life, etc., so as to improve biological stability, improve Bioavailability, the effect of not easy hydrolysis separation

Inactive Publication Date: 2005-08-10
EAST CHINA NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, insulin is a protein polypeptide drug, which is easily hydrolyzed by proteases in the body, resulting in unstable physical and chemical properties and reduced bioavailability; the molecular weight is small, 5.8KD, and it is easily filtered by the glomerulus, resulting in a short half-life in vivo
For example, the clinical plan for treating patients with type I diabetes is: 0.5~1U / Kg, three times a day, such injections for a long time have brought pain and inconvenience to the patients; 1. High-dose insulin injection will cause the body to produce antibodies, so that the normal physiological function of insulin cannot be exerted, and even cause allergies and insulin resistance in severe cases.

Method used

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  • Mono methoxy polyethylene glycol-insulin complex substance and its preparation method
  • Mono methoxy polyethylene glycol-insulin complex substance and its preparation method
  • Mono methoxy polyethylene glycol-insulin complex substance and its preparation method

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1 Preparation method of mPEG-modified insulin complex. In this example, mPEG is mPEG-ALD with a molecular weight of 5KD.

[0025] The first step is to mix 0.75ml of 0.16% insulin solution with 0.6ml of 200mM boric acid-borax buffer at pH 7.4;

[0026] In the second step, add 0.96 mg of mPEG-ALD with a molecular weight of 5KD to the solution in the first step, and mix well;

[0027] Step 3 Add 0.15ml of 0.75% NaBH to the solution in the second step 3 CN solution, mixed into a reaction solution;

[0028] The fourth step is to place the reaction solution obtained in the third step at 4-40°C for 0.5-24 hours;

[0029] Step 5 Use glacial acetic acid to adjust the pH to 4.2 to stop the reaction, prepare the crude product solution of mPEG-modified insulin complex, take 20ul of the reaction solution for SDS-PAGE electrophoresis, and check the degree of modification. The results are shown in figure 1 ;

[0030] The sixth step is to dilute the crude product solution ...

Embodiment 2

[0033] Example 2 Preparation method of mPEG-modified insulin complex. In this example, mPEG is mPEG-ALD with a molecular weight of 5KD.

[0034] The first step is to mix 0.75ml of 0.16% insulin solution with 0.6ml of 200mM boric acid-borax buffer at pH 7.4;

[0035] In the second step, add 3.84 mg of mPEG-ALD with a molecular weight of 5KD to the solution in the first step, and mix well;

[0036] The third step to the seventh step is carried out according to the corresponding operation steps in embodiment 1;

[0037] In the eighth step, the single-point modification absorption peak solution collected in the seventh step is concentrated by ultrafiltration with a Millipore Amicon Ultra-15 ultrafiltration tube with a molecular weight cut-off of 1-5KD, and freeze-dried to obtain 0.30-0.35 mg of single-point modification mPEG The pure product of the modified insulin complex has a molecular weight of 10.8KD.

Embodiment 3

[0038] Example 3 Preparation method of mPEG-modified insulin complex. In this example, mPEG is mPEG-ALD with a molecular weight of 5KD.

[0039] The first step is to mix 0.75ml of 0.16% insulin solution with 0.6ml of 200mM boric acid-borax buffer at pH 7.4;

[0040] In the second step, add 9.6 mg of mPEG-ALD with a molecular weight of 5KD to the solution in the first step, and mix well;

[0041] The third step to the seventh step is carried out according to the corresponding operation steps in embodiment 1;

[0042] In the eighth step, the single-point modification absorption peak solution collected in the seventh step is concentrated by ultrafiltration with a Millipore Amicon Ultra-15 ultrafiltration tube with a molecular weight cut-off of 1-5KD, and freeze-dried to obtain 0.27-0.31 mg of single-point modification mPEG The pure product of the modified insulin complex has a molecular weight of 10.8KD.

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Abstract

A methoxy polyethylene glycol (mPEG) modified insulin compound is prepared from INS protein (insulin) and methoxy polyethylene glycol derivative through modifying reaction to obtain the solution of coarse product, ion exchange chromatography, collecting, centrifugal ultrafiltering, concentrating, and freeze drying to obtain high-purity product.

Description

technical field [0001] The present invention relates to a monomethoxypolyethylene glycol-insulin complex and a preparation method thereof, specifically, to a monomethoxypolyethylene glycol derivative, i.e., an mPEG-modified insulin complex and a preparation method thereof, It belongs to the technical field of peptide or protein modification compound and its preparation. Background technique [0002] Insulin (INS) is a protein hormone synthesized and secreted by pancreatic beta cells, consisting of two polypeptide chains, A and B, connected by two disulfide bonds, and contains 51 amino acid residues. The relative molecular weight of the biologically active insulin monomer is 5.8KD. Insulin is the earliest (1921) discovered protein with hormonal effects, and it is also the first protein to be crystallized, the sequence of amino acid residues determined, artificially chemically synthesized, and mass-produced by genetic engineering techniques to be put on th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/62C07K17/02C08G65/00
Inventor 郁正艳吴自荣黄静楼旻
Owner EAST CHINA NORMAL UNIV
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