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Monomodified PEGylated insulin and its preparation method

A technology of PEGylation and monomethoxy polyethylene glycol, which is applied in the field of polymer modified protein and polypeptide, can solve the problems of slow reaction speed, complicated steps, low yield and the like, and achieve the effects of low cost and simple operation.

Inactive Publication Date: 2009-06-03
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Boc protection of non-modified sites will increase production costs and complicate steps
The reaction rate of acid anhydride and amino group is slow and the yield is low
In addition, organic solvents and high pH can reduce insulin activity, which is not conducive to post-processing of products

Method used

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  • Monomodified PEGylated insulin and its preparation method
  • Monomodified PEGylated insulin and its preparation method
  • Monomodified PEGylated insulin and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] (1) Dissolve 1 part of insulin in pH 3.0 acetate buffer (containing NaCl), add 1 part of mPEG750-aldehyde and 10 parts of NaBH 3 CN, after stirring for 10 hours at 20°C, add a small molecule amino acid to terminate the reaction;

[0043] (2) dialyze the reaction solution of (1), remove small molecular salts, and concentrate;

[0044] (3) Put the concentrated solution on the SP Sepharose FF chromatography column, carry out gradient elution with a pH3.8 buffer solution containing 0-1M NaCl, and detect the absorption peak at 220nm UV;

[0045] (4) The collected eluate from peak 3 was concentrated, dialyzed, and freeze-dried to obtain a monomodified pegylated insulin, PheB1-PEG-insulin.

Embodiment 2

[0047] (1) Dissolve 1 part of insulin in pH4.0 acetate buffer (containing NaCl), add 2 parts of mPEG750-aldehyde and 5 parts of NaBH 3 CN, after stirring at 20°C for 8 hours, add a small molecule amino acid to terminate the reaction;

[0048] (2) dialyze the reaction solution of (1), remove small molecular salts, and concentrate;

[0049] (3) Put the concentrated solution on the SP Sepharose FF chromatography column, perform gradient elution with a pH4.0 buffer solution containing 0-1M NaCl, and detect the absorption peak at 220nm UV;

[0050] (4) The collected eluate from peak 3 was concentrated, dialyzed, and freeze-dried to obtain a monomodified pegylated insulin, PheB1-PEG-insulin.

Embodiment 3

[0052] (1) Dissolve 1 part of insulin in pH3.8 acetate buffer (containing NaCl), add 10 parts of mPEG750-aldehyde and 30 parts of NaBH 3 CN, after stirring at 20°C for 0.5 hours, add a small molecule amino acid to terminate the reaction;

[0053] (2) dialyze the reaction solution of (1), remove small molecular salts, and concentrate;

[0054] (3) Put the concentrated solution on the SP Sepharose FF chromatography column, perform gradient elution with a pH 2.0 buffer solution containing 0-1M NaCl, and detect the absorption peak at 220nm UV;

[0055] (4) The collected eluate from peak 3 was concentrated, dialyzed, and freeze-dried to obtain a monomodified pegylated insulin, PheB1-PEG-insulin.

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Abstract

The invention discloses single-decorative polietilenglicol insulin and making method with molecular weight at 6550-10800, which comprises the following steps: dissolving insulin in the acetate buffer with pH value at 3.0-5.0; adding mPEG-aldehyde and NaBH3CN; stirring; adding small-molecular amino acid to terminate reaction; dialyzing; condensing in the SP Sepharose FF chromatographic column; proceeding gradient elution through buffer with NaCl; collecting peak-3 eluent; condensing; dialyzing; freezing; drying to obtain PheB1-PEG-insulin.

Description

technical field [0001] The invention belongs to the technical field of polymer modified protein polypeptides, and specifically relates to monomodified pegylated insulin and a preparation method thereof. Background technique [0002] Insulin is an important polypeptide regulating hormone secreted by the beta cells of the vertebrate pancreas to control blood sugar. The primary structure of insulin from different species is slightly different. In 1955, Sanger et al. clarified the primary structure of bovine insulin. It consists of two peptide chains containing 51 amino acids, of which the A chain contains 21 amino acid residues, and the B chain contains 30 amino acid residues. The A chain and the B chain are covalently linked by two disulfide bonds. In addition, The A chain itself also has an intrachain disulfide bond. The secondary structure mainly has two configurations: α-helix and β-sheet. Its molecular weight is 5734. Since its introduction in 1921, it has been the dr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K47/48A61K38/28C07K17/08A61P3/10A61K47/60
Inventor 印春华窦怀智张敏
Owner FUDAN UNIV
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