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mpeg-mal-aGLP-1 composite

A technology of mpeg-mal-aglp-1 and complexes, applied in the field of monomethoxypolyethylene glycol-human glucagon-like peptide-1 derivative complexes, can solve the problem of polyethylene glycol-modified protein/ Difficult to control the site and degree of modification of the polypeptide, difficult to purify and quality control, cumbersome preparation steps, etc., to achieve the effect of low cost, simple purification and high yield

Inactive Publication Date: 2013-10-02
EAST CHINA NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since the PEG-polypeptide / protein complex is a complex linked together by an amide bond formed by the amino acid free amino group of the protein / polypeptide and the succinimide ester bond of PEG, if the protein / polypeptide has multiple Amino acids, the position and degree of modification of proteins / polypeptides modified by polyethylene glycol are often difficult to control, resulting in difficulties in subsequent purification and quality control, cumbersome preparation steps, low yields, and high costs

Method used

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Examples

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Embodiment 1

[0028] Preparation of embodiment 1mPEG-MAL-aGLP-1 complex

[0029] In this embodiment, the structural formula of the aGLP-1 derivative is: aGLP-1(7-36), namely Seq ID No.2, and the sequence of Seq ID No.2 is as follows:

[0030] His-Xaa2-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Xaa20-Glu-Phe-Ile-Ala-Trp- Leu-Val-Xaa28-Gly-Xaa30.

[0031] Where Xaa2=D-Ala, Xaa20=Ser, Xaa28=Ala, Xaa30=Cys, and the molecular weight of mPEG-MAL is 2KD.

[0032] In the first step, 1.0 mg of aGLP-1 was dissolved in 0.5 ml of 100 mM, pH 7.4 phosphate buffer, and then mixed with 0.5 ml of 200 mM phosphate buffer of pH 6.0 to 9.0;

[0033] In the second step, add 0.6 mg of mPEG-MAL with a molecular weight of 2KD to the solution in the first step, and mix evenly;

[0034] In the third step, the solution obtained in the second step is placed at 4°C to 30°C for mPEG modification reaction for 0.5 to 24 hours;

[0035] In the fourth step, the solution treated in the third step...

Embodiment 2

[0041] Preparation of embodiment 2mPEG-MAL-aGLP-1 complex

[0042] In this example, the structural formula of the aGLP-1 derivative used is shown in Table 1, and the molecular weight of mPEG-MAL is 2KD.

[0043] In this example, the mPEG-MAL-aGLP-1 complex was prepared according to the experimental conditions and procedures in Example 1.

[0044] Table 1aGLP-1 derivative structure

[0045]

Embodiment 3

[0046] Embodiment 3 Preparation of mPEG-MAL-aGLP-1 complex

[0047] In this example, the structural formula of the aGLP-1 derivative is: aGLP-1(7-36), namely Seq ID No.2, wherein Xaa2=Ser, Xaa20=Ser, Xaa28=Ala, Xaa30=Cys, mPEG- The molecular weight of MAL is 20KD.

[0048] In the first step, 1.0 mg of aGLP-1 was dissolved in 0.5 ml of 100 mM, pH 7.4 phosphate buffer, and then mixed with 0.5 ml of 200 mM phosphate buffer of pH 6.0 to 9.0;

[0049] In the second step, add 6.0 mg of mPEG-MAL with a molecular weight of 20KD to the solution in the first step, and mix evenly;

[0050] The third step to the fifth step are carried out according to the corresponding operation steps in the first embodiment.

[0051] In the sixth step, the eluate collected in the fifth step was concentrated with a Millipore Amicon Ultra-15 ultrafiltration tube with a molecular weight cut-off of 1 to 5 KD, and freeze-dried to obtain 0.7 mg of aGLP-1 derivative with a molecular weight of 23.5 KD pure co...

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Abstract

The invention discloses an mPEG-MAL-aGLP-1 composite. The mPEG-MAL-aGLP-1 composite is formed by connecting human glucagon-like peptide-1 derivative aGLP-1 with monomethoxypolyethylene glycol-maleimide mPEG-MAL by virtue of an addition reaction, wherein a sulfydryl -SH of amino acid of a terminal C of aGLP-1 and a terminal double bond of mPEG-MAL form a covalent bond, so that aGLP-1 and mPEG-MAL are connected; aGLP-1 is of any structure shown in Seq ID No.2, Seq ID No.3 or Seq ID No.4. The mPEG-MAL-aGLP-1 composite avoids polymorphism of a modification site, so that follow-up purification is extremely simple, yield is high, and cost is low; a half-life period of the composite is further prolonged, continuous blood sugar level reducing time is 24 hours, and administration can be realized in an oral manner, so that the mPEG-MAL-aGLP-1 composite can be applied to treatment on diabetes.

Description

technical field [0001] The invention belongs to the technical field of peptide or protein modification complex and its preparation, in particular to a monomethoxypolyethylene glycol modified human glucagon-like peptide-1 (Glucagon-like Peptide1, GLP-1) derivative (GLP-1 derivative, aGLP-1), that is, a monomethoxypolyethylene glycol-human glucagon-like peptide-1 derivative complex. Background technique [0002] GLP-1 was first identified as a glucose-dependent enterocrine hormone peptide in 1987, mainly secreted by L cells of the distal ileum, colon and rectum. This 31-amino acid polypeptide signals through G protein-coupled receptors on target cells, resulting in glucose-dependent insulinotropic release, stimulation of somatostatin release, inhibition of glucagon secretion, and inhibition of gastric emptying and gastric acid Secretion and other physiological functions, it is a polypeptide drug for treating diabetes, especially type II diabetes, and has extensive social bene...

Claims

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Application Information

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IPC IPC(8): C07K14/605C07K1/107A61K38/26A61K47/48A61P3/10
Inventor 黄静吴自荣金丽潘盈盈杨芳
Owner EAST CHINA NORMAL UNIV
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