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N-terminal site-specific modified PEGylated growth hormone antagonist and its preparation method

A growth hormone, polyethylene glycol technology, applied in the field of biomedicine, can solve problems such as reducing affinity, and achieve the effect of reducing loss

Active Publication Date: 2013-05-08
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This may be due to the modification of 4-6 5kDa PEG molecules on the 8 lysine residues and the N-terminal residues of the B2036 molecule, which reduces the affinity of the first binding site of Pegvisomant to GHR

Method used

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  • N-terminal site-specific modified PEGylated growth hormone antagonist and its preparation method
  • N-terminal site-specific modified PEGylated growth hormone antagonist and its preparation method
  • N-terminal site-specific modified PEGylated growth hormone antagonist and its preparation method

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Determination of the preparation conditions of embodiment 1M-P20K-GHA

[0024] GHA was replaced into 50 mM NaAc-HAc buffer (pH 5.5). Adjust the GHA concentration to 2.2 mg / mL (ie 0.1 mM), according to the GHA:PEG aldehyde (20kDa):NaCNBH3 molar ratio of 1:4:40, 1:4:80, 1:6:80, 1:6:60 , 1:6:120, 1:8:40, 1:8:80, and 1:8:160, react overnight at 4°C (~16 hours). The modified product was detected with a Superdex200 gel filtration column (1cm×30cm), and the eluent was 20mM phosphate buffer (pH7.2). The results of 20kDa PEG aldehyde modification of GHA are as follows figure 1 As shown, the yield was measured by the ratio of the peak area of ​​M-P20K-GHA to the total peak area. With 20kDa PEG aldehyde and NaCNBH 3 With the increase of the reaction ratio, the yield of M-P20K-GHA also increased. However, a multi-modified GHA, D-P20K-GHA, appeared and increased as the reaction ratio increased. In addition, the residual amount of GHA decreased significantly as the reaction rat...

Embodiment 2

[0025] Determination of the preparation conditions of embodiment 2M-P40K-GHA

[0026] GHA was replaced into 50 mM NaAc-HAc buffer (pH 5.5). Adjust GHA concentration to 2.2mg / mL (ie 0.1mM), according to GHA:PEG aldehyde (40kDa):NaCNBH 3 The molar ratios were 1:4:40, 1:4:80, 1:6:60, 1:6:120, 1:8:40, and 1:8:80, respectively reacted at 4°C overnight (~16 hours). The obtained binding product was detected by Superdex200 gel filtration column (1cm×30cm), and the eluent was 20mM phosphate buffer (pH7.2). Such as figure 2 As shown, it is basically the same as the 20kDa PEG aldehyde modification of GHA, and the 40kDa PEG aldehyde modification of GHA, with GHA:PEG aldehyde (40kDa):NaCNBH 3 With the increase of the molar ratio, the yield of M-P40K-GHA increased, and the residual amount of GHA gradually decreased. Although the molar ratio of GHA:PEG aldehyde (40kDa):NaCNBH3 is 1:8:80, more GHA modified products can be obtained, but due to the higher yield of D-P40K-GHA, GHA:PEG aldeh...

Embodiment 3

[0027] Example 3 Separation, purification and identification of M-P20K-GHA and M-P40K-GHA

[0028] As described in Example 1, select GHA:PEG aldehyde (20kDa):NaCNBH 3 A molar ratio of 1:8:80 was used to prepare M-P20K-GHA. As described in Example 2, select GHA:PEG aldehyde (40kDa):NaCNBH 3 A molar ratio of 1:4:80 was used to prepare M-P40K-GHA. The modified product was separated and purified by Superdex200 gel filtration column (1.6cm×60cm), and the eluent was 20mM phosphate buffer (pH7.2). Such as image 3 As shown, three elution peaks appeared after elution, corresponding to the double-modified product of PEG (two PEGs bound to each GHA), the single-modified product of PEG (one PEG bound to each GHA) and the unmodified The GHA. The two PEG-modified single-modified products were collected separately, concentrated and identified by SDS-PAGE and gel filtration chromatography. The eluate of the modified product was collected and concentrated for subsequent product identifi...

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Abstract

The invention relates to the biological medicine field, and concretely relates to an N-terminal site-specific modified PEGylated growth hormone antagonist (GHA) and its preparation method. The method comprises the following steps: 1, respectively carrying out site-specific modification of the N-terminal of GHA with PEG aldehydes having molecular weights of 20kDa and 40kDa; 2, separating and purifying two PEG modification products obtained in step 1 through gel filtration chromatography; and 3, preliminarily evaluating the pharmacodynamics of the two PEG modification products, wherein the PEG aldelyde having a molecular weight of 20kDa is optimized PEG. The modification products have high drug effects.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a polyethylene glycol (PEG) growth hormone antagonist with site-directed modification of the N-terminus, and a preparation method thereof. Background technique [0002] The main role of growth hormone is to promote growth, which involves regulating the growth of the body and regulating the metabolism of proteins, carbohydrates and lipids. The metabolic effects of growth hormone are mediated by insulin-like growth factor-I (IGF-I). Pituitary somatotropin adenoma is one of the common pituitary tumors. Due to excessive secretion of growth hormone (GH) and increased IGF-I concentration, it leads to gigantism and acromegaly, and its mortality rate can be as high as 20%-30%. The current treatment method is mainly to reduce or inhibit the secretion of growth hormone, so as to reduce the level of growth hormone and prevent the excessive secretion of growth hormone, but the treatment effect is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/61C07K1/107
Inventor 胡涛吴玲苏志国马光辉
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI