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Method for preparing polyethylene glycol (PEG) and nerve growth factor (NGF) conjugate

A nerve growth factor and polyethylene glycol technology, applied in the field of biochemistry, can solve the problems of short plasma half-life and pain, and achieve the effect of reducing the frequency of administration and improving the half-life

Active Publication Date: 2013-03-13
SINOBIOWAY BIOMEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition to the disadvantages of being easily hydrolyzed by proteases and short plasma half-life like other protein drugs, there are also side effects of local pain after injection

Method used

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  • Method for preparing polyethylene glycol (PEG) and nerve growth factor (NGF) conjugate
  • Method for preparing polyethylene glycol (PEG) and nerve growth factor (NGF) conjugate
  • Method for preparing polyethylene glycol (PEG) and nerve growth factor (NGF) conjugate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Preparation of Nerve Growth Factor Modified by mPEG-Succinimidyl Valerate [mPEG-SVA]

[0049] Nerve growth factor PBS solution with a concentration of 0.68 mg / mL (the PBS solution of the company’s marketed product Enjingfu) and mPEG-SVA (manufactured by Laysan Bio) dissolved in a PBS solution with a pH of 7.4, with a material ratio of 1 :10 (NGF:PEG) and stirred at room temperature for 24 hours. The PEG-modified protein is run on a gel chromatography column (such as KW802.5) was purified by chromatography using 0.05M PBS buffer solution. The results are shown in Figure 1. It can be seen from the figure that the chromatographic peak 1 is the nerve growth factor modified by mPEG-SVA, and the chromatographic peak 2 is Excess mPEG-SVA during the modification reaction. PEG-modified proteins were identified by SDS-PAGE, the results are shown in figure 2 , where lanes 1-3 represent purified PEG-modified mouse nerve growth factor, and lane M is a molecular marker...

Embodiment 2

[0050] Example 2: Preparation of Nerve Growth Factor Modified by mPEG-aldyhyde [mPEG-AD]

[0051] Nerve growth factor PBS solution with a concentration of 0.68 mg / mL (the PBS solution of the company’s marketed product Enjingfu) and mPEG-AD (manufactured by Laysan Bio) dissolved in a PBS solution with a pH of 6, with a material ratio of 1: 10 (NGF:PEG) ratio mixed, add 10 times the amount of NGF NaBCNH 3 , stirred at room temperature for 24 hours. The PEG-modified protein is run on a gel chromatography column (such as KW802.5) was purified by chromatography using 0.05M PBS buffer solution. PEG-modified proteins were identified by SDS-PAGE. Eluted from the column corresponding to bound a PEG (PEG 1 -NGF) or two PEGs (PEG 2 -NGF) or three PEGs (PEG 3 -NGF) NGF protein. These proteins were pooled, concentrated, and the protein concentration (7 ng / mL) was determined by colorimetric analysis. PEG-NGF was stored in 0.05M PBS (pH=7.0) buffer at -20°C.

Embodiment 3

[0052] Example 3: Preparation of Nerve Growth Factor Modified by mPEG-hydrazine [mPEG-HZ]

[0053] Method 1: Nerve growth factor solution with a concentration of 0.68 mg / mL (the PBS solution of the company’s marketed product Enjingfu) and mPEG-HZ (produced by Laysan Bio) dissolved in a PBS solution with a pH of 6 The ratio of 1:10 (NGF:PEG) was mixed, and EDAC was added with 10 times the amount of NGF, and stirred at room temperature for 24 hours. The PEG-modified protein is run on a gel chromatography column (such as KW802.5) was purified by chromatography using 0.05M PBS buffer solution. PEG-modified proteins were identified by SDS-PAGE. Eluted from the column corresponding to bound a PEG (PEG 1 -NGF) or two PEGs (PEG 2 -NGF) or three PEGs (PEG 3 -NGF) NGF protein. The proteins were pooled, concentrated and the protein concentration was determined by colorimetric analysis. PEG-NGF was stored in 0.05M PBS (pH=7.0) buffer at -20°C.

[0054] Method 2: Nerve growth fact...

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Abstract

The invention relates to a method for preparing a polyethylene glycol (PEG) and nerve growth factor (NGF) conjugate. The method specifically comprises the following steps of: conjugating: dissolving an NGF and PEG into buffer liquid with the pH of 4-10, and stirring for 24 hours at room temperature, wherein the mass ratio of the NGF to the PEG is 1: 10; carrying out purification and recovery, thereby obtaining the PEG and NGF conjugate; and concentrating the recovered protein. The invention also discloses the PEG and NGF conjugate prepared according to the method, and compositions containing the PEG and NGF conjugate. The invention further discloses application of the PEG and NGF conjugate in medicine preparation. The method is simple and practicable; and after the NGF and the PEG are conjugated, the activity of the NGF is still kept, and the half life of the NGF in vivo can be prolonged, so that the administration frequency is reduced, and the pain of patients is reduced.

Description

technical field [0001] The present invention relates to the field of biochemistry, more specifically, the present invention relates to the preparation method of polyethylene glycol and nerve growth factor conjugate. Background technique [0002] Since the early 1980s, protein drugs have become one of the most important types of drugs in the international pharmaceutical market. Clinically, protein drugs not only have the advantages of specific action sites and clear curative effects, but also have many disadvantages, such as being easily hydrolyzed by proteases in the gastrointestinal tract, and are generally limited to injection administration; the plasma half-life is short and requires repeated treatment. Multiple injections; strong antigenicity, easy to cause adverse reactions such as allergies; low solubility, etc. As a result, it is difficult for patients to achieve therapeutically effective blood levels of the protein. [0003] These problems can be overcome by conjug...

Claims

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Application Information

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IPC IPC(8): C07K14/48C07K1/107A61K38/18A61K47/48
Inventor 任宏伟林昳陈星陈胜亮
Owner SINOBIOWAY BIOMEDICINE
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