Conjugate of branched chair polymacrogol-interferon, and its preparing method

A technology of polyethylene glycol and interferon, applied in the direction of interferon, cytokines/lymphokines/interferon, pharmaceutical formulations, etc., can solve the problems of reducing the biological activity of interferon, prolonging the half-life is not obvious, and improving interferon, etc. Achieve long retention time, high antiviral activity, and extended half-life

Inactive Publication Date: 2012-07-25
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] At present, there are existing literature reports on PEG-modified interferon, but some have several problems. The first is that the biological activity of interferon is reduced after PEG is combined with interferon, and the dosage of interferon must be increased in use; secondly, after the formation of PEG When an interferon conjugate is used, the linker formed may be hydrolyzed in vivo, resulting in an insignificant extension of the half-life

Method used

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  • Conjugate of branched chair polymacrogol-interferon, and its preparing method
  • Conjugate of branched chair polymacrogol-interferon, and its preparing method
  • Conjugate of branched chair polymacrogol-interferon, and its preparing method

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Embodiment 1

[0025] Embodiment 1. Preparation of branched chain polyethylene glycol-composite interferon

[0026] Composite interferon was dissolved with 5mM sodium acetate buffer of pH4.0, and was prepared as a solution of 2mg / ml; by interferon: PEG was added mPEG2-NHS (Mw=10000, laboratory Homemade, the same below), adjust the pH to 9.0 with sodium hydroxide, react at 4°C for 24 hours, add 0.2ml of 0.5 M glycine to terminate the reaction; after 5 minutes, use 10 times the volume of 20mM sodium acetate buffer at pH4.0 Dilute the reaction solution; put the diluted reaction solution on a carboxymethylcellulose column (Waterman CM-52), wash the column with 6 times the volume of sodium acetate (or sodium phosphate) buffer solution with a pH of 4.0, and wash the column with 0.3M Sodium chloride-sodium acetate (or sodium phosphate) buffer was used for elution, and the eluate containing branched-chain polyethylene glycol-composite interferon was collected and detected by SDS-PAGE electrophoresis...

Embodiment 2

[0027] Embodiment 2. Preparation of branched-chain polyethylene glycol-interferon α-2a

[0028] Dissolve interferon α-2a with 5mM sodium acetate buffer solution pH4.0, and prepare a solution of 5 mg / ml; add mPEG2-NHS (Mw=20000) according to the ratio of interferon: PEG is 1:35 , adjust the pH to 8.0 with sodium hydroxide, react at 25°C for 12 hours, add 0.5ml of 0.5M glycine to terminate the reaction; after 5 minutes, dilute the reaction solution with 6 times the volume of 20mM pH4.0 sodium acetate buffer; The diluted reaction solution was applied to the (Waterman CM-52) column, washed with 6 times the volume of sodium acetate buffer solution with pH 4.0, and then eluted with sodium acetate buffer solution containing 0.3M sodium chloride, and the branched chain was collected. The eluate of polyethylene glycol-interferon α-2a was detected by SDS-PAGE electrophoresis. Branched-chain polyethylene glycol-interferon α-2a was further purified with Superdex200, the eluent was sodium...

Embodiment 3

[0029] Embodiment 3. Preparation of branched-chain polyethylene glycol-interferon α-2b

[0030] Interferon α-2b is dissolved with 5mM sodium acetate buffer solution of pH4.5, and is prepared into a solution of 3mg / ml; Add mPEG2-NHS (Mw=10000, Mw=10000, Laboratory self-made, the same below), adjust the pH to 8.0 with sodium hydroxide, react at 25°C for 5 hours, add 0.5ml of 0.5 M glycine to terminate the reaction; after 5 minutes, use 6 times the volume of 20mM sodium acetate pH4.0 Dilute the reaction solution with buffer; put the diluted reaction solution on the (Waterman CM-52) column, wash the column with 6 times the volume of sodium acetate buffer at pH 4.5, and then wash the column with sodium acetate buffer containing 0.3 M sodium chloride For elution, the eluate containing branched-chain polyethylene glycol-interferon α-2b was collected and detected by SDS-PAGE electrophoresis. Branched-chain polyethylene glycol-interferon α-2b was further purified with Superdex 200, th...

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Abstract

A compound of the branch chains PEG-interferon and its structural formula are disclosed. Said compound has high antiviral activity, long stay time in human body and long plasma half-life period (40-80 hr).

Description

field of invention [0001] The invention relates to a branched-chain PEG-interferon conjugate in which interferon is chemically modified with branched-chain polyethylene glycol (abbreviated: branched-chain PEG), and a preparation method thereof. Background technique [0002] Many natural and recombinant proteins have medicinal properties and, if purified and formulated, can be administered parenterally for a variety of medical indications. However, proteins administered by parenteral routes are likely to be immunogenic, and usually have a short plasma half-life. Therefore, it is difficult for a patient to achieve a therapeutically effective plasma concentration of a pharmaceutical protein. [0003] These problems can be overcome by combining proteins with high molecular weight polymers such as polyethylene glycol. Davis et al. disclosed in US Patent No. 4,179,337 a technical method for combining polyethylene glycol with proteins (enzyme and insulin) to obtain a conjugate wit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K47/48A61K38/21C07K14/555C08G65/00A61P31/12A61K47/60
Inventor 苏志国马光辉雷建都李兴奇郑春杨翟艳琴
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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