Preparation and application of recombinant consensus interferon mutant polyethylene glycol conjugate

A technology of polyethylene glycol and polyethylene glycol propionaldehyde, which is applied to the preparation methods of interferon and peptides, and medical preparations of non-active ingredients, etc., which can solve the problems of short half-life in vivo, low biological activity, and strong immunogenicity and other issues to achieve the effect of low immunogenicity

Active Publication Date: 2012-05-16
BEIJING KAWIN TECH SHARE HLDG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Ganfujin is the same as natural alpha interferon, and has the disadvantages of poor stability, short half-life in vivo, and strong immunogenicity.
At the same time, the first cysteine ​​at the N-terminal needs to form a disulfide bond with the 99th cysteine, which cannot be effectively modified at the N-terminus
Although the U.S. Patent US005985265A of Amgen Company discloses the method for modifying the N-terminus of the rejuvenation protein, the biological activity retained after modification rate and modification is significantly lower than that of other proteins (recombinant human granulocyte colony-stimulating Factor G-CSF and recombinant human growth hormone GH, etc.)

Method used

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  • Preparation and application of recombinant consensus interferon mutant polyethylene glycol conjugate
  • Preparation and application of recombinant consensus interferon mutant polyethylene glycol conjugate
  • Preparation and application of recombinant consensus interferon mutant polyethylene glycol conjugate

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0063] Example 1, Modification conditions of recombinant integrated interferon variant PEGylated conjugate (PEG20-IFN-SA)

[0064] IFN-SA solution (concentration: 10mg / mL, buffer: 100mM PB, pH7.0) was prepared by Chongqing Fujin Biopharmaceutical Co., Ltd., mPEG-ButyrALD-20KD (purity>95%, relative molecular mass: 20×10 3 ) was purchased from Beijing Jiankai Technology Co., Ltd., sodium cyanoborohydride (CH3BrNa) solution was purchased from Sigma Company, and SDS-PAGE related reagents and solutions and various analytical grades were all products of Shanghai Sangong. Constant temperature magnetic stirrer (Jiangsu Zhongda Instrument Factory), electrophoresis instrument, SP Sepharose F.F., Superdex75 molecular sieve, AKTA explore chromatography system, and SDS-PAGE electrophoresis system are all products of Pharmacia.

[0065] 1. The influence of reaction pH value on the modified product

[0066] The IFN-SA solution was prepared with 100mmol / L PB (pH4.0, pH5.0 and pH5.5) buffer s...

example 2

[0076] Example 2, Separation and Purification of Recombinant Integrated Interferon Variant PEGylated Conjugate (PEG20-IFN-SA)

[0077] The modified sample PEG-IFN-SA was dialyzed overnight with 5×DDW, and the dialyzed sample was applied to SP Sepharose F.F chromatographic column. The SP Sepharose F.F chromatography column was equilibrated with BufferA (10mM acetic acid-sodium acetate pH5.5) and loaded on the sample, respectively, with elution 1 (10mM acetic acid-sodium acetate 0.15M NaCl pH5.5), elution 2 (10mM acetic acid -sodium acetate 0.2M NaCl pH5.5), elution 3 (10mM acetic acid-sodium acetate 0.25M NaCl pH5.5), elution 4 (10mM acetic acid-sodium acetate 0.35M NaCl pH5.5), elution 5 (10mM Acetic acid-sodium acetate (0.45MNaCl pH5.5) was used for elution, the breakthrough peak and each elution peak were collected respectively, and samples were taken for SDS-PAGE electrophoresis analysis. Collect the PEG-IFN-SA sample purified by SPSepharose F.F chromatography, and put it ...

example 3、10

[0079] Example 3, 10KD and 40 polyethylene glycol modified recombinant integrated interferon variants

[0080] Select respectively the monomethoxypolyethylene glycol butyraldehyde of 10KD and the branched chain monomethoxypolyethylene glycol propionaldehyde of 40KD (both are the products of Beijing Jiankai Biological Company) according to the method of example 1 and example 2 to IFN -SA was modified at the N-terminal single site specificity, and PEG10-IFN-SA and PEG40-IFN-SA were obtained after separation and purification (see image 3 ).

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Abstract

The invention relates to a recombinant consensus interferon mutant polyethylene glycol conjugate, and a preparation method and applications thereof, and is characterized in that a recombinant consensus interferon mutant is chemically modified at a single locus at an amino terminal by an aldehyde group-activated polyethylene glycol molecule with a molecular weight of 10 KD-40 KD. The modified recombinant consensus interferon mutant polyethylene glycol conjugate has the characteristics of prolonged in-vivo half life, reduced immunogenicity, and long-term antiviral activity in vivo. The invention also discloses a preparation method of the recombinant consensus interferon mutant polyethylene glycol conjugate, and provides applications of its combined medicaments in the treatment and prevention of viral infection or hyperplastic diseases, or the regulation of immune functions.

Description

technical field [0001] The invention belongs to the fields of biotechnology and pharmacy, and in particular relates to a polyethylene glycol conjugate of recombinantly integrated interferon variants and its preparation method and application. The present invention is an extension of the invention patent titled "Human Alpha Interferon Derivatives with Antiviral Activity" (Patent No. ZL01102915.3). Background technique [0002] As early as 1957, lssacs et al. discovered that virus-infected cells produced a factor that could resist virus infection and interfere with virus replication, hence the name interferon (IFN). Interferon is a group of glycoproteins encoded by the genome of susceptible cells after inactivated or live viruses act on susceptible cells. Its activity and antigenicity depend on the protein in the molecule, and have nothing to do with its sugar group. According to its source and structure, IEN can be divided into IFN-α, IFN-β, IFN-γ, which are produced by leu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/555C07K1/16A61K38/21A61K47/48A61P31/12A61P35/00A61P37/04
CPCA61K47/48215C07K14/555A61K47/60A61P31/12A61P35/00A61P37/00A61P37/02A61P37/04
Inventor 范开罗华张继兰李祥张益
Owner BEIJING KAWIN TECH SHARE HLDG
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