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Recombinant human interferon beta-1b modified by polyethylene glycol and preparation method of recombinant human interferon beta-1b

A technology of recombinant human interferon and polyethylene glycol, which is applied in the field of protein chemistry, can solve problems such as difficulties, stability and physiological activity cannot be guaranteed, and achieve the effects of simple separation, improved pharmacological and pharmacokinetic properties, and increased use efficiency

Active Publication Date: 2015-06-03
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

The stability and physiological activity of the products obtained by traditional amino modification cannot be guaranteed, which makes it difficult for these products to apply for new drugs. At present, there are no polyethylene glycol-modified interferon-β drugs on the market at home and abroad.

Method used

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  • Recombinant human interferon beta-1b modified by polyethylene glycol and preparation method of recombinant human interferon beta-1b
  • Recombinant human interferon beta-1b modified by polyethylene glycol and preparation method of recombinant human interferon beta-1b
  • Recombinant human interferon beta-1b modified by polyethylene glycol and preparation method of recombinant human interferon beta-1b

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Nitrogen terminal oxidation of embodiment 1 recombinant human interferon beta-1b

[0056] Weigh 2 mg of IFN-β-1b lyophilized powder and dissolve it in 2 mL of 50 mM PB, pH 4.0, 0.1% SDS buffer solution. Add 200 μL of 1 mg / mL NaIO 4 Aqueous solution, NaIO 4 The molar feed ratio with IFN-β-1b is 10:1. React for 1h at 25°C. Then, 100 μL of ethylene glycol was added to the reaction system to terminate the reaction, and the reaction was carried out at room temperature for 10 min. The protein peak was collected by gel column chromatography, and the sample was added with achromatic magenta color development reagent for color development. The color development result shows that the oxidized IFN-β-1b sample is bright red, which is the characteristic color of achromatic magenta to detect the aldehyde group, and the color development result of the unoxidized sample is colorless, indicating that NaIO 4 Oxidation of IFN-β-1b produces aldehyde-based products.

Embodiment 2

[0057] Example 2 polyethylene glycol hydrazide 5000 (mPEG 5k -HZ) Preparation of site-directed single-modification recombinant human interferon β-1b

[0058] Take the oxidized recombinant human interferon β-1b solution, add polyethylene glycol hydrazide powder, the molecular weight is 5000Da, and the molar feed ratio of polyethylene glycol hydrazide and recombinant human interferon β-1b is 10:1. The reaction was shaken at pH 4.5 and 25° C. for 24 h.

[0059] After the reaction is completed, take the reaction solution and carry out chromatographic separation through the Sephacryl S-200HR column, the mobile phase is containing 20mM PB, 0.1M Na 2 SO 4 buffer system (pH7.4). The flow rate is 0.8mL / min, and the single loading volume is 500μL. The detection wavelength was 280nm, and the peak was collected. The collected sample was dialyzed overnight in 20mM PB, 5% mannitol system, and concentrated by ultrafiltration.

[0060] The results of the reaction and separation were dete...

Embodiment 3

[0061] Example 3 polyethylene glycol hydrazide 20000 (mPEG 20k -HZ) Preparation of site-directed single-modification recombinant human interferon β-1b

[0062] Take the oxidized recombinant human interferon β-1b solution, add polyethylene glycol hydrazide powder, the molecular weight is 20000Da, and the molar feed ratio of polyethylene glycol hydrazide and recombinant human interferon β-1b is 10:1. The reaction was shaken at pH 4.5 and 25° C. for 24 h.

[0063] After the reaction is completed, take the reaction solution and carry out chromatographic separation through the Sephacryl S-200HR column, the mobile phase is containing 20mM PB, 0.1M Na 2 SO 4 buffer system (pH7.4). The flow rate is 0.8mL / min, and the single loading volume is 500μL. The detection wavelength was 280nm, and the peak was collected. The collected sample was dialyzed overnight in 20mM PB, 5% mannitol system, and concentrated by ultrafiltration.

[0064] The results of the reaction and separation were d...

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Abstract

The invention relates to a recombinant human interferon beta-1b (IFN-beta-1b) modified by polyethylene glycol and preparation method of the recombinant human interferon beta-1b modified by polyethylene glycol. The preparation method comprises the following steps of: oxidizing serine (Ser) at a nitrogen terminal of the recombinant human interferon beta-1b into an aldehyde group, and then covalently coupling the aldehyde group with polyethylene glycol derivatives (PEG-NH2, PEG-O-NH2, PEG-HZ) capable of reacting with aldehyde group to obtain the recombinant human interferon beta-1b product subjected to site-specific modification of polyethylene glycol at the nitrogen terminal. The recombinant human interferon beta-1b modified by polyethylene glycol still has the physiological activity of the recombinant human interferon beta-1b, but is superior to original protein in aspects such as immunogenicity, pharmacology, pharmacokinetics, pharmacodynamics and the like.

Description

technical field [0001] The invention belongs to the field of protein chemistry, and relates to a polyethylene glycol-modified protein and a preparation method, in particular to a polyethylene glycol-point-modified product of the nitrogen terminal of recombinant human interferon beta-1b and a preparation method. Background technique [0002] With the development of biotechnology, biomacromolecular therapeutic drugs such as peptides and proteins are emerging, and have been more and more widely used in the treatment of diseases. Protein and peptide drugs have the advantages of high activity, low toxicity, clear biological function and strong specificity. However, there are also disadvantages such as easy hydrolysis by enzymes, short circulation half-life, high immunogenicity and low solubility. Therefore, the development of safe, effective, and highly bioavailable drug delivery systems is of great significance for improving the clinical effectiveness of protein drugs. After m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/565C07K1/113A61K38/21A61P31/12A61P35/00A61P35/02A61P25/00A61P29/00A61P37/02
Inventor 张竞周展苏志国马光辉
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI