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Human interferon alpha derivatives and preparation and use of pegylated products thereof

A technology of pegylation and interferon alpha, applied in the field of biomedicine, can solve the problems of fast plasma clearance, limitation, poor stability, etc., and achieve the effect of treating or preventing viral infections or tumor diseases

Active Publication Date: 2012-10-03
BEIJING SIHUAN PHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The clinical therapeutic effect of the existing human interferon α preparation is not ideal yet, and its antiviral specific activity is only 1×10 8 IU / mg, there is a disadvantage of greater side effects in large doses
[0004] However, whether it is human interferon α-2a, human interferon α-1b or human interferon α-1a, as a protein drug, due to poor stability, high plasma clearance rate, short half-life in vivo, and easy antigen-antibody reaction, etc., Very limited in clinical treatment
Genetic engineering technology makes it possible to synthesize human recombinant proteins on a large scale, which largely solves the immunogenicity problem caused by heterologous proteins, but still cannot overcome the shortcomings of fast plasma clearance and low bioavailability
The result of these shortcomings is that frequent injections of human interferon are required to achieve an effective plasma therapeutic concentration; moreover, each injection will lead to large fluctuations in blood drug concentration, forming a "peak-valley" effect of drug concentration.
This may increase the cost of treatment as well as the inconvenience of administration and the risk of adverse reactions

Method used

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  • Human interferon alpha derivatives and preparation and use of pegylated products thereof
  • Human interferon alpha derivatives and preparation and use of pegylated products thereof
  • Human interferon alpha derivatives and preparation and use of pegylated products thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Secretion and expression of Glu-Phe-Met-IFNα-2a in methanolic yeast

[0036] 1. Acquisition and design of target genes

[0037] The amino acid sequence of Glu-Phe-Met-IFNα-2a (hereinafter referred to as IFN-D) is shown in SEQ ID NO: 1, and the sequence length is 168 amino acids.

[0038] After obtaining the cDNA of IFNα-2a through Genebank, the corresponding codons were changed to yeast preference. And the corresponding nucleotide sequence of Glu-Phe-Met was added at the N-terminus. The cDNA sequence was used to construct the PIC9K expression plasmid of Pichia pastoris. The constructed expression plasmid was transformed into GS115 host bacteria to achieve secretory expression. So adding KEX to the design 2 The enzyme recognition site CTC GAG AAA AGA, wherein CTC GAG is the XhoI restriction site. At the same time, a double stop codon TGA TAA and a NotI restriction sequence GCG GCCGC were introduced into the 3'??? end. SEQ ID NO: 2 is the cDNA sequence of ...

Embodiment 2I

[0047] The purification of embodiment 2IFN-D

[0048] 1. Cationic gel column (such as CM Sepharose F.F. gel) chromatography:

[0049] Acetate buffer with pH 3.8-4.6 was used for column loading and elution, and electrophoresis monitoring was used to collect target objects. Then use the pH7.5~8.5 Tris-HCl buffer solution to dialyze the target substance.

[0050] 2. Anion gel column (such as DEAE Sepharose F.F. gel) chromatography:

[0051] The pH7.5-8.5 Tris-HCl buffer solution is used for column loading and elution, and the target substance is collected. Then dialyze the target substance with pH 7.5-8.5 phosphate buffer solution.

[0052] 3. SDS-PAGE detection: The pass-through solution and the target peak of the above CM column and DEAE column were taken for detection. The test results showed that the fermentation broth was purified by the above column chromatography, and IFN-D with a purity of more than 95% was obtained. see attached results figure 2 shown.

Embodiment 3

[0053] Example 3 Preparation and purification process of PEG coupling modification sample:

[0054] 1. Dialyze the IFN-D sample with phosphate buffer, then add equimolar ALD-PEG20KD for modification at 2-15°C, and the reaction time is 24-36 hours. The obtained modified sample was tested by SDS-PAGE. The test results showed that after PEG coupling modification, the molecular weight increased from the original 19,000 Daltons to nearly 40,000 Daltons, and the modification rate reached more than 40%, and the target compound was obtained mPEG(20KD)-IFN-D. The results are attached image 3 shown.

[0055] 2. Purification of mPEG(20KD)-IFN-D cationic gel column (such as SP Sepharose F.F. gel):

[0056] Adjust the conductivity of the modified sample to 4.0-5.0 to terminate the reaction, and put it on the SP column for purification. Use acetate buffer for elution to collect target compounds. Dialyze the target substance against a phosphate buffered saline solution.

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Abstract

The invention relates to human interferon alpha derivatives and preparation and use of pegylated products thereof. The human interferon alpha derivatives are formed by bonding three amino acids to N terminals of human interferon alpha and have a structural formula of R3-R2-R1-interferon, wherein the interferon is the human interferon alpha, and a first position amino acid R1 bonded to the N terminals of the human interferon alpha is a sculpture amino acid or glycine; the second position amino R2 is an aromatic amino acid or glycine; and the third position amino acid R3 is an acidic amino acidor glycine. The invention also provides pegylated derivatives of the human interferon alpha derivatives, a preparation method thereof and use thereof in the preparation of medicaments for treating and preventing viral infection or lung cancer. The human interferon alpha derivatives have the advantages of high specific activity and high pegylation rate. The pegylated derivatives of the human interferon alpha derivatives have the characteristics of high biological activity and no non-N terminal modified isomers and have the advantages of prolonging in-vivo half-life, reducing plasma clearance and the like.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to the preparation and application of human interferon alpha derivatives and their pegylated modifications. Background technique [0002] Human interferon (IFN for short) is a kind of active protein with broad-spectrum anti-virus, anti-proliferation and immunoregulatory functions existing in the body. It is one of the earliest cytokines used in clinical treatment. Human interferon can be divided into three types: Types, namely human interferon-α, human interferon-β, and human interferon-γ, can be further divided into several subtypes according to the amino acid sequence of each type of IFN. A large number of clinical studies have proved that human interferon-α is an important anti-tumor and anti-viral drug. At present, the most widely used clinically in my country is mainly recombinant human interferon α-1a, α-2a and α-1b. [0003] It has been found that the type I family of h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/56C12N15/21C12N15/81C07K1/107A61K38/21A61P31/12A61P35/00
Inventor 夏中宁张丽杰舒军丁成刚
Owner BEIJING SIHUAN PHARMA
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