Polyethylene glycol modified porcine alpha-interferon, preparation method and application thereof

A polyethylene glycol and interferon technology, applied in the field of biomedicine, can solve the problems of undetectable interferon activity, short activity retention time, inconvenient drug use, etc., to achieve long retention time in the body, avoid recognition and removal, reduce The effect of enzymatic hydrolysis

Inactive Publication Date: 2011-11-02
BEIJING DAWEIJIA BIOTECH SHARE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Schering-Plough uses 12kD linear monomethoxypolyethylene glycol succinimidyl carbonate for modification, the in vivo activity retention time is too short, basically no interferon activity can be detected in the bo

Method used

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  • Polyethylene glycol modified porcine alpha-interferon, preparation method and application thereof
  • Polyethylene glycol modified porcine alpha-interferon, preparation method and application thereof
  • Polyethylene glycol modified porcine alpha-interferon, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] The preparation method of the porcine alpha-interferon of embodiment 1 polyethylene glycol modification

[0052] 1) Preparation of pig α-interferon borate solution

[0053] Take commercially available porcine α-interferon (dissolved in PBS buffer, purchased from Beijing Luyuan Chuangsheng Biotechnology Co., Ltd.), at 4°C, dialyze with PBS buffer, concentrate, and then replace the buffer , replace porcine α-interferon into boric acid buffer (pH 9.0). Store at 4°C. The protein concentration was determined by the LOWRY method, and the protein concentration was adjusted to 10 mg / ml.

[0054] 2) Preparation of PEG hydrochloric acid solution

[0055] Take 100mg of methoxypolyethylene glycol-amide-propionic acid succinimide ester (mPEG-NHS, molecular weight 20kD, purchased from Beijing Jiankai Technology Co., Ltd.), dissolve it in 0.5ml of 0.1M hydrochloric acid solution, and set aside.

[0056] 3) PEG modification reaction

[0057] The PEG hydrochloric acid solution was ...

Embodiment 2

[0062] The preparation method of the porcine alpha-interferon of embodiment 2 polyethylene glycol modification

[0063] Operate in the same way as in Example 1, the only difference being:

[0064] The pH of the boric acid buffer in step 1) is 8.4, and the concentration of porcine α-interferon protein is adjusted to 2.5 mg / ml.

[0065] In step 2), 100mg of methoxypolyethylene glycol carboxylic acid (mPEG-COOH, molecular weight 30kD, purchased from Beijing Jiankai Technology Co., Ltd.) was taken and dissolved in 1ml of 0.05M hydrochloric acid solution.

[0066] In step 3), the PEG hydrochloric acid solution was added to 10 ml of the borate solution containing porcine α-interferon, and stirred at 500 rpm for 4 hours at 4°C. The pH value was adjusted to 4.8 by adding glacial acetic acid to terminate the reaction. Add 50 ml of sodium chloride solution.

[0067] SDS-PAGE electrophoresis detection results and figure 1 same.

[0068] After cation exchange chromatography, a single...

Embodiment 3

[0069] The preparation method of the porcine alpha-interferon of embodiment 3 polyethylene glycol modification

[0070] Operate in the same way as in Example 1, the only difference being:

[0071] The pH of the boric acid buffer in step 1) is 8.7, and the concentration of porcine α-interferon protein is adjusted to 5 mg / ml.

[0072] Step 2) get 100mg methoxy polyethylene glycol amine (mPEG-NH 2 , molecular weight 40kD, purchased from Beijing Jiankai Technology Co., Ltd.), dissolved in 0.5ml of 0.1M hydrochloric acid solution.

[0073] In step 3), the PEG hydrochloric acid solution was added to 6 ml of borate solution containing porcine α-interferon, and stirred at 800 rpm for 4 hours at 4°C. The pH value was adjusted to 4.5 by adding glacial acetic acid to terminate the reaction. Add 50 ml of sodium chloride solution.

[0074] SDS-PAGE electrophoresis detection results and figure 1 same.

[0075] After cation exchange chromatography, a single PEG-modified porcine α-inter...

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Abstract

The invention discloses a polyethylene glycol modified porcine alpha-interferon and a preparation method thereof. The preparation method comprises the following steps: coupling polyethylene glycol with amino groups on lysine of porcine alpha-interferon under alkaline condition, and then performing cation exchange chromatography for separating and purifying, thereby acquiring the polyethylene glycol modified porcine alpha-interferon. The invention also discloses a long-acting injection containing the polyethylene glycol modified porcine alpha-interferon. The polyethylene glycol modified porcine alpha-interferon provided by the invention has the advantages that the modified rate is about 50%, the purity is above 97%, and the existing time in vivo is long.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a polyethylene glycol-modified pig alpha-interferon and a preparation method thereof, and a long-acting injection containing the polyethylene glycol-modified pig alpha-interferon. Background technique [0002] Porcine α-interferon is a low-molecular-weight glycoprotein produced by white blood cells through antiviral responses when the body is infected with viruses. It has biological activities such as antiviral, antitumor, and immune regulation. Porcine α-interferon is used to treat various porcine viral diseases. In clinical application, its circulating half-life in the body is short, has immunogenicity and antigenicity, is easy to be cleared by the circulatory system, and requires multiple administrations. Pigs bring great stress and affect the therapeutic effect. Also increase labor cost simultaneously, use inconvenience. [0003] Polyethylene glycol (PEG) is a neutral p...

Claims

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Application Information

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IPC IPC(8): C07K14/56C07K1/18C07K1/113A61K38/21A61K47/48A61K9/08A61P31/12A61P35/00A61P37/02
Inventor 赵宝凯刘美廖峰
Owner BEIJING DAWEIJIA BIOTECH SHARE CO LTD
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