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Recombinant human interferon-alpha 1b compound and process for preparation

A technology of recombinant human interferon and complex, which is applied in the field of modified complex of peptide or protein and its preparation, can solve the problems such as the inability of the drug to play a normal pharmacological action, the reduction of bioavailability, the poor biological stability, etc., so as to improve the bioavailability degree, long half-life and stable properties

Active Publication Date: 2006-12-13
SHANGHAI INST OF BIOLOGICAL PROD CO LTD +1
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, recombinant human interferon-α1b is a biologically active protein, which is easily degraded by proteases in the body, resulting in poor biological stability; at the same time, the molecular weight of recombinant human interferon-α1b is (relatively) small, 19.329KD, and is easily detected by renal failure. Globular filtration, thus short half-life in vivo
If you want to cure the disease, you need repeated long-term injections of drugs. For example, the clinical plan for the treatment of hepatitis B is: 4 million units / time, intramuscular injection, once a day, and continuous use for 3 months is a course of treatment. Some patients need multiple courses of treatment. Bring pain and inconvenience to the patient; In addition, because the patient receives long-term, repeated, high-dose administration, it will produce antibodies in the body, so that the given drug cannot normally exert its pharmacological effect and the bioavailability is reduced

Method used

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  • Recombinant human interferon-alpha 1b compound and process for preparation
  • Recombinant human interferon-alpha 1b compound and process for preparation
  • Recombinant human interferon-alpha 1b compound and process for preparation

Examples

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Embodiment 1

[0022] Example 1 Preparation method of mPEG-modified recombinant human interferon-α1b complex. In this example, the mPEG modifier is mPEG-SPA, a derivative of mPEG, with a molecular weight of 5KD.

[0023] The first step is to dissolve 1.0mg of recombinant human interferon-α1b protein in 0.5ml of 100mM, pH7.0 phosphate buffer, and mix with 0.5ml of 200mM boric acid-borax buffer of pH8.4;

[0024] Step 2 Add 0.25mg mPEG-SPA to the solution in the first step, and mix to form a reaction solution;

[0025] The third step is to place the reaction solution obtained in the second step at 4-40°C for 0.5-24 hours;

[0026] Step 4 Use glacial acetic acid to adjust the pH to 4.2 to terminate the reaction, prepare the crude mPEG-modified recombinant human interferon-α1b complex solution, take 20 μl of the reaction solution for SDS-PAGE electrophoresis, and check the degree of modification. The results are shown in figure 1 ;

[0027] The fifth step is to dilute the crude product soluti...

Embodiment 2

[0030] Example 2 Preparation method of mPEG-modified recombinant human interferon-α1b complex. In this example, the mPEG modifier is mPEG-SPA, a derivative of mPEG, with a molecular weight of 5KD.

[0031]The first step is to dissolve 1.0mg of recombinant human interferon-α1b protein in 0.5ml of 100mM, pH7.0 phosphate buffer, and mix with 0.5ml of 200mM boric acid-borax buffer of pH8.4;

[0032] Step 2 Add 1.25mg mPEG-SPA to the solution in the first step, and mix well to form a reaction solution;

[0033] The third step to the sixth step are carried out according to the corresponding operation steps in Example 1;

[0034] Step 7 Concentrate the collected absorption peak solution with MilliporeAmicon Ultra-15 ultrafiltration tube with a molecular weight cut-off of 1-5KD, freeze-dry to obtain 0.5-0.7 mg of single-point modified mPEG-modified recombinant human interferon-α1b The pure compound has a molecular weight of 24.329KD.

Embodiment 3

[0035] Example 3 Preparation method of mPEG-modified recombinant human interferon-α1b complex. In this example, the mPEG modifier is mPEG-SPA, a derivative of mPEG, with a molecular weight of 5KD.

[0036] The first step is to dissolve 1.0mg of recombinant human interferon-α1b protein in 0.5ml of 100mM, pH7.0 phosphate buffer, and mix with 0.5ml of 200mM boric acid-borax buffer of pH8.4;

[0037] Step 2 Add 2.5mg mPEG-SPA to the solution in the first step, and mix well to form a reaction solution;

[0038] The third step to the sixth step are carried out according to the corresponding operation steps in Example 1;

[0039] Step 7 Concentrate the collected absorption peak solution with MilliporeAmicon Ultra-15 ultrafiltration tube with a molecular weight cut-off of 1-5KD, and freeze-dry to obtain 0.4-0.5 mg of single-point modified mPEG-modified recombinant human interferon-α1b The pure compound has a molecular weight of 24.329KD.

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Abstract

The invention relates to a mPEG modified recombinant human interferon -alpha1 b complex and a method for preparing the same, and belongs to the technical field of a modified complex of a peptide or a protein and preparation therefore. The complex comprises a recombinant human interferon -alpha1 b protein and a mPEG derivative, the two are being connected together by the amido bond formed by a free amino of the former and an active group activated by hydroxyl succimide of the latter. The crude product solution of the mPEG modified recombinant human interferon -alpha1 b complex is prepared by modification reaction of the recombinant human interferon -alpha1 b protein with mPEG finishing agent. After ion exchange chromatography, complex portion collection, centrifugal ultrafiltration and concentration, and freeze drying, the pure product of mPEG modified recombinant human interferon -alpha1 b complex is obtained. The complex has an antiviral biological activity of the natural recombinant human interferon -alpha1 b, and has an advantage of high in vivo biological stability.

Description

technical field [0001] The invention relates to a recombinant human interferon-α1b complex and a preparation method thereof, to be precise, to an mPEG (monomethoxypolyethylene glycol) modified recombinant human interferon-α1b complex and a preparation method thereof, belonging to Technical field of peptide or protein modification complex and its preparation. Background technique [0002] Human interferon-α1b (hIFN-α1b) is a protein composed of 166 amino acids produced by human leukocytes, with a (relative) molecular weight of 19.329KD. At present, recombinant human interferon-α1b (rhIFN-α1b) can be prepared by genetic engineering technology. In 1987, recombinant human interferon-α1b was approved for marketing as the first genetically engineered drug to enter industrialization in my country (Guo Baoyu edited "Gene Engineering Pharmacy", Second Military Medical University Press, October 2000, first edition P151). Recombinant human interferon-α1b has a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/56C07K17/02A61K38/21A61P31/12
Inventor 吴自荣徐帆洪杨宇峰马相虎黄静路煜马明浩吴腾捷
Owner SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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