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Method for recovering interferon from polyethylene glycol modified interferon reaction liquid

A technology of interferon reaction and polyethylene glycol, applied in the preparation method of interferon, peptide, cytokine/lymphokine/interferon, etc. Separation effect, small sample volume effect

Inactive Publication Date: 2010-07-07
ANHUI ANKE BIOTECHNOLOGY (GRP) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The disadvantages of protein and peptide drugs are: easy to be hydrolyzed by enzymes and cleared by the kidneys, the clinical half-life is short, and multiple injections are required to maintain the drug concentration
However, when eluted with high-concentration salt, it often leads to protein denaturation and precipitation. At the same time, when eluted with high-concentration salt, some impurities such as denatured protein, endotoxin, reaction by-products or PEG modifier hydrolyzed off groups will also be eluted together. , so that the purity, specific activity, and endotoxin level of the recovered protein are affected. The protein recovered by this method is often discarded because it has no use value, which not only increases the production cost of the PEGylated protein, but also causes considerable degree of waste

Method used

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  • Method for recovering interferon from polyethylene glycol modified interferon reaction liquid
  • Method for recovering interferon from polyethylene glycol modified interferon reaction liquid
  • Method for recovering interferon from polyethylene glycol modified interferon reaction liquid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Recovering IFNα-2b using anionic medium Q-sepharose

[0039] 1. Preparation of PEG-modified IFNα-2b reaction solution

[0040] Add boric acid buffer solution to the IFNα-2b stock solution, adjust the pH value to 7.0-9.0, add mPEG-NHS (molecular weight 20KD) according to the feeding ratio of 2.0-3.0:1, react at room temperature for 2hrs, and make A solution.

[0041] 2. Recovery of IFNα-2b and purification of mono-PEGylated IFNα-2b:

[0042] 2.1. Preparation of relevant working fluids

[0043] Solution B (sample loading buffer): phosphate buffer (disodium hydrogen phosphate-sodium dihydrogen phosphate), pH7.5, 20mM.

[0044] Solution C (reaction solution after replacement treatment): fully replace solution A with solution B (dialysis or ultrafiltration) to obtain solution C.

[0045] Solution D (IFN eluent): NaCl was added to solution B, and the final concentration of NaCl was 100 mM.

[0046] Solution E (PEG-IFN purification working solution): Tris-HCl bu...

Embodiment 2

[0065] Example 2: Recovering IFNα-2b using anionic medium Q-sepharose

[0066] Solution B (sample buffer): Tris-hydrochloric acid as sample buffer, pH 8.0, 50mM;

[0067] Solution D (IFN eluent): add NaCl to solution B, and the final concentration of NaCl is 20 mM.

[0068] All the other conditions are the same as in Example 1.

[0069] For the test and identification results, see the attached Figure 2A , 2B 、 2C, because iodine staining is specific for PEG, it is attached Figure 2A 3, 4, and 5 are not stained.

[0070] The test results of various quality control indicators showed that compared with the reference substance, the recovered IFNα-2b by this method was consistent with it, and complied with the provisions of the Chinese Pharmacopoeia.

Embodiment 3

[0071] Example 3: Recovering IFNα-2b using anionic medium Q-sepharose

[0072] 1. Preparation of PEG-modified IFNα-2b reaction solution

[0073] Add boric acid buffer solution to IFNα-2b stock solution, adjust the pH value to 7.0-9.0, add mPEG-NHS (molecular weight 40KD) according to the feeding ratio of 2.0-3.0:1, react at room temperature for 2hrs, and make A solution.

[0074] All the other conditions are the same as in Example 1.

[0075] The test results of various quality control indicators showed that compared with the reference substance, the recovered IFNα-2b by this method was consistent with it, and complied with the provisions of the Chinese Pharmacopoeia.

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Abstract

The invention relates to a method for recovering interferon from a polyethylene glycol modified interferon reaction liquid, belonging to the field of separating and purifying each component in the polyethylene glycol modified interferon reaction liquid. The method of the invention comprises the following steps: a, regulating the pH value of the polyethylene glycol modified interferon reaction liquid to 5.0-8.0 with acid or alkali; b, replacing the obtained reaction liquid into a sample loading buffer liquid with the same pH, regulating the ion strength of the sample loading buffer liquid, and carrying out ion-exchange chromatography so that the polyethylene glycol modified interferon penetrates and the dissociated interferon is absorbed; and c, after treating and regenerating the ion-exchange chromatography columns, eluting with low salt to obtain the eluting peak which is the recovered dissociated interferon. The method of the invention does not affect the yield and purity of PEG IFN, and simultaneously, each quality control index of the recovered IFN from the reaction liquid is consistent with a comparison product, and the IFN can be recycled.

Description

technical field [0001] The invention belongs to the field of separation and purification of components in a polyethylene glycol-modified interferon reaction solution, and in particular relates to a method for recovering interferon from a polyethylene glycol-modified interferon reaction solution. Background technique [0002] Interferon (Interferon, IFN) was discovered by British scientists Isaacs and LinderMann in 1957. It is a class of proteins with broad-spectrum antiviral activity. Among them, α-interferon is mainly used clinically to treat viral diseases such as hepatitis B and hepatitis C. It is also used in combination with some cancer chemotherapy drugs. [0003] The disadvantages of protein and peptide drugs are: easy to be hydrolyzed by enzymes and cleared by the kidneys, and the clinical half-life is short, requiring multiple injections to maintain the drug concentration. After proteins and polypeptides are modified with polyethylene glycol (PEG), the obtained pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/18C07K14/56
Inventor 宋礼华许培戎隆富程婷邬婧
Owner ANHUI ANKE BIOTECHNOLOGY (GRP) CO LTD
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