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Polyethylene glycol modified protein and method for purification of modified product

A polyethylene glycol and protein technology, applied in the field of modification and purification of polyethylene glycol, can solve the problems of low product yield and many steps, and achieve the effect of high product yield, simple method and reduced impact

Active Publication Date: 2019-11-19
ZONHON BIOPHARMA INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Purification needs to be purified by ion exchange first, then concentrated by ultrafiltration, and then purified by gel filtration after concentration. There are many steps and the product yield is also low.

Method used

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  • Polyethylene glycol modified protein and method for purification of modified product
  • Polyethylene glycol modified protein and method for purification of modified product
  • Polyethylene glycol modified protein and method for purification of modified product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1 is used for the buffer replacement of the asparaginase of PEG modification, modification reaction and modification product purification

preparation example 1

[0068] Step 1: Buffer Exchange

[0069] The freeze-dried powder of asparaginase was dissolved in 20 mM Tris-Hcl (pH 8.0) buffer solution to prepare a solution with a protein concentration of 5 mg / ml. Then the sample was loaded through the sample pump of the AKTA chromatography system, and the sample was drawn into the Q ion exchange column (purchased from GE, HiTrap Q HP 5mL). After loading the sample, equilibrate the chromatographic column with equilibration buffer A. After equilibrating for 5 column volumes, perform one-step elution with elution buffer B, and collect the elution peaks.

[0070] (A solution: 20mM phosphate buffer (pH8.0); B solution: 20mM phosphate buffer + 0.2M sodium chloride (pH7.5)

[0071] Step 2: Modification reaction and purification of modified products

[0072] Using M-SPA-5000 (purchased from Beijing Jiankai Technology Co., Ltd.) as a PEG modifier, the asparaginase solution of the elution peak collected in the first step was asparaginase: PEG modi...

Embodiment 2

[0091] Embodiment 2: Carry out the PEG modification buffer replacement of asparaginase by conventional method, modification reaction and modification product purification

[0092] Step 1: Buffer Exchange

[0093] The freeze-dried powder of asparaginase was dissolved in 20 mM Tris-Hcl (pH 8.0) buffer solution to prepare a solution with a protein concentration of 5 mg / ml. The dissolved asparaginase protein solution was buffer exchanged with an ultrafiltration system (Sartorius Vivaflow 20P0), and finally replaced into a pH 7.0 phosphate buffer. After the ultrafiltration, it was found that the retentate was turbid, indicating that a precipitate was formed. Next, filter the retentate with a 0.22um membrane, and the filtered solution is clarified.

[0094] Step 2: Modification reaction and purification of modified products

[0095] Use M-SPA5000 as a PEG modifier, add the modifier to the asparaginase solution after ultrafiltration and filtration, and react according to the molar...

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Abstract

The invention discloses a technological process for modification of proteins with PEG and purification. The technological process comprises the following steps: proteins are dissolved in a buffer solution 1, the protein solution is absorbed into an ion exchange chromatographic column, an equilibration buffer is used for equilibration, an elution buffer is used for elution, proteins after elution are collected, and the like. Compared with traditional processes for modification with PEG and purification, the process has the advantages of simple process steps, higher yield of products, and low influences on activity of proteins.

Description

technical field [0001] The invention relates to a method for modifying and purifying polyethylene glycol, in particular to a method and application for purifying polyethylene glycol-modified proteins and modified products, and more specifically to a method for modifying and purifying asparaginase with polyethylene glycol. Background technique [0002] Since the emergence of polyethylene glycol (PEG) modification technology in the 1970s, in the past three decades, many proteins have been modified by PEG for drug development, reaction catalysis, drug delivery, etc., and have achieved significant social and economic benefits. benefit. Well-known international biological companies are actively promoting the technology of PEG-modified proteins and polypeptide drugs. Products that have been on the market, such as PEG-modified adenosine deaminase in 1990, are used to treat deficient diseases (Enzon’s ) on the market, it not only reduces the immunogenicity and antigenicity of the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/96C12N9/82
Inventor 马永王俊吴鼎龙闻兴元王和徐春林陈一飞王耀方
Owner ZONHON BIOPHARMA INST
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