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Method for modifying hirudin by polyethyleneglycol assisted by anion exchange column

An anion-exchange column and polyethylene glycol technology, applied in the field of protein modification, can solve the problems of high ligand cost, increased drug cost, and limited use range, etc., and achieve the effects of improving efficiency, shortening operation time, and simplifying operation steps

Inactive Publication Date: 2008-10-08
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method has a series of advantages such as protecting the active site and strong specificity, its disadvantages are that the steps are cumbersome, and it is necessary to find a ligand that has an affinity with the target protein, and some ligands are expensive, which increases the cost of the drug. , which limits its use

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] 4ml of hirudin solution dissolved in 0.02M pH8 phosphate buffer a, the concentration of which is 1.5mg / ml, is loaded on a Hitrap Q HP 5ml prepacked ion exchange column equilibrated with the same buffer solution (the microbead structure in the column is 6 % Highly cross-linked agarose, the pore size is 34 μm, the charged group is quaternary amino group), after no eluate flows out, then activate mPEG 5kDa (SC-PEG) with 4ml of succinimide dissolved in buffer a 5000 ) solution, loaded on the column, the molar ratio of sample amount to hirudin was 9:1, reacted for 1 h, and eluted after the reaction. A linear gradient of 0-30% liquid b is used for elution, wherein liquid b is liquid a containing 1M NaCl. Elution time 100min. Each eluate was collected manually, and each eluted fraction was verified by electrophoresis, and the electrophoresis staining method was iodine staining. Staining solution is 0.1M containing 5% BaCl 2 The I / KI solution. Dyeing time 4min, decolorizati...

Embodiment 2

[0019] 4ml of hirudin solution dissolved in 0.02M pH 8 phosphate buffer a at a concentration of 1.5mg / ml was applied to a Hitrap Q HP 5ml prepacked ion exchange column equilibrated with the same buffer until no eluate came out , then 4ml of succinimide-activated mPEG 20kDa (SC-PEG 20000 ) solution, loaded on the column, the molar ratio of sample amount to hirudin was 9:1, reacted for 1 h, and eluted after the reaction. The elution adopts a linear gradient of 0-30% liquid b, wherein liquid b is liquid a containing 1M NaCl. Elution time 60min. Each eluate was collected manually, and each eluted fraction was verified by electrophoresis, and the electrophoresis staining method was iodine staining. Staining solution is 0.1M containing 5% BaCl 2 The I / KI solution. Dyeing time 4min, decolorization time 12h. The collected single modified product is mPEG 20000 - hirudin, which has a molecular weight of 27 kDa. In the determination of specific activity, the Lowry method was used ...

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Abstract

Disclosed is a method to assist polyethylene glycol(PEG) to modify hirudin with anion exchange column, belonging to protein modification technical field. The method is characterized in that ion exchange column is introduced to assit protein modification with PEG; the ph value for the reaction is 6-9 and the reaction time is 15-120min; the mol ration of hirudin to PEG is 1:3-1:9; the ion exchange column is a strong anion exchange column and the quaternary ammonium groups are the charged groups.The substrate in the column is sephadex or agarose, with average particle size of 30-90 micrometre. The method simplifies the reaction process and separation process in protein modification and correspondingly redues the consumption of sample in the process. The eluted hirudin can be recycled after being desalted. Compared with conventional liquid phase modification method, the method can produce hirudin with great activity in vitro, which is only modified by PEG.

Description

technical field [0001] The invention belongs to the technical field of protein modification, relates to polyethylene glycol solid-phase modification of protein, and particularly relates to a method for modifying hirudin by using anion exchange column to assist polyethylene glycol modification. Background technique [0002] Polyethylene glycol (PEG) modification is a chemical reaction in which protein drugs are modified with neutral, chemically inert, and hydrophilic PEG polymers. Since the 1970s, scientists have found that some medicinal proteins have greatly improved their medicinal properties after being modified with PEG, such as prolonged half-life in vivo, reduced immunogenicity, and so on. [0003] Some PEG-protein conjugates that are already on the market, such as Adagen, Oncospar, PEG-Intron, etc., are all prepared based on the first-generation PEGylation technology, and their characteristics are: (1) The molecular weight of the modifier is generally very low; (2) D...

Claims

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Application Information

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IPC IPC(8): C07K1/107C07K1/04
Inventor 修志龙李雪芹李晓晖
Owner DALIAN UNIV OF TECH
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