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Method for removing endotoxin in antibody protein

A technology of endotoxin and protein, which is applied to the preparation method of peptides, immunoglobulin, chemical instruments and methods, etc., can solve the problems of difficult screening of endotoxin removal methods, and achieve good clinical application prospects, simple processing methods, and safety Good results

Inactive Publication Date: 2018-09-28
SHANGHAI WUXI BIOLOGIC TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The present invention aims at the defects of the prior art, and provides a method for removing endotoxin, so as to solve the problem of difficult screening of existing endotoxin removal methods

Method used

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  • Method for removing endotoxin in antibody protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 The cultivation and purification process of monoclonal antibody is complicated, and endotoxin may be introduced in many steps. An example is given to illustrate.

[0035] Step 1 Protein A affinity chromatography of cell supernatant

[0036] Chromatography filler: MabSelect SuRe (an alkali-resistant protein A filler) of GE Company.

[0037] Chromatography flow rate: 150~300cm / h.

[0038] Rinsing: equilibrate the chromatography column with equilibrium solution 20mM phosphate, 150mM NaCl, pH7.2~7.4.

[0039] Disinfection: Rinse the chromatography column with 0.5M NaOH for 15 minutes.

[0040] Equilibrium: Rinse the chromatography column with equilibrium solution until the pH drops to 7.2-7.4.

[0041] Sample loading: The supernatant obtained from cell culture was loaded onto the column, and after sample loading was completed, the chromatography column was washed with an equilibrium solution until the 280nm ultraviolet absorption of the effluent returned to th...

Embodiment 2

[0063] Example 2 Endotoxin removal method: In view of the possibility of endotoxin infection risk in the existing process, the present invention provides a fast and efficient purification method for endotoxin-exceeding protein samples.

[0064] 1. Protein A affinity chromatography

[0065] Chromatography filler: MabSelect SuRe of GE Company.

[0066] Chromatography flow rate: 150~300cm / h.

[0067] Rinsing: equilibrate the chromatography column with equilibrium solution 20mM phosphate, 150mM NaCl, pH7.2~7.4.

[0068] Disinfection: Rinse the chromatography column with 0.5M NaOH for 15 minutes.

[0069] Equilibrium: Rinse the chromatography column with equilibrium solution until the pH drops to 7.2-7.4.

[0070] Sample loading: load the protein sample containing a certain amount of endotoxin (the endotoxin content exceeds the standard) to the column. After loading the sample, wash the column with the balance solution until the 280nm ultraviolet absorption of the effluent retur...

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Abstract

The invention provides a method for removing endotoxin in an antibody protein. A to-be-treated protein sample is subjected to affinity chromatography. The method comprises the following steps: (1) rinsing an affinity chromatographic column by using an equilibrium liquid, and carrying out disinfection, re-equilibrating and sampling, wherein the equilibrium liquid is an NaCl-containing phosphate buffer solution with the pH value of 7.2-7.4 and the electric conductivity of 16.0-18.0mS / cm; (2) flushing the chromatographic column by using a flushing liquid, and flushing the chromatographic column by using an equilibrium liquid, wherein the flushing liquid is an equilibrium liquid in which Arg is added; and (3) carrying out elution by using an eluant, and collecting the eluant, wherein the eluant is a sodium acetate buffer solution with the pH value of 3.3-3.5 and the electric conductivity of 0.24-0.44mS / cm. By using the method, endotoxin in a protein sample can be effectively removed underthe condition that the property of the antibody protein is not changed, the purity of the protein is not affected in the process, and the recovery rate is high. The method provided by the invention issimple, low in cost and short in time consumption; and the sample treated by using the method has the endotoxin content of smaller than 0.1EU / mg and is good in safety and clinic application prospect.

Description

technical field [0001] The invention relates to the technical field of biological products, in particular to a method for removing endotoxin of a monoclonal antibody. Background technique [0002] Endotoxin is a kind of lipopolysaccharide from the outer membrane of Gram-negative bacteria, which is released after the bacteria are lysed, and is also called "pyrogen". Their relative molecular mass is generally 10,000 to 25,000, and they form associations in aqueous solution, and their molecular mass can reach 500,000 to 1 million. Its chemical composition is mainly phospholipopolysaccharide-protein complex, and its toxic composition is mainly lipid A. [0003] Endotoxin is not a protein, so it is very heat-resistant, and has the characteristics of small size and strong chemical stability, so it is difficult to remove. Only heating at 160°C for 2-4 hours, or heating at 240°C for 1 hour, or heating and boiling with strong acid, strong alkali or strong oxidant for 30 minutes can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/00C07K1/22
CPCC07K1/22C07K16/00
Inventor 席宏星赵普亚陈晓悦蔡洁行周伟昌陈智胜
Owner SHANGHAI WUXI BIOLOGIC TECH CO LTD
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