Method for removing bacterial endotoxin in protein solution

A bacterial endotoxin and protein solution technology, applied in the biological field, can solve the problems of expensive equipment, disease deterioration, bacterial endotoxin release, etc., and achieve the effects of simple and practical operation, improved safety, and prevention of adsorption

Active Publication Date: 2015-01-28
CHENGDU OLYMVAX BIOPHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Even after the purification process, the bacterial endotoxin has been reduced, but the bacterial endotoxin content is still very high, which is the main reason for the vaccine's febrile reaction
Most antibiotics used clinically in the treatment of bacterial infections (mainly Gram-negative bacterial infections) lead to the release of a large number of bacterial endotoxins due to bactericidal/bacteriostatic reasons, making the condition worse
[0004] At present, the removal methods of bacterial endotoxin in the solution include: (1) Ultrafiltration technology separates the

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Embodiment 1: bacterial endotoxin removal method in PT solution, it comprises the following steps:

[0013] S1. Preparation of aluminum hydroxide gel: add 2mol / L sodium hydroxide to 4.5% aluminum chloride solution, react for 2 hours at a pH of 7.0 and a temperature of 2°C, and collect the gel by centrifugation at 4000rpm;

[0014] S2. Remove bacterial endotoxin in the PT solution: add 0.3% of the aluminum hydroxide gel prepared in step S1 to the PT solution with a pH of 7.0, stir at 100rpm for 2.5 hours at a temperature of 4°C, and use 4000rpm The supernatant after removing endotoxin was collected by centrifugation.

Embodiment 2

[0015] Embodiment 2: bacterial endotoxin removal method in PT solution, it comprises the following steps:

[0016] S1. Preparation of aluminum hydroxide gel: Add 2mol / L sodium hydroxide to 4.5% aluminum chloride solution, react for 1.5 hours at a pH of 7.3 and a temperature of 5°C, and collect the gel by centrifugation at 4000rpm;

[0017] S2. Remove bacterial endotoxin in the PT solution: add 0.3% of the aluminum hydroxide gel prepared in step S1 to the PT solution with a pH of 8.0, stir at 100 rpm for 2 hours at a temperature of 4 ° C, and use 4000 rpm The supernatant after removing endotoxin was collected by centrifugation.

Embodiment 3

[0018] Embodiment 3: bacterial endotoxin removal method in PT solution, it comprises the following steps:

[0019] S1. Preparation of aluminum hydroxide gel: add 2mol / L sodium hydroxide to 4.5% aluminum chloride solution, react for 1 hour at pH 7.5 and temperature 8°C, and collect the gel by centrifugation at 4000rpm;

[0020] S2. Remove bacterial endotoxin in the PT solution: add 0.3% of the aluminum hydroxide gel prepared in step S1 to the PT solution with a pH of 9.0, stir at 100rpm for 1.5 hours at a temperature of 4°C, and use 4000rpm The supernatant after removing endotoxin was collected by centrifugation.

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PUM

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Abstract

The invention discloses a method for removing bacterial endotoxin in a protein solution. The method comprises the following steps of S1, preparing aluminum hydroxide gel, namely adding 2mol/L sodium hydroxide into 4.5% of aluminum chloride solution to obtain a solution with the pH value of 7.0-7.5, reacting at the temperature of 2-8 DEG C for 1-2 hours, and centrifugally collecting gel at the speed of 4000rpm; and S2, removing bacterial endotoxin in the protein solution, adding the aluminum hydroxide gel prepared in the step S1 into the protein solution, stirring at the temperature of 4 DEG C and the speed of 100rpm for 1.5-2.5 hours, and centrifugally collecting at the speed of 4000rpm to obtain a supernatant solution without endotoxin. According to the method, electrostatic adsorption is mainly used as the way of adsorbing proteins by using the aluminum hydroxide gel, the adsorption of aluminum hydroxide to the proteins can be reduced or avoided when phosphate radicals and high-concentration NaCl (contained in a PB buffer solution) exist, and 99% of bacterial endotoxin can be removed by using the buffer solution.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for removing bacterial endotoxin from a protein solution. Background technique [0002] Bacterial endotoxin (Endotoxin) is a unique structure on the cell wall of Gram-negative bacteria. Endotoxin is an exogenous pyrogen. The main chemical component of bacterial endotoxin is lipopolysaccharide (LPS), and its toxic components are mainly lipids. A. Endotoxin is heat-resistant and stable, and its activity can only be destroyed at 180°C for 2 hours. The toxic effects of endotoxins of various bacteria are weak and roughly the same, which can cause fever, endotoxin shock and disseminated intravascular coagulation. Usually, LPS exists in the complete form on the outer membrane of bacterial cells. When it is released from the bacteria, its activity is 50-100 times that of endotoxin on the cell wall. It has a wide range of biological activities and can cause severe inflam...

Claims

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Application Information

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IPC IPC(8): C07K1/14C07K14/235
Inventor 陈道远陈元芬李靖苏淑
Owner CHENGDU OLYMVAX BIOPHARM
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