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Method for producing tetanus toxoid raw liquid through chromatography purification method

A tetanus toxoid and chromatographic purification technology, applied in the biological field, can solve the problems of low purity of toxoid stock solution, large detoxification volume, and difficulty in purification, and achieve the effects of saving equipment, reducing detoxification volume, and single component

Active Publication Date: 2015-02-04
CHENGDU OLYMVAX BIOPHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Although this process has the advantage of being relatively stable, the preparation of the toxoid stock solution has low purity (darker color, high content of miscellaneous proteins), large detoxification volume, inconvenient operation, and impurities are easily mixed with toxin proteins during the detoxification process. Cross-linked, difficult to purify

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  • Method for producing tetanus toxoid raw liquid through chromatography purification method
  • Method for producing tetanus toxoid raw liquid through chromatography purification method

Examples

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Effect test

Embodiment 1

[0026] Example 1: A method for producing tetanus toxoid stock solution by chromatographic purification, which includes the following steps:

[0027] S1. Pre-treatment: filter the tetanus culture broth, and concentrate the filtrate to 1 / 20 of its original volume through a 30KD ultrafiltration membrane package. Wash the concentrated solution repeatedly with 15mmol / LPB buffer until the permeate is at 280nm The lower absorbance reaches a constant level, and the concentrated solution after washing is collected;

[0028] S2. Ion exchange chromatography: load the above concentrate on a well-balanced Q-Sepharose Fast Flow chromatography column, eluting with 15mmol / L PB, 0.16mol / L NaCl solution, and collecting the elution peak is the IEX toxin target protein;

[0029] S3. Hydrophobic chromatography: add the same amount of 15mmol / LPB, 1.5mmol / L ammonium sulfate to the eluent collected in step S2, and load the sample on a well-balanced Phenyl FF chromatography column, and use PB buffer to elut...

Embodiment 2

[0034] Example 2: A method for producing tetanus toxoid stock solution by chromatographic purification, which includes the following steps:

[0035] S1. Pre-treatment: Filter the tetanus culture solution, and concentrate the filtrate to 1 / 10 of the original volume by ultrafiltration with a 30KD ultrafiltration membrane package. Wash the concentrated solution repeatedly with 25mmol / L PB buffer until the permeate is in The absorbance at 280nm is constant, and the concentrated solution after washing is collected;

[0036] S2. Ion exchange chromatography: load the above concentrate on a well-balanced Q-Sepharose Fast Flow chromatography column, eluting with 25mmol / L PB, 0.16mol / L NaCl solution, and collecting the elution peak is the IEX toxin target protein;

[0037] S3. Hydrophobic chromatography: add the same amount of 25mmol / L PB and 2.5mmol / L ammonium sulfate to the eluent collected in step S2, and load the sample on a well-balanced Phenyl FF chromatography column, and use PB buffer...

Embodiment 3

[0042] Example 3: A method for producing tetanus toxoid stock solution by chromatographic purification, which includes the following steps:

[0043] S1. Pre-treatment: Filter the tetanus culture solution, and concentrate the filtrate to 1 / 15 of its original volume through a 30KD ultrafiltration membrane package. Wash the concentrated solution repeatedly with 20mmol / L PB buffer until the permeate is in The absorbance at 280nm is constant, and the concentrated solution after washing is collected;

[0044] S2. Ion exchange chromatography: load the above concentrated solution on a well-balanced Q-Sepharose Fast Flow chromatography column, eluting with 20mmol / L PB, 0.16mol / L NaCl solution, and collecting the elution peak is the IEX toxin target protein;

[0045] S3. Hydrophobic chromatography: add the same amount of 20mmol / L PB and 2mmol / L ammonium sulfate to the eluent collected in step S2, and load the sample on a well-balanced Phenyl FF chromatography column, eluting with PB buffer, ...

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Abstract

The present invention discloses a method for producing a tetanus toxoid raw liquid through a chromatography purification method, and belongs to the technical field of biology. The production method comprises: filtering a bacillus tetani culture solution, carrying out ultrafiltration concentration on the filtrate through a 30 KD ultrafiltration membrane to achieve 1 / 20-1 / 10 of the original volume, washing the concentrated solution, collecting, and carrying out ion exchange chromatography, hydrophobic chromatography, primary membrane filtration, detoxification, ultrafiltration and secondary membrane filtration on the concentrated solution to obtain the tetanus toxoid raw liquid. The method of the present invention has advantages of simple process, easy operation, high production efficiency and low cost. The tetanus toxoid raw liquid produced by the method of the present invention has advantages of single tetanus toxoid component and high purity so as to obtain the good immunogenicity and reducing the occurrence of adverse events following immunization.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a method for producing a tetanus toxoid stock solution by a chromatography purification method. Background technique [0002] Tetanus is usually a fatal infectious disease caused by the toxin-producing Clostridium tetani (Clostridium tetani). In many parts of the world, tetanus is still a serious public health problem, especially in some of the poorest areas in tropical developing countries. The morbidity and mortality of tetanus in these areas are mainly caused by MNT. In 2002, the total number of deaths due to tetanus in the world was approximately 213,000. Among them, about 180,000 newborns died of tetanus, and the number of pregnant women who died of tetanus may be as high as 15,000-30,000. The continued emphasis on improving vaccine coverage, production hygiene and monitoring, as well as specific measures in high-risk areas, has led to a continuous decline in the incidenc...

Claims

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Application Information

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IPC IPC(8): C07K14/33C07K1/36C07K1/34C07K1/18
CPCC07K14/33
Inventor 陈道远马礼耕罗力心陈克平李靖
Owner CHENGDU OLYMVAX BIOPHARM
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