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Refining method of tetanus toxoid

A technology of tetanus toxoid and refining method, which is applied in the field of preparation of tetanus toxoid, can solve problems such as inability to effectively separate TT monomer, and achieve the effect of improving immunogenicity and purity

Inactive Publication Date: 2018-05-08
BEIJING BIOLOGICAL PROD INST CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The product purified by this method is a mixture of TT monomers, dimers and multimers, and the TT monomer cannot be effectively separated

Method used

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  • Refining method of tetanus toxoid
  • Refining method of tetanus toxoid
  • Refining method of tetanus toxoid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The present embodiment provides the refining method of tetanus toxoid, and it comprises the steps:

[0043] (a) prepare the tetanus toxoid stock solution to be purified:

[0044] In this embodiment, the tetanus toxoid stock solution prepared as follows is used as the tetanus toxoid before purification, and the Clostridium tetani strain is cultivated in a suitable medium to produce tetanus toxin, and the tetanus toxin is added Appropriate amount of formaldehyde solution, detoxified at a suitable temperature to produce tetanus toxoid, tetanus toxoid is ultrafiltered and ammonium sulfate precipitated to obtain refined tetanus toxoid, the refined tetanus toxoid is adjusted to a suitable pH and then sterilized and filtered Obtain the tetanus toxoid stock solution for the experiment.

[0045] The obtained tetanus toxoid stock solution was subjected to high-performance liquid chromatography analysis, wherein the high-performance liquid chromatography system was Agilent 1100Se...

Embodiment 2

[0057] The present embodiment adopts the method substantially identical with embodiment 1 to purify TT stoste, presses the same method test with embodiment 1, and in this stoste, tetanus toxoid monomer content is only 71.61%, dimer content is 25.77%, more The polymer content was 2.62%. Chromatography was carried out in the same manner as in Example 1. The eluents from the 6th to 14min TT monomers were combined, and the samples were tested in the same way as in Example 1 to test the purity of TT monomer after purification. HPLC such as image 3 Shown, the purity reaches 96.04%, and its yield is 48.62%. The dimer content was 3.96%. Polymer content was 0.

Embodiment 3

[0059] The present embodiment adopts the substantially same method as in Example 1 to purify the TT stock solution, and tests in the same method as in Example 1. In this stock solution, the tetanus toxoid monomer content is only 77.43%, and the dimer content is 19.37%. Body content is 3.20, through the same method as in Example 1, carry out chromatography, the eluate containing TT monomer collected at the 7th ~ 15min is combined, and the TT monomer content after sampling is tested by the same method as in Example 1, Its HPLC such as Figure 4 Shown, the purity reaches 97.36%, and its yield is 41.69%. The dimer content was 2.64%. Polymer content was 0.

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Abstract

The invention provides a refining method of tetanus toxoid. The refining method comprises the steps of preparing a to-be-purified tetanus toxoid stock solution; adding the to-be-purified tetanus toxoid stock solution into a Fractogel EMD DEAE (M) gel chromatographic column; adopting equilibrium liquid to balance the chromatographic column added with the sample, after balancing, adopting a first eluant to elute needed tetanus toxoid monomers, and collecting the eluant containing the tetanus toxoid monomers. By means of the refining method, the purity of the tetanus toxoid monomers can be obviously improved, the yield of the tetanus toxoid monomers reaches 40% or above, the tetanus toxoid monomers obtained through the method serve as carrier protein of conjugate vaccines, the immunogenicityof the vaccines can be obviously improved, and safer and more effective protein is provided for the polysaccharide-protein conjugate vaccines and other vaccines with TT being conjugate vaccine components.

Description

technical field [0001] The invention belongs to the field of tetanus toxoid preparation and relates to a method for refining the tetanus toxoid. Background technique [0002] Tetanus toxoid (TT or TTd), as the main component of monovalent or adsorbed DTP vaccine, has been widely used in the vaccination of infants and suitable populations. With the rapid development of bacterial conjugate vaccines at home and abroad, b Haemophilus influenzae type conjugate vaccines, meningococcal conjugate vaccines, and pneumococcal conjugate vaccines have been successfully marketed with TT as the carrier protein (Tontini M, Berti F, Romano MR, et al. Comparison of CRM197, diphtheria toxoid and tetanus toxoid as protein carriers for meningococcalglycoconjugate vaccines [J]. Vaccine, 2013, 31(42): 4827-4833. WHO. Recommendations to assure the quality, safety and efficacy of pneumococcal conjugate vaccines [S]. Geneva, 19 to 23 October, 2009). Safety has been confirmed by long-term clinical tr...

Claims

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Application Information

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IPC IPC(8): C07K14/33C07K1/16
CPCC07K14/33
Inventor 李世慧韩俊杰张萍肖詹蓉王珣周第
Owner BEIJING BIOLOGICAL PROD INST CO LTD
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