Method for detecting content of each group of free polysaccharide in meningococcus polysaccharide conjugate vaccine finished product

Abstract

The invention relates to a method for measuring the free polysaccharide content of each group of meningococcal polysaccharide-conjugated vaccine finished products, belonging to the field of biotechnology. In this method, proteinase K treatment is carried out in the preparation of the test product, the amount of proteinase K added is 2 to 8 times the protein content in the enzymatic hydrolysis reaction system, and cold phenol treatment is used to separate the proteinase K with saturated cold phenol solution of sodium acetate. Combined polysaccharides and free polysaccharides in the conjugate are combined with immunoelectrophoresis detection technology to measure the content of free polysaccharides in each group of the finished conjugated vaccine. The method of the invention eliminates the influence of carrier proteins of each group of conjugates on electrophoretic mobility and other related factors in immunoelectrophoresis, effectively separates the bound polysaccharides and free polysaccharides in the conjugates, and establishes a method for detecting the combination of polysaccharides of more than 4 groups of meningococci. The suitable detection system for the free polysaccharides of each group of vaccine products has the characteristics of strong durability, high accuracy and precision. A method for evaluating the quality of finished vaccines with more than 4 groups of meningococcal polysaccharide conjugate vaccines has been established.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to a method for determining the content of free polysaccharides in each group of finished meningococcal polysaccharide conjugated vaccines. Background technique [0002] Neisseria meningitidis (n. meningitidis ), referred to as meningococcus, meningococcal infection is a common cause of bacterial meningitis in children, with a high mortality rate. Due to the use of meningococcal capsular polysaccharide vaccine, the incidence rate was effectively controlled. However, capsular polysaccharide is a T cell-independent antigen, and re-immunization cannot be boosted, and it cannot induce immune responses in children under 2 years old, which has certain limitations. Covalently binding the protein carrier to the capsular polysaccharide by chemical means can make it a T cell-dependent antigen, thereby having an immune memory response, thus providing immune protection for infants...

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Problems solved by technology

[0004] Because the properties of different bacterial capsular polysaccharides and different polysaccharide conjugates are quite different, for the content of free polysaccharides in the stock solution of individual conjugates, group A can be obtained by measuring phosphorus content after being treated with cold phenol, and group C, Y or W135 can also be obtained by using cold After phenol treatment, it was obtained by measuring the sialic acid content; the free polysaccharide content of each group of the A+C conjugate vaccine could also be obtained by measuring the phosphorus content of the A group and the sialic acid content of the C group respectively, and in A, C, Y, W135 The conjugates of groups A, C, Y, and W135 in the finished product of group conjugate vaccines are mixed together, and groups C, Y, and W135 all contain sialic acid, so the free polysaccharide content of groups C, Y, and W135 cannot be obtained by measuring the content of sialic acid. Since there are many influencing factors in the process of producing more than 3 groups of polysaccharide conjugated vaccine products, and the stability of free polysaccharides of groups A, C, Y or W135 is different, and the content of free polysaccharides in each group is different from that in the finished conjugate vaccine. The corresponding group antigens are closely related to the immunogenicity of the body, thus affecting the immune protection effect of the final product. Therefore, it is necessary to separately measure the free polysaccharide content of each group of the finished product of the meningococcal polysaccharide conjugate vaccine of groups A, C, Y, and W135. To ensure the quality of the final product

[0005] "Methodological Validation of Quantitative Detection of Group C Meningococcal Polysaccharide Content in Quadrivalent Meningococcal Polysaccharide Vaccine by Rocket Immunoelectrophoresis" (Lin Yun et al., published in International Journal of Biological Products, Volume 31, Issue 6, December 2008) A method for detecting the polysaccharide content of group C meningococcus in the 4-valent meningococcal polysaccharide vaccine was introduced, which solved the technical problem of determining the polysaccharide content of each group of meningococcal polysaccharide vaccines for groups A, C, Y, and W135. The method is not suitable for the determination of the polysaccharide content of each group in the finished conjugated vaccine, because experiments have proved that the carrier protein of each group in the conjugated vaccine will affect the rocket immunoelectrophoresis, resulting in results that cannot truly and accurately reflect the polysaccharide content, so the three groups The above polysaccharide conjugate vaccine products include A, C, Y, W135 groups of meningococcal polysaccharide conjugate vaccines. The determination of the free polysaccharide content of each group of the finished polysaccharide conjugate vaccine is a technical difficulty. At present, there is no method for the determination of the free polysaccharide content of each group of the polysaccharide conjugate vaccine products. General method, it is necessary to establish a method to accurately measure the free polysaccharide content of each group such as A, C, Y, W135 in the finished product of this type of polysaccharide conjugate vaccine

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