Method for determining content of polysaccharide of each group of meningococcus polysaccharide conjugate vaccine finished products

Abstract

The invention relates to a method for determining the content of the polysaccharide of each group of meningococcus polysaccharide conjugate vaccine finished products and belongs to the technical field of biology. The method comprises the following steps of: treating by using protease K during preparation of a detection product sample, wherein the total size of an enzymolysis reaction system is 420 mu l; and by using the total size as reference, adding the protease K, a protease K buffer solution which occupies one tenth of the total size and an ultra-filtration concentrated solution which occupies one sixth of the total size, uniformly mixing the protease K, the protease K buffer solution and the ultra-filtration concentrated solution, and then incubating, wherein the addition amount of the protease K is two to eight times of the content of proteins in the enzymolysis reaction system. By adoption of the method, influence of each group of conjugate carrier proteins on immunoelectrophoresis is eliminated, an appropriate detection system for detecting the polysaccharide of each of more than four groups of meningococcus polysaccharide conjugate vaccine finished products is established, and the content of the polysaccharide of each of more than four groups of meningococcus polysaccharide conjugate vaccine finished products can be accurately and quickly determined. The method has the characteristics of high durability, high accuracy and high precision. A method for evaluating the quality of meningococcus polysaccharide conjugate vaccine is 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 polysaccharides in each group of finished meningococcal polysaccharide conjugate vaccines. Background technique

[0002] The polysaccharide (antigen) content of the finished product of meningococcal polysaccharide conjugate vaccine is one of the basic conditions to ensure the effectiveness of the vaccine. For the polysaccharide content of the stock solution of the single conjugate, the A group can be obtained by measuring the phosphorus content, and the C, Y, or W135 group can be obtained by measuring the sialic acid content, and the finished products of meningococcal polysaccharide conjugate vaccines with more than 4 groups, such as A and C , Y, and W135 groups in the finished product conjugate vaccines of groups A, C, Y, and W135 are mixed together, and groups C, Y, and W135 all contain sialic acid, so it is impossible to obta...

Examples

Embodiment 1

[0031] Example 1 Determination of Group A polysaccharide content in the finished product of Group A, C, Y, W135 meningococcal polysaccharide conjugate vaccines

[0032]The determination of the polysaccharide content of group A in the finished product of group A, C, Y, and W135 meningococcal polysaccharide conjugate vaccines was repeated three times, namely repeat 1, repeat 2, and repeat 3, and each repeat was carried out according to the following steps:

[0033] (1) Preparation of test products for inspection

[0034] ①Pretreatment: Take 12 bottles of finished meningococcal polysaccharide conjugate vaccines of groups A, C, Y, and W135 (the labeled volume is 0.5ml per bottle), and reconstitute with 6ml of water for injection (that is, reconstitute with water for injection according to the labeled volume) , take 5.0ml in an ultrafiltration cup, centrifuge ultrafiltration at 5000×g 4°C for 40min, collect the concentrate, add water for injection to 1.0ml, and obtain the ultrafil...

Embodiment 2

[0045] Example 2 Determination of the polysaccharide content of group A in the finished product of meningococcal polysaccharide conjugate vaccine of group A, C, Y, W135

[0046] Except that the operations of the following steps are different, other operations are the same as those in Embodiment 1, and will not be repeated here.

[0047] (1) Preparation of test products for inspection

[0048] ②Protease K treatment: Take 70 μl of the ultrafiltration concentrate, add 4 μl of proteinase K, 42 μl of proteinase K buffer and supplement water for injection to 420 μl, mix well and incubate at 37°C for 6 hours to obtain the test product. The amount of proteinase K added is 4 times of the protein content in the enzymolysis reaction system.

[0049] (2) Preparation of antiserum agarose gel plate

[0050] Weigh 0.1521g of agarose, add 10.0ml of pH8.6 barbiturate electrophoresis buffer, heat and swell completely, put in 56℃ water bath for 20min, add 0.3ml of meningococcal A polysacch...

Embodiment 3

[0051] Example 3 Determination of the polysaccharide content of group A in the finished product of meningococcal polysaccharide conjugate vaccine of group A, C, Y, W135

[0052] Except that the operations of the following steps are different, other operations are the same as those in Embodiment 1, and will not be repeated here.

[0053] (1) Preparation of test products for inspection

[0054] ②Protease K treatment: Take 70 μl of the ultrafiltration concentrate, add 8 μl of proteinase K, 42 μl of proteinase K buffer and make up to 420 μl with water for injection, mix well and incubate at 37°C for 8 hours to obtain the test product. The amount of proteinase K added is 8 times of the protein content in the enzymolysis reaction system.

[0055] (2) Preparation of antiserum agarose gel plate

[0056] Weigh 0.1522g of agarose, add 10.0ml of pH8.6 barbiturate electrophoresis buffer, heat and swell completely, put in 56℃ water bath for 30min, add 0.3ml of meningococcal A polysac...

technical field

[0001] The invention belongs to the field of biotechnology, and more specifically relates to a method for determining the content of polysaccharides in each group of finished meningococcal polysaccharide conjugate vaccines. Background technique

[0002] The polysaccharide (antigen) content of the finished product of meningococcal polysaccharide conjugate vaccine is one of the basic conditions to ensure the effectiveness of the vaccine. For the polysaccharide content of the stock solution of the single conjugate, the A group can be obtained by measuring the phosphorus content, and the C, Y, or W135 group can be obtained by measuring the sialic acid content, and the finished products of meningococcal polysaccharide conjugate vaccines with more than 4 groups, such as A and C , Y, and W135 groups in the finished product conjugate vaccines of groups A, C, Y, and W135 are mixed together, and groups C, Y, and W135 all contain sialic acid, so it is impossible to obta...

Examples

Embodiment 1

[0031] Example 1 Determination of Group A polysaccharide content in the finished product of Group A, C, Y, W135 meningococcal polysaccharide conjugate vaccines

[0032]The determination of the polysaccharide content of group A in the finished product of group A, C, Y, and W135 meningococcal polysaccharide conjugate vaccines was repeated three times, namely repeat 1, repeat 2, and repeat 3, and each repeat was carried out according to the following steps:

[0033] (1) Preparation of test products for inspection

[0034] ①Pretreatment: Take 12 bottles of finished meningococcal polysaccharide conjugate vaccines of groups A, C, Y, and W135 (the labeled volume is 0.5ml per bottle), and reconstitute with 6ml of water for injection (that is, reconstitute with water for injection according to the labeled volume) , take 5.0ml in an ultrafiltration cup, centrifuge ultrafiltration at 5000×g 4°C for 40min, collect the concentrate, add water for injection to 1.0ml, and obtain the ultrafil...

Embodiment 2

[0045] Example 2 Determination of the polysaccharide content of group A in the finished product of meningococcal polysaccharide conjugate vaccine of group A, C, Y, W135

[0046] Except that the operations of the following steps are different, other operations are the same as those in Embodiment 1, and will not be repeated here.

[0047] (1) Preparation of test products for inspection

[0048] ②Protease K treatment: Take 70 μl of the ultrafiltration concentrate, add 4 μl of proteinase K, 42 μl of proteinase K buffer and supplement water for injection to 420 μl, mix well and incubate at 37°C for 6 hours to obtain the test product. The amount of proteinase K added is 4 times of the protein content in the enzymolysis reaction system.

[0049] (2) Preparation of antiserum agarose gel plate

[0050] Weigh 0.1521g of agarose, add 10.0ml of pH8.6 barbiturate electrophoresis buffer, heat and swell completely, put in 56℃ water bath for 20min, add 0.3ml of meningococcal A polysacch...

Embodiment 3

[0051] Example 3 Determination of the polysaccharide content of group A in the finished product of meningococcal polysaccharide conjugate vaccine of group A, C, Y, W135

[0052] Except that the operations of the following steps are different, other operations are the same as those in Embodiment 1, and will not be repeated here.

[0053] (1) Preparation of test products for inspection

[0054] ②Protease K treatment: Take 70 μl of the ultrafiltration concentrate, add 8 μl of proteinase K, 42 μl of proteinase K buffer and make up to 420 μl with water for injection, mix well and incubate at 37°C for 8 hours to obtain the test product. The amount of proteinase K added is 8 times of the protein content in the enzymolysis reaction system.

[0055] (2) Preparation of antiserum agarose gel plate

[0056] Weigh 0.1522g of agarose, add 10.0ml of pH8.6 barbiturate electrophoresis buffer, heat and swell completely, put in 56℃ water bath for 30min, add 0.3ml of meningococcal A polysac...

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