WO2012088762A1

WO2012088762A1 - Method for determining free polysaccharide content in meningococcal polysaccharide conjugate

Info

Publication number: WO2012088762A1
Application number: PCT/CN2011/002180
Authority: WO
Inventors: 王婷婷; 魏文进; 何佳琦
Original assignee: 北京民海生物科技有限公司; 深圳康泰生物制品股份有限公司
Priority date: 2010-12-30
Filing date: 2011-12-26
Publication date: 2012-07-05
Also published as: CN102175841A

Abstract

Provided is a method for determining free polysaccharide content in meningococcal Groups A, C, W135, and Y polysaccharide conjugate, comprising the following steps: 1) sampling: respectively taking 1 ml of a solution of meningococcal polysaccharide conjugate to be tested and a solution of a derivative corresponding to the meningococcal polysaccharide conjugate to be tested as Samples 1 and 3; and 2) centrifugation: additionally adding the solution of the meningococcal polysaccharide conjugate to be tested and the solution of the derivative corresponding to the meningococcal polysaccharide conjugate to be tested to a centrifugal tube; and adding a sodium deoxycholate solution to each centrifugal tube, standing for 30 min in an ice bath, then adding a hydrochloric acid solution, fully shaking, centrifuging, and collecting a supernatant from each centrifugal tube, as Samples 2 and 4; 3) calculating the polysaccharide concentration; and 4) calculating the free polysaccharide content. Through the present invention, the free polysaccharide content in meningococcal Groups A, C, W135, and Y polysaccharide conjugate can be accurately and conveniently determined, so as to evaluate the quality of a product.

Description

Method for determining free polysaccharide content in meningococcal polysaccharide conjugates

The invention belongs to the field of biotechnology, and relates to a method for determining a free polysaccharide, in particular to a method for determining the content of free polysaccharide in a polysaccharide combination of eight, C, W135 and Y meningococcus. Background technique

Meningococcal infection is the leading cause of bacterial meningitis and fulminant sepsis in children. According to statistics, the annual incidence of meningococcal meningitis in the world is about 500,000, especially in developing countries. Worldwide, most meningitis diseases are caused by meningococcal bacteria such as group A, group B, group C, group Y and group W135, and safe and effective vaccination is an ideal measure to prevent and control meningitis.

At present, meningitis vaccines at home and abroad mainly include polysaccharide vaccines and polysaccharide conjugate vaccines, among which: the antigen of the polysaccharide vaccine is a hapten, which is a non-T cell-dependent antigen, and the immune response induced by it does not involve T lymphocytes. It is not possible to produce immune memory. Therefore, WHO has suggested that the meningococcal polysaccharide vaccine is generally not used for the inoculation of infants and young children under 2 years old. The meningococcal polysaccharide conjugate vaccine is a combination of the extracted meningococcal capsular polysaccharide and protein carrier. The antigen is a T cell-dependent antigen, and the induced immune response is involved in T lymphocytes, which can stimulate the body to produce high levels of antibodies, enhance the immune memory of infants and young children, and thereby improve the immune effect.

The content of free polysaccharide in the polysaccharide conjugate vaccine is one of the key indicators for the quality control of polysaccharide conjugate vaccine. The free polysaccharide content exceeding the prescribed limit will have an adverse effect on the clinical effect of the vaccine. Therefore, the free polysaccharide content is an important indicator reflecting the quality of the combined vaccine. In foreign literature reports, hydrophobic chromatography and HPLC methods were used to determine the free polysaccharides in polysaccharide conjugates. However, due to the influence of similar molecular size on the impurities during the chromatographic process, the error of the determination results was large.

Chinese Zhu Wei et al. introduced the determination of group A meningococcal polysaccharide-tetanus toxoid by ethanol precipitation method in the establishment of a method for determination of free polysaccharide in group A meningococcal polysaccharide-tetanus toxoid. The method of free polysaccharides, but the method is cumbersome to operate. It takes several days for a sample to obtain the results. At the same time, there may be some error in the detection of samples with low free polysaccharide content. Chinese Patent Application No. 200610048876.9 discloses a method for determining a free polysaccharide of a group A and C meningococcal polysaccharide conjugate, which requires adding a cold phenol solution to the sample for shaking, but the cold phenol is more toxic, to the skin and mucous membranes. It is strongly corrosive. Long-term exposure can inhibit central nervous system or impair liver and kidney function. Inhalation of phenol can also cause headache, dizziness, fatigue, blurred vision, pulmonary edema and other symptoms.

In addition, due to the difference in polysaccharide conjugates, the difference in properties is also large. Therefore, the determination of free polysaccharides in polysaccharide conjugate vaccines has always been a technical difficulty. At present, there is no use of sodium deoxycholate extraction method to determine the content of free polysaccharides in polysaccharide conjugates. Report. Summary of the invention

The invention provides a method for overcoming the lack of free polysaccharide content in the prior method, which can accurately determine the content of free polysaccharide in the polysaccharide combination of A, C, W135 and Y meningococcal polysaccharides, and the operation method is simple and rapid.

The invention provides a method for determining a free polysaccharide of a meningococcal polysaccharide conjugate, comprising the following steps: sampling, centrifuging, calculating a polysaccharide concentration, calculating a free polysaccharide content, wherein during centrifugation, a meningococcal polysaccharide conjugate and a test are to be tested A sodium deoxycholate solution is added to the corresponding derivative of the meningococcal polysaccharide conjugate. :

Preferably, the method for determining a free polysaccharide in a meningococcal polysaccharide conjugate provided by the present invention comprises the following steps:

1) sampling: taking the meningococcal polysaccharide conjugate 1ml as sample 1, taking 1ml of the derivative solution corresponding to the meningococcal polysaccharide conjugate to be tested as sample 3;

2) Centrifugation: Another 1 ml of meningococcal polysaccharide conjugate is placed in a 1.5 ml centrifuge tube, and 1 ml of the derivative solution corresponding to the meningococcal polysaccharide conjugate to be tested is placed in another 1.5 ml centrifuge tube; Add ΙΟΟμΙ 1% DOC (sodium deoxycholate) solution to the ice for 30 minutes, then add 0.5μ of 0.5M HC1 solution, shake well, centrifuge at 5000 ~ 6000rpm for 15 ~ 20 minutes, collect the supernatant separately from each centrifuge tube. Lml, wherein the conjugate supernatant is sample 2, and the derivative supernatant is sample 4;

3) Calculate the polysaccharide concentration:

Group A meningococcal polysaccharide conjugates were used to determine the phosphorus content, and the phosphorus content was used to calculate the polysaccharide concentrations of sample 1, sample 2, sample 3, and sample 4; group C, group W135, and group Y meningococcal polysaccharide conjugates were used to determine the sialic acid content. The polysaccharide concentrations of Sample 1, Sample 2, Sample 3, and Sample 4 were calculated from the sialic acid content, and the polysaccharide concentrations of Sample 1, Sample 2, Sample 3, and Sample 4 were respectively Cl, C2, C3, and C4.

4) Calculate the free polysaccharide content: where P is the recovery rate and H is the free polysaccharide content

P=—X 100%, H=— ÷ P x draw %.

C3 CI

The meningococcal polysaccharide conjugate is a group A, group C, group W135 or group Y meningococcal polysaccharide conjugate.

Further preferably, the method for determining the free polysaccharide content of the group A meningococcal polysaccharide conjugate provided by the invention comprises the following steps:

1) Sampling: Take 1 ml of the meningococcal polysaccharide conjugate to be tested as sample 1, and take 1 ml of the derivative solution corresponding to the meningococcal polysaccharide conjugate to be tested as sample 3;

2) Centrifugation: Another 1 ml of meningococcal polysaccharide conjugate is placed in a 1.5 ml centrifuge tube, and 1 ml of the derivative solution corresponding to the meningococcal polysaccharide conjugate to be tested is placed in another 1.5 ml centrifuge tube; Add ΙΟΟμΙ 1% sodium deoxycholate solution to the tube for 30 minutes, then add 0.5 μl of hydrochloric acid solution to 25 μl, shake well, centrifuge at 5000 ~ 6000 rpm for 15-20 minutes, and collect 15 ml of supernatant from each centrifuge tube. Wherein the conjugate supernatant is sample 2, and the derivative supernatant is sample 4;

3) Calculate the concentration of polysaccharide: Group A meningococcal polysaccharide conjugate to determine the phosphorus content, according to "A group polysaccharide concentration = phosphorus content / 0.08", calculate the concentration of polysaccharides of sample 1, sample 2, sample 3, sample 4, sample 1, Sample 2, sample 3, sample 4 polysaccharide concentration is Cl, C2, C3, C4;

4) Calculate the free polysaccharide content: where P is the recovery rate and H is the free polysaccharide content.

P= ― X 100%, H=— ÷ P X 100%.

C3 C1

The group A polysaccharide concentration = phosphorus content / 0.08 in the formula and the coefficient in the formula 0.08 reference from "Embrane Miao", Zhang Yanling.

Further preferably, the method for determining the free polysaccharide content of the group C meningococcal polysaccharide conjugate provided by the invention comprises the following steps:

1) Sampling: Take 1 ml of the meningococcal polysaccharide conjugate to be tested as sample 1, and take the lml of the derivative solution corresponding to the meningococcal polysaccharide conjugate to be sample 3;

2) Centrifugation: Another 1 ml of meningococcal polysaccharide conjugate is placed in a 1.5 ml centrifuge tube for the brain to be examined. 1 ml of the derivative solution corresponding to the Mesangococcus polysaccharide conjugate was placed in another 1.5 ml centrifuge tube; ΙΟΟμΙ 1% sodium deoxycholate solution was added to each centrifuge tube, and the ice bath was kept for 30 minutes, and then 0.5 Μ hydrochloric acid was added. Solution 25μ1, fully shaken, centrifuged at 5000 ~ 6000rpm for 15 ~ 20 minutes, each supernatant tube separately collected 1ml of the supernatant, wherein the supernatant of the combination is sample 2, the supernatant of the derivative is sample 4;

3) Calculate the concentration of polysaccharide: Group C meningococcal polysaccharide conjugate to determine the sialic acid content, according to "C group polysaccharide concentration = sialic acid content / 0.8", calculate the sample 1, sample 2, sample 3, sample 4 ^ polysaccharide concentration, sample 1, sample 2, sample 3, sample 4 polysaccharide concentration is Cl, C2, C3, C4;

P= — X 100%, Η=— ÷ Ρ χ 100%.

C3 CI

The C group polysaccharide concentration = sialic acid content / 0.8 in the formula and the coefficient in the formula are referenced from "Vaccine", Zhang Yanling.

Further preferably, the method for determining the free polysaccharide content of the W135 group meningococcal polysaccharide conjugate provided by the invention comprises the following steps:

2) Centrifugation: Another 1 ml of meningococcal polysaccharide conjugate is placed in a 1.5 ml centrifuge tube, and 1 ml of the derivative solution corresponding to the meningococcal polysaccharide conjugate to be tested is placed in another 1.5 ml centrifuge tube; Add ΙΟΟμΙ 1% sodium deoxycholate solution to the tube for 30 minutes, then add 0.5 μl of hydrochloric acid solution to 25 μl, shake well, centrifuge at 5000 ~ 6000 rpm for 15-20 minutes, and collect 1 ml of supernatant from each centrifuge tube. Wherein the conjugate supernatant is sample 2, and the derivative supernatant is sample 4;

3) Calculate the concentration of polysaccharide: Determine the sialic acid content of the W135 group meningococcal polysaccharide conjugate, and calculate the polysaccharide concentration of sample 1, sample 2, sample 3, sample 4 according to "W135 group polysaccharide concentration = sialic acid content / 0.56". 1, sample 2, sample 3, sample 4 polysaccharide concentration is Cl, C2, C3, C4;

P= ― X 100%, Η=— ÷ Ρ χ 100%.

C3 CI

The W135 group polysaccharide concentration = phosphorus content / 0.56 formula and the coefficient of the formula 0.56 are referenced from "vaccine", Zhang Yanling. Further preferably, the method for determining the free polysaccharide content of the Y group meningococcal polysaccharide conjugate provided by the invention comprises the following steps:

1) Sampling: Take 1 ml of meningococcal polysaccharide conjugate to be tested as sample 1, and take 1 ml of the derivative solution corresponding to the meningococcal polysaccharide conjugate to be sample 3;

2) Centrifugation: Another 1 ml of meningococcal polysaccharide conjugate is placed in a 1.5 ml centrifuge tube, and 1 ml of the derivative solution corresponding to the meningococcal polysaccharide conjugate to be tested is placed in another 1.5 ml centrifuge tube; Add ΙΟΟμΙ 1% sodium deoxycholate solution to the tube for 30 minutes, then add 0.5 μl of hydrochloric acid solution to 25 μl, shake well, centrifuge at 5000-6000 rpm for 15-20 minutes, and collect 1 ml of supernatant from each centrifuge tube. The conjugate supernatant is sample 2, and the derivative supernatant is sample 4;

3) Calculate the concentration of polysaccharide: Y group meningococcal polysaccharide conjugate to determine the sialic acid content, according to "Y group polysaccharide concentration = sialic acid content / 0.56", calculate the concentration of polysaccharides of sample 1, sample 2, sample 3, sample 4, sample 1, sample 2, sample 3, sample 4 polysaccharide concentration is Cl, C2, C3, C4;

P= ― X 100%, H=— ÷ P x l00%.

C3 CI

The W135 group polysaccharide concentration = phosphorus content / 0.56 formula and the coefficient in the formula 0.56 reference from "vaccine" (Zhang Yanling).

among them:

The concentration unit of the DOC is 1% (lg/100 ml);

The concentration unit of the hydrochloric acid is mol/L, abbreviated as M;

The phosphorus content in the polysaccharide conjugate is determined by the phosphorus assay in the third edition of Appendix VIIA of the 2010 edition of the Chinese Pharmacopoeia. The sialic acid content in the polysaccharide conjugate is used in the 2010 edition of the Chinese Pharmacopoeia. Determination by resorcinol colorimetry.

The beneficial effects of the invention are:

1. The present invention provides a method for accurately and rapidly determining the content of free polysaccharides in a combination of group A, group C, group W135 and group Y meningococcus, thereby evaluating the quality of the product.

2, sodium deoxycholate, molecular formula: C ₂₄ H4 ₀ O ₄ ' Na, molecular weight: 414.56, traits: white crystalline powder. Similar to bile smell, it has a strong bitter taste. Easy to absorb moisture. Soluble in water, slightly soluble in anhydrous alcohol, insoluble in ether. Low toxicity, half lethal dose (rat, oral) 1370mg/kg. Irritating. The invention utilizes the characteristics of DOC (sodium deoxycholate) which can precipitate proteins under acidic conditions. After the sample is treated by DOC and centrifuged at high speed, the conjugate in the conjugate vaccine is precipitated and the polysaccharide remains in the supernatant under the same conditions. In this way, the bound polysaccharide is separated from the free polysaccharide, and the content of the free polysaccharide can be calculated by separately measuring the polysaccharide content of the centrifugation supernatant and the stock solution.

3. Compared with the patent 200610048876.9, although the recovery rate is not much different, it overcomes the unfavorable factor that the cold phenol is harmful to the human body. At the same time, the method is not only applicable to the free polysaccharide content of the polysaccharide combination of group A and group C meningococcus. The determination of free polysaccharides in W135, Y meningococcal polysaccharide conjugates can also be determined quickly and accurately. detailed description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

In the present invention, the recovery ratio P is between 80 and 120%, and higher than 90% means high accuracy.

Example 1

1. Sampling: Take 1ml of group A meningococcal polysaccharide conjugate to be tested as sample 1 and take the lml of the derivative solution corresponding to the group A meningococcal polysaccharide conjugate as sample 3;

2. Centrifugation: Take another 1 ml of the group A meningococcal polysaccharide conjugate in a 1.5 ml centrifuge tube, and take 1 ml of the derivative solution corresponding to the group A meningococcal polysaccharide conjugate to be tested in another 1.5 ml centrifuge tube. Add ΙΟΟμΙ 1% DOC solution to each centrifuge tube, keep icolo for 30 minutes, then add 0.5μH0.5 solution to 25μ1, shake well, centrifuge at 5000rpm for 15-20 minutes, collect supernatant 1ml separately from each centrifuge tube. , wherein the conjugate supernatant is sample 2, and the derivative supernatant is sample 4.

3. Calculate the polysaccharide concentration: Determine the phosphorus content of sample 1, sample 2, sample 3, sample 4, and calculate the polysaccharide concentration of sample 1, sample 2, sample 3, sample 4 according to the formula "group A polysaccharide concentration = phosphorus content / 0.08". Cl, C2, C3 and C4.

The phosphorus content in the sample was determined by the phosphorus method in the third edition of Appendix VIIA of the 2010 edition of the Chinese Pharmacopoeia.

4. Calculate the free polysaccharide content: where P is the recovery rate and H is the free polysaccharide content.

5, using the method provided in Example 1 of the present invention to determine three batches of meningococcal group A polysaccharide conjugate samples The test results are shown in Table 1.

Table 1: Results of three batches of meningococcal group A polysaccharide conjugate samples

The results in Table 1 show that: The method provided by the present invention can accurately determine the content of free polysaccharide in the polysaccharide combination of meningococcal group A.

Example 2

1. Sampling: Take 1ml of group C meningococcal polysaccharide conjugate as sample 1, take 1ml of the derivative solution corresponding to the group C meningococcal polysaccharide conjugate as sample 3;

2. Centrifugation: Take another 1 ml of the group C meningococcal polysaccharide conjugate in a 1.5 ml centrifuge tube, and take 1 ml of the derivative solution corresponding to the group C meningococcal polysaccharide conjugate to be tested in another 1.5 ml centrifuge tube. Add ΙΟΟμΙ 1% DOC solution to each centrifuge tube, hold for 30 minutes in ice bath, add 25μ1 of 0.5M HCL solution, shake well, centrifuge at 5000rpm for 15-20 minutes, collect supernatant 1ml separately from each centrifuge tube. , wherein the conjugate supernatant is sample 2, and the derivative supernatant is sample 4;

3. Calculate the concentration of polysaccharide: Determine the sialic acid content of sample 1, sample 2, sample 3, sample 4, and calculate the sample 1, sample 2, sample 3, sample 4 according to the formula "C group polysaccharide concentration = sialic acid content / 0.8" Polysaccharide concentration Cl, C2, C3, C4;

The content of sialic acid in the sample was determined by the sialic acid content determination method in the third edition of Appendix VI of the 2010 edition of the Chinese Pharmacopoeia.

P=—X 100%, Η=— ÷ Ρ χ 100%.

C3 CI

5. The samples of the three groups of meningococcal group C polysaccharide conjugates were determined by the method provided in Example 2 of the present invention, and the test results are shown in Table 2: Table 2: Results of three batches of meningococcal group C polysaccharide conjugate samples

The results in Table 2 show that: This method can accurately determine the free polysaccharide content of the meningococcal group C polysaccharide combination.

Example 3

1. Sampling: Take 1ml of the W135 group meningococcal polysaccharide conjugate as sample 1, and take 1ml of the derivative solution corresponding to the W135 group meningococcal polysaccharide conjugate as sample 3;

2. Centrifugation: Take another 1 ml of the W135 group meningococcal polysaccharide conjugate in a 1.5 ml centrifuge tube, and take 1 ml of the derivative solution corresponding to the W135 group meningococcal polysaccharide conjugate to be placed in another 1.5 ml centrifuge tube. Add ΙΟΟμΙ 1% DOC solution to each centrifuge tube, hold for 30 minutes in an ice bath, then add 0.5 μL of 0.5 Μ HCL solution, shake well, centrifuge at 5000 rpm for 15-20 minutes, and collect supernatant 1 ml separately from each centrifuge tube. , wherein the conjugate supernatant is sample 2, and the derivative supernatant is sample 4;

3. Calculate the concentration of polysaccharide: sialic acid content of sample 1, sample 2, sample 3, sample 4 (the sialic acid content in the sample is determined in the same manner as in Example 2), according to the formula "W135 group polysaccharide concentration = sialic acid content / 0.56 "Computation sample 1, sample 2, sample 3, sample 4 polysaccharide concentration Cl, C2, C3, C4;

P=—X 100%, Η=— ÷ Ρ χ 100%.

C3 CI

5. Three batches of meningococcal W135 group polysaccharide composition samples were determined by the method provided in Example 1 of the present invention, and the test results are shown in Table 3:

Table 3: Three batches of meningococcal bacteria W135 group polysaccharide conjugate sample determination results

20100407 233.7 22.9 196.3 189.5 96.5 10.2

20100412 286.5 26.0 225.6 218.9 97.0 9.4 Table 3 shows that: This method can accurately determine the free polysaccharide content of meningococcal W135 group polysaccharide conjugate.

Example 4

1. Sampling: Take the group Y meningococcal polysaccharide conjugate lml as sample 1 and take the lml of the derivative solution corresponding to the group Y meningococcal polysaccharide conjugate as sample 3;

2. Centrifugation: Take another 1 ml of the Y group meningococcal polysaccharide conjugate in a 1.5 ml centrifuge tube, and take 1 ml of the derivative solution corresponding to the Y group meningococcal polysaccharide conjugate to be placed in another 1.5 ml centrifuge tube. Add ΙΟΟμΙ 1% DOC solution to each centrifuge tube, keep ice for 30 minutes, then add 0.5μH0.5 solution to 25μ1, shake well, centrifuge at 5000rpm for 15-20 minutes, collect supernatant 1ml separately from each centrifuge tube. , wherein the conjugate supernatant is sample 2, and the derivative supernatant is sample 4;

3. Calculate the concentration of polysaccharide: Determination of the sialic acid content of sample 1, sample 2, sample 3, sample 4 (the determination method of sialic acid in the sample is the same as in Example 2), according to the formula "Y group polysaccharide concentration = sialic acid content / 0.56 "Computation sample 1, sample 2, sample 3, sample 4 polysaccharide concentration Cl, C2, C3, C4;

P=—X 100%, H=— ÷ P x 100%.

C3 CI

5. Using the method provided in Example 1 of the present invention, three batches of meningococcal Y group polysaccharide conjugate samples were determined, and the test results are shown in Table 4:

Table 4: Results of three batches of meningococcal Y group polysaccharide conjugate samples

The results in Table 4 show that: This method can accurately determine the free polysaccharide content of the meningococcal Y group polysaccharide combination. Example 5

1. Sampling: Take 1ml of the W135 group meningococcal polysaccharide conjugate as sample 1 and take 1ml of the derivative solution corresponding to the W135 group meningococcal polysaccharide conjugate as sample 3;

2. Centrifugation: Another 1 ml of the W135 group meningococcal polysaccharide conjugate is placed in a 1.5 ml centrifuge tube, and 1 ml of the derivative solution corresponding to the W135 group meningococcal polysaccharide conjugate is placed in another 1.5 ml centrifuge tube. Add ΙΟΟμΙ 1% DOC solution to each centrifuge tube, keep in ice bath for 30 minutes, then add 0.5μH 0.5ml HCL solution, shake well, centrifuge at 6000rpm for 15-20 minutes, and collect 1ml of supernatant from each centrifuge tube. Wherein the conjugate supernatant is sample 2, and the derivative supernatant is sample 4;

3. Calculate the concentration of polysaccharide: sialic acid content of sample 1, sample 2, sample 3, sample 4 (the sialic acid content in the sample is determined in the same manner as in Example 2), according to the formula "W135 group polysaccharide concentration = sialic acid content / 0.56 ,, calculate the concentration of the sample 1, sample 2, sample 3, sample 4, Cl, C2, C3, C4;

P=—X 100%, Η=— ÷ Ρ χ 100%.

C3 CI

5. Three batches of meningococcal W135 group polysaccharide composition samples were determined by the method provided in Example 1 of the present invention, and the test results are shown in Table 5:

Table 5: Results of three batches of meningococcal W135 group polysaccharide conjugate samples

The results in Table 5 show that: This method can accurately determine the free polysaccharide content of the polysaccharide conjugate of meningococcal W135 group.

Example ό

1. Sampling: Take 1ml of the meningococcal polysaccharide conjugate of the sputum group as sample 1, and take the lml of the derivative solution corresponding to the Y group meningococcal polysaccharide conjugate as sample 3;

2, centrifugation: Another group of Y group meningococcal polysaccharide conjugate lml placed in a 1.5ml centrifuge tube, Take 1 ml of the derivative solution corresponding to the Y group meningococcal polysaccharide conjugate to be placed in another 1.5 ml centrifuge tube; add 100 μl 1% DOC solution to each centrifuge tube, keep ice for 30 minutes, then add 0.5 25 μl of M HCL solution, fully shaken, centrifuged at 6000 rpm for 15-20 minutes, and each supernatant was separately collected 1 ml of the supernatant, wherein the supernatant of the conjugate was sample 2, and the supernatant of the derivative was sample 4;

P=—X 100%, Η=— ÷ Ρ χ 100%.

C3 CI

5. Three batches of meningococcal Y group polysaccharide conjugate samples were determined by the method provided in Example 1 of the present invention, and the test results are shown in Table 6:

Table 6: Results of three batches of meningococcal sputum polysaccharide conjugate samples

The results in Table 6 show that: This method can accurately determine the free polysaccharide content of the meningococcal polysaccharide combination. Industrial applicability

The invention utilizes the characteristics of sodium deoxycholate (DOC) to precipitate proteins under acidic conditions. After the sample is treated by DOC and centrifuged at high speed, the conjugate in the conjugate vaccine is precipitated and the polysaccharide remains in the supernatant under the same conditions. In this way, the bound polysaccharide is separated from the free polysaccharide, and the content of the free polysaccharide can be calculated by separately measuring the polysaccharide content of the centrifugation supernatant and the stock solution. The method can accurately and quickly determine the content of the polysaccharides in the polysaccharide combination of the sputum group, the C group, the W135 group and the Y group meningococcus, thereby evaluating the quality of the product.

Claims

Claim

A method for determining a free polysaccharide content of a meningococcal polysaccharide conjugate, comprising sampling, centrifuging, calculating a polysaccharide concentration, and calculating a free polysaccharide content, characterized in that the meningococcal polysaccharide conjugate and the meninges to be tested are centrifuged A sodium deoxycholate solution is added to the derivative corresponding to the inflammatory combination of the bacterium.

The assay method according to claim 1, wherein the meningococcal polysaccharide composition is selected from the group consisting of group A, group C, group W135 or group Y.

The method according to claim 2, wherein the meningococcal polysaccharide composition is a group A meningococcal polysaccharide conjugate, and the method for measuring the free polysaccharide content in the conjugate comprises the following steps:

2) Centrifugation: Another 1 ml of meningococcal polysaccharide conjugate is placed in a 1.5 ml centrifuge tube, and 1 ml of the derivative solution corresponding to the meningococcal polysaccharide conjugate to be tested is placed in another 1.5 ml centrifuge tube; Add ΙΟΟμΙ 1% sodium deoxycholate solution to the tube for 30 minutes, then add 0.5 μl of hydrochloric acid solution to 25 μl, shake well, centrifuge at 5000-6000 rpm for 15-20 minutes, and collect 1 ml of supernatant from each centrifuge tube. sample 2 was combined supernatant, the supernatant of derivatives of 4 ^samples;.:

3) Calculate the concentration of polysaccharide: Group A meningococcal polysaccharide conjugate to determine the phosphorus content, according to "group A polysaccharide concentration = phosphorus content / 0.08", calculate the concentration of the sample 1, sample 2, sample 3, sample 4, Cl, C2 C3, C4;

4) Calculate the free polysaccharide content: where P is the recovery rate and H is the free polysaccharide content'

The method according to claim 2, wherein the meningococcal polysaccharide composition is a group C meningococcal polysaccharide conjugate, and the method for measuring the free polysaccharide content in the conjugate comprises the following steps:

1) Sampling: Take 1 ml of meningococcal polysaccharide conjugate to be sampled as sample 1, and take 1 ml of the derivative solution corresponding to the meningococcal polysaccharide conjugate to be sample 3; 2) Centrifugation: Another 1 ml of meningococcal polysaccharide conjugate is placed in a 1.5 ml centrifuge tube, and 1 ml of the derivative solution corresponding to the meningococcal polysaccharide conjugate to be tested is placed in another 1.5 ml centrifuge tube; Add ΙΟΟμΙ 1% sodium deoxycholate solution to the tube, keep ice for 30 minutes, then add 0.5 Μ.. hydrochloric acid solution 25μ1, shake well, centrifuge at 5000 ~ 6000rpm for 15 ~ 20 minutes, separately collect the supernatant lml in each centrifuge tube , wherein the conjugate supernatant is sample 2, and the derivative supernatant is sample 4;

3) Calculate the concentration of polysaccharide: Group C meningococcal polysaccharide conjugate to determine the sialic acid content, according to "C group polysaccharide concentration = sialic acid content / 0.8", calculate the concentration of the sample 1, sample 2, sample 3, sample 4, Cl, C2, C3, C4;

P=—X 100%, H=— ÷ P x 100%.

C3 CI

5. The method according to claim 2, wherein the meningococcal polysaccharide composition is a W135 group meningococcal polysaccharide conjugate, and the method for determining the free polysaccharide content in the conjugate comprises the following steps:

2) Centrifugation: Another 1 ml of meningococcal polysaccharide conjugate is placed in a 1.5 ml centrifuge tube, and 1 ml of the derivative solution corresponding to the meningococcal polysaccharide conjugate to be tested is placed in another 1.5 ml centrifuge tube; Add ΙΟΟμΙ 1% sodium deoxycholate solution to the tube for 30 minutes, then add 0.5 μl hydrochloric acid solution 25 μl, shake well, centrifuge at 5000 ~ 6000 rpm for 15 ~ 20 minutes, separate each tube. Collect the supernatant lml, Wherein the conjugate supernatant is sample 2, and the derivative supernatant is sample 4;

3) Calculate the polysaccharide concentration: The sialic acid content of the W135 group meningococcal polysaccharide conjugate is determined. According to the "W135 group polysaccharide concentration = sialic acid content / 0.56", the polysaccharide concentration Cl of sample 1, sample 2, sample 3, sample 4 is calculated. C2, C3, C4;

P=—X 100%, H=— ÷ P x 100%.

C3 CI

The method according to claim 2, wherein the meningococcal polysaccharide conjugate is a group Y meningococcal polysaccharide conjugate, and the method for measuring a free polysaccharide content in the conjugate is Including the following steps:

2) Centrifugation: Another 1 ml of meningococcal polysaccharide conjugate is placed in a 1.5 ml centrifuge tube, and 1 ml of the derivative solution corresponding to the meningococcal polysaccharide conjugate to be tested is placed in another L5 ml centrifuge tube; Add ΙΟΟμΙ 1% sodium deoxycholate solution, keep ice for 30 minutes, then add 0.5μl hydrochloric acid solution 25μ1, shake well, centrifuge at 5000 ~ 6000rpm for 15 ~ 20 minutes, collect 15ml of supernatant separately from each centrifuge tube, where combined The supernatant is sample 2, and the derivative supernatant is sample 4;

3) Calculate the polysaccharide concentration: Y group meningococcal polysaccharide conjugate to determine the sialic acid content, according to "Y group polysaccharide concentration = sialic acid content / 0.56,, calculate the sample 1, sample 2, sample 3, sample 4 polysaccharide concentration Cl , C2, C3, C4;

P=—X 100%, Η=— ÷ Ρ χ 100%.

C3 CI