Method for detecting the activation degree of polysaccharides activated by cyanogen bromide

Abstract

The invention discloses a method for detecting the activation degree of polysaccharide activated by cyanogen bromide. The method comprises the following steps: pre-treating a sample to form a test sample, and obtaining a cyanate test sample and an iminocarbonate test sample; obtaining a cyanate standard sample and an iminocarbonate standard sample, and forming a cyanate standard sample absorbance curve and an iminocarbonate standard sample absorbance curve; measuring the absorbance of the cyanate test sample, and obtaining the content of cyanate in the test sample according to the absorbance curve of the cyanate standard sample; measuring the absorbance of the iminocarbonate test sample, and obtaining the content of iminocarbonate in the test sample according to the absorbance curve of the iminocarbonate standard sample; and calculating the activation degree of the test sample, that is, the activation degree of the polysaccharide activated by cyanogen bromide in the sample, according to the contents of cyanate and iminocarbonate in the test sample. The method for detecting the activation degree of polysaccharide activated by cyanogen bromide can effectively measure the activation degree of polysaccharide activated by cyanogen bromide.

Description

Technical Field

[0001] The present invention relates to a method for detecting the activation degree, and particularly to a method for detecting the activation degree of cyanogen bromide-activated polysaccharide. Background Art

[0002] A polysaccharide conjugate vaccine refers to a polysaccharide-protein conjugate vaccine prepared by covalently binding a polysaccharide to a protein carrier by a chemical method. The polysaccharide conjugate vaccine may specifically include Haemophilus influenzae type b conjugate vaccine, meningococcal conjugate vaccine, pneumococcal conjugate vaccine, etc. Immunogenicity refers to the property of being able to induce an immune response, that is, the property that an antigen can stimulate specific immune cells, activate, proliferate, and differentiate the immune cells, and finally produce immune effector substances such as antibodies and sensitized lymphocytes.

[0003] To a certain extent, the quality of a polysaccharide conjugate vaccine can be evaluated through immunogenicity. By measuring the activation degree of cyanogen bromide-activated polysaccharide, which is the raw material for preparing the polysaccharide conjugate vaccine, the derivatization rate of the polysaccharide conjugate vaccine can be evaluated, the content of free polysaccharide in the polysaccharide conjugate vaccine can be reduced, and the immunogenicity can be improved. Therefore, how to detect the activation degree of cyanogen bromide-activated polysaccharide is a problem to be solved.

[0004] The information disclosed in this background art section is only intended to enhance the overall understanding of the present invention and should not be regarded as an admission or any form of suggestion that this information constitutes prior art already known to those of ordinary skill in the art. Summary of the Invention

[0005] An object of the present invention is to provide a method for detecting the activation degree of cyanogen bromide-activated polysaccharide, which can effectively and correctly detect the activation degree of cyanogen bromide-activated polysaccharide.

[0006] To achieve the above object, an embodiment of the present invention provides a method for detecting the activation degree of cyanogen bromide-activated polysaccharide, including the following steps:

[0007] The sample is pretreated to form a test sample, and a cyanate test sample and an imidocarbonate test sample are obtained;

[0008] A cyanate standard sample and an imidocarbonate standard sample are obtained, and a cyanate standard sample absorbance curve and an imidocarbonate standard sample absorbance curve are formed;

[0009] The absorbance of the cyanate test sample is measured, and the content of cyanate in the test sample is obtained according to the cyanate standard sample absorbance curve;

[0010] The absorbance of the imidocarbonate test sample is measured, and the content of imidocarbonate in the test sample is obtained according to the imidocarbonate standard sample absorbance curve; and

[0011] According to the contents of cyanate ester and imidocarbonate in the test sample, the activation degree of the test sample, that is, the activation degree of cyanogen bromide-activated polysaccharide in the sample, is calculated. Before the implementation of the method, the determination of the contents of cyanate ester and imidocarbonate is subjected to methodological verification in terms of method linearity, accuracy, precision, and specificity to ensure the accuracy of the results.

[0012] In one or more embodiments of the present invention, the steps of the pretreatment include:

[0013] Centrifuging or dialyzing the sample.

[0014] In one or more embodiments of the present invention, the conditions of the centrifuging include: the centrifugation parameter is 6000 - 8000g, and the number of centrifugations is at least nine times.

[0015] In one or more embodiments of the present invention, the conditions of the dialyzing include: using a sodium chloride solution with a concentration of less than 0.9 wt% as the dialysis solution, using a regenerated cellulose membrane as the dialysis membrane, and dialyzing for at least 24 h under the condition of a temperature of 2 - 8°C.

[0016] In one or more embodiments of the present invention, the steps of obtaining the imidocarbonate test sample include:

[0017] Adding a part of the test sample into a container, adding a hydrochloric acid solution, reacting at 20 - 60°C for at least 15 min, then adding a sodium acetate solution and mixing evenly, and after centrifuging, taking the supernatant as the imidocarbonate test sample.

[0018] In one or more embodiments of the present invention, the steps of obtaining the cyanate ester test sample include:

[0019] Taking a part of the test sample, namely obtaining the cyanate ester test sample.

[0020] In one or more embodiments of the present invention, the steps of measuring the absorbance of the cyanate ester test sample include:

[0021] After adding a dimethylbarbituric acid pyridine solution to the cyanate ester test sample, stirring at a temperature not exceeding 60°C for at least 5 min, and then measuring its absorbance.

[0022] In one or more embodiments of the present invention, the concentration of the dimethylbarbituric acid pyridine solution is 0.8% - 2%.

[0023] In one or more embodiments of the present invention, the steps of measuring the absorbance of the imidocarbonate test sample include:

[0024] After adding the reduced ninhydrin reagent to the imidocarbonate test sample, heat it in a boiling water bath for 10 - 30 min, then add the formaldehyde dilution solution, and measure its absorbance after mixing evenly.

[0025] In one or more embodiments of the present invention, the method for measuring the absorbance includes: using ultraviolet-visible spectrophotometry to measure the absorbance at a wavelength of 500 - 600 nm.

[0026] Compared with the prior art, the detection method for the activation degree of cyanogen bromide-activated polysaccharide according to the embodiments of the present invention calculates the activation degree of cyanogen bromide-activated polysaccharide in the sample by measuring the contents of cyanate ester and imidocarbonate in the sample. The detection method for the activation degree of cyanogen bromide-activated polysaccharide of the present invention can effectively detect the activation degree of cyanogen bromide-activated polysaccharide. Detailed Embodiments

[0027] The following describes the detailed embodiments of the present invention in detail, but it should be understood that the protection scope of the present invention is not limited by the detailed embodiments.

[0028] Unless otherwise clearly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "having" etc. will be understood to include the stated elements or components, without excluding other elements or other components.

[0029] The detection method for the activation degree of cyanogen bromide-activated polysaccharide according to the preferred embodiment of the present invention includes the following steps:

[0030] S1. Pretreat the sample to form a test sample, and obtain the cyanate ester test sample and the imidocarbonate test sample.

[0031] The sample can be considered as a sample with cyanogen bromide-activated polysaccharide. After cyanogen bromide activates the polysaccharide, cyanate ester groups (-OCN) will be generated. If -OH exists in the ortho position, the cyanate ester groups will rearrange to form linear or cyclic imidocarbonates. And the cyanate ester groups may also hydrolyze under strong acid or strong base conditions to form carbamates, which cannot participate in the derivatization and binding processes. Therefore, the determination of the activation degree of the activated polysaccharide is to detect the ratio of the contents of cyanate ester and imidocarbonate to the polysaccharide content. There is a certain amount of cyanogen bromide in the sample of cyanogen bromide-activated polysaccharide, and in the subsequent steps, it is necessary to measure the absorbances of the cyanate ester test sample and the imidocarbonate test sample. During this measurement process, cyanogen bromide will affect the result of the absorbance test. Therefore, it is necessary to pretreat the sample to remove the interference of cyanogen bromide in the sample.

[0032] Since in this detection method, the reduced ninhydrin reagent is used to react with the imidocarbonate and measure the content of the imidocarbonate, but due to the following color reactions of the reduced ninhydrin reagent,

[0033] 1) Ninhydrin reagent + sodium acetate: shows brick red color;

[0034] 2) Ninhydrin reagent + hydrochloric acid: does not show color. When sodium hydroxide is added, the color gradually turns blue-violet;

[0035] 3) Ninhydrin reagent + sodium hydroxide: shows blue-violet color.

[0036] 4) Ninhydrin reagent + cyanogen bromide: shows yellow color. When sodium hydroxide is gradually added, there is no obvious change. It can be considered that the reaction between cyanogen bromide and ninhydrin reagent is irreversible. Therefore, it can be considered that cyanogen bromide has an impact on the determination of the content of imine carbonate.

[0037] It should be noted that the raw material of the polysaccharide conjugate vaccine (polysaccharide raw material) is activated and derivatized with cyanogen bromide, and then subsequent steps are carried out to finally produce the product, namely the polysaccharide conjugate vaccine. Among them, the raw material after cyanogen bromide activation treatment can be called activated polysaccharide, and after further derivatization treatment, polysaccharide derivatives can be obtained. That is, cyanogen bromide-activated polysaccharide can specifically be considered as the activated polysaccharide obtained by activating the raw material of the polysaccharide conjugate vaccine with cyanogen bromide.

[0038] Specifically, for example, the polysaccharide conjugate vaccine of meningococcus group ACYW135 is made from the refined polysaccharide of meningococcus group A, which is activated and derivatized with cyanogen bromide, and the Neisseria meningitidis of groups C, Y, and W135 are derivatized and then covalently bound to the CRM197 carrier protein to form a polysaccharide-protein conjugate, and then freeze-dried after adding a suitable protective agent. It is used to prevent epidemic cerebrospinal meningitis caused by Neisseria meningitidis of groups A, C, Y, and W135. In the 13-valent pneumococcal polysaccharide conjugate vaccine, the 13 types of refined polysaccharides (type 1, type 3, type 4, type 5, type 6A, type 6B, type 7F, type 9V, type 14, type 18C, type 19A, type 19F, type 23F) are activated and derivatized with cyanogen bromide, covalently bound to the tetanus carrier protein to form a polysaccharide-protein conjugate, and after being adsorbed by a certain amount of adjuvant, it is used to prevent various diseases caused by Streptococcus pneumoniae of the above serotypes.

[0039] In the specific implementation manner, the cyanogen bromide-activated polysaccharide can be the activated polysaccharide formed by activating the refined polysaccharide of meningococcus group A with cyanogen bromide, or the activated polysaccharide obtained by activating the refined polysaccharides of the above-mentioned pneumococcal types with cyanogen bromide.

[0040] Removing cyanogen bromide from the sample can be considered that the content of cyanogen bromide in the test sample is extremely low, and its impact on the subsequent test steps can be ignored. Or removing cyanogen bromide from the sample can also be considered that the test sample does not contain cyanogen bromide, that is, it has no impact on the subsequent test steps. The impact of the above two situations on the subsequent test process can be ignored.

[0041] In S1, the specific steps for sample pretreatment to form the test sample can be: putting the sample into an ultrafiltration centrifuge tube for centrifugation; or subjecting the sample to dialysis treatment. The results of a large number of experiments have proved that both methods can achieve the purpose of removing cyanogen bromide without interference in this test, and the two methods can be selected according to the experimental conditions when treating the sample.

[0042] Among them, when putting the sample into an ultrafiltration centrifuge tube for centrifugation, it is because cyanogen bromide is a small molecule and can pass through the filter membrane in the ultrafiltration centrifuge tube, while components to be detected such as cyanate ester and imidocarbonate will not pass through the filter membrane. The conditions for centrifugation include: the centrifugation parameters are 6000 - 8000g, 10 - 30 min / time, and the number of ultrafiltration centrifugation times is at least nine times. The reason for the number of ultrafiltration centrifugation times being nine times is that, first, a relatively large centrifugation parameter of 6000 - 8000g is required. Considering that there may be cyanogen bromide residues on the filter membrane in the ultrafiltration centrifuge tube, it is necessary that the cyanogen bromide residue amount in the retentate of two consecutive centrifugations has no obvious change, or the filtrate of two consecutive centrifugations has basically no cyanogen bromide residue, then it can be determined that all the cyanogen bromide in the activated sugar has been removed. For the ultrafiltration centrifugation of pneumococcal activated polysaccharides of types 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F, appropriate samples were taken at the 3rd, 4th, 5th, 6th, 7th, 8th, and 9th centrifugations to measure the cyanogen bromide content in the filtrate and retentate. The results of a large number of experiments have proved (some data are shown in Table 1) that the average cyanogen bromide content in the retentate at the 7th centrifugation is 20.3869 ng / ml, and the average cyanogen bromide content in the retentate at the 8th centrifugation is 19.1977 ng / ml, that is, the cyanogen bromide residue amount in the retentate of the two centrifugations has no obvious change, it can be determined that there is basically no cyanogen bromide residue in the retentate or the influence of the remaining cyanogen bromide on the subsequent detection steps can be ignored, and the 8th time can be determined as the end point of sample ultrafiltration centrifugation. However, in order to reduce the influence of cyanogen bromide residue on the experiment, according to the different addition amounts of cyanogen bromide in the process, the number of centrifugation times is generally selected as nine times or more.

[0043] Table 1

[0044] Concentration (ng / ml) The third retentate of activated sugar 11074 The fourth filtrate of activated sugar 8745 The fifth retentate of activated sugar 457 The sixth filtrate of activated sugar 418 The seventh retentate of activated sugar 20 The eighth retentate of activated sugar 19 The eighth filtrate of activated sugar 25

[0045] In addition, in this detection method, dimethylbarbituric acid reacts with cyanate ester to determine the content of cyanate ester. However, in the preliminary method exploration, the sample was only centrifuged once to treat the sample and determine the cyanate ester respectively, and it was found that the cyanate ester contents in the filtrate and retentate were different, and the filtrate also showed color. It was speculated that cyanogen bromide might also react with dimethylbarbituric acid. Therefore, the following experiments were done on the sample after centrifugation once to verify the conjecture:

[0046] 1) Dimethylbarbituric acid pyridine solution + cyanogen bromide solution with a certain concentration;

[0047] 2) Pyridine solution of dimethylbarbituric acid + filtered solution of activated sugar;

[0048] 3) Pyridine solution of dimethylbarbituric acid + retentate of activated sugar.

[0049] It was found that the colors of the solutions after the reactions in the three experiments were different. It can be considered that a large amount of residual cyanogen bromide also has an impact on the determination of cyanate ester content.

[0050] The purpose of dialyzing the sample is also to remove cyanogen bromide from the sample to obtain the test sample. The specific steps can be as follows: After placing the sample in a dialysis bag, put it into a container filled with dialysis fluid and dialyze for more than 24 h at 2 - 8°C. Among them, the dialysis bag can be made of dialysis membrane, the dialysis fluid is sodium chloride solution with a concentration not exceeding 0.9 wt%, and the dialysis membrane is a 3.5 - 7KD regenerated cellulose membrane. The dialysis fluid can be appropriately replaced during dialysis to remove cyanogen bromide as much as possible. In the experiment, types 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F of pneumococcal activated polysaccharides were dialyzed. Appropriate samples were taken before replacing the dialysis fluid at the 4th h, 8th h, and 24th h to measure the cyanogen bromide content in the dialysis fluid. The results of a large number of experiments (part of the experimental data is shown in Table 2) prove that the cyanogen bromide content in the dialysis fluid measured after 8 h of dialysis ranges from 1000 to 4000 ng / ml, while dialysis for more than 24 h can reduce the cyanogen bromide content in the sample to 10 - 20 ng / ml, and the residual cyanogen bromide in the retention fluid is about 20 ng / ml, that is, the impact on the subsequent detection steps can be ignored.

[0051] Table 2

[0052]

[0053] In S1, the steps to obtain the cyanate ester test sample can be: directly measure a certain amount of the test sample to obtain the cyanate ester test sample.

[0054] The steps to obtain the imidocarbonate test sample can be: Add part of the test sample into a container, add hydrochloric acid solution, react at 20 - 60°C for at least 15 min, then add sodium acetate solution, mix evenly and make up the volume to a certain amount. After centrifugation, take the supernatant as the imidocarbonate test sample. The concentration of hydrochloric acid can be 0.1 mol / L, and the concentration of sodium acetate solution can be 0.2 mol / L.

[0055] S2. Obtain the cyanate ester standard sample and the imidocarbonate standard sample, and form the absorbance curve of the cyanate ester standard sample and the absorbance curve of the imidocarbonate standard sample.

[0056] The purpose of obtaining the cyanate ester standard sample and the imide carbonate standard sample is to form the absorbance curves of the cyanate ester standard sample and the imide carbonate standard sample. Thus, when the absorbances of the cyanate ester and imide carbonate test samples are measured, they can be substituted into the absorbance curves of the cyanate ester and imide carbonate standard samples, so as to obtain the contents of cyanate ester and imide carbonate in the test sample, that is, the contents of cyanate ester and imide carbonate in the sample.

[0057] The cyanate ester standard sample and the imide carbonate standard sample can be obtained by directly purchasing the cyanate ester standard sample and the imide carbonate standard sample sold on the market, or by preparing the cyanate ester standard sample and the imide carbonate standard sample.

[0058] The cyanate ester standard sample can be a cyanogen bromide standard solution. Both cyanogen bromide and cyanate ester can react with pyridine to open the ring, and the resulting glutaconaldehyde reacts with dimethylbarbituric acid to form a colored glutaconaldehyde derivative. Therefore, the cyanogen bromide standard solution can be selected as the cyanate ester standard sample.

[0059] The cyanogen bromide standard solution can specifically be a 0.1 μmol / ml cyanogen bromide solution, and its preparation process can be as follows: Weigh accurately 0.424 g of cyanogen bromide and dissolve it in 10 ml of acetonitrile to obtain the stock solution of cyanogen bromide reference substance (400 μmol / ml). Accurately measure 500 μl of the stock solution of cyanogen bromide reference substance (400 μmol / ml) into a 200 ml volumetric flask, dilute it to the mark with purified water, shake well. Then accurately measure 1 ml of the above solution and add 9 ml of purified water, mix well, and the cyanogen bromide standard solution is obtained. To ensure the accuracy of the experiment, the cyanogen bromide standard solution is prepared and used immediately.

[0060] In S2, the steps to form the absorbance curve of the cyanate ester standard sample can be as follows: Respectively measure 0.00 ml, 0.20 ml, 0.40 ml, 0.80 ml, 1.20 ml, 1.60 ml, and 2.00 ml of the cyanogen bromide standard solution and place them in glass sample bottles respectively, add purified water to make up to 2.0 ml, then add 2 ml of dimethylbarbituric acid pyridine solution respectively, stir vigorously at a temperature not exceeding 60 °C for more than 5 min to obtain multiple cyanate ester test groups. Then, according to the ultraviolet-visible spectrophotometry, measure the absorbance at 588 nm, and use the concentration of cyanogen bromide and the corresponding absorbance values in multiple cyanate ester test groups to make a regression curve, and the absorbance curve of the cyanate ester standard sample can be obtained.

[0061] The preparation process of the dimethylbarbituric acid pyridine solution can be as follows: Weigh 500 mg of dimethylbarbituric acid, add 5 ml of purified water to dissolve it, then add 20 - 57.5 ml of pyridine, shake well, and the dimethylbarbituric acid pyridine solution is obtained. That is, the concentration of the dimethylbarbituric acid pyridine solution can be 0.8% - 2%.

[0062] Since imidocarbonate undergoes hydrolysis under acidic conditions, releasing ammonium ions (NH 4 + ), which reacts with ninhydrin to produce a color reaction. That is, measuring the content of imidocarbonate is to measure the content of NH 4 + . Therefore, we choose ammonium chloride as the standard for detecting the content of imidocarbonate. That is, an ammonium ion standard solution can be used as the imidocarbonate standard sample.

[0063] The concentration of the ammonium ion standard solution can be 2 μmol / ml. Its preparation process can be as follows: Accurately weigh 0.535 g of ammonium chloride, add an appropriate amount of 0.2 mol / L sodium acetate solution to dissolve it, transfer it to a 250 ml volumetric flask, wash it more than 3 times, and incorporate the washing liquid into the volumetric flask; make up the volume to the mark with 0.2 mol / L sodium acetate solution, mix well, and obtain the stock solution of ammonium ion reference substance (ammonium ion concentration is 40 μmol / ml). Accurately measure 500 μl of the stock solution of ammonium ion reference substance into a 10 ml volumetric flask, make up the volume to the mark with 0.2 mol / L sodium acetate solution, mix well, and obtain the ammonium ion standard solution.

[0064] In the preparation process of the ammonium ion standard solution, the preparation process of the 0.2 mol / L sodium acetate solution can be as follows: Weigh 1.64 g of anhydrous sodium acetate, dissolve it with an appropriate amount of purified water, adjust the pH to 5.0 - 7.0, and then make up the volume to 100 ml, mix well, and obtain the 0.2 mol / L sodium acetate solution.

[0065] In S2, the specific steps to form the absorbance curve of the imidocarbonate standard sample can be as follows: Respectively measure 0.00 ml, 0.20 ml, 0.40 ml, 0.60 ml, 0.80 ml, 1.00 ml of the ammonium ion standard solution and place them in test tubes respectively, and then add 0.2 mol / L sodium acetate solution to make up to 1.0 ml to obtain multiple imidocarbonate test groups. Add 1 ml of 2 mol / L sodium acetate solution and 1.5 ml of reduced ninhydrin reagent to each of the multiple imidocarbonate test groups, and mix well. Keep it in a boiling water bath for 10 - 30 min, and then add 6.5 ml of formaldehyde dilution solution and mix well. Then, according to the ultraviolet-visible spectrophotometry method, measure the absorbance at 570 nm. Make a regression curve with the ammonium ion concentration and its corresponding absorbance in the multiple imidocarbonate test groups, and the absorbance curve of the imidocarbonate standard sample can be obtained.

[0066] In the above steps, the preparation method of the reduced ninhydrin reagent can be as follows: Weigh 0.8 - 1 g of reduced ninhydrin and 4 - 5 g of ninhydrin hydrate, dissolve them with ethylene glycol monomethyl ether and make up the volume to 100 ml, mix well, and obtain the reduced ninhydrin reagent. The preparation method of the formaldehyde dilution solution can be as follows: Accurately measure 8 ml of formaldehyde, add isopropanol to make up to 500 ml with 250 ml of water, mix well, and obtain the formaldehyde dilution solution.

[0067] S3. Measure the absorbance of the cyanate test sample, and obtain the content of cyanate in the test sample according to the absorbance curve of the cyanate standard sample.

[0068] In S3, add a certain amount of 0.8% - 2% dimethylbarbituric acid pyridine solution to the cyanate test sample, and stir for at least 5 min under the condition that the temperature does not exceed 60°C. Preferably, stir vigorously for about 30 min. Measure the absorbance of the above mixture at 588 nm according to the ultraviolet-visible spectrophotometry, which is the absorbance of the cyanate test sample. The addition amount of the dimethylbarbituric acid pyridine solution is determined according to the amount of the cyanate test sample.

[0069] In S3, substitute the absorbance of the obtained cyanate test sample into the absorbance curve of the cyanate standard sample, and the content of cyanate in the cyanate test sample can be obtained. Then, according to the mass ratio of the cyanate test sample to the test sample, the content of cyanate in the test sample can be obtained, which is the content of cyanate in the sample.

[0070] S4. Measure the absorbance of the imidocarbonate test sample, and obtain the content of imidocarbonate in the test sample according to the absorbance curve of the imidocarbonate standard sample.

[0071] In S4, the steps for measuring the absorbance of the imidocarbonate test sample can be as follows: Add a certain amount of 2 mol / L sodium acetate solution and a certain amount of reduced ninhydrin reagent to the imidocarbonate test sample. After mixing, heat in a boiling water bath for 10 - 30 min, add a certain amount of formaldehyde dilution solution and mix well. Measure the absorbance of the above mixture at 570 nm according to the ultraviolet-visible spectrophotometry. The addition amounts of the 2 mol / L sodium acetate solution, the reduced ninhydrin reagent, and the formaldehyde dilution solution in the above process are determined according to the amount of the imidocarbonate test sample.

[0072] In S4, substitute the absorbance of the obtained imidocarbonate test sample into the absorbance curve of the imidocarbonate standard sample, and the content of cyanate in the imidocarbonate test sample can be obtained. Then, according to the volume ratio or mass ratio of the imidocarbonate test sample to the test sample, the content of imidocarbonate in the test sample can be obtained, which is the content of imidocarbonate in the sample.

[0073] S5. Calculate the activation degree of the test sample according to the contents of cyanate and imidocarbonate in the test sample, that is, the activation degree of the sample.

[0074] Among them, the activation degree of the sample can be understood as the activation degree of the polysaccharide activated by cyanogen bromide in the sample.

[0075] In this method, the calculation method of the activation degree can be:

[0076] Activation degree (%) = [Content of cyanate ester (μg / ml) + Content of imidocarbonate (μg / ml)] / Content of polysaccharide (μg / ml) × 100

[0077] Among them, the content of cyanate ester (μg / ml) = C 1 × 42

[0078] The content of imidocarbonate (μg / ml) = C 2 × 59 × n

[0079] C 1 is the concentration of cyanate ester in the test sample;

[0080] 42 is the relative molecular weight of cyanate ester;

[0081] C 2 is the concentration of imidocarbonate in the test sample;

[0082] 59 is the relative molecular weight of imidocarbonate;

[0083] n is the dilution factor for determining the content of imidocarbonate in the test sample.

[0084] In this detection method, the activation degree of the sample is the same as that of the test sample. The difference between the sample and the test sample lies in the content of cyanogen bromide.

[0085] It should be noted that the above-mentioned S1 - S5 are only the detection sequences of one of the detection methods for the activation degree of cyanogen bromide-activated polysaccharides in the present invention. In other embodiments, it can also be adjusted to a suitable test sequence.

[0086] After the raw material of the polysaccharide conjugate vaccine (polysaccharide raw material) is activated and derivatized with cyanogen bromide, subsequent steps are carried out, and finally the product, namely the polysaccharide conjugate vaccine, is produced. Among them, the raw material after being activated with cyanogen bromide can be called cyanogen bromide-activated polysaccharide, and after being further derivatized, a polysaccharide derivative can be obtained. The detection method of the present invention can detect the activation rate of cyanogen bromide-activated polysaccharide so as to explore the derivatization rate of the subsequent polysaccharide derivative, further control the polysaccharide-protein ratio, reduce the content of free polysaccharide in the polysaccharide conjugate vaccine, and make the final polysaccharide conjugate vaccine have better immunogenicity.

[0087] Next, the method for evaluating the influence of the activation degree of the polysaccharide conjugate vaccine on the derivatization rate based on the activation degree of cyanogen bromide-activated polysaccharide of the present invention will be elaborated in detail with specific examples.

[0088] Example 1,

[0089] Take the activated polysaccharide of Streptococcus pneumoniae type 1 and ultrafiltrate and centrifuge it at 6000g for 30 min for 15 times to remove cyanogen bromide.

[0090] ① Take 2 ml of the retentate after removing cyanogen bromide as sample 1 to measure the content of cyanate ester.

[0091] ② Take 2 ml of the retentate after removing cyanogen bromide as Sample 2 for determining the content of imidocarbonate.

[0092] ③ Take an appropriate amount of the retentate after removing cyanogen bromide as Sample 3 and use the anthrone method to determine the polysaccharide content of the activated polysaccharide.

[0093] Sample 1, determination of cyanate content

[0094] Measure 0.00 ml, 0.20 ml, 0.40 ml, 0.80 ml, 1.20 ml, 1.60 ml, 2.00 ml of the cyanogen bromide reference working solution and place them in glass sample bottles respectively, add purified water to make up to 2.0 ml. Additionally, take 2.0 ml of the test sample solution (Sample 1) and place it in a glass sample bottle. Add 2 ml of 0.8% dimethylbarbituric acid pyridine solution, stir vigorously at 40 °C for 15 min. Then, measure the absorbance of the above samples at 588 nm according to the ultraviolet-visible spectrophotometry method. Use the concentration of the cyanogen bromide reference working solution and its corresponding absorbance to make a regression curve, and substitute the absorbance of the test sample solution into the regression curve to calculate its corresponding concentration (μmol / ml).

[0095] Sample 2, determination of imidocarbonate content

[0096] Take Sample 2, add 2 ml of HCl, hydrolyze at 37 °C for 50 min, transfer to a 10 ml volumetric flask, add 0.2 mol / L sodium acetate to volume to the scale, mix well, centrifuge at 6500 g for 5 min, and take the supernatant as the test sample to determine the content of ammonium ions generated by the hydrolysis of imidocarbonate.

[0097] Measure 0.00 ml, 0.20 ml, 0.40 ml, 0.60 ml, 0.80 ml, 1.00 ml of the ammonium ion reference working solution and place them in test tubes respectively, add 0.2 mol / L sodium acetate to make up to 1.0 ml. Additionally, take 1.0 ml of the above test sample solution and place it in a test tube. Add 1 ml of 2 mol / L sodium acetate and 1.5 ml of the reduced ninhydrin reagent, mix well, heat in a boiling water bath for 15 min, add 6.5 ml of the formaldehyde dilution solution and mix well. Take 0.5 ml and dilute it 10 times with the formaldehyde dilution solution. Then, measure the absorbance of each tube at 570 nm according to the ultraviolet-visible spectrophotometry method. Use the concentration of the ammonium ion reference working solution and its corresponding absorbance to make a regression curve, and substitute the absorbance of the test sample solution into the regression curve to calculate its corresponding concentration (μmol / ml).

[0098] Result calculation

[0099] Content of cyanate (μg / ml) = C 1 × 42

[0100] Content of imidocarbonate (μg / ml) = C2 ×59×n

[0101] Activation degree (%) = [Content of cyanate ester (μg / ml) + Content of imide carbonate (μg / ml)] / Content of polysaccharide (μg / ml) × 100

[0102] C 1 is the concentration of cyanate ester in the test sample;

[0103] 42 is the relative molecular weight of cyanate ester;

[0104] C 2 is the concentration of imide carbonate in the test sample;

[0105] 59 is the relative molecular weight of imide carbonate;

[0106] n is the dilution factor for determining the content of imide carbonate in the test sample.

[0107] The measured content of cyanate ester is 0.8736 μg / ml, the content of imide carbonate is 18.9980 μg / ml, and the content of polysaccharide is 382.3 μg / ml. The activation degree is calculated to be 5.20% according to the formula.

[0108] Example 2

[0109] Take the activated polysaccharide of Streptococcus pneumoniae type 3 and ultrafilter and centrifuge it at 6500 g for 30 min for 12 times to remove cyanogen bromide.

[0110] ① Take 2 ml of the retentate after removing cyanogen bromide as sample 1 to measure the content of cyanate ester.

[0111] ② Take 2 ml of the retentate after removing cyanogen bromide as sample 2 to determine the content of imide carbonate.

[0112] ③ Take an appropriate amount of the retentate after removing cyanogen bromide as sample 3 and use the anthrone method to measure the polysaccharide content of the activated polysaccharide.

[0113] Sample 1, determination of cyanate ester content

[0114] Measure 0.00 ml, 0.20 ml, 0.40 ml, 0.80 ml, 1.20 ml, 1.60 ml, 2.00 ml of the cyanogen bromide reference working solution and place them in glass sample bottles respectively, add purified water to make up to 2.0 ml. Take another 2.0 ml of the test sample solution (sample 1) and place it in a glass sample bottle. Add 2 ml of 0.8% dimethylbarbituric acid pyridine solution, stir vigorously at 40 °C for 15 min, and measure the absorbance of the above samples at 588 nm according to the ultraviolet-visible spectrophotometry. Make a regression curve with the concentration of the cyanogen bromide reference working solution and its corresponding absorbance, and substitute the absorbance of the test sample solution into the regression curve to calculate its corresponding concentration (μmol / ml).

[0115] Sample 2, Determination of Imidocarbonate Content

[0116] Take 2 samples and add 2 ml of HCl. Hydrolyze at 40 °C for 35 min, transfer to a 10-ml volumetric flask, add 0.2 mol / L sodium acetate to volume, mix well, centrifuge at 6500 g for 5 min, and take the supernatant as the test sample to determine the content of ammonium ions produced by the hydrolysis of imidocarbonate.

[0117] Measure 0.00 ml, 0.20 ml, 0.40 ml, 0.60 ml, 0.80 ml, and 1.00 ml of the ammonium ion reference working solution and place them in test tubes respectively. Add 0.2 mol / L sodium acetate to make up to 1.0 ml. Take 1.0 ml of the above test sample solution and place it in a test tube. Add 1 ml of 2 mol / L sodium acetate and 1.5 ml of the reduced ninhydrin reagent, mix well, heat in a boiling water bath for 20 min, add 6.5 ml of the formaldehyde dilution solution and mix well. Take 0.5 ml and dilute it 10 times with the formaldehyde dilution solution. Then, measure the absorbance of each tube at 570 nm according to the ultraviolet-visible spectrophotometry method. Make a regression curve with the concentration of the ammonium ion reference working solution and its corresponding absorbance, and substitute the absorbance of the test sample solution into the regression curve to calculate its corresponding concentration (μmol / ml).

[0118] Result Calculation

[0119] Content of cyanate (μg / ml) = C 1 × 42

[0120] Content of imidocarbonate (μg / ml) = C 2 × 59 × n

[0121] Activation degree (%) = [Content of cyanate (μg / ml) + Content of imidocarbonate (μg / ml)] / Content of polysaccharide (μg / ml) × 100

[0122] C 1 is the concentration of cyanate in the test sample;

[0123] 42 is the relative molecular mass of cyanate;

[0124] C 2 is the concentration of imidocarbonate in the test sample;

[0125] 59 is the relative molecular mass of imidocarbonate;

[0126] n is the dilution factor for determining the content of imidocarbonate in the test sample.

[0127] The measured content of cyanate is 0.7856 μg / ml, the content of imidocarbonate is 23.7885 μg / ml, and the content of polysaccharide is 1746.3 μg / ml. The activation degree is calculated to be 1.41% according to the formula.

[0128] Example 3

[0129] Take the activated polysaccharide of Streptococcus pneumoniae type 4 and ultrafiltrate and centrifuge it at 7000 g for 20 min for 15 times to remove cyanogen bromide.

[0130] ① Take 2 ml of the retentate after removing cyanogen bromide as sample 1 to measure the cyanate ester content.

[0131] ② Take 2 ml of the retentate after removing cyanogen bromide as sample 2 to determine the imidocarbonate content.

[0132] ③ Take an appropriate amount of the retentate after removing cyanogen bromide as sample 3 and use the anthrone method to measure the polysaccharide content of the activated polysaccharide.

[0133] Sample 1, Determination of Cyanate Ester Content

[0134] Measure 0.00 ml, 0.20 ml, 0.40 ml, 0.80 ml, 1.20 ml, 1.60 ml, 2.00 ml of the cyanogen bromide reference working solution and place them in glass sample bottles respectively, add purified water to make up to 2.0 ml. Take 2.0 ml of the test solution (sample 1) and place it in a glass sample bottle. Add 2 ml of 0.8% dimethylbarbituric acid pyridine solution, stir vigorously at 50 °C for 10 min. Measure the absorbance of the above samples by ultraviolet-visible spectrophotometry at 588 nm. Use the concentration of the cyanogen bromide reference working solution and its corresponding absorbance to make a regression curve, and substitute the absorbance of the test solution into the regression curve to calculate its corresponding concentration (μmol / ml).

[0135] Sample 2, Determination of Imidocarbonate Content

[0136] Take sample 2, add 2 ml of HCl, hydrolyze it at 45 °C for 30 min, transfer it to a 10 ml volumetric flask, add 0.2 mol / L sodium acetate to make up to the scale, mix well, centrifuge at 6500 g for 5 min, and take the supernatant as the test solution to measure the content of ammonium ions produced by the hydrolysis of imidocarbonate.

[0137] Measure 0.00 ml, 0.20 ml, 0.40 ml, 0.60 ml, 0.80 ml, 1.00 ml of the ammonium ion reference working solution and place them in test tubes respectively, add 0.2 mol / L sodium acetate to make up to 1.0 ml. Take 1.0 ml of the above test solution and place it in a test tube. Add 1 ml of 2 mol / L sodium acetate and 1.5 ml of reduced ninhydrin reagent, mix well, heat in a boiling water bath for 25 min, add 6.5 ml of formaldehyde dilution solution and mix well. Take 0.5 ml and dilute it 10 times with formaldehyde dilution solution. Measure the absorbance of each tube by ultraviolet-visible spectrophotometry at 570 nm. Use the concentration of the ammonium ion reference working solution and its corresponding absorbance to make a regression curve, and substitute the absorbance of the test solution into the regression curve to calculate its corresponding concentration (μmol / ml).

[0138] Result calculation

[0139] Content of cyanate (μg / ml) = C 1 × 42

[0140] Content of imidocarbonate (μg / ml) = C 2 × 59 × n

[0141] Activation degree (%) = [Content of cyanate (μg / ml) + Content of imidocarbonate (μg / ml)] / Content of polysaccharide (μg / ml) × 100

[0142] C 1 is the concentration of cyanate in the test sample;

[0143] 42 is the relative molecular weight of cyanate;

[0144] C 2 is the concentration of imidocarbonate in the test sample;

[0145] 59 is the relative molecular weight of imidocarbonate;

[0146] n is the dilution factor for determining the content of imidocarbonate in the test sample.

[0147] The measured content of cyanate is 0.7014 μg / ml, the content of imidocarbonate is 10.2707 μg / ml, and the content of polysaccharide is 629.4 μg / ml. The activation degree is calculated to be 1.74% according to the formula.

[0148] Example 4

[0149] Take 5700 g of activated polysaccharide of Streptococcus pneumoniae type 5, and remove cyanogen bromide by ultrafiltration and centrifugation for 12 times for 20 min.

[0150] ① Take 2 ml of the retentate after removing cyanogen bromide as sample 1 to measure the content of cyanate.

[0151] ② Take 2 ml of the retentate after removing cyanogen bromide as sample 2 to determine the content of imidocarbonate.

[0152] ③ Take an appropriate amount of the retentate after removing cyanogen bromide as sample 3 and use the anthrone method to measure the polysaccharide content of the activated polysaccharide.

[0153] Sample 1, determination of cyanate content

[0154] Measure 0.00 ml, 0.20 ml, 0.40 ml, 0.80 ml, 1.20 ml, 1.60 ml, 2.00 ml of the cyanogen bromide reference working solution respectively into glass sample bottles, add purified water to make up to 2.0 ml. Take another 2.0 ml of the test solution (sample 1) and place it in a glass sample bottle. Add 2 ml of 1% dimethylbarbituric acid pyridine solution, stir vigorously at 45 °C for 20 min. Measure the absorbance of the above samples at 588 nm according to the ultraviolet-visible spectrophotometry. Use the concentration of the cyanogen bromide reference working solution and its corresponding absorbance to make a regression curve, and substitute the absorbance of the test solution into the regression curve to calculate its corresponding concentration (μmol / ml).

[0155] Sample 2, determination of the content of imidocarbonate

[0156] Take sample 2, add 2 ml of HCl, hydrolyze at 25 °C for 50 min, transfer to a 10 ml volumetric flask, add 0.2 mol / L sodium acetate to make up to the mark, mix well, centrifuge at 6500 g for 5 min, and take the supernatant as the test sample to determine the content of ammonium ions produced by the hydrolysis of imidocarbonate.

[0157] Measure 0.00 ml, 0.20 ml, 0.40 ml, 0.60 ml, 0.80 ml, 1.00 ml of the ammonium ion reference working solution respectively into test tubes, add 0.2 mol / L sodium acetate to make up to 1.0 ml. Take another 1.0 ml of the above test solution and place it in a test tube. Add 1 ml of 2 mol / L sodium acetate and 1.5 ml of reduced ninhydrin reagent, mix well, heat in a boiling water bath for 10 min, add 6.5 ml of formaldehyde dilution solution and mix well. Take 0.5 ml and dilute it 10 times with the formaldehyde dilution solution. Measure the absorbance of each of the above tubes at 570 nm according to the ultraviolet-visible spectrophotometry. Use the concentration of the ammonium ion reference working solution and its corresponding absorbance to make a regression curve, and substitute the absorbance of the test solution into the regression curve to calculate its corresponding concentration (μmol / ml).

[0158] Result calculation

[0159] Content of cyanate (μg / ml) = C 1 × 42

[0160] Content of imidocarbonate (μg / ml) = C 2 × 59 × n

[0161] Activation degree (%) = [Content of cyanate (μg / ml) + Content of imidocarbonate (μg / ml)] / Content of polysaccharide (μg / ml) × 100

[0162] C 1 is the concentration of cyanate in the test sample;

[0163] 42 is the relative molecular weight of cyanate ester;

[0164] C 2 is the concentration of imidocarbonate in the test sample;

[0165] 59 is the relative molecular weight of imidocarbonate;

[0166] n is the dilution factor for determining the content of imidocarbonate in the test sample.

[0167] The measured content of cyanate ester is 0.6708 μg / ml, the content of imidocarbonate is 14.9385 μg / ml, and the content of polysaccharide is 940.5 μg / ml. The activation degree is calculated to be 1.66% according to the formula.

[0168] Example 5

[0169] Take the activated polysaccharide of Streptococcus pneumoniae type 6A and ultrafiltrate and centrifuge it at 8000 g for 10 min for 12 times to remove cyanogen bromide.

[0170] ① Take 2 ml of the retentate after removing cyanogen bromide as sample 1 to measure the content of cyanate ester.

[0171] ② Take 2 ml of the retentate after removing cyanogen bromide as sample 2 to determine the content of imidocarbonate.

[0172] ③ Take an appropriate amount of the retentate after removing cyanogen bromide as sample 3 and use the anthrone method to determine the polysaccharide content of the activated polysaccharide.

[0173] Sample 1, determination of cyanate ester content

[0174] Measure 0.00 ml, 0.20 ml, 0.40 ml, 0.80 ml, 1.20 ml, 1.60 ml, 2.00 ml of the working solution of cyanogen bromide reference substance and place them in glass sample bottles respectively, add purified water to make up to 2.0 ml. Take another 2.0 ml of the test sample solution (sample 1) and place it in a glass sample bottle. Add 2 ml of 1% dimethylbarbituric acid pyridine solution, stir vigorously at 30 °C for 30 min, and measure the absorbance of the above samples at 588 nm according to the ultraviolet-visible spectrophotometry. Make a regression curve with the concentration of the working solution of cyanogen bromide reference substance and its corresponding absorbance, and substitute the absorbance of the test sample solution into the regression curve to calculate its corresponding concentration (μmol / ml).

[0175] Sample 2, determination of imidocarbonate content

[0176] Take sample 2, add 2 ml of HCl, hydrolyze it at 20 °C for 60 min, transfer it to a 10 ml volumetric flask, add 0.2 mol / L sodium acetate to make up to the mark, mix well, centrifuge at 6500 g for 5 min, and take the supernatant as the test sample to determine the content of ammonium ions produced by the hydrolysis of imidocarbonate.

[0177] Measure 0.00 ml, 0.20 ml, 0.40 ml, 0.60 ml, 0.80 ml, and 1.00 ml of the ammonium ion reference working solution respectively into test tubes, and make up to 1.0 ml with 0.2 mol / L sodium acetate. Additionally, take 1.0 ml of the above-mentioned test sample solution and place it in a test tube. Add 1 ml of 2 mol / L sodium acetate and 1.5 ml of the reduced ninhydrin reagent, mix well, then place it in a boiling water bath for 30 min. Add 6.5 ml of the formaldehyde dilution solution and mix well. Take 0.5 ml and dilute it 10 times with the formaldehyde dilution solution. Then, measure the absorbance of each of the above tubes at 570 nm according to the ultraviolet-visible spectrophotometry method. Use the concentration of the ammonium ion reference working solution and its corresponding absorbance to make a regression curve, and substitute the absorbance of the test sample solution into the regression curve to calculate its corresponding concentration (μmol / ml).

[0178] Result calculation

[0179] Content of cyanate (μg / ml) = C 1 ×42

[0180] Content of imidocarbonate (μg / ml) = C 2 ×59×n

[0181] Activation degree (%) = [Content of cyanate (μg / ml) + Content of imidocarbonate (μg / ml)] / Content of polysaccharide (μg / ml) × 100

[0182] C 1 is the concentration of cyanate in the test sample;

[0183] 42 is the relative molecular weight of cyanate;

[0184] C 2 is the concentration of imidocarbonate in the test sample;

[0185] 59 is the relative molecular weight of imidocarbonate;

[0186] n is the dilution factor for determining the content of imidocarbonate in the test sample.

[0187] The measured content of cyanate is 0.6006 μg / ml, the content of imidocarbonate is 26.0898 μg / ml, and the content of polysaccharide is 1906.7 μg / ml. The activation degree is calculated to be 1.40% according to the formula.

[0188] Example 6

[0189] Take the activated polysaccharide of Streptococcus pneumoniae type 6B and ultrafiltrate and centrifuge it at 8000 g for 10 min for 10 times to remove cyanogen bromide.

[0190] ① Take 2 ml of the retentate after removing cyanogen bromide as sample 1 to measure the content of cyanate.

[0191] ② Take 2 ml of the retentate after removing cyanogen bromide as Sample 2 to determine the content of imidocarbonate.

[0192] ③ Take an appropriate amount of the retentate after removing cyanogen bromide as Sample 3 and use the anthrone method to determine the polysaccharide content of the activated polysaccharide.

[0193] Sample 1, determination of cyanate content

[0194] Measure 0.00 ml, 0.20 ml, 0.40 ml, 0.80 ml, 1.20 ml, 1.60 ml, 2.00 ml of the cyanogen bromide reference working solution and place them in glass sample bottles respectively, add purified water to make up to 2.0 ml. Take another 2.0 ml of the test solution (Sample 1) and place it in a glass sample bottle. Add 2 ml of 1% dimethylbarbituric acid pyridine solution, stir vigorously at 35 °C for 30 min. Measure the absorbance of the above samples at 588 nm according to the ultraviolet-visible spectrophotometry. Use the concentration of the cyanogen bromide reference working solution and its corresponding absorbance to make a regression curve, and substitute the absorbance of the test solution into the regression curve to calculate its corresponding concentration (μmol / ml).

[0195] Sample 2, determination of imidocarbonate content

[0196] Take Sample 2, add 2 ml of HCl, hydrolyze at 60 °C for 20 min, transfer to a 10 ml volumetric flask, add 0.2 mol / L sodium acetate to volume to the mark, mix well, centrifuge at 6500 g for 5 min, and take the supernatant as the test solution to determine the content of ammonium ions produced by the hydrolysis of imidocarbonate.

[0197] Measure 0.00 ml, 0.20 ml, 0.40 ml, 0.60 ml, 0.80 ml, 1.00 ml of the ammonium ion reference working solution and place them in test tubes respectively, add 0.2 mol / L sodium acetate to make up to 1.0 ml. Take another 1.0 ml of the above test solution and place it in a test tube. Add 1 ml of 2 mol / L sodium acetate and 1.5 ml of the reduced ninhydrin reagent, mix well, heat in a boiling water bath for 15 min, add 6.5 ml of the formaldehyde dilution solution and mix well. Take 0.5 ml and dilute it 10 times with the formaldehyde dilution solution. Measure the absorbance of each tube at 570 nm according to the ultraviolet-visible spectrophotometry. Use the concentration of the ammonium ion reference working solution and its corresponding absorbance to make a regression curve, and substitute the absorbance of the test solution into the regression curve to calculate its corresponding concentration (μmol / ml).

[0198] Result calculation

[0199] Content of cyanate (μg / ml) = C 1 × 42

[0200] Content of imidocarbonate (μg / ml) = C 2 × 59 × n

[0201] Degree of activation (%) = [Content of cyanate ester (μg / ml) + Content of imidocarbonate (μg / ml)] / Content of polysaccharide (μg / ml) × 100

[0202] C 1 is the concentration of cyanate ester in the test sample;

[0203] 42 is the relative molecular weight of cyanate ester;

[0204] C 2 is the concentration of imidocarbonate in the test sample;

[0205] 59 is the relative molecular weight of imidocarbonate;

[0206] n is the dilution factor for determining the content of imidocarbonate in the test sample.

[0207] The measured content of cyanate ester is 0.6426 μg / ml, the content of imidocarbonate is 21.3267 μg / ml, and the content of polysaccharide is 1552.2 μg / ml. The degree of activation is calculated to be 1.42% according to the formula.

[0208] Example 7

[0209] Take 10 ml of the activated polysaccharide of Streptococcus pneumoniae type 7F and place it in a beaker containing dialysis fluid. Dialyze at 2 - 8 °C for 24 h. Appropriately change the dialysis fluid during the dialysis process to ensure that all cyanogen bromide is removed.

[0210] ① Take 2 ml of the retained solution after removing cyanogen bromide as Sample 1 to measure the content of cyanate ester.

[0211] ② Take 2 ml of the retained solution after removing cyanogen bromide as Sample 2 to determine the content of imidocarbonate.

[0212] ③ Take an appropriate amount of the retained solution after removing cyanogen bromide as Sample 3 and use the anthrone method to measure the polysaccharide content of the activated polysaccharide.

[0213] Sample 1, determination of cyanate ester content

[0214] Measure 0.00 ml, 0.20 ml, 0.40 ml, 0.80 ml, 1.20 ml, 1.60 ml, 2.00 ml of the cyanogen bromide reference working solution and place them in glass sample bottles respectively. Make up to 2.0 ml with purified water. Take another 2.0 ml of the test sample solution (Sample 1) and place it in a glass sample bottle. Add 2 ml of 1.5% dimethylbarbituric acid pyridine solution, stir vigorously at 60 °C for 5 min. Measure the absorbance of the above samples at 588 nm according to the ultraviolet-visible spectrophotometry. Make a regression curve with the concentration of the cyanogen bromide reference working solution and its corresponding absorbance, and substitute the absorbance of the test sample solution into the regression curve to calculate its corresponding concentration (μmol / ml).

[0215] Example 2, Determination of the content of imidocarbonate

[0216] Take a sample 2, add 2 ml of HCl, hydrolyze it at 30 °C for 45 min, transfer it to a 10-ml volumetric flask, add 0.2 mol / L sodium acetate to volume to the mark, mix well, centrifuge at 6500 g for 5 min, and take the supernatant as the test sample to determine the content of ammonium ions produced by the hydrolysis of imidocarbonate.

[0217] Measure 0.00 ml, 0.20 ml, 0.40 ml, 0.60 ml, 0.80 ml, 1.00 ml of the working solution of ammonium ion reference substance into test tubes respectively, make up to 1.0 ml with 0.2 mol / L sodium acetate, and take 1.0 ml of the above test sample solution and place it in a test tube. Add 1 ml of 2 mol / L sodium acetate and 1.5 ml of reduced ninhydrin reagent, mix well, heat in a boiling water bath for 20 min, add 6.5 ml of formaldehyde dilution solution and mix well. Take 0.5 ml and dilute it 10 times with formaldehyde dilution solution. Then, measure the absorbance of each of the above tubes at 570 nm according to the ultraviolet-visible spectrophotometry. Make a regression curve with the concentration of the working solution of ammonium ion reference substance and its corresponding absorbance, and substitute the absorbance of the test sample solution into the regression curve to calculate its corresponding concentration (μmol / ml).

[0218] Result calculation

[0219] Content of cyanate (μg / ml) = C 1 × 42

[0220] Content of imidocarbonate (μg / ml) = C 2 × 59 × n

[0221] Activation degree (%) = [Content of cyanate (μg / ml) + Content of imidocarbonate (μg / ml)] / Content of polysaccharide (μg / ml) × 100

[0222] C 1 is the concentration of cyanate in the test sample;

[0223] 42 is the relative molecular weight of cyanate;

[0224] C 2 is the concentration of imidocarbonate in the test sample;

[0225] 59 is the relative molecular weight of imidocarbonate;

[0226] n is the dilution factor for determining the content of imidocarbonate in the test sample.

[0227] The measured content of cyanate is 0.7392 μg / ml, the content of imidocarbonate is 27.0957 μg / ml, and the content of polysaccharide is 1598.0 μg / ml. The activation degree is calculated to be 1.74% according to the formula.

[0228] Example 8

[0229] Take 10 ml of activated polysaccharide of type 9V pneumococcus and place it in a beaker containing dialysis solution. Dialyze at 2 - 8 °C for 24 h. Appropriately change the dialysis solution during dialysis to ensure that all cyanogen bromide is removed.

[0230] ① Take 2 ml of the retained solution after removing cyanogen bromide as sample 1 to measure the cyanate ester content.

[0231] ② Take 2 ml of the retained solution after removing cyanogen bromide as sample 2 to determine the imidocarbonate content.

[0232] ③ Take an appropriate amount of the retained solution after removing cyanogen bromide as sample 3 and use the anthrone method to measure the polysaccharide content of the activated polysaccharide.

[0233] Sample 1, determination of cyanate ester content

[0234] Measure 0.00 ml, 0.20 ml, 0.40 ml, 0.80 ml, 1.20 ml, 1.60 ml, 2.00 ml of the cyanogen bromide reference working solution respectively and place them in glass sample bottles. Make up to 2.0 ml with purified water. Take 2.0 ml of the test solution (sample 1) and place it in a glass sample bottle. Add 2 ml of 1.5% dimethylbarbituric acid pyridine solution, stir vigorously at 55 °C for 10 min. Measure the absorbance of the above samples at 588 nm according to the ultraviolet-visible spectrophotometry method. Use the concentration of the cyanogen bromide reference working solution and its corresponding absorbance to make a regression curve, and substitute the absorbance of the test solution into the regression curve to calculate its corresponding concentration (μmol / ml).

[0235] Sample 2, determination of imidocarbonate content

[0236] Take sample 2, add 2 ml of HCl, hydrolyze at 40 °C for 35 min, transfer to a 10 ml volumetric flask, add 0.2 mol / L sodium acetate to volume to the mark, mix well, centrifuge at 6500 g for 5 min, and take the supernatant as the test sample to determine the content of ammonium ions produced by the hydrolysis of imidocarbonate.

[0237] Measure 0.00 ml, 0.20 ml, 0.40 ml, 0.60 ml, 0.80 ml, and 1.00 ml of the ammonium ion reference working solution respectively into test tubes, and make up to 1.0 ml with 0.2 mol / L sodium acetate. Take 1.0 ml of the above-mentioned test sample solution and place it in a test tube. Add 1 ml of 2 mol / L sodium acetate and 1.5 ml of the reduced ninhydrin reagent, mix well, heat in a boiling water bath for 15 min, add 6.5 ml of the formaldehyde dilution solution and mix well. Take 0.5 ml and dilute it 10 times with the formaldehyde dilution solution. Then, measure the absorbance of each of the above tubes at 570 nm according to the ultraviolet-visible spectrophotometry method. Use the concentration of the ammonium ion reference working solution and its corresponding absorbance to make a regression curve, and substitute the absorbance of the test sample solution into the regression curve to calculate its corresponding concentration (μmol / ml).

[0238] Result calculation

[0239] Content of cyanate (μg / ml) = C 1 ×42

[0240] Content of imidocarbonate (μg / ml) = C 2 ×59×n

[0241] Activation degree (%) = [Content of cyanate (μg / ml) + Content of imidocarbonate (μg / ml)] / Content of polysaccharide (μg / ml) × 100

[0242] C 1 is the concentration of cyanate in the test sample;

[0243] 42 is the relative molecular weight of cyanate;

[0244] C 2 is the concentration of imidocarbonate in the test sample;

[0245] 59 is the relative molecular weight of imidocarbonate;

[0246] n is the dilution factor for measuring the content of imidocarbonate in the test sample.

[0247] The measured content of cyanate is 1.0355 μg / ml, the content of imidocarbonate is 41.4393 μg / ml, and the content of polysaccharide is 2003.9 μg / ml. Calculate the activation degree to be 2.12% according to the formula.

[0248] Example 9

[0249] Take 10 ml of the activated polysaccharide of Streptococcus pneumoniae type 14 and put it into a beaker containing dialysis fluid. Dialyze at 2 - 8 °C for 24 h. Appropriately change the dialysis fluid during dialysis to ensure that all cyanogen bromide is removed.

[0250] ① Take 2 ml of the retained solution after removing cyanogen bromide as sample 1 to measure the content of cyanate.

[0251] ② Take 2 ml of the retained solution after removing cyanogen bromide as Sample 2 for determining the content of imide carbonate.

[0252] ③ Take an appropriate amount of the retained solution after removing cyanogen bromide as Sample 3 and use the anthrone method to determine the polysaccharide content of the activated polysaccharide.

[0253] Sample 1, Determination of cyanate content

[0254] Measure 0.00 ml, 0.20 ml, 0.40 ml, 0.80 ml, 1.20 ml, 1.60 ml, 2.00 ml of the working solution of cyanogen bromide reference substance and place them in glass sample bottles respectively, add purified water to make up to 2.0 ml. Additionally, take 2.0 ml of the test solution (Sample 1) and place it in a glass sample bottle. Add 2 ml of 1.5% dimethylbarbituric acid pyridine solution, stir vigorously at 30 °C for 25 min. Then, measure the absorbance of the above samples at 588 nm according to the ultraviolet-visible spectrophotometry method. Use the concentration of the working solution of cyanogen bromide reference substance and its corresponding absorbance to make a regression curve, and substitute the absorbance of the test solution into the regression curve to calculate its corresponding concentration (μmol / ml).

[0255] Sample 2, Determination of imide carbonate content

[0256] Take Sample 2, add 2 ml of HCl, hydrolyze at 25 °C for 60 min, transfer to a 10 ml volumetric flask, add 0.2 mol / L sodium acetate to volume to the mark, mix well, centrifuge at 6500 g for 5 min, and take the supernatant as the test sample to determine the content of ammonium ions produced by the hydrolysis of imide carbonate.

[0257] Measure 0.00 ml, 0.20 ml, 0.40 ml, 0.60 ml, 0.80 ml, 1.00 ml of the working solution of ammonium ion reference substance and place them in test tubes respectively, add 0.2 mol / L sodium acetate to make up to 1.0 ml. Additionally, take 1.0 ml of the above test solution and place it in a test tube. Add 1 ml of 2 mol / L sodium acetate and 1.5 ml of reduced ninhydrin reagent, mix well, heat in a boiling water bath for 20 min, add 6.5 ml of formaldehyde dilution solution and mix well. Take 0.5 ml and dilute it 10 times with the formaldehyde dilution solution. Then, measure the absorbance of each tube at 570 nm according to the ultraviolet-visible spectrophotometry method. Use the concentration of the working solution of ammonium ion reference substance and its corresponding absorbance to make a regression curve, and substitute the absorbance of the test solution into the regression curve to calculate its corresponding concentration (μmol / ml).

[0258] Result calculation

[0259] Content of cyanate (μg / ml) = C 1 × 42

[0260] Content of imide carbonate (μg / ml) = C2 ×59×n

[0261] Degree of activation (%) = [Content of cyanate ester (μg / ml) + Content of imidocarbonate (μg / ml)] / Content of polysaccharide (μg / ml) × 100

[0262] C 1 is the concentration of cyanate ester in the test sample;

[0263] 42 is the relative molecular weight of cyanate ester;

[0264] C 2 is the concentration of imidocarbonate in the test sample;

[0265] 59 is the relative molecular weight of imidocarbonate;

[0266] n is the dilution factor for determining the content of imidocarbonate in the test sample.

[0267] The measured content of cyanate ester is 0.2028 μg / ml, the content of imidocarbonate is 47.0951 μg / ml, and the content of polysaccharide is 2276.1 μg / ml. The degree of activation is calculated to be 2.08% according to the formula.

[0268] Example 10

[0269] Take 10 ml of 18C type pneumococcal activated polysaccharide and place it in a beaker containing dialysis fluid. Dialyze at 2 - 8°C for 24 h. Appropriately change the dialysis fluid during dialysis to ensure that all cyanogen bromide is removed.

[0270] ① Take 2 ml of the retained solution after removing cyanogen bromide as sample 1 to measure the content of cyanate ester.

[0271] ② Take 2 ml of the retained solution after removing cyanogen bromide as sample 2 to measure the content of imidocarbonate.

[0272] ③ Take an appropriate amount of the retained solution after removing cyanogen bromide as sample 3 and use the anthrone method to measure the polysaccharide content of the activated polysaccharide.

[0273] Sample 1, determination of cyanate ester content

[0274] Measure 0.00 ml, 0.20 ml, 0.40 ml, 0.80 ml, 1.20 ml, 1.60 ml, 2.00 ml of the cyanogen bromide reference working solution respectively into glass sample bottles, add purified water to make up to 2.0 ml. Take another 2.0 ml of the test solution (sample 1) and place it in a glass sample bottle. Add 2 ml of 2% dimethylbarbituric acid pyridine solution, stir vigorously at 45 °C for 20 min. Measure the absorbance of the above samples at 588 nm according to the ultraviolet-visible spectrophotometry. Use the concentration of the cyanogen bromide reference working solution and its corresponding absorbance to make a regression curve, and substitute the absorbance of the test solution into the regression curve to calculate its corresponding concentration (μmol / ml).

[0275] Sample 2, determination of the content of iminocarbonate

[0276] Take sample 2, add 2 ml of HCl, hydrolyze at 37 °C for 45 min, transfer to a 10 ml volumetric flask, add 0.2 mol / L sodium acetate to volume to the mark, mix well, centrifuge at 6500 g for 5 min, and take the supernatant as the test sample to determine the content of ammonium ions produced by the hydrolysis of iminocarbonate.

[0277] Measure 0.00 ml, 0.20 ml, 0.40 ml, 0.60 ml, 0.80 ml, 1.00 ml of the ammonium ion reference working solution respectively into test tubes, add 0.2 mol / L sodium acetate to make up to 1.0 ml. Take another 1.0 ml of the above test solution and place it in a test tube. Add 1 ml of 2 mol / L sodium acetate and 1.5 ml of the reduced ninhydrin reagent, mix well, heat in a boiling water bath for 25 min, add 6.5 ml of the formaldehyde dilution solution and mix well. Take 0.5 ml and dilute it 10 times with the formaldehyde dilution solution. Measure the absorbance of each of the above tubes at 570 nm according to the ultraviolet-visible spectrophotometry. Use the concentration of the ammonium ion reference working solution and its corresponding absorbance to make a regression curve, and substitute the absorbance of the test solution into the regression curve to calculate its corresponding concentration (μmol / ml).

[0278] Result calculation

[0279] Content of cyanate (μg / ml) = C 1 × 42

[0280] Content of iminocarbonate (μg / ml) = C 2 × 59 × n

[0281] Activation degree (%) = [content of cyanate (μg / ml) + content of iminocarbonate (μg / ml)] / content of polysaccharide (μg / ml) × 100

[0282] C 1 is the concentration of cyanate in the test sample;

[0283] 42 is the relative molecular weight of cyanate ester;

[0284] C 2 is the concentration of imidocarbonate of the test sample;

[0285] 59 is the relative molecular weight of imidocarbonate;

[0286] n is the dilution factor for determining the content of imidocarbonate in the test sample.

[0287] The measured content of cyanate ester is 0.6594 μg / ml, the content of imidocarbonate is 34.0607 μg / ml, and the content of polysaccharide is 2615.7 μg / ml. The activation degree is calculated to be 1.33% according to the formula.

[0288] Example 11

[0289] Take 10 ml of the activated polysaccharide of Streptococcus pneumoniae type 19A and place it in a beaker containing dialysis fluid. Dialyze at 2 - 8 °C for 24 h. Replace the dialysis fluid appropriately during the dialysis process to ensure that all cyanogen bromide is removed.

[0290] ① Take 2 ml of the retained solution after removing cyanogen bromide as sample 1 to measure the content of cyanate ester.

[0291] ② Take 2 ml of the retained solution after removing cyanogen bromide as sample 2 to determine the content of imidocarbonate.

[0292] ③ Take an appropriate amount of the retained solution after removing cyanogen bromide as sample 3 and use the anthrone method to determine the polysaccharide content of the activated polysaccharide.

[0293] Sample 1, determination of cyanate ester content

[0294] Measure 0.00 ml, 0.20 ml, 0.40 ml, 0.80 ml, 1.20 ml, 1.60 ml, 2.00 ml of the cyanogen bromide reference working solution and place them in glass sample bottles respectively. Add purified water to make up to 2.0 ml. Take another 2.0 ml of the test sample solution (sample 1) and place it in a glass sample bottle. Add 2 ml of 2% dimethylbarbituric acid pyridine solution, stir vigorously at 30 °C for 30 min. Measure the absorbance of the above samples at 588 nm according to the ultraviolet - visible spectrophotometry. Make a regression curve with the concentration of the cyanogen bromide reference working solution and its corresponding absorbance. Substitute the absorbance of the test sample solution into the regression curve to calculate its corresponding concentration (μmol / ml).

[0295] Sample 2, determination of imidocarbonate content

[0296] Take sample 2, add 2 ml of HCl, hydrolyze at 30 °C for 50 min, transfer to a 10 ml volumetric flask, add 0.2 mol / L sodium acetate to volume to the scale, mix well, centrifuge at 6500 g for 5 min, and take the supernatant as the test sample to determine the content of ammonium ions produced by the hydrolysis of imidocarbonate.

[0297] Measure 0.00 mL, 0.20 mL, 0.40 mL, 0.60 mL, 0.80 mL, and 1.00 mL of the ammonium ion reference working solution respectively into test tubes, and make up to 1.0 mL with 0.2 mol / L sodium acetate. Separately, take 1.0 mL of the above-mentioned test sample solution and place it in a test tube. Add 1 mL of 2 mol / L sodium acetate and 1.5 mL of the reduced ninhydrin reagent, mix well, heat in a boiling water bath for 20 min, add 6.5 mL of the formaldehyde dilution solution and mix well. Take 0.5 mL and dilute it 10 times with the formaldehyde dilution solution. Then, for each of the above tubes, measure the absorbance at 570 nm according to the ultraviolet-visible spectrophotometry method. Use the concentration of the ammonium ion reference working solution and its corresponding absorbance to make a regression curve, and substitute the absorbance of the test sample solution into the regression curve to calculate its corresponding concentration (μmol / mL).

[0298] Result calculation

[0299] Content of cyanate (μg / mL) = C 1 × 42

[0300] Content of imidocarbonate (μg / mL) = C 2 × 59 × n

[0301] Activation degree (%) = [Content of cyanate (μg / mL) + Content of imidocarbonate (μg / mL)] / Content of polysaccharide (μg / mL) × 100

[0302] C 1 is the concentration of cyanate in the test sample;

[0303] 42 is the relative molecular mass of cyanate;

[0304] C 2 is the concentration of imidocarbonate in the test sample;

[0305] 59 is the relative molecular mass of imidocarbonate;

[0306] n is the dilution factor for determining the content of imidocarbonate in the test sample.

[0307] The measured content of cyanate is 0.9156 μg / mL, the content of imidocarbonate is 29.1991 μg / mL, and the content of polysaccharide is 1449.3 μg / mL. Calculate the activation degree to be 2.08% according to the formula.

[0308] Example 12

[0309] Take 10 mL of the activated polysaccharide of Streptococcus pneumoniae type 19F and place it in a beaker containing the dialysis solution. Dialyze at 2 - 8 °C for 24 h. Appropriately change the dialysis solution during dialysis to ensure that all cyanogen bromide is removed.

[0310] ① Take 2 ml of the reserved solution after removing cyanogen bromide as Sample 1 to measure the cyanate ester content.

[0311] ② Take 2 ml of the reserved solution after removing cyanogen bromide as Sample 2 to determine the imide carbonate content.

[0312] ③ Take an appropriate amount of the reserved solution after removing cyanogen bromide as Sample 3 and use the anthrone method to measure the polysaccharide content of the activated polysaccharide.

[0313] Sample 1, Determination of Cyanate Ester Content

[0314] Measure 0.00 ml, 0.20 ml, 0.40 ml, 0.80 ml, 1.20 ml, 1.60 ml, 2.00 ml of the cyanogen bromide reference working solution and place them in glass sample bottles respectively. Add purified water to make up to 2.0 ml. Take another 2.0 ml of the test solution (Sample 1) and place it in a glass sample bottle. Add 2 ml of 2% dimethylbarbituric acid pyridine solution, stir vigorously at 35 °C for 20 min. Measure the absorbance of the above samples at 588 nm according to the ultraviolet-visible spectrophotometry. Use the concentration of the cyanogen bromide reference working solution and its corresponding absorbance to make a regression curve, and substitute the absorbance of the test solution into the regression curve to calculate its corresponding concentration (μmol / ml).

[0315] Sample 2, Determination of Imide Carbonate Content

[0316] Take Sample 2, add 2 ml of HCl, hydrolyze at 35 °C for 45 min, transfer to a 10 ml volumetric flask, add 0.2 mol / L sodium acetate to make up to the mark, mix well, centrifuge at 6500 g for 5 min, and take the supernatant as the test solution to determine the content of ammonium ions produced by the hydrolysis of imide carbonate.

[0317] Measure 0.00 ml, 0.20 ml, 0.40 ml, 0.60 ml, 0.80 ml, 1.00 ml of the ammonium ion reference working solution and place them in test tubes respectively. Add 0.2 mol / L sodium acetate to make up to 1.0 ml. Take another 1.0 ml of the above test solution and place it in a test tube. Add 1 ml of 2 mol / L sodium acetate and 1.5 ml of the reduced ninhydrin reagent, mix well, heat in a boiling water bath for 30 min, add 6.5 ml of the formaldehyde dilution solution and mix well. Take 0.5 ml and dilute it 10 times with the formaldehyde dilution solution. Measure the absorbance of each tube at 570 nm according to the ultraviolet-visible spectrophotometry. Use the concentration of the ammonium ion reference working solution and its corresponding absorbance to make a regression curve, and substitute the absorbance of the test solution into the regression curve to calculate its corresponding concentration (μmol / ml).

[0318] Result Calculation

[0319] The content of cyanate ester (μg / ml) = C 1 × 42

[0320] Content of imidocarbonate (μg / ml) = C 2 ×59×n

[0321] Degree of activation (%) = [Content of cyanate (μg / ml) + Content of imidocarbonate (μg / ml)] / Content of polysaccharide (μg / ml) × 100

[0322] C 1 is the concentration of cyanate in the test sample;

[0323] 42 is the relative molecular weight of cyanate;

[0324] C 2 is the concentration of imidocarbonate in the test sample;

[0325] 59 is the relative molecular weight of imidocarbonate;

[0326] n is the dilution factor for determining the content of imidocarbonate in the test sample.

[0327] The measured content of cyanate is 0.8988 μg / ml, the content of imidocarbonate is 26.6031 μg / ml, and the content of polysaccharide is 1449.3 μg / ml. The degree of activation is calculated to be 1.90% according to the formula.

[0328] Example 13

[0329] Take 10 ml of 23F pneumococcal activated polysaccharide and place it in a beaker containing dialysis fluid. Dialyze at 2 - 8°C for 24 h. Replace the dialysis fluid appropriately during dialysis to ensure that all cyanogen bromide is removed.

[0330] ① Take 2 ml of the retained solution after removing cyanogen bromide as sample 1 to measure the content of cyanate.

[0331] ② Take 2 ml of the retained solution after removing cyanogen bromide as sample 2 to determine the content of imidocarbonate.

[0332] ③ Take an appropriate amount of the retained solution after removing cyanogen bromide as sample 3 and use the anthrone method to measure the polysaccharide content of the activated polysaccharide.

[0333] Sample 1, determination of cyanate content

[0334] Measure 0.00 ml, 0.20 ml, 0.40 ml, 0.80 ml, 1.20 ml, 1.60 ml, 2.00 ml of the cyanogen bromide reference working solution respectively into glass sample bottles, add purified water to make up to 2.0 ml. Additionally, take 2.0 ml of the test solution (Sample 1) and place it in a glass sample bottle. Add 2 ml of 2% dimethylbarbituric acid pyridine solution, stir vigorously at 60 °C for 10 min. Measure the absorbance of the above samples at 588 nm according to the ultraviolet-visible spectrophotometry. Use the concentration of the cyanogen bromide reference working solution and its corresponding absorbance to make a regression curve, and substitute the absorbance of the test solution into the regression curve to calculate its corresponding concentration (μmol / ml).

[0335] Sample 2, determination of imidocarbonate content

[0336] Take Sample 2, add 2 ml of HCl, hydrolyze at 50 °C for 15 min, transfer to a 10-ml volumetric flask, add 0.2 mol / L sodium acetate to volume to the mark, mix well, centrifuge at 6500 g for 5 min, and take the supernatant as the test sample to determine the content of ammonium ions produced by the hydrolysis of imidocarbonate.

[0337] Measure 0.00 ml, 0.20 ml, 0.40 ml, 0.60 ml, 0.80 ml, 1.00 ml of the ammonium ion reference working solution respectively into test tubes, add 0.2 mol / L sodium acetate to make up to 1.0 ml. Additionally, take 1.0 ml of the above test solution and place it in a test tube. Add 1 ml of 2 mol / L sodium acetate and 1.5 ml of the reduced ninhydrin reagent, mix well, heat in a boiling water bath for 20 min, add 6.5 ml of the formaldehyde dilution solution and mix well. Take 0.5 ml and dilute it 10 times with the formaldehyde dilution solution. Measure the absorbance of each of the above tubes at 570 nm according to the ultraviolet-visible spectrophotometry. Use the concentration of the ammonium ion reference working solution and its corresponding absorbance to make a regression curve, and substitute the absorbance of the test solution into the regression curve to calculate its corresponding concentration (μmol / ml).

[0338] Result calculation

[0339] Content of cyanate (μg / ml) = C 1 × 42

[0340] Content of imidocarbonate (μg / ml) = C 2 × 59 × n

[0341] Activation degree (%) = [Content of cyanate (μg / ml) + Content of imidocarbonate (μg / ml)] / Content of polysaccharide (μg / ml) × 100

[0342] C 1 is the concentration of cyanate in the test sample;

[0343] 42 is the relative molecular weight of cyanate ester;

[0344] C 2 is the concentration of imidocarbonate in the test sample;

[0345] 59 is the relative molecular weight of imidocarbonate;

[0346] n is the dilution factor for determining the content of imidocarbonate in the test sample.

[0347] The measured content of cyanate ester is 0.7056 μg / ml, the content of imidocarbonate is 30.2552 μg / ml, and the content of polysaccharide is 2002.1 μg / ml. The activation degree is calculated to be 1.55% according to the formula.

[0348] In the present invention, the determination result of the activation degree is calculated by detecting the ratio of the content of cyanate ester and imidocarbonate in the test sample to the content of polysaccharide in the test sample. To ensure the accuracy of the result, a series of methodological validations are carried out for the detection methods of the content of cyanate ester, imidocarbonate and polysaccharide. The results of the three methodological validations all meet the requirements of each index in the General Principles 9101 "Guidance for Validation of Analytical Methods" of the current edition of the Chinese Pharmacopoeia.

[0349] Through the above Examples 1 to 13, it can be proved that the detection method of the present invention can accurately detect the activation degrees of various cyanogen bromide-activated polysaccharides.

[0350] In summary, the detection method of the activation degree of cyanogen bromide-activated polysaccharides of the present invention can effectively measure the activation degree of cyanogen bromide-activated polysaccharides, evaluate the derivatization rate of polysaccharide derivatives through the activation degree of cyanogen bromide-activated polysaccharides, further evaluate the immunogenicity of polysaccharide conjugate vaccines, and increase the quality control method for evaluating the immunogenicity of polysaccharide conjugate vaccines. Further, according to the correlation between the activation degree of cyanogen bromide-activated polysaccharides and the immunogenicity of polysaccharide conjugate vaccines, the activation conditions are optimized, and a higher derivatization rate of polysaccharide derivatives is obtained by controlling a certain activation degree range. The higher the derivatization rate, the lower the content of free polysaccharide, and the better the immunogenicity of the polysaccharide conjugate vaccine.

[0351] The foregoing description of specific exemplary embodiments of the invention has been presented for purposes of illustration and exemplification. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many changes and variations are possible in light of the above teaching. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the invention and its practical application so that those skilled in the art can implement and utilize the various different exemplary embodiments of the invention as well as various different selections and changes. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for detecting the activation degree of polysaccharides activated by cyanogen bromide, characterized in that: The following steps are involved: The sample is pretreated to form a test sample, and a cyanate test sample and an imide carbonate test sample are obtained; Obtaining a cyanate ester standard sample and an imide carbonate ester standard sample, and forming an absorbance curve of the cyanate ester standard sample and an absorbance curve of the imide carbonate ester standard sample; Determining the absorbance of the cyanate test sample, and obtaining the content of cyanate in the test sample according to the absorbance curve of the cyanate standard sample; Determining the absorbance of the iminocarbonate test sample, and obtaining the content of iminocarbonate in the test sample according to the absorbance curve of the iminocarbonate standard sample; as well as Calculate the activation degree of the sample according to the content of cyanate and iminocarbonate in the sample, that is, the activation degree of the cyanogen bromide-activated polysaccharide in the sample; The calculation formula of the activation degree is: Activation degree = [cyanate content + imide carbonate content] / polysaccharide content × 100%.

2. The method for detecting the degree of activation of polysaccharides activated by cyanogen bromide according to claim 1, characterized in that: The pre-processing steps include: The sample is subjected to centrifugation or dialysis.

3. The method for detecting the degree of activation of polysaccharides activated by cyanogen bromide according to claim 2, characterized in that: The centrifugal treatment conditions include: centrifugal parameters of 6000-8000g, and the number of centrifugation is at least nine times.

4. The method for detecting the degree of activation of polysaccharides activated by cyanogen bromide according to claim 2, characterized in that: The dialysis treatment conditions include: using a sodium chloride solution with a concentration of less than 0.9 wt % as a dialysis fluid, using a regenerated cellulose membrane as a dialysis membrane, and dialysis at a temperature of 2 to 8° C. for at least 24 hours.

5. The method for detecting the degree of activation of polysaccharides activated by cyanogen bromide according to claim 1, characterized in that: The steps for obtaining an iminocarbonate test sample include: A portion of the test sample is added into a container, and a hydrochloric acid solution is added, and the mixture is reacted at 20-60° C. for at least 15 minutes, and then a sodium acetate solution is added and mixed evenly. After centrifugation, the supernatant is taken as the iminocarbonate test sample.

6. The method for detecting the degree of activation of polysaccharides activated by cyanogen bromide according to claim 1, characterized in that: The steps for obtaining a cyanate ester test sample include: Taking a portion of the test sample, the cyanate test sample is obtained.

7. The method for detecting the activation degree of cyanogen bromide activated polysaccharide according to claim 1, characterized in that: The step of determining the absorbance of the cyanate test sample comprises: After adding the dimethylbarbituric acid pyridine solution to the cyanate test sample, stirring is performed for at least 5 minutes at a temperature not exceeding 60° C., and then the absorbance is measured.

8. The method for detecting the degree of activation of polysaccharides activated by cyanogen bromide according to claim 7, characterized in that: The concentration of the dimethylbarbituric acid pyridine solution is 0.8% to 2%.

9. The method for detecting the activation degree of cyanogen bromide activated polysaccharide according to claim 1, characterized in that: The step of determining the absorbance of the iminocarbonate test sample comprises: After adding reduced ninhydrin reagent to the iminocarbonate test sample, heat it in a boiling water bath for 10 to 30 minutes, then add the formaldehyde dilution solution, mix well and measure the absorbance.

10. The method for detecting the activation degree of cyanogen bromide activated polysaccharide according to claim 7 or 9, characterized in that: The absorbance determination method comprises: adopting ultraviolet-visible spectrophotometry to determine the absorbance at a wavelength of 500-600nm.

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