Method for detecting content of monosaccharide components in bacterial capsular polysaccharide or conjugate thereof

Abstract

The invention discloses a method for detecting the content of monosaccharide components in bacterial capsular polysaccharide or a conjugate thereof, and belongs to the field of biological product detection. Taking pneumococcus as an example, different hydrolysis methods are adopted for different serotypes of capsular polysaccharides, connected glucosidic bonds are broken through hydrolysis, released monosaccharide components and 1-phenyl-3-methyl-5-pyrazolone are subjected to derivatization under the alkaline condition to generate derivatives with ultraviolet absorption, and then the capsular polysaccharides with different serotypes are obtained. And then detecting the composition and the content of each monosaccharide generated by polysaccharide hydrolysis through liquid chromatography. According to the method, each monosaccharide component can be quickly and effectively hydrolyzed, meanwhile, various polysaccharides with different structural characteristics can be separated and detected by adopting a derivatization method, whether the monosaccharide composition of each serotype polysaccharide accords with a theoretical structure or not can be qualitatively analyzed, and the content of each monosaccharide component can be quantitatively determined to calculate the content of the polysaccharides; and a better technical support is provided for quality control of the antigen component capsular polysaccharide of the bacterial vaccine.

Description

Technical Field

[0001] The invention discloses a method for detecting the content of each monosaccharide component and the content of polysaccharide in a bacterial capsular polysaccharide or a conjugate thereof, and belongs to the field of biological product detection. Background Art

[0002] In the quality control of polysaccharide and polysaccharide protein conjugate vaccines, the analysis of monosaccharide composition of each serotype polysaccharide is one of the important items for structural characterization and quality control. At present, the quantitative detection methods of each monosaccharide component in polysaccharides include the following types:

[0003] (1) Chemical colorimetric method

[0004] Some characteristic components in polysaccharides have light absorption within a specific wavelength range, or after chemical treatment, form substances with light absorption. The absorbance value is correlated with the content and can be used for content detection. For example, uronic acid of serotypes 1, 3, 5, and 9V, aminohexose of serotypes 4, 5, 9V, and 14, methyl pentose of serotypes 6B, 7F, 18C, and 23F, and O-acetyl of type 1, can all form substances with ultraviolet absorption under certain conditions, and then their content can be determined. The limitation of this method is that it has low sensitivity and is greatly interfered by other substances. In addition, this method can only determine the content of a certain characteristic functional group, and cannot fully characterize the components of each monosaccharide, and some polysaccharides do not contain the above-mentioned characteristic functional groups.

[0005] (2) High performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD)

[0006] Anion chromatography is commonly used for the analysis of monosaccharides, disaccharides, oligosaccharides, and polysaccharides. For example, after acid hydrolysis, the capsular polysaccharide of type 6A pneumococcus produces four monosaccharide components: D-glucose, D-galactose, L-rhamnose, and D-ribitol. The HPAEC-PAD method can be used to simultaneously determine the content of the four monosaccharide components. This method has the advantages of being simple, rapid, highly sensitive, and not requiring derivatization. However, its limitations lie in the fact that most serotypes of pneumococcal capsular polysaccharides have complex monosaccharide compositions, especially for samples containing aminohexose and uronic acid. It is difficult to achieve baseline separation of the monosaccharide components. At the same time, the amino acids produced by the hydrolysis of the conjugate carrier protein will interfere with the detection to a certain extent. In addition, this method uses a strongly alkaline solution as the mobile phase and requires a dedicated ion chromatograph, which has high requirements for reagents and the environment.

[0007] (3) Gas chromatography

[0008] Gas chromatography is also a commonly used method for detecting the content of carbohydrate compounds. It has the advantages of good selectivity, low sample consumption, high resolution, and high sensitivity. However, since carbohydrate substances usually contain polar groups such as NH, OH, and SH, the internal hydrogen bonding of these groups enhances the attraction within the molecule, making general sugars and sugar alcohols have the characteristics of high boiling point, low volatility, difficulty in vaporization, and poor stability. When using GC analysis, derivatization treatments such as methylation, acylation, and trimethylsilylation are usually required to convert carbohydrate substances into volatile substances. The operation process is cumbersome and the experimental requirements are high. In addition, the monosaccharide components after derivatization will produce isomers. Each monosaccharide component will produce two or even more chromatographic peaks after derivatization, which will cause certain interference to quantification.

[0009] (4) Pre-column derivatization HPLC

[0010] The method of pre-column derivatization is mainly to make the sugar chain carry ultraviolet or fluorescent groups to improve the sensitivity of detection. At the same time, it can make the sugar chain carry hydrophobic groups, reduce the polarity of the sugar chain, and retain the sugar chain on the reverse phase column, which is convenient for the separation of the sugar chain. The reducing end of the sugar chain is often used to react with derivatization reagents such as 2-aminopyridine (2-AP), 1-phenyl-3-methyl-5-pyrazolone (PMP), hydrazide compounds, etc. to generate monosaccharide derivatives, which are then separated and detected by reverse phase chromatography. PMP is one of the best derivatization reagents for reducing sugars. It can quantitatively condense with monosaccharides under alkaline conditions to generate monosaccharide-PMP derivatives. The derivatives are not easy to cleave and produce isomeric peaks and have strong absorption at a wavelength of 245nm. Therefore, they are often used for the detection of sugar compounds in food, medicine, and the environment.

[0011] The monosaccharide composition of each serotype capsular polysaccharide is one of the key quality attributes in the production process of pneumococcal capsular polysaccharide vaccines and conjugate vaccines. It can not only be used to qualitatively determine whether the structure of the specific serotype polysaccharide is incorrect, but also can be used to quantitatively calculate the content of each monosaccharide component and then calculate the polysaccharide content. Therefore, the detection of monosaccharide components of each type of capsular polysaccharide is of great significance. However, due to the large number of pneumococcal capsular polysaccharide serotypes, the complex structure of polysaccharides, and the large differences, there are problems such as difficulty in polysaccharide hydrolysis, easy degradation of monosaccharide components, and difficulty in achieving baseline separation of monosaccharide components in liquid chromatography. There are few reports on systematic detection methods for the monosaccharide composition of pneumococcal capsular polysaccharides or their conjugates. In the prior art, there are a few reports on how to detect the content of pneumococcal polysaccharides (e.g. CN106018832B, CN112179859A) and the molecular size of polysaccharides (e.g. CN113433122A), but there are no reports on how to systematically, comprehensively and accurately detect the monosaccharide components of various types of capsular polysaccharides.

[0012] In the related technical field, there are reports on the use of anion chromatography to detect the sialic acid content of meningococcal capsular polysaccharides and the content of other plant polysaccharides, and there are also reports on the use of PMP pre-column derivatization high-performance liquid chromatography to analyze and detect the monosaccharide composition of foods and medicinal plants such as wolfberry, fungi, and Sichuan Achyranthes bidentata. However, there are no reports on the use of PMP pre-column derivatization high-performance liquid chromatography for the detection of monosaccharide components of bacterial capsular polysaccharides, and the reported detection methods only target the detection of a few 3-5 monosaccharide components. Furthermore, since (1) bacterial capsular polysaccharides and medicinal plant polysaccharides differ greatly in structure and molecular weight, the detection methods applicable to plant or food polysaccharides may not be applicable to bacterial capsular polysaccharides; (2) there are dozens of serotypes of pneumococcal capsular polysaccharides, it is still unknown whether the PMP pre-column derivatization HPLC method can comprehensively detect each serotype and distinguish them at the same time; (3) in particular, there is interference caused by carrier proteins in the capsular polysaccharide conjugate samples. In view of the above difficulties, technicians in this field cannot reasonably predict whether the PMP pre-column derivatization HPLC method is applicable to the detection of monosaccharide components of bacterial capsular polysaccharides. Summary of the invention

[0013] To solve the above problems, the present invention proposes for the first time to use PMP pre-column derivatization HPLC method to determine the monosaccharide composition and content in pneumococcal capsular polysaccharides or their conjugates. First, according to the structural differences between polysaccharides of different serotypes, different acid hydrolysis methods are used (for polysaccharide or conjugate samples containing uronic acid: "anhydrous HCl / Meth+trifluoroacetic acid" two-step acid hydrolysis method; for samples containing phosphodiester bonds: "hydrofluoric acid+trifluoroacetic acid" two-step acid hydrolysis method; for samples not containing the above two: trifluoroacetic acid one-step acid hydrolysis method), so as to break the glycosidic bonds to the greatest extent to release each monosaccharide component, while minimizing the degradation of each monosaccharide under acidic conditions; then, the hydrolyzed monosaccharides react with PMP derivatization under alkaline conditions, and according to the difference in the peak time of each monosaccharide standard, a standard curve is established with samples of different concentrations of each monosaccharide standard mixture, thereby obtaining each monosaccharide component and its content (concentration) in the analyte; finally, based on the content detection results of characteristic monosaccharides in the measured samples, the polysaccharide content in the sample can be further calculated. Therefore, the present invention provides a method for detecting the content of each monosaccharide component in a sample of bacterial capsular polysaccharide or a conjugate thereof with a carrier protein (referred to as a conjugate or a conjugate) and a method for detecting the polysaccharide content. The applicable bacterial types include but are not limited to Gram-positive bacteria, such as pneumococcus.

[0014] The specific technical solution provided by the present invention includes the following contents:

[0015] The present invention provides a method for detecting the content of each monosaccharide component in a bacterial capsular polysaccharide or a bacterial capsular polysaccharide and carrier protein conjugate sample, which comprises the following steps:

[0016] 1) Sample hydrolysis: Depending on whether the polysaccharide structure of the sample to be tested contains uronic acid or phosphodiester bonds, the connected glycosidic bonds are broken by a method selected from a) to c) below to release the monosaccharide components:

[0017] a) Samples containing neither uronic acid nor phosphodiester bonds: hydrolyzed with trifluoroacetic acid;

[0018] b) Samples containing uronic acid: first treated with anhydrous hydrogen chloride-methanol solution, then hydrolyzed with trifluoroacetic acid;

[0019] c) Samples containing phosphodiester bonds: first treated with hydrofluoric acid, then hydrolyzed with trifluoroacetic acid;

[0020] 2) Derivatization reaction: The hydrolyzed sample is blown dry, dissolved in water, and then reacted with 1-phenyl-3-methyl-5-pyrazolone (PMP) under alkaline conditions to generate a monosaccharide derivative with ultraviolet absorption. After neutralization with hydrochloric acid, the excess PMP is extracted with an organic solvent to remove it.

[0021] 3) Liquid chromatography detection: Determine the monosaccharide components (monosaccharide components) of the sample according to the peak time of the standard monosaccharide, quantify according to the external standard method, substitute the peak area of ​​the monosaccharide component into the standard curve of each monosaccharide, and calculate the content of each monosaccharide component.

[0022] For step 1), the hydrolysis methods a) to c) can be respectively performed as follows:

[0023] a) Samples without uronic acid or phosphodiester bonds: blow dry the sample, add 2-4 mol / L trifluoroacetic acid, and hydrolyze at 100-120°C for 2-4 hours (wherein, the "blow dry" can be blow dried by a nitrogen blow dryer or a vacuum centrifugal concentrator, the same below);

[0024] b) Samples containing uronic acid: blow dry the sample, add 2-4 mol / L anhydrous hydrogen chloride-methanol solution, incubate in a water bath at 70-90℃ for 12-24h, then blow dry, add 2-4 mol / L trifluoroacetic acid, and continue hydrolysis at 100-120℃ for 2-4h;

[0025] c) Samples containing phosphodiester bonds: blow dry the sample, add 40-50% (v / v) hydrofluoric acid solution, incubate in a water bath at 60-80℃ for 2-4h, then blow dry, add 2-4mol / L trifluoroacetic acid, and continue hydrolysis at 100-120℃ for 2-4h;

[0026] For step 2), the preferred implementation includes the following operations: PMP concentration 0.4-0.6 mol / L, sodium hydroxide concentration 0.3-0.5 mol / L, reaction temperature 60-80°C, reaction time 30-90 min; hydrochloric acid concentration and sodium hydroxide concentration are kept consistent; the organic solvent can be selected from: chloroform, toluene, isoamyl acetate, and n-hexane.

[0027] For step 3), the preferred liquid chromatography analysis implementation method is as follows: using a DAD diode array detector or an ultraviolet detector, a detection wavelength of 240-250nm, preferably 245nm, an injection volume of 10-50μL, a flow rate of 0.8-1.2mL / min, mobile phase A: 10-100mM ammonium acetate buffer, mobile phase B: acetonitrile solution, gradient solution elution; wherein the pH of the ammonium acetate buffer is selected to be 5.5-6.0, preferably 5.5-5.7; wherein the gradient solution elution is preferably operated as follows: with the elution time 0→25min→50min→51min→60min, the concentration gradient of mobile phase B is: 20%→20%→30%→20%→20% (volume percentage), and the remainder is mobile phase A, and the total volume of the two is 100%.

[0028] For step 3), the preferred formula for calculating the monosaccharide content is ci=(yb) / a×D, where:

[0029] ci: the content of a characteristic monosaccharide component of the sample to be tested, in μg / mL;

[0030] y: Liquid phase detection peak area of ​​a characteristic monosaccharide component of the sample to be tested, unit: mAU*s;

[0031] a: The slope of the standard curve established based on the different concentrations and peak areas of the characteristic monosaccharide standards;

[0032] b: The intercept of the standard curve established based on different concentrations and peak areas of characteristic monosaccharide standards;

[0033] D: Dilution factor, that is, the multiple by which the sample solution to be tested is diluted.

[0034] The monosaccharides applicable to the present invention can be selected from one or more of the following: D-glucose, D-galactose, L-rhamnose, D-mannose, L-fucose, D-glucuronic acid, D-galacturonic acid, DN-acetylglucosamine, DN-acetylgalactosamine, DN-acetylmannosamine, LN-acetylfucosamine.

[0035] Optionally, for freeze-dried samples, before step 1), water is added to dissolve the sample and dilute it to 50-500 μg / mL; preferably, it is diluted to 200±100 μg / mL.

[0036] The bacteria applicable to the present invention include but are not limited to Gram-positive bacteria, such as pneumococcus. Pneumococcal capsular polysaccharides or their conjugates include but are not limited to 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F pneumococcal capsular polysaccharides and monovalent or multivalent conjugates thereof with carrier proteins, the carrier proteins are selected from but are not limited to CRM197, diphtheria toxoid, tetanus toxoid, etc., and the combination of polysaccharides and proteins adopts but is not limited to cyanogen bromide, CDAP method, etc. For pneumococcal capsular polysaccharides or their conjugates, the sample containing neither uronic acid nor phosphodiester bonds is selected from one or more of the following serotype pneumococcal capsular polysaccharides or their conjugates: 4, 7F, 12F, 14, 17F, and 33F serotypes; the sample containing uronic acid is selected from one or more of the following serotype pneumococcal capsular polysaccharides or their conjugates: 1, 2, 3, 5, 8, 9N, 9V, and 22F serotypes; the sample containing phosphodiester bonds is selected from one or more of the following serotype pneumococcal capsular polysaccharides or their conjugates: 6A, 6B, 10A, 11A, 15B, 18C, 19A, 19F, 20, and 23F serotypes.

[0037] The present invention also provides a method for detecting the content of capsular polysaccharide in a sample of bacterial capsular polysaccharide or a conjugate of bacterial capsular polysaccharide and carrier protein, which comprises the following steps:

[0038] S1) According to the chemical structure of the capsular polysaccharide in the sample to be tested, a characteristic monosaccharide is selected and its mass percentage in the capsular polysaccharide is determined, which is represented by "wi";

[0039] S2) detecting the component content of the characteristic monosaccharide in the sample to be tested according to the above-mentioned method for detecting the content of the monosaccharide component, which is represented by "ci";

[0040] S3) Based on the measured content of characteristic monosaccharide components, the content of capsular polysaccharides is calculated using the formula: ci / wi.

[0041] The above polysaccharide content detection method is applicable to bacteria including but not limited to Gram-positive bacteria, such as pneumococcus. For pneumococcus, the selection of characteristic monosaccharides in step S1) may be specifically referred to Table 4 below.

[0042] Beneficial technical effects of the present invention:

[0043] The present invention adopts a PMP pre-column derivatization high performance liquid chromatography method to detect various monosaccharide components and contents of bacterial capsular polysaccharides and conjugate samples after hydrolysis, and proposes three different hydrolysis methods according to the different structures of capsular polysaccharides of different serotypes and the difficulty of hydrolysis, which can effectively hydrolyze various types of pneumococcal capsular polysaccharides, break glycosidic bonds to the greatest extent to release various monosaccharide components, and reduce the degradation of various monosaccharides under acidic conditions as much as possible, so as to ensure the maximum recovery rate of various monosaccharide components, and at least ensure that a recovery rate of approximately 100% of one monosaccharide component is obtained to calculate the content of the polysaccharide.

[0044] The present invention adopts a pre-column derivatization operation for the detection of monosaccharide samples after hydrolysis. On the one hand, the interference of amino acids generated in the hydrolysis process of carrier protein can be avoided, so that in addition to polysaccharide samples, the method is also suitable for the detection of various monosaccharide components and contents in polysaccharide-protein conjugate samples. On the other hand, the method can overcome the shortcomings of ion chromatography requiring special instruments and equipment and having a poor separation effect. Only a conventional C18 reverse phase chromatographic column and a matching liquid chromatograph are required to complete the detection. At the same time, the elution conditions are optimized. In one detection, a total of 10 monosaccharide components, including neutral monosaccharides, acidic monosaccharides and alkaline monosaccharides, can be separated simultaneously. The linearity, precision, accuracy and the like of the methodological verification all meet the requirements.

[0045] The method provided by the present invention is widely applicable to the separation and identification of various monosaccharide components in bacterial polysaccharide and polysaccharide-protein conjugate samples and the detection of monosaccharide and polysaccharide contents, providing a reliable basis for quality control in the production process of polysaccharide vaccines and polysaccharide-protein conjugate vaccines, and is particularly applicable to the detection of pneumococcal capsular polysaccharide vaccines and conjugate vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the hydrolysis derivatization chromatogram of the monosaccharide standard mixed solution (50 μg / mL) in Example 1.

[0047] Figure 2 This is the chromatogram of the hydrolysis and derivatization of the 7F type pneumococcal capsular polysaccharide solution in Example 1.

[0048] Figure 3 This is the chromatogram of the hydrolysis and derivatization of the 9V type pneumococcal capsular polysaccharide solution in Example 2.

[0049] Figure 4 This is the chromatogram of the hydrolysis and derivatization of the 19F type pneumococcal capsular polysaccharide solution in Example 3.

[0050] Figure 5 This is the chromatogram of the hydrolysis and derivatization of the 6A+6B type pneumococcal polysaccharide protein conjugate stock solution of Example 4.

[0051] Figure 6 This is the overlay chromatogram of the hydrolysis derivatization of the 6-needle system suitability sample in Example 5.

[0052] Figure 7 This is the hydrolysis derivatization chromatogram of the blank solution of Example 5.

[0053] Figure 8 This is the chromatogram of the hydrolysis and derivatization of the carrier protein solution in Example 5.

[0054] Fig. 9 The chromatograms are superimposed with the mobile phases of ammonium acetate buffers of different concentrations in Example 5.

[0055] Fig.10 Overlay chromatograms of the mobile phases for ammonium acetate buffers of different pH values ​​for Example 5.

[0056] Fig.11 The chromatograms are superimposed under different flow rate conditions of Example 5.

[0057] Fig.12 This is an overlay chromatogram of the sample stability study in Example 5.

[0058] Fig.13 Overlay chromatograms for the method optimization of Example 5. DETAILED DESCRIPTION

[0059] The present invention provides a method for detecting the composition and content of each monosaccharide in a bacterial capsular polysaccharide or a conjugate thereof, which is suitable for detecting capsular polysaccharides of each serotype of pneumococcus and conjugates thereof. The method prepares a monosaccharide hydrolyzate of a pneumococcal capsular polysaccharide or a conjugate thereof sample by an acid hydrolysis method, wherein each monosaccharide component in the hydrolyzate undergoes a derivatization reaction with PMP under alkaline conditions, each monosaccharide derivative is subjected to a C18 reverse phase chromatography column, an ammonium acetate / acetonitrile solution gradient elution, and a diode array or ultraviolet detector is used for detection, the monosaccharide composition and content produced by the polysaccharide hydrolysis are determined, and the content of the polysaccharide in the sample is calculated according to the mass fraction of the characteristic monosaccharide.

[0060] The method comprises the following steps:

[0061] 1) Sample preparation: Prepare / dilute various types of capsular polysaccharides or the combination of capsular polysaccharides and carrier proteins into solutions of a certain concentration;

[0062] The concentration of the sample solution ensures that the monosaccharide concentration produced by hydrolysis is within the range of the standard curve, preferably within the range of 50-200 ug / mL;

[0063] 2) Sample hydrolysis: According to the structural characteristics of different serotype capsular polysaccharides (reference: K Aaron Geno et al., Clin Microbiol Rev. 2015 Jul;28(3):871-99.), confirm whether the corresponding serotype polysaccharide of the sample to be tested contains uronic acid and phosphodiester bonds. The samples are divided into three types and the one-step or two-step acid hydrolysis method is used respectively. The specific methods are as follows:

[0064] (1) For polysaccharide or conjugate samples that do not contain uronic acid or phosphodiester bonds (such as pneumococcal serotype 4, 7F, 12F capsular polysaccharides or their conjugates, etc.): a one-step acid hydrolysis method is used, specifically using trifluoroacetic acid treatment: after the sample is blown dry, 2-4 mol / L trifluoroacetic acid is added and hydrolyzed at 100-120°C for 2-4 hours;

[0065] (2) For samples of polysaccharides or conjugates containing uronic acid (e.g., pneumococcal serotype 1, 3, 5, 9V capsular polysaccharides or their conjugates, etc.): a two-step acid hydrolysis method is used, that is, before the trifluoroacetic acid treatment, an anhydrous hydrogen chloride-methanol (anhydrous HCl / Meth) hydrolysis step is added to fully break the glycosidic bonds containing the uronic acid structure, preferably 2-4 mol / L HCl / Meth, 70-90°C for 12-24h, and then the trifluoroacetic acid treatment is performed (parameters are the same as above);

[0066] (3) For samples of polysaccharides or conjugates containing phosphodiester bonds (e.g., pneumococcal serotype 6A, 6B, 19A, 19F capsular polysaccharides or their conjugates, etc.): a two-step acid hydrolysis method is used, that is, before the trifluoroacetic acid treatment, a hydrofluoric acid hydrolysis step is added to break the phosphodiester bonds, preferably at a concentration of 40-50% (v / v) and 60-80°C for 2-4h, followed by trifluoroacetic acid treatment (parameters are the same as above).

[0067] The specific hydrolysis method is shown in the table below:

[0068] Table 1: Different hydrolysis methods of pneumococcal capsular polysaccharides or their conjugates of different serotypes

[0069]

[0070] ※ “Others” refers to polysaccharide or conjugate samples that do not contain uronic acid or phosphodiester bonds in the polysaccharide structure.

[0071] 3) Derivatization: After drying each monosaccharide hydrolyzate with nitrogen, dissolve it in water and react with PMP under alkaline conditions. After the reaction is completed, add acid to neutralize it and use an organic solvent to extract and remove the unreacted PMP reagent;

[0072] The base is sodium hydroxide solution, the concentration is 0.3-0.5 mol / L, the acid is hydrochloric acid, the concentration is consistent with the base concentration, and the amount added is also consistent, the concentration of the derivatizing agent PMP is 0.4-0.6 mol / L, the extractant is chloroform, toluene, isoamyl acetate, n-hexane and other organic solvents, preferably chloroform;

[0073] 4) Liquid phase detection: Inject the prepared samples and standards into the high performance liquid chromatography system for detection and analysis;

[0074] The chromatographic column is a C18 reverse phase chromatographic column, the detector is a diode array detector (DAD) or an ultraviolet detector, the detection wavelength is 240-250 nm, preferably 245 nm, the injection volume is 10-50 μL, the flow rate is 0.8-1.2 mL / min, the mobile phase A is ammonium acetate buffer, the mobile phase B is acetonitrile solution, wherein the concentration range of the ammonium acetate buffer solution is 10-100 mmol / L, the pH is 5.5-6.0 (preferably 5.5-5.7), and the gradient elution (with the elution time 0→25min→50min→51min→60min, the concentration gradient of the mobile phase B is: 20%→20%→30%→20%→20%, the above ratio is the volume percentage occupied by the mobile phase B, and the balance is the mobile phase A, and the two together are 100%);

[0075] 5) The content of each monosaccharide in the sample to be tested is calculated according to formula 1):

[0076] c i =(yb) / a×D…………………………1)

[0077] c i ——The content of a characteristic monosaccharide component of the sample to be tested, in μg / mL;

[0078] y——the peak area of ​​a characteristic monosaccharide component of the sample to be tested, in mAU*s;

[0079] a——The slope of the standard curve established based on the different concentrations and peak areas of the characteristic monosaccharide standards;

[0080] b——The intercept of the standard curve established based on the different concentrations and peak areas of the characteristic monosaccharide standards;

[0081] D——dilution factor, i.e. the multiple by which the sample solution to be tested is diluted;

[0082] The monosaccharides include D-glucose, D-galactose, L-rhamnose, D-mannose, L-fucose, D-glucuronic acid, D-galacturonic acid, DN-acetylglucosamine, DN-acetylgalactosamine, DN-acetylmannosamine, and LN-acetylfucosamine;

[0083] 6) The content of polysaccharides in the sample to be tested is calculated according to formula 2):

[0084] c 多 =c i / w i ……………………………………2)

[0085] c 多 ——The content of polysaccharides in the sample to be tested, in μg / mL;

[0086] c i ——The content of a characteristic monosaccharide in the sample to be tested measured in formula 1, such as glucuronic acid, glucose, galactose, rhamnose, etc., in μg / mL;

[0087] w i ——The mass fraction of a characteristic monosaccharide in the sample to be tested, in %, e.g., 0.232 μg of rhamnose can be released after complete hydrolysis of 1 μg of type 6B pneumococcal capsular polysaccharide, and the mass fraction of rhamnose in type 6B polysaccharide is 23.2%.

[0088] In the following, representative polysaccharide samples are selected for three types of capsular polysaccharides (i.e., polysaccharides containing phosphodiester bonds, polysaccharides containing uronic acid, and polysaccharides without the above two characteristics), and a polysaccharide-protein conjugate sample is selected to further explain and illustrate the present invention through specific examples.

[0089] Example 1: Detection of monosaccharide composition and content of 7F type pneumococcal capsular polysaccharide

[0090] 1. Preparation of monosaccharide standard solution

[0091] Accurately weigh 20 mg each of monosaccharide standard D-glucose, D-galactose, L-rhamnose, D-glucuronic acid, D-galacturonic acid, DN-acetylglucosamine, DN-acetylgalactosamine, DN-acetylmannosamine and 10 mg of D-mannose, place in the same 10 mL volumetric flask, add appropriate amount of water to dissolve and dilute to scale, shake well to make the concentration 1 mg / mL, mannose concentration 500 μg / mL, as monosaccharide standard stock solution. Dilute to 200, 100, 50, 25, 12.5, 6.25 μg / mL (mannose concentration halved) before use as monosaccharide standard linear working solution.

[0092] 2. Preparation of internal standard fucose solution

[0093] Accurately weigh 20 mg of the monosaccharide standard L-fucose, place it in a 10 mL volumetric flask, add an appropriate amount of water to dissolve and dilute to the scale, shake well to make the concentration of 2 mg / mL.

[0094] 3. Sample preparation

[0095] Accurately weigh 50 mg of freeze-dried 7F capsular polysaccharide sample into a 10 mL volumetric flask, add water to dissolve and dilute to the scale to a concentration of 5 mg / mL, and perform two 5x dilutions (1 mL sample + 4 mL water) to a final concentration of 200 μg / mL.

[0096] 4. Hydrolysis of samples and standards

[0097] 1) Use a pipette to accurately draw 500 μL of sample solution and monosaccharide standard solution of different concentrations, place them in a high-temperature resistant screw-cap tube, add 10 μL of fucose internal standard solution (2 mg / mL), and blow dry with nitrogen gas;

[0098] 2) Add 500 μL of 2 mol / L trifluoroacetic acid (TFA), tighten the tube cap, heat and hydrolyze in an oven at 120°C for 2 h, cool to room temperature, and blow dry with nitrogen to remove the trifluoroacetic acid;

[0099] 3) Add 300 μL of water to dissolve for derivatization.

[0100] 5. Derivatization reaction

[0101] Pipette 0.4 mol / L sodium hydroxide solution 0.3 mL, add to each of the above hydrolysis sample tubes, add 0.5 mol / L PMP methanol solution 0.4 mL, incubate in 70 ℃ water bath for 60 min, cool to room temperature, add 0.4 mol / L hydrochloric acid solution 0.3 mL to neutralize, mix well, transfer to a centrifuge tube, add 1 mL of chloroform, shake thoroughly, centrifuge at 5000 rpm for 5 min, discard the lower layer solution, repeat the extraction 3 times, take the upper layer solution for the last time, filter through 0.45 μm filter membrane, and take the filtrate for detection.

[0102] 6. Liquid phase detection

[0103] Run Agilent 1260 Infinity II liquid chromatograph, inject and detect the samples prepared by hydrolysis and derivatization. The following analysis conditions are for reference. Other conditions should be verified for their applicability:

[0104] The chromatographic conditions are as follows:

[0105] Chromatographic column: Shim-pack GIST C18 (4.6*250mm, 5μm);

[0106] Injection volume: 20 μL, flow rate: 1.0 mL / min, column temperature: 30 °C, detection wavelength: 245 nm, detection time: 60 min;

[0107] Mobile phase: Gradient elution with ammonium acetate buffer and acetonitrile solution (Phase A: 20mmol / L ammonium acetate buffer (pH5.5), Phase B: acetonitrile solution)

[0108] The gradient elution program is shown in Table 2:

[0109] Table 2: Gradient elution schedule

[0110]

[0111] 7. Result calculation

[0112] The monosaccharide components in the sample were qualitatively determined according to the retention time of the monosaccharide standard, and quantitatively determined according to the external standard method. The peak area of ​​each monosaccharide component in the sample was substituted into the standard curve to calculate the content of each monosaccharide component, and then the content of 7F type capsular polysaccharide was calculated based on the mass fraction of the D-galactose monosaccharide component.

[0113] See the attached chromatograms of monosaccharide standards and samples. Figure 1 and 2 , the results are shown in Table 3.

[0114] Example 2: Detection of Monosaccharide Composition and Content of Type 9V Pneumococcal Capsular Polysaccharide

[0115] 1. Preparation of monosaccharide standard solution

[0116] Accurately weigh 20 mg each of monosaccharide standard D-glucose, D-galactose, L-rhamnose, D-glucuronic acid, D-galacturonic acid, DN-acetylglucosamine, DN-acetylgalactosamine, DN-acetylmannosamine and 10 mg of D-mannose, place in the same 10 mL volumetric flask, add appropriate amount of water to dissolve and dilute to scale, shake well to make the concentration 1 mg / mL, mannose concentration 500 μg / mL, as monosaccharide standard stock solution. Dilute to 200, 100, 50, 25, 12.5, 6.25 μg / mL (mannose concentration halved) before use as monosaccharide standard linear working solution.

[0117] 2. Preparation of internal standard fucose solution

[0118] Accurately weigh 20 mg of the monosaccharide standard L-fucose, place it in a 10 mL volumetric flask, add an appropriate amount of water to dissolve and dilute to the scale, shake well to make the concentration of 2 mg / mL.

[0119] 3. Sample preparation

[0120] Accurately weigh 50 mg of freeze-dried 9V capsular polysaccharide sample into a 10 mL volumetric flask, add water to dissolve and dilute to the scale to a concentration of 5 mg / mL, and perform two 5x dilutions (1 mL sample + 4 mL water) to a final concentration of 200 μg / mL.

[0121] 4. Hydrolysis of samples and standards

[0122] 1) Use a pipette to accurately draw 500 μL of sample solution, place it in a high temperature resistant screw cap tube, add 10 μL of fucose internal standard solution (2 mg / mL), and blow dry with nitrogen gas;

[0123] 2) Add 500 μL of 2 mol / L anhydrous hydrogen chloride-methanol solution, tighten the tube cap, place in a water bath at 80°C for 24 h, cool to room temperature, and blow dry with nitrogen to remove hydrogen chloride;

[0124] 3) Add 500 μL of 2 mol / L trifluoroacetic acid, tighten the tube cap, place in a 100°C water bath for 2 h, cool to room temperature, and blow dry with nitrogen to remove the trifluoroacetic acid;

[0125] 4) Add 300 μL of water to dissolve for derivatization.

[0126] 5. Derivatization reaction

[0127] Pipette 0.4 mol / L sodium hydroxide solution 0.3 mL, add to each of the above hydrolysis sample tubes, add 0.5 mol / L PMP methanol solution 0.4 mL, incubate in 70 ℃ water bath for 60 min, cool to room temperature, add 0.4 mol / L hydrochloric acid solution 0.3 mL to neutralize, mix well, transfer to a centrifuge tube, add 1 mL of chloroform, shake thoroughly, centrifuge at 5000 rpm for 5 min, discard the lower layer solution, repeat the extraction 3 times, take the upper layer solution for the last time, filter through 0.45 μm filter membrane, and take the filtrate for detection.

[0128] 6. Liquid phase detection

[0129] Run Agilent 1260 Infinity II liquid chromatograph, inject and detect the samples prepared by hydrolysis and derivatization. The following analysis conditions are for reference. Other conditions should be verified for their applicability:

[0130] The chromatographic conditions are as follows:

[0131] Chromatographic column: Shim-pack GIST C18 (4.6*250mm, 5μm);

[0132] Injection volume: 20 μL, flow rate: 1.0 mL / min, column temperature: 30 °C, detection wavelength: 245 nm, detection time: 60 min;

[0133] Mobile phase: Gradient elution with ammonium acetate buffer and acetonitrile solution (Phase A: 20mmol / L ammonium acetate buffer (pH5.5), Phase B: acetonitrile solution)

[0134] The gradient elution program was the same as that in Table 2 above.

[0135] 7. Result calculation

[0136] The monosaccharide components in the sample were qualitatively determined according to the retention time of the monosaccharide standard, and quantitatively determined according to the external standard method. The peak area of ​​each monosaccharide component in the sample was substituted into the standard curve to calculate the content of each monosaccharide component, and then the content of 9V type capsular polysaccharide was calculated based on the mass fraction of the D-galactose monosaccharide component.

[0137] See attached chromatogram Figure 3 , the results are shown in Table 3.

[0138] Example 3: Detection of Monosaccharide Composition and Content of 19F Pneumococcal Capsular Polysaccharide

[0139] 1. Preparation of monosaccharide standard solution

[0140] Accurately weigh 20 mg each of monosaccharide standard D-glucose, D-galactose, L-rhamnose, D-glucuronic acid, D-galacturonic acid, DN-acetylglucosamine, DN-acetylgalactosamine, DN-acetylmannosamine and 10 mg of D-mannose, place in the same 10 mL volumetric flask, add appropriate amount of water to dissolve and dilute to scale, shake well to make the concentration 1 mg / mL, mannose concentration 500 μg / mL, as monosaccharide standard stock solution. Dilute to 200, 100, 50, 25, 12.5, 6.25 μg / mL (mannose concentration halved) before use as monosaccharide standard linear working solution.

[0141] 2. Preparation of internal standard fucose solution

[0142] Accurately weigh 20 mg of the monosaccharide standard L-fucose, place it in a 10 mL volumetric flask, add an appropriate amount of water to dissolve and dilute to the scale, shake well to make the concentration of 2 mg / mL.

[0143] 3. Sample preparation

[0144] Accurately weigh 50 mg of freeze-dried 19F capsular polysaccharide sample into a 10 mL volumetric flask, add water to dissolve and dilute to the scale to a concentration of 5 mg / mL, and perform two 5x dilutions (1 mL sample + 4 mL water) to a final concentration of 200 μg / mL.

[0145] 4. Hydrolysis of samples and standards

[0146] 1) Use a pipette to accurately draw 500 μL of sample solution, place it in a high temperature resistant screw cap tube, add 10 μL of fucose internal standard solution (2 mg / mL), and blow dry with nitrogen gas;

[0147] 2) Add 200 μL of 48% (v / v) hydrofluoric acid, tighten the cap, place in a 65°C water bath for 2 h, cool to room temperature, and blow dry with nitrogen to remove the hydrofluoric acid;

[0148] 3) Add 500 μL of 2 mol / L trifluoroacetic acid, tighten the tube cap, place in a 100°C water bath for 2 h, cool to room temperature, and blow dry with nitrogen to remove the trifluoroacetic acid;

[0149] 4) Add 300 μL of water to dissolve for derivatization.

[0150] 5. Derivatization reaction

[0151] Pipette 0.4 mol / L sodium hydroxide solution 0.3 mL, add to each of the above hydrolysis sample tubes, add 0.5 mol / L PMP methanol solution 0.4 mL, incubate in 70 ℃ water bath for 60 min, cool to room temperature, add 0.4 mol / L hydrochloric acid solution 0.3 mL to neutralize, mix well, transfer to a centrifuge tube, add 1 mL of chloroform, shake thoroughly, centrifuge at 5000 rpm for 5 min, discard the lower layer solution, repeat the extraction 3 times, take the upper layer solution for the last time, filter through 0.45 μm filter membrane, and take the filtrate for detection.

[0152] 6. Liquid phase detection

[0153] Run Agilent 1260 Infinity II liquid chromatograph, inject and detect the samples prepared by hydrolysis and derivatization. The following analysis conditions are for reference. Other conditions should be verified for their applicability:

[0154] The chromatographic conditions are as follows:

[0155] Chromatographic column: Shim-pack GIST C18 (4.6*250mm, 5μm);

[0156] Injection volume: 20 μL, flow rate: 1.0 mL / min, column temperature: 30 °C, detection wavelength: 245 nm, detection time: 60 min;

[0157] Mobile phase: Gradient elution with ammonium acetate buffer and acetonitrile solution (Phase A: 20mmol / L ammonium acetate buffer (pH5.5), Phase B: acetonitrile solution)

[0158] The gradient elution program was the same as that in Table 2 above.

[0159] 7. Result calculation

[0160] The monosaccharide components in the sample were qualitatively determined according to the retention time of the monosaccharide standard, and quantitatively determined according to the external standard method. The peak area of ​​each monosaccharide component in the sample was substituted into the standard curve to calculate the content of each monosaccharide component, and then the content of 19F type capsular polysaccharide was calculated based on the mass fraction of the L-rhamnose monosaccharide component.

[0161] See attached chromatogram Figure 4 , the results are shown in Table 3.

[0162] Example 4: Detection of Monosaccharide Composition and Content of 6A+6B Pneumococcal Polysaccharide Protein Conjugate Stock Solution

[0163] 1. Preparation of monosaccharide standard solution

[0164] Accurately weigh 20 mg each of monosaccharide standard D-glucose, D-galactose, L-rhamnose, D-glucuronic acid, D-galacturonic acid, DN-acetylglucosamine, DN-acetylgalactosamine, DN-acetylmannosamine and 10 mg of D-mannose, place in the same 10 mL volumetric flask, add appropriate amount of water to dissolve and dilute to scale, shake well to make the concentration 1 mg / mL, mannose concentration 500 μg / mL, as monosaccharide standard stock solution. Dilute to 200, 100, 50, 25, 12.5, 6.25 μg / mL (mannose concentration halved) before use as monosaccharide standard linear working solution.

[0165] 2. Preparation of internal standard fucose solution

[0166] Accurately weigh 20 mg of the monosaccharide standard L-fucose, place it in a 10 mL volumetric flask, add an appropriate amount of water to dissolve and dilute to the scale, shake well to make the concentration of 2 mg / mL.

[0167] 3. Sample preparation

[0168] The 6A+6B type pneumococcal polysaccharide protein conjugate is used, which is a divalent conjugate of the pneumococcal capsular polysaccharide of the two serotypes 6A and 6B and the carrier protein TT (i.e., the 6A and 6B type capsular polysaccharides are conjugated to one carrier protein). The conjugate stock solution sample itself is a liquid and does not need to be dissolved or diluted. It is sufficient to ensure that the content of each monosaccharide component is within the linear range after hydrolysis.

[0169] 4. Hydrolysis of samples and standards

[0170] 1) Use a pipette to accurately draw 500 μL of sample solution, place it in a high temperature resistant screw cap tube, add 10 μL of fucose internal standard solution (2 mg / mL), and blow dry with nitrogen gas;

[0171] 2) Add 200 μL of 48% (v / v) hydrofluoric acid, tighten the cap, place in a 65°C water bath for 2 h, cool to room temperature, and blow dry with nitrogen to remove the hydrofluoric acid;

[0172] 3) Add 500 μL of 2 mol / L trifluoroacetic acid, tighten the tube cap, place in a 100°C water bath for 2 h, cool to room temperature, and blow dry with nitrogen to remove the trifluoroacetic acid;

[0173] 4) Add 300 μL of water to dissolve for derivatization.

[0174] 5. Derivatization reaction

[0175] Pipette 0.4 mol / L sodium hydroxide solution 0.3 mL, add to each of the above hydrolysis sample tubes, add 0.5 mol / L PMP methanol solution 0.4 mL, incubate in 70 ℃ water bath for 60 min, cool to room temperature, add 0.4 mol / L hydrochloric acid solution 0.3 mL to neutralize, mix well, transfer to a centrifuge tube, add 1 mL of chloroform, shake thoroughly, centrifuge at 5000 rpm for 5 min, discard the lower layer solution, repeat the extraction 3 times, take the upper layer solution for the last time, filter through 0.45 μm filter membrane, and take the filtrate for detection.

[0176] 6. Liquid phase detection

[0177] Run Agilent 1260 Infinity II liquid chromatograph, inject and detect the samples prepared by hydrolysis and derivatization. The following analysis conditions are for reference. Other conditions should be verified for their applicability:

[0178] The chromatographic conditions are as follows:

[0179] Chromatographic column: Shim-pack GIST C18 (4.6*250mm, 5μm);

[0180] Injection volume: 20 μL, flow rate: 1.0 mL / min, column temperature: 30 °C, detection wavelength: 245 nm, detection time: 60 min;

[0181] Mobile phase: Gradient elution with ammonium acetate buffer and acetonitrile solution (Phase A: 20mmol / L ammonium acetate buffer (pH5.5), Phase B: acetonitrile solution)

[0182] The gradient elution program was the same as that in Table 2 above.

[0183] 7. Result calculation

[0184] The monosaccharide components in the sample were qualitatively determined according to the retention time of the monosaccharide standard, and quantitatively determined according to the external standard method. The peak area of ​​each monosaccharide component in the sample was substituted into the standard curve to calculate the concentration of each monosaccharide component, and then the content of the 6A+6B type pneumococcal polysaccharide protein conjugate stock solution was calculated based on the mass fraction of the L-rhamnose monosaccharide component.

[0185] See attached chromatogram Figure 5, the results are shown in Table 3.

[0186] Table 3: Content of monosaccharide components in pneumococcal capsular polysaccharide and polysaccharide protein conjugate samples

[0187]

[0188] Note: The polysaccharide contents of 7F, 9V, 19F, and 6A+6B conjugate samples were calculated as the mass percentages of characteristic monosaccharide contents of D-galactose, D-galactose, L-rhamnose, and L-rhamnose, respectively (according to the structural formulas of their polysaccharides, the mass percentages were 29.4%, 19.5%, 26.7%, and 23.2%, respectively). The specific calculation method is: polysaccharide content = characteristic monosaccharide content / mass percentage of characteristic monosaccharides.

[0189] In addition to the several serotypes selected as representatives in Examples 1 to 4 of the present application, the present invention is applicable to the detection of monosaccharide components and polysaccharide content of pneumococcal capsular polysaccharides of all serotypes. The detection of monosaccharide components in polysaccharide samples or conjugate samples of different serotypes is divided into three types according to whether they contain uronic acid and phosphodiester bonds, namely: (1) serotypes not containing the above two, (2) serotypes containing uronic acid, and (3) serotypes containing phosphodiester bonds. The monosaccharide component detection is performed by referring to the methods in Examples 7F, 9V, and 19F, respectively. Then, according to the detection results of the monosaccharide components in the tested samples, the polysaccharide content in the tested samples is calculated based on the characteristic monosaccharides of each serotype polysaccharide and its mass percentage in the polysaccharide (see Table 4 for details).

[0190] Table 4: Characteristic monosaccharides and their mass percentages of pneumococcal capsular polysaccharides of different serotypes

[0191]

[0192] In Table 4, the selection of characteristic monosaccharides of each serotype polysaccharide is based on the type of monosaccharide components reflected in the structural formula of each serotype polysaccharide (K Aaron Geno et al., Clin Microbiol Rev. 2015 Jul; 28(3): 871-99.), and based on the recovery rate of each monosaccharide component after polysaccharide hydrolysis, a monosaccharide with a higher recovery rate is selected, mainly based on common galactose and rhamnose, and glucose components are avoided as much as possible as characteristic monosaccharides (reason: some samples may contain some culture medium component impurities, including glucose, so glucose components are avoided as much as possible for calculation). In addition, the structure of type 1 polysaccharide is relatively special. It contains two galacturonic acids connected together. It is difficult to completely break its glycosidic bond to release monosaccharide components by hydrolysis, so the content of type 1 polysaccharide is quantitatively calculated based on polysaccharide standards.

[0193] Example 5: Methodological Investigation

[0194] (1) System applicability

[0195] A mixed working solution of monosaccharide standards with a concentration of 50 μg / mL was prepared, and hydrolysis and derivatization were performed according to the above method. The injection was repeated 5 times, and the sample was injected again after the detection was completed. The relative standard deviation (RSD) of the peak area of ​​each monosaccharide standard was calculated 6 times. Except for the two uronic acid samples, the RSD values ​​of the peak area of ​​each monosaccharide were less than 1%.

[0196] See the attached superimposed chromatogram for 6-injection Figure 6 , the results are shown in Table 5 below.

[0197] Table 5: System suitability test results

[0198]

[0199] (2) Exclusivity

[0200] The blank sodium chloride solution, carrier protein solution, monosaccharide standard solution and capsular polysaccharide & conjugate sample solution were prepared and tested according to the above method. The chromatograms of the carrier protein solution and the blank solution are shown in the attached Figure 7 and 8 There is no obvious chromatographic peak at the position of monosaccharide chromatographic peak. The chromatograms of standard and sample solutions are attached. Figure 1-5 The retention time of each monosaccharide component in the sample is consistent with the retention time of the monosaccharide standard.

[0201] (3) Precision

[0202] Different analysts prepared 6 portions of 7F capsular polysaccharide solution with a concentration of 200 μg / mL, and carried out sample preparation and liquid chromatography detection according to the above conditions. The specific experimental results are shown in Table 6 below. The relative standard error of the content of each monosaccharide component in the 6 samples prepared by each analyst was between 1.91% and 4.05%, and the relative standard error of the content of each monosaccharide component in the 12 samples prepared by two analysts was between 2.60% and 3.09%. The experimental deviation was small and the precision met the requirements.

[0203] Table 6: Precision test results

[0204]

[0205] (4) Detection limit and quantification limit

[0206] The detection limit refers to the minimum concentration or amount of a substance that can be detected in a sample by an analytical method within a given reliability level. The detection limit is the amount when the response signal generated by the detector caused by the substance is three times the noise value, that is, when the signal-to-noise ratio is S / N=3.

[0207] The limit of quantitation refers to the lowest amount of the substance in the sample that can be quantitatively detected, and the test result should have a certain degree of accuracy. The limit of quantitation shows whether a certain analytical method has a quick quota detection capability. The response signal generated by the detector is ten times the noise value, that is, the amount when the signal-to-noise ratio S / N=10 is the limit of quantitation.

[0208] The monosaccharide standard mixed solution was diluted 2-fold in sequence, sampled and tested, and the signal-to-noise ratio of each monosaccharide component at different concentrations was calculated to obtain the detection limit and quantification limit of each monosaccharide component, as shown in Table 7 below:

[0209] Table 7: Detection limit and quantification limit of each monosaccharide component

[0210]

[0211] (5) Standard curve

[0212] The standard solutions of each monosaccharide series were prepared into mixed standard solutions of 6.25, 12.5, 25, 50, 100, and 200 μg / mL (D-mannose concentration was halved), and the samples were prepared by hydrolysis and subsequent derivatization reaction according to the conditions of hydrolysis 1 above, and then detected by liquid chromatography injection. The monosaccharides were qualitatively analyzed by retention time, and the standard working curve was established with the concentration of each monosaccharide standard as the x-axis and the response signal as the y-axis. The linear correlation coefficient R of each monosaccharide standard was 0.04477 W / m. 2 ≥0.998, the linear equation is shown in Table 8 below.

[0213] Table 8: Linear equations for each monosaccharide component

[0214]

[0215] (6) Accuracy

[0216] 7F type pneumococcal capsular polysaccharide was used as the spiked sample. After the sample was hydrolyzed, 0.5, 1.0, and 1.5 times the mass concentration of the mixed standard solution were added, respectively. Pre-column derivatization HPLC analysis was performed according to the above method. The recovery rate was calculated based on the peak area of ​​each monosaccharide. The spiked recoveries of the six monosaccharides in 7F type capsular polysaccharide were between 93% and 105%, and the RSD was between 1% and 8%, which met the requirements. The results are shown in Table 9 below.

[0217] Table 9: Results of the recovery of 7F capsular polysaccharide spiked

[0218]

[0219] (7) Durability & stability

[0220] The concentration (10mM, 20mM, 50mM, 100mM), pH value (pH5.3, pH5.5, pH5.7), and flow rate (0.8mL / min, 1.0mL / min, 1.2mL / min) of ammonium acetate buffer solution were changed to investigate whether the separation of each monosaccharide component was affected. The chromatogram is shown in the attached figure. Figure 9-11 As shown, the concentration of ammonium acetate buffer has little effect on the separation effect of each monosaccharide component, but as the concentration of the buffer decreases, the PMP peak will become wider. The flow rate in the range of 0.8-1.2mL / min has little effect on the separation degree of each monosaccharide component, but has a greater effect on the peak area. As the flow rate decreases, the separation degree between each monosaccharide component is slightly improved, and the peak area will gradually increase. The pH of the buffer solution has a great influence on the separation effect of each monosaccharide component. When the pH is increased to 5.7, the peak time and separation effect do not change significantly, but after the pH drops to 5.3, the retention time of each neutral monosaccharide and acidic monosaccharide component is significantly advanced, but the retention time of the alkaline monosaccharide component does not change significantly, resulting in a decrease in separation, especially for alkaline monosaccharides. Therefore, the lower limit of the pH value of the ammonium acetate buffer solution and the flow rate of the liquid phase should be strictly controlled during the actual detection process.

[0221] The monosaccharide standard working solution was sampled and analyzed at 0h, 18h, 36h, 48h and 72h respectively. The results showed that the relative standard deviations (RSD) of the peak areas of D-glucose, D-galactose, L-rhamnose, D-mannose, D-glucuronic acid, D-galacturonic acid, DN-acetylglucosamine, DN-acetylgalactosamine and DN-acetylmannosamine were 0.79%, 0.21%, 0.98%, 2.13%, 24.12%, 30.33%, 1.05%, 1.13% and 0.60%, respectively, indicating that the stability of the monosaccharide derivatized sample solutions of D-glucuronic acid and D-galacturonic acid was poor, while the stability of the other monosaccharide derivatized sample solutions was good within 72h. Therefore, in the actual detection process, samples containing uronic acid should be detected in time (try to detect within 36h after sample preparation), while samples without uronic acid can be left for a period of time. The results are shown in Table 10 and Attached Fig.12 shown.

[0222] Table 10: Stability test results

[0223]

[0224] (8) Optimization of elution conditions

[0225] The liquid phase detection conditions used in the above-mentioned embodiments have undergone a series of optimizations, especially the composition and proportion of the mobile phase. Two buffers, phosphate and ammonium acetate, were investigated as mobile phase A, respectively. At the same time, different proportions of acetonitrile in the organic phase were investigated, and finally the gradient elution conditions that can successfully separate 10 monosaccharide components were obtained (the concentration gradient of mobile phase B: 20%→20%→30%→20%→20%, and the corresponding concentration gradient of mobile phase A: 80%→80%→70%→80%→80%). Specifically, in terms of elution conditions, the present invention compares the isocratic elution chromatogram when phosphate buffer is used as mobile phase A, the isocratic elution chromatogram when ammonium acetate buffer is used as mobile phase A, and the gradient elution chromatogram when ammonium acetate buffer is used as mobile phase A (the gradient elution conditions are as described above), such as Fig.13 As shown, ammonium acetate buffer can be used in conjunction with mass spectrometry, and gradient elution can significantly shorten the detection time, while achieving good separation and detection effects of different monosaccharides.

[0226] In the examples of the present invention, the PMP pre-column derivatization high performance liquid chromatography method is used to determine the various monosaccharide components and contents of the pneumococcal capsular polysaccharide and the conjugate samples after hydrolysis. Different hydrolysis methods are innovatively used to ensure the maximum recovery rate of each monosaccharide component in view of the different structures of the capsular polysaccharide of different serotypes and the difficulty of hydrolysis. Although the detection process requires derivatization operation, it overcomes the shortcomings of ion chromatography that special instruments and equipment are required and the separation effect is poor. Only a conventional C18 reverse phase chromatographic column and a matching liquid chromatograph are required to complete the detection, and the linearity, precision, accuracy, etc. of the methodological verification meet the requirements. Therefore, the method of the present invention is suitable for the separation and identification of each monosaccharide component in the pneumococcal capsular polysaccharide and the conjugate samples and the detection of the content, and provides a reliable basis for quality control in the vaccine production process.

[0227] The above embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention. The protection scope of the present invention is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present invention within the essence and protection scope of the present invention, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present invention.

Claims

1. A method for detecting the content of each monosaccharide component in a sample of bacterial capsular polysaccharide or a conjugate of bacterial capsular polysaccharide and carrier protein, characterized in that: The method comprises the following steps: 1) Sample hydrolysis: Depending on whether the polysaccharide structure of the sample to be tested contains uronic acid or phosphodiester bonds, the connected glycosidic bonds are broken by a method selected from a) to c) below to release the monosaccharide components: a) Samples without uronic acid or phosphodiester bonds: hydrolyzed with trifluoroacetic acid; b) Samples containing uronic acid: first treated with anhydrous hydrogen chloride-methanol solution, then hydrolyzed with trifluoroacetic acid; c) Samples containing phosphodiester bonds: first treated with hydrofluoric acid, then hydrolyzed with trifluoroacetic acid; 2) Derivatization reaction: The hydrolyzed sample is blown dry, dissolved in water, and then reacted with 1-phenyl-3-methyl-5-pyrazolone (PMP) under alkaline conditions to generate a monosaccharide derivative with ultraviolet absorption. After neutralization with hydrochloric acid, the excess PMP is extracted with an organic solvent to remove it. 3) Liquid chromatography detection: Determine the monosaccharide components of the sample according to the peak time of the standard monosaccharide, quantify according to the external standard method, substitute the peak area of ​​the monosaccharide component into the standard curve of each monosaccharide, and calculate the content of each monosaccharide component.

2. The detection method according to claim 1, characterized in that The sample hydrolysis method a) in step 1) specifically includes the following operations: blow-dry the sample, add 2-4 mol / L trifluoroacetic acid, and hydrolyze at 100-120° C. for 2-4 hours.

3. The detection method according to claim 1, characterized in that The sample hydrolysis method b) in step 1) specifically includes the following operations: blow dry the sample, add 2-4 mol / L anhydrous hydrogen chloride-methanol solution, water bath at 70-90°C for 12-24h, then blow dry, add 2-4 mol / L trifluoroacetic acid, and continue hydrolysis at 100-120°C for 2-4h.

4. The detection method according to claim 1, characterized in that The sample hydrolysis method c) in step 1) specifically includes the following operations: blow dry the sample, add 40-50% (v / v) hydrofluoric acid solution, and place in a water bath at 60-80°C for 2-4 hours, then blow dry, add 2-4 mol / L trifluoroacetic acid, and continue hydrolysis at 100-120°C for 2-4 hours.

5. The detection method according to any one of claims 1 to 4, characterized in that: The derivatization reaction in step 2) adopts the following conditions: PMP concentration 0.4-0.6 mol / L, sodium hydroxide concentration 0.3-0.5 mol / L, reaction temperature 60-80°C, reaction time 30-90 min, and the concentration of hydrochloric acid and sodium hydroxide are kept consistent.

6. The detection method according to any one of claims 1 to 4, characterized in that: The organic solvent used for extraction in step 2) can be selected from one or more of the following: chloroform, toluene, isoamyl acetate, and n-hexane.

7. The detection method according to any one of claims 1 to 4, characterized in that: In step 3), the liquid chromatography analysis adopts the following conditions: using a DAD diode array detector or a UV detector, a detection wavelength of 240-250 nm, an injection volume of 10-50 μL, a flow rate of 0.8-1.2 mL / min, mobile phase A: 10-100 mM ammonium acetate buffer, pH 5.5-6.0, mobile phase B: acetonitrile solution, and gradient solution elution.

8. The detection method according to claim 7, characterized in that The gradient solution elution in step 3) is as follows: with the elution time 0→25min→50min→51min→60min, the concentration gradient of mobile phase B is: 20%→20%→30%→20%→20%, and the corresponding concentration gradient of mobile phase A is: 80%→80%→70%→80%→80%.

9. The detection method according to any one of claims 1 to 4, characterized in that: The calculation formula of monosaccharide content in step 3) is as follows: c i =(yb) / a×D, where: c i : The content of a characteristic monosaccharide component in the sample to be tested, in μg / mL; y: Liquid phase detection peak area of ​​a characteristic monosaccharide component of the sample to be tested, unit: mAU*s; a: The slope of the standard curve established based on the different concentrations and peak areas of the characteristic monosaccharide standards; b: The intercept of the standard curve established based on different concentrations and peak areas of characteristic monosaccharide standards; D: Dilution factor, that is, the multiple by which the sample solution to be tested is diluted.

10. The detection method according to any one of claims 1 to 4, characterized in that: The monosaccharide is selected from one or more of the following: D-glucose, D-galactose, L-rhamnose, D-mannose, L-fucose, D-glucuronic acid, D-galacturonic acid, DN-acetylglucosamine, DN-acetylgalactosamine, DN-acetylmannosamine, and LN-acetylfucosamine.

11. The detection method according to any one of claims 1 to 4, characterized in that: For freeze-dried samples, before step 1), add water to dissolve the sample and dilute it to 50-500 μg / mL.

12. The detection method according to any one of claims 1 to 4, characterized in that: The bacteria are selected from Gram-positive bacteria.

13. The detection method according to claim 12, characterized in that: The Gram-positive bacteria are selected from pneumococci.

14. The detection method according to claim 13, characterized in that: The sample containing neither uronic acid nor phosphodiester bonds is selected from one or more of the following serotype pneumococcal capsular polysaccharides or their combinations: serotypes 4, 7F, 12F, 14, 17F, and 33F.

15. The detection method according to claim 13, characterized in that: The sample containing uronic acid is selected from one or more of the following serotype pneumococcal capsular polysaccharides or their combinations: serotypes 1, 2, 3, 5, 8, 9N, 9V, and 22F.

16. The detection method according to claim 13, characterized in that: The sample containing phosphodiester bonds is selected from one or more of the following serotype pneumococcal capsular polysaccharides or their combinations: 6A, 6B, 10A, 11A, 15B, 18C, 19A, 19F, 20, and 23F serotypes.

17. A method for detecting the polysaccharide content in bacterial capsular polysaccharides or a combination of bacterial capsular polysaccharides and carrier proteins, characterized in that: The steps include: S1) According to the chemical structure formula of the capsular polysaccharide in the sample to be tested, a characteristic monosaccharide is selected and its content percentage in the polysaccharide is determined, and the percentage of the monosaccharide content is expressed as "w i "express; S2) detecting the component content of the characteristic monosaccharide in the sample to be tested according to the method described in any one of claims 1 to 16, wherein "c i "express; S3) Based on the measured content of characteristic monosaccharide components, the content of capsular polysaccharide of the corresponding serotype is calculated using the formula: i / w i .

18. The detection method according to claim 17, characterized in that: The bacteria are selected from Gram-positive bacteria.

19. The detection method according to claim 18, characterized in that: The Gram-positive bacteria are selected from pneumococci.

20. The detection method according to claim 19, characterized in that Based on the specific serotype of pneumococcus, the step S1) adopts one or more embodiments selected from the following a) to w): a) Serotype 2: L-rhamnose is selected as the characteristic monosaccharide, and its mass percentage in the capsular polysaccharide of this serotype is 47.0%; b) Serotype 3: D-glucuronic acid was selected as the characteristic monosaccharide, which accounted for 53.9% of the mass percentage of the capsular polysaccharide of this serotype; c) Serotype 4: D-galactose was selected as the characteristic monosaccharide, which accounted for 21.2% by mass in the capsular polysaccharide of this serotype; d) Serotype 5: D-glucuronic acid was selected as the characteristic monosaccharide, and its mass percentage in the capsular polysaccharide of this serotype was 21.1%; e) Serotype 6A: L-rhamnose was selected as the characteristic monosaccharide, which accounted for 23.2% by mass in the capsular polysaccharide of this serotype; f) Serotype 6B: L-rhamnose was selected as the characteristic monosaccharide, which accounted for 23.2% by mass in the capsular polysaccharide of this serotype; g) Serotype 7F: D-galactose was selected as the characteristic monosaccharide, which accounted for 29.4% by mass in the capsular polysaccharide of this serotype; h) Serotype 8: D-glucuronic acid was selected as the characteristic monosaccharide, which accounted for 26.4% by mass in the capsular polysaccharide of this serotype; i) Serotype 9N: D-galactose was selected as the characteristic monosaccharide, which accounted for 19.5% by mass in the capsular polysaccharide of this serotype; j) Serotype 9V: D-galactose was selected as the characteristic monosaccharide, which accounted for 19.5% by mass in the capsular polysaccharide of this serotype; k) Serotype 10A: D-galactose was selected as the characteristic monosaccharide, which accounts for 43.7% by mass in the capsular polysaccharide of this serotype; l) Serotype 11A: D-galactose is selected as the characteristic monosaccharide, which accounts for 36.2% by mass in the capsular polysaccharide of this serotype; m) Serotype 12F: D-galactose is selected as the characteristic monosaccharide, and its mass percentage in the capsular polysaccharide of this serotype is 15.0%; n) Serotype 14: D-galactose was selected as the characteristic monosaccharide, which accounts for 52.2% by mass in the capsular polysaccharide of this serotype; o) Serotype 15B: D-galactose was selected as the characteristic monosaccharide, which accounts for 46.1% by mass of the capsular polysaccharide of this serotype; p) Serotype 17F: L-rhamnose was selected as the characteristic monosaccharide, which accounted for 39.2% by mass in the capsular polysaccharide of this serotype; q) Serotype 18C: L-rhamnose was selected as the characteristic monosaccharide, which accounted for 16.2% by mass in the capsular polysaccharide of this serotype; r) Serotype 19A: L-rhamnose was selected as the characteristic monosaccharide, which accounts for 26.7% by mass in the capsular polysaccharide of this serotype; s) Serotype 19F: L-rhamnose was selected as the characteristic monosaccharide, which accounted for 26.7% by mass in the capsular polysaccharide of this serotype; t) Serotype 20: D-galactose is selected as the characteristic monosaccharide, which accounts for 27.6% by mass in the capsular polysaccharide of this serotype; u) Serotype 22F: L-rhamnose was selected as the characteristic monosaccharide, which accounted for 29.7% by mass in the capsular polysaccharide of this serotype; v) Serotype 23F: L-rhamnose was selected as the characteristic monosaccharide, which accounted for 41.4% by mass in the capsular polysaccharide of this serotype; w) Serotype 33F: D-glucose was selected as the characteristic monosaccharide, which accounted for 16.0% by mass in the capsular polysaccharide of this serotype.

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