Method for detecting polysaccharide molecule size of multivalent bacterial vaccine

Abstract

The invention provides a method for detecting the size of polysaccharide molecules of a multivalent bacterial vaccine, which is characterized by comprising the following steps in sequence: 1) providing a mixed solution of bacterial polysaccharides containing various serotypes; 2) performing a gel filtration method on the mixed solution to obtain a continuously collected multi-tube eluent; and 3) carrying out rate turbidimetry on the multi-tube eluent to obtain the amount of each type of bacterial polysaccharide in each tube, and respectively obtaining the KD value of each type of bacterial polysaccharide according to the amount of each type of bacterial polysaccharide. According to the method, the detection efficiency of the molecular size of the bacterial polysaccharide is greatly improved, the operation is simple, sensitive and accurate, the automation degree is high, and a guarantee is provided for research, development, production and quality control of bacterial vaccines.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for detecting the size of polysaccharide molecules of a multivalent bacterial vaccine. Background Art

[0002] Molecular size determination is an important quality control indicator to ensure the effectiveness and stability of bacterial polysaccharides and conjugate vaccines, and is closely related to the immunogenicity of vaccines. The traditional method for determining the molecular size of polysaccharides is to first elute the target polysaccharide by gel filtration, and then use chemical methods to analyze the content of specific chemical groups in the sample eluate.

[0003] Size Exclusion Chromatography (SEC), also known as size exclusion chromatography, gel exclusion chromatography, molecular sieve chromatography, etc., is a liquid chromatography technique that separates components based on their molecular size. Its separation principle is the molecular sieve mechanism of the gel chromatography column. Taking pneumococcal polysaccharide (PnPs) as an example, PnPs is the main virulence factor of pneumococcus and has serotype specificity. PnPs has a relatively large relative molecular mass, generally ranging from tens of thousands to millions. It is composed of 10000 polymerized sugar repeating units, which can produce protective effects in the body and is widely used in vaccine development and production. PnPs are TI-2 antigens, and their immunogenicity is closely related to molecular size. Molecular size determination is one of the important quality control items of PnPs. The "Chinese Pharmacopoeia" and "European Pharmacopoeia" have been included in the provisions of the use of molecular exclusion chromatography to determine the molecular size of various types of PnPs, and stipulate the chromatographic column model and K of various types of PnPs. D Detection limit. After PnPs enter the chromatographic column, each component enters the corresponding pore according to its molecular size. Molecules larger than all pore sizes cannot enter the interior of the filler particles and are not retained during the chromatography process. They are eluted to the outside of the column by the mobile phase first, which is manifested as a short retention time. Molecules smaller than all pore sizes can freely enter all pore sizes on the surface of the filler and stay in the chromatographic column for a longer time, which is manifested as a long retention time. The remaining molecules are eluted in sequence according to their molecular size. After the components are separated in the chromatographic column, they enter the UV-visible spectrophotometer for detection. The data processing system records and processes the chromatographic signals, and calculates K based on the UV absorption peak at 206nm. D value.

[0004] In daily scientific research and production, the ultraviolet absorption peaks of polysaccharide samples of different serotypes or different batches may be chaotic peaks or the peak tops are not obvious. Generally, more than two chemical methods are used to analyze the content of specific chemical groups in the sample eluent. However, the chemical method has many disadvantages. First, concentrated sulfuric acid must be used in the chemical method and the amount is quite large. Concentrated sulfuric acid is highly corrosive and irritating. It not only has a strong irritating and corrosive effect on tissues such as skin and mucous membranes, but also causes pollution to water bodies and soil; secondly, different types require different detection methods. The standards, spectrophotometer detection wavelengths and calculation methods used during the period are different. Ice water baths and boiling water baths need to be used repeatedly in the experiment, which is time-consuming and labor-intensive; finally, the chemical method is limited by the detection sensitivity, requiring a relatively large amount of SEC loading (several times or even more than ten times that of the rate turbidimetry method), and the pressure on the gel column also increases accordingly, which is bound to reduce the separation effect and service life of the gel column. Summary of the invention

[0005] In order to solve the problems existing in the above-mentioned prior art, the present invention provides a new method for detecting the size of bacterial polysaccharides of bacterial vaccines.

[0006] Specifically, the present invention provides:

[0007] (1) A method for detecting the size of polysaccharide molecules in a multivalent bacterial vaccine, characterized in that it comprises the following steps performed in sequence:

[0008] 1) providing a mixed solution containing bacterial polysaccharides of multiple serotypes;

[0009] 2) performing gel filtration on the mixed solution to obtain multiple tubes of eluate that are continuously collected;

[0010] 3) Performing rate turbidimetry on the eluates from the multiple tubes to obtain the amount of each type of bacterial polysaccharide in each tube, and obtaining the K of each type of bacterial polysaccharide from the amount of each type of bacterial polysaccharide. D value.

[0011] (2) The method according to (1), wherein the concentration of each type of bacterial polysaccharide in the solution of step 1) is 400-2000 μg / mL.

[0012] (3) The method according to (1), wherein step 2) comprises adding the solution of step 1) to a gel filtration separation column, eluting the various types of bacterial polysaccharides with an eluent and collecting all the eluents in equal parts; and

[0013] Step 3) includes detecting the amount of each type of bacterial polysaccharide in each aliquot of the collected eluate by rate turbidimetry, wherein specific antibodies for each type of bacterial polysaccharide are contacted with each aliquot of the eluate to react with the corresponding bacterial polysaccharide to form each type of antigen-antibody complex, detecting the turbidity value of each type of antigen-antibody complex, and obtaining the amount of each type of bacterial polysaccharide from the turbidity value.

[0014] (4) The method according to (1), wherein step 3) further comprises performing rate turbidimetry detection using a series of concentrations of reference solutions containing each type of bacterial polysaccharide and specific antibodies for each type of bacterial polysaccharide to obtain a standard curve.

[0015] (5) The method according to (4), wherein the reference solution contains the bacterial polysaccharide of multiple serotypes.

[0016] (6) The method according to (1), wherein the bacterial polysaccharide is selected from pneumococcal polysaccharide, meningococcal polysaccharide, typhoid Vi polysaccharide and bacterial polysaccharide intermediates.

[0017] (7) The method according to (6), wherein the pneumococcal polysaccharide includes types 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F pneumococcal polysaccharides; the meningococcal polysaccharides include group A, C, Y, and W135 meningococcal polysaccharides; and the bacterial polysaccharide intermediates include bacterial polysaccharide degradation products, derivatives, esterification products, and conjugates.

[0018] (8) The method according to (1), wherein the gel filtration method is size exclusion chromatography.

[0019] (9) The method according to (1), wherein the rate turbidimetry is performed using a rate turbidimeter.

[0020] Compared with the prior art, the present invention has the following advantages and positive effects:

[0021] The present invention breaks the long-standing inertial thinking in this field, and innovatively proposes to combine the SEC method with the rate turbidimetry method, using specific antibodies for polysaccharides, and relying on an automated immunoassay system to directly quantify the polysaccharide content at each elution volume without interference, which greatly improves the detection efficiency of the size of bacterial polysaccharide molecules. It is simple to operate, sensitive and accurate, and has a high degree of automation, providing a guarantee for the development, production and quality control of bacterial vaccines. Specifically, the method of the present invention has the following advantages:

[0022] 1. The detection efficiency is higher. By cleverly combining the rate turbidimetry and the specific antibody of polysaccharide, the present invention can detect bacterial polysaccharides of multiple serotypes or different types of bacterial polysaccharides at the same time, and can also detect a mixture of bacterial polysaccharides of multiple serotypes, or even a mixture of bacterial polysaccharides of different types (such as a mixture of various types of pneumococcal polysaccharides and various types of meningococcal polysaccharides), while the traditional method can only detect a sample of one serotype of a bacterial polysaccharide at a time. The present invention is several times or even dozens of times more efficient than the traditional method. Taking pneumococcal polysaccharide as an example, when the traditional method is used to detect polysaccharides of 13 serotypes, the respective loading time of all samples, the time for balancing the washing gel column, and the time for detecting each sample by chemical method are required, and the sum of these times takes about 15 days. In contrast, since the rate turbidimetry used by the present invention can detect a mixture of different bacterial polysaccharides, the SEC method is mixed and loaded, and the detection is completed in only 1 day, which greatly improves the detection efficiency.

[0023] 2. Faster detection speed: For detecting a single type of bacterial polysaccharide, the present invention can detect the same number of serotype samples within 20 minutes as the traditional method would take at least 2 hours to complete.

[0024] 3. Stronger specificity. In the rate turbidimetry method, highly specific, non-cross-reactive antibodies (immune serum) are used to quantify the polysaccharide content at each elution volume. They only react specifically with the same type of polysaccharide antigens and do not cross-react with other types of polysaccharide antigens or impurities. Their specificity is much higher than that of traditional methods / chemical methods. Conventional methods must use different detection methods to quantify polysaccharides due to interference from differences between serotypes of bacteria (such as pneumococci). For example, pneumococcal polysaccharide type 4 uses the lichen phenol method, and the remaining types use the anthrone sulfuric acid method.

[0025] 4. Higher degree of automation. The eluate continuously collected by the gel column system does not need to be added with any reagents or processed in any way, and is directly tested by the rate turbidimeter. The instrument program setting allows large batches of samples to be tested simultaneously. The traditional method relies on manual sample addition, sample boiling, and value reading, which is time-consuming, labor-intensive, and prone to errors.

[0026] 5. Less burden on the gel column. The present invention adopts a highly specific immunochemical method, and the sample loading amount is much lower than the traditional method, which greatly reduces the burden on the gel column. In order to increase the degree of color development, the traditional method requires a higher sample loading concentration, generally requiring 2 to 5 mg.

[0027] 6. More environmentally friendly. The buffer and washing solution used in the present invention are phosphate buffer, which is harmless and pollution-free. The concentrated sulfuric acid used in the traditional method not only damages the environment, but also harms human health.

[0028] 7. Broader spectrum. The present invention is not only applicable to pneumococcal polysaccharides, but also to meningococcal polysaccharides, typhoid Vi polysaccharides and other bacterial capsular polysaccharides. In addition, the present invention is not only applicable to polysaccharides, but also to intermediates of vaccine research and development such as degradation products, derivatives, esterification products and conjugates. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG1 is an elution curve obtained by detecting various types of pneumococcal polysaccharides using the method of the present invention in Example 1 and Experimental Example 1; Figure 1A is the elution curve of type 1 polysaccharide, Figure 1B is the elution curve of type 3 polysaccharide. Figure 1C is the elution curve of type 4 polysaccharide. Figure 1D is the elution curve of type 5 polysaccharide. Figure 1E is the elution curve of type 6A polysaccharide. Figure 1F is the elution curve of type 6B polysaccharide. Figure 1G is the elution curve of 7F type polysaccharide, Figure 1H This is the elution curve of 9V type polysaccharide. Fig. 1I is the elution curve of type 14 polysaccharide. Figure 1J is the elution curve of 18C polysaccharide. Figure 1K is the elution curve of 19A type polysaccharide, Figure 1L is the elution curve of 19F polysaccharide. Figure 1M This is the elution curve of 23F polysaccharide.

[0030] FIG. 2 is a result of repeatability verification of the method of the present invention in Experimental Example 1; Figure 2A is the elution curve of type 1 polysaccharide, Figure 2B is the elution curve of type 3 polysaccharide. Figure 2C is the elution curve of type 4 polysaccharide. Figure 2D is the elution curve of type 5 polysaccharide. Figure 2E is the elution curve of type 6A polysaccharide. Figure 2F is the elution curve of type 6B polysaccharide. Figure 2G is the elution curve of 7F type polysaccharide, Figure 2H This is the elution curve of 9V type polysaccharide. Fig.2I is the elution curve of type 14 polysaccharide. Figure 2J is the elution curve of 18C polysaccharide. Figure 2K is the elution curve of 19A type polysaccharide, Figure 2L is the elution curve of 19F polysaccharide. Figure 2M This is the elution curve of 23F polysaccharide.

[0031] FIG3 is an elution curve obtained by detecting various groups of meningococcal polysaccharides using the method of the present invention in Example 2; wherein Figure 3A is the elution curve of group A polysaccharide. Figure 3B C

[0032] Elution curve of polysaccharide Figure 3C is the elution curve of W135 group polysaccharide, Figure 3D This is the elution curve of group Y polysaccharide.

[0033] FIG4 is a result of repeatability verification of the method of the present invention in Example 2; wherein Figure 4A is the elution curve of group A polysaccharide. Figure 4B is the elution curve of group C polysaccharide. Figure 4C is the elution curve of W135 group polysaccharide, Figure 4D This is the elution curve of group Y polysaccharide.

[0034] Figure 5 This is the repeatability verification result of the method of the present invention in Example 3.

[0035] FIG6 is a result of repeatability verification of the method of the present invention in Example 4; wherein Fig. 6A is the elution curve of 18C pneumococcal polysaccharide degradation product. Figure 6B is the elution curve of 18C pneumococcal polysaccharide derivative, Figure 6C is the elution curve of 18C pneumococcal polysaccharide ester. Fig.6D This is the elution curve of 18C pneumococcal polysaccharide conjugate. DETAILED DESCRIPTION

[0036] The present invention will be further illustrated below through the description of specific implementation methods and with reference to the accompanying drawings, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not deviate from the basic idea of ​​the present invention, they are all within the scope of the present invention.

[0037] The present invention improves upon conventional methods and innovatively combines gel filtration with rate turbidimetry to accurately obtain the molecular size of bacterial polysaccharides with simple operation and higher efficiency.

[0038] Therefore, the present invention provides a method for detecting the size of polysaccharide molecules of bacterial vaccines, characterized in that it comprises the following steps performed in sequence:

[0039] 1) providing a solution containing at least one serotype of bacterial polysaccharide;

[0040] 2) separating various types of bacterial polysaccharides in the solution by gel filtration;

[0041] 3) Detect the amount of each type of bacterial polysaccharide by rate turbidimetry, and obtain the K of each type of bacterial polysaccharide from the amount of each type of bacterial polysaccharide. D value.

[0042] Since the present invention skillfully combines the gel filtration method with the rate turbidimetry, the present invention can detect the content of each serotype polysaccharide in a mixture of bacterial polysaccharides of multiple serotypes without cross-reaction, thereby obtaining the K of each polysaccharide. D value. In this way, there is no need to operate each serotype polysaccharide separately, which greatly improves the detection efficiency for the detection of bacterial polysaccharides with multiple serotypes (for example, pneumococcal polysaccharides have 13 serotypes, and the conventional method needs to be operated 13 times respectively, and each operation is complicated and time-consuming). For example, when detecting 13 serotypes of polysaccharides by traditional methods, the time for loading all samples, the time for balancing and washing the gel column, and the time for detecting each sample by chemical methods are required, and the sum of these times takes about 15 days. In contrast, since the rate turbidimetry used in the present invention can detect mixtures of different bacterial polysaccharides, the SEC method is mixed and loaded, and the detection is completed in only 1 day, which greatly improves the detection efficiency.

[0043] Therefore, in some embodiments, the bacterial polysaccharide solution in step 1) is a mixed solution containing bacterial polysaccharides of multiple serotypes.

[0044] Preferably, in the solution of step 1), the concentration of each type of bacterial polysaccharide is 400-2000 μg / mL.

[0045] In some specific embodiments, step 2) includes adding the solution of step 1) to a gel filtration separation column, eluting the various types of bacterial polysaccharides with an eluent and collecting all the eluents in equal parts.

[0046] As used herein, the term "equilibrium collection" refers to collecting the eluate into multiple containers, each container collecting the same volume of eluate, until substantially all of the eluate is collected. For example, as will be understood and routinely performed by those skilled in the art, the eluate is collected using multiple centrifuge tubes, each centrifuge tube having the same volume of eluate, until substantially all of the eluate is collected.

[0047] The eluent used in the present invention includes various eluents commonly used in the art for separating bacterial polysaccharides by gel filtration.

[0048] Taking pneumococcal polysaccharide as an example, the basic principle of using rate scattering turbidimetry for detection is to allow pneumococcal polysaccharide to react specifically with the corresponding type of antibody or antiserum in a buffer system at 37°C to form an antigen-antibody complex. Under the condition of excess antibody, the linear relationship between the turbidity value (instrument response value) of the complex and the polysaccharide concentration is used to make a standard curve, thereby detecting the corresponding polysaccharide concentration in the test sample.

[0049] Therefore, in some specific embodiments, step 3) includes detecting the amount of each type of bacterial polysaccharide in each aliquot of the collected eluate by rate turbidimetry, wherein specific antibodies for each type of bacterial polysaccharide are contacted with each aliquot of the eluate to react with the corresponding bacterial polysaccharide to form each type of antigen-antibody complex, and the turbidity value of each type of antigen-antibody complex is detected, and the amount of each type of bacterial polysaccharide is obtained from the turbidity value.

[0050] The turbidity value can be detected by various known methods.

[0051] In some specific embodiments, step 3) further comprises performing rate turbidimetry detection using a reference solution containing each type of bacterial polysaccharide of a series of concentrations and a specific antibody for each type of bacterial polysaccharide to obtain a standard curve, wherein the reference solution may contain bacterial polysaccharides of multiple serotypes at the same time.

[0052] The inventors of the present invention skillfully combined the gel filtration method with the rate turbidimetry, making the method of the present invention easy to operate, more efficient, more specific, and universal. In the rate turbidimetry, highly specific antibodies (or immune serum) are used for polysaccharide quantification, and the antibodies (or immune serum) only react specifically with the same type of polysaccharide antigens and do not cross-react with other types of polysaccharide antigens or impurities. Therefore, the method of the present invention can detect various types of bacterial polysaccharides in a mixture of bacterial polysaccharides of multiple serotypes, and has universal applicability, and can be used to detect the size of various bacterial polysaccharides, without the need to use different detection methods for different polysaccharides or different types of polysaccharides as in chemical methods.

[0053] The bacterial polysaccharide is selected from the group consisting of pneumococcal polysaccharide, meningococcal polysaccharide, typhoid Vi polysaccharide and bacterial polysaccharide intermediates.

[0054] The pneumococcal polysaccharide includes but is not limited to 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F type pneumococcal polysaccharide. The method of the present invention is not only applicable to the 13 serotypes of pneumococcal polysaccharides, but also to other pneumococcal polysaccharides. At present, 92 serotypes of pneumococcal polysaccharides have been found. If specific antiserum is available, all pneumococcal polysaccharides are suitable for determining the molecular size using the method of the present invention.

[0055] The meningococcal polysaccharides include group A, C, Y, and W135 meningococcal polysaccharides; the bacterial polysaccharide intermediates include degradation products, derivatives, esterification products, and conjugates of various types of pneumococcal polysaccharides and other bacterial polysaccharides, such as degradation products, derivatives, esterification products, and conjugates of type 18C pneumococcal polysaccharides.

[0056] The polysaccharide degradation product is a polysaccharide fragment with a molecular weight reduced to 50,000 to 150,000 Da by physical or chemical methods. The polysaccharide derivative is a polysaccharide or polysaccharide fragment obtained by modifying the bacterial capsular polysaccharide or the polysaccharide degradation product with a chemical reagent (such as dibutyl amine (DAB)) in order to increase the site for covalent binding with a carrier protein (such as tetanus toxoid (TT)) in the next step. The esterified product is a polysaccharide or polysaccharide fragment after the polysaccharide derivative is activated by a chemical reagent (such as disuccinimidyl adipic acid (SIDEAA)), which is an intermediate product before covalent binding with a carrier protein. The conjugate is a product obtained by covalently binding the chemically modified polysaccharide derivative with a carrier protein and then purifying it.

[0057] In some embodiments, the gel filtration method is a molecular exclusion chromatography method. The rate turbidimetry method is performed using a rate turbidimeter.

[0058] The present invention is further explained or illustrated by way of examples below, but these examples should not be construed as limiting the scope of protection of the present invention.

[0059] Example

[0060] Unless otherwise specified, the experimental methods used in the following examples were all performed using conventional experimental procedures, operations, materials and conditions in the art.

[0061] The sources of materials, reagents and instruments used in the examples are as follows:

[0062] 1. Pneumococcal polysaccharides: Types 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F were purchased from the American Type Culture Collection. The types are shown in Table 1.

[0063] 2. Pneumococcal immune sera: All types of sera were purchased from the Danish Serum Institute (SSI). The types and dilution multiples are shown in Table 1.

[0064]

[0065] 3.AKTA Purifier liquid chromatography system, XK16 / 100 chromatography column, Sepharose CL-4B and Sepharose CL-2B gel fillers, and blue dextran 2000 were all purchased from GE Company, USA.

[0066] 4. Waters 2414 differential refractometer was purchased from Waters Company, USA.

[0067] 5. Sodium chloride was purchased from Sinopharm Chemical Co., Ltd.; Vitamin B 12 Purchased from Sigma, USA.

[0068] 6-rate turbidimeter IMMAGE 800 and the matching UDR kit, sample rack, reaction cup, buffer, diluent, and washing solution were purchased from Beckman Coulter, USA.

[0069] Example 1: Detection of the molecular size of pneumococcal polysaccharide

[0070] 1. Solution Preparation

[0071] 1) Reference material: Prepare various types of PnPs with 0.85% sodium chloride solution to prepare a 13-valent mixed solution with a final concentration of 100.0 μg / mL.

[0072] 2) Serum: Dilute each type of immune serum with 0.85% sodium chloride solution according to the dilution multiple shown in Table 1 to the working concentration for later use.

[0073] 3) Monovalent solutions of various types: Various types of PnPs were prepared into monovalent solutions with a final concentration of 5 mg / mL using 0.85% sodium chloride solution.

[0074] 4) Sample A: PnPs of types 1, 3, 4, 6A, 7F, 14, 18C, 19A, 19F, and 23F were prepared into mixed solutions with a final mass concentration of 1 mg / ml for each type using 0.85% sodium chloride solution.

[0075] 5) Sample B: PnPs of types 5, 6B and 9V were prepared into mixed solutions with a final mass concentration of 1 mg / ml for each type using 0.85% sodium chloride solution.

[0076] 2. Method Development

[0077] 1) Determination of V by SEC o and V i (Sepharose CL-4B gel column system and Sepharose CL-2B gel column system were measured separately): Use 0.85% NaCl solution as the mobile phase of the system, with a flow rate of 0.8 mL / min. Take 0.7 mL of 2 mg / mL blue dextran 2000 solution and 1 mg / mL vitamin B 12 The solution was 0.3 mL, and the two were mixed and injected. The absorption signal at 206 nm was collected to obtain the V o and V i . V o is the empty flow volume (ml), V i is the column bed volume (ml).

[0078] 2) SEC injection: Sample A uses Sepharose CL-4B gel column system, and sample B uses Sepharose CL-2B gel column system. Take 1 ml of each and inject at room temperature. Collect the eluent 5 ml / tube continuously. The eluent and elution conditions are the same as 1).

[0079] 3) Determination of sugar content by rate turbidimetry: Add 1 mL of diluted reference solution (concentrations are 2.0, 4.0, 8.0, 16.0, and 20.0 μg / mL) to the sample rack of the rate turbidimeter, add working concentrations of various types of immune serum to the UDR kit, sample and test with a rate turbidimeter, obtain a standard curve, and the R value should be greater than 0.985. Then insert 5 mL / tube of the eluate collected in 2) in sequence into the sample rack of the rate turbidimeter for testing to obtain the content of each type of polysaccharide in each tube (see Figure 1 for the results).

[0080] Calculate K D Value: K D =(V e -V o ) / (V i -V o ), where K D V is the distribution coefficient of the test product; e V is the total volume of the eluent at the top of the main peak of the test sample (ml); o V is the empty flow volume (ml); i is the column volume (ml). K of pneumococcal polysaccharide types 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F D The values ​​are 0.07, 0.07, 0.11, 0.58, 0.28, 0.49, 0.15, 0.44, 0.07, 0.07, 0.41, 0.20, and 0.07 respectively.

[0081] Draw the elution curve: Use the sugar content of the eluate in each tube of the test sample as the ordinate and the tube number corresponding to the eluate 5 mL / tube as the abscissa to draw the elution curve of the sample (Figure 1).

[0082] Experimental Example 1: Verification of the Applicability of the Method

[0083] 1. Specificity verification: Take 200 μl of each type of monovalent solution, 1 mL of sample A, and 1 mL of sample B, respectively, and conduct the test according to the method of Example 1, draw the elution curve of each type of monovalent solution and the elution curve of multivalent mixture, and observe whether the mixed PnPs will interfere with the detection of polysaccharide molecular size.

[0084] 2. Repeatability verification: Take 1 mL of sample A and 1 mL of sample B respectively, and conduct the test according to the method of Example 1. Repeat the test for each sample 3 times.

[0085] The results are as follows: SepharoseCL-4B gel column V o =66.76mL, V i =185.62mL; SepharoseCL-2B gel column V o =66.90mL, V i =175.022mL.

[0086] 1. The specificity verification results show that the content of each type of PnPs shows a trend of gradually decreasing with the extension of elution time, which is consistent with the content distribution characteristics of polymers, and the monovalent elution curve and the multivalent mixed elution curve basically overlap, proving that the mixed PnPs have no interference with the detection of polysaccharide molecular size, indicating that the method has good specificity, see Figure 1.

[0087] 2. The results of repeatability verification showed that the content distribution trend lines of each type of PnPs measured three times basically overlapped, indicating that the method has good repeatability, see Figure 2.

[0088] Comparative Example 1: Comparison with chemical method

[0089] The molecular sizes of samples A and B were measured using the established method, and the results were compared with those of the SEC-chemical method (hereinafter referred to as the conventional method).

[0090] 1. Anthrone sulfuric acid method (see the Chinese Pharmacopoeia Committee. Pharmacopoeia of the People's Republic of China (Volume 3) [S]. Beijing: China Medical Science and Technology Press, 2020)

[0091] 1) Applicable types: 1, 3, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F pneumococcal polysaccharides.

[0092] 2) Principle: The original solution of the conjugate can undergo a dehydration reaction under the action of concentrated sulfuric acid to generate furfural or hydroxymethylfurfural. The generated furfural or hydroxymethylfurfural can react with anthrone to generate blue-green furfural derivatives. Within a certain range, the depth of color is proportional to the sugar content and can be used for the quantification of polysaccharides.

[0093] 3) Operation steps

[0094] Dilute the anhydrous glucose standard with 0.85% to 0.90% sodium chloride solution to prepare a 0 to 100 μg / ml glucose standard solution. Add 225 ml of sulfuric acid to 75 ml of 0.85% to 0.90% sodium chloride solution, weigh 0.3 g of anthraquinone and add it to 10 ml of ethanol, mix the above solutions to prepare anthraquinone mixed solution. Accurately measure 1.0 ml of glucose standard solutions of different concentrations and monovalent sugar type test solutions, add 4.0 ml of anthraquinone mixed solution, mix well, place in a boiling water bath for 20 minutes and then in a 40°C water bath for 10 minutes, measure the absorbance at a wavelength of 620 nm, and calculate the polysaccharide content by linear regression method based on the absorbance corresponding to the concentration of the glucose standard solution.

[0095] The total process of the anthrone sulfuric acid method takes about 3 hours.

[0096] 2. Lichenol method

[0097] 1) Applicable type: Type 4 pneumococcal polysaccharide.

[0098] 2) Principle: In sulfuric acid solution, pneumococcal polysaccharides are hydrolyzed into monosaccharides, which are further oxidized to produce hydroxymethylfurfural, which reacts with lichenol to produce a brown-yellow color-developing group with a maximum absorbance at 425nm. Within a certain range, the depth of color is proportional to the content of polysaccharides and can be used for the quantification of polysaccharides.

[0099] 3) Operation steps

[0100] Accurately measure 0mL, 0.10mL, 0.25mL, 0.50mL, 0.75mL, and 1.0mL of the orcinol reference solution into glass stopper test tubes, add water to make up to 1ml; accurately measure 1.0mL of the diluted monovalent sugar type test sample into a test tube. Mix 1.6% orcinol solution and sulfuric acid solution in a volume ratio of 1:7, add 5.0mL to each tube in an ice water bath, boil in water for 45min, and cool in ice water for 10min. Measure the absorbance at a wavelength of 425nm. Perform a linear regression on the corresponding absorbance values ​​of the orcinol reference solution series concentration (μg / ml) to obtain the linear regression equation. Substitute the absorbance value of the test solution into the linear regression equation, and calculate the orcinol content of the test sample according to the dilution multiple.

[0101] The total process of the orcinol method takes about 5 hours.

[0102] The samples were tested by the method described in Example 1 of the present invention and the conventional method, and the K values ​​of pneumococcal polysaccharides of types 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F were measured. DThe values ​​are 0.07, 0.07, 0.11, 0.58, 0.28, 0.49, 0.15, 0.44, 0.07, 0.07, 0.41, 0.20, and 0.07, respectively, and the results of the two methods are consistent.

[0103] The results show that the method of the present invention can obtain results comparable to those of conventional methods when detecting the molecular size of bacterial polysaccharides, and can therefore replace conventional methods to detect the molecular size of bacterial polysaccharides in a simpler, more efficient and environmentally friendly manner.

[0104] Example 2: Detection of the molecular size of meningococcal polysaccharides

[0105] Reference substance is 100 μ g / mL meningococcal A, C, Y, W135 group polysaccharide mixed solution (all types of polysaccharide are purchased from the National Institute of Biological Products Control, UK, and the models are 13 / 246, 08 / 214, 16 / 206, 16 / 152 respectively); serum is specific meningococcal A, C, Y and W135 group antiserum (purchased from BD, the models are 222281, 222301, 228811, 222531 respectively, and the dilution multiples are 8, 16, 16, 16 times respectively); test sample is meningococcal A, C, Y, W135 group polysaccharide mixed solution (all types of final concentrations are 1 mg / ml). Instrument parameter settings and operating methods are the same as in Example 1, and Sepharose CL-4B gel column system is adopted for loading.

[0106] The method was validated for suitability. The final concentration of each type of monovalent solution was 5 mg / mL.

[0107] The specificity verification results showed that the monovalent elution curves of each group of meningococcal polysaccharides and the multivalent mixed elution curves basically overlapped, as shown in Figure 3. The repeatability verification results showed that the content distribution trend lines of each group of meningococcal polysaccharides measured three times basically overlapped, as shown in Figure 4.

[0108] The samples were tested by the method described in Example 1 of the present invention and the conventional method (Pharmacopoeia method: phosphorus determination method for group A, sialic acid determination method for groups C, Y, and W135, general rules 3103 and 3102 of the Pharmacopoeia of the People's Republic of China 2020 edition, Part IV), and the K values ​​of group A, C, Y, and W135 meningococcal polysaccharides were measured. D The values ​​are 0.36, 0.32, 0.32 and 0.32 respectively, and the two results are consistent.

[0109] Example 3: Detection of Typhoid Vi Polysaccharide Molecular Size

[0110] The reference substance is 100 μg / mL typhoid Vi polysaccharide solution (purchased from the National Biological Products Inspection Institute of the United Kingdom, model 16126), the serum is a 6-fold diluted specific typhoid Vi antiserum (purchased from the National Biological Products Inspection Institute of the United Kingdom, model 16 / 138), and the test substance is 1 mg / ml typhoid Vi polysaccharide solution. The instrument parameter settings and operating methods are the same as in Example 1, and the Sepharose CL-4B gel column system is used for sample loading.

[0111] The method was validated for applicability. The results of repeatability validation showed that the content distribution trend lines of typhoid Vi polysaccharide measured three times were basically overlapping, as shown in Figure 2. Figure 5 .

[0112] The samples were tested using the method described in Example 1 of the present invention and the conventional method (Pharmacopoeia method: oxyacetyl method, Pharmacopoeia of the People's Republic of China 2020 edition, Part IV, General Rule 3117), and the K of typhoid Vi polysaccharide was measured. D The value is 0.03, and the two results are consistent.

[0113] Example 4: Detection of the molecular size of pneumococcal polysaccharide intermediates

[0114] Reference material, serum, instrument parameter setting and operation method are the same as in Example 1, using Sepharose CL-4B gel column system. 18C type pneumococcal polysaccharide degradation products, derivatives, esterification products, and conjugates are all provided by Lanzhou Institute of Biological Products Co., Ltd. in accordance with the pneumococcal conjugate vaccine manufacturing and verification procedures in the European Pharmacopoeia 8.0, and the various biochemical test results and molecular sizes meet the relevant requirements of the pneumococcal conjugate vaccine manufacturing and verification procedures in the European Pharmacopoeia 8.0. The 18C type pneumococcal polysaccharide degradation product is a method of crushing with a high-pressure homogenizer to degrade the pneumococcal capsule polysaccharide to obtain a polysaccharide fragment with a molecular weight reduced to 50000 to 150000 Da. The polysaccharide derivative is a polysaccharide fragment modified by dibutyl amine (DAB) on the 18C type pneumococcal polysaccharide degradation product. The esterification product is a polysaccharide fragment after the polysaccharide derivative is activated by disuccinimidyl adipic acid (SIDEAA). The conjugate is a product obtained by covalently combining a chemically modified polysaccharide derivative with a carrier protein tetanus toxoid (TT) and then purifying it.

[0115] The method was validated for applicability, and the results of repeatability validation showed that the content distribution trend lines of three repeated measurements of 18C pneumococcal polysaccharide degradation products, derivatives, esterification products, and conjugates were basically overlapping, as shown in Figure 6.

[0116] The samples were tested by the method described in Example 1 of the present invention and the conventional method, respectively, and the K values ​​of the degradation products, derivatives, esterification products, and conjugates of 18C pneumococcal polysaccharide were measured. DThe values ​​are 0.41, 0.41, 0.41, and 0.03 respectively, and the two results are consistent.

Claims

1. A method for detecting the molecular size of polysaccharides in a multivalent bacterial vaccine, characterized in that, it comprises the following steps carried out in sequence: 1) Provide a mixed solution containing bacterial polysaccharides of multiple serotypes; 2) Perform gel filtration on the mixed solution to obtain multiple tubes of eluate collected continuously; 3) Perform rate nephelometry on the multi-tube eluate to obtain the amount of polysaccharide of each type of bacteria in each tube, and obtain the K value of polysaccharide of each type of bacteria from the amount of polysaccharide of each type of bacteria respectively. D value.

2. The method according to claim 1, wherein in the solution described in step 1), the concentration of each type of bacterial polysaccharide is 400 - 2000 μg / mL.

3. The method according to claim 1, wherein step 2) includes adding the solution described in step 1) to a gel filtration separation column, eluting each type of bacterial polysaccharide with an eluent and collecting all the eluate in aliquots; and step 3) includes detecting the amount of each type of bacterial polysaccharide in each aliquot of the collected eluate by rate nephelometry, wherein the specific antibody of each type of bacterial polysaccharide is contacted with each aliquot of the eluate, so that it reacts with the corresponding bacterial polysaccharide to form antigen - antibody complexes of each type, detecting the turbidity value of the antigen - antibody complexes of each type, and obtaining the amount of each type of bacterial polysaccharide from this turbidity value.

4. The method according to claim 1, wherein step 3) further includes performing rate nephelometry detection with a reference solution containing each type of bacterial polysaccharide at a series of concentrations and the specific antibody of each type of bacterial polysaccharide to obtain a standard curve.

5. The method according to claim 4, wherein the reference solution contains bacterial polysaccharides of multiple serotypes.

6. The method according to claim 1, wherein the bacterial polysaccharide is selected from pneumococcal polysaccharide, meningococcal polysaccharide, typhoid Vi polysaccharide and bacterial polysaccharide intermediate products.

7. The method according to claim 6, wherein the pneumococcal polysaccharide includes pneumococcal polysaccharides of types 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F; the meningococcal polysaccharide includes meningococcal polysaccharides of groups A, C, Y, W135; and the bacterial polysaccharide intermediate products include bacterial polysaccharide degradation products, derivatives, esterified products, conjugated products.

8. The method according to claim 1, wherein the gel filtration method is size - exclusion chromatography.

9. The method according to claim 1, wherein the rate nephelometry is carried out using a rate nephelometer.

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