Pretreatment method for determining content of hexosamine in pneumococcal polysaccharide

Abstract

The invention discloses a bacterial polysaccharide hydrolysis method and application thereof, and belongs to the field of vaccine preparation. According to the bacterial polysaccharide hydrolysis method, polysaccharide is hydrolyzed to generate monosaccharide in a microwave heating mode, the hydrolysis time can be saved, the hydrolysis efficiency can be improved, further degradation of the hydrolyzed monosaccharide is effectively reduced by adjusting and controlling the reaction power, the reaction temperature, the reaction time and the like, and the yield is improved. Therefore, the accuracy of identifying the polysaccharide structure is improved; the hydrolysis method disclosed by the invention can be applied to streptococcus pneumoniae polysaccharide, and monosaccharide hexosamine generated after streptococcus pneumoniae glycosaminoglycan is hydrolyzed is subjected to specific determination, so that the accuracy of glycosaminoglycan content determination is improved. The method disclosed by the invention is suitable for a vaccine preparation process and a vaccine quality control process, and has important significance for improving the quality controllability of a vaccine product.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine development, and particularly relates to a pretreatment method for determining the content of hexosamine in pneumococcal polysaccharide. Background Art

[0002] Pneumococcal infection is one of the main factors leading to pneumonia morbidity and even death worldwide. The most common manifestations are pneumonia, febrile bacteremia, and meningitis. Dissemination in the respiratory tract can lead to middle ear infection, sinusitis, or recurrent bronchitis. Streptococcus pneumoniae is a Gram-positive bacterium with a relatively thick capsule, and the composition and structure of the capsular polysaccharide are often related to the pathogenicity and serotype of the bacterium. There are more than 90 different serotypes, among which more than 20 are used to prepare vaccines for preventing diseases caused by Streptococcus pneumoniae infection.

[0003] In the preparation process of pneumococcal polysaccharide-protein, corresponding quantitative or qualitative detection needs to be carried out for each step of the process. For the polysaccharide purification process, quantitative detection of the purified polysaccharide is required. By degrading the purified polysaccharide and detecting the content of hexosamine in the degraded substance, the integrity of the purification degree of pneumococcal aminopolysaccharide can be obtained.

[0004] In the purification process of Streptococcus pneumoniae capsular polysaccharide, the content of hexosamine is a very important quality index. Hexosamine is an important component in the structure of Streptococcus pneumoniae capsular polysaccharide. During the vaccine development process, most of the repeating units of key sugar types contain hexosamine. To a certain extent, the content of hexosamine represents the integrity of the repeating units of pneumococcal polysaccharide, and hexosamine is also a specific indicator substance of Streptococcus pneumoniae capsular polysaccharide.

[0005] In national pharmacopoeias and literature reports, the detection method for the content of aminopolysaccharide in pneumococcal polysaccharide is mostly to hydrolyze pneumococcal polysaccharide into monosaccharides first and then determine the content by chemical colorimetry. In the prior art, pneumococcal polysaccharide hydrolysis usually adopts the method of acid hydrolysis by water bath heating. However, the polysaccharide structure is complex, and it is difficult to hydrolyze it fully by water bath heating. Moreover, the water bath heating process is slow, time-consuming, and the stability of the hydrolyzed monosaccharides is different, which is extremely likely to cause further degradation of the monosaccharides, thus affecting the accuracy of the detection results of the content of hydrolyzed monosaccharides and further affecting the accuracy of obtaining the content of aminopolysaccharide in pneumococcal polysaccharide.

[0006] Therefore, a detection method that can effectively degrade polysaccharides and maintain the stability of the structure of the degraded hexosamine is needed, so as to accurately detect the content of Streptococcus pneumoniae capsular polysaccharide. Summary of the Invention

[0007] Aiming at the problems of incomplete hydrolysis and instability of monosaccharides during the detection of aminohexose in polysaccharides by the existing chemical colorimetric method, the purpose of the present invention is to provide a pretreatment method for the determination of aminohexose content in polysaccharides. By pretreating the polysaccharides, the glycosidic bonds are opened to achieve complete hydrolysis of the polysaccharides. The reaction process is efficient and mild, and the aminohexose obtained by hydrolysis has high stability, effectively improving the accuracy of the detection result of aminohexose content.

[0008] The present invention provides a pretreatment method for the determination of aminohexose content in Streptococcus pneumoniae polysaccharides, which degrades the capsular polysaccharides by using a rapid heating method. On the one hand, rapid heating can make the polysaccharide degradation more complete, and on the other hand, stable reaction conditions can ensure the stability of aminohexose.

[0009] The present invention relates to a hydrolysis method for pneumococcal polysaccharides, which hydrolyzes pneumococcal capsular polysaccharides by microwave assistance under acidic conditions.

[0010] Specifically, the reaction conditions for the microwave hydrolysis are: reaction power: 100 - 400W, preferably 300W.

[0011] Specifically, the reaction conditions for the microwave hydrolysis are: reaction time is 5 - 60 min, preferably 10 - 30 min.

[0012] Specifically, the reaction conditions for the microwave hydrolysis are: hydrolysis temperature is 60 - 140 °C, preferably 80 - 130 °C.

[0013] Specifically, the microwave hydrolysis conditions are acidic; preferably, the acid is any one or more of hydrochloric acid, trifluoroacetic acid, and sulfuric acid.

[0014] Specifically, the concentration of the acid is 6M - 12M.

[0015] The present invention relates to a pneumococcal polysaccharide pretreatment method for the determination of aminohexose content in Streptococcus pneumoniae polysaccharide vaccines, which hydrolyzes the pneumococcal polysaccharides by using any one of the foregoing methods.

[0016] Specifically, for the method for determining the aminohexose content in Streptococcus pneumoniae polysaccharides, the hydrolysis step is as follows:

[0017] S1. Drawing of the standard curve: Weigh an appropriate amount of glucosamine hydrochloride reference substance and prepare a standard substance solution containing glucosamine; Detect the absorbance of each concentration standard working solution at 530 nm and draw the standard curve.

[0018] S2. Hydrolyze the pneumococcal polysaccharides. After the reaction ends, adjust the pH to neutral with sodium hydroxide solution. After the acetylation reaction and the color reaction, detect the absorbance of the sample at 530 nm and substitute it into the standard curve equation to calculate the concentration value.

[0019] Specifically, the hydrolysis described in S2 is the hydrolysis of pneumococcal capsular polysaccharide under acidic conditions assisted by microwave.

[0020] Specifically, the reaction power of the microwave hydrolysis is 100 - 400 W, preferably 300 W; and / or, the reaction time of the microwave hydrolysis is 5 - 60 min, preferably 10 - 30 min; and / or, the reaction hydrolysis temperature of the microwave hydrolysis is 60 - 140 °C, preferably 80 - 130 °C.

[0021] Specifically, the reagent in the acetylation reaction in S2 is: the acetylation reagent is any one of acetylacetone reagent and acetylacetone - sodium carbonate solution.

[0022] Specifically, the acetylation reaction temperature is 80 - 100 °C; and / or, the acetylation reaction time is 30 - 120 minutes.

[0023] The present invention also provides a method for determining the content of aminohexose in polysaccharide, which is ion exchange chromatography, used to support the results of chemical colorimetry with data. It is characterized in that the specific steps are as follows:

[0024] Preferably, the test sample in the ion exchange chromatography is the neutral hydrolysis solution obtained by acid hydrolysis of pneumococcal polysaccharide. Specifically, the acid hydrolysis solution is obtained by hydrolyzing pneumococcal polysaccharide, and after the reaction ends, the pH is adjusted to neutral with sodium hydroxide solution. Preferably, the ion exchange chromatography is carried out under the following chromatographic conditions: Chromatographic column: CarbopacTM PA10, 4×250 mm Eluent A: 18 mM sodium hydroxide solution

[0025] Eluent B: 100 mM sodium hydroxide solution

[0026] Eluent C: A mixed solution of 100 mM sodium hydroxide and 500 mM sodium acetate

[0027] Flow rate: 1.0 mL / min

[0028] Column temperature: 30 °C

[0029] Injection volume: 25 μL

[0030] Detector: Electrochemical detector

[0031] Electrode: Gold electrode

[0032] The present invention also provides a method for determining the content of aminohexose in polysaccharide by nuclear magnetic resonance method, which is used to support the results of chemical colorimetry with data. The method selects the internal standard method for content determination, uses the internal standard substance with known content as a reference, and obtains the absolute content of the aminohexose to be measured through the proportional relationship of the integral areas of the quantitative peaks in the nuclear magnetic spectrum.

[0033] Preferably, the nuclear magnetic resonance method selects a nuclear magnetic resonance instrument of 400 MHz or above;

[0034] Preferably, the nuclear magnetic resonance method selects a 5 mm broadband probe and a liquid probe with higher sensitivity;

[0035] Preferably, the internal standard substance is selected from maleic acid, dimethyl sulfone, and dimethyl sulfoxide with accurately labeled contents;

[0036] Preferably, the test nuclide of the nuclear magnetic resonance method is hydrogen nucleus;

[0037] Preferably, the pulse angle of the nuclear magnetic resonance method is selected from 30 degrees, 45 degrees, and 90 degrees;

[0038] Preferably, the test temperature of the nuclear magnetic resonance method is 25 - 80 °C;

[0039] Preferably, the relaxation time of the nuclear magnetic resonance method is 10 - 60 seconds;

[0040] Preferably, the number of scans of the nuclear magnetic resonance method is 16 - 64 times.

[0041] The method of the present invention provides a content determination method for aminohexose contained in the repeating unit structures of serotypes including 4, 5, 9V, 14, 19A, 19F, etc., and the method is reliable and accurate.

[0042] The beneficial effects of the present invention:

[0043] First, the hydrolysis method of the bacterial polysaccharide described in the present invention can be specifically applied to the pretreatment for the determination of the aminohexose content in Streptococcus pneumoniae capsular polysaccharide. Through the microwave-assisted hydrolysis method, the reaction time is significantly shortened compared with the traditional water bath heating method, the hydrolysis is more complete, and the efficiency of the pretreatment process is improved.

[0044] Second, the polysaccharide hydrolysis method provided by the present invention explores suitable microwave reaction conditions by controlling the microwave hydrolysis time, temperature, power, etc., can open the glycosidic bonds that are difficult to open, so that the polysaccharide is fully degraded into aminohexose without being further degraded, the structure of the hydrolyzed aminohexose is stable, and the ion exchange chromatography method and nuclear magnetic resonance quantitative method are used to characterize and verify this method, effectively improving the detection accuracy and reliability of aminohexose.

[0045] III. The present invention provides a method for determining the content of hexosamine contained in the repeating unit structures such as various pneumococcal protein types in the serotypes of polysaccharide or polysaccharide conjugate vaccines, which can be widely applied to the vaccine preparation process. Detailed implementation manners

[0046] To better understand the present invention, the beneficial effects of the present invention are further illustrated through the following examples, but they do not constitute a limitation to the present invention.

[0047] Method for determining the content of hexosamine

[0048] Experimental instruments and equipment: ultraviolet spectrophotometer, ion chromatograph, nuclear magnetic resonance spectrometer (NMR), microwave hydrolysis instrument, water bath.

[0049] Experimental reagents: glucosamine hydrochloride, purchased from the National Institutes for Food and Drug Control.

[0050] Experimental steps:

[0051] S1. Drawing of the standard curve: Weigh an appropriate amount of glucosamine hydrochloride reference substance, prepare a standard solution containing 500 μg / mL of glucosamine, and dilute it into standard working solutions of 50, 125, 250, 375, and 500 μg / mL respectively. Detect the absorbance of each concentration standard working solution at 530 nm and draw the standard curve.

[0052] S2. Hydrolysis of polysaccharide: Under certain power (100 - 400 W), temperature (5 - 60 min), reaction time (60 - 140 °C) and acidic conditions, microwave heat-treat the polysaccharide to release the hexosamine in the pneumococcal capsular polysaccharide. After the reaction ends, adjust the pH to neutral with sodium hydroxide solution; set a water bath heat-treatment as a comparative example.

[0053] S3. Determining the concentration of hexosamine in the sample by acetylation reaction and color reaction: Take a certain amount of the standard working solution and the sample solution, add hydrochloric acid solution for hydrolysis, add 5 g / L thymolphthalein-ethanol solution, dropwise add 200 g / L sodium hydroxide solution until just turning blue, then dropwise add 0.25 mol / L hydrochloric acid solution until just turning colorless, add purified water to a final volume of 10 mL. Take 1 mL of the solution, add 1 mL of acetylacetone-sodium carbonate solution, heat in a water bath at 90 °C for 45 min, add 2.5 mL of anhydrous ethanol, add 1 mL of p-dimethylaminobenzaldehyde solution, add anhydrous ethanol to 10 mL, let it stand in the dark for 90 min, detect the absorbance at 530 nm, and substitute it into the standard curve equation to calculate the concentration value.

[0054] Method for determining the content of methyl pentose

[0055] Accurately weigh 0.1 g of methyl pentose (rhamnose), dissolve it in water and make up the volume to 100 ml, shake well to prepare a 1 mg / ml methyl pentose reference stock solution (store at -20 °C, valid for 3 months, dilute 50-fold before use to obtain a 20 μg / ml methyl pentose reference solution).

[0056] Accurately measure 0 ml, 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml and 1.0 ml of the 20 μg / ml methyl pentose reference solution into stoppered test tubes respectively, make up the volume to 1 ml with water. Using the blank tube in the reference tube group (methyl pentose content is 0) as the control, in an ice bath, while continuously stirring, add 4.5 ml of pre-cooled sulfuric acid solution dropwise. Stopper the tube, warm the test tube to room temperature, then place it in a water bath at 100 °C for at least 5 minutes, cool to room temperature. Add 0.1 ml of 3% mercaptoalanine (cysteine) hydrochloride solution to each tube, mix well, stopper the tube, and let it stand at room temperature for 1 - 2 hours. Measure the absorbance A value at wavelengths of 396 nm and 430 nm for each tube, using the blank tube as the control at the same time.

[0057] Take the test sample and dilute it with water to a solution with a methyl pentose concentration lower than 20 μg / ml. Accurately measure 1.0 ml of the test sample solution into a stoppered test tube, measure two tubes in parallel, and perform the same operation starting from "in an ice bath, while continuously stirring, add 4.5 ml of pre-cooled sulfuric acid solution dropwise".

[0058] Use the series of concentrations of the reference solution (μg / ml) to perform a linear regression on their corresponding corrected absorbance values (A396 - A430nm) to obtain the linear regression equation. Substitute the corrected absorbance value (A396 - A430nm) of the test sample solution into the linear regression equation, calculate the methyl pentose content of the test sample according to the dilution factor, and then calculate the percentage content of methyl pentose based on the dry weight of the polysaccharide.

[0059] Example 1: Comparison of the content determination methods for the pretreatment of the aminopolysaccharide of Streptococcus pneumoniae type 4 capsular polysaccharide by water bath heating method and microwave hydrolysis method.

[0060] Use the above aminoglycoside content determination method to pretreat the Streptococcus pneumoniae type 4 capsular polysaccharide under the condition of a reaction power of 300 w, and the treatment results are shown in Table 1.

[0061] Table 1

[0062]

[0063] From the results of Example 1, it can be concluded that by using microwave-assisted hydrolysis for the pretreatment of type 4 pneumococcal capsular polysaccharide, the content of aminohexose obtained by hydrolysis is much higher than that of the traditional water bath heating treatment method. This indicates that the use of microwave-assisted hydrolysis can improve the hydrolysis efficiency of amino polysaccharides, making the hydrolysis of capsular polysaccharides more complete and thorough. Therefore, when measuring the content of aminohexose obtained after pretreatment by the method of the present invention, the measurement accuracy is greatly improved. At the same time, the structure of the aminohexose obtained by its hydrolysis is stable, facilitating subsequent research.

[0064] Example 2: Comparison of the content determination methods for the pretreatment of type 5 Streptococcus pneumoniae capsular polysaccharide amino polysaccharide by water bath heating method and microwave hydrolysis method.

[0065] Using the above-mentioned aminohexose content determination method to pretreat type 5 Streptococcus pneumoniae capsular polysaccharide under the condition of a reaction power of 300 w, the treatment results are shown in Table 2.

[0066] Table 2

[0067]

[0068] By performing microwave hydrolysis pretreatment on type 5 pneumococcal capsular polysaccharide and measuring the peak area of the total amino sugar after hydrolysis, it is found that the microwave hydrolysis method can also achieve good treatment effects for the pretreatment of type 5 pneumococcal capsular polysaccharide. Compared with the water bath heating treatment method, the polysaccharide hydrolysis by the microwave hydrolysis method is more complete. Although the effect under the water bath heating hydrolysis condition of 5 h is also better than that of 1 h of water bath heating, this method consumes too much hydrolysis time and has low efficiency. The method of the present invention can fully hydrolyze the polysaccharide in a shorter time, with thorough hydrolysis and high efficiency.

[0069] Example 3: Comparison of the content determination methods for the pretreatment of type 9V Streptococcus pneumoniae capsular polysaccharide amino polysaccharide by water bath heating method and microwave hydrolysis method.

[0070] Using the above-mentioned aminohexose content determination method to pretreat type 9V Streptococcus pneumoniae capsular polysaccharide under the condition of a reaction power of 300 w, the treatment results are shown in Table 3.

[0071] Table 3

[0072]

[0073]

[0074] Performing microwave-assisted hydrolysis pretreatment on type 9V Streptococcus pneumoniae capsular polysaccharide can also achieve the technical effects of more complete polysaccharide hydrolysis and higher efficiency, indicating that the method of the present invention is also applicable to the hydrolysis pretreatment process for the content determination of type 9V Streptococcus pneumoniae capsular polysaccharide.

[0075] Example 4: Comparison of content determination methods for the pretreatment of capsular polysaccharide amino polysaccharide of Streptococcus pneumoniae type 14 by water bath heating method and microwave hydrolysis method.

[0076] The capsular polysaccharide of Streptococcus pneumoniae type 14 was pretreated under the condition of a reaction power of 300 w using the above-mentioned method for determining the content of aminohexose, and the treatment results are shown in Table 4.

[0077] Table 4

[0078]

[0079]

[0080] The capsular polysaccharide of Streptococcus pneumoniae type 14 was subjected to microwave-assisted hydrolysis treatment. The content of aminohexose obtained by hydrolysis was higher than that obtained by water bath heating treatment, and the hydrolysis efficiency was higher and more thorough, indicating that this method is suitable for the pretreatment of the content determination of capsular polysaccharide of Streptococcus pneumoniae type 14.

[0081] Example 5: Comparison of content determination methods for the pretreatment of capsular polysaccharide amino polysaccharide of Streptococcus pneumoniae type 19F by water bath heating method and microwave hydrolysis method.

[0082] In this specific embodiment, the capsular polysaccharide of Streptococcus pneumoniae type 19F was pretreated under the condition of a reaction power of 300 w using the above-mentioned pretreatment method for determining the content of aminohexose, and the contents of aminohexose and methylpentose in the hydrolysis products were measured simultaneously. The treatment results are shown in Table 5.

[0083] Table 5

[0084]

[0085]

[0086] It can be seen from the results in Table 5 that the pretreatment effect on the capsular polysaccharide amino polysaccharide of Streptococcus pneumoniae type 19F is good. The degree of microwave hydrolysis of amino polysaccharide is higher than that of water bath heating treatment, and the hydrolysis efficiency is higher. At the same time, the content of methylpentose in the hydrolysis products was measured, and it was found that the content of methylpentose obtained by microwave hydrolysis was lower than that by water bath heating hydrolysis, indicating that the method of the present invention is particularly suitable for the pretreatment in the determination of aminohexose content.

[0087] Example 6: Comparison of content determination methods for the pretreatment of capsular polysaccharide amino polysaccharide of Streptococcus pneumoniae type 19A by water bath heating method and microwave hydrolysis method.

[0088] Using the above methods for determining the content of hexosamine and methyl pentose, the capsular polysaccharide of Streptococcus pneumoniae type 19A was pretreated under the condition of a reaction power of 300 W, and the treatment results are shown in Table 6.

[0089] Table 6

[0090]

[0091]

[0092] It can be seen from the results in Table 6 that the effect of microwave hydrolysis treatment on the capsular polysaccharide of Streptococcus pneumoniae type 19A is better than that of water bath heating treatment. Similarly, different from the hydrolysis result of hexosamine, the hydrolysis degree of methyl pentose is lower than that of the control group treated by water bath heating, indicating that the method of the present invention is particularly suitable for the pretreatment in the determination of hexosamine content.

[0093] The results of the above specific embodiments show that by pretreating the capsular polysaccharide of Streptococcus pneumoniae by microwave-assisted hydrolysis, the amino polysaccharides in different types of capsular polysaccharides of Streptococcus pneumoniae can be fully hydrolyzed, and the hexosamine structure after hydrolysis is stable and will not be further degraded, proving that this method is feasible for the pretreatment of the determination of hexosamine content after the degradation of capsular polysaccharides of different serotypes of Streptococcus pneumoniae, and has universality for various types of capsular polysaccharides of Streptococcus pneumoniae. Compared with the conventional water bath heating method for degrading polysaccharides, the polysaccharides are hydrolyzed thoroughly by the method of the present invention, and the obtained hydrolysis products have a stable structure, which is helpful for the subsequent accurate determination of hexosamine content; on the contrary, the same treatment method is not suitable for degrading polysaccharides to obtain methyl pentose, indicating that the pretreatment method of the present invention is particularly suitable for the determination of hexosamine content, and the determination method is effective and has high accuracy.

Claims

1. A method for hydrolyzing Streptococcus pneumoniae capsular polysaccharide, characterized in that, the Streptococcus pneumoniae capsular polysaccharide is hydrolyzed by microwave assistance under acidic conditions.

2. The hydrolysis method according to claim 1, characterized in that, the reaction conditions for the microwave hydrolysis are: the reaction time is 10 - 60 min, preferably 10 - 30 min.

3. The hydrolysis method according to claim 2, characterized in that, the reaction conditions for the microwave hydrolysis are: the hydrolysis temperature is 60 - 140 °C, preferably 80 - 130 °C.

4. The hydrolysis method according to any one of claims 1 - 3, characterized in that, the microwave hydrolysis conditions are acidic; preferably, the acid is any one or more of hydrochloric acid, trifluoroacetic acid, and sulfuric acid.

5. The hydrolysis method according to claim 4, characterized in that, the concentration of the acid is 6M - 12M.

6. A method for determining the content of hexosamine in Streptococcus pneumoniae polysaccharide vaccine, characterized in that, it comprises the following steps: (1) Hydrolyzing the Streptococcus pneumoniae capsular polysaccharide by any one of the methods of claims 1 - 5; (2) Determining the content of hexosamine in the product after polysaccharide hydrolysis.

7. The method for determining the content of hexosamine in Streptococcus pneumoniae polysaccharide according to claim 6, characterized in that, the content determination is carried out by chemical colorimetry.

8. The method for determining the content of hexosamine in Streptococcus pneumoniae polysaccharide according to claim 7, characterized in that, the method steps are as follows: S1. Standard curve drawing: Weigh an appropriate amount of glucosamine hydrochloride reference substance, prepare a standard solution containing glucosamine, detect the absorbance of each concentration standard working solution at 530 nm, and draw a standard curve; S2. Sample content determination: Hydrolyze the Streptococcus pneumoniae capsular polysaccharide sample by any one of the methods of claims 1 - 5. After the reaction ends, adjust the pH to neutral with sodium hydroxide solution, followed by acetylation reaction and color reaction; Detect the absorbance at 530 nm, and substitute it into the standard curve equation to calculate the concentration value.

9. The determination method according to claim 8, characterized in that, the reagent in the acetylation reaction in S2 is: the acetylation reagent is any one of acetylacetone reagent and acetylacetone - sodium carbonate solution; and / or, the acetylation reaction temperature is 80 - 100 °C; and / or, the acetylation reaction time is 30 - 120 minutes.

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