A process for the preparation of a group a meningococcal capsular polysaccharide conjugate

Abstract

This application relates to the technical field of vaccine conjugates, specifically to a method for preparing a polysaccharide conjugate of group A meningococcal capsular polysaccharide, including the preparation of polysaccharide derivatives, mixing ratio, conjugation reaction, aging treatment, ultrafiltration and sterilization steps. This application eliminates the pH adjustment and conjugate purification steps in the activation process by optimizing the process parameters, but the quality of the harvested polysaccharide conjugate still meets the national standards, thereby simplifying the preparation process of polysaccharide conjugates and shortening the preparation cycle of polysaccharide conjugates.

Description

Technical Field

[0001] This application relates to the technical field of vaccine conjugates, and more specifically, it relates to a method for preparing a group A meningococcal capsular polysaccharide conjugate. Background Technology

[0002] Epidemic meningitis is an epidemic disease caused by Neisseria meningitidis, which causes inflammation of the meninges. The strains that cause human disease are mainly classified into groups A, B, C, Y and W135. In my country, more than 90% of meningococcal cases are caused by group A Neisseria meningitidis.

[0003] Vaccines are an effective way to prevent and control meningococcal infection. Group A meningococcal conjugate vaccine is made by extracting the bacterial capsular polysaccharide of Group A meningococcal strains, coupling the capsular polysaccharide with specific proteins or polypeptides to form a polysaccharide conjugate, and then formulating it into a vaccine to prevent invasive infection by this strain of meningococcus.

[0004] The existing preparation of group A meningococcal conjugate vaccines requires first converting capsular polysaccharides into polysaccharide derivatives, then coupling these polysaccharide derivatives with carrier proteins under the mediation of a cross-linking agent to form a conjugate. The conjugate is then subjected to ultrafiltration and purification to obtain a polysaccharide conjugate that meets national standards. Therefore, the entire preparation process of group A meningococcal capsular polysaccharide conjugates is cumbersome and time-consuming, resulting in a long production cycle for group A meningococcal conjugate vaccines. Summary of the Invention

[0005] To shorten the production cycle of group A meningococcal conjugate vaccine, this application provides a method for preparing group A meningococcal capsular polysaccharide conjugates. By optimizing the process parameters, the conjugates can still meet national standards even after the purification process is omitted, thereby simplifying the preparation process and shortening the preparation cycle.

[0006] The technical solution adopted in this application is as follows:

[0007] A method for preparing a group A meningococcal capsular polysaccharide conjugate includes the following steps:

[0008] ① Preparation of polysaccharide derivatives: The polysaccharide of group A meningococcal capsular polysaccharide was dissolved in physiological saline to obtain polysaccharide solution a. The pH of polysaccharide solution a was adjusted to 10.5±0.2 with sodium hydroxide solution. An activator was added for activation to obtain polysaccharide solution b. Adipic hydrazine was added to polysaccharide solution b for derivatization. The pH of the derivatization reaction was maintained at 9.0±0.2 to obtain polysaccharide solution c. The solution was stirred at low temperature at 2-8℃ for 6-20h. After ultrafiltration, the polysaccharide derivative stock solution was obtained.

[0009] ② Mixing ratio: Under the condition of temperature control at 4-8℃, the polysaccharide derivative stock solution obtained in step ① is prepared into a derivative solution with a pH of 6.0±0.3 using physiological saline and hydrochloric acid solution, and the concentration of polysaccharide derivative in the derivative solution is 0.5-5.0mg / mL. Diphtheria CRM197 protein is added and stirred to mix, wherein the weight ratio of polysaccharide derivative to diphtheria CRM197 protein is 1:(0.8-2.5), and an equilibrium solution is obtained;

[0010] ③ Combination reaction: Under the condition of temperature control at 4-8℃, carbodiimide is added to the equilibrium solution obtained in step ② to carry out the combination reaction and obtain the initial reactant;

[0011] ④ Aging treatment: Place the initial reactant obtained in step ③ in a sterile environment at 2-8℃ and let it stand for 16-20 hours to perform aging treatment and obtain the aged reactant.

[0012] ⑤ Ultrafiltration: The aged reactants obtained in step ④ are subjected to ultrafiltration to remove excess residual reagents from the binding reaction process, and a concentrated solution of the binding compound is obtained.

[0013] ⑥ Sterilization: The concentrated solution of the conjugate obtained in step ⑤ is sterilized by using a sterile filter membrane to obtain the stock solution of group A meningococcal capsular polysaccharide conjugate.

[0014] By adopting the above technical solution, this application uses physiological saline (sodium chloride concentration of 0.154 mol / L) to dissolve Group A meningococcal capsular polysaccharide (hereinafter referred to as Group A polysaccharide), which can effectively dissolve Group A polysaccharide with a molecular weight of 50-100 kDa. In the process of activating Group A polysaccharide with hydrogen bromide, this application omits the pH adjustment step, but it has little impact on the activation effect of Group A polysaccharide. When derivatizing Group A polysaccharide with adipic hydrazide, this application sets the pH of the derivatization reaction to 9.5 ± 0.2. At this time, the activated Group A polysaccharide can be derivatized quickly and effectively, and the polysaccharide derivative obtained thereby has better activity so as to bind to diphtheria CRM197 protein.

[0015] Based on this, the polysaccharide derivative obtained in step ① of this application is prepared into a derivative solution using physiological saline and hydrochloric acid solution. The physiological saline solution can ensure that the derivative solution is homogeneous and stable. Subsequently, the reaction concentration of the polysaccharide derivative and its weight ratio with diphtheria CRM197 protein are adjusted to ensure that the binding reaction proceeds effectively. After ultrafiltration, the polysaccharide yield of the obtained conjugate can reach 85% or more, the protein yield can reach 90% or more, and other quality requirements also meet national standards.

[0016] Therefore, the preparation method of this application eliminates the pH adjustment and purification steps of the activation process by optimizing various process parameters, but the quality of the harvested polysaccharide conjugate still meets the national standards, thereby simplifying the preparation process of polysaccharide conjugate and shortening the preparation cycle of polysaccharide conjugate.

[0017] Preferably, in step ①, the physiological saline is preheated to 30±2℃ to dissolve the capsular polysaccharide of group A meningococcus.

[0018] By adopting the above technical solution, the preheated saline promotes the rapid dissolution of group A polysaccharides, thereby shortening the dissolution time of the polysaccharides.

[0019] Preferably, in step ①, the supernatant of the polysaccharide solution a is taken after centrifugation and the pH is adjusted.

[0020] By adopting the above technical solution, centrifugation can remove a small amount of undissolved group A polysaccharides and other impurities from polysaccharide solution a, thereby obtaining polysaccharide conjugates with higher purity and more stable performance.

[0021] Preferably, in step ①, the activation temperature is 25±2℃.

[0022] By adopting the above technical solution, if the temperature is too high, the activation rate of group A polysaccharides by hydrogen bromide is too fast, which may result in incomplete activation of group A polysaccharides; if the temperature is too low, the activation rate of group A polysaccharides by hydrogen bromide is slow. Therefore, this application prefers an activation temperature of 25±2℃, which helps hydrogen bromide to effectively activate group A polysaccharides.

[0023] Preferably, in step ①, the polysaccharide solution c is ultrafiltered with physiological saline after stirring.

[0024] By adopting the above technical solution, the solvent used for dissolving group A polysaccharides and ultrafiltration is kept consistent, which can avoid the introduction of other solvents into the reaction system, thereby ensuring that the reaction process is simple and controllable.

[0025] Preferably, in step ①, the activator is a hydrogen bromide solution, and the weight ratio of the group A meningococcal capsular polysaccharide to the hydrogen bromide in the hydrogen bromide solution is 1:(1.2-1.8).

[0026] By adopting the above technical solution, hydrogen bromide has a good activation effect on group A polysaccharides. After activation, it can also be quickly volatilized and removed, reducing its interference with the subsequent derivatization reaction of group A polysaccharides. On this basis, the addition of an appropriate excess of hydrogen bromide in this application helps to activate as many group A polysaccharides as possible.

[0027] Preferably, in step ①, the activator is carbodiimide, and the carbodiimide is added to a concentration of 0.01-0.2 mol / L.

[0028] By adopting the above technical solution, the carbodiimide at this concentration can activate the carboxyl group, promote the formation of amides and esters, and thus activate group A polysaccharides. After the group A polysaccharides are activated, the carbodiimide can also work together with adipic hydrazide to participate in the derivatization reaction of group A polysaccharides, thereby improving the derivatization effect of group A polysaccharides.

[0029] Preferably, in step ①, the weight ratio of the group A meningococcal capsular polysaccharide to the adipic hydrazide is 1:(2-5).

[0030] By adopting the above technical solution, the weight ratio of adipic hydrazide can effectively promote the derivatization of group A polysaccharides. If the amount is too small, the derivatization of group A polysaccharides will not be thorough. If the amount is too large, it will waste reagents. The preferred ratio in this application is 1: (2-5), at which point the derivatization rate of polysaccharide derivatives is higher.

[0031] Preferably, in step ③, the concentration of carbodiimide in the initial reactant is 10-100 mmol / L.

[0032] By adopting the above technical solution, this concentration of carbodiimide can effectively promote the binding reaction between group A polysaccharides and diphtheria CRM197 protein. The resulting polysaccharide conjugate has a higher polysaccharide yield and protein yield, and is therefore preferred as a further alternative.

[0033] Preferably, in step ③, trehalose is also added to the equilibrium solution, and the concentration of trehalose is added to 1-5 mmol / L.

[0034] By adopting the above technical solution, this application adds an appropriate amount of trehalose to the equilibrium solution. Its effect on the binding of group A polysaccharide to diphtheria CRM197 protein is negligible. In addition, it can increase the stability of the polysaccharide conjugate to a certain extent, thereby obtaining a polysaccharide conjugate with higher polysaccharide yield and protein yield.

[0035] In summary, this application has the following beneficial effects:

[0036] 1. This application eliminates the pH adjustment and purification steps of the activation process by optimizing various process parameters, but the quality of the harvested polysaccharide conjugates still meets national standards, thereby simplifying the preparation process of polysaccharide conjugates and shortening the preparation cycle of polysaccharide conjugates.

[0037] 2. In this application, adding an appropriate amount of trehalose to the equilibrium solution can increase the stability of the polysaccharide conjugate to a certain extent, thereby obtaining a polysaccharide conjugate with higher polysaccharide and protein yields. Detailed Implementation

[0038] The present application will be further described in detail below with reference to embodiments and comparative examples.

[0039] Preparation examples of raw materials and / or intermediates

[0040] Extraction of polysaccharides from the capsular region of Group A meningococci

[0041] Group A meningococcal strains were purchased from a bacterial culture bank and, after six passages, a culture medium containing Group A meningococcal strains was harvested. 1 L of this culture medium was sterilized with formaldehyde, ensuring sterilization safety without damaging the bacterial polysaccharides. The sterilized culture medium was centrifuged to remove bacterial cells, and the supernatant was collected. The supernatant was concentrated to 1 / 8 of its original volume using an ultrafiltration membrane with a molecular weight cutoff of 50-100 KD. Hexadecyltrimethylammonium bromide was added and mixed thoroughly. The mixture was stirred at room temperature for 60 min, incubated at 4°C for 8 h, and then centrifuged at 15000 rpm to collect the precipitate. Calcium chloride solution was added to the precipitate to a final concentration of 1 mol / L to dissociate the polysaccharide hexadecyltrimethylammonium bromide. Insoluble matter was removed by filtration, and ethanol was added to a final concentration of 30% (v / v). The mixture was incubated at 4°C for at least 3 h, centrifuged to remove nucleic acid precipitates, and the clear supernatant was collected. Add cold ethanol to the supernatant to a final concentration of 75% (v / v) and shake thoroughly. Centrifuge to collect the precipitate, then wash twice each with anhydrous ethanol and acetone. Store the precipitate (group A meningococcal capsular polysaccharide) at -20°C or below for later use.

[0042] The diphtheria CRM197 protein, carbodiimide (EDAC), sodium chloride, and hydrochloric acid were all commercially available products, with sodium chloride and hydrochloric acid being of analytical grade. The distilled water was prepared on-site by a pure water machine and was also of analytical grade.

[0043] Physiological saline: 0.9 wt% sodium chloride aqueous solution, with a molar concentration of 0.154 mol / L. Example

[0044] Example 1

[0045] A method for preparing a group A meningococcal capsular polysaccharide conjugate includes the following steps:

[0046] ① Preparation of polysaccharide derivatives

[0047] Weigh 1.0 g of group A meningococcal capsular polysaccharide, place it in a beaker, add 200 mL of physiological saline (25℃), and stir with a magnetic stirrer for 30 min at room temperature at a stirring speed of 400 r / min to dissolve it into polysaccharide solution a;

[0048] The pH of polysaccharide solution a was adjusted to 10.5 using a sodium hydroxide solution with a molar concentration of 0.1 mol / L, and this pH was allowed to fluctuate within the range of ±0.2 (fluctuations within this range have little effect on product performance). 1.2 L of hydrogen bromide solution with a mass concentration of 1 g / L was added for activation to obtain polysaccharide solution b.

[0049] Add 3.0 L of adipic hydrazide solution with a molar concentration of 1 g / L to polysaccharide solution b for derivatization for 15 min. During this period, the pH of the derivatization reaction is maintained at 9.0, and the pH is allowed to fluctuate within ±0.2 (fluctuations within this range have little effect on product performance), to obtain polysaccharide solution c.

[0050] The polysaccharide solution c was transferred to a sterile cold storage and stirred at a low temperature of 4℃ for 10 hours with a stirring speed of 600 r / min. The temperature of the cold storage could fluctuate within the range of 2-8℃, and the stirring time could fluctuate within the range of 6-20 hours depending on the stirring state of the polysaccharide solution, thus obtaining polysaccharide solution d.

[0051] The polysaccharide solution d was then subjected to ultrafiltration using an ultrafiltration machine equipped with a 50kD ultrafiltration membrane pack. 20L of physiological saline (4℃) was added, and the ultrafiltration machine was started. The liquid volume was reduced to 5L. The ultrafiltration membrane pack was replaced and the above process was repeated 6 times. After ultrafiltration, the polysaccharide derivative stock solution was obtained.

[0052] ② Mixing ratio

[0053] Under the condition of temperature control at 4-8℃, the polysaccharide derivative stock solution obtained in step ① was prepared into a derivative solution with pH 6.0 using physiological saline (4℃) and hydrochloric acid solution with a molar concentration of 0.1mol / L (4℃), and the concentration of polysaccharide derivative in the derivative solution was 1.2mg / mL. The pH of the derivative solution was allowed to fluctuate within ±0.2 (fluctuations within this range have little impact on product performance). Diphtheria CRM197 protein was added and stirred to mix, with the weight ratio of polysaccharide derivative to diphtheria CRM197 protein being 1:1, to obtain an equilibrium solution.

[0054] ③ Combination reaction

[0055] Under the condition of temperature control at 4-8℃, carbodiimide was added to the equilibrium solution obtained in step ② to carry out the binding reaction. The concentration of carbodiimide added to the reactants was 40 mmol / L. The reaction temperature was controlled within the range of 4-8℃ for 3 hours to obtain the initial reactants.

[0056] ④ Time-sensitive processing

[0057] The initial reactant obtained in step ③ is placed in a sterile refrigerator at 2-8℃ and allowed to stand for 16 hours for aging treatment. The aging treatment time can be adjusted within the range of 16-20 hours as needed to obtain the aged reactant.

[0058] ⑤ Ultrafiltration

[0059] The aged reactants obtained in step ④ were subjected to ultrafiltration. The ultrafiltration process was carried out 6 times with an ultrafiltration machine and a 100kD ultrafiltration membrane. Each time, the reactants were diluted with 5 times the volume of distilled water to remove excess residual reagents from the binding reaction process, and the conjugate concentrate was obtained.

[0060] ⑥. Sterilization

[0061] The concentrate obtained in step ⑤ was sterilized by filtration through a 0.22 μm filter membrane to obtain the stock solution of group A meningococcal capsular polysaccharide conjugate.

[0062] The group A meningococcal capsular polysaccharide conjugate stock solution is an intermediate product in the preparation of the group A meningococcal conjugate vaccine. It is usually stored temporarily in stock solution form, and its preparation method for the conjugate vaccine will not be discussed further here.

[0063] Examples 2-4

[0064] Examples 2-4 are based on the method of Example 1, but the process parameters in step ② are adjusted. See Table 1 below for details of the adjustments.

[0065] Table 1. Adjustment of Process Parameters for Examples 1-4

[0066] Example 1 Example 2 Example 3 Example 4 Reaction concentration of polysaccharide derivatives (mg / mL) 1.2 0.5 1.6 5.0 The weight ratio of polysaccharide derivatives to diphtheria CRM197 protein 1:1 1:0.8 1:1.5 1:2.5

[0067] Example 5

[0068] Based on the method of Example 1, this embodiment preheats the physiological saline in step ① to 30°C to dissolve the polysaccharide of group A meningococcus capsularis. The preheating temperature can fluctuate within the range of ±2°C, and the fluctuation within this range has little impact on the product performance.

[0069] Example 6

[0070] Based on the method of Example 1, in this embodiment, the polysaccharide solution a in step ① is centrifuged at 4000 r / min for 5 min, and the supernatant is taken for pH adjustment.

[0071] Example 7

[0072] Based on the method of Example 1, this embodiment maintains the activation temperature in step ① at 25°C, and the activation temperature is allowed to fluctuate within the range of ±2°C. Fluctuations within this range have little impact on the product performance.

[0073] Example 8

[0074] This embodiment is based on the method of Example 1, except that the physiological saline used for ultrafiltration is replaced with a sodium chloride aqueous solution with a molar concentration of 0.2 mol / L.

[0075] Examples 9-10

[0076] Examples 9-10 are based on the method of Example 1, but with adjustments made to the amount of hydrogen bromide added in step ①. Specifically, in Example 9, 1.5 L of a 1 g / L hydrogen bromide solution was added for activation; in Example 10, 1.8 L of a 1 g / L hydrogen bromide solution was added for activation.

[0077] Examples 11-13

[0078] Examples 11-13 are based on the method of Example 1, except that the activator hydrogen bromide solution in step ① is replaced with carbodiimide. Specifically, in Example 11, the concentration of carbodiimide is added to 0.01 mol / L; in Example 12, the concentration of carbodiimide is added to 0.1 mol / L; and in Example 13, the concentration of carbodiimide is added to 0.2 mol / L.

[0079] Examples 14-16

[0080] Examples 14-16 are based on the method of Example 1, but with adjustments made to the amount of adipic acid hydrazine added in step ①. Specifically, in Example 14, 2.0 L of a 1 g / L adipic acid hydrazine solution was added for derivatization; in Example 15, 5.0 L of a 1 g / L adipic acid hydrazine solution was added for derivatization; and in Example 16, 6.0 L of a 1 g / L adipic acid hydrazine solution was added for derivatization.

[0081] Examples 17-19

[0082] Examples 17-19 are based on the method of Example 1, but with adjustments made to the concentration of carbodiimide in the initial reactant of step ③. Specifically, the concentration of carbodiimide in the initial reactant in Example 17 is 10 mmol / L; the concentration of carbodiimide in the initial reactant in Example 18 is 60 mmol / L; and the concentration of carbodiimide in the initial reactant in Example 19 is 100 mmol / L.

[0083] Examples 20-22

[0084] Based on the method of Example 1, this embodiment adds trehalose to the equilibrium solution in step ③. Specifically, the concentration of trehalose in the equilibrium solution of Example 20 is 1 mmol / L; the concentration of trehalose in the equilibrium solution of Example 21 is 3 mmol / L; and the concentration of trehalose in the equilibrium solution of Example 22 is 5 mmol / L.

[0085] Comparative Example

[0086] Comparative Example 1

[0087] This comparative example is based on the method of Example 1, except that the physiological saline in steps ① and ② is replaced with a sodium chloride aqueous solution with a molar concentration of 0.2 mol / L. Specifically, the group A meningococcal capsular polysaccharide is dissolved using a sodium chloride aqueous solution with a molar concentration of 0.2 mol / L (25°C); ultrafiltration is performed using a sodium chloride aqueous solution with a molar concentration of 0.2 mol / L (5°C); and a derivative solution with a pH of 6.0 is prepared using a sodium chloride aqueous solution with a molar concentration of 0.2 mol / L (4°C) and a hydrochloric acid solution with a molar concentration of 0.1 mol / L (4°C).

[0088] Comparative Example 2

[0089] Based on the method of Example 1, this comparative example maintains the pH during the activation reaction in step ① at 10.5±0.2 and the pH during the derivatization reaction at 9.0±0.2.

[0090] Comparative Example 3

[0091] Based on the method of Example 1, this comparative example further includes purification before sterilization in step ⑥. Specifically, the conjugate concentrate was purified by column chromatography using an AKTApure 150M protein purifier. The purification packing material was Sepharose 4Fast Flow, the linear flow rate was 30-50 cm / h, and the sample loading volume did not exceed 5% of the column volume. The substance at the A280 absorption peak near V0 with kD ≤ 0.2 was collected to obtain the purified conjugate solution.

[0092] Performance testing

[0093] Detection methods / test methods

[0094] The polysaccharide conjugate stock solutions of Group A meningococcal capsular polysaccharide obtained in Examples 1-22 and Comparative Examples 1-3 were tested according to the testing standards in the 2015 edition of the Chinese Pharmacopoeia. The specific test results are shown in Table 2 below.

[0095] Table 2 shows the polysaccharide yield and protein yield of the polysaccharide conjugates from Examples 1-22 and Comparative Examples 1-3.

[0096] Polysaccharide yield / % Protein yield / % Example 1 86.8 91.7 Example 2 87.0 91.5 Example 3 88.5 93.2 Example 4 87.1 92.0 Example 5 86.9 91.7 Example 6 89.1 93.0 Example 7 87.2 92.4 Example 8 85.9 90.0 Example 9 87.5 92.5 Example 10 87.2 92.1 Example 11 85.0 90.1 Example 12 91.8 94.2 Example 13 92.0 94.7 Example 14 88.1 93.0 Example 15 87.5 92.4 Example 16 87.3 92.4 Example 17 85.4 90.1 Example 18 88.1 93.0 Example 19 86.0 91.1 Example 20 91.8 95.4 Example 21 93.5 97.8 Example 22 92.0 95.9 Comparative Example 1 83.1 81.8 Comparative Example 2 82.7 80.4 Comparative Example 3 86.0 91.4

[0097] The endotoxin content of the stock solutions of the group A meningococcal capsular polysaccharide conjugates prepared in Examples 1-22 above was ≤1.0 EU / μg (standard ≤10 EU / μg), the free polysaccharide content was ≤9.7% (standard ≤20%), the free protein content was ≤0.9% (standard ≤5%), the recovery rate of eluent with a molecular size KD value <0.2 was ≥90.3% (standard ≥60%), the EDAC residue was ≤0.5 μmol / L (standard ≤5 μmol / L), the cyanide residue was ≤0.4 ng / mg (standard ≤5 ng / mg), and sterility was not detected in any of them (standard not detected). Therefore, the quality of the group A meningococcal capsular polysaccharide conjugates prepared in the examples of this application meets the national testing standards.

[0098] By comparing the test results of Example 1 with those of Comparative Examples 1-4 in Table 2, it can be seen that the present application uses physiological saline to dissolve the polysaccharide of Group A meningococcal capsularis, which can effectively dissolve Group A polysaccharides with a molecular weight of 50-100 kDa. In the process of activating Group A polysaccharides with hydrogen bromide, the present application omits the pH adjustment step, but it has little impact on the activation effect of Group A polysaccharides. When derivatizing Group A polysaccharides with adipic hydrazide, the present application sets the pH of the derivatization reaction to be maintained at 9.0 ± 0.2. At this time, the activated Group A polysaccharides can be derivatized quickly and effectively. The polysaccharide derivatives obtained are prepared into derivative solutions using physiological saline and hydrochloric acid solutions. Then, the reaction concentration of the polysaccharide derivatives and the weight ratio with diphtheria CRM197 protein are adjusted to ensure that the binding reaction proceeds effectively. After ultrafiltration, the polysaccharide yield can reach 85% or more, the protein yield can reach 90% or more, and other quality requirements also meet national standards.

[0099] Therefore, the preparation method of this application, as a whole scheme, eliminates the purification step of the conjugate by optimizing various process parameters, but the quality of the harvested polysaccharide conjugate still meets the national standards. This simplifies the preparation process of polysaccharide conjugate, shortens the preparation cycle of polysaccharide conjugate, and has good application prospects.

[0100] In Example 5, the dissolution time of the group A meningococcal capsular polysaccharide was 18 minutes, which was significantly less than the 30 minutes required for stirring at room temperature.

[0101] Comparing the detection results of Example 1 and Example 6, the polysaccharide yield and protein yield of Example 6 were both higher than those of Example 1. This shows that centrifugation can remove a small amount of undissolved group A polysaccharides and other impurities from polysaccharide solution a, thereby yielding polysaccharide conjugates with higher purity and more stable performance.

[0102] Comparing the detection results of Example 1 and Example 7, it can be seen that the preferred activation temperature of this application is 25±2℃, which helps hydrogen bromide to effectively activate group A polysaccharides, thereby obtaining polysaccharide conjugates with higher polysaccharide and protein yields.

[0103] Comparing the detection results of Example 1 and Example 8, it can be seen that using the same solvent for dissolving group A meningococcal capsular polysaccharide and ultrafiltration can avoid the introduction of other solvents into the reaction system, thereby ensuring that the reaction process is simple and controllable, and obtaining polysaccharide conjugates with higher polysaccharide and protein yields.

[0104] Comparing the detection results of Example 1 with those of Examples 9-10, it can be seen that the activation efficiency of hydrogen bromide on group A polysaccharides is limited. When hydrogen bromide is added in this application at a weight ratio of group A meningococcal capsular polysaccharide to hydrogen bromide of 1:(1.2-1.8), it helps to activate as much group A polysaccharide as possible, thereby further improving the polysaccharide yield and protein yield of the polysaccharide conjugate.

[0105] Comparing the detection results of Example 1 with those of Examples 11-13, it can be seen that the present application uses carbodiimide as an activator. When carbodiimide is added to a concentration of 0.01-0.2 mol / L, it can activate carboxyl groups, promoting the formation of amides and esters, thereby activating group A polysaccharides. After the group A polysaccharides are activated, the carbodiimide can also cooperate with adipic hydrazide to participate in the derivatization reaction of group A polysaccharides, improving the derivatization effect of group A polysaccharides, thereby improving the polysaccharide yield and protein yield of the polysaccharide conjugate. Among these, a concentration of 0.1-0.2 mol / L of carbodiimide is further preferred, and its effect is better than that of hydrogen bromide as an activator.

[0106] Comparing the detection results of Example 1 with those of Examples 14-16, it can be seen that the activation efficiency of hydrogen bromide on group A polysaccharides is limited. In the preferred step ① of this application, the weight ratio of group A meningococcal capsular polysaccharide to adipic hydrazide is 1:(2-5). At this time, the derivatization rate of polysaccharide derivatives is relatively high, which can further improve the polysaccharide yield and protein yield of polysaccharide conjugates.

[0107] Comparing the detection results of Example 1 with those of Examples 17-19, it can be seen that when the concentration of carbodiimide in the initial reactant is set at 10-100 mmol / L, the carbodiimide can effectively promote the binding reaction between group A polysaccharide and diphtheria CRM197 protein. A further preferred concentration is 40-60 mmol / L.

[0108] Comparing the detection results of Example 1 with those of Examples 20-22, it can be seen that the addition of an appropriate amount of trehalose to the equilibrium solution in this application has a negligible effect on the binding of group A polysaccharides to diphtheria CRM197 protein. In addition, it can increase the stability of the polysaccharide conjugate to a certain extent, effectively improving the polysaccharide yield and protein yield of the polysaccharide conjugate.

[0109] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A process for the preparation of a group A meningococcal capsular polysaccharide conjugate characterised in that, Prepare according to the following steps: ① Preparation of polysaccharide derivatives: The polysaccharide of group A meningococcal capsular polysaccharide was dissolved in physiological saline to obtain polysaccharide solution a. The pH of polysaccharide solution a was adjusted to 10.5±0.2 with sodium hydroxide solution. An activator was added for activation to obtain polysaccharide solution b. Adipic hydrazine was added to polysaccharide solution b for derivatization. The pH of the derivatization reaction was maintained at 9.0±0.2 to obtain polysaccharide solution c. The solution was stirred at low temperature at 2-8℃ for 6-20h. After ultrafiltration, the polysaccharide derivative stock solution was obtained. ② Mixing ratio: Under the condition of temperature control at 4-8℃, the polysaccharide derivative stock solution obtained in step ① is prepared into a derivative solution with a pH of 6.0±0.3 using physiological saline and hydrochloric acid solution, and the concentration of polysaccharide derivative in the derivative solution is 0.5-5.0mg / mL. Diphtheria CRM197 protein is added and stirred to mix, wherein the weight ratio of polysaccharide derivative to diphtheria CRM197 protein is 1:(0.8-2.5), and an equilibrium solution is obtained; ③ Combination reaction: Under the condition of temperature control at 4-8℃, carbodiimide is added to the equilibrium solution obtained in step ② to carry out the combination reaction and obtain the initial reactant; ④ Aging treatment: Place the initial reactant obtained in step ③ in a sterile environment at 2-8℃ and let it stand for 16-20 hours to perform aging treatment and obtain the aged reactant. ⑤ Ultrafiltration: The aged reactants obtained in step ④ are subjected to ultrafiltration to remove excess residual reagents from the binding reaction process, and a concentrated solution of the binding compound is obtained. ⑥ Sterilization: The concentrated solution of the conjugate obtained in step ⑤ is sterilized by using a sterile filter membrane to obtain the stock solution of group A meningococcal capsular polysaccharide conjugate.

2. The method of claim 1, wherein: In step ①, the physiological saline is preheated to 30±2℃ to dissolve the capsular polysaccharide of group A meningococcus.

3. The method of claim 1, wherein: In step ①, the supernatant of the polysaccharide solution a is taken after centrifugation and the pH is adjusted.

4. The method of claim 1, wherein: In step ①, the activation temperature is 25±2℃.

5. The method of claim 1, wherein: In step ①, the polysaccharide solution c is stirred and then subjected to ultrafiltration with physiological saline.

6. The method of claim 1, wherein: In step ①, the activator is a hydrogen bromide solution, and the weight ratio of the group A meningococcal capsular polysaccharide to the hydrogen bromide in the hydrogen bromide solution is 1:(1.2-1.8).

7. The method of claim 1, wherein: In step ①, the activator is carbodiimide, and the carbodiimide is added to a concentration of 0.01-0.2 mol / L.

8. The method of claim 1, wherein: In step ①, the weight ratio of the group A meningococcal capsular polysaccharide to the adipic hydrazide is 1:(2-5).

9. The method of claim 1, wherein: In step ③, the concentration of carbodiimide in the initial reactant is 10-100 mmol / L.

10. The method of claim 1, wherein: In step ③, trehalose is also added to the equilibrium solution, and the concentration of trehalose is added to 1-5 mmol / L.