WO2017036136A1

WO2017036136A1 - A-group and c-group neisseria meningitidis polysaccharide conjugate vaccine activating process

Info

Publication number: WO2017036136A1
Application number: PCT/CN2016/078081
Authority: WO
Inventors: 吴强; 伍长华; 谭勇
Original assignee: 成都欧林生物科技股份有限公司
Priority date: 2015-08-31
Filing date: 2016-03-31
Publication date: 2017-03-09
Also published as: CN105031634A

Abstract

Disclosed is an A-group and C-group Neisseria meningitidis polysaccharide conjugate vaccine activating process, comprising the following steps: (1) cultivating Neisseria meningitidis; (2) extracting crude polysaccharide by using the cultivated Neisseria meningitidis; (3) purifying the crude polysaccharide to prepare refined Neisseria meningitidis polysaccharide; and (4) activating the refined polysaccharide by using CDAP, and then reacting with ADH to generate a polysaccharide derivative.

Description

Title: Inventive name: Group A Group C meningococcal polysaccharide conjugate vaccine activation process

[0001] The present invention relates to a method for cultivating a strain, and specifically relates to a group A group C meningococcal polysaccharide-binding vaccine activation process.

Background technique

[0002] Epidemic cerebrospinal meningitis (abbreviated as ECM, the same below) is an acute respiratory infection caused by Neisseria mening gitidis. For more than 100 years, it has been prevalent or scattered around the world, causing sepsis and meningitis after infection with pathogens. The susceptible population is mainly children, with the highest rate of fulminant deaths, up to 40<3⁄4~60<3⁄4. The incidence rate in every continent in the world is between 1/100,000 and 10/10 million, and the total case fatality rate is 5%~15<3⁄4. Up to 20% of meningitis patients have neurological sequelae, including mental impairment and deafness. Serological classification according to the capsular polysaccharide type can be divided into 13 serotypes, of which group A, group B, and group C account for about 90% of the epidemic group. Group A meningococcal is a major epidemic of the major pathogenic serogroups, especially in the so-called African “Brain Brain Epidemic Belt”, which is a major epidemic every 7-14 years.

[0003] There were five national epidemic epidemics in China in 1938, 1949, 1959, 1967, and 1977; among them, the most prevalent in the spring of 1967, the incidence rate was as high as 403/100,000, and the mortality rate was 5.49<3⁄4. In the past 90% of cases in China, the disease of group A was caused by disease. Now, group B or group C germs have convulsions. Since the beginning of 2003, the incidence of C group cerebral palsy has increased significantly. At present, group A and group C account for more than 50% of all serogroups, and group C still has a tendency to increase further. Therefore, the focus of prevention of epidemic work in China is to prevent group A and group C.

[0004] The most effective way to prevent epidemic cerebrospinal meningitis is to vaccinate. Studies have shown that the capsular polysaccharide of group A group C meningococcus has good immunogenicity, and the capsular polysaccharide can be directly prepared into a vaccine, and the immunized population can obtain immune protection. Since 2007, China has included meningococcal vaccines into national immunization programs, and has universally vaccinated suitable children across the country. The meningococcal polysaccharide vaccine currently included in the national immunization program includes group A meningococcal polysaccharide vaccine, group A group C meningococcal polysaccharide vaccine.

[0005] At present, all domestically produced conjugate vaccines (such as Haemophilus influenzae type b conjugate vaccine, group A C group meningococcal conjugate vaccine) are all prepared by the same chemical combination method, that is, cyanogen bromide (CNBr) activates the ortho-hydroxy group of the polysaccharide, and the activated polysaccharide reacts with adipic hydrazide (ADH) to form a polysaccharide-ADH derivative. The polysaccharide-ADH derivative is then covalently bound to the carrier protein under the catalysis of carbodiimide (EDAC). A major disadvantage of this process is the use of cyanogen bromide during the activation of the polysaccharide. Cyanogen bromide is a highly toxic and active substance that emits highly toxic gases such as hydrogen cyanide and hydrogen cyanide when exposed to heat. It is prone to explosion when exposed to acid. The toxic effect of cyanogen bromide is similar to that of hydrocyanic acid, which is highly irritating to the eyes and skin. For people, at very low concentrations, it can irritate the eyes, throat and tears, cough; can not endure at a concentration of 0.05mg / L (20PPM) for 1 minute; if under 120mg / m ³ conditions, contact After 30 minutes, he died. During the First World War, hydrogen bromide was used as a poison gas for the Australian army.

[0006] With the increased awareness of environmental protection and the emphasis on the working environment of workers, people have been looking for appropriate ways to avoid the use of highly toxic chemicals such as cyanogen bromide in the vaccine manufacturing process. The use of modern technology to improve traditional production processes to reduce or eliminate toxic chemical agents is important for protecting human health and reducing environmental pollution.

technical problem

[0007] The object of the present invention is to solve the problems in the prior art that the use of highly toxic chemical reagents such as cyanogen bromide in the manufacture of a vaccine leads to problems that are not conducive to protecting human health and the environment, and provides a group C that solves the above problems. Group meningococcal polysaccharide conjugate vaccine activation process.

Problem solution

Technical solution

[0008] In order to solve the above disadvantages, the technical solution of the present invention is as follows:

[0009] A group C group meningococcal polysaccharide conjugate vaccine activation process, comprising the following steps:

[0010] (1) cultivating meningococcal bacteria;

[0011] (2) extracting the crude polysaccharide using the cultured meningococcus;

[0012] (3) the crude polysaccharide is purified to make meningococcal refined sugar;

[0013] (4) activation and derivatization of refined sugar, the activation and derivatization process of the refined sugar is as follows:

[0014] (41) Weigh meningococcal refined sugar and dissolved in water for injection, formulated into a concentration of 5mg / ml of polysaccharide solution, and adjusted to pH 9.0;

[0015] (42) Dissolving CDAP in acetonitrile to a concentration of 100 mg/ml of CDAP solution; then pressing polysaccharide: CD AP = 1: 0.5 weight ratio, adding CDAP solution to the polysaccharide solution, stirring at room temperature for 2 to 5 minutes to prepare a CDA mixture;

[4316] (43) Dissolve ADH with 0.2 mol / L of NaHC0 ₃ into a concentration of 100 mg / ml of ADH solution, according to the weight ratio of polysaccharide: ADH = 1: 3.5, add ADH solution to the CDAP mixture, stir at room temperature 1 hour

[0017] (44) An O.05 mol/L sodium chloride solution was used as the ultrafiltrate, and the mixture was filtered through an ultrafiltration membrane package, and finally freeze-dried to obtain a polysaccharide-ADH derivative.

[0018] All the chemicals used in the present invention are non-toxic substances, thereby effectively ensuring human health and avoiding environmental pollution.

[0019] The method of the invention optimizes the proportion and concentration of each chemical substance added to the reaction enthalpy, effectively improves the ADH derivative rate, and the derivatization effect is better.

Further, in order to make the cultured meningococcal bacteria less, it is easier to purify the extracted polysaccharide, and the extraction rate of the polysaccharide in the capsule is higher. The specific process of the step (1) is as follows

[0021] (11) Preparation of purified working seed strains: Group A strain of N. meningitidis is inoculated onto a purification medium, and incubated at 38 ° C for 20 hours to select robust and no-bacteria-contaminated The strain is inoculated on the purification medium for secondary culture, and then incubated at 38 ° C for 12 h. The robust and bacteria-free bacteria are added to the sterile skim milk, and the mixture is lyophilized to obtain purified. Working seed strains;

[0022] (12) Dissolving the working seed strain in sterile water, inoculation on 10% sheep blood agar medium, and culturing in the environment of 35~37 ° C for 16~20 hours, then selecting a vigorous and no-bacteria-contaminated strain; inoculate the purified seed after inoculation into a semi-integrated liquid medium for activation culture; the conditions of the activation culture are temperature 35 to 37 ° C, pH 7.0 to 7.2, stirring speed 140~150r/min, the culture medium is 6~8h; finally, the activated purified seeds are cultured in three generations to prepare a suitable amount of production seeds; wherein the medium for expansion culture is semi-integrated liquid medium. Medium; cultivation conditions are temperature 35~37 ° C, p H7.0~7.2, stirring speed 140~150r/min;

[0023] (13) The seed for production is inoculated into the fermenter for 6 to 12 hours; the medium in the fermenter is a semi-integrated medium, the temperature is 35 to 37 ° C, and the pH of the medium is 7.0 to 7.2. The stirring speed in the fermenter is 140 to 150 r/min, and the inoculum is 10 to 150,000/ml. [0024] As a preferred method, the specific process of the step (2) is as follows:

[0025] (21) the cultured meningococcal bacteria are sterilized by formaldehyde, and the sterilized culture liquid is centrifuged to collect the supernatant;

[0026] (22) adding a final concentration of CTAB to CTAB to the supernatant of 1.0 g / L ,, and then stirring well, standing overnight, and collecting the polysaccharide by centrifugation;

[0027] (23) The precipitated polysaccharide was stirred with a calcium chloride solution for 3 hours to dissociate the polysaccharide and CTAB, and the supernatant was collected by centrifugation;

[0028] (24) Ethanol was added to the supernatant until the final concentration of ethanol reached 25%, at 2 to 8. C is allowed to stand overnight, and the supernatant is collected by centrifugation; then ethanol is added to the supernatant until the final concentration of ethanol reaches 75 to 80%, and the polysaccharide is precipitated by shaking sufficiently, and then allowed to stand for 18 hours or more, and the precipitate is collected by centrifugation. ;

[0029] (25) After washing three times with each of anhydrous ethanol and acetone, it is dried, and after drying, it is a crude polysaccharide.

[0030] This step is effective to increase the rate of polysaccharide precipitation in the meningococcal capsule, thereby effectively increasing the yield of the crude polysaccharide.

[0031] In order to improve the recovery and purity of the refined sugar, the specific process of the step (3) is as follows:

[0032] (31) The crude polysaccharide is dissolved in a 8% to 12% saturated neutral sodium acetate solution, and then the volume ratio is 1:

Add a cold phenol solution in a ratio of 0.8 to 1.2, mix by shaking, collect the supernatant by centrifugation, and extract 1 to 3 times to clarify the supernatant;

[0033] (32) collecting the supernatant, ultrafiltration membrane ultrafiltration to remove residual phenol and nucleic acid;

[0034] (33) adding a NaCl solution to the ultra-filtered polysaccharide solution until the final concentration of NaCl is 0.2 to 0.4 mol/L

Then, add ethanol to ethanol to a final concentration of 70 to 78%, and mix well and let stand at 2 to 8 ° C overnight; [0035] (34) Collect the precipitate by centrifugation, and then wash twice with absolute ethanol and acetone. , after drying, it is refined sugar.

[0036] In the present invention, CDAP is 1-cyano-2-methylaminopyridine tetrafluoroborate; ADH is adipic hydrazide; C

TAB is cetyltrimethylammonium bromide.

Advantageous effects of the invention

Beneficial effect

[0037] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0038] 1. The setting of the step (1) of the present invention effectively makes the cultivated group A meningococcal bacteria less dead, mentioning The extracted polysaccharide is easier to purify; and the extraction rate of the polysaccharide in the capsule is higher;

[0039] 2. The present invention effectively increases the rate of polysaccharide precipitation in the meningococcal capsule by the setting of step (2)

, thereby effectively increasing the yield of the crude polysaccharide;

[0040] 3. The invention effectively improves the recovery rate and purity of the refined sugar by the setting of the step (3);

[0041] 4. The invention optimizes the proportion and concentration of each chemical substance added to the reaction enthalpy in the step (4), thereby effectively improving the ADH derivative rate, and the derivatization effect is better;

[0042] 5. All the chemicals used in the present invention are non-toxic substances, thereby effectively ensuring human health.

And avoid environmental pollution.

Embodiments of the invention

The present invention will be further described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto.

Embodiment 1

[0045] A group C group meningococcal polysaccharide conjugate vaccine activation process, comprising the following steps:

[0046] (1) cultivating meningococcal bacteria;

[0047] (11) Preparation of purified working seed strains: Group A group of N. meningitidis strains are inoculated on a purification medium, and incubated at 38 ° C for 20 hours to select robust and non-organic contamination. The strain is inoculated on the purification medium for secondary culture, and then incubated at 38 ° C for 12 h. The robust and bacteria-free bacteria are added to the sterile skim milk, and the mixture is lyophilized to obtain purified. Working seed strains;

[0048] (12) Dissolving the working seed strain in sterile water, inoculation on 10% sheep blood agar medium, and culturing in the environment of 35~37 ° C for 16~20 hours, then selecting a vigorous and no-bacteria-contaminated strain; inoculate the purified seed after inoculation into a semi-integrated liquid medium for activation culture; the conditions of the activation culture are temperature 35 to 37 ° C, pH 7.0 to 7.2, stirring speed 140~150r/min, the culture medium is 6~8h; finally, the activated purified seeds are cultured in three generations to prepare a suitable amount of production seeds; wherein the medium for expansion culture is semi-integrated liquid medium. Medium; cultivation conditions are temperature 35~37 ° C, p H7.0~7.2, stirring speed 140~150r/min;

[0049] (13) The seed for production is inoculated into the fermenter for 6 to 12 hours; the medium in the fermenter is a semi-integrated medium, the temperature is 35 to 37 ° C, and the pH of the medium is 7.0 to 7.2. The stirring speed in the fermenter is 140 ~150r/min, the inoculation amount is 10~150,000/ml.

[0050] In this embodiment, specific cultivation conditions in the above steps are as follows:

[0051] Step (12) Working seed culture strain cultivation condition is 35 ° C, culture 18 hours; activation culture conditions are temperature 37 ° C, pH 7.2, stirring speed 140 r / min, culture daytime 6~8h; the incubation conditions for the expansion culture are temperature 36 ° C, pH 7.0, stirring speed 145r / min;

[0052] The fermentation condition of the fermenter culture in the step (13) is a temperature of 37 ° C, a pH of the medium of 7.0, a stirring speed of 145 r / min, and an inoculum amount of 120,000 / ml;

[0053] and the purification medium used in the step (11) is a chocolate medium added with an antibiotic, which is effective for the purpose of purification; the medium used in the step (12) is the chocolate without the antibiotic added. The medium, that is, 10% sheep blood agar medium, can effectively achieve the effect of the seed strain of the seed by the culture of the medium.

[0054] The medium used for the culture for activation and amplification is a semi-integrated liquid medium, and the ratio of the medium is: beef leachate 1 L, peptone 10 g, sodium chloride 5 g, sterile defibrinated horse blood, sheep blood Or rabbit blood 100ml, glucose 3g, hydrochloric acid casein hydrolysate 2g, pH 7.0~7.2.

[0055] (2) extracting the crude polysaccharide using the cultured meningococcus;

[0056] (21) the cultured meningococcal bacteria are sterilized by formaldehyde, and the sterilized culture liquid is centrifuged to collect the supernatant;

[0057] (22) The final concentration of CTAB to CTAB is added to the supernatant until l.Og / L ,, and then fully stirred, allowed to stand overnight, and the polysaccharide is collected by centrifugation;

[0058] (23) The precipitated polysaccharide was stirred with a calcium chloride solution for 3 hours to dissociate the polysaccharide and CTAB, and the supernatant was collected by centrifugation;

[0059] (24) Ethanol was added to the supernatant until the final concentration of ethanol reached 25%, at 2-8. C is allowed to stand overnight, and the supernatant is collected by centrifugation; then ethanol is added to the supernatant until the final concentration of ethanol reaches 75 to 80%, and the polysaccharide is precipitated by shaking sufficiently, and then allowed to stand for 18 hours or more, and the precipitate is collected by centrifugation. ;

[0060] (25) After washing three times with each of anhydrous ethanol and acetone, it is dried, and after drying, it is a crude polysaccharide.

[0061] (3) the crude polysaccharide is purified to make meningococcal refined sugar;

[0062] (31) The crude polysaccharide is dissolved in a 10% saturated neutral sodium acetate solution, and then the cold phenol solution is added in a ratio of 1:1 by volume, vortexed and mixed, and the supernatant is collected by centrifugation and extracted. Clear the supernatant 1 to 3 times; [0063] (32) collecting the supernatant, ultrafiltration membrane ultrafiltration to remove residual phenol and nucleic acid;

[0064] (33) Add 4mol / L NaCl solution to the ultra-filtered polysaccharide solution until the final concentration of NaCl is 0.3m ol / L, then add 95% ethanol to ethanol, the final concentration of 75%, fully mixed After 2 to 8 ° C, let stand overnight

, precipitation;

[0065] (34) The precipitate was collected by centrifugation, and then washed twice with each of anhydrous ethanol and acetone, and dried to obtain refined sugar.

[0066] (4) Activation and derivatization of refined sugar, the activation and derivatization of the refined sugar is as follows:

[0067] (41) Weigh meningococcal refined sugar and dissolved in water for injection, formulated into a concentration of 5mg / ml of polysaccharide solution, and adjusted to pH 9.0;

[0068] (42) Dissolving CDAP in acetonitrile to a concentration of 100 mg/ml of CDAP solution; then pressing polysaccharide: CD

AP = 1: 0.5 weight ratio, add CDAP solution to the polysaccharide solution, stir at room temperature for 2 to 5 minutes to make C

DAP mixture;

[43] (43) ADH was dissolved in 0.2 mol / L of NaHC0 ₃ into a concentration of 100 mg / ml of ADH solution, according to the weight ratio of polysaccharide: ADH = 1: 3.5, add ADH solution to the CDAP mixture, stir at room temperature 1 hour

[0070] (44) An O.05 mol/L sodium chloride solution was used as the ultrafiltrate, and the mixture was filtered through an ultrafiltration membrane package, and finally freeze-dried to obtain a polysaccharide-ADH derivative.

Example 2

[0072] This embodiment differs from Example 1 only in that the polysaccharide and C in the step (42) described in the present embodiment.

The weight ratio of DAP is 1: 0.6; the weight ratio of polysaccharide to ADH in the step (43) is 1:4.

Example 3

[0074] The difference between this embodiment and the embodiment 1 is only the ratio and concentration of the chemical substances added to the reaction enthalpy in the present embodiment, and the specific settings are as follows:

[0075] The concentration of the polysaccharide solution in step (41) is 10 mg / ml, and the pH is adjusted to 9.0 with sodium hydroxide;

[0076] The concentration of the CDAP solution in the step (42) is 80 mg/ml, and the polysaccharide: CDAP=1: 0.7;

[0077] The concentration of the ADH solution in the step (43) is 80 mg/ml of the ADH solution, and the polysaccharide: ADH=1:5.

Example 4

[0079] This embodiment is a comparative embodiment of Embodiment 1, and the method used in the step (4) in this embodiment is different, and The specific method is as follows:

[0080] The cyanogen bromide was dissolved in acetonitrile to a solution of 100 mg/ml. Group A meningococcal saccharin was dissolved in water for injection to prepare a solution having a concentration of 5 mg/ml, and the pH was adjusted to 9.0. The cyanogen bromide solution was added in a ratio of polysaccharide: cyanogen bromide = 1:0.5 (w/w). The temperature was maintained at 23 ± 3 ° C and the pH was 10.8 ± 0.2 for 30 minutes for activation. After the activation was completed, the adipic dihydrazide solution was added in a ratio of polysaccharide: adipic hydrazide = 1:3.5 (w/w). Derivatization was carried out by maintaining the temperature at 23 ± 3 ° C and reacting at pH 8.5 ± 0.2 for 15 minutes. After the reaction, the O.05 mol/L sodium chloride solution was used as the ultrafiltrate, and ultrafiltration was used to remove the cyanogen bromide by ultrafiltration, which is the reference substance of the polysaccharide-ADH derivative.

[0081] The samples of Examples 1 to 4 were sampled three times, and the ADH derivatization rate was measured. The detection results of the first sampling, the second sampling, and the third sampling of Example 1 were 1.30%, 1.26%, and 1.33%, respectively; The detection results of the first sampling, the second sampling, and the third sampling of Example 2 were 1.22%, 1.20%, and 1.17%, respectively; the first sampling, the second sampling, and the third sampling of Example 3 were performed. The detection results were 0.97%, 0.87%, 0.9 4%, respectively; the detection results of the first sampling, the second sampling, and the third sampling of Example 4 were 1.12%, 1.08%, and 1.05%, respectively; According to the structure, all the chemical substances used in the step (4) of the present invention are non-toxic substances, which effectively ensure human health and avoid environmental pollution; and the chemical substances mentioned in the step (4) are added. The ratio and concentration effectively increase the ADH derivatization rate and make the derivatization effect better.

Example 5

[0083] The difference between this embodiment and the embodiment 1 is that the specific cultivation conditions in each step (1) are different, and the specific settings are as follows:

[0084] The working condition of the working seed batch strain in the step (12) is 36 ° C, and the culture is carried out for 16 hours; the conditions of the activated culture are the temperature 37 ° C, the pH 7.0, the stirring speed 145 r / min, and the culture The incubation conditions were 8 °C, pH 7.0, and stirring speed 150 r/min _{. The} fermentation conditions of the fermenter culture in the step (13) were 37 ° C and the pH of the medium was 7.0. The stirring speed is 150r/min, and the inoculation amount is 150,000/ml.

Example 6

[0086] This embodiment differs from the first embodiment only in that the ratio and concentration of each chemical substance added to the reaction enthalpy in each step (3) are different, and the specific settings are as follows: [0087] in step (31), the crude polysaccharide is dissolved in 8% saturated neutral sodium acetate solution, and then the cold phenol solution is added in a ratio of 1:1.2 by volume, vortexed and mixed, and the supernatant is collected by centrifugation, and The supernatant is clarified by extracting 1 to 3 times;

[0088] In step (33), the concentration of the NaCl solution is 2 mol/L until the final concentration of NaCl is 0.4 mol/L, and then 95% ethanol is added to a final concentration of ethanol of 70%, and fully mixed after 2 to 8 ° C. Allow to stand overnight.

Example 7

[0090] The difference between this embodiment and the embodiment 1 is that the proportions and concentrations of the chemical substances added to the reaction enthalpy in the step (3) in the embodiment are different, and the specific settings are as follows:

[0091] in step (31), the crude polysaccharide is dissolved in a 15% saturated neutral sodium acetate solution, and then the volume ratio is

The ratio of 1:2 was added to the cold phenol solution, and the mixture was shaken and mixed, and the supernatant was collected by centrifugation, and extracted 1 to 3 times to clarify the supernatant;

[0092] The concentration of the NaCl solution in the step (33) is 3 mol / L, until the final concentration of NaCl is 0.5 mol / L, and then adding 95% ethanol to the final concentration of ethanol is 60%, fully mixed after 2 ~ 8 ° C Allow to stand overnight.

[0093] The fine sugars prepared in Example 1, Example 6, and Example 7 were sampled, and the purity and recovery rate of the refined sugar were examined. The test results were as follows: The purity of Example 1 was 99%, and the recovery rate was 80%. The purity of Example 2 is

97%, the recovery was 81%; the purity of Example 3 was 89%, and the recovery was 70%. It can be seen from the above test results that after purification by the respective reagents of the present invention, the purity of the purified refined sugar can be over 95%, and the recovery rate is also higher.

The above embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any changes made by the design principles of the present invention and non-inventive work on the basis of the present invention should belong to the present invention. Within the scope of protection.

Claims

Claim

[Claim 1] Group A Group C meningococcal polysaccharide conjugate vaccine activation process, comprising the steps of:

(1) cultivating meningococcal bacteria;

(2) extracting the crude polysaccharide by using cultured meningococcus;

(3) purifying the crude polysaccharide to make meningococcal refined sugar;

(4) Activation and derivatization of refined sugar, the activation and derivatization of the refined sugar is as follows:

(41) Weighing meningococcal refined sugar and dissolving it in water for injection, preparing a polysaccharide solution having a concentration of 5 mg/ml, and adjusting the pH to 9.0;

(42) Dissolve CDAP in acetonitrile to a concentration of 100 mg / ml of CDAP solution; then add the CDAP solution to the polysaccharide solution at a weight ratio of polysaccharide: CDAP = 1: 0.4 to 0.6, and stir at room temperature for 2 to 5 minutes to make CDAP. Mixture

(43) Dissolve ADH with 0.2 mol/L NaHCO ₃ into a concentration of 100 mg/ml ADH solution, add ADH solution to CDAP mixture according to the weight ratio of polysaccharide: ADH=1:3~5, stir at room temperature. Xiao Yan;

(44) O.05 mol/L sodium chloride solution was used as the ultrafiltrate, which was filtered through an ultrafiltration membrane package, and finally freeze-dried to obtain a polysaccharide-ADH derivative.

The group A group C meningococcal polysaccharide conjugate vaccine activation process according to claim 1, wherein the specific process of the step (1) is as follows:

(11) Preparation of purified working seed strains: Inoculate Group A N. meningitidis strains onto the purification medium, and incubate at 38 ° C for 20 h to select robust and no-bacteria-contaminated strains. Inoculate on the purification medium for secondary culture, and then incubate at 38 °C for 12 h, select the robust and bacteria-free bacteria into the sterile skim milk, mix and freeze to prepare the purified working seeds. Batch of bacteria;

(12) Dissolve the working seed strain in sterile water, inoculate it on 10% sheep blood agar medium, and incubate it in the environment of 35~37 °C for 16~20 hours, then select robust and The fungus contaminated with the bacteria; inoculate the purified seed after inoculation into semi-integrated liquid medium for activation culture; the conditions of activation culture are temperature 35~37 ° C, pH 7.0~7.2, stirring The speed is 140~150r/min, and the culture time is 6~8h; finally, the activated purified seeds are subjected to three generations of expansion culture to prepare a suitable amount of production seeds; wherein the medium for expansion culture is semi-integrated liquid culture. Base conditions; incubation conditions are temperature ₃₅ ~ ₃₇ ° _C , _p H7.0~7

.2, stirring speed 140~150r/min;

(13) Inoculating the production seeds into the fermenter for 6~12 hours; the medium in the fermenter is semi-integrated medium, the temperature is 35~37°C, the medium pH is 7.0~7.2, in the fermenter The stirring speed was 140 to 150 r/min, and the inoculum amount was 10 to 150,000/ml.

The group A group C meningococcal polysaccharide conjugate vaccine activation process according to claim 1, wherein the specific process of the step (2) is as follows:

(21) The cultured meningococcal bacteria are sterilized by formaldehyde, and the sterilized culture liquid is centrifuged to collect the supernatant;

(22) Adding CTAB to CTAB to a final concentration of 1.0 g/L 上 in the supernatant, then stirring well, allowing to stand overnight, and collecting the polysaccharide by centrifugation;

(23) The precipitated polysaccharide was stirred with a calcium chloride solution for 3 hours to dissociate the polysaccharide and CTAB, and the supernatant was collected by centrifugation;

(24) Add ethanol to the supernatant until the final concentration of ethanol reaches 25%, stand at 2~8 °C overnight, collect the supernatant by centrifugation; add ethanol to the supernatant until the final concentration of ethanol 75~80%, fully shaken to precipitate the polysaccharide, then allowed to stand for more than 18 hours, and collect the precipitate by centrifugation;

(25) After washing three times with absolute ethanol and acetone, it is dried, and after drying, it is a crude polysaccharide.

The group A group C meningococcal polysaccharide conjugate vaccine activation process according to claim 1, wherein the specific process of the step (3) is as follows:

(31) Dissolving the crude polysaccharide in 8%~12% saturated neutral sodium acetate solution, then adding cold phenol solution in a ratio of 1:0.8~1.2 by volume, vortexing and mixing, and collecting the supernatant by centrifugation, and The supernatant is clarified by extracting 1 to 3 times;

(32) collecting the supernatant, and ultrafiltration membrane ultrafiltration to remove residual phenol and nucleic acid;

(33) Add NaCl solution to the ultra-filtered polysaccharide solution until the final concentration of NaCl is 0.2~0.4mol/L, then add ethanol to the final concentration of ethanol to 70~78%, mix well. After standing at 2~8 °C overnight;

(34) The precipitate was collected by centrifugation, and then washed twice with absolute ethanol and acetone, respectively, and dried to obtain refined sugar.