CN117756958A - Method for preparing streptococcus pneumoniae capsular polysaccharide or polysaccharide degradation thereof - Google Patents
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- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention relates to the technical field of biological products, in particular to a method for preparing streptococcus pneumoniae capsular polysaccharide or polysaccharide degradation thereof. The method for preparing streptococcus pneumoniae capsular polysaccharide or polysaccharide degradation thereof provided by the invention comprises the following steps: after sterilizing a fermentation culture of streptococcus pneumoniae, separating a supernatant, and sequentially performing a first ultrafiltration treatment, a precipitation treatment and a second ultrafiltration treatment on the supernatant; or sequentially performing a first ultrafiltration treatment, a precipitation treatment, a degradation treatment and a second ultrafiltration treatment on the supernatant; the precipitant used in the precipitation treatment comprises a calcium salt, and the precipitation treatment is carried out at a pH of 2.4-3.6. The method has the advantages of simple process and short preparation period, remarkably reduces the time and material cost, ensures that the quality control indexes of impurities such as nucleic acid, protein and the like of the prepared refined polysaccharide and degraded polysaccharide, specific groups and the like are better than the standard requirements, and can be used for preparing streptococcus pneumoniae capsular polysaccharide vaccines or conjugate vaccines.
Description
Technical Field
The invention relates to the technical field of biological products, in particular to a method for preparing streptococcus pneumoniae capsular polysaccharide or polysaccharide degradation thereof.
Background
Streptococcus pneumoniae infection is one of the leading causes of death worldwide, and is also the main cause of pneumonia, meningitis, and otitis media. With the increasing use of antibiotics, resistant strains are increasingly attracting attention in vaccine development. The study shows that the capsular polysaccharide on the surface of bacteria can protect organisms against bacterial infection, and the polysaccharide is a non-thymus dependent antigen, so that the secondary immunity can not lead the reduced antibody to reach the level of primary immunity, the production of immune memory cells can not be induced, and the capsular polysaccharide has little protective effect on infants and old people, and the age group crowd is a high risk group of streptococcus pneumoniae infection. The streptococcus pneumoniae capsular polysaccharide is coupled to a protein carrier, so that the streptococcus pneumoniae capsular polysaccharide becomes thymus-dependent antigen, B cells can be activated to generate antibodies under the assistance of T cells and macrophages, immune protection is provided for infants, immune memory is induced, and the immunity is enhanced again. Studies have shown that the introduction of streptococcus pneumoniae vaccines significantly reduces the burden of global streptococcus pneumoniae disease.
Streptococcus pneumoniae capsular polysaccharides are a series of high molecular weight homologs made up of repeating units, typically obtained by fermentation culturing streptococcus pneumoniae and extraction purification from the fermentation culture. On the one hand, when preparing streptococcus pneumoniae capsular polysaccharide vaccines, the problems of complex polysaccharide purification process, long purification period, safety or health risk of using reagents and the like exist, for example: in the existing preparation method of streptococcus pneumoniae refined polysaccharide, organic reagents such as phenol, ethanol and the like are often adopted, so that the health of human bodies is endangered and the polysaccharide is difficult to completely remove; the process is complex and the time is long when the streptococcus pneumoniae refined polysaccharide is prepared; in addition, chromatography systems (e.g., china patent application CN104815327A, CN108079286A, CN112646050A, CN116970095A, etc.) are often used in the preparation of purified polysaccharides, and the apparatus and materials are expensive, greatly increasing the production cost. Chinese patent application CN116970095A discloses a process for removing protein and nucleic acid impurities by a two-step precipitation method, wherein sodium Deoxycholate (DOC) is mixed with a first ultrafiltrate under the condition of proper neutral salt, the pH value of the solution is controlled to be 4.5-5.0, the effect of adsorbing and precipitating protein by sodium deoxycholate can be effectively exerted, impurities such as protein and nucleic acid are further removed by calcium salt precipitation, supernatant is collected by centrifugation, and the supernatant is washed, filtered and freeze-dried to obtain refined polysaccharide. However, the method still has the problems of complex process, large consumption of sodium deoxycholate, long purification period and the like. Therefore, a purification method with simple and efficient process and higher safety is required to be developed.
On the other hand, when preparing streptococcus pneumoniae capsular polysaccharide conjugate vaccine, the polysaccharide often has larger molecular weight, and the too large molecular weight is unfavorable for the combination of the polysaccharide, and the too small molecular weight can affect immunogenicity, so that the polysaccharide macromolecule needs to be degraded into the polysaccharide with small molecular weight by a certain method so as to meet the requirements and the application of the streptococcus pneumoniae conjugate vaccine. The existing capsular polysaccharide degradation process is to dissolve and degrade the streptococcus pneumoniae refined polysaccharide freeze-dried preparation, has complex operation and is not suitable for large-scale production and application.
Disclosure of Invention
the invention provides a method for preparing streptococcus pneumoniae capsular polysaccharide or polysaccharide degradation thereof.
In the research and development process of the streptococcus pneumoniae capsular polysaccharide preparation method, a simple and rapid streptococcus pneumoniae polysaccharide preparation method is discovered accidentally, the method can prepare high-quality refined polysaccharide by only carrying out one-step precipitation (one-step calcium salt acid precipitation) under an acidic condition and matching with a conventional ultrafiltration step, and various indexes of the prepared refined polysaccharide are higher than the Chinese pharmacopoeia standard, so that the material and time cost is obviously reduced, and the purification efficiency is improved.
Specifically, the invention provides the following technical scheme:
The invention provides a method for preparing streptococcus pneumoniae capsular polysaccharides or degradation polysaccharides thereof, comprising the following steps: after sterilizing a fermentation culture of streptococcus pneumoniae, separating a supernatant, and sequentially performing a first ultrafiltration treatment, a precipitation treatment and a second ultrafiltration treatment on the supernatant;
or sequentially performing a first ultrafiltration treatment, a precipitation treatment, a degradation treatment and a second ultrafiltration treatment on the supernatant;
Wherein the precipitating agent used in the precipitating treatment comprises a calcium salt, and the precipitating treatment is carried out under the condition of pH 2.4-3.6.
The preparation method of the invention is divided into two implementation modes of degradation treatment and degradation treatment, the molecular weight of the capsular polysaccharide (refined polysaccharide) prepared by the method without degradation treatment is relatively large, the capsular polysaccharide is suitable for preparing streptococcus pneumoniae capsular polysaccharide vaccines, the polysaccharide (degraded polysaccharide) with reduced molecular weight can be obtained by degradation treatment, and the capsular polysaccharide is more suitable for preparing streptococcus pneumoniae capsular polysaccharide conjugate vaccines after being combined with carrier proteins (such as TT, DT, CRM and the like).
The invention discovers that on the basis of the one-step calcium hydrochloric acid precipitation method, the degradation treatment can be arranged after the one-step calcium hydrochloric acid precipitation, and the degradation treatment is not needed to be carried out after the prepared freeze-dried refined polysaccharide is redissolved, so that the process flow is greatly simplified, and the preparation efficiency is improved.
The use of calcium salt as precipitant and the precipitation treatment (calcium salt acid precipitation) under acidic condition with pH of 2.4-3.6 are key to the above preparation method. The method is matched with the calcium salt acid precipitation, and ultrafiltration treatments are respectively arranged before and after the calcium salt acid precipitation, wherein the primary effect of the first ultrafiltration treatment is to remove small molecular impurities in supernatant fluid after fermentation culture sterilization treatment, so that the calcium salt acid precipitation is more beneficial to better play a role in removing impurities, the impurity removal efficiency is improved, and the primary effect of the second ultrafiltration treatment is to concentrate polysaccharide solution and wash and filter the polysaccharide solution, so as to further remove the small molecular impurities and salt ions in the polysaccharide solution.
Preferably, the calcium salt is calcium chloride.
Preferably, the final concentration of the calcium salt in the precipitation system is 80-200 mmol/L.
Preferably, calcium chloride with a final concentration of 80-200 mmol/L is added to the first ultrafiltration concentrate obtained by the first ultrafiltration treatment, and the pH is adjusted to 2.4-3.6 by using an acid solution to perform precipitation treatment.
preferably, the precipitation treatment is to collect the supernatant after 1-5 hours of treatment at 2-8 ℃ to obtain the first supernatant.
Unlike some available streptococcus pneumoniae capsular polysaccharide purifying methods, which require elevated temperature and acid precipitation (which is easy to degrade polysaccharide and lower molecular weight to affect immunogenicity), the present invention can realize good impurity eliminating effect only at low temperature.
for embodiments comprising a degradation treatment step, the degradation treatment is performed using a chemical treatment or a physical treatment.
wherein, the chemical treatment method preferably adopts trifluoroacetic acid for degradation treatment to obtain degradation treatment products.
The physical treatment method is preferably a degradation treatment by adopting an ultrasonic treatment method or a high-pressure homogenization treatment method, so as to obtain a degradation treated product.
Specifically, the ultrasonic treatment method can be referred to in Chinese patent CN108079286B, and the high-pressure homogenization treatment method can be referred to in Chinese patent application CN106413747A.
In some embodiments of the invention, the degradation treatment is performed with trifluoroacetic acid, wherein the final concentration of trifluoroacetic acid is 0.2-2mol/L. Preferably, the degradation treatment is a treatment at 25-60 ℃ for 2-10 hours. After the degradation treatment was completed, the supernatant was collected.
the degradation treatment described above degrades the macromolecular polysaccharide into a degraded polysaccharide of suitable molecular weight suitable for use in conjugate vaccine preparation without affecting its immunogenicity.
the method further comprises the steps of: and (3) regulating the pH value of the first supernatant obtained by precipitation treatment or the degradation treatment product to 6.8-7.5, collecting the supernatant to obtain a second supernatant, and carrying out second ultrafiltration treatment on the second supernatant.
in the present invention, the pH adjustment to acidity can be performed using acid solutions, including, but not limited to, glacial acetic acid, hydrochloric acid, phosphoric acid, etc.; phosphoric acid is preferred. The pH is adjusted to neutral or alkaline by alkali liquor, including but not limited to sodium hydroxide, potassium hydroxide and the like; sodium hydroxide is preferred.
in the above method, the first ultrafiltration treatment is performed using an ultrafiltration membrane having a pore size of 100 to 150KD, preferably 100 KD.
In some embodiments of the invention, the first ultrafiltration treatment is an isovolumetric ultrafiltration after concentration 3-5 times using a membrane packet having a pore size of 100 KD. Preferably, the volume of purified water used for ultrafiltration is 4-6 times the volume of the concentrate.
In the method, for the second supernatant which is not degraded, the second ultrafiltration treatment is performed by using an ultrafiltration membrane with the pore diameter of 100-150 KD; for the second supernatant obtained by the degradation treatment, the second ultrafiltration treatment is carried out using an ultrafiltration membrane having a pore size of 30 to 50KD (preferably 30 KD).
Preferably, in the second ultrafiltration treatment, the volume of water for injection used for washing and filtering is 8-15 times the volume of the supernatant.
Preferably, after the second ultrafiltration treatment, the method further comprises the step of drying the second ultrafiltration concentrate obtained by the second ultrafiltration treatment.
the drying may be freeze drying.
in the present invention, the separation supernatant may be subjected to conventional solid-liquid separation methods, including, but not limited to, centrifugation.
The method of preparation according to the present invention is not particularly limited in principle, and the fermentation culture of Streptococcus pneumoniae may be sterilized by using sodium deoxycholate which is currently used (for example, the sterilization method used in WO2023202607A1 and CN 114106210A), or may be sterilized by using other agents which kill Streptococcus pneumoniae and release capsular polysaccharides.
in some embodiments of the invention, the fermentation culture of Streptococcus pneumoniae is sterilized by adding sodium deoxycholate to a final concentration of 0.06% -0.24%.
in other embodiments of the invention, beta Propiolactone (BPL) is used to sterilize a fermentation culture of streptococcus pneumoniae. For the final concentration of beta-propiolactone in the treatment system, it may be added in the amounts usual in the art. Preferably, the final concentration of the beta-propiolactone in the treatment system is not less than 0.01%. Exemplary final beta-propiolactone concentrations may be 0.01%, 0.025%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, etc. More preferably, the final concentration of the beta-propiolactone in the treatment system is not less than 0.05%. In some embodiments the final concentration of beta-propiolactone in the treatment system is 0.05-1%. Preferably 0.05-0.5%. The temperature of the treatment is preferably 2-8 ℃. The treatment time is preferably 8 to 16 hours. More preferably 10-14h. The treatment is preferably a low temperature incubation treatment after uniform mixing.
preferably, the treatment is performed by adding beta-propiolactone (BPL) at the late stage of logarithmic growth of the streptococcus pneumoniae fermentation culture. After adding beta-propiolactone (BPL), stirring and mixing uniformly, and then incubating at low temperature.
in the case of sterilization treatment using beta-propiolactone, the above-described preparation method preferably does not use sodium deoxycholate.
In the existing production method of streptococcus pneumoniae capsular polysaccharide, sodium deoxycholate is used as a cracking agent for cracking thalli to release capsular polysaccharide, which plays a very important role in extracting capsular polysaccharide, and no substitute reagent can be used for preparing streptococcus pneumoniae capsular polysaccharide at present. The beta-propiolactone has the obvious effects of inactivating streptococcus pneumoniae and promoting the streptococcus pneumoniae to release capsular polysaccharide, can be used for treating the streptococcus pneumoniae to extract capsular polysaccharide, and is different from deoxycholate sodium, the beta-propiolactone does not crack thalli, thereby obviously reducing the release of impurities such as protein, nucleic acid and the like in thalli, being more beneficial to reducing the impurity content and the removal difficulty in the purification process of the capsular polysaccharide, being easy to hydrolyze, and being nontoxic and harmless to hydrolysate, so that the beta-propiolactone does not cause residues or health risks in capsular polysaccharide products. Beta-propiolactone is currently used as a virus inactivating agent for preparing virus vaccines, and has not been reported so far regarding the function of promoting streptococcus pneumoniae to release capsular polysaccharide and reducing release of intracellular proteins, nucleic acids and other impurities. In the method, beta-propiolactone is used for replacing sodium deoxycholate to treat the fermentation culture of streptococcus pneumoniae, so that the problems of health risk or side effect caused by sodium deoxycholate residues are solved, the content of impurities such as protein in the extracted capsular polysaccharide is obviously reduced, the subsequent purification and impurity removal are facilitated, the recovery rate of polysaccharide is higher, and the recovery rate is obviously higher than that of the fermentation culture of streptococcus pneumoniae treated by formaldehyde.
In the present invention, the fermentation culture is a culture (e.g., fermentation broth) comprising Streptococcus pneumoniae obtained by fermentation culture of Streptococcus pneumoniae.
The preparation method of streptococcus pneumoniae capsular polysaccharide in the invention can be used for different types of streptococcus pneumoniae, including but not limited to: 1. types 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F.
Preferably, the streptococcus pneumoniae is type 2, 5, 6A, 10A, 11A, 19F.
The invention provides the streptococcus pneumoniae capsular polysaccharide or the polysaccharide degradation thereof, which are prepared by the method for preparing the streptococcus pneumoniae capsular polysaccharide or the polysaccharide degradation thereof.
the invention provides a method for preparing streptococcus pneumoniae capsular polysaccharide or degrading polysaccharide thereof or application of the streptococcus pneumoniae capsular polysaccharide or degrading polysaccharide thereof in preparing streptococcus pneumoniae capsular polysaccharide-containing products.
Preferably, the streptococcus pneumoniae capsular polysaccharide product is a vaccine.
the vaccine comprises streptococcus pneumoniae capsular polysaccharide vaccine or streptococcus pneumoniae capsular polysaccharide conjugate vaccine.
The invention provides a streptococcus pneumoniae capsular polysaccharide vaccine or streptococcus pneumoniae capsular polysaccharide conjugate vaccine, which comprises the streptococcus pneumoniae capsular polysaccharide or degradation polysaccharide thereof.
the streptococcus pneumoniae capsular polysaccharide vaccine or streptococcus pneumoniae capsular polysaccharide conjugate vaccine may be a monovalent or multivalent vaccine.
wherein the multivalent vaccine comprises capsular polysaccharides of at least two types of streptococcus pneumoniae types 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F.
in particular, the streptococcus pneumoniae capsular polysaccharide vaccine may be 23-valent, 24-valent, 25-valent, 26-valent, 27-valent, 28-valent, 29-valent, 30-valent or even higher-valent streptococcus pneumoniae capsular polysaccharide vaccine. Exemplary are streptococcus pneumoniae capsular polysaccharide vaccines at valency 23.
The streptococcus pneumoniae capsular polysaccharide conjugate vaccine may be a 10-valent, 11-valent, 12-valent, 13-valent, 14-valent, 15-valent, 16-valent, 17-valent, 18-valent, 19-valent, 20-valent or even higher-valent streptococcus pneumoniae capsular polysaccharide conjugate vaccine. Exemplary are, for example, a Streptococcus pneumoniae capsular polysaccharide conjugate vaccine at 13, a Streptococcus pneumoniae capsular polysaccharide conjugate vaccine at 20, and a Streptococcus pneumoniae capsular polysaccharide conjugate vaccine at 24.
Illustratively, the 23-valent streptococcus pneumoniae capsular polysaccharide vaccine comprises streptococcus pneumoniae serotypes of type 1, type 3, type 4, type 5, type 6A, type 6B, type 7F, type 8, type 9V, type 10A, type 11A, type 12F, type 14, type 15B, type 18C, type 19A, type 19F, type 22F, type 23F, and type 33F.
illustratively, the 13-valent streptococcus pneumoniae capsular polysaccharide conjugate vaccine comprises streptococcus pneumoniae serotypes of types 4, 5, 6A, 6B, 7F, 9V, 18C, 19A, 19F, 23F, and the like.
Illustratively, the 20-valent streptococcus pneumoniae capsular polysaccharide conjugate vaccine comprises streptococcus pneumoniae serotypes of type 1, type 3, type 4, type 5, type 6B, type 7F, type 8, type 9V, type 10A, type 11A, type 12F, type 14, type 15B, type 18C, type 19A, type 19F, type 22F, type 23F, and type 33F.
Illustratively, the 24-valent streptococcus pneumoniae capsular polysaccharide conjugate vaccine comprises streptococcus pneumoniae serotypes of type 1, type 3, type 4, type 5, type 6A, type 6B, type 7F, type 8, type 9N, type 9V, type 10A, type 11A, type 12F, type 14, type 15B, type 18C, type 19A, type 19F, type 22F, type 23F, and type 33F.
The invention provides a preparation method of streptococcus pneumoniae capsular polysaccharide vaccine or streptococcus pneumoniae capsular polysaccharide conjugate vaccine, which comprises the following steps: the streptococcus pneumoniae capsular polysaccharide or the degradation polysaccharide thereof is prepared by the method for preparing the streptococcus pneumoniae capsular polysaccharide or the degradation polysaccharide thereof, and the streptococcus pneumoniae capsular polysaccharide or the degradation polysaccharide thereof is used as an immunogen to prepare a streptococcus pneumoniae capsular polysaccharide vaccine or a streptococcus pneumoniae capsular polysaccharide conjugate vaccine.
the beneficial effects of the invention at least comprise:
(1) The preparation method has the advantages of simple process and short preparation period, and remarkably shortens the purification process time in the preparation of the refined polysaccharide; the polysaccharide is directly degraded in the later purification stage in the process of preparing the polysaccharide, so that the process flow is greatly simplified. According to measurement and calculation, compared with the technical scheme disclosed in Chinese patent application CN116970095A, when the refined polysaccharide is prepared, 2 days are required for the method disclosed in patent application CN116970095A, but only 1 day is required for the method disclosed by the invention, and the process time is shortened by 50%; in the preparation of the degraded polysaccharide: the method disclosed in the patent application CN116970095A takes 3 days, but the method only takes 2 days, the process shortening time reaches more than 30%, and the time cost is greatly reduced; in terms of material cost, whether refined polysaccharide is prepared or polysaccharide is degraded, the method of the invention is reduced by at least 50% by taking the feeding amount of sodium deoxycholate as an example.
(2) The quality control indexes of impurities such as nucleic acid, protein and the like, specific groups and the like of the streptococcus pneumoniae capsular polysaccharide and the degradation polysaccharide prepared by the method are superior to the standard (23-valent streptococcus pneumoniae polysaccharide vaccine) of the pharmacopoeia of the people's republic of China (2020 edition), are superior to the quality indexes of the prior art, and can be used for preparing the streptococcus pneumoniae capsular polysaccharide vaccine or capsular polysaccharide conjugate vaccine.
(3) The method of the invention avoids the use of toxic and harmful reagents such as phenol, acetone, ethanol and the like in the preparation process, and reduces the harm to human health and environment; meanwhile, the operation of a chromatography is avoided, the cost is reduced, and the method is more beneficial to industrialized mass production.
Detailed Description
the present invention provides a process for preparing streptococcus pneumoniae capsular polysaccharides or their degradation polysaccharides.
In some embodiments of the invention there is provided a method of preparing streptococcus pneumoniae capsular polysaccharides comprising the steps of:
(1) Sterilizing Streptococcus pneumoniae fermentation broth, standing at 4deg.C for 6-12 hr, centrifuging, and collecting supernatant;
(2) Ultrafiltering the supernatant obtained in the step (1) by using a membrane with the aperture of 100KD to obtain a first ultrafiltered concentrated solution, wherein the volume of the concentrated solution is 1/5-1/4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times of the volume of the concentrated solution;
(3) Adding CaCl with final concentration of 80-200mmol/L into the first ultrafiltration concentrated solution2the solution is fully stirred, the pH value is regulated to 2.4-3.6 by acid liquor, the solution is fully stirred and then is kept stand for 1-5 hours at the temperature of 2-8 ℃, then the supernatant is collected by centrifugation, the pH value is regulated to 7.00-7.50 by alkali liquor, and the supernatant is collected by centrifugation again;
(4) Ultrafiltering the supernatant obtained in the step (3) by using a membrane with the aperture of 100KD to obtain polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in ultrafiltration is more than 10 times of the volume of the supernatant;
(5) And (3) freeze-drying the polysaccharide ultrafiltration concentrated solution obtained in the step (4), and collecting refined polysaccharide after the freeze-drying is finished.
In some embodiments of the invention there is provided a method of preparing a degraded polysaccharide of streptococcus pneumoniae capsular polysaccharides comprising the steps of:
(1) Sterilizing Streptococcus pneumoniae fermentation broth, standing at 4deg.C for 6-12 hr, centrifuging, and collecting supernatant;
(2) Ultrafiltering the supernatant obtained in the step (1) by using a membrane with the aperture of 100KD to obtain a first ultrafiltered concentrated solution, wherein the volume of the concentrated solution is 1/5-1/4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times of the volume of the concentrated solution;
(3) Adding CaCl with final concentration of 80-200mmol/L into the first ultrafiltration concentrated solution2the solution is fully stirred, the pH value is regulated to 2.4-3.6 by acid liquor, the solution is fully stirred and then is kept stand for 1-5 hours at the temperature of 2-8 ℃, then the supernatant is collected by centrifugation, the pH value is regulated to 7.00-7.50 by alkali liquor, and the supernatant is collected by centrifugation again;
(4) Adding trifluoroacetic acid with the final concentration of 0.2-2mol/L into the supernatant obtained in the step (3), standing for 2-10h at 25-60 ℃, adjusting the pH to 7.00-7.50 with alkali liquor, and centrifuging again to collect the supernatant;
(5) Ultrafiltering the supernatant obtained in the step (4) by using a membrane with the aperture of 30KD to obtain polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in ultrafiltration is more than 10 times of the volume of the supernatant;
(6) And (3) freeze-drying the polysaccharide ultrafiltration concentrated solution obtained in the step (5), and collecting degraded polysaccharide after the freeze-drying.
for the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 preparation of 6A Streptococcus pneumoniae capsular polysaccharide
1. Preparation of 6A Streptococcus pneumoniae capsular polysaccharide
(1) Sterilizing 6A Streptococcus pneumoniae fermentation broth with 0.1% DOC, standing at 4deg.C for 12 hr, and then standing at 12000gcentrifuging for 30 min, and collecting supernatant;
(2) Ultrafiltering the supernatant obtained in the step (1) by using a membrane with the aperture of 100KD to obtain an ultrafiltered concentrated solution, wherein the volume of the concentrated solution is 1/5-1/4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times of the volume of the concentrated solution;
(3) Adding CaCl with a final concentration of 200mmol/L into the ultrafiltration concentrated solution obtained in the step (2)2stirring, regulating pH to 3.00+ -0.1 with phosphoric acid, stirring, standing at 2-8deg.C for 1 hr, and standing at 12000gCentrifuging for 30 min, collecting supernatant, adjusting pH of the supernatant to 7.00-7.50 with NaOH solution, and 12000 againgcentrifuging for 30 min, and collecting supernatant;
(4) Ultrafiltering the supernatant obtained in the step (3) by using a membrane with the aperture of 100KD to obtain polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in ultrafiltration is more than 10 times of the volume of the supernatant;
(5) And (3) freeze-drying the ultrafiltration concentrated solution obtained in the step (4), and collecting refined polysaccharide after the completion of the freeze-drying.
2. Assay of 6A Streptococcus pneumoniae capsular polysaccharide
The detection of the content of capsular polysaccharide is carried out according to the turbidimetry (3.3.2) in the three parts of the pharmacopoeia of the people's republic of China 2020 edition; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the methylpentanose is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China.
The recovery rate was calculated as follows: recovery = refined polysaccharide content/polysaccharide content in supernatant after fermentation broth sterilization x 100%.
The results of the relevant assays are shown in Table 1.
TABLE 1 recovery and quality control index of 6A Streptococcus pneumoniae capsular polysaccharide
note that: in Table 1, 001, 002, 003 represent three experiments, respectively, as follows.
EXAMPLE 2 preparation of 6A Streptococcus pneumoniae capsular polysaccharide degradation polysaccharide
1. Preparation of 6A streptococcus pneumoniae capsular polysaccharide degradation polysaccharide
(1) Sterilizing 6A Streptococcus pneumoniae fermentation broth with 0.1% DOC, standing at 4deg.C for 12 hr, centrifuging at 12000 g for 30 min, and collecting supernatant;
(2) Ultrafiltering the supernatant obtained in the step (1) by using a membrane with the aperture of 100KD to obtain an ultrafiltered concentrated solution, wherein the volume of the concentrated solution is 1/5-1/4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times of the volume of the concentrated solution;
(3) Adding CaCl with final concentration of 80mmol/L (6A-2308001L), 120mmol/L (6A-2308002L) and 200mmol/L (6A-2308003L) into the ultrafiltration concentrated solution obtained in the step (2)2the solution is fully stirred, the pH value is regulated to 3.00+/-0.1 by phosphoric acid, and is fully stirred, and then is stood for 1h at the temperature of 2-8 ℃ and is 12000gcentrifuging for 30 min, and collecting supernatant;
(4) Adding trifluoroacetic acid with the final concentration of 0.25mol/L into the supernatant obtained in the step (3), standing for 3 hours at 40 ℃, adjusting the pH to 7.00-7.50, and centrifuging again to collect the supernatant;
(5) Ultrafiltering the supernatant solution obtained in the step (4) by using a membrane with the aperture of 30KD to obtain polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in ultrafiltration is more than 10 times of the volume of the supernatant solution;
(6) And freeze-drying the ultrafiltration concentrated solution, and collecting degradation polysaccharide after the completion of the freeze-drying.
2. Detection of degradation polysaccharide of 6A streptococcus pneumoniae capsular polysaccharide
The detection of the degradation polysaccharide of the streptococcus pneumoniae capsular polysaccharide of 6A refers to the 2020 edition of the pharmacopoeia of the people's republic of China, wherein the detection of the capsular polysaccharide content is carried out according to the speed turbidimetry (3.3.2) in the three parts of the pharmacopoeia of the people's republic of China of 2020 edition; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the methylpentanose is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in Table 2.
TABLE 2 recovery and quality control index of 6A Streptococcus pneumoniae capsular polysaccharide degradation polysaccharide
example 35 preparation of Streptococcus pneumoniae capsular polysaccharide
1. Preparation of streptococcus pneumoniae capsular polysaccharide
(1) Sterilizing Streptococcus pneumoniae fermentation broth 5 with 0.1% DOC, standing at 4deg.C for 12 hr, and 12000gcentrifuging for 30 min, and collecting supernatant;
(2) Ultrafiltering the supernatant obtained in the step (1) by using a membrane with the aperture of 100KD to obtain an ultrafiltered concentrated solution, wherein the volume of the concentrated solution is 1/5-1/4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times of the volume of the concentrated solution;
(3) Adding CaCl with final concentration of 80mmol/L into the ultrafiltration concentrated solution obtained in the step (2)2The solution is fully stirred, the pH value is regulated to 3.00+/-0.1 by phosphoric acid, and is fully stirred, and then is stood for 5 hours at the temperature of 2-8 ℃ and is 12000gCentrifuging for 30min, collecting supernatant, adjusting pH to 7.00-7.50 with NaOH, and 12000 againgcentrifuging for 30 min, and collecting supernatant;
(4) Ultrafiltering the supernatant obtained in the step (3) by using a membrane with the aperture of 100KD to obtain polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in ultrafiltration is more than 10 times of the volume of the supernatant;
(5) And (3) freeze-drying the ultrafiltration concentrated solution obtained in the step (4), and collecting refined polysaccharide after the completion of the freeze-drying.
2. Assay of streptococcus pneumoniae capsular polysaccharides
The detection of the content of capsular polysaccharide is carried out according to the turbidimetry (3.3.2) in the three parts of the pharmacopoeia of the people's republic of China 2020 edition; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the uronic acid content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the hexosamine content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in Table 3.
table 35 index of recovery and quality control of monovalent Streptococcus pneumoniae capsular polysaccharides
Example 45 preparation of Streptococcus pneumoniae capsular polysaccharide-degrading polysaccharide
1. preparation of 5-type streptococcus pneumoniae capsular polysaccharide degradation polysaccharide
(1) Sterilizing the streptococcus pneumoniae fermentation broth with 0.1% DOC, standing at 4 ℃ for 12 hours, centrifuging 12000 and g for 30 min, and collecting supernatant;
(2) Ultrafiltering the supernatant obtained in the step (1) by using a membrane with the aperture of 100KD to obtain an ultrafiltered concentrated solution, wherein the volume of the concentrated solution is 1/5-1/4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times of the volume of the concentrated solution;
(3) Adding CaCl with final concentration of 100mmol/L into the ultrafiltration concentrated solution obtained in the step (2)2The solution is fully stirred, the pH is regulated to be 2.50+/-0.1 (5-2308001L), 3.00+/-0.1 (5-2308002L), 3.50+/-0.1 (5-2308003L) by phosphoric acid respectively, the solution is fully stirred and then is kept stand for 5 hours at the temperature of 2-8 ℃, and then the solution is centrifuged for 30 minutes by 12000 g, and the supernatant is collected;
(4) Adding trifluoroacetic acid with the final concentration of 1.5 mol/L into the supernatant obtained in the step (3), standing for 10 hours at 25 ℃, adjusting the pH to 7.00-7.50, and centrifuging again to collect the supernatant;
(5) Ultrafiltering the supernatant obtained in the step (4) by using a membrane with the aperture of 30KD to obtain polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in ultrafiltration is more than 10 times of the volume of the supernatant;
(6) And (3) freeze-drying the ultrafiltration concentrated solution obtained in the step (5), and collecting degraded polysaccharide after the freeze-drying is finished.
2. Detection of polysaccharide degradation of streptococcus pneumoniae capsular polysaccharide
The detection of the polysaccharide degradation of streptococcus pneumoniae capsular polysaccharide refers to the 2020 edition of the pharmacopoeia of the people's republic of China, wherein the detection of the capsular polysaccharide content is carried out according to the medium speed turbidimetry (3.3.2) in the 2020 edition of the pharmacopoeia of the people's republic of China; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the uronic acid content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the hexosamine content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in Table 4.
Table 45 recovery and quality control index of Streptococcus pneumoniae capsular polysaccharide degradation polysaccharide
EXAMPLE 5 preparation of 10A Streptococcus pneumoniae capsular polysaccharide
1. Preparation of 10A Streptococcus pneumoniae capsular polysaccharide
(1) Sterilizing 10A Streptococcus pneumoniae fermentation broth with 0.1% DOC, standing at 4deg.C for 12 hr, and 12000gcentrifuging for 30 min, and collecting supernatant;
(2) Ultrafiltering the supernatant obtained in the step (1) by using a membrane with the aperture of 100KD to obtain an ultrafiltered concentrated solution, wherein the volume of the concentrated solution is 1/5-1/4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times of the volume of the concentrated solution;
(3) Adding CaCl with final concentration of 80mmol/L into the ultrafiltration concentrated solution obtained in the step (2)2The solution is fully stirred and then the pH is regulated to 2.50+/-0.1 (10A-2308001), 3.00+/-0.1 (10A-2308002), 3.50+/-0.1 (10A-2308003) and 4.00+/-0.1 (10A-2308004) by phosphoric acid, and then the solution is fully stirred and then is kept stand for 3 hours at the temperature of 2-8 ℃ and then 12000gCentrifuging for 30min, collecting supernatant, adjusting pH to 7.00-7.50 with NaOH, and 12000 againgcentrifuging for 30 min, and collecting supernatant;
(4) Ultrafiltering the supernatant solution obtained in the step (3) by using a membrane with the aperture of 100KD to obtain polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in ultrafiltration is more than 10 times of the volume of the supernatant;
(5) And (3) freeze-drying the ultrafiltration concentrated solution obtained in the step (4), and collecting refined polysaccharide after the completion of the freeze-drying.
2. assay for capsular polysaccharide of streptococcus pneumoniae 10A
The detection of the content of capsular polysaccharide is carried out according to the turbidimetry (3.3.2) in the three parts of the pharmacopoeia of the people's republic of China 2020 edition; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the hexosamine content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in Table 5. The results show that in the calcium salt acid precipitation of the step (3), the pH is controlled within the range of 2.4-3.6, and the content of protein impurities in the prepared refined polysaccharide is obviously reduced and is obviously lower than that of the group with the pH of 4.0.
TABLE 5 recovery and quality control index of Streptococcus pneumoniae capsular polysaccharide 10A
EXAMPLE 6 preparation of 10A Streptococcus pneumoniae capsular polysaccharide degradation polysaccharide
1. preparation of 10A streptococcus pneumoniae capsular polysaccharide degradation polysaccharide
(1) Sterilizing the 10A streptococcus pneumoniae fermentation broth by 0.1% DOC, standing at 4 ℃ for 12 hours, centrifuging for 30 min by 12000 and g, and collecting supernatant;
(2) Ultrafiltering the supernatant obtained in the step (1) by using a membrane with the aperture of 100KD to obtain an ultrafiltered concentrated solution, wherein the volume of the concentrated solution is 1/5-1/4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times of the volume of the concentrated solution;
(3) Adding CaCl with final concentration of 80mmol/L into the ultrafiltration concentrated solution obtained in the step (2)2The solution is fully stirred, the pH value is regulated to 3.00+/-0.1 by phosphoric acid, the solution is fully stirred and then is kept stand for 3 hours at the temperature of 2-8 ℃, and then the solution is centrifuged for 30 min by 12000 g, and the supernatant is collected;
(4) Adding trifluoroacetic acid with the final concentration of 0.5mol/L into the supernatant obtained in the step (3), standing for 3 hours at the temperature of 30 ℃, adjusting the pH to 7.00-7.50, and centrifuging again to collect the supernatant;
(5) Ultrafiltering the supernatant obtained in the step (4) by using a membrane with the aperture of 30KD to obtain polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in ultrafiltration is more than 10 times of the volume of the supernatant;
(6) And (3) freeze-drying the ultrafiltration concentrated solution obtained in the step (5), and collecting refined polysaccharide after the completion of the freeze-drying.
2. Detection of degradation polysaccharide of 10A streptococcus pneumoniae capsular polysaccharide
The detection of the degradation polysaccharide of the streptococcus pneumoniae capsular polysaccharide of 10A refers to the 2020 edition of the pharmacopoeia of the people's republic of China, wherein the detection of the capsular polysaccharide content is carried out according to the medium speed turbidimetry (3.3.2) in the 2020 edition of the pharmacopoeia of the people's republic of China; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the hexosamine content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in Table 6.
TABLE 6 recovery and quality control index of polysaccharide degradation from Streptococcus pneumoniae capsular polysaccharide of 10A type
EXAMPLE 7 preparation of Streptococcus pneumoniae capsular polysaccharide
1. Preparation of 11A Streptococcus pneumoniae capsular polysaccharide
(1) After sterilization by 0.1% DOC, the 11A streptococcus pneumoniae fermentation broth is left to stand for 12 hours at 4 ℃, and then 12000gcentrifuging for 30 min, and collecting supernatant;
(2) Ultrafiltering the supernatant obtained in the step (1) by using a membrane with the aperture of 100KD to obtain an ultrafiltered concentrated solution, wherein the volume of the concentrated solution is 1/5-1/4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times of the volume of the concentrated solution;
(3) Adding CaCl with final concentration of 80mmol/L into the ultrafiltration concentrated solution obtained in the step (2)2The solution is fully stirred, the pH is regulated to 3.00+/-0.1, and is fully stirred, and then is stood for 2 hours at the temperature of 2-8 ℃ and is 12000gCentrifuging for 30min, collecting supernatant, adjusting pH to 7.00-7.50 with NaOH, and 12000 againgcentrifuging for 30 min, and collecting supernatant;
(4) Ultrafiltering the supernatant solution obtained in the step (3) by using a membrane with the aperture of 100KD to obtain polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in ultrafiltration is more than 10 times of the volume of the supernatant;
(5) And (3) freeze-drying the ultrafiltration concentrated solution obtained in the step (4), and collecting degraded polysaccharide after the freeze-drying is finished.
2. assay of capsular polysaccharide from Streptococcus pneumoniae 11A
The detection of the content of capsular polysaccharide is carried out according to the turbidimetry (3.3.2) in the three parts of the pharmacopoeia of the people's republic of China 2020 edition; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the O-acetyl content is carried out according to the three parts (general rule 3117) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in Table 7.
TABLE 7 recovery and quality control index of Streptococcus pneumoniae capsular polysaccharide
Example 8 preparation of Streptococcus pneumoniae capsular polysaccharide degradation polysaccharide
1. Preparation of 11A streptococcus pneumoniae capsular polysaccharide degradation polysaccharide
(1) Sterilizing the 11A streptococcus pneumoniae fermentation broth by 0.1% DOC, standing at 4 ℃ for 12 hours, centrifuging for 30 min by 12000 and g, and collecting supernatant;
(2) Ultrafiltering the supernatant obtained in the step (1) by using a membrane with the aperture of 100KD to obtain an ultrafiltered concentrated solution, wherein the volume of the concentrated solution is 1/5-1/4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times of the volume of the concentrated solution;
(3) Adding CaCl with final concentration of 100mmol/L into the ultrafiltration concentrated solution obtained in the step (2)2The solution is fully stirred, the pH is regulated to 3.00+/-0.1, the solution is fully stirred and then is kept stand for 2 hours at the temperature of 2-8 ℃, and then the solution is centrifuged for 30 minutes by 12000 and g, and the supernatant is collected;
(4) Adding trifluoroacetic acid with the final concentration of 0.5mol/L into the supernatant obtained in the step (3), standing for 6 hours at 40 ℃, adjusting the pH to 7.00-7.50, and centrifuging again to collect the supernatant;
(5) Ultrafiltering the supernatant solution obtained in the step (4) by using a membrane with the aperture of 30KD to obtain polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in ultrafiltration is more than 10 times of the volume of the supernatant solution;
(6) And (3) freeze-drying the ultrafiltration concentrated solution obtained in the step (5), and collecting refined polysaccharide after the completion of the freeze-drying.
2. detection of degradation polysaccharide of 11A streptococcus pneumoniae capsular polysaccharide
The detection of the degrading polysaccharide of the streptococcus pneumoniae capsular polysaccharide of 11A refers to the 2020 edition of the pharmacopoeia of the people's republic of China, wherein the detection of the capsular polysaccharide content is carried out according to the medium speed turbidimetry (3.3.2) in the 2020 edition of the pharmacopoeia of the people's republic of China; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the O-acetyl content is carried out according to the three parts (general rule 3117) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in Table 8.
Table 8 recovery rate and quality control index of Streptococcus pneumoniae capsular polysaccharide degradation polysaccharide
EXAMPLE 9 preparation of Streptococcus pneumoniae capsular polysaccharide 19F
1. Preparation of 19F type streptococcus pneumoniae capsular polysaccharide
(1) After sterilization by 0.1% DOC, the 19F streptococcus pneumoniae fermentation broth is left to stand for 6 hours at 4 ℃ and then 12000gcentrifuging for 30 min, and collecting supernatant;
(2) Ultrafiltering the supernatant obtained in the step (1) by using a membrane with the aperture of 100KD to obtain an ultrafiltered concentrated solution, wherein the volume of the concentrated solution is 4-5 times of the volume of the supernatant, and the volume of purified water used for ultrafiltration is 10 times of the volume of the concentrated solution;
(3) Adding CaCl with final concentration of 80mmol/L into the ultrafiltration concentrated solution obtained in the step (2)2The solution is fully stirred, the pH is regulated to 3.00+/-0.1, and is fully stirred, and then is stood for 2 hours at the temperature of 2-8 ℃ and is 12000gCentrifuging for 30min, collecting supernatant, adjusting pH to 7.00-7.50 with NaOH, and 12000 againgcentrifuging for 30 min, and collecting supernatant;
(4) Ultrafiltering the supernatant obtained in the step (3) by using a membrane with the aperture of 100KD to obtain polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in ultrafiltration is 10 times of that of the supernatant;
(5) And (3) freeze-drying the ultrafiltration concentrated solution obtained in the step (4), and collecting refined polysaccharide after the completion of the freeze-drying.
2. Assay of 19F Streptococcus pneumoniae capsular polysaccharide
The detection of the content of capsular polysaccharide is carried out according to the turbidimetry (3.3.2) in the three parts of the pharmacopoeia of the people's republic of China 2020 edition; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the hexosamine content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the methylpentanose is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in Table 9.
TABLE 9 recovery and quality control index of Streptococcus pneumoniae capsular polysaccharide 19F
EXAMPLE 10 preparation of 19F Streptococcus pneumoniae capsular polysaccharide-degrading polysaccharide
1. Preparation of 19F streptococcus pneumoniae capsular polysaccharide degradation polysaccharide
(1) After sterilization by 0.1% DOC, the 19F streptococcus pneumoniae fermentation broth is kept stand for 6 hours h hours at 4 ℃, and then is centrifuged for 30 minutes by 12000 g, and the supernatant is collected;
(2) Ultrafiltering the supernatant obtained in the step (1) by using a membrane with the aperture of 100KD to obtain an ultrafiltered concentrated solution, wherein the volume of the concentrated solution is 1/5-1/4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times of the volume of the concentrated solution;
(3) Adding CaCl with final concentration of 80mmol/L into the ultrafiltration concentrated solution obtained in the step (2)2The solution is fully stirred, the pH is regulated to 3.00+/-0.1, the solution is fully stirred and then is kept stand for 2 hours at the temperature of 2-8 ℃, and then the solution is centrifuged for 30 minutes by 12000 and g, and the supernatant is collected;
(4) Adding trifluoroacetic acid with the final concentration of 0.25mol/L into the supernatant obtained in the step (3), standing for 3 hours at 25 ℃, adjusting the pH to 7.00-7.50, and centrifuging again to collect the supernatant;
(5) Ultrafiltering the supernatant obtained in the step (4) by using a membrane with the aperture of 30KD to obtain polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in ultrafiltration is more than 10 times of the volume of the supernatant;
(6) And (3) freeze-drying the ultrafiltration concentrated solution obtained in the step (5), and collecting degraded polysaccharide after the freeze-drying is finished.
2. detection of degradation polysaccharide of 19F streptococcus pneumoniae capsular polysaccharide
the detection of the degradation polysaccharide of the streptococcus pneumoniae capsular polysaccharide of 19F is referred to the 2020 edition of the pharmacopoeia of the people's republic of China, wherein the detection of the capsular polysaccharide content is carried out according to the medium speed turbidimetry (3.3.2) in the 2020 edition of the pharmacopoeia of the people's republic of China; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the hexosamine content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the methylpentanose is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in Table 10.
TABLE 10 recovery and quality control index of capsular polysaccharide degradation polysaccharide from Streptococcus pneumoniae 19F
EXAMPLE 11 preparation of Streptococcus pneumoniae capsular polysaccharide
1. Purification of streptococcus pneumoniae capsular polysaccharides
(1) Sterilizing Streptococcus pneumoniae 2 fermentation broth with 0.1% DOC, standing at 4deg.C for 12 hr, and 12000gcentrifuging for 30 min, and collecting supernatant;
(2) Ultrafiltering the supernatant obtained in the step (1) by using a membrane with the aperture of 100KD to obtain an ultrafiltered concentrated solution, wherein the volume of the concentrated solution is 1/5-1/4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times of the volume of the concentrated solution;
(3) Adding CaCl with final concentration of 80mmol/L into the ultrafiltration concentrated solution obtained in the step (2)2the solution is fully stirred, the pH is regulated to 3.00+/-0.1, and is fully stirred, and then is stood for 4 hours at the temperature of 2-8 ℃ and is 12000gCentrifuging for 30min, collecting supernatant, adjusting pH to 7.00-7.50 with NaOH, and 12000 againgcentrifuging for 30 min, and collecting supernatant;
(4) Ultrafiltering the supernatant obtained in the step (3) by using a membrane with the aperture of 100KD to obtain polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in ultrafiltration is more than 10 times of the volume of the supernatant;
(5) And (3) freeze-drying the ultrafiltration concentrated solution obtained in the step (4), and collecting refined polysaccharide after the completion of the freeze-drying.
2. Assay of streptococcus pneumoniae capsular polysaccharides
The detection of the content of capsular polysaccharide is carried out according to the turbidimetry (3.3.2) in the three parts of the pharmacopoeia of the people's republic of China 2020 edition; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the uronic acid content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the methylpentanose is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in Table 11.
Table 11 recovery and quality control index of monovalent Streptococcus pneumoniae capsular polysaccharide
EXAMPLE 12 preparation of Streptococcus pneumoniae capsular polysaccharide-degrading polysaccharide
1. Preparation of 2-type streptococcus pneumoniae capsular polysaccharide degradation polysaccharide
(1) 2, sterilizing the streptococcus pneumoniae fermentation broth by 0.1% DOC, standing for 12 hours at 4 ℃, centrifuging for 30 min by 12000 and g, and collecting supernatant;
(2) Ultrafiltering the supernatant obtained in the step (1) by using a membrane with the aperture of 100KD to obtain an ultrafiltered concentrated solution, wherein the volume of the concentrated solution is 1/5-1/4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times of the volume of the concentrated solution;
(3) Adding CaCl with final concentration of 80mmol/L into the ultrafiltration concentrated solution obtained in the step (2)2The solution is fully stirred, the pH is regulated to 3.00+/-0.1, the solution is fully stirred and then is kept stand for 2 hours at the temperature of 2-8 ℃, and then the solution is centrifuged for 30 minutes by 12000 and g, and the supernatant is collected;
(4) Adding trifluoroacetic acid with the final concentration of 0.5mol/L into the supernatant obtained in the step (3), standing for 3 hours at 40 ℃, adjusting the pH to 7.00-7.50, and centrifuging again to collect the supernatant;
(5) Ultrafiltering the supernatant solution obtained in the step (4) by using a membrane with the aperture of 30KD to obtain polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in ultrafiltration is more than 10 times of the volume of the supernatant solution;
(6) And (3) freeze-drying the ultrafiltration concentrated solution obtained in the step (5), and collecting degraded polysaccharide after the freeze-drying is finished.
2. Detection of polysaccharide degradation of streptococcus pneumoniae capsular polysaccharide
The detection of the polysaccharide degradation of streptococcus pneumoniae capsular polysaccharide refers to the 2020 edition of the pharmacopoeia of the people's republic of China, wherein the detection of the capsular polysaccharide content is carried out according to the medium speed turbidimetry (3.3.2) in the 2020 edition of the pharmacopoeia of the people's republic of China; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the uronic acid content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the methylpentanose is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in Table 12.
table 12 recovery rate and quality control index of degrading polysaccharide of Streptococcus pneumoniae capsular polysaccharide
It should be noted that the sterilization method in the above embodiments is only illustrative, and the improvement of the purification method of capsular polysaccharide and the improvement of the effect thereof of the present invention are not dependent on the specific sterilization method. Experiments prove that the purification effect equivalent to the above-mentioned examples can be obtained by using the capsular polysaccharide purification method of the present invention in combination with substituting sodium deoxycholate for beta-propiolactone as a bactericide, and the following examples using 0.1% beta-propiolactone are provided as examples. In addition, the invention also tries to carry out sterilization treatment by adopting beta-propiolactone with other concentrations, and the result shows that beta-propiolactone with the final concentration not lower than 0.01 percent can realize better effects of killing streptococcus pneumoniae and releasing capsular polysaccharide.
EXAMPLE 13 preparation of Streptococcus pneumoniae capsular polysaccharide 14
1. Purification of streptococcus pneumoniae capsular polysaccharide 14
(1) Adding beta-propiolactone with final concentration of 0.1% into Streptococcus pneumoniae fermentation broth of 14 type, stirring, incubating at 4deg.C for 12 hr, and 12000gcentrifuging for 30 min, and collecting supernatant;
(2) Ultrafiltering the supernatant obtained in the step (1) by using a membrane with the aperture of 100KD to obtain an ultrafiltered concentrated solution, wherein the volume of the concentrated solution is 1/5-1/4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times of the volume of the concentrated solution;
(3) Adding CaCl with final concentration of 80mmol/L (14-2308001), 120mmol/L (14-2308002) and 200mmol/L (14-2308003) into the ultrafiltration concentrated solution obtained in the step (2)2The solution is fully stirred, the pH is regulated to 3.00+/-0.1, and is fully stirred, and then is stood for 5 hours at the temperature of 2-8 ℃ and is 12000gCentrifuging for 30min, collecting supernatant, adjusting pH to 7.00-7.50 with NaOH, and 12000 againgcentrifuging for 30 min, and collecting supernatant;
(4) Ultrafiltering the supernatant obtained in the step (3) by using a membrane with the aperture of 100KD to obtain polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in ultrafiltration is more than 10 times of the volume of the supernatant;
(5) And (3) freeze-drying the ultrafiltration concentrated solution obtained in the step (4), and collecting refined polysaccharide after the completion of the freeze-drying.
2. Assay for capsular polysaccharide of streptococcus pneumoniae 14
The detection of the content of capsular polysaccharide is carried out according to the turbidimetry (3.3.2) in the three parts of the pharmacopoeia of the people's republic of China 2020 edition; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the uronic acid content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the methylpentanose is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in Table 13.
TABLE 13 recovery and quality control indicators for Streptococcus pneumoniae capsular polysaccharide
Comparative example 1 preparation of 6A Streptococcus pneumoniae capsular polysaccharide
1. Preparation of 6A Streptococcus pneumoniae capsular polysaccharide
steps (1) and (2) are the same as in example 1, steps (3) - (6) are as follows:
(3) Adding NaCl to the ultrafiltration concentrated solution obtained in the step (2) until the concentration is 0.3m 1/L, adding 0.5% (w/v) sodium deoxycholate after complete dissolution, dropwise adding glacial acetic acid while stirring to adjust the pH to 5.0, fully stirring, standing at 2-8 ℃ for 12 hours, centrifuging at 12000g for 30min, collecting supernatant, discarding precipitate, and clarifying and filtering the supernatant with a 0.8 mu m filter membrane if small chips exist;
(4) Adjusting the pH of the supernatant obtained in the step (3) to be neutral by using sodium hydroxide or potassium hydroxide, and then adding Na with the final concentration of 0.01mol/L into the obtained feed liquid2HPO4and NaH with final concentration of 0.01mol/L2PO4as a buffer system, after complete dissolution, sodium acetate was added to a final concentration of 0.6mol/L, caCl2The final concentration is 0.25mol/L, stirring until dissolving, dripping glacial acetic acid into the feed liquid to adjust the pH to 5.3-5.5, fully stirring, standing at 2-8 ℃ for 12 hours, centrifuging 12000 g for 30min after standing is finished, collecting supernatant, discarding precipitate, and clarifying and filtering the supernatant with a 0.8 mu m filter membrane if small scraps exist;
(5) Ultrafiltering the supernatant obtained in the step (4) with purified water, collecting polysaccharide solution after the conductivity of the permeation end is lower than 10 mu s/cm, and collecting the polysaccharide solution after sterilization and filtration;
(6) And further freeze-drying the polysaccharide solution to obtain streptococcus pneumoniae capsular polysaccharide.
2. Assay of 6A Streptococcus pneumoniae capsular polysaccharide
The detection of the content of capsular polysaccharide is carried out according to the turbidimetry (3.3.2) in the three parts of the pharmacopoeia of the people's republic of China 2020 edition; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the methylpentanose is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in Table 14.
TABLE 14 recovery and quality control index of 6A Streptococcus pneumoniae capsular polysaccharide
The results show that compared with the quality control index of the 6A streptococcus pneumoniae refined polysaccharide prepared by the method of comparative example 1, the protein content of the refined polysaccharide prepared by the method of example 1 is significantly reduced, and other quality control indexes of the 6A streptococcus pneumoniae refined polysaccharide prepared by example 1 are not inferior to those of comparative example 1, and the process time is shortened.
Comparative example 2 preparation of 6A Streptococcus pneumoniae capsular polysaccharide degradation polysaccharide
1. Preparation of 6A streptococcus pneumoniae capsular polysaccharide degradation polysaccharide
(1) 3.0+/-0.15 g of the refined polysaccharide obtained in the comparative example 1 is taken and dissolved in 1.0L of 0.15mol/L sodium chloride solution, and the solution is stirred until the polysaccharide is completely dissolved;
(2) Homogenizing the refined polysaccharide solution in the step (1) by a high-pressure homogenizer at 600+/-50 bar, circulating for 2 times, and harvesting the degraded polysaccharide solution;
(3) And freeze-drying the degradation polysaccharide solution, and collecting the degradation polysaccharide after the freeze-drying is finished.
2. Detection of degradation polysaccharide of 6A streptococcus pneumoniae capsular polysaccharide
the detection of the polysaccharide degradation of the streptococcus pneumoniae capsular polysaccharide of 6A refers to the 2020 edition of the pharmacopoeia of the people's republic of China, wherein the detection of the capsular polysaccharide content is carried out according to the medium speed turbidimetry (3.3.2) in the 2020 edition of the pharmacopoeia of the people's republic of China; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the methylpentanose is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in Table 15.
TABLE 15 recovery and quality control index of 6A Streptococcus pneumoniae capsular polysaccharide degradation polysaccharide
The results show that the quality control index of the degradation polysaccharide of the streptococcus pneumoniae capsular polysaccharide 6A prepared by the method of the embodiment 2 of the invention is obviously reduced compared with that of the degradation polysaccharide prepared by the comparative example 2, and other quality control indexes are not inferior to those of the comparative example 2, and the process flow is simplified and the process time is shortened.
Comparative example 35 preparation of Streptococcus pneumoniae capsular polysaccharide
1. Preparation of streptococcus pneumoniae capsular polysaccharide
Steps (1) and (2) are the same as in example 3, steps (3) - (6) being as follows:
(3) Adding NaCl to the ultrafiltration concentrated solution obtained in the step (2) until the concentration is 0.3m 1/L, adding 0.5% (w/v) sodium deoxycholate after complete dissolution, dropwise adding glacial acetic acid while stirring to adjust the pH to 5.0, fully stirring, standing at 2-8 ℃ for 12 hours, centrifuging at 12000g for 30min, collecting supernatant, discarding precipitate, and clarifying and filtering the supernatant with a 0.8 mu m filter membrane if small fragments exist;
(4) Adjusting the pH of the supernatant obtained in the step (3) to be neutral by sodium hydroxide or potassium hydroxide, and then adding Na with the final concentration of 0.01mol/L into the obtained feed liquid2HPO4and NaH with final concentration of 0.01mol/L2PO4as a buffer system, after complete dissolution, sodium acetate was added to a final concentration of 0.6mol/L, caCl2The final concentration is 0.25mol/L, stirring until dissolving, dripping glacial acetic acid into the feed liquid to adjust the pH to 5.3-5.5, fully stirring, standing at 2-8 ℃ for 12 hours, standing, centrifuging at 12000 g for 30min, collecting supernatant, discarding precipitate, and clarifying and filtering the supernatant with a 0.8 mu m filter membrane if small scraps exist;
(5) Ultrafiltering the supernatant obtained in the step (4) with purified water, collecting polysaccharide solution after the conductivity of the permeation end is lower than 10 mu s/cm, and collecting the polysaccharide solution after sterilization and filtration;
(6) And further freeze-drying the polysaccharide solution to obtain streptococcus pneumoniae capsular polysaccharide.
2. Assay of streptococcus pneumoniae capsular polysaccharides
The detection of the content of capsular polysaccharide is carried out according to the turbidimetry (3.3.2) in the three parts of the pharmacopoeia of the people's republic of China 2020 edition; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the uronic acid content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the hexosamine content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in Table 16.
Table 16 recovery and quality control index of Streptococcus pneumoniae capsular polysaccharide
The results show that the quality control index of the refined polysaccharide of streptococcus pneumoniae prepared in the embodiment 3 of the invention is no worse than that of the refined polysaccharide prepared in the comparative embodiment 3, and the process time is shortened.
Comparative example 45 preparation of Streptococcus pneumoniae capsular polysaccharide degradation polysaccharide
1. preparation of 5-type streptococcus pneumoniae capsular polysaccharide degradation polysaccharide
(1) Taking 3.0+/-0.15 g of refined polysaccharide obtained in comparative example 3, dissolving in 1.0L of 0.15mol/L sodium chloride solution, and stirring until the polysaccharide is completely dissolved;
(2) Homogenizing the refined polysaccharide solution in the step (1) by a high-pressure homogenizer at 1100+/-50 bar, circulating for 3 times, and harvesting the degraded polysaccharide solution;
(3) And freeze-drying the degradation polysaccharide solution, and collecting the degradation polysaccharide after the freeze-drying is finished.
2. Detection of polysaccharide degradation of streptococcus pneumoniae capsular polysaccharide
The detection of the polysaccharide degradation of streptococcus pneumoniae capsular polysaccharide refers to the 2020 edition of the pharmacopoeia of the people's republic of China, wherein the detection of the capsular polysaccharide content is carried out according to the medium speed turbidimetry (3.3.2) in the 2020 edition of the pharmacopoeia of the people's republic of China; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the uronic acid content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the hexosamine content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in Table 17.
Table 17 recovery rate and quality control index of degrading polysaccharide of Streptococcus pneumoniae capsular polysaccharide
The results show that the quality control index of the polysaccharide degradation of the streptococcus pneumoniae capsular polysaccharide prepared in the embodiment 4 of the invention is reduced compared with the protein content of the polysaccharide degradation prepared in the comparative example 4, the hexosamine content is increased, and other quality control indexes are not inferior to those of the comparative example 4, and the process flow is simplified and the process time is shortened.
comparative example 5 preparation of 10A Streptococcus pneumoniae capsular polysaccharide
1. Preparation of 10A Streptococcus pneumoniae capsular polysaccharide
Steps (1) and (2) are the same as in example 5, steps (3) - (6) are as follows:
(3) Adding NaCl to the ultrafiltration concentrated solution obtained in the step (2) until the concentration is 0.3mol/L, adding 0.5% (w/v) sodium deoxycholate after complete dissolution, dropwise adding glacial acetic acid while stirring to adjust the pH to 5.0, fully stirring, standing at 2-8 ℃ for 12 hours, centrifuging at 12000g for 30min, collecting supernatant, discarding precipitation, and clarifying and filtering the supernatant with a 0.8 mu m filter membrane if small fragments exist;
(4) Adjusting the pH of the supernatant obtained in the step (3) to be neutral by using sodium hydroxide or potassium hydroxide, and then adding Na with the final concentration of 0.01mol/L into the obtained feed liquid2HPO4and NaH with final concentration of 0.01mol/L2PO4as a buffer system, after complete dissolution, sodium acetate was added to a final concentration of 0.6mol/L, caCl2The final concentration is 0.25mol/L, stirring until dissolving, dripping glacial acetic acid into the feed liquid to adjust the pH to 5.3-5.5, fully stirring, standing at 2-8 ℃ for 12 hours, centrifuging at 12000 g for 30min after standing is finished, collecting supernatant, discarding precipitate, and clarifying and filtering the supernatant with a 0.8 mu m filter membrane if small scraps exist;
(5) Ultrafiltering the supernatant obtained in the step (4) with purified water, collecting polysaccharide solution after the conductivity of the permeation end is lower than 10 mu s/cm, and collecting the polysaccharide solution after sterilization and filtration;
(6) And further freeze-drying the polysaccharide solution to obtain streptococcus pneumoniae capsular polysaccharide.
2. assay for capsular polysaccharide of streptococcus pneumoniae 10A
The detection of the content of capsular polysaccharide is carried out according to the turbidimetry (3.3.2) in the three parts of the pharmacopoeia of the people's republic of China 2020 edition; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the hexosamine content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in Table 18.
TABLE 18 recovery and quality control index of monovalent Streptococcus pneumoniae capsular polysaccharide of 10A type
The results show that the quality control index of the 10A streptococcus pneumoniae refined polysaccharide prepared in the embodiment 5 of the invention is reduced compared with the protein content and the total phosphorus content of the comparative example 5, the hexosamine content is increased compared with the comparative example 5, and other quality control indexes are not inferior to the comparative example, and the process time is shortened.
comparative example 6 preparation of polysaccharide degradation of Streptococcus pneumoniae capsular polysaccharide of 10A type
1. preparation of 10A streptococcus pneumoniae capsular polysaccharide degradation polysaccharide
(1) 3.0+/-0.15 g of the refined polysaccharide obtained in the comparative example 5 is taken and dissolved in 1.0L of 0.15mol/L sodium chloride solution, and the solution is stirred until the polysaccharide is completely dissolved;
(2) Homogenizing the refined polysaccharide solution in the step (1) by a high-pressure homogenizer at 600+/-50 bar, circulating for 3 times, and harvesting the degraded polysaccharide solution;
(3) And freeze-drying the degradation polysaccharide solution, and collecting the degradation polysaccharide after the freeze-drying is finished.
2. Detection of degradation polysaccharide of 10A streptococcus pneumoniae capsular polysaccharide
The detection of the degradation polysaccharide of the streptococcus pneumoniae capsular polysaccharide of 10A refers to the 2020 edition of the pharmacopoeia of the people's republic of China, wherein the detection of the capsular polysaccharide content is carried out according to the medium speed turbidimetry (3.3.2) in the 2020 edition of the pharmacopoeia of the people's republic of China; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the hexosamine content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in Table 19.
TABLE 19 recovery and quality control index of polysaccharide degradation from Streptococcus pneumoniae capsular polysaccharide of 10A type
The results show that the quality control index of the 10A streptococcus pneumoniae capsular polysaccharide degradation polysaccharide prepared in the embodiment 6 of the invention is reduced compared with the protein content of the degradation polysaccharide prepared in the comparative example 6, and other quality control indexes are not inferior to those of the comparative example 6, so that the process flow is simplified, and the process time is shortened.
comparative example 7 preparation of Streptococcus pneumoniae capsular polysaccharide
1. Preparation of 11A Streptococcus pneumoniae capsular polysaccharide
Steps (1) and (2) are the same as in example 7, steps (3) - (6) are as follows:
(3) Adding NaCl to the ultrafiltration concentrated solution obtained in the step (2) until the concentration is 0.3m 1/L, adding 0.5% (w/v) sodium deoxycholate after complete dissolution, dropwise adding glacial acetic acid while stirring to adjust the pH to 5.0, fully stirring, standing at 2-8 ℃ for 12 hours, centrifuging at 12000g for 30min, collecting supernatant, discarding precipitate, and clarifying and filtering the supernatant with a 0.8 mu m filter membrane if small fragments exist;
(4) Adjusting the pH of the supernatant obtained in the step (3) to be neutral by using sodium hydroxide or potassium hydroxide, and then adding Na with the final concentration of 0.01mol/L into the obtained feed liquid2HPO4and NaH with final concentration of 0.01mol/L2PO4as a buffer system, after complete dissolution, sodium acetate was added to a final concentration of 0.6mol/L, caCl2the final concentration is 0.25mol/L, stirring until dissolving, dripping glacial acetic acid into the feed liquid to adjust the pH to 5.3-5.5, fully stirring, standing at 2-8 ℃ for 12 hours, standing, centrifuging at 12000 g for 30min, collecting supernatant, discarding precipitate, and clarifying and filtering the supernatant with a 0.8 mu m filter membrane if small scraps exist;
(5) Ultrafiltering the supernatant obtained in the step (4) with purified water, collecting polysaccharide solution after the conductivity of the permeation end is lower than 10 mu s/cm, and collecting the polysaccharide solution after sterilization and filtration;
(6) And further freeze-drying the polysaccharide solution to obtain streptococcus pneumoniae capsular polysaccharide.
2. assay of capsular polysaccharide from Streptococcus pneumoniae 11A
The detection of the content of capsular polysaccharide is carried out according to the turbidimetry (3.3.2) in the three parts of the pharmacopoeia of the people's republic of China 2020 edition; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the O-acetyl content is carried out according to the three parts (general rule 3117) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in Table 20.
Table 20 recovery and quality control index of Streptococcus pneumoniae capsular polysaccharide
The results show that the quality control index of the 11A streptococcus pneumoniae refined polysaccharide prepared by the method of the embodiment 7 is reduced compared with the protein content, the nucleic acid and the total phosphorus content of the comparative example 7, the O-acetyl content is increased compared with the comparative example 7, other quality control indexes are not inferior to the comparative example, and the process time is shortened.
Comparative example 8 preparation of Streptococcus pneumoniae capsular polysaccharide degradation polysaccharide
1. Preparation of 11A streptococcus pneumoniae capsular polysaccharide degradation polysaccharide
(1) Taking 3.0+/-0.15 g of refined polysaccharide obtained in the comparative example 7, dissolving in 1.0L of 0.15mol/L sodium chloride solution, and stirring until the polysaccharide is completely dissolved;
(2) Homogenizing the refined polysaccharide solution in the step (1) by a high-pressure homogenizer at 600+/-50 bar, circulating for 3 times, and harvesting the degraded polysaccharide solution;
(3) And freeze-drying the degradation polysaccharide solution, and collecting the degradation polysaccharide after the freeze-drying is finished.
2. detection of degradation polysaccharide of 11A streptococcus pneumoniae capsular polysaccharide
The detection of the degrading polysaccharide of the streptococcus pneumoniae capsular polysaccharide of 11A refers to the 2020 edition of the pharmacopoeia of the people's republic of China, wherein the detection of the capsular polysaccharide content is carried out according to the medium speed turbidimetry (3.3.2) in the 2020 edition of the pharmacopoeia of the people's republic of China; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the O-acetyl content is carried out according to the three parts (general rule 3117) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in Table 21.
table 21 recovery rate and quality control index of Streptococcus pneumoniae capsular polysaccharide degradation polysaccharide
The results show that the quality control index of the 11A streptococcus pneumoniae capsular polysaccharide degradation polysaccharide prepared by the method of the embodiment 8 is reduced compared with that of the degradation polysaccharide prepared by the comparative embodiment 8, and the protein content, the nucleic acid and the total phosphorus content of the degradation polysaccharide are not inferior to those of the comparative embodiment 8, so that the process flow is simplified, and the process time is shortened.
comparative example 9 preparation of 19F Streptococcus pneumoniae capsular polysaccharide
1. purification of 19F Streptococcus pneumoniae capsular polysaccharide
steps (1) and (2) are the same as in example 9, steps (3) - (6) are as follows:
(3) Adding NaCl to the ultrafiltration concentrated solution obtained in the step (2) until the concentration is 0.3m 1/L, adding 0.5% (w/v) sodium deoxycholate after complete dissolution, dropwise adding glacial acetic acid while stirring to adjust the pH to 5.0, fully stirring, standing at 2-8 ℃ for 12 hours, centrifuging for 30min at 12000g, collecting supernatant, discarding precipitation, and clarifying and filtering the supernatant with a 0.8 mu m filter membrane if small fragments exist;
(4) Adjusting the pH of the supernatant obtained in the step (3) to be neutral by using sodium hydroxide or potassium hydroxide, and then adding Na with the final concentration of 0.01mol/L into the obtained feed liquid2HPO4and NaH with final concentration of 0.01mol/L2PO4as a buffer system, after complete dissolution, sodium acetate was added to a final concentration of 0.6mol/L, caCl2The final concentration is 0.25mol/L, stirring until dissolving, dripping glacial acetic acid into the feed liquid to adjust the pH to 5.3-5.5, fully stirring, standing at 2-8 ℃ for 12 hours, centrifuging 12000 g for 30min after standing is finished, collecting supernatant, discarding precipitate, and clarifying and filtering the supernatant with a 0.8 mu m filter membrane if small scraps exist;
(5) Ultrafiltering the supernatant obtained in the step (4) with purified water, collecting polysaccharide solution after the conductivity of the permeation end is lower than 10 mu s/cm, and collecting the polysaccharide solution after sterilization and filtration.
(6) And further freeze-drying the polysaccharide solution to obtain streptococcus pneumoniae capsular polysaccharide.
2. Assay of 19F Streptococcus pneumoniae capsular polysaccharide
The detection of the content of capsular polysaccharide is carried out according to the turbidimetry (3.3.2) in the three parts of the pharmacopoeia of the people's republic of China 2020 edition; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the hexosamine content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the methylpentanose is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in Table 22.
TABLE 22 recovery and quality control indicators for Streptococcus pneumoniae capsular polysaccharide 19F
The results show that the quality control index of the refined polysaccharide of the streptococcus pneumoniae 11A prepared in the embodiment 9 of the invention is reduced compared with the protein content of the refined polysaccharide prepared in the comparative embodiment 9, and other quality control indexes are not inferior to those of the comparative embodiment 9, and the process time is shortened.
comparative example 10 preparation of capsular polysaccharide degradation polysaccharide of Streptococcus pneumoniae 19F
1. Preparation of 19F streptococcus pneumoniae capsular polysaccharide degradation polysaccharide
(1) Taking 3.0+/-0.15 g of refined polysaccharide obtained in comparative example 9, dissolving in 1.0L of 0.15mol/L sodium chloride solution, and stirring until the polysaccharide is completely dissolved;
(2) Homogenizing the refined polysaccharide solution in the step (1) by a high-pressure homogenizer at 600+/-50 bar, circulating for 2 times, and harvesting the degraded polysaccharide solution;
(3) And freeze-drying the degradation polysaccharide solution, and collecting the degradation polysaccharide after the freeze-drying is finished.
2. detection of degradation polysaccharide of 19F streptococcus pneumoniae capsular polysaccharide
The detection of the degradation polysaccharide of the streptococcus pneumoniae capsular polysaccharide of 19F is referred to the 2020 edition of the pharmacopoeia of the people's republic of China, wherein the detection of the capsular polysaccharide content is carried out according to the medium speed turbidimetry (3.3.2) in the 2020 edition of the pharmacopoeia of the people's republic of China; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the hexosamine content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the methylpentanose is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in Table 23.
TABLE 23 recovery and quality control index of Streptococcus pneumoniae capsular polysaccharide degradation polysaccharide 19F
The results show that the quality control index of the 11A streptococcus pneumoniae capsular polysaccharide degradation polysaccharide prepared in the embodiment 10 of the invention is reduced compared with the protein content and the total phosphorus content of the degradation polysaccharide prepared in the comparative example 10, and other quality control indexes are not inferior to those of the comparative example 10, so that the process flow is simplified, and the process time is shortened.
comparative example 11 preparation of Streptococcus pneumoniae capsular polysaccharide
1. Preparation of 2-type streptococcus pneumoniae capsular polysaccharide degradation polysaccharide
steps (1) and (2) are the same as in example 11, steps (3) - (6) are as follows:
(3) Adding NaCl to the concentration of 0.3m 1/L into the ultrafiltration concentrated solution prepared in the step (2), adding 0.5% (w/v) sodium deoxycholate after complete dissolution, dropwise adding glacial acetic acid while stirring to adjust the pH to 5.0, fully stirring, standing at 2-8 ℃ for 12 hours, centrifuging at 12000g for 30min, collecting supernatant, discarding precipitate, and clarifying and filtering the supernatant with a 0.8 mu m filter membrane if small fragments exist;
(4) Adjusting the pH of the supernatant obtained in the step (3) to be neutral by using sodium hydroxide or potassium hydroxide, and then adding Na with the final concentration of 0.01mol/L into the obtained feed liquid2HPO4and NaH with final concentration of 0.01mol/L2PO4as a buffer system, after complete dissolution, sodium acetate was added to a final concentration of 0.6mol/L, caCl2the final concentration is 0.25mol/L, stirring until dissolving, dripping glacial acetic acid into the feed liquid to adjust the pH to 5.3-5.5, fully stirring, standing at 2-8 ℃ for 12 hours, standing, centrifuging at 12000 g for 30min, collecting supernatant, discarding precipitate, and clarifying and filtering the supernatant with a 0.8 mu m filter membrane if small scraps exist;
(5) Ultrafiltering the supernatant obtained in the step (4) with purified water, collecting polysaccharide solution after the conductivity of the permeation end is lower than 10 mu s/cm, and collecting the polysaccharide solution after sterilization and filtration.
(6) And further freeze-drying the polysaccharide solution to obtain streptococcus pneumoniae capsular polysaccharide.
2. Assay of streptococcus pneumoniae capsular polysaccharides
The detection of the capsular polysaccharide of the streptococcus pneumoniae is referred to the 2020 edition of the pharmacopoeia of the people's republic of China, wherein the detection of the content of the capsular polysaccharide is carried out according to the medium speed turbidimetry (3.3.2) in the 2020 edition of the pharmacopoeia of the people's republic of China; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the uronic acid content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the methylpentanose is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in Table 24.
Table 24 recovery and quality control index of Streptococcus pneumoniae capsular polysaccharide
The results show that the quality control index of the streptococcus pneumoniae refined polysaccharide 2 prepared in the embodiment 11 of the invention is reduced compared with the nucleic acid content in the comparative example 11, the methylpentanose content is increased compared with the comparative example 11, and other quality control indexes are not inferior to the comparative example 11, and the process time is shortened.
Comparative example 12 preparation of Streptococcus pneumoniae capsular polysaccharide degradation polysaccharide
1. Preparation of 2-type streptococcus pneumoniae capsular polysaccharide degradation polysaccharide
(1) 3.0+/-0.15 g of the refined polysaccharide obtained in the comparative example 11 is taken and dissolved in 1.0L of 0.15mol/L sodium chloride solution, and the solution is stirred until the polysaccharide is completely dissolved;
(2) Homogenizing the refined polysaccharide solution in the step (1) by a high-pressure homogenizer at 800+/-50 bar, circulating for 3 times, and harvesting the degraded polysaccharide solution. The method comprises the steps of carrying out a first treatment on the surface of the
(3) And freeze-drying the degradation polysaccharide solution, and collecting the degradation polysaccharide after the freeze-drying is finished.
2. Detection of polysaccharide degradation of streptococcus pneumoniae capsular polysaccharide
The detection of the polysaccharide degradation of streptococcus pneumoniae capsular polysaccharide refers to the 2020 edition of the pharmacopoeia of the people's republic of China, wherein the detection of the capsular polysaccharide content is carried out according to the medium speed turbidimetry (3.3.2) in the 2020 edition of the pharmacopoeia of the people's republic of China; the detection of the protein content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the uronic acid content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the methylpentanose is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three 3.1.2.10 parts of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in Table 25.
Table 25 recovery rate and quality control index of degrading polysaccharide of Streptococcus pneumoniae capsular polysaccharide
The results show that the quality control index of the polysaccharide degradation of the streptococcus pneumoniae capsular polysaccharide prepared in the embodiment 12 of the invention is reduced compared with the protein content, the nucleic acid, the total nitrogen and the total phosphorus content of the polysaccharide degradation prepared in the comparative example 12, and other quality control indexes are not inferior to those of the comparative example 12, so that the process flow is simplified, and the process time is shortened.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (12)
1. A method of preparing streptococcus pneumoniae capsular polysaccharides or degradation polysaccharides thereof, the method comprising: after sterilizing a fermentation culture of streptococcus pneumoniae, separating a supernatant, and sequentially performing a first ultrafiltration treatment, a precipitation treatment and a second ultrafiltration treatment on the supernatant;
or sequentially performing a first ultrafiltration treatment, a precipitation treatment, a degradation treatment and a second ultrafiltration treatment on the supernatant;
Wherein the precipitating agent used in the precipitating treatment comprises a calcium salt, and the precipitating treatment is carried out under the condition of pH 2.4-3.6.
2. the method of preparing streptococcus pneumoniae capsular polysaccharides or their degradation polysaccharides according to claim 1, wherein the calcium salt is calcium chloride;
and/or the final concentration of the calcium salt in the precipitation system is 80-200mmol/L.
3. The method for producing streptococcus pneumoniae capsular polysaccharides or their degradation polysaccharides according to claim 2, wherein the precipitation treatment is to collect the supernatant after 1-5 hours at 2-8 ℃ to obtain a first supernatant.
4. A method for preparing streptococcus pneumoniae capsular polysaccharides or polysaccharide degradation thereof according to any one of claims 1-3, wherein the degradation treatment is performed by chemical treatment or physical treatment.
5. The method for preparing streptococcus pneumoniae capsular polysaccharides or polysaccharide degradation thereof according to claim 4, wherein the chemical treatment method is degradation treatment with trifluoroacetic acid to obtain degradation treated product;
And/or the physical treatment method is degradation treatment by adopting an ultrasonic treatment method or a high-pressure homogenization treatment method, so as to obtain a degradation treatment product.
6. the method for producing streptococcus pneumoniae capsular polysaccharides or polysaccharide degradation according to claim 5, wherein the final concentration of trifluoroacetic acid is 0.2-2mol/L and/or the degradation treatment is 2-10h at 25-60 ℃.
7. The method for preparing streptococcus pneumoniae capsular polysaccharides or their degradation polysaccharides according to any of claims 1-3, 5, 6, further comprising: and (3) regulating the pH value of the first supernatant obtained by precipitation treatment or the degradation treatment product to 6.8-7.5, collecting the supernatant to obtain a second supernatant, and carrying out second ultrafiltration treatment on the second supernatant.
8. The method for preparing streptococcus pneumoniae capsular polysaccharide or polysaccharide degradation thereof according to any one of claims 1-3, 5, 6, wherein the first ultrafiltration treatment is performed using an ultrafiltration membrane with a pore size of 100-150 KD;
and/or, for the second supernatant obtained without degradation treatment, the second ultrafiltration treatment is carried out using an ultrafiltration membrane having a pore size of 100-150 KD; for the second supernatant obtained by the degradation treatment, the second ultrafiltration treatment is carried out by using an ultrafiltration membrane with a pore size of 30-50 KD.
9. the streptococcus pneumoniae capsular polysaccharide or the polysaccharide degradation thereof prepared by the method for preparing streptococcus pneumoniae capsular polysaccharide or polysaccharide degradation thereof according to any one of claims 1-8.
10. The method for preparing streptococcus pneumoniae capsular polysaccharides or degradation polysaccharides thereof according to any one of claims 1-8 or the use of streptococcus pneumoniae capsular polysaccharides or degradation polysaccharides thereof according to claim 9 in the preparation of products containing streptococcus pneumoniae capsular polysaccharides.
11. a streptococcus pneumoniae capsular polysaccharide vaccine or streptococcus pneumoniae capsular polysaccharide conjugate vaccine, characterized in that the vaccine comprises a streptococcus pneumoniae capsular polysaccharide or a degrading polysaccharide thereof according to claim 9.
12. A method of preparing a streptococcus pneumoniae capsular polysaccharide vaccine or a streptococcus pneumoniae capsular polysaccharide conjugate vaccine, the method comprising: the streptococcus pneumoniae capsular polysaccharide or the polysaccharide degradation thereof prepared by the method for preparing the streptococcus pneumoniae capsular polysaccharide or the polysaccharide degradation thereof according to any one of claims 1-8, wherein the streptococcus pneumoniae capsular polysaccharide or the polysaccharide degradation thereof is used as an immunogen to prepare a streptococcus pneumoniae capsular polysaccharide vaccine or a streptococcus pneumoniae capsular polysaccharide conjugate vaccine.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102660602A (en) * | 2012-04-17 | 2012-09-12 | 江苏康泰生物医学技术有限公司 | Method for rapidly purifying bacteria capsular polysaccharide |
| CN105669873A (en) * | 2016-01-26 | 2016-06-15 | 北京民海生物科技有限公司 | Hydrolysis method of different serotypes of streptococcus pneumoniae capsular polysaccharides |
| CN106146679A (en) * | 2015-04-23 | 2016-11-23 | 中国医学科学院医学生物学研究所 | A kind of method of purification of bacterial capsular polysaccharide |
| US20210070890A1 (en) * | 2019-09-06 | 2021-03-11 | Serum Institute Of India Private Limited | Method for obtaining purified bacterial polysaccharides |
| CN113603804A (en) * | 2021-09-01 | 2021-11-05 | 沈阳礼慧医药科技有限公司 | Refining method of streptococcus pneumoniae capsular polysaccharide |
| CN114853917A (en) * | 2022-04-22 | 2022-08-05 | 兰州生物制品研究所有限责任公司 | Methods for preparing pneumococcal capsular polysaccharide and pneumococcal vaccine |
| CN116970095A (en) * | 2022-04-11 | 2023-10-31 | 北京民海生物科技有限公司 | Preparation method of pneumococcal capsular polysaccharide |
-
2024
- 2024-02-22 CN CN202410196193.6A patent/CN117756958A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102660602A (en) * | 2012-04-17 | 2012-09-12 | 江苏康泰生物医学技术有限公司 | Method for rapidly purifying bacteria capsular polysaccharide |
| CN106146679A (en) * | 2015-04-23 | 2016-11-23 | 中国医学科学院医学生物学研究所 | A kind of method of purification of bacterial capsular polysaccharide |
| CN105669873A (en) * | 2016-01-26 | 2016-06-15 | 北京民海生物科技有限公司 | Hydrolysis method of different serotypes of streptococcus pneumoniae capsular polysaccharides |
| US20210070890A1 (en) * | 2019-09-06 | 2021-03-11 | Serum Institute Of India Private Limited | Method for obtaining purified bacterial polysaccharides |
| CN113603804A (en) * | 2021-09-01 | 2021-11-05 | 沈阳礼慧医药科技有限公司 | Refining method of streptococcus pneumoniae capsular polysaccharide |
| CN116970095A (en) * | 2022-04-11 | 2023-10-31 | 北京民海生物科技有限公司 | Preparation method of pneumococcal capsular polysaccharide |
| CN114853917A (en) * | 2022-04-22 | 2022-08-05 | 兰州生物制品研究所有限责任公司 | Methods for preparing pneumococcal capsular polysaccharide and pneumococcal vaccine |
Non-Patent Citations (2)
| Title |
|---|
| 李小波;张锐;谢媛媛;程尊平;陈娟;江山;: "酸沉淀法纯化14血清型肺炎链球菌荚膜多糖工艺的建立", 中国生物制品学杂志, no. 08, 20 August 2016 (2016-08-20) * |
| 焦瑞身等: "《生物工程概论》", 30 April 1991, 化学工业出版社, pages: 211 * |
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