A method for preparing pneumococcal capsular polysaccharide or its degraded polysaccharide

Abstract

The present invention relates to the technical field of biological products, and in particular to a method for preparing pneumococcal capsular polysaccharides or their degradable polysaccharides. The method for preparing pneumococcal capsular polysaccharides or their degradable polysaccharides provided by the present invention includes: after the fermentation culture of pneumococcal is sterilized, the supernatant is separated, and the supernatant is sequentially subjected to a first ultrafiltration treatment, a precipitation treatment, a second ultrafiltration treatment; or, the supernatant is sequentially subjected to a first ultrafiltration treatment, a precipitation treatment, a degradation treatment, and a second ultrafiltration treatment; the precipitant used in the precipitation treatment comprises a calcium salt, and the precipitation treatment is carried out under pH 2.4-3.6. The method has the advantages of simple process and short preparation cycle, significantly reduces time and material costs, and the quality control indicators such as nucleic acid, protein and other impurities and specific groups of the refined polysaccharides and degraded polysaccharides are better than the standard requirements, and can be used to prepare pneumococcal capsular polysaccharide vaccines or combined vaccines.

Description

Technical Field

[0001] The present invention relates to the technical field of biological products, and in particular to a method for preparing pneumococcal capsular polysaccharide or its degraded polysaccharide. Background Art

[0002] Pneumococcal infection is one of the major causes of death worldwide and is also the main cause of pneumonia, meningitis, and otitis media. With the extensive use of antibiotics, drug-resistant strains are increasing day by day, and the development of vaccines has attracted much attention. Studies have found that the capsular polysaccharides on the surface of bacteria can protect the body against bacterial infections. Since polysaccharides are non-thymus-dependent antigens, re-immunization cannot make the reduced antibodies reach the level of primary immunization, cannot induce the production of immune memory cells, and has almost no protective effect on infants and the elderly, who are at high risk of pneumococcal infection. The capsular polysaccharides of pneumococci are coupled to protein carriers to make them thymus-dependent antigens, which can activate B cells to produce antibodies with the assistance of T cells and macrophages, provide immune protection for infants, induce immune memory, and re-immunization has a reinforcing immune effect. Studies have shown that the introduction of pneumococcal vaccines has significantly reduced the burden of pneumococcal disease worldwide.

[0003] The pneumococcal capsular polysaccharide is a series of high molecular weight homologues composed of repeating units, which are usually obtained by fermenting and culturing pneumococcal capsular polysaccharides and extracting and purifying them from the fermentation culture. On the one hand, when preparing pneumococcal capsular polysaccharide vaccines, there are problems such as complex polysaccharide purification process, long purification cycle, and safety or health risks in the use of reagents. For example, the existing pneumococcal refined polysaccharide preparation methods often use organic reagents such as phenol and ethanol, which are harmful to human health and difficult to completely remove; the preparation of pneumococcal refined polysaccharides is often complicated and time-consuming; in addition, chromatography systems are often used in the preparation of refined polysaccharides (for example: Chinese patent applications CN104815327A, CN108079286A, CN112646050A, CN116970095A, etc.), and the instruments and materials are expensive, which greatly increases the production cost. Chinese patent application CN116970095A discloses a process of removing protein and nucleic acid impurities by a "two-step precipitation method". By mixing sodium deoxycholate (DOC) with the first ultrafiltrate under suitable neutral salt conditions and controlling the pH of the solution to 4.5-5.0, sodium deoxycholate can efficiently exert its adsorption and precipitation effect on protein. Then, calcium salt precipitation is used to further remove protein, nucleic acid and other impurities. The supernatant is collected by centrifugation, filtered and then lyophilized to obtain refined polysaccharides. However, this method still has problems such as complex process, large amount of sodium deoxycholate, and long purification cycle. Therefore, it is necessary to develop a purification method with simple process, high efficiency and higher safety.

[0004] On the other hand, when preparing pneumococcal capsular polysaccharide conjugate vaccines, polysaccharides often have a large molecular weight, which is not conducive to the binding of polysaccharides, and a small molecular weight will affect the immunogenicity. Therefore, it is necessary to use a certain method to degrade the polysaccharide macromolecules into small molecular weight polysaccharides to meet the needs and applications of pneumococcal conjugate vaccines. The existing capsular polysaccharide degradation process is to dissolve the pneumococcal refined polysaccharide freeze-dried preparation and then degrade it. The operation is complicated and not suitable for large-scale production and application. Summary of the invention

[0005] The invention provides a method for preparing pneumococcal capsular polysaccharide or its degraded polysaccharide.

[0006] During the research and development process of the preparation method of pneumococcal capsular polysaccharide, the present invention accidentally discovered a simple and rapid preparation method of pneumococcal polysaccharide. This method only requires one-step precipitation (one-step calcium hydrochloric acid precipitation) using calcium salt under acidic conditions in combination with a conventional ultrafiltration step to prepare high-quality refined polysaccharide. All indicators of the refined polysaccharide are higher than the Chinese Pharmacopoeia standards, which significantly reduces material and time costs and improves purification efficiency.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing pneumococcal capsular polysaccharide or a degraded polysaccharide thereof, the method comprising: sterilizing a fermentation culture of pneumococcal bacteria, separating a supernatant, and sequentially subjecting the supernatant to a first ultrafiltration treatment, a precipitation treatment, and a second ultrafiltration treatment;

[0009] Alternatively, the supernatant is sequentially subjected to a first ultrafiltration treatment, a precipitation treatment, a degradation treatment, and a second ultrafiltration treatment;

[0010] The precipitant used in the precipitation treatment comprises calcium salt, and the precipitation treatment is carried out under pH 2.4-3.6.

[0011] The preparation method of the present invention is divided into two implementation modes: no degradation treatment and degradation treatment. The capsular polysaccharide (refined polysaccharide) obtained by the method without degradation treatment has a relatively large molecular weight and is suitable for preparing a pneumococcal capsular polysaccharide vaccine. The polysaccharide (degraded polysaccharide) with a reduced molecular weight can be obtained by degradation treatment, and is more suitable for preparing a pneumococcal capsular polysaccharide conjugate vaccine after being combined with a carrier protein (such as TT, DT, CRM197, etc.).

[0012] The present invention finds that, based on the above-mentioned "one-step calcium hydrochloric acid precipitation" method, the degradation treatment can be set after the "one-step calcium hydrochloric acid precipitation", without the need to re-dissolve the obtained freeze-dried refined polysaccharide before performing the degradation treatment, which greatly simplifies the process flow and improves the preparation efficiency.

[0013] The key to achieving the effect of the above preparation method is to use calcium salt as a precipitant and perform precipitation treatment (calcium hydrochloride acid precipitation) under acidic conditions of pH 2.4-3.6. In conjunction with the above "calcium hydrochloride acid precipitation", an ultrafiltration treatment is set before and after it. The main function of the first ultrafiltration treatment is to remove small molecular impurities in the supernatant after the fermentation culture is sterilized, which is more conducive to the "calcium hydrochloride acid precipitation" to better play the role of impurity removal and improve the efficiency of impurity removal. The main function of the second ultrafiltration treatment is to concentrate the polysaccharide solution and perform diafiltration on the polysaccharide solution to further remove small molecular impurities and salt ions in the polysaccharide solution.

[0014] Preferably, the calcium salt is calcium chloride.

[0015] Preferably, the final concentration of the calcium salt in the precipitation system is 80-200 mmol / L.

[0016] Preferably, calcium chloride having a final concentration of 80-200 mmol / L is added to the first ultrafiltration concentrate obtained in the first ultrafiltration treatment, and the pH is adjusted to 2.4-3.6 with an acid solution for precipitation treatment.

[0017] Preferably, the precipitation treatment is to collect the supernatant after treating at 2-8°C for 1-5h to obtain the first supernatant.

[0018] Unlike some existing methods for purifying pneumococcal capsular polysaccharides that require temperature-raising acid precipitation (temperature-raising acid precipitation easily leads to degradation of polysaccharides, resulting in a decrease in molecular weight and affecting its immunogenicity), the precipitation treatment of the present invention only needs to be carried out under low temperature conditions to achieve a better impurity removal effect.

[0019] For the embodiment comprising a degradation treatment step, the degradation treatment is performed by a chemical treatment method or a physical treatment method.

[0020] Among them, the chemical treatment method is preferably to use trifluoroacetic acid for degradation treatment to obtain a degraded product.

[0021] The physical treatment method preferably uses an ultrasonic treatment method or a high-pressure homogenization treatment method to perform degradation treatment to obtain a degraded product.

[0022] Specifically, the ultrasonic treatment method can be found in Chinese patent CN108079286B, and the high-pressure homogenization treatment method can be found in Chinese patent application CN106413747A.

[0023] In some embodiments of the present invention, trifluoroacetic acid is used for degradation treatment, wherein the final concentration of trifluoroacetic acid is 0.2-2 mol / L. Preferably, the degradation treatment is carried out at 25-60° C. for 2-10 h. After the degradation treatment is completed, the supernatant is collected.

[0024] The above degradation treatment degrades the macromolecular polysaccharide into a degraded polysaccharide of suitable molecular weight that is suitable for the preparation of conjugate vaccine and does not affect its immunogenicity.

[0025] The method described above also includes: adjusting the pH of the first supernatant obtained by precipitation treatment or the degradation product to 6.8-7.5, collecting the supernatant to obtain a second supernatant, and subjecting the second supernatant to a second ultrafiltration treatment.

[0026] In the present invention, the pH can be adjusted to acidic by using acidic solution, including but not limited to glacial acetic acid, hydrochloric acid, phosphoric acid, etc., preferably phosphoric acid. The pH can be adjusted to neutral or alkaline by using alkaline solution, including but not limited to sodium hydroxide, potassium hydroxide, etc., preferably sodium hydroxide.

[0027] In the method described above, the first ultrafiltration treatment is performed using an ultrafiltration membrane with a pore size of 100-150 KD (preferably 100 KD).

[0028] In some embodiments of the present invention, the first ultrafiltration treatment is to use a membrane package with a pore size of 100KD to concentrate 3-5 times and then ultrafilter with equal volume. Preferably, the volume of purified water used for ultrafiltration is 4-6 times the volume of the concentrate.

[0029] In the method described above, for the second supernatant obtained without degradation treatment, the second ultrafiltration treatment is carried out using an ultrafiltration membrane with a pore size of 100-150KD; for the second supernatant obtained after degradation treatment, the second ultrafiltration treatment is carried out using an ultrafiltration membrane with a pore size of 30-50KD (preferably 30KD).

[0030] Preferably, in the second ultrafiltration treatment, the volume of water for injection used for diafiltration is 8-15 times the volume of the supernatant.

[0031] Preferably, after the second ultrafiltration treatment, the method further comprises the step of drying the second ultrafiltration concentrate obtained by the second ultrafiltration treatment.

[0032] The drying may be freeze-drying.

[0033] In the present invention, the separation of the supernatant can be carried out by conventional solid-liquid separation methods, including but not limited to centrifugal separation.

[0034] In principle, the preparation method described in the present invention has no special restrictions on the method of sterilization. The currently commonly used sodium deoxycholate (for example, the sterilization method used in WO2023202607A1 and CN114106210A) can be used to sterilize the fermentation culture of Streptococcus pneumoniae, or other reagents that can kill Streptococcus pneumoniae and release capsular polysaccharides can be used for sterilization.

[0035] In some embodiments of the present invention, sodium deoxycholate is added to the fermentation culture of Streptococcus pneumoniae for sterilization, and the final concentration of sodium deoxycholate is 0.06%-0.24%.

[0036] In other embodiments of the present invention, β-propiolactone (BPL) is used to sterilize the fermentation culture of Streptococcus pneumoniae. For the final concentration of β-propiolactone in the treatment system, the β-propiolactone dosage commonly used in the art can be added. Preferably, the final concentration of β-propiolactone in the treatment system is not less than 0.01%. Exemplary final concentrations of β-propiolactone 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 β-propiolactone in the treatment system is not less than 0.05%. In some specific embodiments, the final concentration of β-propiolactone in the treatment system is 0.05-1%. Preferably, it is 0.05-0.5%. The temperature of the treatment is preferably 2-8°C. The time of the treatment is preferably 8-16h. More preferably, it is 10-14h. The treatment is preferably a low-temperature incubation treatment after uniform mixing.

[0037] Preferably, β-propiolactone (BPL) is added to the fermentation culture of Streptococcus pneumoniae for treatment in the late logarithmic growth stage. After adding β-propiolactone (BPL), the mixture is stirred and mixed, and then incubated at low temperature.

[0038] When β-propiolactone is used for sterilization, the above-mentioned preparation method preferably does not use sodium deoxycholate.

[0039] In the existing pneumococcal capsular polysaccharide production method, sodium deoxycholate plays a very important role in the extraction of capsular polysaccharides as a lysing agent for lysing the thalline to release the capsular polysaccharide, and there is currently no alternative reagent for the preparation of pneumococcal capsular polysaccharides. The present invention unexpectedly found in the research and development process that β-propiolactone has a significant effect of inactivating pneumococcal pneumococcus and promoting pneumococcal pneumococcus to release capsular polysaccharides, which can be used to treat pneumococcus and extract capsular polysaccharides, and unlike sodium deoxycholate, β-propiolactone does not lyse the thalline, thereby significantly reducing the release of impurities such as proteins and nucleic acids in the thalline, which is more conducive to reducing the impurity content and removal difficulty in the pneumococcal polysaccharide purification process, and β-propiolactone is easily hydrolyzed, and the hydrolyzate is non-toxic and harmless, so it will not cause residues or health risks in the capsular polysaccharide product. β-propiolactone is currently used as a virus inactivator for the preparation of viral vaccines, and there is no relevant report on its function of promoting pneumococcal pneumococcus to release capsular polysaccharides and reducing the release of impurities such as intracellular proteins and nucleic acids. In the above method, β-propiolactone is used instead of sodium deoxycholate to treat the fermentation culture of Streptococcus pneumoniae, which not only solves the health risks or side effects caused by the residual sodium deoxycholate, but also significantly reduces the content of impurities such as protein in the extracted capsular polysaccharide, is more conducive to the subsequent purification and removal of impurities, and has a higher polysaccharide recovery rate, which is significantly higher than that of the fermentation culture of Streptococcus pneumoniae treated with formaldehyde.

[0040] In the present invention, the fermentation culture is a culture (eg, fermentation broth) containing Streptococcus pneumoniae obtained by fermentation culture of Streptococcus pneumoniae.

[0041] The method for preparing the pneumococcal capsular polysaccharide of the present invention can be used for different types of pneumococcal bacteria, including but not limited to types 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F.

[0042] Preferably, the Streptococcus pneumoniae is type 2, 5, 6A, 10A, 11A, or 19F.

[0043] The present invention provides Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide prepared by the above method for preparing Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide.

[0044] The present invention provides the above-mentioned method for preparing Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide or the use of the Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide in preparing a product containing Streptococcus pneumoniae capsular polysaccharide.

[0045] Preferably, the product containing Streptococcus pneumoniae capsular polysaccharide is a vaccine.

[0046] The vaccine comprises a Streptococcus pneumoniae capsular polysaccharide vaccine or a Streptococcus pneumoniae capsular polysaccharide conjugate vaccine.

[0047] The present invention provides a Streptococcus pneumoniae capsular polysaccharide vaccine or a Streptococcus pneumoniae capsular polysaccharide conjugate vaccine, wherein the vaccine comprises the Streptococcus pneumoniae capsular polysaccharide or the degraded polysaccharide thereof as described above.

[0048] The above-mentioned Streptococcus pneumoniae capsular polysaccharide vaccine or Streptococcus pneumoniae capsular polysaccharide conjugate vaccine may be a monovalent or multivalent vaccine.

[0049] Wherein, the multivalent vaccine comprises at least two types of capsular polysaccharides 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, and 33F.

[0050] Specifically, the pneumococcal capsular polysaccharide vaccine can be a 23-valent, 24-valent, 25-valent, 26-valent, 27-valent, 28-valent, 29-valent, 30-valent or even higher-valent pneumococcal capsular polysaccharide vaccine. Exemplary is a 23-valent pneumococcal capsular polysaccharide vaccine.

[0051] The pneumococcal 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 pneumococcal capsular polysaccharide conjugate vaccine. Exemplary are 13-valent pneumococcal capsular polysaccharide conjugate vaccine, 20-valent pneumococcal capsular polysaccharide conjugate vaccine, and 24-valent pneumococcal capsular polysaccharide conjugate vaccine.

[0052] Exemplarily, the 23-valent pneumococcal capsular polysaccharide vaccine contains pneumococcal serotypes such as 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.

[0053] Exemplarily, the 13-valent pneumococcal capsular polysaccharide conjugate vaccine contains pneumococcal serotypes such as 4, 5, 6A, 6B, 7F, 9V, 18C, 19A, 19F, and 23F.

[0054] Exemplarily, the 20-valent pneumococcal capsular polysaccharide conjugate vaccine contains pneumococcal serotypes such as 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.

[0055] Exemplarily, the 24-valent pneumococcal capsular polysaccharide conjugate vaccine contains pneumococcal serotypes such as 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.

[0056] The present invention provides a method for preparing a pneumococcal capsular polysaccharide vaccine or a pneumococcal capsular polysaccharide conjugate vaccine. The method comprises: preparing pneumococcal capsular polysaccharide or a degraded polysaccharide thereof by using the method for preparing pneumococcal capsular polysaccharide or a degraded polysaccharide thereof, and preparing the pneumococcal capsular polysaccharide vaccine or a pneumococcal capsular polysaccharide conjugate vaccine by using the pneumococcal capsular polysaccharide or a degraded polysaccharide thereof as an immunogen.

[0057] The beneficial effects of the present invention include at least:

[0058] (1) The preparation method of the present invention has the advantages of simple process and short preparation cycle. In the preparation of refined polysaccharides, the purification process time is significantly shortened; in the preparation of degraded polysaccharides, the polysaccharides are directly degraded in the later stage of purification, which greatly simplifies the process flow. According to calculations, in terms of time cost, compared with the technical solution disclosed in Chinese patent application CN116970095A, when preparing refined polysaccharides, the method disclosed in patent application CN116970095A requires 2 days, while the method of the present invention only requires 1 day, and the process time is shortened by 50%; when preparing degraded polysaccharides: the method disclosed in patent application CN116970095A requires 3 days, while the method of the present invention only requires 2 days, and the process time is shortened by more than 30%, that is, the time cost is greatly reduced; in terms of material cost, whether it is preparing refined polysaccharides or degrading polysaccharides, taking the feeding amount of sodium deoxycholate as an example, the method of the present invention has at least reduced by 50%.

[0059] (2) The method of the present invention is used to prepare pneumococcal capsular polysaccharides and degraded polysaccharides, and the quality control indicators of impurities such as nucleic acids and proteins and specific groups are better than the standards of the "Pharmacopoeia of the People's Republic of China" (2020 edition) (23-valent pneumococcal polysaccharide vaccine), and are better than the quality indicators of existing processes. It can be used for the preparation of pneumococcal capsular polysaccharide vaccine or capsular polysaccharide conjugate vaccine.

[0060] (3) The method of the present invention avoids the use of toxic and harmful reagents such as phenol, acetone and ethanol during the preparation process, reducing the harm to human health and the environment; at the same time, it avoids the use of chromatography operations, reduces costs, and is more conducive to industrial-scale production. DETAILED DESCRIPTION

[0061] The present invention provides a method for preparing pneumococcal capsular polysaccharide or its degraded polysaccharide.

[0062] In some embodiments of the present invention, a method for preparing a pneumococcal capsular polysaccharide is provided, the method comprising the following steps:

[0063] (1) After sterilization, the fermentation broth of Streptococcus pneumoniae was allowed to stand at 4°C for 6-12 hours, and then the supernatant was collected by centrifugation;

[0064] (2) ultrafiltration of the supernatant obtained in step (1) using a membrane with a pore size of 100 KD to obtain a first ultrafiltration concentrate, the volume of which after concentration is 1 / 5 to 1 / 4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times the volume of the concentrate;

[0065] (3) Add CaCl2 solution with a final concentration of 80-200 mmol / L to the first ultrafiltration concentrate, stir thoroughly, adjust the pH to 2.4-3.6 with acid solution, stir thoroughly, let stand at 2-8°C for 1-5 hours, then centrifuge to collect the supernatant, adjust the pH to 7.00-7.50 with alkali solution, and centrifuge again to collect the supernatant;

[0066] (4) ultrafiltration of the supernatant obtained in step (3) using a membrane with a pore size of 100 KD to obtain a polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in the ultrafiltration is at least 10 times the volume of the supernatant;

[0067] (5) The polysaccharide ultrafiltration concentrate obtained in step (4) is freeze-dried, and the purified polysaccharide is collected after completion.

[0068] In some embodiments of the present invention, a method for preparing a degraded polysaccharide of a capsular polysaccharide of Streptococcus pneumoniae is provided, the method comprising the following steps:

[0069] (1) After sterilization, the fermentation broth of Streptococcus pneumoniae was allowed to stand at 4°C for 6-12 hours, and then the supernatant was collected by centrifugation;

[0070] (2) ultrafiltration of the supernatant obtained in step (1) using a membrane with a pore size of 100 KD to obtain a first ultrafiltration concentrate, the volume of which after concentration is 1 / 5 to 1 / 4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times the volume of the concentrate;

[0071] (3) Add CaCl2 solution with a final concentration of 80-200 mmol / L to the first ultrafiltration concentrate, stir thoroughly, adjust the pH to 2.4-3.6 with acid solution, stir thoroughly, let stand at 2-8°C for 1-5 hours, then centrifuge to collect the supernatant, adjust the pH to 7.00-7.50 with alkali solution, and centrifuge again to collect the supernatant;

[0072] (4) adding trifluoroacetic acid at a final concentration of 0.2-2 mol / L to the supernatant obtained in step (3), standing at 25-60°C for 2-10 h, adjusting the pH to 7.00-7.50 with alkaline solution, and centrifuging again to collect the supernatant;

[0073] (5) The supernatant obtained in step (4) is ultrafiltered using a membrane with a pore size of 30KD to obtain a polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in the ultrafiltration is at least 10 times the volume of the supernatant;

[0074] (6) The polysaccharide ultrafiltration concentrate obtained in step (5) is freeze-dried, and after completion, the degraded polysaccharide is collected.

[0075] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0076] Example 1 Preparation of 6A Streptococcus pneumoniae capsular polysaccharide

[0077] 1. Preparation of 6A Streptococcus pneumoniae capsular polysaccharide

[0078] (1) The 6A pneumococcal fermentation liquid was sterilized with 0.1% DOC and then allowed to stand at 4°C for 12 hours. g Centrifuge for 30 min and collect the supernatant;

[0079] (2) The supernatant obtained in step (1) is ultrafiltered using a membrane with a pore size of 100 KD to obtain an ultrafiltration concentrate, the volume of which after concentration is 1 / 5 to 1 / 4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times the volume of the concentrate;

[0080] (3) Add a CaCl2 solution with a final concentration of 200 mmol / L to the ultrafiltration concentrate obtained in step (2), stir thoroughly, and adjust the pH to 3.00 ± 0.1 with phosphoric acid. Stir thoroughly, let stand at 2-8°C for 1 hour, and then heat at 12000 g Centrifuge for 30 min, collect the supernatant, adjust the pH of the supernatant to 7.00-7.50 with NaOH solution, and then centrifuge again at 12000 g Centrifuge for 30 min and collect the supernatant;

[0081] (4) ultrafiltration of the supernatant obtained in step (3) using a membrane with a pore size of 100 KD to obtain a polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in the ultrafiltration is at least 10 times the volume of the supernatant;

[0082] (5) The ultrafiltration concentrate obtained in step (4) is freeze-dried, and the purified polysaccharide is collected after completion.

[0083] 2. Detection of Capsular Polysaccharide of Type 6A Streptococcus Pneumococcus

[0084] The determination of capsular polysaccharide content is carried out according to the rate turbidimetry (3.3.2) in the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III; the determination of protein content is carried out according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0731); the determination of nucleic acid content is carried out according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401); the determination of total nitrogen content is carried out according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0704); the determination of phosphorus content is carried out according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 3103); the determination of methyl pentose is carried out according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401); the determination of the molecular size of capsular polysaccharide is carried out according to the first method in 3.1.2.10 of the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III.

[0085] The recovery rate was calculated as follows: recovery rate = refined polysaccharide content / polysaccharide content in the supernatant after fermentation broth sterilization × 100%.

[0086] The relevant test results are shown in Table 1.

[0087] Table 1 Recovery rate and quality control indexes of 6A Streptococcus pneumoniae capsular polysaccharide

[0088]

[0089] Note: 001, 002, and 003 in Table 1 represent three batches of experiments, the same below.

[0090] Example 2 Preparation of 6A Streptococcus pneumoniae capsular polysaccharide degradable polysaccharide

[0091] 1. Preparation of degraded polysaccharide of 6A type pneumococcal capsular polysaccharide

[0092] (1) After the fermentation broth of type 6A pneumococcus was sterilized with 0.1% DOC, it was allowed to stand at 4°C for 12 hours, then centrifuged at 12,000 g for 30 minutes, and the supernatant was collected;

[0093] (2) The supernatant obtained in step (1) is ultrafiltered using a membrane with a pore size of 100 KD to obtain an ultrafiltration concentrate, the volume of which after concentration is 1 / 5 to 1 / 4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times the volume of the concentrate;

[0094] (3) Add CaCl2 solution with a final concentration of 80mmol / L (6A-2308001L), 120mmol / L (6A-2308002L), and 200mmol / L (6A-2308003L) to the ultrafiltration concentrate obtained in step (2), stir thoroughly, and adjust the pH to 3.00±0.1 with phosphoric acid. Stir thoroughly, let stand at 2-8℃ for 1h, and then heat at 12000℃. g Centrifuge for 30 min and collect the supernatant;

[0095] (4) adding trifluoroacetic acid at a final concentration of 0.25 mol / L to the supernatant obtained in step (3), standing at 40°C for 3 h, adjusting the pH to 7.00-7.50, and centrifuging again to collect the supernatant;

[0096] (5) ultrafiltration of the supernatant solution obtained in step (4) using a membrane with a pore size of 30KD to obtain a polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in the ultrafiltration is at least 10 times the volume of the supernatant;

[0097] (6) The ultrafiltration concentrate is freeze-dried and the degraded polysaccharides are collected after completion.

[0098] 2. Degradation of polysaccharide from the capsular polysaccharide of Streptococcus pneumoniae type 6A

[0099] Degradation of 6A pneumococcal capsular polysaccharide The determination of polysaccharides refers to the 2020 edition of the Pharmacopoeia of the People's Republic of China, among which the determination of capsular polysaccharide content is carried out according to the rate turbidimetry (3.3.2) in Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; the determination of protein content is carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0731); the determination of nucleic acid content is carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401) ; The total nitrogen content was tested in accordance with the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 0704); the phosphorus content was tested in accordance with the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 3103); the methyl pentose content was tested in accordance with the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 0401); the molecular size of the capsular polysaccharide was determined in accordance with the first method in 3.1.2.10 of the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III. The relevant test results are shown in Table 2.

[0100] Table 2 Recovery rate and quality control indexes of degraded polysaccharide of 6A Streptococcus pneumoniae capsular polysaccharide

[0101]

[0102] Example 3 Preparation of Type 5 Streptococcus pneumoniae Capsular Polysaccharide

[0103] 1. Preparation of Streptococcus pneumoniae type 5 capsular polysaccharide

[0104] (1) The fermentation broth of Streptococcus pneumoniae type 5 was sterilized with 0.1% DOC and then allowed to stand at 4°C for 12 hours. g Centrifuge for 30 min and collect the supernatant;

[0105] (2) The supernatant obtained in step (1) is ultrafiltered using a membrane with a pore size of 100 KD to obtain an ultrafiltration concentrate, the volume of which after concentration is 1 / 5 to 1 / 4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times the volume of the concentrate;

[0106] (3) Add a CaCl2 solution with a final concentration of 80 mmol / L to the ultrafiltration concentrate obtained in step (2), stir thoroughly, and adjust the pH to 3.00 ± 0.1 with phosphoric acid. Stir thoroughly, and let stand at 2-8°C for 5 h, then heat at 12000 g Centrifuge for 30 min, collect the supernatant, adjust the pH to 7.00-7.50 with NaOH, and then resuspend at 12000 g Centrifuge for 30 min and collect the supernatant;

[0107] (4) ultrafiltration of the supernatant obtained in step (3) using a membrane with a pore size of 100 KD to obtain a polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in the ultrafiltration is at least 10 times the volume of the supernatant;

[0108] (5) The ultrafiltration concentrate obtained in step (4) is freeze-dried, and the purified polysaccharide is collected after completion.

[0109] Detection of Capsular Polysaccharide of Type 2 and Type 5 Streptococcus Pneumoniae

[0110] The content of capsular polysaccharide shall be determined according to the rate turbidimetry (3.3.2) in the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III; the content of protein shall be determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0731); the content of nucleic acid shall be determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401); the content of total nitrogen shall be determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0704); phosphorus The content was tested in accordance with the Pharmacopoeia of the People's Republic of China, Part III, 2020 edition (General Rule 3103); the uronic acid content was tested in accordance with the Pharmacopoeia of the People's Republic of China, Part III, 2020 edition (General Rule 0731); the aminohexose content was tested in accordance with the Pharmacopoeia of the People's Republic of China, Part III, 2020 edition (General Rule 0401); the molecular size of the capsular polysaccharide was determined in accordance with the first method in 3.1.2.10 of Part III, 2020 edition of the Pharmacopoeia of the People's Republic of China. The relevant test results are shown in Table 3.

[0111] Table 3 Recovery rate and quality control indexes of type 5 monovalent Streptococcus pneumoniae capsular polysaccharide

[0112]

[0113] Example 4 Preparation of degraded polysaccharide of Streptococcus pneumoniae type 5 capsular polysaccharide

[0114] Preparation of degraded polysaccharide of 1.5-streptococcus pneumoniae capsular polysaccharide

[0115] (1) After the fermentation broth of Streptococcus pneumoniae type 5 was sterilized with 0.1% DOC, it was allowed to stand at 4°C for 12 hours, then centrifuged at 12,000 g for 30 minutes, and the supernatant was collected;

[0116] (2) The supernatant obtained in step (1) is ultrafiltered using a membrane with a pore size of 100 KD to obtain an ultrafiltration concentrate, the volume of which after concentration is 1 / 5 to 1 / 4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times the volume of the concentrate;

[0117] (3) Add a CaCl2 solution with a final concentration of 100 mmol / L to the ultrafiltration concentrate obtained in step (2), stir thoroughly and adjust the pH to 2.50±0.1 (5-2308001L), 3.00±0.1 (5-2308002L), and 3.50±0.1 (5-2308003L) with phosphoric acid, stir thoroughly and let stand at 2-8°C for 5 h, then centrifuge at 12000 g for 30 min, and collect the supernatant;

[0118] (4) Add trifluoroacetic acid at a final concentration of 1.5 mol / L to the supernatant obtained in step (3), let stand at 25°C for 10 h, adjust the pH to 7.00-7.50, and centrifuge again to collect the supernatant;

[0119] (5) The supernatant obtained in step (4) is ultrafiltered using a membrane with a pore size of 30KD to obtain a polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in the ultrafiltration is at least 10 times the volume of the supernatant;

[0120] (6) The ultrafiltration concentrate obtained in step (5) is freeze-dried, and the degraded polysaccharides are collected after the freeze-drying.

[0121] Detection of degraded polysaccharides of capsular polysaccharides of Streptococcus pneumoniae type 2 and 5

[0122] The determination of degradable polysaccharides of type 5 pneumococcal capsular polysaccharides shall refer to the 2020 edition of the Pharmacopoeia of the People's Republic of China, among which the determination of capsular polysaccharide content shall be carried out according to the rate turbidimetry (3.3.2) in Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; the determination of protein content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0731); the determination of nucleic acid content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401); the determination of total nitrogen content shall be carried out according to Part 2 of the Pharmacopoeia of the People's Republic of China. 020 edition, Part III (General Rule 0704); phosphorus content was tested in accordance with the 2020 edition, Part III of the Pharmacopoeia of the People's Republic of China (General Rule 3103); uronic acid content was tested in accordance with the 2020 edition, Part III of the Pharmacopoeia of the People's Republic of China (General Rule 0731); hexosamine content was tested in accordance with the 2020 edition, Part III of the Pharmacopoeia of the People's Republic of China (General Rule 0401); the molecular size of capsular polysaccharides was determined in accordance with the first method in 3.1.2.10 of the 2020 edition, Part III of the Pharmacopoeia of the People's Republic of China. The relevant test results are shown in Table 4.

[0123] Table 4 Recovery rate and quality control indexes of degraded polysaccharide of Streptococcus pneumoniae type 5 capsular polysaccharide

[0124]

[0125] Example 5 Preparation of 10A Streptococcus pneumoniae capsular polysaccharide

[0126] 1. Preparation of 10A Streptococcus pneumoniae capsular polysaccharide

[0127] (1) The fermentation broth of Streptococcus pneumoniae type 10A was sterilized with 0.1% DOC and then allowed to stand at 4°C for 12 hours. g Centrifuge for 30 min and collect the supernatant;

[0128] (2) The supernatant obtained in step (1) is ultrafiltered using a membrane with a pore size of 100 KD to obtain an ultrafiltration concentrate, the volume of which after concentration is 1 / 5 to 1 / 4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times the volume of the concentrate;

[0129] (3) Add a CaCl2 solution with a final concentration of 80 mmol / L to the ultrafiltration concentrate obtained in step (2), stir thoroughly and adjust the pH to 2.50±0.1 (10A-2308001), 3.00±0.1 (10A-2308002), 3.50±0.1 (10A-2308003), 4.00±0.1 (10A-2308004) with phosphoric acid, stir thoroughly and let stand at 2-8°C for 3 h, then heat at 12000 °C. gCentrifuge for 30 min, collect the supernatant, adjust the pH to 7.00-7.50 with NaOH, and then resuspend at 12000 g Centrifuge for 30 min and collect the supernatant;

[0130] (4) ultrafiltration of the supernatant solution obtained in step (3) using a membrane with a pore size of 100 KD to obtain a polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in the ultrafiltration is at least 10 times the volume of the supernatant;

[0131] (5) The ultrafiltration concentrate obtained in step (4) is freeze-dried, and the purified polysaccharide is collected after completion.

[0132] 2. Detection of Capsular Polysaccharide of Type 10A Streptococcus Pneumococcus

[0133] The content of capsular polysaccharide was tested according to the rate turbidimetry (3.3.2) in Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; the content of protein was tested according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0731); the content of nucleic acid was tested according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401); the content of total nitrogen was tested according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0704); the content of phosphorus was tested according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 3103); the content of hexosamine was tested according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401); the molecular size of capsular polysaccharide was determined according to the first method in 3.1.2.10 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China. The relevant test results are shown in Table 5. The results showed that when the pH in the calcium hydrochloric acid precipitation in step (3) was controlled within the range of 2.4-3.6, the protein impurity content in the obtained refined polysaccharide was significantly reduced and was significantly lower than that in the group with a pH of 4.0.

[0134] Table 5 Recovery rate and quality control indexes of 10A Streptococcus pneumoniae capsular polysaccharide

[0135]

[0136] Example 6 Preparation of 10A Streptococcus pneumoniae capsular polysaccharide degradable polysaccharide

[0137] 1. Preparation of degraded polysaccharide of 10A Streptococcus pneumoniae capsular polysaccharide

[0138] (1) After the fermentation broth of type 10A pneumococcal bacteria was sterilized with 0.1% DOC, it was allowed to stand at 4°C for 12 hours, then centrifuged at 12,000 g for 30 minutes, and the supernatant was collected;

[0139] (2) The supernatant obtained in step (1) is ultrafiltered using a membrane with a pore size of 100 KD to obtain an ultrafiltration concentrate, the volume of which after concentration is 1 / 5 to 1 / 4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times the volume of the concentrate;

[0140] (3) Add a CaCl2 solution with a final concentration of 80 mmol / L to the ultrafiltration concentrate obtained in step (2), stir thoroughly, and adjust the pH to 3.00±0.1 with phosphoric acid. Stir thoroughly, let stand at 2-8°C for 3 h, and then centrifuge at 12000 g for 30 min to collect the supernatant;

[0141] (4) adding trifluoroacetic acid at a final concentration of 0.5 mol / L to the supernatant obtained in step (3), standing at 30°C for 3 h, adjusting the pH to 7.00-7.50, and centrifuging again to collect the supernatant;

[0142] (5) The supernatant obtained in step (4) is ultrafiltered using a membrane with a pore size of 30KD to obtain a polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in the ultrafiltration is at least 10 times the volume of the supernatant;

[0143] (6) The ultrafiltration concentrate obtained in step (5) is freeze-dried, and the purified polysaccharide is collected after completion.

[0144] 2. Detection of degraded polysaccharide of 10A Streptococcus pneumoniae capsular polysaccharide

[0145] The assay of degradable polysaccharides of capsular polysaccharides of type 10A pneumococcus shall refer to the 2020 edition of the Pharmacopoeia of the People's Republic of China, among which the assay of capsular polysaccharide content shall be carried out according to the rate turbidimetry (3.3.2) in Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; the assay of protein content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0731); the assay of nucleic acid content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401); The total nitrogen content was tested in accordance with the Pharmacopoeia of the People's Republic of China, Part III, 2020 edition (General Rule 0704); the phosphorus content was tested in accordance with the Pharmacopoeia of the People's Republic of China, Part III, 2020 edition (General Rule 3103); the hexosamine content was tested in accordance with the Pharmacopoeia of the People's Republic of China, Part III, 2020 edition (General Rule 0401); the molecular size of the capsular polysaccharide was determined in accordance with the first method in 3.1.2.10 of Part III, 2020 edition of the Pharmacopoeia of the People's Republic of China. The relevant test results are shown in Table 6.

[0146] Table 6 Recovery rate and quality control indexes of degraded polysaccharide of 10A Streptococcus pneumoniae capsular polysaccharide

[0147]

[0148] Example 7 Preparation of Type 11A Streptococcus pneumoniae Capsular Polysaccharide

[0149] 1. Preparation of 11A Streptococcus pneumoniae capsular polysaccharide

[0150] (1) The fermentation broth of Streptococcus pneumoniae type 11A was sterilized with 0.1% DOC and then allowed to stand at 4°C for 12 hours. g Centrifuge for 30 min and collect the supernatant;

[0151] (2) The supernatant obtained in step (1) is ultrafiltered using a membrane with a pore size of 100 KD to obtain an ultrafiltration concentrate, the volume of which after concentration is 1 / 5 to 1 / 4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times the volume of the concentrate;

[0152] (3) Add a CaCl2 solution with a final concentration of 80 mmol / L to the ultrafiltration concentrate obtained in step (2), stir thoroughly and adjust the pH to 3.00 ± 0.1, stir thoroughly and let stand at 2-8°C for 2 h, then heat at 12000 g Centrifuge for 30 min, collect the supernatant, adjust the pH to 7.00-7.50 with NaOH, and then resuspend at 12000 g Centrifuge for 30 min and collect the supernatant;

[0153] (4) ultrafiltration of the supernatant solution obtained in step (3) using a membrane with a pore size of 100 KD to obtain a polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in the ultrafiltration is at least 10 times the volume of the supernatant;

[0154] (5) The ultrafiltration concentrate obtained in step (4) is freeze-dried, and the degraded polysaccharides are collected after completion.

[0155] 2. Detection of Capsular Polysaccharide of Type 11A Streptococcus Pneumococcus

[0156] The content of capsular polysaccharide was tested according to the rate turbidimetry (3.3.2) in Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; the content of protein was tested according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0731); the content of nucleic acid was tested according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401); the content of total nitrogen was tested according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0704); the content of phosphorus was tested according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 3103); the content of O-acetyl was tested according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 3117); the molecular size of capsular polysaccharide was determined according to the first method in 3.1.2.10 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China. The relevant test results are shown in Table 7.

[0157] Table 7 Recovery rate and quality control indexes of 11A Streptococcus pneumoniae capsular polysaccharide

[0158]

[0159] Example 8 Preparation of degraded polysaccharide of 11A Streptococcus pneumoniae capsular polysaccharide

[0160] 1. Preparation of degraded polysaccharide of 11A Streptococcus pneumoniae capsular polysaccharide

[0161] (1) After the fermentation broth of Streptococcus pneumoniae type 11A was sterilized with 0.1% DOC, it was allowed to stand at 4°C for 12 hours, then centrifuged at 12,000 g for 30 minutes, and the supernatant was collected;

[0162] (2) The supernatant obtained in step (1) is ultrafiltered using a membrane with a pore size of 100 KD to obtain an ultrafiltration concentrate, the volume of which after concentration is 1 / 5 to 1 / 4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times the volume of the concentrate;

[0163] (3) Add a CaCl2 solution with a final concentration of 100 mmol / L to the ultrafiltration concentrate obtained in step (2), stir thoroughly and adjust the pH to 3.00±0.1, stir thoroughly and let stand at 2-8°C for 2 h, then centrifuge at 12000 g for 30 min, and collect the supernatant;

[0164] (4) adding trifluoroacetic acid at a final concentration of 0.5 mol / L to the supernatant obtained in step (3), standing at 40°C for 6 h, adjusting the pH to 7.00-7.50, and centrifuging again to collect the supernatant;

[0165] (5) ultrafiltration of the supernatant solution obtained in step (4) using a membrane with a pore size of 30KD to obtain a polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in the ultrafiltration is at least 10 times the volume of the supernatant;

[0166] (6) The ultrafiltration concentrate obtained in step (5) is freeze-dried, and the purified polysaccharide is collected after completion.

[0167] 2. Detection of degraded polysaccharide of capsular polysaccharide of Streptococcus pneumoniae type 11A

[0168] The assay of degradable polysaccharides of capsular polysaccharides of type 11A pneumococcus shall refer to the 2020 edition of the Pharmacopoeia of the People's Republic of China, among which the assay of capsular polysaccharide content shall be carried out according to the rate turbidimetry (3.3.2) in Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; the assay of protein content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0731); the assay of nucleic acid content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401); The total nitrogen content was tested in accordance with the Pharmacopoeia of the People's Republic of China, Part III, 2020 edition (General Rule 0704); the phosphorus content was tested in accordance with the Pharmacopoeia of the People's Republic of China, Part III, 2020 edition (General Rule 3103); the O-acetyl content was tested in accordance with the Pharmacopoeia of the People's Republic of China, Part III, 2020 edition (General Rule 3117); the molecular size of the capsular polysaccharide was determined in accordance with the first method in 3.1.2.10 of Part III, 2020 edition of the Pharmacopoeia of the People's Republic of China. The relevant test results are shown in Table 8.

[0169] Table 8 Recovery rate and quality control indexes of degraded polysaccharide of 11A Streptococcus pneumoniae capsular polysaccharide

[0170]

[0171] Example 9 Preparation of 19F Streptococcus pneumoniae capsular polysaccharide

[0172] 1. Preparation of 19F Streptococcus pneumoniae capsular polysaccharide

[0173] (1) The 19F pneumococcal fermentation liquid was sterilized with 0.1% DOC and then allowed to stand at 4°C for 6 h. g Centrifuge for 30 min and collect the supernatant;

[0174] (2) The supernatant obtained in step (1) is ultrafiltered using a membrane with a pore size of 100 KD to obtain an ultrafiltration concentrate, the volume of which after concentration is 4 to 5 times the volume of the supernatant, and the volume of purified water used for ultrafiltration is 10 times the volume of the concentrate;

[0175] (3) Add a CaCl2 solution with a final concentration of 80 mmol / L to the ultrafiltration concentrate obtained in step (2), stir thoroughly and adjust the pH to 3.00 ± 0.1, stir thoroughly and let stand at 2-8°C for 2 h, then heat at 12000 g Centrifuge for 30 min, collect the supernatant, adjust the pH to 7.00-7.50 with NaOH, and then resuspend at 12000 g Centrifuge for 30 min and collect the supernatant;

[0176] (4) ultrafiltration of the supernatant obtained in step (3) using a membrane with a pore size of 100 KD to obtain a polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in the ultrafiltration is 10 times the volume of the supernatant;

[0177] (5) The ultrafiltration concentrate obtained in step (4) is freeze-dried, and the purified polysaccharide is collected after completion.

[0178] 2. Detection of 19F Streptococcus pneumoniae capsular polysaccharide

[0179] The content of capsular polysaccharide shall be determined according to the rate turbidimetry (3.3.2) in the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III; the content of protein shall be determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0731); the content of nucleic acid shall be determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401); the content of total nitrogen shall be determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0704); phosphorus The content was tested in accordance with the Pharmacopoeia of the People's Republic of China, Part III, 2020 edition (General Rule 3103); the content of hexosamine was tested in accordance with the Pharmacopoeia of the People's Republic of China, Part III, 2020 edition (General Rule 0401); the content of methylpentose was tested in accordance with the Pharmacopoeia of the People's Republic of China, Part III, 2020 edition (General Rule 0401); the molecular size of capsular polysaccharides was determined in accordance with the first method in 3.1.2.10 of Part III, 2020 edition of the Pharmacopoeia of the People's Republic of China. The relevant test results are shown in Table 9.

[0180] Table 9 Recovery rate and quality control indexes of 19F Streptococcus pneumoniae capsular polysaccharide

[0181]

[0182] Example 10 Preparation of 19F Streptococcus pneumoniae capsular polysaccharide degradable polysaccharide

[0183] 1. Preparation of 19F type Streptococcus pneumoniae capsular polysaccharide degradable polysaccharide

[0184] (1) The 19F pneumococcal fermentation broth was sterilized with 0.1% DOC and then allowed to stand at 4°C for 6 h. It was then centrifuged at 12,000 g for 30 min and the supernatant was collected.

[0185] (2) The supernatant obtained in step (1) is ultrafiltered using a membrane with a pore size of 100 KD to obtain an ultrafiltration concentrate, the volume of which after concentration is 1 / 5 to 1 / 4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times the volume of the concentrate;

[0186] (3) Add a CaCl2 solution with a final concentration of 80 mmol / L to the ultrafiltration concentrate obtained in step (2), stir thoroughly and adjust the pH to 3.00±0.1, stir thoroughly and let stand at 2-8°C for 2 h, then centrifuge at 12000 g for 30 min, and collect the supernatant;

[0187] (4) Add trifluoroacetic acid at a final concentration of 0.25 mol / L to the supernatant obtained in step (3), let stand at 25°C for 3 h, adjust the pH to 7.00-7.50, and centrifuge again to collect the supernatant;

[0188] (5) The supernatant obtained in step (4) is ultrafiltered using a membrane with a pore size of 30KD to obtain a polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in the ultrafiltration is at least 10 times the volume of the supernatant;

[0189] (6) The ultrafiltration concentrate obtained in step (5) is freeze-dried, and the degraded polysaccharides are collected after the freeze-drying.

[0190] 2. Detection of degraded polysaccharide of 19F Streptococcus pneumoniae capsular polysaccharide

[0191] The assay of degradable polysaccharides of 19F type pneumococcal capsular polysaccharide shall refer to the 2020 edition of the Pharmacopoeia of the People's Republic of China, among which the assay of capsular polysaccharide content shall be carried out according to the rate turbidimetry (3.3.2) in Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; the assay of protein content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0731); the assay of nucleic acid content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401); the assay of total nitrogen content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 05 ... 》2020 edition, Part III (General Rule 0704); phosphorus content was tested in accordance with the 2020 edition, Part III of the Pharmacopoeia of the People's Republic of China (General Rule 3103); hexosamine content was tested in accordance with the 2020 edition, Part III of the Pharmacopoeia of the People's Republic of China (General Rule 0401); methyl pentose was tested in accordance with the 2020 edition, Part III of the Pharmacopoeia of the People's Republic of China (General Rule 0401); the molecular size of capsular polysaccharides was determined in accordance with the first method in 3.1.2.10 of the 2020 edition, Part III of the Pharmacopoeia of the People's Republic of China. The relevant test results are shown in Table 10.

[0192] Table 10 Recovery rate and quality control index of degraded polysaccharide of 19F Streptococcus pneumoniae capsular polysaccharide

[0193]

[0194] Example 11 Preparation of Type 2 Streptococcus pneumoniae capsular polysaccharide

[0195] Purification of Capsular Polysaccharide of Streptococcus pneumoniae Type 1 and Type 2

[0196] (1) The fermentation broth of Streptococcus pneumoniae type 2 was sterilized with 0.1% DOC and then allowed to stand at 4°C for 12 hours. g Centrifuge for 30 min and collect the supernatant;

[0197] (2) The supernatant obtained in step (1) is ultrafiltered using a membrane with a pore size of 100 KD to obtain an ultrafiltration concentrate, the volume of which after concentration is 1 / 5 to 1 / 4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times the volume of the concentrate;

[0198] (3) Add a CaCl2 solution with a final concentration of 80 mmol / L to the ultrafiltration concentrate obtained in step (2), stir thoroughly and adjust the pH to 3.00 ± 0.1, stir thoroughly and let stand at 2-8°C for 4 h, then heat at 12000 g Centrifuge for 30 min, collect the supernatant, adjust the pH to 7.00-7.50 with NaOH, and then resuspend at 12000 g Centrifuge for 30 min and collect the supernatant;

[0199] (4) ultrafiltration of the supernatant obtained in step (3) using a membrane with a pore size of 100 KD to obtain a polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in the ultrafiltration is at least 10 times the volume of the supernatant;

[0200] (5) The ultrafiltration concentrate obtained in step (4) is freeze-dried, and the purified polysaccharide is collected after completion.

[0201] 2. Detection of Capsular Polysaccharide of Streptococcus pneumoniae Type 2

[0202] The content of capsular polysaccharide shall be tested according to the rate turbidimetry (3.3.2) in the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III; the content of protein shall be tested according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0731); the content of nucleic acid shall be tested according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401); the content of total nitrogen shall be tested according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0704); The phosphorus content was tested in accordance with the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 3103); the uronic acid content was tested in accordance with the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 0731); the methyl pentose content was tested in accordance with the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 0401); the molecular size of the capsular polysaccharide was determined in accordance with the first method in 3.1.2.10 of Part III of the Pharmacopoeia of the People's Republic of China, 2020 edition. The relevant test results are shown in Table 11.

[0203] Table 11 Recovery rate and quality control indexes of type 2 monovalent Streptococcus pneumoniae capsular polysaccharide

[0204]

[0205] Example 12 Preparation of degraded polysaccharide of Streptococcus pneumoniae type 2 capsular polysaccharide

[0206] Preparation of degraded polysaccharide of capsular polysaccharide of Streptococcus pneumoniae type 1 and 2

[0207] (1) After the fermentation broth of Streptococcus pneumoniae type 2 was sterilized with 0.1% DOC, it was allowed to stand at 4°C for 12 hours, then centrifuged at 12,000 g for 30 minutes, and the supernatant was collected;

[0208] (2) The supernatant obtained in step (1) is ultrafiltered using a membrane with a pore size of 100 KD to obtain an ultrafiltration concentrate, the volume of which after concentration is 1 / 5 to 1 / 4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times the volume of the concentrate;

[0209] (3) Add a CaCl2 solution with a final concentration of 80 mmol / L to the ultrafiltration concentrate obtained in step (2), stir thoroughly and adjust the pH to 3.00±0.1, stir thoroughly and let stand at 2-8°C for 2 h, then centrifuge at 12000 g for 30 min, and collect the supernatant;

[0210] (4) adding trifluoroacetic acid at a final concentration of 0.5 mol / L to the supernatant obtained in step (3), standing at 40°C for 3 h, adjusting the pH to 7.00-7.50, and centrifuging again to collect the supernatant;

[0211] (5) ultrafiltration of the supernatant solution obtained in step (4) using a membrane with a pore size of 30KD to obtain a polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in the ultrafiltration is at least 10 times the volume of the supernatant;

[0212] (6) The ultrafiltration concentrate obtained in step (5) is freeze-dried, and the degraded polysaccharides are collected after the freeze-drying.

[0213] 2. Degradation of polysaccharide from capsular polysaccharide of Streptococcus pneumoniae type 2

[0214] The determination of degradable polysaccharides of type 2 pneumococcal capsular polysaccharides shall refer to the 2020 edition of the Pharmacopoeia of the People's Republic of China, among which the determination of capsular polysaccharide content shall be carried out according to the rate turbidimetry (3.3.2) in Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; the determination of protein content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0731); the determination of nucleic acid content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401); the determination of total nitrogen content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China The content of phosphorus was determined in accordance with the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0704); the content of uronic acid was determined in accordance with the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 3103); the content of uronic acid was determined in accordance with the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0731); the content of methyl pentose was determined in accordance with the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401); the molecular size of capsular polysaccharides was determined in accordance with the first method in 3.1.2.10 of the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III. The relevant test results are shown in Table 12.

[0215] Table 12 Recovery rate and quality control indexes of degraded polysaccharide of Streptococcus pneumoniae type 2 capsular polysaccharide

[0216]

[0217] It should be noted that the sterilization method in the above embodiment is only used as an exemplary description, and the improvement of the capsular polysaccharide purification method of the present invention and its effect enhancement do not depend on a specific sterilization method. The present invention has been verified by experiments that the sodium deoxycholate sterilization treatment is replaced with β-propiolactone as a bactericide, and the capsular polysaccharide purification method of the present invention can also achieve a purification effect equivalent to the above embodiment. As an example, an embodiment of sterilization treatment using 0.1% β-propiolactone is provided below. In addition, the present invention also attempts to use other concentrations of β-propiolactone for sterilization treatment, and the results show that β-propiolactone with a final concentration of not less than 0.01% can achieve a good effect of killing Streptococcus pneumoniae and releasing capsular polysaccharides.

[0218] Example 13 Preparation of Type 14 Streptococcus pneumoniae Capsular Polysaccharide

[0219] 1. Purification of Capsular Polysaccharide of Streptococcus pneumoniae Type 14

[0220] (1) Add β-propiolactone to the fermentation broth of Streptococcus pneumoniae type 14 at a final concentration of 0.1%, stir evenly, incubate at 4°C for 12 hours, and then incubate at 12,000 g Centrifuge for 30 min and collect the supernatant;

[0221] (2) The supernatant obtained in step (1) is ultrafiltered using a membrane with a pore size of 100 KD to obtain an ultrafiltration concentrate, the volume of which after concentration is 1 / 5 to 1 / 4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times the volume of the concentrate;

[0222] (3) Add CaCl2 solutions with final concentrations of 80 mmol / L (14-2308001), 120 mmol / L (14-2308002), and 200 mmol / L (14-2308003) to the ultrafiltration concentrate obtained in step (2), stir thoroughly and adjust the pH to 3.00 ± 0.1, stir thoroughly and let stand at 2-8 °C for 5 h, then heat at 12000 g Centrifuge for 30 min, collect the supernatant, adjust the pH to 7.00-7.50 with NaOH, and then resuspend at 12000 g Centrifuge for 30 min and collect the supernatant;

[0223] (4) ultrafiltration of the supernatant obtained in step (3) using a membrane with a pore size of 100 KD to obtain a polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in the ultrafiltration is at least 10 times the volume of the supernatant;

[0224] (5) The ultrafiltration concentrate obtained in step (4) is freeze-dried, and the purified polysaccharide is collected after completion.

[0225] 2. Detection of Capsular Polysaccharide of Streptococcus pneumoniae Type 14

[0226] The content of capsular polysaccharide shall be tested according to the rate turbidimetry (3.3.2) in the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III; the content of protein shall be tested according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0731); the content of nucleic acid shall be tested according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401); the content of total nitrogen shall be tested according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0704); The phosphorus content was tested in accordance with the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 3103); the uronic acid content was tested in accordance with the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 0731); the methyl pentose content was tested in accordance with the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 0401); the molecular size of the capsular polysaccharide was determined in accordance with the first method in 3.1.2.10 of Part III of the Pharmacopoeia of the People's Republic of China, 2020 edition. The relevant test results are shown in Table 13.

[0227] Table 13 Recovery rate and quality control indexes of type 14 Streptococcus pneumoniae capsular polysaccharide

[0228]

[0229] Comparative Example 1 Preparation of 6A Streptococcus pneumoniae capsular polysaccharide

[0230] 1. Preparation of 6A Streptococcus pneumoniae capsular polysaccharide

[0231] Steps (1) and (2) are the same as those in Example 1, and steps (3) to (6) are as follows:

[0232] (3) Add NaCl to the ultrafiltration concentrate obtained in step (2) to a concentration of 0.3 mol / L. After complete dissolution, add 0.5% (w / v) sodium deoxycholate. While stirring, add glacial acetic acid dropwise to adjust the pH to 5.0. After fully stirring, let stand at 2-8°C for 12 hours, then centrifuge at 12000 g for 30 minutes, collect the supernatant, discard the precipitate, and clarify and filter with a 0.8 μm filter membrane if there are small debris in the supernatant;

[0233] (4) Adjust the pH of the supernatant obtained in step (3) to neutral with sodium hydroxide or potassium hydroxide, then add Na2HPO4 with a final concentration of 0.01 mol / L and NaH2PO4 with a final concentration of 0.01 mol / L as a buffer system to the obtained feed liquid, and after complete dissolution, add sodium acetate with a final concentration of 0.6 mol / L and CaCl2 with a final concentration of 0.25 mol / L, stir until dissolved, add glacial acetic acid dropwise to the feed liquid to adjust the pH to 5.3-5.5, stir thoroughly and let stand at 2-8°C for 12 hours, after standing, centrifuge at 12000g for 30 minutes, collect the supernatant, discard the precipitate, and clarify and filter with a 0.8 μm filter membrane if there are small debris in the supernatant;

[0234] (5) ultrafiltration of the supernatant obtained in step (4) with purified water, collecting the polysaccharide solution when the conductivity at the permeate end is lower than 10 μs / cm, and collecting the polysaccharide solution after sterilization filtration;

[0235] (6) The polysaccharide solution is further freeze-dried to obtain Streptococcus pneumoniae capsular polysaccharide.

[0236] 2. Detection of Capsular Polysaccharide of Type 6A Streptococcus Pneumococcus

[0237] The content of capsular polysaccharide was tested according to the rate turbidimetry (3.3.2) in Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; the content of protein was tested according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0731); the content of nucleic acid was tested according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401); the content of total nitrogen was tested according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0704); the content of phosphorus was tested according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 3103); the content of methyl pentose was tested according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401); the molecular size of capsular polysaccharide was determined according to the first method in 3.1.2.10 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China. The relevant test results are shown in Table 14.

[0238] Table 14 Recovery rate and quality control indexes of 6A type pneumococcal capsular polysaccharide

[0239]

[0240] The results show that compared with the quality control indicators of the refined polysaccharide of type 6A Streptococcus pneumoniae 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 the other quality control indicators of the refined polysaccharide of type 6A Streptococcus pneumoniae prepared in Example 1 are not inferior to those of Comparative Example 1, and the process time is shortened.

[0241] Comparative Example 2 Preparation of 6A Streptococcus pneumoniae capsular polysaccharide degraded polysaccharide

[0242] 1. Preparation of degraded polysaccharide of 6A type pneumococcal capsular polysaccharide

[0243] (1) Take 3.0±0.15 g of the refined polysaccharide obtained in Comparative Example 1 and dissolve it in 1.0 L of 0.15 mol / L sodium chloride solution, and stir until it is completely dissolved;

[0244] (2) homogenizing the refined polysaccharide solution in step (1) with a high-pressure homogenizer at a pressure of 600±50 bar, circulating twice, and harvesting the degraded polysaccharide solution;

[0245] (3) The degraded polysaccharide solution is freeze-dried, and the degraded polysaccharide is collected after the freeze-drying.

[0246] 2. Degradation of polysaccharide from the capsular polysaccharide of Streptococcus pneumoniae type 6A

[0247] The determination of degradable polysaccharides of 6A type pneumococcal capsular polysaccharides refers to the 2020 edition of the Pharmacopoeia of the People's Republic of China, among which the determination of capsular polysaccharide content is carried out according to the rate turbidimetry (3.3.2) in Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; the determination of protein content is carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0731); the determination of nucleic acid content is carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401) ; The total nitrogen content was tested in accordance with the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 0704); the phosphorus content was tested in accordance with the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 3103); the methyl pentose content was tested in accordance with the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 0401); the molecular size of the capsular polysaccharide was determined in accordance with the first method in 3.1.2.10 of the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III. The relevant test results are shown in Table 15.

[0248] Table 15 Recovery rate and quality control index of degraded polysaccharide of 6A type pneumococcal capsular polysaccharide

[0249]

[0250] The results show that the quality control index of the degraded polysaccharide of 6A type pneumoniae capsular polysaccharide obtained by the method of Example 2 of the present invention is significantly reduced in protein content compared with the degraded polysaccharide obtained in Comparative Example 2, and other quality control indicators are not inferior to those of Comparative Example 2, and the process flow is simplified and the process time is shortened.

[0251] Comparative Example 3 Preparation of Type 5 Streptococcus pneumoniae Capsular Polysaccharide

[0252] 1. Preparation of Streptococcus pneumoniae type 5 capsular polysaccharide

[0253] Steps (1) and (2) are the same as those in Example 3, and steps (3) to (6) are as follows:

[0254] (3) Add NaCl to the ultrafiltration concentrate obtained in step (2) to a concentration of 0.3 mol / L. After complete dissolution, add 0.5% (w / v) sodium deoxycholate. While stirring, add dropwise glacial acetic acid to adjust the pH to 5.0. After fully stirring, let stand at 2-8°C for 12 hours, then centrifuge at 12000 g for 30 minutes, collect the supernatant, discard the precipitate, and clarify and filter with a 0.8 μm filter membrane if there are small debris in the supernatant;

[0255] (4) Adjust the pH of the supernatant obtained in step (3) to neutral with sodium hydroxide or potassium hydroxide, then add Na2HPO4 with a final concentration of 0.01 mol / L and NaH2PO4 with a final concentration of 0.01 mol / L as a buffer system to the obtained feed liquid. After complete dissolution, add sodium acetate with a final concentration of 0.6 mol / L and CaCl2 with a final concentration of 0.25 mol / L, stir until dissolved, add glacial acetic acid dropwise to the feed liquid to adjust the pH to 5.3-5.5, stir thoroughly and let stand at 2-8°C for 12 hours. After standing, centrifuge at 12000 g for 30 minutes, collect the supernatant, discard the precipitate, and clarify and filter with a 0.8 μm filter membrane if there are small debris in the supernatant;

[0256] (5) ultrafiltration of the supernatant obtained in step (4) with purified water, collecting the polysaccharide solution when the conductivity at the permeate end is lower than 10 μs / cm, and collecting the polysaccharide solution after sterilization filtration;

[0257] (6) The polysaccharide solution is further freeze-dried to obtain Streptococcus pneumoniae capsular polysaccharide.

[0258] Detection of Capsular Polysaccharide of Type 2 and Type 5 Streptococcus Pneumoniae

[0259] The content of capsular polysaccharide shall be determined according to the rate turbidimetry (3.3.2) in the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III; the content of protein shall be determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0731); the content of nucleic acid shall be determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401); the content of total nitrogen shall be determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0704); phosphorus The content was tested in accordance with the Pharmacopoeia of the People's Republic of China, Part III, 2020 edition (General Rule 3103); the uronic acid content was tested in accordance with the Pharmacopoeia of the People's Republic of China, Part III, 2020 edition (General Rule 0731); the aminohexose content was tested in accordance with the Pharmacopoeia of the People's Republic of China, Part III, 2020 edition (General Rule 0401); the molecular size of the capsular polysaccharide was determined in accordance with the first method in 3.1.2.10 of Part III, 2020 edition of the Pharmacopoeia of the People's Republic of China. The relevant test results are shown in Table 16.

[0260] Table 16 Recovery and quality control indicators of type 5 Streptococcus pneumoniae capsular polysaccharide

[0261]

[0262] The results show that the quality control indicators of the refined polysaccharide of type 5 Streptococcus pneumoniae obtained in Example 3 of the present invention are not inferior to those of the refined polysaccharide obtained in Comparative Example 3, and the process time is shortened.

[0263] Comparative Example 4 Preparation of degraded polysaccharide of Streptococcus pneumoniae type 5 capsular polysaccharide

[0264] Preparation of degraded polysaccharide of 1.5-streptococcus pneumoniae capsular polysaccharide

[0265] (1) Take 3.0±0.15 g of the refined polysaccharide obtained in Comparative Example 3 and dissolve it in 1.0 L of 0.15 mol / L sodium chloride solution, and stir until it is completely dissolved;

[0266] (2) homogenizing the refined polysaccharide solution in step (1) with a high-pressure homogenizer at a pressure of 1100±50 bar, circulating for 3 times, and harvesting the degraded polysaccharide solution;

[0267] (3) The degraded polysaccharide solution is freeze-dried, and the degraded polysaccharide is collected after the freeze-drying.

[0268] Detection of degraded polysaccharides of capsular polysaccharides of Streptococcus pneumoniae type 2 and 5

[0269] The determination of degradable polysaccharides of type 5 pneumococcal capsular polysaccharides shall refer to the 2020 edition of the Pharmacopoeia of the People's Republic of China, among which the determination of capsular polysaccharide content shall be carried out according to the rate turbidimetry (3.3.2) in Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; the determination of protein content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0731); the determination of nucleic acid content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401); the determination of total nitrogen content shall be carried out according to Part 2 of the Pharmacopoeia of the People's Republic of China. 020 edition, Part III (General Rule 0704); phosphorus content was tested in accordance with the 2020 edition, Part III of the Pharmacopoeia of the People's Republic of China (General Rule 3103); uronic acid content was tested in accordance with the 2020 edition, Part III of the Pharmacopoeia of the People's Republic of China (General Rule 0731); hexosamine content was tested in accordance with the 2020 edition, Part III of the Pharmacopoeia of the People's Republic of China (General Rule 0401); the molecular size of capsular polysaccharides was determined in accordance with the first method in 3.1.2.10 of the 2020 edition, Part III of the Pharmacopoeia of the People's Republic of China. The relevant test results are shown in Table 17.

[0270] Table 17 Recovery rate and quality control index of degraded polysaccharide of type 5 Streptococcus pneumoniae capsular polysaccharide

[0271]

[0272] The results show that the quality control indicators of the degraded polysaccharide of type 5 pneumococcal capsular polysaccharide prepared in Example 4 of the present invention are lower in protein content and higher in aminohexose content than those of the degraded polysaccharide prepared in Comparative Example 4, and other quality control indicators are not inferior to those of Comparative Example 4, and the process flow is simplified and the process time is shortened.

[0273] Comparative Example 5 Preparation of 10A Streptococcus pneumoniae capsular polysaccharide

[0274] 1. Preparation of 10A Streptococcus pneumoniae capsular polysaccharide

[0275] Steps (1) and (2) are the same as those in Example 5, and steps (3) to (6) are as follows:

[0276] (3) Add NaCl to the ultrafiltration concentrate obtained in step (2) to a concentration of 0.3 mol / L. After complete dissolution, add 0.5% (w / v) sodium deoxycholate. While stirring, add dropwise glacial acetic acid to adjust the pH to 5.0. After fully stirring, let stand at 2-8°C for 12 hours, then centrifuge at 12000 g for 30 minutes, collect the supernatant, discard the precipitate, and clarify and filter with a 0.8 μm filter membrane if there are small debris in the supernatant;

[0277] (4) Adjust the pH of the supernatant obtained in step (3) to neutral with sodium hydroxide or potassium hydroxide, then add Na2HPO4 with a final concentration of 0.01 mol / L and NaH2PO4 with a final concentration of 0.01 mol / L as a buffer system to the obtained feed liquid. After complete dissolution, add sodium acetate with a final concentration of 0.6 mol / L and CaCl2 with a final concentration of 0.25 mol / L, stir until dissolved, add glacial acetic acid dropwise to the feed liquid to adjust the pH to 5.3-5.5, stir thoroughly and let stand at 2-8°C for 12 hours. After standing, centrifuge at 12000 g for 30 min, collect the supernatant, discard the precipitate, and clarify and filter with a 0.8 μm filter membrane if there are small debris in the supernatant;

[0278] (5) ultrafiltration of the supernatant obtained in step (4) with purified water, collecting the polysaccharide solution when the conductivity at the permeate end is lower than 10 μs / cm, and collecting the polysaccharide solution after sterilization filtration;

[0279] (6) The polysaccharide solution is further freeze-dried to obtain Streptococcus pneumoniae capsular polysaccharide.

[0280] 2. Detection of Capsular Polysaccharide of Type 10A Streptococcus Pneumococcus

[0281] The determination of the content of capsular polysaccharide is carried out according to the rate turbidimetry (3.3.2) in Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; the determination of the content of protein is carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0731); the determination of the content of nucleic acid is carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401); the determination of the content of total nitrogen is carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0704); the determination of the content of phosphorus is carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 3103); the determination of the content of hexosamine is carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401); the determination of the molecular size of capsular polysaccharide is carried out according to the first method in 3.1.2.10 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China. The relevant test results are shown in Table 18.

[0282] Table 18 Recovery and quality control indicators of 10A type monovalent Streptococcus pneumoniae capsular polysaccharide

[0283]

[0284] The results show that the quality control indicators of the refined polysaccharide of type 10A Streptococcus pneumoniae obtained in Example 5 of the present invention are lower in protein content and total phosphorus content than those in Comparative Example 5, and the aminohexose content is increased compared with Comparative Example 5, and other quality control indicators are not inferior to those of the Comparative Example, and the process time is shortened.

[0285] Comparative Example 6 Preparation of 10A Streptococcus pneumoniae capsular polysaccharide degraded polysaccharide

[0286] 1. Preparation of degraded polysaccharide of 10A Streptococcus pneumoniae capsular polysaccharide

[0287] (1) Take 3.0±0.15 g of the refined polysaccharide obtained in Comparative Example 5 and dissolve it in 1.0 L of 0.15 mol / L sodium chloride solution, and stir until it is completely dissolved;

[0288] (2) homogenizing the refined polysaccharide solution in step (1) with a high-pressure homogenizer at a pressure of 600±50 bar, circulating for 3 times, and harvesting the degraded polysaccharide solution;

[0289] (3) The degraded polysaccharide solution is freeze-dried, and the degraded polysaccharide is collected after the freeze-drying.

[0290] 2. Detection of degraded polysaccharide of 10A Streptococcus pneumoniae capsular polysaccharide

[0291] The assay of degradable polysaccharides of capsular polysaccharides of type 10A pneumococcus shall refer to the 2020 edition of the Pharmacopoeia of the People's Republic of China, among which the assay of capsular polysaccharide content shall be carried out according to the rate turbidimetry (3.3.2) in Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; the assay of protein content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0731); the assay of nucleic acid content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401); The total nitrogen content was tested in accordance with the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0704); the phosphorus content was tested in accordance with the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 3103); the aminohexose content was tested in accordance with the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401); the molecular size of the capsular polysaccharide was determined in accordance with the first method in 3.1.2.10 of the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III. The relevant test results are shown in Table 19.

[0292] Table 19 Recovery rate and quality control index of degraded polysaccharide of 10A Streptococcus pneumoniae capsular polysaccharide

[0293]

[0294] The results show that the quality control index of the degraded polysaccharide of 10A pneumococcal capsular polysaccharide prepared in Example 6 of the present invention is lower than that of the degraded polysaccharide prepared in Comparative Example 6 in terms of protein content, and other quality control indicators are not inferior to those of Comparative Example 6, and the process flow is simplified and the process time is shortened.

[0295] Comparative Example 7 Preparation of Type 11A Streptococcus pneumoniae Capsular Polysaccharide

[0296] 1. Preparation of 11A Streptococcus pneumoniae capsular polysaccharide

[0297] Steps (1) and (2) are the same as those in Example 7, and steps (3) to (6) are as follows:

[0298] (3) Add NaCl to the ultrafiltration concentrate obtained in step (2) to a concentration of 0.3 mol / L. After complete dissolution, add 0.5% (w / v) sodium deoxycholate. While stirring, add dropwise glacial acetic acid to adjust the pH to 5.0. After fully stirring, let stand at 2-8°C for 12 hours, then centrifuge at 12000 g for 30 minutes, collect the supernatant, discard the precipitate, and clarify and filter with a 0.8 μm filter membrane if there are small debris in the supernatant;

[0299] (4) Adjust the pH of the supernatant obtained in step (3) to neutral with sodium hydroxide or potassium hydroxide, then add Na2HPO4 with a final concentration of 0.01 mol / L and NaH2PO4 with a final concentration of 0.01 mol / L as a buffer system to the obtained feed solution, and after complete dissolution, add sodium acetate with a final concentration of 0.6 mol / L and CaCl2 with a final concentration of 0.25 mol / L, stir until dissolved, add glacial acetic acid dropwise to the feed solution to adjust the pH to 5.3-5.5, stir thoroughly and let stand at 2-8°C for 12 hours, then centrifuge at 12000 g for 30 minutes, collect the supernatant, discard the precipitate, and clarify and filter with a 0.8 μm filter membrane if there are small debris in the supernatant;

[0300] (5) ultrafiltration of the supernatant obtained in step (4) with purified water, collecting the polysaccharide solution when the conductivity at the permeate end is lower than 10 μs / cm, and collecting the polysaccharide solution after sterilization filtration;

[0301] (6) The polysaccharide solution is further freeze-dried to obtain Streptococcus pneumoniae capsular polysaccharide.

[0302] 2. Detection of Capsular Polysaccharide of Type 11A Streptococcus Pneumococcus

[0303] The content of capsular polysaccharide was tested according to the rate turbidimetry (3.3.2) in Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; the content of protein was tested according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0731); the content of nucleic acid was tested according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401); the content of total nitrogen was tested according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0704); the content of phosphorus was tested according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 3103); the content of O-acetyl was tested according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 3117); the molecular size of capsular polysaccharide was determined according to the first method in 3.1.2.10 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China. The relevant test results are shown in Table 20.

[0304] Table 20 Recovery rate and quality control indexes of 11A Streptococcus pneumoniae capsular polysaccharide

[0305]

[0306] The results show that the quality control indicators of the refined polysaccharide of type 11A Streptococcus pneumoniae obtained by the method of Example 7 of the present invention are lower in protein content, nucleic acid, and total phosphorus content than those of Comparative Example 7, and the O-acetyl content is increased compared with Comparative Example 7, and other quality control indicators are not inferior to those of the Comparative Example, and the process time is shortened.

[0307] Comparative Example 8 Preparation of degraded polysaccharide of 11A Streptococcus pneumoniae capsular polysaccharide

[0308] 1. Preparation of degraded polysaccharide of 11A Streptococcus pneumoniae capsular polysaccharide

[0309] (1) Take 3.0±0.15 g of the refined polysaccharide obtained in Comparative Example 7 and dissolve it in 1.0 L of 0.15 mol / L sodium chloride solution, and stir until it is completely dissolved;

[0310] (2) homogenizing the refined polysaccharide solution in step (1) with a high-pressure homogenizer at a pressure of 600±50 bar, circulating for 3 times, and harvesting the degraded polysaccharide solution;

[0311] (3) The degraded polysaccharide solution is freeze-dried, and the degraded polysaccharide is collected after the freeze-drying.

[0312] 2. Detection of degraded polysaccharide of capsular polysaccharide of Streptococcus pneumoniae type 11A

[0313] The assay of degradable polysaccharides of capsular polysaccharides of type 11A pneumococcus shall refer to the 2020 edition of the Pharmacopoeia of the People's Republic of China, among which the assay of capsular polysaccharide content shall be carried out according to the rate turbidimetry (3.3.2) in Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; the assay of protein content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0731); the assay of nucleic acid content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401); The total nitrogen content was tested in accordance with the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0704); the phosphorus content was tested in accordance with the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 3103); the O-acetyl content was tested in accordance with the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 3117); the molecular size of the capsular polysaccharide was determined in accordance with the first method in 3.1.2.10 of the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III. The relevant test results are shown in Table 21.

[0314] Table 21 Recovery rate and quality control index of degraded polysaccharide of 11A Streptococcus pneumoniae capsular polysaccharide

[0315]

[0316] The results show that the quality control indicators of the degraded polysaccharide of type 11A pneumococcal capsular polysaccharide obtained by the method of Example 8 of the present invention are lower than those of the degraded polysaccharide obtained in Comparative Example 8 in terms of protein content, nucleic acid, and total phosphorus content, and other quality control indicators are not inferior to those of Comparative Example 8, and the process flow is simplified and the process time is shortened.

[0317] Comparative Example 9 Preparation of 19F Streptococcus pneumoniae capsular polysaccharide

[0318] 1. Purification of 19F Streptococcus pneumoniae capsular polysaccharide

[0319] Steps (1) and (2) are the same as those in Example 9, and steps (3) to (6) are as follows:

[0320] (3) Add NaCl to the ultrafiltration concentrate obtained in step (2) to a concentration of 0.3 mol / L. After complete dissolution, add 0.5% (w / v) sodium deoxycholate. While stirring, add dropwise glacial acetic acid to adjust the pH to 5.0. After fully stirring, let stand at 2-8°C for 12 hours, then centrifuge at 12000g for 30 minutes, collect the supernatant, discard the precipitate, and clarify and filter with a 0.8 μm filter membrane if there are small debris in the supernatant;

[0321] (4) Adjust the pH of the supernatant obtained in step (3) to neutral with sodium hydroxide or potassium hydroxide, then add Na2HPO4 with a final concentration of 0.01 mol / L and NaH2PO4 with a final concentration of 0.01 mol / L as a buffer system to the obtained feed liquid. After complete dissolution, add sodium acetate with a final concentration of 0.6 mol / L and CaCl2 with a final concentration of 0.25 mol / L, stir until dissolved, add glacial acetic acid dropwise to the feed liquid to adjust the pH to 5.3-5.5, stir thoroughly and let stand at 2-8°C for 12 hours. After standing, centrifuge at 12000g for 30 minutes, collect the supernatant, discard the precipitate, and clarify and filter with a 0.8 μm filter membrane if there are small debris in the supernatant;

[0322] (5) The supernatant obtained in step (4) is ultrafiltered with purified water, and the polysaccharide solution is collected when the conductivity at the permeate end is lower than 10 μs / cm, and the polysaccharide solution is collected after sterilization filtration.

[0323] (6) The polysaccharide solution is further freeze-dried to obtain Streptococcus pneumoniae capsular polysaccharide.

[0324] 2. Detection of 19F Streptococcus pneumoniae capsular polysaccharide

[0325] The content of capsular polysaccharide shall be determined according to the rate turbidimetry (3.3.2) in the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III; the content of protein shall be determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0731); the content of nucleic acid shall be determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401); the content of total nitrogen shall be determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0704); phosphorus The content was tested in accordance with the Pharmacopoeia of the People's Republic of China, Part III, 2020 edition (General Rule 3103); the content of hexosamine was tested in accordance with the Pharmacopoeia of the People's Republic of China, Part III, 2020 edition (General Rule 0401); the content of methyl pentose was tested in accordance with the Pharmacopoeia of the People's Republic of China, Part III, 2020 edition (General Rule 0401); the molecular size of capsular polysaccharides was determined in accordance with the first method in 3.1.2.10 of Part III, 2020 edition of the Pharmacopoeia of the People's Republic of China. The relevant test results are shown in Table 22.

[0326] Table 22 Recovery rate and quality control indexes of 19F Streptococcus pneumoniae capsular polysaccharide

[0327]

[0328] The results show that the quality control index of the refined polysaccharide of type 11A Streptococcus pneumoniae prepared in Example 9 of the present invention is lower than that of the refined polysaccharide prepared in Comparative Example 9 in terms of protein content, and other quality control indicators are not inferior to those of Comparative Example 9, and the process time is shortened.

[0329] Comparative Example 10 Preparation of 19F type Streptococcus pneumoniae capsular polysaccharide degraded polysaccharide

[0330] 1. Preparation of 19F type Streptococcus pneumoniae capsular polysaccharide degradable polysaccharide

[0331] (1) Take 3.0±0.15 g of the refined polysaccharide obtained in Comparative Example 9 and dissolve it in 1.0 L of 0.15 mol / L sodium chloride solution, and stir until it is completely dissolved;

[0332] (2) homogenizing the refined polysaccharide solution in step (1) with a high-pressure homogenizer at a pressure of 600±50 bar, circulating twice, and harvesting the degraded polysaccharide solution;

[0333] (3) The degraded polysaccharide solution is freeze-dried, and the degraded polysaccharide is collected after the freeze-drying.

[0334] 2. Detection of degraded polysaccharide of 19F Streptococcus pneumoniae capsular polysaccharide

[0335] The assay of degradable polysaccharides of 19F type pneumococcal capsular polysaccharide shall refer to the 2020 edition of the Pharmacopoeia of the People's Republic of China, among which the assay of capsular polysaccharide content shall be carried out according to the rate turbidimetry (3.3.2) in Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; the assay of protein content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0731); the assay of nucleic acid content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401); the assay of total nitrogen content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 05 ... 》2020 edition, Part III (General Rule 0704); phosphorus content was tested in accordance with the 2020 edition, Part III of the Pharmacopoeia of the People's Republic of China (General Rule 3103); aminohexose content was tested in accordance with the 2020 edition, Part III of the Pharmacopoeia of the People's Republic of China (General Rule 0401); methyl pentose was tested in accordance with the 2020 edition, Part III of the Pharmacopoeia of the People's Republic of China (General Rule 0401); the molecular size of capsular polysaccharides was determined in accordance with the first method in 3.1.2.10 of the 2020 edition, Part III of the Pharmacopoeia of the People's Republic of China. The relevant test results are shown in Table 23.

[0336] Table 23 Recovery rate and quality control index of 19F type pneumococcal capsular polysaccharide degradation polysaccharide

[0337]

[0338] The results show that the quality control indicators of the degraded polysaccharide of type 11A pneumococcal capsular polysaccharide obtained in Example 10 of the present invention are lower in protein content and total phosphorus content than those of the degraded polysaccharide obtained in Comparative Example 10, and other quality control indicators are not inferior to those of Comparative Example 10, and the process flow is simplified and the process time is shortened.

[0339] Comparative Example 11 Preparation of Type 2 Streptococcus pneumoniae capsular polysaccharide

[0340] Preparation of degraded polysaccharide of capsular polysaccharide of Streptococcus pneumoniae type 1 and 2

[0341] Steps (1) and (2) are the same as those in Example 11, and steps (3) to (6) are as follows:

[0342] (3) Add NaCl to the ultrafiltration concentrate obtained in step (2) to a concentration of 0.3 mol / L. After complete dissolution, add 0.5% (w / v) sodium deoxycholate. While stirring, add dropwise glacial acetic acid to adjust the pH to 5.0. After fully stirring, let stand at 2-8°C for 12 hours, then centrifuge at 12000 g for 30 minutes, collect the supernatant, discard the precipitate, and clarify and filter with a 0.8 μm filter membrane if there are small debris in the supernatant;

[0343] (4) Adjust the pH of the supernatant obtained in step (3) to neutral with sodium hydroxide or potassium hydroxide, then add Na2HPO4 with a final concentration of 0.01 mol / L and NaH2PO4 with a final concentration of 0.01 mol / L as a buffer system to the obtained feed solution, and after complete dissolution, add sodium acetate with a final concentration of 0.6 mol / L and CaCl2 with a final concentration of 0.25 mol / L, stir until dissolved, add glacial acetic acid dropwise to the feed solution to adjust the pH to 5.3-5.5, stir thoroughly and let stand at 2-8°C for 12 hours, then centrifuge at 12000 g for 30 minutes, collect the supernatant, discard the precipitate, and clarify and filter with a 0.8 μm filter membrane if there are small debris in the supernatant;

[0344] (5) The supernatant obtained in step (4) is ultrafiltered with purified water, and the polysaccharide solution is collected when the conductivity at the permeate end is lower than 10 μs / cm, and the polysaccharide solution is collected after sterilization filtration.

[0345] (6) The polysaccharide solution is further freeze-dried to obtain Streptococcus pneumoniae capsular polysaccharide.

[0346] 2. Detection of Capsular Polysaccharide of Streptococcus pneumoniae Type 2

[0347] The determination of capsular polysaccharide of type 2 pneumococcus shall refer to the 2020 edition of the Pharmacopoeia of the People's Republic of China, among which the determination of capsular polysaccharide content shall be carried out according to the rate turbidimetry (3.3.2) in Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; the determination of protein content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0731); the determination of nucleic acid content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401); the determination of total nitrogen content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0501); The content of phosphorus was determined in accordance with the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0704); the content of uronic acid was determined in accordance with the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 3103); the content of uronic acid was determined in accordance with the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0731); the content of methyl pentose was determined in accordance with the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401); the molecular size of capsular polysaccharides was determined in accordance with the first method in 3.1.2.10 of the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III. The relevant test results are shown in Table 24.

[0348] Table 24 Recovery rate and quality control indexes of type 2 Streptococcus pneumoniae capsular polysaccharide

[0349]

[0350] The results show that the quality control index of the refined polysaccharide of Streptococcus pneumoniae type 2 obtained in Example 11 of the present invention is lower in nucleic acid content than that in Comparative Example 11, and the methyl pentose content is increased compared with that in Comparative Example 11, and other quality control indicators are not inferior to those of Comparative Example 11, and the process time is shortened.

[0351] Comparative Example 12 Preparation of degraded polysaccharide of Streptococcus pneumoniae type 2 capsular polysaccharide

[0352] Preparation of degraded polysaccharide of capsular polysaccharide of Streptococcus pneumoniae type 1 and 2

[0353] (1) Take 3.0±0.15 g of the refined polysaccharide obtained in Comparative Example 11 and dissolve it in 1.0 L of 0.15 mol / L sodium chloride solution, and stir until it is completely dissolved;

[0354] (2) The refined polysaccharide solution in step (1) is homogenized by a high-pressure homogenizer at a pressure of 800±50 bar, and the mixture is circulated for 3 times to obtain the degraded polysaccharide solution. ;

[0355] (3) The degraded polysaccharide solution is freeze-dried, and the degraded polysaccharide is collected after the freeze-drying.

[0356] 2. Degradation of polysaccharide from capsular polysaccharide of Streptococcus pneumoniae type 2

[0357] The determination of degradable polysaccharides of type 2 pneumococcal capsular polysaccharides shall refer to the 2020 edition of the Pharmacopoeia of the People's Republic of China, among which the determination of capsular polysaccharide content shall be carried out according to the rate turbidimetry (3.3.2) in Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; the determination of protein content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0731); the determination of nucleic acid content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401); the determination of total nitrogen content shall be carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China The content of phosphorus was determined in accordance with the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0704); the content of uronic acid was determined in accordance with the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 3103); the content of uronic acid was determined in accordance with the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0731); the content of methyl pentose was determined in accordance with the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401); the molecular size of capsular polysaccharides was determined in accordance with the first method in 3.1.2.10 of the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III. The relevant test results are shown in Table 25.

[0358] Table 25 Recovery rate and quality control index of degraded polysaccharide of type 2 Streptococcus pneumoniae capsular polysaccharide

[0359]

[0360] The results show that the quality control indicators of the degraded polysaccharide of type 2 Streptococcus pneumoniae capsular polysaccharide obtained in Example 12 of the present invention are lower than those of the degraded polysaccharide obtained in Comparative Example 12 in terms of protein content, nucleic acid, total nitrogen and total phosphorus content, and other quality control indicators are not inferior to those of Comparative Example 12. In addition, the process flow is simplified and the process time is shortened.

[0361] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide, characterized in that: The method comprises: sterilizing the fermentation culture of Streptococcus pneumoniae to lyse the bacteria, separating the supernatant, and sequentially subjecting the supernatant to a first ultrafiltration treatment, a precipitation treatment, and a second ultrafiltration treatment to obtain a refined polysaccharide; Alternatively, the supernatant is sequentially subjected to a first ultrafiltration treatment, a precipitation treatment, a degradation treatment, and a second ultrafiltration treatment to obtain a degraded polysaccharide; Wherein, the precipitation treatment is to use calcium salt as a precipitant to perform one-step precipitation under acidic conditions; the precipitation treatment is to collect the supernatant after treating at 2-8°C and pH 2.4-3.1 for 1-5 hours to obtain a first supernatant; The calcium salt is calcium chloride; The final concentration of the calcium salt in the precipitation system is 80-200 mmol / L; After adjusting the pH of the first supernatant or the degradation product to 6.8-7.5, collecting the supernatant to obtain a second supernatant, and subjecting the second supernatant to a second ultrafiltration treatment; The first ultrafiltration treatment is performed using an ultrafiltration membrane with a pore size of 100-150KD; For the second supernatant obtained without degradation treatment, the second ultrafiltration treatment is carried out using an ultrafiltration membrane with a pore size of 100-150KD; for the second supernatant obtained after degradation treatment, the second ultrafiltration treatment is carried out using an ultrafiltration membrane with a pore size of 30-50KD.

2. The method for preparing Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide according to claim 1, characterized in that: The degradation treatment is carried out by chemical treatment or physical treatment.

3. The method for preparing Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide according to claim 2, characterized in that: The chemical treatment method is to use trifluoroacetic acid for degradation treatment to obtain a degradation product; And / or, the physical treatment method is to use ultrasonic treatment or high-pressure homogenization treatment to perform degradation treatment to obtain a degraded product.

4. The method for preparing Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide according to claim 3, characterized in that: In the degradation treatment with trifluoroacetic acid, the final concentration of trifluoroacetic acid is 0.2-2 mol / L, and / or the degradation treatment is carried out at 25-60° C. for 2-10 hours.

5. The Streptococcus pneumoniae capsular polysaccharide or the degraded polysaccharide thereof prepared by the method for preparing Streptococcus pneumoniae capsular polysaccharide or the degraded polysaccharide thereof according to any one of claims 1 to 4.

6. The method for preparing Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide according to any one of claims 1 to 4 or the use of Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide according to claim 5 in preparing a product containing Streptococcus pneumoniae capsular polysaccharide.

7. A Streptococcus pneumoniae capsular polysaccharide vaccine or a Streptococcus pneumoniae capsular polysaccharide conjugate vaccine, characterized in that: The vaccine comprises the pneumococcal capsular polysaccharide or the degraded polysaccharide thereof according to claim 5.

8. A method for preparing a pneumococcal capsular polysaccharide vaccine or a pneumococcal capsular polysaccharide conjugate vaccine, characterized in that: The method comprises: preparing Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide by the method for preparing Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide according to any one of claims 1 to 4, and preparing Streptococcus pneumoniae capsular polysaccharide vaccine or Streptococcus pneumoniae capsular polysaccharide conjugate vaccine by using the Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide as an immunogen.

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