Method for preparing streptococcus pneumoniae capsular polysaccharide or degraded polysaccharide thereof

Abstract

The present invention relates to the technical field of biological products, in particular to a method for preparing a Streptococcus pneumoniae capsular polysaccharide or a degraded polysaccharide thereof. Said method comprises: sterilizing a fermentation culture of Streptococcus pneumoniae, separating a supernatant; and sequentially performing first ultrafiltration treatment, precipitation treatment and second ultrafiltration treatment on the supernatant, or sequentially performing first ultrafiltration treatment, precipitation treatment, degradation treatment and second ultrafiltration treatment on the supernatant, a precipitant used in the precipitation treatment comprising a calcium salt, and the precipitation treatment being performed under a condition that the pH is 2.4-3.6. The method has the advantages of simple process and short preparation period, thus remarkably reducing the cost in time and materials; in addition, quality control indexes, including impurities such as nucleic acids and proteins and specific groups, of the prepared refined polysaccharide and degraded polysaccharide are superior to standards, so that said polysaccharide and degraded polysaccharide can be used for preparing Streptococcus pneumoniae capsular polysaccharide vaccines or conjugate vaccines.

Description

A method for preparing Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide

[0001] Cross-references

[0002] This application claims priority to Chinese Patent Application No. 2024101961936, filed on February 22, 2024, entitled “A Method for Preparing Streptococcus Pneumoniae Capsular Polysaccharide or Its Degraded Polysaccharide,” the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to the technical field of biological products, and in particular to a method for preparing Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide. Background Art

[0004] Pneumococcal infection is a leading cause of death worldwide and a major cause of pneumonia, meningitis, and otitis media. With the widespread use of antibiotics, drug-resistant strains are increasing, necessitating the development of vaccines. Studies have shown that capsular polysaccharides on the bacterial surface can protect the body against bacterial infection. However, because polysaccharides are thymus-independent antigens, secondary immunization fails to restore reduced antibodies to the same level as in the initial immunization, fails to induce the production of immune memory cells, and offers little protection for infants and the elderly, age groups at high risk for pneumococcal infection. However, conjugating pneumococcal capsular polysaccharides to a protein carrier renders them thymus-dependent antigens. These antigens can activate B cells to produce antibodies with the assistance of T cells and macrophages, providing immune protection in infants and young children, inducing immune memory, and providing a booster effect with secondary immunization. Studies have shown that the introduction of pneumococcal vaccines has significantly reduced the global burden of pneumococcal disease.

[0005] The pneumococcal capsular polysaccharide is a series of high-molecular-weight homologues with varying molecular weights, consisting of repeating units. These homologues are typically obtained by fermenting and culturing pneumococcal capsular polysaccharides and extracting and purifying them from the fermentation culture. On the one hand, the preparation of pneumococcal capsular polysaccharide vaccines is subject to complex polysaccharide purification processes, long purification cycles, and safety or health risks associated with the use of reagents. For example, existing methods for preparing pneumococcal refined polysaccharides often employ organic reagents such as phenol and ethanol, which are harmful to human health and difficult to completely remove. The preparation of pneumococcal refined polysaccharides often involves complex processes and takes a long time. Furthermore, chromatography systems (e.g., Chinese patent applications CN104815327A, CN108079286A, CN112646050A, and CN116970095A) are often used, requiring expensive instruments and materials, significantly increasing production costs. Chinese patent application CN116970095A discloses a process for removing protein and nucleic acid impurities by a “two-step precipitation method”. By mixing sodium deoxycholate (DOC) with a first ultrafiltrate under suitable neutral salt conditions and controlling the pH of the solution to 4.5-5.0, the effect of sodium deoxycholate adsorbing and precipitating proteins can be efficiently exerted. Then, by calcium salt precipitation, impurities such as proteins and nucleic acids are further removed. The supernatant is collected by centrifugation, and then the supernatant is filtered and 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.

[0006] On the other hand, when preparing pneumococcal capsular polysaccharide conjugate vaccines, polysaccharides often have a large molecular weight. Excessively large molecular weights hinder polysaccharide conjugates, while too small molecular weights compromise immunogenicity. Therefore, a method is needed to degrade the large polysaccharide molecules into smaller ones to meet the needs and applications of pneumococcal conjugate vaccines. Existing capsular polysaccharide degradation processes involve dissolving a lyophilized preparation of purified pneumococcal polysaccharide before degradation, resulting in complex operations and unsuitable for large-scale production applications. Summary of the Invention

[0007] The present invention provides a method for preparing Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide.

[0008] During the research and development of a method for preparing pneumococcal capsular polysaccharide, the present invention accidentally discovered a simple and rapid method for preparing 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, significantly reducing material and time costs and improving purification efficiency.

[0009] Specifically, the present invention provides the following technical solutions:

[0010] The present invention provides a method for preparing a capsular polysaccharide of Streptococcus pneumoniae or a degraded polysaccharide thereof, the method comprising: sterilizing a fermentation culture of Streptococcus pneumoniae, separating a supernatant, and sequentially subjecting the supernatant to a first ultrafiltration treatment, a precipitation treatment, and a second ultrafiltration treatment;

[0011] Alternatively, the supernatant is sequentially subjected to a first ultrafiltration treatment, a precipitation treatment, a degradation treatment, and a second ultrafiltration treatment;

[0012] The precipitant used in the precipitation treatment comprises calcium salt, and the precipitation treatment is carried out at a pH of 2.4-3.6.

[0013] The preparation method of the present invention is divided into two embodiments: no degradation treatment and degradation treatment. The capsular polysaccharide (refined polysaccharide) prepared by the method without degradation treatment has a relatively large molecular weight and is suitable for preparing a pneumococcal capsular polysaccharide vaccine. The degradation treatment can obtain a polysaccharide with a reduced molecular weight (degraded polysaccharide), which is more suitable for combining with a carrier protein (such as TT, DT, CRM197, etc.) for preparing a pneumococcal capsular polysaccharide conjugate vaccine.

[0014] The present invention found 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 redissolve the obtained freeze-dried refined polysaccharide before degradation treatment, which greatly simplifies the process flow and improves the preparation efficiency.

[0015] The key to achieving the above-mentioned preparation method's effectiveness is that calcium salt is used as a precipitant and precipitation is performed under acidic conditions of pH 2.4-3.6 (calcium hydrochloride precipitation). Ultrafiltration is performed before and after the calcium hydrochloride precipitation, with the primary effect of the first ultrafiltration being to remove small molecule impurities from the sterilized supernatant of the fermentation culture, thereby further facilitating the calcium hydrochloride precipitation and allowing the impurity removal efficiency to be improved. The secondary ultrafiltration is then performed to concentrate the polysaccharide solution and perform diafiltration on the polysaccharide solution, further removing small molecule impurities and salt ions from the polysaccharide solution.

[0016] Preferably, the calcium salt is calcium chloride.

[0017] Preferably, the final concentration of the calcium salt in the precipitation system is 80-200 mmol / L.

[0018] 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.

[0019] Preferably, the precipitation treatment is to collect the supernatant after treating at 2-8° C. for 1-5 hours to obtain the first supernatant.

[0020] Unlike some existing methods for purifying Streptococcus pneumoniae capsular polysaccharides that require elevated temperature acid precipitation (which can easily lead to degradation of the polysaccharide, resulting in a decrease in molecular weight and affecting its immunogenicity), the precipitation treatment of the present invention can achieve better impurity removal effects simply by being performed under low temperature conditions.

[0021] For the embodiment comprising a degradation treatment step, the degradation treatment is performed by a chemical treatment method or a physical treatment method.

[0022] Among them, the chemical treatment method preferably uses trifluoroacetic acid for degradation treatment to obtain a degraded product.

[0023] The physical treatment method preferably adopts ultrasonic treatment method or high-pressure homogenization treatment method to perform degradation treatment to obtain a degraded product.

[0024] 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.

[0025] In some embodiments of the present invention, trifluoroacetic acid is used for degradation, wherein the final concentration of trifluoroacetic acid is 0.2-2 mol / L. Preferably, the degradation treatment is performed at 25-60° C. for 2-10 hours. After the degradation treatment is completed, the supernatant is collected.

[0026] The above degradation treatment degrades the macromolecular polysaccharide into degraded polysaccharide of suitable molecular weight that is suitable for the preparation of conjugate vaccine and does not affect the immunogenicity thereof.

[0027] The above method further comprises: 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.

[0028] In the present invention, the pH can be adjusted to acidic using an 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 using an alkaline solution, including but not limited to sodium hydroxide, potassium hydroxide, etc., preferably sodium hydroxide.

[0029] In the method described above, the first ultrafiltration treatment is performed using an ultrafiltration membrane with a pore size of 100-150KD (preferably 100KD).

[0030] 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 ultrafiltration of equal volume. Preferably, the volume of purified water used for ultrafiltration is 4-6 times the volume of the concentrate.

[0031] 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).

[0032] Preferably, in the second ultrafiltration treatment, the volume of water for injection used for diafiltration is 8-15 times the volume of the supernatant.

[0033] Preferably, after the second ultrafiltration treatment, the method further comprises the step of drying the second ultrafiltration concentrate obtained by the second ultrafiltration treatment.

[0034] The drying may be freeze-drying.

[0035] 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.

[0036] The preparation method described in the present invention is not particularly limited in principle to 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. Alternatively, other reagents that can kill Streptococcus pneumoniae and release capsular polysaccharides can also be used for sterilization.

[0037] 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%.

[0038] In other embodiments of the present invention, β-propiolactone (BPL) is used to sterilize the fermentation culture of Streptococcus pneumoniae. The final concentration of β-propiolactone in the treatment system can be added in an amount commonly used in the art. 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 treatment temperature is preferably 2-8°C. The treatment time is preferably 8-16 hours. More preferably, it is 10-14 hours. The treatment is preferably a low-temperature incubation treatment after uniform mixing.

[0039] Preferably, β-propiolactone (BPL) is added to the fermentation culture of Streptococcus pneumoniae at the late logarithmic growth stage. After adding β-propiolactone (BPL), the mixture is stirred and mixed, and then incubated at low temperature.

[0040] When β-propiolactone is used for sterilization, the above-mentioned preparation method preferably does not use sodium deoxycholate.

[0041] In the existing pneumococcal capsular polysaccharide production method, sodium deoxycholate plays a very important role as a cleavage agent for lysing the bacterium to release capsular polysaccharides for the extraction of capsular polysaccharides. Currently, there is no alternative reagent available for pneumococcal capsular polysaccharide preparation. The present invention unexpectedly discovered during the research and development process that β-propiolactone has a significant effect of inactivating pneumococcus and promoting pneumococcus to release capsular polysaccharides. It can be used to treat pneumococcus and extract capsular polysaccharides. Unlike sodium deoxycholate, β-propiolactone does not lyse the bacterium, thereby significantly reducing the release of impurities such as protein and nucleic acid in the bacterium, which is more conducive to reducing the impurity content and removal difficulty during the pneumococcal polysaccharide purification process. Moreover, β-propiolactone is easily hydrolyzed, and the hydrolyzate is non-toxic and harmless, so it does 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. Regarding its ability to promote pneumococcus to release capsular polysaccharides and reduce the release of impurities such as intracellular protein and nucleic acid, there is no relevant report yet. 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 residual sodium deoxycholate, but also significantly reduces the content of impurities such as protein in the extracted capsular polysaccharide, is more conducive to subsequent purification and removal of impurities, and has a higher polysaccharide recovery rate, which is significantly higher than that of treating the fermentation culture of Streptococcus pneumoniae with formaldehyde.

[0042] In the present invention, the fermentation culture is a culture (eg, fermentation broth) containing Streptococcus pneumoniae obtained by fermentation culture of Streptococcus pneumoniae.

[0043] The method for preparing the pneumococcal capsular polysaccharide of the present invention can be used for different types of pneumococcus, 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.

[0044] Preferably, the Streptococcus pneumoniae is type 2, 5, 6A, 10A, 11A, or 19F.

[0045] 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.

[0046] 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.

[0047] Preferably, the product containing Streptococcus pneumoniae capsular polysaccharide is a vaccine.

[0048] The vaccine includes a Streptococcus pneumoniae capsular polysaccharide vaccine or a Streptococcus pneumoniae capsular polysaccharide conjugate vaccine.

[0049] The present invention provides a Streptococcus pneumoniae capsular polysaccharide vaccine or a Streptococcus pneumoniae capsular polysaccharide conjugate vaccine, which comprises the Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide as described above.

[0050] The aforementioned Streptococcus pneumoniae capsular polysaccharide vaccine or Streptococcus pneumoniae capsular polysaccharide conjugate vaccine may be a monovalent or multivalent vaccine.

[0051] The multivalent vaccine comprises capsular polysaccharides of at least two types of Streptococcus pneumoniae 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.

[0052] 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, for example, a 23-valent pneumococcal capsular polysaccharide vaccine.

[0053] The pneumococcal capsular polysaccharide conjugate vaccine can 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, for example, a 13-valent pneumococcal capsular polysaccharide conjugate vaccine, a 20-valent pneumococcal capsular polysaccharide conjugate vaccine, or a 24-valent pneumococcal capsular polysaccharide conjugate vaccine.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] The present invention provides a method for preparing a Streptococcus pneumoniae capsular polysaccharide vaccine or a Streptococcus pneumoniae capsular polysaccharide conjugate vaccine. The method comprises: preparing Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide by utilizing the method for preparing Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide; and preparing the Streptococcus pneumoniae capsular polysaccharide vaccine or the Streptococcus pneumoniae capsular polysaccharide conjugate vaccine using the Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide as an immunogen.

[0059] The beneficial effects of the present invention include at least:

[0060] (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 polysaccharide is 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 reduces it by at least 50%.

[0061] (2) The method of the present invention is used to prepare pneumococcal capsular polysaccharide and degraded polysaccharide, 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, and can be used for the preparation of pneumococcal capsular polysaccharide vaccine or capsular polysaccharide conjugate vaccine.

[0062] (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

[0063] The present invention provides a method for preparing Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide.

[0064] In some embodiments of the present invention, a method for preparing a capsular polysaccharide of Streptococcus pneumoniae is provided, the method comprising the following steps:

[0065] (1) Sterilize the fermentation broth of Streptococcus pneumoniae and let it stand at 4°C for 6-12 hours, then centrifuge and collect the supernatant;

[0066] (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;

[0067] (3) Adding a CaCl2 solution with a final concentration of 80-200 mmol / L to the first ultrafiltration concentrate, stirring thoroughly, and adjusting the pH to 2.4-3.6 with acid solution. Stir thoroughly, and let it stand at 2-8°C for 1-5 hours. Then, centrifuge and collect the supernatant. Adjust the pH to 7.00-7.50 with alkaline solution, and centrifuge again to collect the supernatant.

[0068] (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;

[0069] (5) freeze-drying the polysaccharide ultrafiltration concentrate obtained in step (4), and collecting the refined polysaccharide after completion.

[0070] In some embodiments of the present invention, a method for preparing a degraded polysaccharide of Streptococcus pneumoniae capsular polysaccharide is provided, the method comprising the following steps:

[0071] (1) Sterilize the fermentation broth of Streptococcus pneumoniae and let it stand at 4°C for 6-12 hours, then centrifuge and collect the supernatant;

[0072] (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;

[0073] (3) Adding a CaCl2 solution with a final concentration of 80-200 mmol / L to the first ultrafiltration concentrate, stirring thoroughly, and adjusting the pH to 2.4-3.6 with acid solution. Stir thoroughly, and let it stand at 2-8°C for 1-5 hours. Then, centrifuge and collect the supernatant. Adjust the pH to 7.00-7.50 with alkaline solution, and centrifuge again to collect the supernatant.

[0074] (4) adding trifluoroacetic acid to a final concentration of 0.2-2 mol / L to the supernatant obtained in step (3), allowing the mixture to stand 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;

[0075] (5) ultrafiltration of the supernatant obtained in step (4) using a 30KD membrane 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;

[0076] (6) freeze-drying the polysaccharide ultrafiltration concentrate obtained in step (5), and collecting the degraded polysaccharide after completion.

[0077] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0078] Example 1 Preparation of 6A Streptococcus pneumoniae capsular polysaccharide

[0079] 1. Preparation of 6A Streptococcus pneumoniae capsular polysaccharide

[0080] (1) The fermentation broth of Streptococcus pneumoniae type 6A was sterilized with 0.1% DOC and allowed to stand at 4°C for 12 hours. The broth was then centrifuged at 12,000 g for 30 minutes, and the supernatant was collected.

[0081] (2) ultrafiltration of the supernatant obtained in step (1) 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;

[0082] (3) Adding a CaCl2 solution with a final concentration of 200 mmol / L to the ultrafiltration concentrate obtained in step (2), stirring thoroughly, and adjusting the pH to 3.00±0.1 with phosphoric acid. After stirring thoroughly, standing at 2-8°C for 1 hour, and then centrifuging at 12000g for 30 minutes, collecting the supernatant, adjusting the pH of the supernatant to 7.00-7.50 with NaOH solution, and centrifuging again at 12000g for 30 minutes, and collecting the supernatant;

[0083] (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;

[0084] (5) The ultrafiltration concentrate obtained in step (4) is freeze-dried, and the refined polysaccharide is collected after the freeze-drying.

[0085] 2. Detection of 6A Streptococcus pneumoniae capsular polysaccharide

[0086] The determination of capsular polysaccharide content is carried out in accordance with 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 in accordance with 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 in accordance with 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 in accordance with 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 in accordance with 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 in accordance with 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 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.

[0087] The recovery rate was calculated as follows: recovery rate = refined polysaccharide content / polysaccharide content in the supernatant of the fermentation broth after sterilization × 100%.

[0088] The relevant test results are shown in Table 1.

[0089] Table 1 Recovery rate and quality control indexes of 6A Streptococcus pneumoniae capsular polysaccharide

[0090] Note: 001, 002, and 003 in Table 1 represent three batches of experiments, the same below.

[0091] Example 2 Preparation of degraded polysaccharide of 6A Streptococcus pneumoniae capsular polysaccharide

[0092] 1. Preparation of degraded polysaccharide of 6A Streptococcus pneumoniae capsular polysaccharide

[0093] (1) The fermentation broth of Streptococcus pneumoniae type 6A was sterilized with 0.1% DOC and allowed to stand at 4°C for 12 hours, then centrifuged at 12,000 g for 30 minutes, and the supernatant was collected;

[0094] (2) ultrafiltration of the supernatant obtained in step (1) 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;

[0095] (3) CaCl2 solution with a final concentration of 80 mmol / L (6A-2308001L), 120 mmol / L (6A-2308002L), and 200 mmol / L (6A-2308003L) was added to the ultrafiltration concentrate obtained in step (2), and the mixture was thoroughly stirred and then adjusted to pH 3.00 ± 0.1 with phosphoric acid. After thorough stirring, the mixture was allowed to stand at 2-8°C for 1 hour, and then centrifuged at 12000g for 30 minutes to collect the supernatant;

[0096] (4) adding trifluoroacetic acid to 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;

[0097] (5) ultrafiltration of the supernatant solution obtained in step (4) using a membrane with a pore size of 30 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;

[0098] (6) The ultrafiltration concentrate is freeze-dried, and the degraded polysaccharides are collected after the drying.

[0099] 2. Assay of degraded polysaccharide of 6A Streptococcus pneumoniae capsular polysaccharide

[0100] Degradation of 6A Streptococcus pneumoniae capsular polysaccharide The assay of polysaccharide was carried out with reference to the 2020 edition of the Pharmacopoeia of the People's Republic of China, where the assay of capsular polysaccharide content was 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 was 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 was carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401). Total nitrogen content was determined in accordance with the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 0704); phosphorus content was determined in accordance with the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 3103); methyl pentose content was determined in accordance with the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 0401); and capsular polysaccharide molecular size was determined in accordance with Method 1, Section 3.1.2.10, Part III, 2020 edition. The relevant test results are shown in Table 2.

[0101] Table 2 Recovery rate and quality control indexes of degraded polysaccharide of 6A Streptococcus pneumoniae capsular polysaccharide

[0102] Example 3 Preparation of Streptococcus pneumoniae type 5 capsular polysaccharide

[0103] Preparation of 1.5 Streptococcus pneumoniae capsular polysaccharide

[0104] (1) The fermentation broth of Streptococcus pneumoniae type 5 was sterilized with 0.1% DOC and allowed to stand at 4°C for 12 hours, then centrifuged at 12,000 g for 30 minutes, and the supernatant was collected;

[0105] (2) ultrafiltration of the supernatant obtained in step (1) 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) adding a CaCl2 solution with a final concentration of 80 mmol / L to the ultrafiltration concentrate obtained in step (2), stirring thoroughly, and adjusting the pH to 3.00±0.1 with phosphoric acid. After stirring thoroughly, standing at 2-8°C for 5 h, and then centrifuging at 12000g for 30 min, collecting the supernatant, adjusting the pH to 7.00-7.50 with NaOH, and centrifuging again at 12000g for 30 min, and collecting 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 refined polysaccharide is collected after the freeze-drying.

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

[0110] The content of capsular polysaccharide was determined by 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 was determined by the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0731); the content of nucleic acid was determined by the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401); the content of total nitrogen was determined by the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0704); phosphorus The content of the test was determined according to the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 3103); the content of uronic acid was determined according to the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 0731); the content of hexosamine was determined according to the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 0401); and the molecular size of the capsular polysaccharide was determined according to 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 3.

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

[0112] Example 4 Preparation of degraded polysaccharide of Streptococcus pneumoniae type 5 capsular polysaccharide

[0113] Preparation of degraded polysaccharide of Streptococcus pneumoniae type 1 and 5 capsular polysaccharide

[0114] (1) The fermentation broth of Streptococcus pneumoniae type 5 was sterilized with 0.1% DOC and allowed to stand at 4°C for 12 hours, then centrifuged at 12,000 g for 30 minutes, and the supernatant was collected;

[0115] (2) ultrafiltration of the supernatant obtained in step (1) 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;

[0116] (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, let stand at 2-8°C for 5 h, then centrifuge at 12000g for 30 min, and collect the supernatant;

[0117] (4) adding trifluoroacetic acid to a final concentration of 1.5 mol / L to the supernatant obtained in step (3), allowing the mixture to stand at 25°C for 10 h, adjusting the pH to 7.00-7.50, and centrifuging again to collect the supernatant;

[0118] (5) ultrafiltration of the supernatant obtained in step (4) using a 30KD membrane 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;

[0119] (6) The ultrafiltration concentrate obtained in step (5) is freeze-dried, and the degraded polysaccharide is collected after the drying.

[0120] Detection of degraded polysaccharides of capsular polysaccharides of Streptococcus pneumoniae types 2 and 5

[0121] The assay of degradable polysaccharides of type 5 pneumococcal capsular polysaccharide was carried out with reference to the 2020 edition of the Pharmacopoeia of the People's Republic of China. The assay of capsular polysaccharide content was 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 was 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 was 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 was carried out according to Part 2 of the Pharmacopoeia of the People's Republic of China. The content of the test samples was determined according to the Pharmacopoeia of the People's Republic of China, 2020 Edition, Part III (General Rule 0704); phosphorus content was determined according to the Pharmacopoeia of the People's Republic of China, 2020 Edition, Part III (General Rule 3103); uronic acid content was determined according to the Pharmacopoeia of the People's Republic of China, 2020 Edition, Part III (General Rule 0731); hexosamine content was determined according to the Pharmacopoeia of the People's Republic of China, 2020 Edition, Part III (General Rule 0401); and the molecular size of capsular polysaccharides was determined according to 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 4.

[0122] Table 4 Recovery rate and quality control indexes of degraded polysaccharides of Streptococcus pneumoniae type 5 capsular polysaccharide

[0123] Example 5 Preparation of Type 10A Streptococcus pneumoniae Capsular Polysaccharide

[0124] 1. Preparation of 10A Streptococcus pneumoniae capsular polysaccharide

[0125] (1) The fermentation broth of Streptococcus pneumoniae type 10A was sterilized with 0.1% DOC and allowed to stand at 4°C for 12 hours, then centrifuged at 12,000 g for 30 minutes, and the supernatant was collected.

[0126] (2) ultrafiltration of the supernatant obtained in step (1) 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;

[0127] (3) Adding a CaCl2 solution with a final concentration of 80 mmol / L to the ultrafiltration concentrate obtained in step (2), stirring thoroughly, and adjusting the pH to 2.50±0.1 (10A-2308001), 3.00±0.1 (10A-2308002), 3.50±0.1 (10A-2308003), and 4.00±0.1 (10A-2308004) with phosphoric acid, stirring thoroughly, and letting it stand at 2-8°C for 3 h, then centrifuging at 12000g for 30 min, collecting the supernatant, adjusting the pH to 7.00-7.50 with NaOH, and centrifuging again at 12000g for 30 min, and collecting the supernatant;

[0128] (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;

[0129] (5) The ultrafiltration concentrate obtained in step (4) is freeze-dried, and the refined polysaccharide is collected after the freeze-drying.

[0130] 2. Detection of 10A Streptococcus pneumoniae capsular polysaccharide

[0131] Capsular polysaccharide content was determined according to the rate turbidimetric method (3.3.2) in the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III. Protein content was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0731). Nucleic acid content was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401). Total nitrogen content was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0704). Phosphorus content was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 3103). Hexosamine content was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401). The molecular size of capsular polysaccharide was determined 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. 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 significantly lower than that of the group with a pH of 4.0.

[0132] Table 5 Recovery rate and quality control indexes of 10A Streptococcus pneumoniae capsular polysaccharide

[0133] Example 6 Preparation of Degraded Polysaccharide of Type 10A Streptococcus Pneumoniae Capsular Polysaccharide

[0134] 1. Preparation of degraded polysaccharide of 10A Streptococcus pneumoniae capsular polysaccharide

[0135] (1) The fermentation broth of Streptococcus pneumoniae type 10A was sterilized with 0.1% DOC and allowed to stand at 4°C for 12 hours, then centrifuged at 12,000 g for 30 minutes, and the supernatant was collected.

[0136] (2) ultrafiltration of the supernatant obtained in step (1) 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;

[0137] (3) adding a CaCl2 solution with a final concentration of 80 mmol / L to the ultrafiltration concentrate obtained in step (2), stirring thoroughly, and adjusting the pH to 3.00 ± 0.1 with phosphoric acid. After stirring thoroughly, standing at 2-8°C for 3 h, and then centrifuging at 12000g for 30 min, collecting the supernatant;

[0138] (4) adding trifluoroacetic acid to 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;

[0139] (5) ultrafiltration of the supernatant obtained in step (4) using a 30KD membrane 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;

[0140] (6) The ultrafiltration concentrate obtained in step (5) is freeze-dried, and the refined polysaccharide is collected after the drying.

[0141] 2. Assay of degraded polysaccharide of 10A Streptococcus pneumoniae capsular polysaccharide

[0142] The assay of degraded polysaccharides of capsular polysaccharides of type 10A Streptococcus pneumoniae was carried out with reference to the 2020 edition of the Pharmacopoeia of the People's Republic of China. The assay of capsular polysaccharide content was 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 was 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 was carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401); Total nitrogen content was determined in accordance with the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 0704); phosphorus content was determined in accordance with the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 3103); hexosamine content was determined in accordance with the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 0401); and capsular polysaccharide molecular size was determined in accordance with Method 1, Section 3.1.2.10, Part III, 2020 edition. The relevant test results are shown in Table 6.

[0143] Table 6 Recovery rate and quality control index of degraded polysaccharide of 10A Streptococcus pneumoniae capsular polysaccharide

[0144] Example 7 Preparation of Streptococcus pneumoniae Type 11A Capsular Polysaccharide

[0145] 1. Preparation of 11A Streptococcus pneumoniae capsular polysaccharide

[0146] (1) The fermentation broth of Streptococcus pneumoniae type 11A was sterilized with 0.1% DOC and allowed to stand at 4°C for 12 hours, then centrifuged at 12,000 g for 30 minutes, and the supernatant was collected.

[0147] (2) ultrafiltration of the supernatant obtained in step (1) 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;

[0148] (3) adding a CaCl2 solution with a final concentration of 80 mmol / L to the ultrafiltration concentrate obtained in step (2), adjusting the pH to 3.00±0.1 after thorough mixing, letting it stand at 2-8°C for 2 h, then centrifuging at 12000g for 30 min, collecting the supernatant, adjusting the pH to 7.00-7.50 with NaOH, centrifuging again at 12000g for 30 min, and collecting the supernatant;

[0149] (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;

[0150] (5) freeze-drying the ultrafiltration concentrate obtained in step (4), and collecting the degraded polysaccharide after completion.

[0151] 2. Detection of Capsular Polysaccharide of Streptococcus pneumoniae Type 11A

[0152] Capsular polysaccharide content was determined according to the rate turbidimetric method (3.3.2) in the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III. Protein content was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0731). Nucleic acid content was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401). Total nitrogen content was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0704). Phosphorus content was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 3103). O-acetyl content was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 3117). The molecular size of capsular polysaccharide was determined 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. The relevant test results are shown in Table 7.

[0153] Table 7 Recovery rate and quality control indexes of 11A Streptococcus pneumoniae capsular polysaccharide

[0154] Example 8 Preparation of Degraded Polysaccharide of Type 11A Streptococcus Pneumoniae Capsular Polysaccharide

[0155] 1. Preparation of degraded polysaccharide of Streptococcus pneumoniae type 11A capsular polysaccharide

[0156] (1) The fermentation broth of Streptococcus pneumoniae type 11A was sterilized with 0.1% DOC and allowed to stand at 4°C for 12 hours, then centrifuged at 12,000 g for 30 minutes, and the supernatant was collected.

[0157] (2) ultrafiltration of the supernatant obtained in step (1) 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;

[0158] (3) Adding a CaCl2 solution with a final concentration of 100 mmol / L to the ultrafiltration concentrate obtained in step (2), adjusting the pH to 3.00 ± 0.1 after thorough mixing, letting it stand at 2-8°C for 2 h, and then centrifuging at 12000g for 30 min to collect the supernatant;

[0159] (4) adding trifluoroacetic acid to 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;

[0160] (5) ultrafiltration of the supernatant solution obtained in step (4) using a membrane with a pore size of 30 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;

[0161] (6) The ultrafiltration concentrate obtained in step (5) is freeze-dried, and the refined polysaccharide is collected after the drying.

[0162] 2. Detection of degraded polysaccharides of Streptococcus pneumoniae type 11A capsular polysaccharide

[0163] The assay of degraded polysaccharides of capsular polysaccharides of type 11A Streptococcus pneumoniae was carried out with reference to the 2020 edition of the Pharmacopoeia of the People's Republic of China. The assay of capsular polysaccharide content was 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 was 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 was carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401); Total nitrogen content was determined in accordance with the Pharmacopoeia of the People's Republic of China (2020 edition), Part III (General Rule 0704); phosphorus content was determined in accordance with the Pharmacopoeia of the People's Republic of China (2020 edition), Part III (General Rule 3103); O-acetyl content was determined in accordance with the Pharmacopoeia of the People's Republic of China (2020 edition), Part III (General Rule 3117); and capsular polysaccharide molecular size was determined in accordance with Method 1, Section 3.1.2.10, Part III, 2020 edition. The relevant test results are shown in Table 8.

[0164] Table 8 Recovery rate and quality control index of degraded polysaccharide of 11A Streptococcus pneumoniae capsular polysaccharide

[0165] Example 9 Preparation of 19F Streptococcus pneumoniae capsular polysaccharide

[0166] 1. Preparation of 19F Streptococcus pneumoniae capsular polysaccharide

[0167] (1) The 19F pneumococcal fermentation broth was sterilized with 0.1% DOC and allowed to stand at 4°C for 6 h, then centrifuged at 12,000 g for 30 min, and the supernatant was collected.

[0168] (2) ultrafiltration of the supernatant obtained in step (1) 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;

[0169] (3) adding a CaCl2 solution with a final concentration of 80 mmol / L to the ultrafiltration concentrate obtained in step (2), adjusting the pH to 3.00±0.1 after thorough mixing, letting it stand at 2-8°C for 2 h, then centrifuging at 12000g for 30 min, collecting the supernatant, adjusting the pH to 7.00-7.50 with NaOH, centrifuging again at 12000g for 30 min, and collecting the supernatant;

[0170] (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;

[0171] (5) The ultrafiltration concentrate obtained in step (4) is freeze-dried, and the refined polysaccharide is collected after the freeze-drying.

[0172] 2. Detection of 19F Streptococcus pneumoniae capsular polysaccharide

[0173] The content of capsular polysaccharide was determined by 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 was determined by the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0731); the content of nucleic acid was determined by the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401); the content of total nitrogen was determined by the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0704); phosphorus The content of the test was determined according to the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 3103); the content of hexosamine was determined according to the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 0401); the content of methylpentose was determined according to the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 0401); and the molecular size of the capsular polysaccharide was determined according to the first method in Section 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 9.

[0174] Table 9 Recovery rate and quality control indexes of 19F Streptococcus pneumoniae capsular polysaccharide

[0175] Example 10 Preparation of 19F Streptococcus pneumoniae Capsular Polysaccharide Degradation Polysaccharide

[0176] 1. Preparation of 19F Streptococcus pneumoniae capsular polysaccharide degradation polysaccharide

[0177] (1) The 19F pneumococcal fermentation broth was sterilized with 0.1% DOC and allowed to stand at 4°C for 6 h, then centrifuged at 12,000 g for 30 min, and the supernatant was collected.

[0178] (2) ultrafiltration of the supernatant obtained in step (1) 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;

[0179] (3) adding a CaCl2 solution with a final concentration of 80 mmol / L to the ultrafiltration concentrate obtained in step (2), adjusting the pH to 3.00 ± 0.1 after thorough mixing, letting it stand at 2-8°C for 2 h, and then centrifuging at 12000g for 30 min to collect the supernatant;

[0180] (4) adding trifluoroacetic acid to a final concentration of 0.25 mol / L to the supernatant obtained in step (3), allowing the mixture to stand at 25°C for 3 h, adjusting the pH to 7.00-7.50, and centrifuging again to collect the supernatant;

[0181] (5) ultrafiltration of the supernatant obtained in step (4) using a 30 kD membrane 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;

[0182] (6) The ultrafiltration concentrate obtained in step (5) is freeze-dried, and the degraded polysaccharide is collected after the drying.

[0183] 2. Assay of degraded polysaccharide of 19F Streptococcus pneumoniae capsular polysaccharide

[0184] The assay of degradable polysaccharides of 19F type pneumococcal capsular polysaccharide was carried out with reference to the 2020 edition of the Pharmacopoeia of the People's Republic of China, wherein the assay of capsular polysaccharide content was 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 was 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 was 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 was 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 was carried out according to Part III of the Pharmacopoeia of the People's Republic of China (General Rule 0401); the assay of protein content was 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 was carried out according to Part III ... protein content was carried out according to Part III of the 202 The content of the test was determined in accordance with the 2020 edition, Part III of the Pharmacopoeia of the People's Republic of China (General Rule 0704). The content of phosphorus was determined in accordance with the 2020 edition, Part III of the Pharmacopoeia of the People's Republic of China (General Rule 3103). The content of hexosamine was determined in accordance with the 2020 edition, Part III of the Pharmacopoeia of the People's Republic of China (General Rule 0401). The content of methylpentose was determined 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 the capsular polysaccharide 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.

[0185] Table 10 Recovery rate and quality control index of degraded polysaccharide of 19F Streptococcus pneumoniae capsular polysaccharide

[0186] Example 11 Preparation of Streptococcus pneumoniae type 2 capsular polysaccharide

[0187] Purification of Streptococcus pneumoniae type 1 and 2 capsular polysaccharide

[0188] (1) After sterilization with 0.1% DOC, the fermentation broth of Streptococcus pneumoniae type 2 was allowed to stand at 4°C for 12 hours, then centrifuged at 12,000 g for 30 minutes, and the supernatant was collected.

[0189] (2) ultrafiltration of the supernatant obtained in step (1) 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;

[0190] (3) adding a CaCl2 solution with a final concentration of 80 mmol / L to the ultrafiltration concentrate obtained in step (2), adjusting the pH to 3.00±0.1 after thorough mixing, letting it stand at 2-8°C for 4 h, then centrifuging at 12000g for 30 min, collecting the supernatant, adjusting the pH to 7.00-7.50 with NaOH, centrifuging again at 12000g for 30 min, and collecting the supernatant;

[0191] (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;

[0192] (5) The ultrafiltration concentrate obtained in step (4) is freeze-dried, and the refined polysaccharide is collected after the freeze-drying.

[0193] 2. Detection of Streptococcus pneumoniae type 2 capsular polysaccharide

[0194] The determination of capsular polysaccharide content is carried out in accordance with 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 in accordance with 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 in accordance with 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 in accordance with the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0704); Phosphorus content was determined according to the Pharmacopoeia of the People's Republic of China (2020 edition), Part III (General Rule 3103); uronic acid content was determined according to the Pharmacopoeia of the People's Republic of China (2020 edition), Part III (General Rule 0731); methyl pentose content was determined according to the Pharmacopoeia of the People's Republic of China (2020 edition), Part III (General Rule 0401); and capsular polysaccharide molecular size was determined according to 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 11.

[0195] Table 11 Recovery and quality control indicators of type 2 monovalent Streptococcus pneumoniae capsular polysaccharide

[0196] Example 12 Preparation of degraded polysaccharide of Streptococcus pneumoniae type 2 capsular polysaccharide

[0197] Preparation of degraded polysaccharides of Streptococcus pneumoniae type 1 and 2 capsular polysaccharides

[0198] (1) After sterilization with 0.1% DOC, the fermentation broth of Streptococcus pneumoniae type 2 was allowed to stand at 4°C for 12 hours, then centrifuged at 12,000 g for 30 minutes, and the supernatant was collected.

[0199] (2) ultrafiltration of the supernatant obtained in step (1) 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;

[0200] (3) adding a CaCl2 solution with a final concentration of 80 mmol / L to the ultrafiltration concentrate obtained in step (2), adjusting the pH to 3.00 ± 0.1 after thorough mixing, letting it stand at 2-8°C for 2 h, and then centrifuging at 12000g for 30 min to collect the supernatant;

[0201] (4) adding trifluoroacetic acid to 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;

[0202] (5) ultrafiltration of the supernatant solution obtained in step (4) using a membrane with a pore size of 30 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;

[0203] (6) The ultrafiltration concentrate obtained in step (5) is freeze-dried, and the degraded polysaccharide is collected after the drying.

[0204] 2. Assay of degraded polysaccharide of Streptococcus pneumoniae type 2 capsular polysaccharide

[0205] The assay 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 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 The determination of the content of the test results was carried out in accordance with the 2020 edition, Part III (General Rule 0704); the determination of phosphorus content was carried out in accordance with the 2020 edition, Part III (General Rule 3103); the determination of uronic acid content was carried out in accordance with the 2020 edition, Part III (General Rule 0731); the determination of methyl pentose content was carried out in accordance with the 2020 edition, Part III (General Rule 0401); and the determination of the molecular size of the capsular polysaccharide was carried out 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 12.

[0206] Table 12 Recovery rate and quality control index of degraded polysaccharide of Streptococcus pneumoniae type 2 capsular polysaccharide

[0207] It should be noted that the sterilization methods in the above embodiments are only exemplary, and the improvement of the capsular polysaccharide purification method of the present invention and its enhanced effect do not depend on a specific sterilization method. The present invention has been verified through experiments that replacing the sodium deoxycholate sterilization treatment with β-propiolactone as a bactericide, combined with 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 using 0.1% β-propiolactone for sterilization is provided below. In addition, the present invention also attempts to use other concentrations of β-propiolactone for sterilization. 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.

[0208] Example 13 Preparation of Streptococcus pneumoniae Type 14 Capsular Polysaccharide

[0209] 1. Purification of Streptococcus pneumoniae type 14 capsular polysaccharide

[0210] (1) β-propiolactone was added to the fermentation broth of Streptococcus pneumoniae type 14 at a final concentration of 0.1%, stirred evenly, incubated at 4°C for 12 hours, and then centrifuged at 12,000 g for 30 minutes, and the supernatant was collected;

[0211] (2) ultrafiltration of the supernatant obtained in step (1) 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;

[0212] (3) adding 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), respectively, and adjusting the pH to 3.00±0.1 after thorough mixing. After thorough mixing, the mixture was allowed to stand at 2-8°C for 5 h, and then centrifuged at 12000g for 30 min, the supernatant was collected, and the pH was adjusted to 7.00-7.50 with NaOH, and centrifuged again at 12000g for 30 min, and the supernatant was collected;

[0213] (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;

[0214] (5) The ultrafiltration concentrate obtained in step (4) is freeze-dried, and the refined polysaccharide is collected after the freeze-drying.

[0215] 2. Detection of Streptococcus pneumoniae type 14 capsular polysaccharide

[0216] The determination of capsular polysaccharide content is carried out in accordance with 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 in accordance with 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 in accordance with 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 in accordance with the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0704); Phosphorus content was determined according to the Pharmacopoeia of the People's Republic of China (2020 edition), Part III (General Rule 3103); uronic acid content was determined according to the Pharmacopoeia of the People's Republic of China (2020 edition), Part III (General Rule 0731); methyl pentose content was determined according to the Pharmacopoeia of the People's Republic of China (2020 edition), Part III (General Rule 0401); and capsular polysaccharide molecular size was determined according to 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 13.

[0217] Table 13 Recovery rate and quality control indexes of 14-type Streptococcus pneumoniae capsular polysaccharide

[0218] Comparative Example 1 Preparation of 6A Streptococcus pneumoniae capsular polysaccharide

[0219] 1. Preparation of 6A Streptococcus pneumoniae capsular polysaccharide

[0220] Steps (1) and (2) are the same as those in Example 1, and steps (3) to (6) are as follows:

[0221] (3) NaCl was added to the ultrafiltration concentrate obtained in step (2) to a concentration of 0.3 mol / L. After complete dissolution, 0.5% (w / v) sodium deoxycholate was added, and glacial acetic acid was added dropwise while stirring to adjust the pH to 5.0. After thorough mixing, the mixture was allowed to stand at 2-8°C for 12 hours, and then centrifuged at 12000g for 30 minutes. The supernatant was collected and the precipitate was discarded. If there were small debris in the supernatant, it was clarified and filtered using a 0.8 μm filter membrane;

[0222] (4) The supernatant obtained in step (3) was adjusted to a neutral pH with sodium hydroxide or potassium hydroxide, and then Na2HPO4 with a final concentration of 0.01 mol / L and NaH2PO4 with a final concentration of 0.01 mol / L were added to the obtained feed solution as a buffer system. After complete dissolution, sodium acetate was added to a final concentration of 0.6 mol / L and CaCl2 was added to a final concentration of 0.25 mol / L, and stirred until dissolved. Glacial acetic acid was added dropwise to the feed solution to adjust the pH to 5.3-5.5. After sufficient stirring, the mixture was allowed to stand at 2-8°C for 12 hours. After standing, the mixture was centrifuged at 12000g for 30 minutes, the supernatant was collected, and the precipitate was discarded. If there were small debris in the supernatant, it could be clarified and filtered with a 0.8 μm filter membrane;

[0223] (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;

[0224] (6) The polysaccharide solution is further freeze-dried to obtain Streptococcus pneumoniae capsular polysaccharide.

[0225] 2. Detection of 6A Streptococcus pneumoniae capsular polysaccharide

[0226] Capsular polysaccharide content was determined according to the rate turbidimetric method (3.3.2) in the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III. Protein content was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0731). Nucleic acid content was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401). Total nitrogen content was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0704). Phosphorus content was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 3103). Methyl pentose content was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401). The molecular size of capsular polysaccharide was determined 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. The relevant test results are shown in Table 14.

[0227] Table 14 Recovery and quality control indicators of 6A Streptococcus pneumoniae capsular polysaccharide

[0228] The results showed that compared with the quality control indicators of the refined polysaccharide of type 6 Streptococcus pneumoniae prepared by the method of Comparative Example 1, the protein content of the refined polysaccharide prepared by the method of Example 1 was significantly reduced, and the other quality control indicators of the refined polysaccharide of type 6 Streptococcus pneumoniae prepared in Example 1 were not inferior to those of Comparative Example 1, and the process time was shortened.

[0229] Comparative Example 2 Preparation of Degraded Polysaccharide of Type 6A Streptococcus Pneumoniae Capsular Polysaccharide

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

[0231] (1) 3.0 ± 0.15 g of the refined polysaccharide obtained in Comparative Example 1 was dissolved in 1.0 L of 0.15 mol / L sodium chloride solution and stirred until completely dissolved;

[0232] (2) homogenizing the refined polysaccharide solution in step (1) using a high-pressure homogenizer at a pressure of 600±50 bar for 2 cycles to harvest the degraded polysaccharide solution;

[0233] (3) The degraded polysaccharide solution is freeze-dried, and the degraded polysaccharide is collected after the freeze-drying.

[0234] 2. Assay of degraded polysaccharide of 6A Streptococcus pneumoniae capsular polysaccharide

[0235] The assay of degradable polysaccharides of 6A type pneumoniae capsular polysaccharide was carried out in accordance with the 2020 edition of the Pharmacopoeia of the People's Republic of China. The assay of capsular polysaccharide content was carried out in accordance with 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 was carried out in accordance with 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 was carried out in accordance with Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401). Total nitrogen content was determined in accordance with the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 0704); phosphorus content was determined in accordance with the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 3103); methyl pentose content was determined in accordance with the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 0401); and capsular polysaccharide molecular size was determined in accordance with Method 1, Section 3.1.2.10, Part III, 2020 edition. Relevant test results are shown in Table 15.

[0236] Table 15 Recovery rate and quality control index of degraded polysaccharide of 6A Streptococcus pneumoniae capsular polysaccharide

[0237] The results showed that the quality control indicators of the degraded polysaccharide of 6A type pneumonia capsular polysaccharide prepared by the method of Example 2 of the present invention were significantly reduced in terms of protein content compared with the degraded polysaccharide prepared in Comparative Example 2, and other quality control indicators were not inferior to those of Comparative Example 2. In addition, the process flow was simplified and the process time was shortened.

[0238] Comparative Example 3 Preparation of Streptococcus pneumoniae type 5 capsular polysaccharide

[0239] Preparation of 1.5 Streptococcus pneumoniae capsular polysaccharide

[0240] Steps (1) and (2) are the same as those in Example 3, and steps (3) to (6) are as follows:

[0241] (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 thorough mixing, 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 the supernatant with a 0.8 μm filter membrane if there are small debris.

[0242] (4) The supernatant obtained in step (3) is adjusted to a neutral pH with sodium hydroxide or potassium hydroxide, and then Na2HPO4 with a final concentration of 0.01 mol / L and NaH2PO4 with a final concentration of 0.01 mol / L are added to the obtained feed solution as a buffer system. After complete dissolution, sodium acetate is added to a final concentration of 0.6 mol / L and CaCl2 is added to a final concentration of 0.25 mol / L, and stirred until dissolved. Glacial acetic acid is added dropwise to the feed solution to adjust the pH to 5.3-5.5, and after sufficient stirring, the mixture is allowed to stand at 2-8°C for 12 hours. After standing, the mixture is centrifuged at 12000g for 30 minutes, the supernatant is collected, and the precipitate is discarded. If there are small debris in the supernatant, it can be clarified and filtered with a 0.8 μm filter membrane;

[0243] (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;

[0244] (6) The polysaccharide solution is further freeze-dried to obtain Streptococcus pneumoniae capsular polysaccharide.

[0245] Detection of Capsular Polysaccharide of Streptococcus pneumoniae Type 2 and Type 5

[0246] The content of capsular polysaccharide was determined by 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 was determined by the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0731); the content of nucleic acid was determined by the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401); the content of total nitrogen was determined by the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0704); phosphorus The content of uronic acid was determined according to 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 according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0731); the content of hexosamine was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401); and the molecular size of capsular polysaccharides was determined 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. The relevant test results are shown in Table 16.

[0247] Table 16 Recovery and quality control indicators of type 5 Streptococcus pneumoniae capsular polysaccharide

[0248] The results showed that the quality control indicators of the refined polysaccharide of Streptococcus pneumoniae type 5 prepared in Example 3 of the present invention were not inferior to those of the refined polysaccharide prepared in Comparative Example 3, and the process time was shortened.

[0249] Comparative Example 4 Preparation of degraded polysaccharide of Streptococcus pneumoniae type 5 capsular polysaccharide

[0250] Preparation of degraded polysaccharide of Streptococcus pneumoniae type 1 and 5 capsular polysaccharide

[0251] (1) 3.0 ± 0.15 g of the refined polysaccharide obtained in Comparative Example 3 was dissolved in 1.0 L of 0.15 mol / L sodium chloride solution and stirred until completely dissolved;

[0252] (2) homogenizing the refined polysaccharide solution in step (1) using a high-pressure homogenizer at a pressure of 1100 ± 50 bar for 3 cycles to harvest the degraded polysaccharide solution;

[0253] (3) The degraded polysaccharide solution is freeze-dried, and the degraded polysaccharide is collected after the freeze-drying.

[0254] Detection of degraded polysaccharides of capsular polysaccharides of Streptococcus pneumoniae types 2 and 5

[0255] The assay of degradable polysaccharides of type 5 pneumococcal capsular polysaccharide was carried out with reference to the 2020 edition of the Pharmacopoeia of the People's Republic of China. The assay of capsular polysaccharide content was 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 was 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 was 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 was carried out according to Part 2 of the Pharmacopoeia of the People's Republic of China. The content of the test samples was determined in accordance with the Pharmacopoeia of the People's Republic of China, 2020 Edition, Part III (General Rule 0704); phosphorus content was determined in accordance with the Pharmacopoeia of the People's Republic of China, 2020 Edition, Part III (General Rule 3103); uronic acid content was determined in accordance with the Pharmacopoeia of the People's Republic of China, 2020 Edition, Part III (General Rule 0731); hexosamine content was determined in accordance with the Pharmacopoeia of the People's Republic of China, 2020 Edition, Part III (General Rule 0401); and the molecular size of capsular polysaccharides 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 17.

[0256] Table 17 Recovery rate and quality control index of degraded polysaccharide of Streptococcus pneumoniae type 5 capsular polysaccharide

[0257] The results showed that the quality control indicators of the degraded polysaccharide of type 5 Streptococcus pneumoniae capsular polysaccharide prepared in Example 4 of the present invention were lower in protein content and higher in hexosamine content than those of the degraded polysaccharide prepared in Comparative Example 4, and other quality control indicators were not inferior to those of Comparative Example 4. In addition, the process flow was simplified and the process time was shortened.

[0258] Comparative Example 5 Preparation of 10A Streptococcus pneumoniae capsular polysaccharide

[0259] 1. Preparation of 10A Streptococcus pneumoniae capsular polysaccharide

[0260] Steps (1) and (2) are the same as those in Example 5, and steps (3) to (6) are as follows:

[0261] (3) NaCl was added to the ultrafiltration concentrate obtained in step (2) to a concentration of 0.3 mol / L. After complete dissolution, 0.5% (w / v) sodium deoxycholate was added, and glacial acetic acid was added dropwise while stirring to adjust the pH to 5.0. After thorough mixing, the mixture was allowed to stand at 2-8°C for 12 hours, and then centrifuged at 12000g for 30 minutes. The supernatant was collected and the precipitate was discarded. If there were any small debris in the supernatant, it was clarified and filtered using a 0.8 μm filter membrane.

[0262] (4) The supernatant obtained in step (3) was adjusted to a neutral pH with sodium hydroxide or potassium hydroxide, and then Na2HPO4 with a final concentration of 0.01 mol / L and NaH2PO4 with a final concentration of 0.01 mol / L were added to the obtained feed solution as a buffer system. After complete dissolution, sodium acetate was added to a final concentration of 0.6 mol / L and CaCl2 was added to a final concentration of 0.25 mol / L, and stirred until dissolved. Glacial acetic acid was added dropwise to the feed solution to adjust the pH to 5.3-5.5, and after sufficient stirring, the solution was allowed to stand at 2-8°C for 12 hours. After the standing was completed, the solution was centrifuged at 12000g for 30 min, the supernatant was collected, and the precipitate was discarded. If there were small debris in the supernatant, it could be clarified and filtered with a 0.8 μm filter membrane;

[0263] (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;

[0264] (6) The polysaccharide solution is further freeze-dried to obtain Streptococcus pneumoniae capsular polysaccharide.

[0265] 2. Detection of 10A Streptococcus pneumoniae capsular polysaccharide

[0266] Capsular polysaccharide content was determined according to the rate turbidimetry method (3.3.2) in the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III. Protein content was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0731). Nucleic acid content was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401). Total nitrogen content was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0704). Phosphorus content was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 3103). Hexosamine content was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401). The molecular size of capsular polysaccharide was determined 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. The relevant test results are shown in Table 18.

[0267] Table 18 Recovery and quality control indicators of 10A monovalent Streptococcus pneumoniae capsular polysaccharide

[0268] The results showed that the quality control indicators of the refined polysaccharide of type 10A Streptococcus pneumoniae prepared in Example 5 of the present invention were lower in protein content and total phosphorus content than those in Comparative Example 5, and the hexosamine content was increased compared with Comparative Example 5, and other quality control indicators were not inferior to those of the Comparative Example, and the process time was shortened.

[0269] Comparative Example 6 Preparation of Degraded Polysaccharide from Type 10A Streptococcus Pneumoniae Capsular Polysaccharide

[0270] 1. Preparation of degraded polysaccharide of 10A Streptococcus pneumoniae capsular polysaccharide

[0271] (1) 3.0 ± 0.15 g of the refined polysaccharide obtained in Comparative Example 5 was dissolved in 1.0 L of 0.15 mol / L sodium chloride solution and stirred until completely dissolved;

[0272] (2) homogenizing the refined polysaccharide solution in step (1) using a high-pressure homogenizer at a pressure of 600±50 bar for 3 cycles to harvest the degraded polysaccharide solution;

[0273] (3) The degraded polysaccharide solution is freeze-dried, and the degraded polysaccharide is collected after the freeze-drying.

[0274] 2. Assay of degraded polysaccharide of 10A Streptococcus pneumoniae capsular polysaccharide

[0275] The assay of degraded polysaccharides of capsular polysaccharides of type 10A Streptococcus pneumoniae was carried out with reference to the 2020 edition of the Pharmacopoeia of the People's Republic of China. The assay of capsular polysaccharide content was 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 was 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 was carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401); Total nitrogen content was determined in accordance with the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 0704); phosphorus content was determined in accordance with the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 3103); hexosamine content was determined in accordance with the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 0401); and capsular polysaccharide molecular size was determined in accordance with Method 1, Section 3.1.2.10, Part III, 2020 edition. The relevant test results are shown in Table 19.

[0276] Table 19 Recovery rate and quality control index of degraded polysaccharide of 10A Streptococcus pneumoniae capsular polysaccharide

[0277] The results showed that the quality control indicators of the degraded polysaccharide of 10A pneumoniae capsular polysaccharide prepared in Example 6 of the present invention were lower than those of the degraded polysaccharide prepared in Comparative Example 6 in terms of protein content, and other quality control indicators were not inferior to those of Comparative Example 6. In addition, the process flow was simplified and the process time was shortened.

[0278] Comparative Example 7 Preparation of Type 11A Streptococcus pneumoniae Capsular Polysaccharide

[0279] 1. Preparation of 11A Streptococcus pneumoniae capsular polysaccharide

[0280] Steps (1) and (2) are the same as those in Example 7, and steps (3) to (6) are as follows:

[0281] (3) NaCl was added to the ultrafiltration concentrate obtained in step (2) to a concentration of 0.3 mol / L. After complete dissolution, 0.5% (w / v) sodium deoxycholate was added, and glacial acetic acid was added dropwise while stirring to adjust the pH to 5.0. After thorough mixing, the mixture was allowed to stand at 2-8°C for 12 hours, and then centrifuged at 12000g for 30 minutes. The supernatant was collected and the precipitate was discarded. If there were any small debris in the supernatant, it was clarified and filtered using a 0.8 μm filter membrane.

[0282] (4) The supernatant obtained in step (3) was adjusted to a neutral pH with sodium hydroxide or potassium hydroxide, and then Na2HPO4 with a final concentration of 0.01 mol / L and NaH2PO4 with a final concentration of 0.01 mol / L were added to the obtained feed solution as a buffer system. After complete dissolution, sodium acetate was added to a final concentration of 0.6 mol / L and CaCl2 was added to a final concentration of 0.25 mol / L, and stirred until dissolved. Glacial acetic acid was added dropwise to the feed solution to adjust the pH to 5.3-5.5, and after sufficient stirring, the mixture was allowed to stand at 2-8°C for 12 hours. After standing, the mixture was centrifuged at 12000g for 30 minutes, the supernatant was collected, and the precipitate was discarded. If there were small debris in the supernatant, it could be clarified and filtered with a 0.8 μm filter membrane;

[0283] (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;

[0284] (6) The polysaccharide solution is further freeze-dried to obtain Streptococcus pneumoniae capsular polysaccharide.

[0285] 2. Detection of Capsular Polysaccharide of Streptococcus pneumoniae Type 11A

[0286] Capsular polysaccharide content was determined according to the rate turbidimetric method (3.3.2) in the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III. Protein content was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0731). Nucleic acid content was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401). Total nitrogen content was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0704). Phosphorus content was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 3103). O-acetyl content was determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 3117). The molecular size of capsular polysaccharide was determined 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. The relevant test results are shown in Table 20.

[0287] Table 20 Recovery and quality control indicators of 11A Streptococcus pneumoniae capsular polysaccharide

[0288] The results showed 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 were lower than those of Comparative Example 7 in terms of protein content, nucleic acid content, and total phosphorus content, and the O-acetyl content was increased compared with Comparative Example 7. Other quality control indicators were not inferior to those of the Comparative Example, and the process time was shortened.

[0289] Comparative Example 8 Preparation of Degraded Polysaccharide of Type 11A Streptococcus Pneumoniae Capsular Polysaccharide

[0290] 1. Preparation of degraded polysaccharide of Streptococcus pneumoniae type 11A capsular polysaccharide

[0291] (1) 3.0 ± 0.15 g of the refined polysaccharide obtained in Comparative Example 7 was dissolved in 1.0 L of 0.15 mol / L sodium chloride solution and stirred until completely dissolved;

[0292] (2) homogenizing the refined polysaccharide solution in step (1) using a high-pressure homogenizer at a pressure of 600±50 bar for 3 cycles to harvest the degraded polysaccharide solution;

[0293] (3) The degraded polysaccharide solution is freeze-dried, and the degraded polysaccharide is collected after the freeze-drying.

[0294] 2. Detection of degraded polysaccharides of Streptococcus pneumoniae type 11A capsular polysaccharide

[0295] The assay of degraded polysaccharides of capsular polysaccharides of type 11A Streptococcus pneumoniae was carried out with reference to the 2020 edition of the Pharmacopoeia of the People's Republic of China. The assay of capsular polysaccharide content was 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 was 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 was carried out according to Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China (General Rule 0401); Total nitrogen content was determined in accordance with the Pharmacopoeia of the People's Republic of China (2020 edition), Part III (General Rule 0704); phosphorus content was determined in accordance with the Pharmacopoeia of the People's Republic of China (2020 edition), Part III (General Rule 3103); O-acetyl content was determined in accordance with the Pharmacopoeia of the People's Republic of China (2020 edition), Part III (General Rule 3117); and capsular polysaccharide molecular size was determined in accordance with Method 1, Section 3.1.2.10, Part III, 2020 edition. The relevant test results are shown in Table 21.

[0296] Table 21 Recovery rate and quality control index of degraded polysaccharide of 11A Streptococcus pneumoniae capsular polysaccharide

[0297] The results show that the quality control indicators of the degraded polysaccharide of type 11A Streptococcus pneumoniae capsular polysaccharide prepared by the method of Example 8 of the present invention are lower than those of the degraded polysaccharide prepared 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. In addition, the process flow is simplified and the process time is shortened.

[0298] Comparative Example 9 Preparation of 19F Streptococcus pneumoniae capsular polysaccharide

[0299] 1. Purification of 19F Streptococcus pneumoniae capsular polysaccharide

[0300] Steps (1) and (2) are the same as those in Example 9, and steps (3) to (6) are as follows:

[0301] (3) NaCl was added to the ultrafiltration concentrate obtained in step (2) to a concentration of 0.3 mol / L. After complete dissolution, 0.5% (w / v) sodium deoxycholate was added, and glacial acetic acid was added dropwise while stirring to adjust the pH to 5.0. After thorough mixing, the mixture was allowed to stand at 2-8°C for 12 hours, and then centrifuged at 12000g for 30 minutes. The supernatant was collected and the precipitate was discarded. If there were any small debris in the supernatant, it was clarified and filtered using a 0.8 μm filter membrane.

[0302] (4) The supernatant obtained in step (3) was adjusted to a neutral pH with sodium hydroxide or potassium hydroxide, and then Na2HPO4 with a final concentration of 0.01 mol / L and NaH2PO4 with a final concentration of 0.01 mol / L were added to the obtained feed solution as a buffer system. After complete dissolution, sodium acetate was added to a final concentration of 0.6 mol / L and CaCl2 was added to a final concentration of 0.25 mol / L, and stirred until dissolved. Glacial acetic acid was added dropwise to the feed solution to adjust the pH to 5.3-5.5, and after sufficient stirring, the mixture was allowed to stand at 2-8°C for 12 hours. After standing, the mixture was centrifuged at 12000g for 30 minutes, the supernatant was collected, and the precipitate was discarded. If there were small debris in the supernatant, it could be clarified and filtered with a 0.8 μm filter membrane;

[0303] (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.

[0304] (6) The polysaccharide solution is further freeze-dried to obtain Streptococcus pneumoniae capsular polysaccharide.

[0305] 2. Detection of 19F Streptococcus pneumoniae capsular polysaccharide

[0306] The content of capsular polysaccharide was determined by 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 was determined by the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0731); the content of nucleic acid was determined by the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401); the content of total nitrogen was determined by the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0704); phosphorus The content of the test was determined according to the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 3103); the content of hexosamine was determined according to the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 0401); the content of methylpentose was determined according to the Pharmacopoeia of the People's Republic of China, 2020 edition, Part III (General Rule 0401); and the molecular size of the capsular polysaccharide was determined according to 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 22.

[0307] Table 22 Recovery and quality control indicators of 19F Streptococcus pneumoniae capsular polysaccharide

[0308] The results showed that the quality control indicators of the refined polysaccharide of type 11A Streptococcus pneumoniae prepared in Example 9 of the present invention were lower in protein content than those of the refined polysaccharide prepared in Comparative Example 9, and other quality control indicators were not inferior to those of Comparative Example 9, and the process time was shortened.

[0309] Comparative Example 10 Preparation of 19F Streptococcus pneumoniae Capsular Polysaccharide Degraded Polysaccharide

[0310] 1. Preparation of 19F Streptococcus pneumoniae capsular polysaccharide degradation polysaccharide

[0311] (1) 3.0 ± 0.15 g of the refined polysaccharide obtained in Comparative Example 9 was dissolved in 1.0 L of 0.15 mol / L sodium chloride solution and stirred until completely dissolved;

[0312] (2) homogenizing the refined polysaccharide solution in step (1) using a high-pressure homogenizer at a pressure of 600±50 bar for 2 cycles to harvest the degraded polysaccharide solution;

[0313] (3) The degraded polysaccharide solution is freeze-dried, and the degraded polysaccharide is collected after the freeze-drying.

[0314] 2. Assay of degraded polysaccharide of 19F Streptococcus pneumoniae capsular polysaccharide

[0315] The assay of degradable polysaccharides of 19F type pneumococcal capsular polysaccharide was carried out with reference to the 2020 edition of the Pharmacopoeia of the People's Republic of China, wherein the assay of capsular polysaccharide content was 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 was 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 was 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 was 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 was carried out according to Part III of the Pharmacopoeia of the People's Republic of China (General Rule 0401); the assay of protein content was 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 was carried out according to Part III ... protein content was carried out according to Part III of the 202 The content of the test was determined in accordance with the 2020 edition, Part III of the Pharmacopoeia of the People's Republic of China (General Rule 0704). The content of phosphorus was determined in accordance with the 2020 edition, Part III of the Pharmacopoeia of the People's Republic of China (General Rule 3103). The content of hexosamine was determined in accordance with the 2020 edition, Part III of the Pharmacopoeia of the People's Republic of China (General Rule 0401). The content of methyl pentose was determined 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 the capsular polysaccharide 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.

[0316] Table 23 Recovery rate and quality control index of degraded polysaccharide of 19F Streptococcus pneumoniae capsular polysaccharide

[0317] The results show that the quality control indicators of the degraded polysaccharide of type 11A Streptococcus pneumoniae capsular polysaccharide prepared in Example 10 of the present invention are lower than those of the degraded polysaccharide prepared in Comparative Example 10 in terms of protein content and total phosphorus content, and other quality control indicators are not inferior to those of Comparative Example 10. In addition, the process flow is simplified and the process time is shortened.

[0318] Comparative Example 11 Preparation of Streptococcus pneumoniae type 2 capsular polysaccharide

[0319] Preparation of degraded polysaccharides of Streptococcus pneumoniae type 1 and 2 capsular polysaccharides

[0320] Steps (1) and (2) are the same as those in Example 11, and steps (3) to (6) are as follows:

[0321] (3) NaCl was added to the ultrafiltration concentrate obtained in step (2) to a concentration of 0.3 mol / L. After complete dissolution, 0.5% (w / v) sodium deoxycholate was added, and glacial acetic acid was added dropwise while stirring to adjust the pH to 5.0. After thorough mixing, the mixture was allowed to stand at 2-8°C for 12 hours, and then centrifuged at 12000g for 30 minutes. The supernatant was collected and the precipitate was discarded. If there were any small debris in the supernatant, it was clarified and filtered using a 0.8 μm filter membrane.

[0322] (4) The supernatant obtained in step (3) was adjusted to a neutral pH with sodium hydroxide or potassium hydroxide, and then Na2HPO4 with a final concentration of 0.01 mol / L and NaH2PO4 with a final concentration of 0.01 mol / L were added to the obtained feed solution as a buffer system. After complete dissolution, sodium acetate was added to a final concentration of 0.6 mol / L and CaCl2 was added to a final concentration of 0.25 mol / L, and stirred until dissolved. Glacial acetic acid was added dropwise to the feed solution to adjust the pH to 5.3-5.5, and after sufficient stirring, the mixture was allowed to stand at 2-8°C for 12 hours. After standing, the mixture was centrifuged at 12000g for 30 minutes, the supernatant was collected, and the precipitate was discarded. If there were small debris in the supernatant, it could be clarified and filtered with a 0.8 μm filter membrane;

[0323] (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.

[0324] (6) The polysaccharide solution is further freeze-dried to obtain Streptococcus pneumoniae capsular polysaccharide.

[0325] 2. Detection of Streptococcus pneumoniae type 2 capsular polysaccharide

[0326] The determination of capsular polysaccharide of type 2 Streptococcus pneumoniae 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 0402); 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 0403); 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 0404); 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 0405). The determination of the content of methyl pentose was carried out in accordance with the Pharmacopoeia of the People's Republic of China, 2020 Edition, Part III (General Rule 0704); the determination of phosphorus content was carried out in accordance with the Pharmacopoeia of the People's Republic of China, 2020 Edition, Part III (General Rule 3103); the determination of uronic acid content was carried out in accordance with the Pharmacopoeia of the People's Republic of China, 2020 Edition, Part III (General Rule 0731); the determination of methyl pentose content was carried out in accordance with the Pharmacopoeia of the People's Republic of China, 2020 Edition, Part III (General Rule 0401); and the determination of the molecular size of the capsular polysaccharide was carried out 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 24.

[0327] Table 24 Recovery and quality control indicators of type 2 Streptococcus pneumoniae capsular polysaccharide

[0328] The results showed that the quality control indicators of the refined polysaccharide of Streptococcus pneumoniae type 2 prepared in Example 11 of the present invention were lower in nucleic acid content than those in Comparative Example 11, and higher in methyl pentose content than those in Comparative Example 11, and other quality control indicators were not inferior to those in Comparative Example 11, and the process time was shortened.

[0329] Comparative Example 12 Preparation of degraded polysaccharide of Streptococcus pneumoniae type 2 capsular polysaccharide

[0330] Preparation of degraded polysaccharides of Streptococcus pneumoniae type 1 and 2 capsular polysaccharides

[0331] (1) 3.0 ± 0.15 g of the refined polysaccharide obtained in Comparative Example 11 was dissolved in 1.0 L of 0.15 mol / L sodium chloride solution and stirred until completely dissolved;

[0332] (2) The purified polysaccharide solution in step (1) was homogenized using a high-pressure homogenizer at a pressure of 800 ± 50 bar, and the mixture was cycled three times to obtain 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. Assay of degraded polysaccharide of Streptococcus pneumoniae type 2 capsular polysaccharide

[0335] The assay 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 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 The determination of the content of the test results was carried out in accordance with the 2020 edition, Part III (General Rule 0704); the determination of phosphorus content was carried out in accordance with the 2020 edition, Part III (General Rule 3103); the determination of uronic acid content was carried out in accordance with the 2020 edition, Part III (General Rule 0731); the determination of methyl pentose content was carried out in accordance with the 2020 edition, Part III (General Rule 0401); and the determination of the molecular size of the capsular polysaccharide was carried out 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 25.

[0336] Table 25 Recovery rate and quality control index of degraded polysaccharide of Streptococcus pneumoniae type 2 capsular polysaccharide

[0337] The results show that the quality control indicators of the degraded polysaccharide of type 2 Streptococcus pneumoniae capsular polysaccharide prepared in Example 12 of the present invention are lower than those of the degraded polysaccharide prepared 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.

[0338] 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 various embodiments of the present invention. Industrial Applicability

[0339] The present invention provides a method for preparing pneumococcal capsular polysaccharide or its degraded polysaccharide. The method for preparing pneumococcal capsular polysaccharide or its degraded polysaccharide provided by the present invention comprises: sterilizing the fermentation culture of pneumococcus, separating the supernatant, and sequentially subjecting the supernatant to a first ultrafiltration treatment, a precipitation treatment, a second ultrafiltration treatment; or, sequentially subjecting the supernatant 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 of the refined polysaccharide and degraded polysaccharide such as nucleic acid, protein and other impurities and specific groups are better than the standard requirements, and can be used to prepare pneumococcal capsular polysaccharide vaccine or conjugate vaccine, with good economic value and application prospects.

Claims

1. A method for preparing Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide, characterized in that: The method comprises: sterilizing a fermentation culture of Streptococcus pneumoniae, separating a supernatant, and sequentially subjecting the supernatant to a first ultrafiltration treatment, a precipitation treatment, and a second ultrafiltration treatment; Alternatively, the supernatant is sequentially subjected to a first ultrafiltration treatment, a precipitation treatment, a degradation treatment, and a second ultrafiltration treatment; The precipitation agent used in the precipitation treatment comprises calcium salt, and the precipitation treatment is carried out at a pH of 2.4-3.

6.

2. The method for preparing Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide according to claim 1, characterized in that: The calcium salt is calcium chloride; And / or, the final concentration of the calcium salt in the precipitation system is 80-200 mmol / L.

3. The method for preparing Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide according to claim 2, characterized in that: The precipitation treatment is performed at 2-8° C. for 1-5 hours and then the supernatant is collected to obtain a first supernatant.

4. The method for preparing Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide according to any one of claims 1 to 3, characterized in that: The degradation treatment is carried out by chemical treatment or physical treatment.

5. The method for preparing Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide according to claim 4, characterized in that: The chemical treatment method is to use trifluoroacetic acid for degradation treatment to obtain a degraded 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.

6. The method for preparing Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide according to claim 5, characterized in that: In the degradation treatment using trifluoroacetic acid, the final concentration of trifluoroacetic acid is 0.2-2 mol / L, and / or the degradation treatment is performed at 25-60° C. for 2-10 hours.

7. The method for preparing Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide according to any one of claims 1 to 3, 5 and 6, characterized in that: The method further 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.

8. The method for preparing Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide according to any one of claims 1 to 3, 5 and 6, characterized in that: The first ultrafiltration treatment is performed using an ultrafiltration membrane with a pore size of 100-150KD; And / or, 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.

9. Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide prepared by the method for preparing Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide according to any one of claims 1 to 8.

10. The method for preparing Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide according to any one of claims 1 to 8, or use of the Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide according to claim 9 in preparing a product containing Streptococcus pneumoniae capsular polysaccharide.

11. A Streptococcus pneumoniae capsular polysaccharide vaccine or a Streptococcus pneumoniae capsular polysaccharide conjugate vaccine, characterized in that: The vaccine comprises the Streptococcus pneumoniae capsular polysaccharide or the degraded polysaccharide thereof according to claim 9.

12. A method for preparing a Streptococcus pneumoniae capsular polysaccharide vaccine or a Streptococcus pneumoniae 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 8, and preparing Streptococcus pneumoniae capsular polysaccharide vaccine or Streptococcus pneumoniae capsular polysaccharide conjugate vaccine using the Streptococcus pneumoniae capsular polysaccharide or its degraded polysaccharide as an immunogen.

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