Method for preparing streptococcus pneumoniae capsular polysaccharide

Abstract

The present invention relates to the technical field of biologics, and in particular to a method for preparing a Streptococcus pneumoniae capsular polysaccharide. The present invention provides a method for preparing a Streptococcus pneumoniae capsular polysaccharide. The method comprises: using β-propiolactone to treat a Streptococcus pneumoniae fermentation culture, separating and collecting a supernatant, purifying the supernatant, and collecting a capsular polysaccharide. In the present invention, β-propiolactone is used to treat the Streptococcus pneumoniae fermentation culture, thereby reducing residual impurities such as proteins, and achieving relatively high polysaccharide yield; in addition, the β-propiolactone is susceptible to hydrolysis, and the hydrolysate is non-toxic and harmless and has higher safety. The Streptococcus pneumoniae capsular polysaccharide prepared by the method of the present invention have quality control indexes meeting requirements, and can be used for preparing a Streptococcus pneumoniae capsular polysaccharide vaccine and a polysaccharide conjugate vaccine.

Description

A preparation method of Streptococcus pneumoniae capsular polysaccharide

[0001] Cross-references

[0002] This application claims priority to Chinese Patent Application No. 2024101961993, filed on February 22, 2024, entitled “A Method for Preparing Streptococcus Pneumoniae Capsular 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. Background Art

[0004] Streptococcus pneumoniae, a Gram-positive coccus with a capsule, is a major pathogen causing pneumonia, meningitis, otitis media, and bacteremia in humans. Diseases caused by Streptococcus pneumoniae are associated with high morbidity and mortality, with infants under five years of age being the primary susceptible population. With the increasing prevalence of drug resistance in Streptococcus pneumoniae, vaccination to prevent infection is gaining increasing attention. Capsular polysaccharides are key virulence factors of Streptococcus pneumoniae. Polysaccharide or polysaccharide conjugate vaccines, prepared by extracting, isolating, purifying, and combining Streptococcus pneumoniae after fermentation and culture, can effectively induce an immune response in the human body, thereby preventing infection and morbidity.

[0005] Currently, the vaccines commonly used in clinical practice are 23-valent pneumococcal polysaccharide vaccine, 13-valent pneumococcal polysaccharide conjugate vaccine and 20-valent pneumococcal polysaccharide conjugate vaccine.

[0006] The main components of the 23-valent pneumococcal polysaccharide vaccine are multiple capsular polysaccharide antigens of Streptococcus pneumoniae. Since the polysaccharide is a T-cell-independent antigen, it can stimulate mature B lymphocytes but not T lymphocytes. Since the immune function of infants and young children under 2 years old is not yet fully developed and their response to T-cell-independent antigens is poor, the polysaccharide vaccine can only be used for immunization of people over 2 years old.

[0007] The 13-valent and 20-valent pneumococcal polysaccharide conjugate vaccines are also vaccines with capsular polysaccharides as protective antigens. The conjugate vaccines use conjugation technology to combine bacterial capsular polysaccharides with specific carrier proteins. After combination, they become T cell-dependent antigens. After vaccination, they can form immune memory and have long-lasting immunity. They are suitable for people of all ages, especially for infants under 2 years old. They have good protective effects and can be used for preventive immunization of children or the elderly.

[0008] At present, in the production process of Streptococcus pneumoniae capsular polysaccharide, it is necessary to add sodium deoxycholate (DOC) solution to the fermentation broth to lyse the bacteria and release the capsular polysaccharide in the bacteria (CN107835821A, and, Ren Keming, Bai Guijie, Zhang Lizhi et al. Establishment of a new method for purifying pneumococcal capsular polysaccharide [J]. Chinese Journal of Biological Products, 2020, 33(10): 1181-1185. DOI: 10.13200 / j.cnki.cjb.003178). Sodium deoxycholate is a bile salt that can quickly activate autolytic enzymes and accelerate the self-lysis of bacteria such as Streptococcus pneumoniae. Adding a sodium deoxycholate solution with a final concentration of 0.1% at the end of the fermentation of Streptococcus pneumoniae can lyse the bacterial cells, thereby releasing the capsular polysaccharide on the surface of the cell wall of Streptococcus pneumoniae. However, in this process, not only are the capsular polysaccharides connected to the cell wall released in large quantities, but the intracellular products of the bacteria are also released into the polysaccharide solution, which greatly increases the difficulty of subsequent purification and increases the content of residual impurities (proteins, nucleic acids, etc.). Moreover, sodium deoxycholate is an animal-derived substance and may contain exogenous factors (such as bovine-derived viruses) or other allergens, which can easily lead to stronger side effects or hypersensitivity reactions. In addition, a certain amount of sodium deoxycholate has a damaging effect on gastric mucosal cells, nasal mucosal cells, and pancreatic acinar cells. Therefore, if sodium deoxycholate residues are present in the vaccine product, when it is injected into the human body, there is also a risk of causing damage to human cells. The current solution is to ensure the safety of vaccine use by establishing quality control methods (such as CN102830185B) and standards. However, there is still a need to develop better solutions that can reduce or even avoid the use of sodium deoxycholate.

[0009] In addition, as the price of pneumococcal vaccine increases, residual protein and nucleic acid impurities in the purified polysaccharide in the preparation will also accumulate, posing new challenges to the safety and quality control of pneumococcal vaccine. Therefore, in order to further reduce the total amount of residual protein and residual nucleic acid in pneumococcal polysaccharide vaccine and pneumococcal polysaccharide conjugate vaccine, it is necessary to reduce the residual protein and nucleic acid content of individual purified polysaccharide types.

[0010] Summary of the Invention

[0011] The present invention provides a method for preparing Streptococcus pneumoniae capsular polysaccharide.

[0012] In the existing pneumococcal capsular polysaccharide production method, sodium deoxycholate plays a very important role in the extraction of pneumococcal capsular polysaccharide as a cleavage agent for lysing thalline to release capsular polysaccharide, and no alternative reagent is currently available for pneumococcal capsular polysaccharide preparation. The present invention unexpectedly found in the research and development process that β-propiolactone (BPL) has the effect of significantly promoting pneumococcal capsular polysaccharide release, can be used for treating pneumococcal capsular polysaccharide, extract capsular polysaccharide, and unlike sodium deoxycholate, β-propiolactone does not lyse thalline, and then significantly reduces the release of impurities such as protein and nucleic acid in thalline, is conducive to reducing the impurity content and removal difficulty in the pneumococcal polysaccharide purification process, and β-propiolactone is easily hydrolyzed, and the hydrolyzate is nontoxic and harmless, therefore does not cause residual or health risks in the pneumococcal polysaccharide product. β-propiolactone is currently used as a virus inactivator for the preparation of viral vaccines, and about its function of promoting pneumococcal capsular polysaccharide release and reducing the release of impurities such as intracellular protein and nucleic acid, there is no relevant report at present.

[0013] Specifically, the present invention provides the following technical solutions:

[0014] The present invention provides a method for preparing capsular polysaccharide of Streptococcus pneumoniae. The method comprises: treating a fermentation culture of Streptococcus pneumoniae with beta-propiolactone, separating and collecting a supernatant, purifying the supernatant, and collecting capsular polysaccharide.

[0015] In the above method, β-propiolactone is used instead of sodium deoxycholate to kill Streptococcus pneumoniae and release polysaccharides, which not only solves the health risks or side effects caused by sodium deoxycholate residues, but also significantly reduces the content of impurities such as protein in the extracted capsular polysaccharide. The content of impurities such as protein in the purified polysaccharide is significantly reduced (especially for types 5, 10A, 14, 7F, and 15B of Streptococcus pneumoniae capsular polysaccharides with high protein content, the protein residue in the capsular polysaccharide obtained by treating the bacteria with β-propiolactone is significantly reduced, and the impurity removal effect is excellent). The quality is significantly better than the purified polysaccharide obtained by lysing the bacteria with sodium deoxycholate and then purifying it, and the method has a higher polysaccharide recovery rate, which is significantly higher than that obtained by treating the fermentation culture of Streptococcus pneumoniae with formaldehyde.

[0016] In the above method, the fermentation culture is a culture (eg, fermentation broth) containing Streptococcus pneumoniae obtained by fermentation culture of Streptococcus pneumoniae.

[0017] The final concentration of β-propiolactone in the treatment system may be the amount of β-propiolactone commonly used in the art.

[0018] Preferably, the final concentration of the β-propiolactone in the treatment system is not less than 0.01%.

[0019] Exemplary final concentrations of β-propiolactone can 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.

[0020] In some embodiments of the present invention, the final concentration of the β-propiolactone in the treatment system is not less than 0.05%.

[0021] In some embodiments of the present invention, the final concentration of β-propiolactone in the treatment system is 0.05-1%, preferably 0.05-0.5%.

[0022] Preferably, the treatment temperature is 2-8°C.

[0023] Preferably, the treatment time is 8-16 hours.

[0024] Preferably, the treatment is mixing evenly and then incubating at low temperature.

[0025] In the above-described preparation method, sodium deoxycholate is preferably not used.

[0026] 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.

[0027] In the present invention, the separation of the supernatant can be carried out by any solid-liquid separation method, for example, the supernatant can be separated by centrifugation.

[0028] Those skilled in the art will appreciate that the preparation of pneumococcal capsular polysaccharide is divided into a stage of extracting pneumococcal capsular polysaccharide from the thalline (i.e., separating pneumococcal capsular polysaccharide from the thalline, the first stage) and a stage of purifying pneumococcal capsular polysaccharide. β-propiolactone, as a reagent that promotes the release of pneumococcal capsular polysaccharide from the thalline, plays a role in the first stage, and the present invention has been shown through experiments that the use of β-propiolactone to treat pneumococcal fermentation culture can not only effectively release pneumococcal capsular polysaccharide, but also the content of protein and nucleic acid in the pneumococcal capsular polysaccharide extract (the supernatant collected after treatment) is significantly reduced compared to the use of sodium deoxycholate. Therefore, the present invention is not particularly limited to the subsequent pneumococcal capsular polysaccharide purification method, as long as it can effectively remove impurities such as proteins and nucleic acids, so that the purity of the pneumococcal capsular polysaccharide obtained meets the requirements (e.g., methods for purifying pneumococcal capsular polysaccharide known in the prior art) can be used.

[0029] The purification described above includes protein removal and nucleic acid removal.

[0030] The purification mentioned above comprises one or more methods selected from ultrafiltration, acid precipitation, salt precipitation, organic solvent precipitation, and chromatography.

[0031] Traditional purification processes for Streptococcus pneumoniae capsular polysaccharide typically use ethanol fractionation to obtain crude polysaccharide, followed by phenol extraction, activated carbon adsorption, or chromatography to obtain refined polysaccharide. While these methods can all yield high-quality Streptococcus pneumoniae capsular polysaccharide, they have drawbacks and limitations, such as the multiple steps involved and the time required. Furthermore, phenol, a highly corrosive reagent, poses serious risks to humans and the environment when used in large quantities.

[0032] Preferably, the purification of the present invention does not use toxic or harmful reagents, and the toxic or harmful reagents include one or more of phenol and acetone.

[0033] Preferably, the purification of the present invention does not use exogenous macromolecules, and the exogenous macromolecules include one or more of nucleases and proteases.

[0034] The ultrafiltration is preferably carried out using an ultrafiltration membrane with a pore size of 100-300KD.

[0035] The acid precipitation is preferably carried out at a pH of 2.5-4.5.

[0036] The above-mentioned salt precipitation is preferably carried out using a precipitant comprising a calcium salt.

[0037] The precipitant used in the above-mentioned organic solvent precipitation can be an organic solvent such as a lower alcohol (eg, ethanol, methanol).

[0038] As an example of a purification method, the purification includes: ultrafiltration of the supernatant to obtain a first ultrafiltration concentrate; subjecting the first ultrafiltration concentrate to a first precipitation treatment to separate the supernatant to obtain a first supernatant; subjecting the first supernatant to a second precipitation treatment to separate the supernatant to obtain a second supernatant;

[0039] Wherein, the first precipitation treatment is carried out at a pH of 2.8-4.2;

[0040] The precipitant used in the second precipitation treatment includes lower alcohol.

[0041] In the above purification method, the supernatant of the fermentation culture of Streptococcus pneumoniae treated with β-propiolactone is first subjected to ultrafiltration to remove small molecular residual substances, and then subjected to a first precipitation treatment (acid precipitation) and a second precipitation treatment (alcohol precipitation) to remove impurities such as proteins and nucleic acids. Finally, it can be concentrated by ultrafiltration and dried to obtain refined polysaccharides.

[0042] Preferably, the first precipitation treatment is performed by standing at 2-8° C. for not less than 1 hour. In the present invention, the first precipitation can be referred to as acid precipitation.

[0043] In some embodiments of the present invention, the first ultrafiltration concentrate can be subjected to constant volume replacement, the replacement solution is water for injection, and the volume of the injection water used is 4-6 times the volume of the concentrate.

[0044] In some embodiments of the present invention, an acid solution is added to the first ultrafiltration concentrate to adjust the pH to 2.8-4.2, and the concentrate is thoroughly stirred and allowed to stand at 2-8° C. for not less than 1 hour, and then the first supernatant is collected by centrifugation.

[0045] In other embodiments of the present invention, after the first ultrafiltration concentrate is subjected to constant volume replacement, an acid solution is added to adjust the pH to 2.8-4.2, and after being fully stirred, it is allowed to stand at 2-8°C for not less than 1 hour, and then the first supernatant is collected by centrifugation.

[0046] Preferably, the precipitant used in the second precipitation treatment includes phosphate buffer salt, sodium salt, calcium salt and lower alcohol.

[0047] Wherein, the phosphate buffer includes disodium hydrogen phosphate and / or sodium dihydrogen phosphate.

[0048] The sodium salt includes sodium chloride and / or sodium acetate.

[0049] The calcium salt includes calcium chloride.

[0050] The lower alcohol includes ethanol.

[0051] Preferably, in the second precipitation treatment, the concentration of disodium hydrogen phosphate is 8-12 mM, the concentration of sodium dihydrogen phosphate is 8-12 mM, the concentration of sodium chloride is 0.1-2.5 M, the concentration of sodium acetate is 0.3-1.2 M, the concentration of calcium chloride is 0.1-0.35 M, and the amount of ethanol used is 20%-30% by volume to the feed liquid. In the present invention, this step can be simply referred to as ethanol precipitation.

[0052] In some embodiments, the ethanol can be provided by 50%, 60%, 70%, 80%, 90% ethanol solution or anhydrous ethanol, preferably anhydrous ethanol.

[0053] Preferably, the second precipitation treatment is performed at a pH of 5.3-5.5.

[0054] Preferably, the second precipitation treatment is a standing treatment, and the treatment time is preferably not less than 3 hours.

[0055] Preferably, after adjusting the pH of the first supernatant to 6.8-7.2 with alkaline solution, phosphate buffer salt, sodium salt and calcium salt are added, and then the pH is adjusted to 5.3-5.5 with acid solution, and then lower alcohol is added to perform the second precipitation treatment.

[0056] The pH can be adjusted to acidic by using an acid solution, including but not limited to glacial acetic acid, hydrochloric acid, phosphoric acid, etc.; preferably glacial acetic acid.

[0057] The pH value may be adjusted to alkaline using an alkali solution, including but not limited to sodium hydroxide, potassium hydroxide, etc.; sodium hydroxide is preferred.

[0058] Preferably, the ultrafiltration is performed by ultrafiltration concentration using a membrane package with a pore size of 100KD to obtain the first ultrafiltration concentrate.

[0059] Preferably, the purification further comprises: ultrafiltration of the second supernatant to obtain a second ultrafiltration concentrate.

[0060] Preferably, the second supernatant is ultrafiltered using a membrane with a pore size of 100-300 KD to obtain a second ultrafiltration concentrate.

[0061] The volume of water for injection used in ultrafiltration can be 5 times, 6 times, 7 times or 8 times or more of the volume of the second supernatant, preferably 8 times or more.

[0062] As another example of the purification method, the purification includes: sequentially subjecting the supernatant to a first ultrafiltration treatment, a precipitation treatment, and a second ultrafiltration treatment;

[0063] The precipitant used in the precipitation treatment includes calcium salt, and the precipitation treatment is carried out at a pH of 2.4-3.6.

[0064] The present invention is in the research and development process of pneumococcal capsular polysaccharide preparation method, accidentally discovered a kind of simple, rapid pneumococcal polysaccharide preparation method, the method only needs to utilize calcium salt to carry out one-step precipitation (one-step calcium hydrochloric acid precipitation) under acidic conditions to cooperate with conventional ultrafiltration step, high-quality refined polysaccharide can be prepared, and each index of obtained refined polysaccharide is all higher than Chinese Pharmacopoeia standard, significantly reduces material and time cost, improves purification efficiency. Using calcium salt as precipitation agent and carrying out precipitation treatment (calcium hydrochloric acid precipitation) under the acidic conditions of pH 2.4-3.6 is the key that above-mentioned preparation method is able to realize its effect. Be coordinated with above-mentioned " calcium hydrochloric acid precipitation ", arrange ultrafiltration treatment respectively before and after it, the main effect of first ultrafiltration treatment is to remove the small molecule impurities in the supernatant after fermentation culture sterilization treatment, and then be more conducive to " calcium hydrochloric acid precipitation " and better play impurity removal effect, improve the removal efficiency of impurities, the main effect of second ultrafiltration treatment is to concentrate polysaccharide solution and polysaccharide solution is carried out diafiltration, further removes small molecule impurities and salt ions in polysaccharide solution.

[0065] Preferably, the calcium salt is calcium chloride.

[0066] In the precipitation treatment, the concentration of calcium salt is 80-200 mmol / L.

[0067] The precipitation treatment is to collect the supernatant after standing at 2-8° C. for 1-5 hours to obtain a first supernatant.

[0068] Preferably, the purification further comprises adjusting the pH of the first supernatant obtained by the precipitation treatment to 6.8-7.5, collecting the supernatant to obtain a second supernatant, and subjecting the second supernatant to a second ultrafiltration treatment.

[0069] Wherein, the first ultrafiltration treatment is carried out using an ultrafiltration membrane with a pore size of 100-150KD (preferably 100KD). 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 to 5 times and then ultrafiltration of equal volume. Preferably, the volume of purified water used for ultrafiltration is 4 to 6 times the volume of the concentrate. The second ultrafiltration treatment is carried out using an ultrafiltration membrane with a pore size of 100-150KD. In some embodiments of the present invention, in the second ultrafiltration treatment, the volume of water for injection used for diafiltration is 8-15 times the volume of the supernatant.

[0070] The pH can be adjusted to acidic by using an acid solution, including but not limited to glacial acetic acid, hydrochloric acid, phosphoric acid, etc.; preferably phosphoric acid.

[0071] The pH value may be adjusted to alkaline using an alkali solution, including but not limited to sodium hydroxide, potassium hydroxide, etc.; sodium hydroxide is preferred.

[0072] The present invention has verified through experiments that the above-mentioned different purification methods combined with β-propiolactone treatment of Streptococcus pneumoniae fermentation culture can achieve good purification effects of Streptococcus pneumoniae capsular polysaccharide. The content of impurities such as protein in the obtained Streptococcus pneumoniae capsular polysaccharide is significantly reduced compared with that when treated with DOC, and both meet the requirements. Quality control indicators such as specific group content also meet the requirements.

[0073] The above-mentioned preparation method further comprises: drying the second ultrafiltration concentrate.

[0074] Preferably, the drying process is a freeze-drying process.

[0075] 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.

[0076] In some embodiments of the present invention, the Streptococcus pneumoniae is one or more of types 5, 10A, 14, 7F, and 15B.

[0077] The present invention also provides Streptococcus pneumoniae capsular polysaccharide prepared by the above-mentioned preparation method of Streptococcus pneumoniae capsular polysaccharide.

[0078] Preferably, the Streptococcus pneumoniae capsular polysaccharide does not contain deoxycholate.

[0079] Further preferably, the Streptococcus pneumoniae capsular polysaccharide does not contain sodium deoxycholate.

[0080] The present invention provides a method for preparing the above-mentioned Streptococcus pneumoniae capsular polysaccharide or use of the Streptococcus pneumoniae capsular polysaccharide in preparing a product containing the Streptococcus pneumoniae capsular polysaccharide.

[0081] Preferably, the product containing Streptococcus pneumoniae capsular polysaccharide is a vaccine.

[0082] The vaccine includes a Streptococcus pneumoniae capsular polysaccharide vaccine or a Streptococcus pneumoniae capsular polysaccharide conjugate vaccine.

[0083] The present invention provides a Streptococcus pneumoniae capsular polysaccharide vaccine or a Streptococcus pneumoniae capsular polysaccharide conjugate vaccine, wherein the vaccine comprises the Streptococcus pneumoniae capsular polysaccharide described above.

[0084] The aforementioned Streptococcus pneumoniae capsular polysaccharide vaccine or Streptococcus pneumoniae capsular polysaccharide conjugate vaccine may be a monovalent or multivalent vaccine.

[0085] 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.

[0086] Preferably, the multivalent pneumococcal capsular polysaccharide vaccine or capsular polysaccharide conjugate vaccine comprises capsular polysaccharide of at least one type of pneumococcal types 5, 7F, 10A, 14, and 15B.

[0087] Specifically, the pneumococcal capsular polysaccharide vaccine or pneumococcal capsular polysaccharide conjugate vaccine can be a 23-valent pneumococcal polysaccharide vaccine, a 13-valent pneumococcal polysaccharide conjugate vaccine, a 20-valent pneumococcal polysaccharide conjugate vaccine, or a 24-valent pneumococcal polysaccharide conjugate vaccine.

[0088] The 23-valent pneumococcal polysaccharide vaccine contains capsular polysaccharides of pneumococcal serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.

[0089] The 13-valent pneumococcal polysaccharide conjugate vaccine contains capsular polysaccharides of pneumococcal serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F.

[0090] The 20-valent pneumococcal polysaccharide conjugate vaccine contains capsular polysaccharides of pneumococcal serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F.

[0091] The 24-valent pneumococcal polysaccharide conjugate vaccine contains capsular polysaccharides of pneumococcal serotypes 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.

[0092] The present invention also provides a method for preparing a Streptococcus pneumoniae capsular polysaccharide vaccine or a Streptococcus pneumoniae capsular polysaccharide conjugate vaccine, the method comprising: preparing Streptococcus pneumoniae capsular polysaccharide using the above-described method for preparing Streptococcus pneumoniae capsular polysaccharide, and using the Streptococcus pneumoniae capsular polysaccharide as an immunogen to prepare the Streptococcus pneumoniae capsular polysaccharide vaccine or the Streptococcus pneumoniae capsular polysaccharide conjugate vaccine.

[0093] The beneficial effects of the present invention include at least the following: β-propiolactone is used to treat the fermentation culture of Streptococcus pneumoniae, which can achieve the effect of inactivating the bacteria and effectively releasing capsular polysaccharides without lysing the bacteria, thereby avoiding the large-scale release of impurities such as intracellular proteins and nucleic acids after bacterial lysis, thereby reducing the difficulty of subsequent purification and reducing residual impurities such as proteins, while achieving a higher polysaccharide yield; furthermore, β-propiolactone is easily hydrolyzed, and the hydrolysis product is non-toxic and harmless, eliminating the health risks and side effects caused by its residue in the finished vaccine, thereby improving safety. Using β-propiolactone instead of sodium deoxycholate to treat Streptococcus pneumoniae avoids the introduction of animal-derived substances and the potential health risks of sodium deoxycholate. The quality control indicators such as the content of impurities such as proteins and nucleic acids and the content of specific groups in the resulting Streptococcus pneumoniae capsular polysaccharide meet the requirements, and can be used to prepare Streptococcus pneumoniae capsular polysaccharide vaccines and polysaccharide conjugate vaccines. DETAILED DESCRIPTION

[0094] The present invention provides a method for preparing Streptococcus pneumoniae capsular polysaccharide. The method comprises: treating a fermentation culture of Streptococcus pneumoniae with beta-propiolactone, separating and collecting a supernatant, purifying the supernatant, and collecting capsular polysaccharide.

[0095] Specifically, the preparation method of the Streptococcus pneumoniae capsular polysaccharide comprises the following steps:

[0096] 1. Add β-propiolactone (BPL) to a final concentration of not less than 0.01% in a fermentation broth of Streptococcus pneumoniae cultured to the late logarithmic growth stage and stir to mix thoroughly. Incubate at low temperature for 8-16 hours, then centrifuge the treated fermentation broth to collect the supernatant.

[0097] 2. Purify the supernatant to obtain refined capsular polysaccharide.

[0098] As for the purification method, as an example, in some embodiments of the present invention, the purification includes the following steps:

[0099] (1) The supernatant collected after the β-propiolactone treatment was concentrated by ultrafiltration using a 100 kD membrane to obtain a first ultrafiltration concentrate, and the concentrate was subjected to constant volume replacement, and the replacement solution was injection water, and the volume of the injection water used was 4-6 times the volume of the concentrate;

[0100] (2) adding glacial acetic acid to the first ultrafiltration concentrate obtained in step (1) after constant volume replacement to adjust the pH to 2.8-4.2, stirring thoroughly, and then standing at 2-8°C for not less than 1 hour, and then centrifuging to collect the supernatant;

[0101] (3) The supernatant obtained in step (2) was adjusted to a neutral pH with a NaOH solution, disodium hydrogen phosphate and sodium dihydrogen phosphate were added to the feed solution, and then sodium chloride, sodium acetate, and calcium chloride were added. After thorough stirring, glacial acetic acid was added to adjust the pH to 5.3-5.5, anhydrous ethanol was added, and the mixture was allowed to stand for not less than 3 hours, and the supernatant was collected by centrifugation;

[0102] (4) ultrafiltration of the supernatant obtained in step (3) using a membrane with a pore size of 100-300 kD to obtain a second ultrafiltration concentrate, wherein the volume of water for injection used in the ultrafiltration is at least 8 times the volume of the supernatant;

[0103] (5) freeze-drying the second ultrafiltration concentrate obtained in step (4) to obtain Streptococcus pneumoniae capsular polysaccharide.

[0104] As an example, in some other embodiments of the present invention, the purification comprises the following steps:

[0105] (1) ultrafiltration of the supernatant collected after treatment with β-propiolactone using a membrane with a pore size of 100 kD to obtain a first ultrafiltration concentrate;

[0106] (2) adding a CaCl2 solution with a final concentration of 80-200 mmol / L to the first ultrafiltration concentrate, adjusting the pH to 2.4-3.6 with acid, standing at 2-8°C for more than 1 hour, then centrifuging to collect the supernatant, adjusting the pH to 7.00-7.50 with alkaline solution, and centrifuging again to collect the supernatant;

[0107] (3) ultrafiltration of the supernatant obtained in step (2) using a membrane with a pore size of 100 kD to obtain a polysaccharide ultrafiltration concentrate;

[0108] (4) freeze-drying the polysaccharide ultrafiltration concentrate obtained in step (3) to obtain Streptococcus pneumoniae capsular polysaccharide.

[0109] 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.

[0110] The calculation method of the capsular polysaccharide yield in the following examples and comparative examples is as follows:

[0111] The complete cell lysis by DOC treatment and the complete release of capsular polysaccharide are counted as 100%. The yield of capsular polysaccharide when the fermentation broth is treated with BPL or formaldehyde is calculated based on the total amount of polysaccharide in the fermentation broth treated with DOC:

[0112] Polysaccharide yield during BPL or formaldehyde treatment = total amount of polysaccharides in the fermentation broth treated with BPL or formaldehyde / total amount of polysaccharides in the fermentation broth treated with DOC.

[0113] Example 1 Preparation method of Streptococcus pneumoniae capsular polysaccharide (1)

[0114] This embodiment provides a method for preparing a capsular polysaccharide of Streptococcus pneumoniae. The capsular polysaccharide is prepared using a fermentation culture of Streptococcus pneumoniae type 5 as a raw material. The specific method is as follows:

[0115] BPL was added to the fermentation broth of type 5 pneumococcus cultured to the late logarithmic growth stage to a final concentration of 0.05% (batch 1-001 of Example 1), 0.1% (batch 1-002 of Example 1-003), and 0.2% (batch 1-003 of Example 1), and the mixture was stirred uniformly and incubated at 2-8° C. for 12 h. The treated fermentation broth was centrifuged at 8000 rpm for 30 min, and the supernatant was collected. The supernatant was purified to prepare a refined polysaccharide. The specific purification process is as follows:

[0116] The supernatant is concentrated by ultrafiltration using a membrane package with a pore size of 100KD to obtain a concentrate, and the concentrate is subjected to constant volume replacement. The replacement solution is water for injection, and the volume of the water for injection used is 4-6 times the volume of the concentrate. This step obtains the first ultrafiltration concentrate;

[0117] Add glacial acetic acid to the first ultrafiltration concentrate to adjust the pH to 4.0, stir thoroughly and let stand at 2-8°C for 1 hour, then centrifuge and collect the supernatant, which is the first supernatant;

[0118] The first supernatant was adjusted to pH 7.0 with 5M NaOH solution, and disodium hydrogen phosphate was added to a concentration of 10 mmol / L, sodium dihydrogen phosphate (monohydrate) to a concentration of 10 mmol / L, sodium chloride to a concentration of 0.15 mol / L, anhydrous sodium acetate to a concentration of 0.3 mol / L, and calcium chloride (dihydrate) to a concentration of 0.25 mol / L according to the volume of the feed liquid. After thorough stirring, glacial acetic acid was added to adjust the pH to 5.3-5.5. 20% (v / v) anhydrous ethanol was added according to the volume of the feed liquid. After stirring, the mixture was allowed to stand at 2-8°C for 3 h, and then the supernatant was collected by centrifugation to obtain the second supernatant.

[0119] The second supernatant is replaced with a membrane package with a pore size of 100KD. The replacement solution is water for injection, and the volume of the water for injection is more than 8 times the volume of the supernatant. The feed liquid is ultrafiltered and concentrated to obtain a capsular polysaccharide stock solution.

[0120] The capsular polysaccharide stock solution is freeze-dried, and the refined polysaccharide is collected after the freeze-drying is completed.

[0121] Comparative Example 1

[0122] In this comparative example, capsular polysaccharide was prepared using fermentation culture of type 5 Streptococcus pneumoniae as raw material (the raw material was the same as that in Example 1, and the fermentation broth of type 5 Streptococcus pneumoniae cultured to the late logarithmic growth stage of the fermentation was divided into three parts and used in Example 1, Comparative Example 1, and Comparative Example 2, respectively). The preparation method of the Streptococcus pneumoniae capsular polysaccharide used was different from that in Example 1 only in that BPL was replaced with 0.1% DOC.

[0123] Comparative Example 2

[0124] In this comparative example, capsular polysaccharide was prepared using a fermentation culture of Streptococcus pneumoniae type 5 as a raw material (the raw material was the same as that in Example 1, and the fermentation broth of Streptococcus pneumoniae type 5 cultured to the late logarithmic growth stage of the fermentation was divided into three parts and used in Example 1, Comparative Example 1, and Comparative Example 2, respectively). The preparation method of the Streptococcus pneumoniae capsular polysaccharide used was different from that in Example 1 only in that BPL was replaced with 1% formaldehyde.

[0125] Experimental Example 1

[0126] The polysaccharide yield and impurity content of the obtained refined capsular polysaccharide prepared by the preparation methods of Example 1 and Comparative Examples 1-2 were tested as follows:

[0127] The determination of capsular polysaccharide content is carried out according to the rate turbidimetry (3.3.2) in the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III; the determination of protein content is carried out according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0731); the determination of nucleic acid content is carried out according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0401); the determination of total nitrogen content is carried out according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0704); the determination of phosphorus content is carried out according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part III (General Rule 0706); The determination of the content of uronic acid 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 the content of uronic acid 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 content of hexosamine 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 weight of the 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.

[0128] The relevant test results are shown in Tables 1 and 2.

[0129] Table 1 Quality control indicators of capsular polysaccharides obtained in Example 1 and Comparative Examples 1 and 2

[0130] Note: Comparative Examples 1-001, 002, and 003 were conducted in parallel for three times, and were compared with Examples 1-001, 002, and 003, respectively. The same applies to other comparative examples.

[0131] Table 2 Polysaccharide content and recovery rate of fermentation broth treated in Example 1 and Comparative Examples 1 and 2

[0132] The results showed that the total amount of polysaccharides after BPL treatment (Example 1) reached over 70% of the total amount of polysaccharides after DOC treatment (Comparative Example 1), while the total amount of polysaccharides after formaldehyde treatment (Comparative Example 2) was only about 40% of the total amount of polysaccharides after DOC treatment. Compared with DOC treatment, the impurity protein content in the refined polysaccharides obtained by BPL and formaldehyde treatments decreased by over 30%, significantly improving their quality compared to those obtained by DOC treatment. Furthermore, the protein content of the refined polysaccharides obtained by BPL treatment was not significantly different from that obtained by formaldehyde treatment. Therefore, BPL can be used as a bactericide for Streptococcus pneumoniae to obtain high-quality refined polysaccharides while achieving a relatively high polysaccharide recovery rate.

[0133] Example 2 Preparation method of Streptococcus pneumoniae capsular polysaccharide (2)

[0134] This embodiment provides a method for preparing a capsular polysaccharide of Streptococcus pneumoniae. The capsular polysaccharide is prepared using a fermentation culture of type 10A Streptococcus pneumoniae as a raw material. The specific method is as follows:

[0135] BPL was added to the fermentation broth of type 10A Streptococcus pneumoniae cultured to the late logarithmic growth stage to a final concentration of 0.05% (batch 2-001 of Example 2), 0.1% (batch 2-002 of Example 2-003), and 0.2% (batch 2-003 of Example 2) and stirred uniformly. The mixture was incubated at 2-8° C. for 8 h. The treated fermentation broth was centrifuged at 8000 rpm for 30 min, and the supernatant was collected. The supernatant was purified to prepare a refined polysaccharide. The specific purification process is as follows:

[0136] The supernatant is concentrated by ultrafiltration using a membrane package with a pore size of 100KD to obtain a concentrate, and the concentrate is subjected to constant volume replacement. The replacement solution is water for injection, and the volume of the water for injection used is 4-6 times the volume of the concentrate. This step obtains the first ultrafiltration concentrate;

[0137] Add glacial acetic acid to the first ultrafiltration concentrate to adjust the pH to 4.0, stir thoroughly and let stand at 2-8°C for 1 hour, then centrifuge and collect the supernatant, which is the first supernatant;

[0138] Adjust the pH of the first supernatant to 7.0 with 5M NaOH solution, then add disodium hydrogen phosphate to a concentration of 10 mmol / L, sodium dihydrogen phosphate (monohydrate) to a concentration of 10 mmol / L, sodium chloride to a concentration of 0.9 mol / L, anhydrous sodium acetate to a concentration of 1.2 mol / L, and calcium chloride (dihydrate) to a concentration of 0.25 mol / L, based on the volume of the feed solution. Stir thoroughly, then add glacial acetic acid to adjust the pH to 5.3-5.5. Add 30% (v / v) anhydrous ethanol based on the volume of the feed solution, stir thoroughly, and let stand at 2-8°C for 3 h. Then, centrifuge and collect the supernatant, which is the second supernatant.

[0139] The second supernatant is replaced with a membrane package with a pore size of 100KD. The replacement solution is water for injection, and the volume of the water for injection is more than 8 times the volume of the supernatant. The feed liquid is ultrafiltered and concentrated to obtain a capsular polysaccharide stock solution.

[0140] The capsular polysaccharide stock solution is freeze-dried, and the refined polysaccharide is collected after the freeze-drying is completed.

[0141] Comparative Example 3

[0142] In this comparative example, capsular polysaccharide was prepared using fermentation culture of type 10A Streptococcus pneumoniae as raw material (the raw material is the same as that in Example 2, the fermentation broth of type 5 Streptococcus pneumoniae cultured to the late logarithmic growth stage of the fermentation was divided into three parts and used in Example 2, Comparative Example 3 and Comparative Example 4, respectively). The preparation method of the Streptococcus pneumoniae capsular polysaccharide used was different from that in Example 2 only in that BPL was replaced with 0.1% DOC.

[0143] Comparative Example 4

[0144] In this comparative example, capsular polysaccharide was prepared using fermentation culture of type 10A Streptococcus pneumoniae as raw material (the raw material is the same as that in Example 2, the fermentation broth of type 5 Streptococcus pneumoniae cultured to the late logarithmic growth stage of the fermentation was divided into three parts and used in Example 2, Comparative Example 3, and Comparative Example 4, respectively). The preparation method of the Streptococcus pneumoniae capsular polysaccharide used was different from that in Example 2 only in that BPL was replaced with 1% formaldehyde.

[0145] Experimental Example 2

[0146] The polysaccharide yield and impurity content of the purified capsular polysaccharide prepared by the preparation methods of Example 2 and Comparative Examples 3-4 were tested as follows:

[0147] 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 hexosamine 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 the molecular weight 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.

[0148] The relevant test results are shown in Tables 3 and 4.

[0149] Table 3 Quality control indicators of capsular polysaccharides obtained in Example 2 and Comparative Examples 3 and 4

[0150] Table 4 Polysaccharide content and recovery rate of fermentation broth treated with Example 2 and Comparative Examples 3 and 4

[0151] The results showed that the total amount of polysaccharides after BPL treatment (Example 2) reached approximately 70% of the total amount of polysaccharides after DOC treatment (Comparative Example 3), while the total amount of polysaccharides after formaldehyde treatment (Comparative Example 4) was only about 45% of the total amount of polysaccharides after DOC treatment. Compared with DOC treatment, the impurity protein content of the refined polysaccharides obtained by BPL and formaldehyde treatments decreased by more than 72%, significantly improving their quality compared to those obtained by DOC treatment. At the same time, the impurity protein content of the refined polysaccharides obtained by BPL treatment was not significantly different from that of the refined polysaccharides obtained by formaldehyde treatment. Therefore, BPL can be used as a bactericide for Streptococcus pneumoniae to obtain high-quality refined polysaccharides while achieving a relatively high polysaccharide recovery rate.

[0152] Example 3 Preparation method of Streptococcus pneumoniae capsular polysaccharide (3)

[0153] This embodiment provides a method for preparing a capsular polysaccharide of Streptococcus pneumoniae. The capsular polysaccharide is prepared using a fermentation culture of Streptococcus pneumoniae type 14 as a raw material. The specific method is as follows:

[0154] BPL was added to the fermentation broth of Streptococcus pneumoniae type 14 cultured to the late logarithmic growth stage to a final concentration of 0.05% (batch 3-001 of Example 3), 0.1% (batch 3-002 of Example 3-003), and 0.2% (batch 3-003 of Example 3) and stirred uniformly. The mixture was incubated at 2-8° C. for 10 h. The treated fermentation broth was centrifuged at 8000 rpm for 30 min, and the supernatant was collected. The supernatant was purified to prepare refined polysaccharide. The specific purification process is as follows:

[0155] The supernatant is concentrated by ultrafiltration using a membrane package with a pore size of 100KD to obtain a concentrate, and the concentrate is subjected to constant volume replacement. The replacement solution is water for injection, and the volume of the water for injection used is 4-6 times the volume of the concentrate. This step obtains the first ultrafiltration concentrate;

[0156] Add glacial acetic acid to the first ultrafiltration concentrate to adjust the pH to 4.0, stir thoroughly and let stand at 2-8°C for 1 hour, then centrifuge and collect the supernatant, which is the first supernatant;

[0157] The first supernatant was adjusted to pH 7.0 with 5M NaOH solution, and disodium hydrogen phosphate was added to a concentration of 10 mmol / L, sodium dihydrogen phosphate (monohydrate) to a concentration of 10 mmol / L, sodium chloride to a concentration of 2.5 mol / L, anhydrous sodium acetate to a concentration of 0.9 mol / L, and calcium chloride (dihydrate) to a concentration of 0.35 mol / L according to the volume of the feed liquid. After thorough stirring, glacial acetic acid was added to adjust the pH to 5.3-5.5. 25% (v / v) anhydrous ethanol was added according to the volume of the feed liquid, and the mixture was stirred and allowed to stand at 2-8°C for 3 h. The supernatant was then collected by centrifugation to obtain the second supernatant.

[0158] The second supernatant is replaced with a membrane package with a pore size of 100KD. The replacement solution is water for injection, and the volume of the water for injection is more than 8 times the volume of the supernatant. The feed liquid is ultrafiltered and concentrated to obtain a capsular polysaccharide stock solution.

[0159] The capsular polysaccharide stock solution is freeze-dried, and the refined polysaccharide is collected after the freeze-drying is completed.

[0160] Comparative Example 5

[0161] In this comparative example, capsular polysaccharide was prepared using fermentation culture of Streptococcus pneumoniae type 14 as raw material (the raw material was the same as that in Example 3, and the fermentation broth of Streptococcus pneumoniae type 14 cultured to the late logarithmic growth stage of the fermentation was divided into three parts and used in Example 3, Comparative Example 5, and Comparative Example 6, respectively). The preparation method of the Streptococcus pneumoniae capsular polysaccharide used was different from that in Example 3 only in that BPL was replaced with 0.1% DOC.

[0162] Comparative Example 6

[0163] In this comparative example, capsular polysaccharide was prepared using fermentation culture of Streptococcus pneumoniae type 14 as raw material (the raw material was the same as that in Example 3, and the fermentation broth of Streptococcus pneumoniae type 14 cultured to the late logarithmic growth stage of the fermentation was divided into three parts and used in Example 3, Comparative Example 5, and Comparative Example 6, respectively). The preparation method of the Streptococcus pneumoniae capsular polysaccharide used was different from that in Example 3 only in that BPL was replaced with 1% formaldehyde.

[0164] Experimental Example 3

[0165] The polysaccharide yield and impurity content of the obtained refined capsular polysaccharide prepared by the preparation methods of Example 3 and Comparative Examples 5-6 were tested as follows:

[0166] 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 weight 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 Tables 5 and 6.

[0167] Table 5 Quality control indicators of capsular polysaccharides obtained in Example 3 and Comparative Examples 5 and 6

[0168] Table 6 Polysaccharide content and recovery rate of fermentation broth treated with Example 3 and Comparative Examples 5 and 6

[0169] The results showed that the total amount of polysaccharides after BPL treatment (Example 3) reached approximately 70% of the total amount of polysaccharides after DOC treatment (Comparative Example 5), while the total amount of polysaccharides after formaldehyde treatment (Comparative Example 6) was only approximately 47% of the total amount of polysaccharides after DOC treatment. The protein content of the refined polysaccharides obtained by BPL treatment was not significantly different from that obtained by formaldehyde treatment. However, compared with DOC treatment, the protein content of the refined polysaccharides obtained by BPL treatment decreased by more than 57%, and its quality was significantly better than that of the refined polysaccharides obtained by DOC treatment. Therefore, BPL can be used as a bactericide for Streptococcus pneumoniae to obtain high-quality refined polysaccharides and achieve a relatively high polysaccharide recovery rate.

[0170] Example 4 Preparation method of Streptococcus pneumoniae capsular polysaccharide (4)

[0171] This example provides a method for preparing a capsular polysaccharide of Streptococcus pneumoniae. The capsular polysaccharide is prepared using a fermentation culture of 7F Streptococcus pneumoniae as a raw material. The specific method is as follows:

[0172] BPL was added to the fermentation broth of 7F type Streptococcus pneumoniae cultured to the late logarithmic growth stage to a final concentration of 0.05% (batch 4-001 of Example), 0.1% (batch 4-002 of Example), and 0.2% (batch 4-003 of Example), stirred evenly, and incubated at 2-8° C. for 16 h. The treated fermentation broth was centrifuged at 8000 rpm for 30 min, and the supernatant was collected. The supernatant was purified to prepare refined polysaccharide. The specific purification process is as follows:

[0173] The supernatant is concentrated by ultrafiltration using a membrane package with a pore size of 100KD to obtain a concentrate, and the concentrate is subjected to constant volume replacement. The replacement solution is water for injection, and the volume of the water for injection used is 4-6 times the volume of the concentrate. This step obtains the first ultrafiltration concentrate;

[0174] Add glacial acetic acid to the first ultrafiltration concentrate to adjust the pH to 4.0, stir thoroughly and let stand at 2-8°C for 1 hour, then centrifuge and collect the supernatant, which is the first supernatant;

[0175] The first supernatant was adjusted to pH 7.0 with 5M NaOH solution, and disodium hydrogen phosphate was added to a concentration of 10 mmol / L, sodium dihydrogen phosphate (monohydrate) to a concentration of 10 mmol / L, sodium chloride to a concentration of 0.3 mol / L, anhydrous sodium acetate to a concentration of 1.2 mol / L, and calcium chloride (dihydrate) to a concentration of 0.2 mol / L according to the volume of the feed liquid. After thorough stirring, glacial acetic acid was added to adjust the pH to 5.3-5.5. 25% (v / v) anhydrous ethanol was added according to the volume of the feed liquid. After stirring, the mixture was allowed to stand at 2-8°C for 3 h, and then the supernatant was collected by centrifugation to obtain the second supernatant.

[0176] The second supernatant is replaced with a membrane package with a pore size of 100KD. The replacement solution is water for injection, and the volume of the water for injection is more than 8 times the volume of the supernatant. The feed liquid is ultrafiltered and concentrated to obtain a capsular polysaccharide stock solution.

[0177] The capsular polysaccharide stock solution is freeze-dried, and the refined polysaccharide is collected after the freeze-drying is completed.

[0178] Comparative Example 7

[0179] In this comparative example, capsular polysaccharide was prepared using a fermentation culture of 7F type Streptococcus pneumoniae as a raw material (the raw material was the same as that in Example 4, and the fermentation broth of 7F type Streptococcus pneumoniae cultured to the late logarithmic growth stage of the fermentation was divided into three parts and used in Example 4, Comparative Example 7, and Comparative Example 8, respectively). The preparation method of the Streptococcus pneumoniae capsular polysaccharide used was different from that in Example 4 only in that BPL was replaced with 0.1% DOC.

[0180] Comparative Example 8

[0181] In this comparative example, capsular polysaccharide was prepared using a fermentation culture of 7F Streptococcus pneumoniae as a raw material (the raw material was the same as that in Example 4, and the fermentation broth of 7F Streptococcus pneumoniae cultured to the late logarithmic growth stage was divided into three portions and used in Example 4, Comparative Example 7, and Comparative Example 8, respectively). The preparation method of the Streptococcus pneumoniae capsular polysaccharide used was different from that in Example 4 only in that BPL was replaced with 1% formaldehyde.

[0182] Experimental Example 4

[0183] The polysaccharide yield and impurity content of the refined capsular polysaccharide prepared by the preparation methods of Example 4 and Comparative Examples 7-8 were tested as follows:

[0184] 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). Methylpentose 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 weight 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 Tables 7 and 8.

[0185] Table 7 Quality control indicators of capsular polysaccharides obtained in Example 4 and Comparative Examples 7 and 8

[0186] Table 8 Polysaccharide content and recovery rate of fermentation broth treated with Example 4 and Comparative Examples 7 and 8

[0187] The results showed that the total amount of polysaccharides after BPL treatment (Example 4) reached approximately 73% of the total amount of polysaccharides after DOC treatment (Comparative Example 7), while the total amount of polysaccharides after formaldehyde treatment (Comparative Example 8) was only approximately 47% of the total amount of polysaccharides after DOC treatment. The protein content of the refined polysaccharides obtained after BPL treatment was not significantly different from that after formaldehyde treatment. However, compared with DOC treatment, the protein content of the refined polysaccharides obtained after BPL treatment decreased by more than 57%, and its quality was significantly better than that of the refined polysaccharides obtained after DOC treatment. Therefore, BPL can be used as a bactericide for Streptococcus pneumoniae to obtain high-quality refined polysaccharides and achieve a relatively high polysaccharide recovery rate.

[0188] Example 5 Preparation method of Streptococcus pneumoniae capsular polysaccharide (5)

[0189] This embodiment provides a method for preparing a capsular polysaccharide of Streptococcus pneumoniae. The capsular polysaccharide is prepared using a fermentation culture of Streptococcus pneumoniae type 15B as a raw material. The specific method is as follows:

[0190] BPL was added to the fermentation broth of Streptococcus pneumoniae type 15B cultured to the late logarithmic growth stage to a final concentration of 0.05% (batch 5-001 of Example 5), 0.1% (batch 5-002 of Example 5-003), and 0.2% (batch 5-003 of Example 5) and stirred uniformly. The mixture was incubated at 2-8° C. for 14 h. The treated fermentation broth was centrifuged at 8000 rpm for 30 min, and the supernatant was collected. The supernatant was purified to prepare refined polysaccharide. The specific purification process is as follows:

[0191] The supernatant is concentrated by ultrafiltration using a membrane package with a pore size of 100KD to obtain a concentrate, and the concentrate is subjected to constant volume replacement. The replacement solution is water for injection, and the volume of the water for injection used is 4-6 times the volume of the concentrate. This step obtains the first ultrafiltration concentrate;

[0192] Add glacial acetic acid to the first ultrafiltration concentrate to adjust the pH to 4.0, stir thoroughly and let stand at 2-8°C for 1 hour, then centrifuge and collect the supernatant, which is the first supernatant;

[0193] The first supernatant was adjusted to pH 7.0 with 5M NaOH solution, and disodium hydrogen phosphate was added to a concentration of 10 mmol / L, sodium dihydrogen phosphate (monohydrate) to a concentration of 10 mmol / L, sodium chloride to a concentration of 0.15 mol / L, anhydrous sodium acetate to a concentration of 0.9 mol / L, and calcium chloride (dihydrate) to a concentration of 0.15 mol / L according to the volume of the feed liquid. After thorough stirring, glacial acetic acid was added to adjust the pH to 5.3-5.5. 30% (v / v) anhydrous ethanol was added according to the volume of the feed liquid. After stirring, the mixture was allowed to stand at 2-8°C for 3 h, and then the supernatant was collected by centrifugation to obtain the second supernatant.

[0194] The second supernatant is replaced with a membrane package with a pore size of 100KD. The replacement solution is water for injection, and the volume of the water for injection is more than 8 times the volume of the supernatant. The feed liquid is ultrafiltered and concentrated to obtain a capsular polysaccharide stock solution.

[0195] The capsular polysaccharide stock solution is freeze-dried, and the refined polysaccharide is collected after the freeze-drying is completed.

[0196] Comparative Example 9

[0197] In this comparative example, capsular polysaccharide was prepared using fermentation culture of Streptococcus pneumoniae type 15B as raw material (the raw material was the same as that in Example 5, and the fermentation broth of Streptococcus pneumoniae type 15B cultured to the late logarithmic growth stage of the fermentation was divided into three parts and used in Example 5, Comparative Example 9, and Comparative Example 10, respectively). The preparation method of the Streptococcus pneumoniae capsular polysaccharide used was different from that in Example 5 only in that BPL was replaced with 0.1% DOC.

[0198] Comparative Example 10

[0199] In this comparative example, capsular polysaccharide was prepared using fermentation culture of Streptococcus pneumoniae type 15B as raw material (the raw material was the same as that in Example 5, and the fermentation broth of Streptococcus pneumoniae type 15B cultured to the late logarithmic growth stage of the fermentation was divided into three parts and used in Example 5, Comparative Example 9, and Comparative Example 10, respectively). The preparation method of the Streptococcus pneumoniae capsular polysaccharide used was different from that in Example 5 only in that BPL was replaced with 1% formaldehyde.

[0200] Experimental Example 5

[0201] The polysaccharide yield and impurity content of the refined capsular polysaccharide prepared by the preparation methods of Example 5 and Comparative Examples 9-10 were tested as follows:

[0202] 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 weight 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 Tables 9 and 10.

[0203] Table 9 Quality control indicators of capsular polysaccharides obtained in Example 5 and Comparative Examples 9 and 10

[0204] Table 10 Polysaccharide content and recovery rate of fermentation broth treated with Example 5 and Comparative Examples 9 and 10

[0205] The results showed that the total amount of polysaccharides after BPL treatment (Example 5) reached approximately 68% of the total amount of polysaccharides after DOC treatment (Comparative Example 9), while the total amount of polysaccharides after formaldehyde treatment (Comparative Example 10) was only approximately 49% of the total amount of polysaccharides after DOC treatment. The protein content of the refined polysaccharides obtained after BPL treatment was not significantly different from that after formaldehyde treatment. However, compared with DOC treatment, the protein content of the refined polysaccharides obtained after BPL treatment decreased by more than 62%, significantly improving their quality. Therefore, BPL can be used as a bactericide for Streptococcus pneumoniae to obtain high-quality refined polysaccharides while achieving a relatively high polysaccharide recovery rate.

[0206] In summary, the protein removal rate of Streptococcus pneumoniae capsular polysaccharide prepared by treating Streptococcus pneumoniae with β-propiolactone was significantly higher than that of DOC treatment and was basically the same as that of formaldehyde treatment, but the release amount of polysaccharide was significantly higher than that of formaldehyde treatment, and all quality indicators of the prepared capsular polysaccharide met the requirements of the Chinese Pharmacopoeia standards.

[0207] Example 6 Preparation Method of Streptococcus Pneumoniae Capsular Polysaccharide (6)

[0208] This embodiment provides a method for preparing a capsular polysaccharide of Streptococcus pneumoniae. The capsular polysaccharide is prepared using a fermentation culture of Streptococcus pneumoniae type 14 as a raw material. The specific method is as follows:

[0209] β-propiolactone was added to a fermentation broth of Streptococcus pneumoniae type 14 cultured to the late logarithmic growth stage to a final concentration of 0.1%, stirred evenly, and incubated at 4°C for 12 hours, followed by centrifugation at 12,000 g for 30 minutes. The supernatant was collected and purified to prepare a refined polysaccharide. The specific purification process was as follows:

[0210] (1) The obtained supernatant is ultrafiltered using a membrane with a pore size of 100 kD to obtain an ultrafiltration concentrate, the volume of which after concentration is 1 / 5 to 1 / 4 of the volume of the supernatant, and the volume of purified water used for ultrafiltration is more than 5 times the volume of the concentrate;

[0211] (2) 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 (1), 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;

[0212] (3) ultrafiltration of the supernatant obtained in step (2) 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;

[0213] (4) freeze-drying the ultrafiltration concentrate obtained in step (3), and collecting the refined polysaccharide after completion.

[0214] Experimental Example 6

[0215] The type 14 Streptococcus pneumoniae capsular polysaccharide prepared by the preparation method of Example 6 was assayed as follows:

[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 11.

[0217] Table 11 Quality control indicators of Streptococcus pneumoniae type 14 capsular polysaccharide

[0218] 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

[0219] The present invention provides a method for preparing a capsular polysaccharide of Streptococcus pneumoniae. The present invention provides a method for preparing a capsular polysaccharide of Streptococcus pneumoniae, the method comprising: treating a fermentation culture of Streptococcus pneumoniae with β-propiolactone, separating and collecting a supernatant, purifying the supernatant, and collecting the capsular polysaccharide. The present invention uses β-propiolactone to treat a fermentation culture of Streptococcus pneumoniae, thereby reducing residual impurities such as protein, while having a higher polysaccharide yield; moreover, β-propiolactone is easily hydrolyzed, and the hydrolysis product is non-toxic and harmless, thus having higher safety. The quality control indicators of the capsular polysaccharide of Streptococcus pneumoniae obtained by the method of the present invention all meet the requirements, and the polysaccharide can be used for the preparation of a capsular polysaccharide vaccine and a polysaccharide conjugate vaccine of Streptococcus pneumoniae, and has good economic value and application prospects.

Claims

1. A method for preparing Streptococcus pneumoniae capsular polysaccharide, characterized in that: The method comprises: treating the fermentation culture of Streptococcus pneumoniae with beta-propiolactone, separating and collecting the supernatant, purifying the supernatant, and collecting capsular polysaccharide.

2. The method for preparing Streptococcus pneumoniae capsular polysaccharide according to claim 1, characterized in that: The final concentration of the β-propiolactone in the treatment system is not less than 0.01%.

3. The method for preparing Streptococcus pneumoniae capsular polysaccharide according to claim 2, characterized in that: The treatment temperature is 2-8° C., and / or the treatment time is 8-16 hours.

4. The method for preparing Streptococcus pneumoniae capsular polysaccharide according to any one of claims 1 to 3, characterized in that: The purification includes protein removal and nucleic acid removal; And / or, the purification comprises one or more methods selected from ultrafiltration, acid precipitation, salt precipitation, organic solvent precipitation, and chromatography.

5. The method for preparing Streptococcus pneumoniae capsular polysaccharide according to claim 4, characterized in that: The purification comprises: ultrafiltration of the supernatant to obtain a first ultrafiltration concentrate; subjecting the first ultrafiltration concentrate to a first precipitation treatment to separate the supernatant to obtain a first supernatant; subjecting the first supernatant to a second precipitation treatment to separate the supernatant to obtain a second supernatant; Wherein, the first precipitation treatment is carried out at a pH of 2.8-4.2; The precipitant used in the second precipitation treatment includes lower alcohol.

6. The method for preparing Streptococcus pneumoniae capsular polysaccharide according to claim 5, characterized in that: The precipitant used in the second precipitation treatment includes phosphate buffer salt, sodium salt, calcium salt and lower alcohol.

7. The method for preparing Streptococcus pneumoniae capsular polysaccharide according to claim 6, characterized in that: The phosphate buffer includes disodium hydrogen phosphate and / or sodium dihydrogen phosphate; and / or, the sodium salt comprises sodium chloride and / or sodium acetate; and / or, the calcium salt comprises calcium chloride; And / or, the lower alcohol includes ethanol.

8. The method for preparing Streptococcus pneumoniae capsular polysaccharide according to any one of claims 5 to 7, characterized in that: The purification further comprises: ultrafiltration of the second supernatant to obtain a second ultrafiltration concentrate.

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

10. The method for preparing the Streptococcus pneumoniae capsular polysaccharide according to any one of claims 1 to 8 or use of the Streptococcus pneumoniae capsular polysaccharide according to claim 9 in preparing a product containing the 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 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 using the preparation method of Streptococcus pneumoniae capsular 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 as an immunogen.

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