A process for the preparation of pneumococcal capsular polysaccharides

Abstract

The present application belongs to the field of biotechnology, and particularly relates to a preparation method of pneumococcal capsular polysaccharide. The method comprises the following steps: (1) providing a pneumococcal fermentation broth, sterilizing and centrifuging the pneumococcal fermentation broth, collecting a first supernatant and ultrafiltering to obtain a first ultrafiltrate; (2) adding a neutral salt to the first ultrafiltrate, mixing in sodium deoxycholate to obtain a mixed solution, and adjusting the pH to 4.5-5.0; then centrifuging to collect a second supernatant; the ratio of the sodium deoxycholate to the first ultrafiltrate is 0.4-0.7 g:100 mL; (3) adjusting the pH of the second supernatant to neutral, precipitating impurities by calcium salt, centrifuging to collect a third supernatant; and then drying the third supernatant to obtain the pneumococcal capsular polysaccharide. The preparation method has the advantages of simple process, low cost, high recovery rate of pneumococcal capsular polysaccharide, easy scale-up, and suitability for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing pneumococcal capsular polysaccharide. Background Technology

[0002] Streptococcus pneumoniae, commonly known as pneumococcus, is the leading cause of respiratory infections. Besides pneumonia, it can also cause invasive diseases such as meningitis, sepsis, bacteremia, and peritonitis. Streptococcus pneumoniae infection is a major cause of death worldwide. With the widespread use of antibiotics, drug-resistant strains are increasing, prompting renewed focus on vaccine development. Capsular polysaccharides are a major virulence factor of Streptococcus pneumoniae, and research indicates that Streptococcus pneumoniae capsular polysaccharide vaccines possess good immunogenicity and safety. In recent years, numerous reports and studies have been published on purification methods for Streptococcus pneumoniae capsular polysaccharides, and vaccine manufacturers and research institutions are continuously innovating and establishing or optimizing purification processes for capsular polysaccharide vaccines. Therefore, establishing a simple and stable capsular polysaccharide purification process is an inevitable trend and urgent need for optimizing pneumococcal vaccine production processes and improving vaccine quality. Currently, the most commonly used vaccines in clinical practice are the 13-valent pneumococcal polysaccharide-protein conjugate vaccine, the 20-valent pneumococcal polysaccharide-protein conjugate vaccine, and the 23-valent pneumococcal polysaccharide vaccine.

[0003] Based on the differences in their capsular polysaccharides, Streptococcus pneumoniae can be classified into 100 serotypes, of which about 30 serotypes are pathogenic to humans and are one of the leading causes of morbidity and mortality in infants and young children. Different serotypes of Streptococcus pneumoniae capsular polysaccharides have different chemical properties; therefore, in production, processes are often used to purify capsular polysaccharides from different serotypes separately. To date, there is still no universal method to purify the capsular polysaccharides of all 23 serotypes of Streptococcus pneumoniae. In particular, some serotypes have more unique capsular polysaccharides, exhibiting greater viscosity, thus increasing the difficulty of purification and resulting in generally low recovery rates.

[0004] Currently, precipitation and gel chromatography are commonly used methods for preparing pneumococcal capsular polysaccharides, or a combination of both. Gel chromatography requires a relatively mild purification environment, thus causing less damage to the polysaccharides; however, it is time-consuming and labor-intensive, and the amount separated each time is relatively small. Furthermore, choosing a suitable gel is difficult when the relative molecular mass is uncertain. Precipitation methods often use ethanol, acetone, and long-chain quaternary ammonium salts as precipitants. This method avoids the disadvantages of gel chromatography, but the purification environment is not mild enough, there are many influencing factors, and it requires the use of large quantities of toxic and harmful reagents. In China, the traditional phenol extraction method is generally used in the preparation of pneumococcal capsular polysaccharides. This method requires a long centrifugation time, and phenol, as a highly corrosive reagent, poses serious harm to humans and the environment when used in large quantities.

[0005] Although the use of ethanol precipitation and CTAB precipitation methods to purify capsular polysaccharides avoids the large-scale use of toxic and harmful chemical reagents such as acetone or phenol, current processes generally suffer from problems such as complex procedures and low polysaccharide recovery rates.

[0006] In the existing technology, Ren Keming et al. (Ren Keming et al. "Establishment of ethanol-free and phenol-free purification process for capsular polysaccharides of type 5 pneumococcus", Advances in Microbiology and Immunology 1 (2020):6.) recovered the polysaccharides by lysing the fermentation broth of pneumococcus with sodium deoxycholate, adding sodium deoxycholate again, and then recovering the polysaccharides by dialysis and chromatography. However, the cost of chromatographic packing materials and other consumables is high, and it requires two additions of sodium deoxycholate and dialysis operations, making the process complex and unsuitable for industrial applications.

[0007] Developing a method for preparing pneumococcal capsular polysaccharide that is simple to produce, highly safe (without using toxic or harmful reagents such as phenol and acetone), low in cost (without using expensive exogenous macromolecules and consumables such as nucleases, proteases, and chromatography packing materials), has a high recovery rate, and meets pharmacopoeia standards is a pressing technical problem that needs to be solved. Summary of the Invention

[0008] The purpose of this invention is to provide a method for preparing pneumococcal capsular polysaccharide, comprising the following steps:

[0009] (1) Provide pneumococcal fermentation broth, sterilize and centrifuge the pneumococcal fermentation broth, collect the first supernatant and ultrafilter it to obtain the first ultrafiltrate;

[0010] (2) Add the neutral salt to the first ultrafiltrate, then mix in sodium deoxycholate to obtain a mixed solution, and adjust the pH to 4.5-5.0; then centrifuge to collect the second supernatant;

[0011] (3) Adjust the pH of the second supernatant to neutral, precipitate impurities by calcium salt, and collect the third supernatant by centrifugation; then dry the third supernatant to obtain pneumococcal capsular polysaccharide.

[0012] In the specific implementation process, the neutral salt may include, but is not limited to, NaCl, KCl, etc.

[0013] In some implementations, the chloride ion concentration of the mixed solution is controlled to be 0.3–0.7 mol / L.

[0014] This invention reveals that sodium deoxycholate achieves optimal adsorption and precipitation of proteins when the chloride ion concentration in the mixed solution is controlled at 0.3–0.7 mol / L and the pH is 4.5–5.0.

[0015] In some implementations, the ratio of sodium deoxycholate to the first ultrafiltrate is 0.4–0.7 g: 100 mL.

[0016] Under the above-mentioned ratio, the adsorption and precipitation effect of sodium deoxycholate can be further improved, and the excessive amount of sodium deoxycholate used is avoided. This avoids the need for additional steps to remove excess sodium deoxycholate, which helps to shorten the process flow of the preparation method and save production costs.

[0017] In some implementations, in step (3), the calcium salt comprises CaCl2 and sodium acetate;

[0018] Based on the volume of the second supernatant, the final concentration of CaCl2 is 0.05–0.35 mol / L and the final concentration of sodium acetate is 0.4–0.9 mol / L.

[0019] In some embodiments, the calcium salt further includes a buffer substance; the buffer substance contains 0.005–0.015 mol / L Na2HPO4 and 0.005–0.015 mol / L NaH2PO4, based on the volume of the second supernatant.

[0020] The above buffer system can ensure the stability of the solution system, protect the pneumococcal capsular polysaccharide from the influence of calcium salt precipitation impurities, and further promote the improvement of recovery rate.

[0021] In some implementation methods, the preparation method of the pneumococcal capsular polysaccharide does not use any toxic or harmful reagents throughout the process. The toxic or harmful reagents include one or more of phenol, acetone, and ethanol.

[0022] In some implementations, the method for preparing the pneumococcal capsular polysaccharide does not use any exogenous macromolecules throughout the process, and the exogenous macromolecules include one or more of nucleases and proteases.

[0023] In some implementations, the method for preparing the pneumococcal capsular polysaccharide does not use chromatographic packing materials or chromatographic equipment throughout the entire process.

[0024] In some embodiments, in the method for preparing pneumococcal capsular polysaccharide, in step (1), the pneumococcal fermentation broth is sterilized and then allowed to stand at 2-8℃ for 6-12 hours, and the first supernatant is collected by centrifugation; the first supernatant is then ultrafiltered with 5-10 times the volume of water to obtain the first ultrafiltrate.

[0025] In some embodiments, in the preparation method of the pneumococcal capsular polysaccharide, after adjusting the pH to 4.5-5.0 in step (2), the mixture is left to stand at 2-8°C for 3-16 hours, and then centrifuged to collect the second supernatant.

[0026] In some embodiments, the preparation method of the pneumococcal capsular polysaccharide involves step (3) precipitating impurities with calcium salts, adjusting the pH to 5.3-5.5, allowing it to stand at 2-8°C for 12-16 hours, and then centrifuging to collect the third supernatant.

[0027] Allowing the mixture to stand under the above conditions can further promote the precipitation of sodium deoxycholate and the calcium salt.

[0028] In some implementations, in step (3), the pH of the second supernatant is adjusted to neutral using sodium hydroxide or potassium hydroxide.

[0029] In some embodiments, the method for preparing pneumococcal capsular polysaccharide involves collecting the first supernatant and / or the second supernatant and / or the third supernatant, and then performing ultrafiltration using a tangential flow ultrafiltration system. In the ultrafiltration, the pore size of the ultrafiltration membrane is 100 kDa, and a constant volume water replenishment method is used. Ultrafiltration is stopped when the conductivity at the permeate end is lower than 10 μS / cm.

[0030] In the specific implementation process, if there are small debris in the second supernatant and / or the third supernatant, a 0.8μm filter membrane can be used for clarification and filtration before ultrafiltration.

[0031] In the specific implementation process, the pH of the solution may be adjusted by using glacial acetic acid, phosphoric acid, or hydrochloric acid, with glacial acetic acid being preferred.

[0032] As a preferred embodiment of the present invention, the method includes the following steps:

[0033] (1) Provide pneumococcal fermentation broth, sterilize and centrifuge the pneumococcal fermentation broth, collect the first supernatant and ultrafilter it, use 5 to 10 times the volume of the supernatant for ultrafiltration to obtain the first ultrafiltrate;

[0034] (2) Add NaCl to the first ultrafiltrate, and then mix in sodium deoxycholate to obtain a mixed solution, wherein the ratio of sodium deoxycholate to the first ultrafiltrate is 0.4 to 0.7 w / v%, the chloride ion concentration of the mixed solution is controlled to be 0.3 to 0.7 mol / L, and the pH is adjusted to 4.5 to 5.0 with glacial acetic acid; let stand at 2 to 8°C for 3 to 16 hours, and then centrifuge to collect the second supernatant;

[0035] (3) Adjust the pH to neutral using sodium hydroxide solution; add 0.005-0.015 mol / L Na2HPO4, 0.005-0.015 mol / L NaH2PO4, 0.05-0.35 mol / L CaCl2 and 0.4-0.9 mol / L sodium acetate to the second supernatant, adjust the pH to 5.3-5.5 using glacial acetic acid, let stand at 2-8℃ for 12-16 hours, centrifuge and collect the third supernatant; then use a tangential flow ultrafiltration system to perform ultrafiltration by constant volume water replenishment, the pore size of the ultrafiltration membrane is 100 KD, and stop ultrafiltration when the conductivity at the permeate end is lower than 10 μs / cm, and obtain pneumococcal capsular polysaccharide by sterilization and freeze drying.

[0036] Those skilled in the art can further combine the above-mentioned preferred embodiments to obtain other preferred embodiments of the method for preparing pneumococcal capsular polysaccharide in this invention.

[0037] This invention utilizes a suitable neutral salt condition to mix sodium deoxycholate with the first ultrafiltrate, controlling the pH of the solution to 4.5–5.0. This allows sodium deoxycholate to efficiently adsorb and precipitate proteins. Further precipitation with calcium salts removes impurities such as proteins and nucleic acids. The supernatant is collected by centrifugation, washed, filtered, and then freeze-dried to obtain purified polysaccharides, significantly improving the uniformity and purity of the prepared pneumococcal capsular polysaccharide. This simple and low-cost process efficiently prepares pneumococcal capsular polysaccharides from pneumococcal fermentation broth with a high recovery rate. The preparation process avoids the use of toxic and harmful reagents such as phenol, acetone, and ethanol, costly exogenous macromolecules such as nucleases and proteases, and costly consumables such as chromatography packing materials. The obtained pneumococcal capsular polysaccharides meet the standards of the Chinese Pharmacopoeia.

[0038] As a preferred embodiment of the present invention, the raw materials for the pneumococcal fermentation broth can be selected from pneumococcal type 2 fermentation broth, pneumococcal type 18C fermentation broth, pneumococcal type 20 fermentation broth, etc.

[0039] The capsular polysaccharides of these types of pneumococcus have high viscosity. When preparing these capsular polysaccharides using existing technologies, there are drawbacks such as complex procedures, long preparation cycles, use of toxic and harmful reagents such as phenol, acetone, and ethanol, use of expensive exogenous macromolecules such as nucleases and proteases, or use of expensive consumables such as chromatography packing materials. The recovery rate is also low. However, the preparation method of this application has a significant advantage in the preparation of capsular polysaccharides of pneumococcus with high viscosity, and the recovery rate is still high.

[0040] Furthermore, the present invention also provides a method for preparing a multivalent pneumococcal vaccine, comprising: preparing pneumococcal capsular polysaccharide using any of the above embodiments, and then using the pneumococcal capsular polysaccharide to prepare a multivalent pneumococcal vaccine.

[0041] In a preferred embodiment of the present invention, the multivalent pneumococcal vaccine is a pneumococcal polysaccharide vaccine or a pneumococcal polysaccharide conjugate vaccine.

[0042] The pneumococcal vaccine contains any one or a combination of pneumococcal serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F.

[0043] Because the preparation method of the present invention is simple and has a high recovery rate of pneumococcal capsular polysaccharide, its application in the preparation of multivalent pneumococcal vaccines can effectively shorten the vaccine preparation process, save economic costs, and is suitable for large-scale industrial production.

[0044] The beneficial effects of this invention are as follows:

[0045] (1) The preparation method of the present invention has the advantages of simple process, high recovery rate of pneumococcal capsular polysaccharide, easy scale-up, and suitability for industrial application. It has a particularly significant advantage in the preparation of pneumococcal capsular polysaccharide of high viscosity type, with a recovery rate of over 58%.

[0046] (2) The pneumococcal capsular polysaccharide prepared by the method of the present invention has good immunogenicity and the quality control indicators such as the content of impurities such as nucleic acid and protein, as well as the content of O-acetyl, methylpentose, uronic acid, and aminohexose, all meet the standards of the Pharmacopoeia of the People's Republic of China (2020 edition); it can be used for the preparation of pneumococcal capsular polysaccharide vaccines and conjugate vaccines.

[0047] (3) The preparation method of the present invention avoids the use of toxic and harmful reagents such as phenol, acetone, and ethanol, thereby reducing the harm to human health and the environment.

[0048] (4) The preparation method of the present invention avoids the use of high-cost exogenous macromolecular substances such as nucleases and proteases, and avoids the use of high-cost consumables such as chromatography packing materials, thus effectively reducing the preparation cost. Detailed Implementation

[0049] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0050] Unless otherwise specified, all methods used in the examples were conventional or performed according to techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents and instruments used without specified manufacturers were all conventional products that could be purchased from legitimate channels.

[0051] Example 1

[0052] This embodiment provides a method for preparing pneumococcal capsular polysaccharide. Specifically, it includes the following steps:

[0053] (1) After sterilizing the type 1 pneumococcal fermentation broth, let it stand at 2-8℃ for 6-12 hours, then centrifuge at 12000g for 30-60min and collect the supernatant. Use a 100KD membrane to ultrafilter the supernatant to obtain an ultrafiltration concentrate. The solution used for ultrafiltration is purified water, and its volume is 5 times that of the supernatant.

[0054] (2) Add NaCl to the ultrafiltration concentrate until the concentration is 0.3 mol / L. After it is completely dissolved, add 0.5% (w / v) sodium deoxycholate. While stirring, add glacial acetic acid dropwise to adjust the pH to 5.0. After stirring thoroughly, let it stand at 2-8℃ for 3-16 hours. Then centrifuge at 12000g for 30min, collect the supernatant, discard the precipitate, and if there are small fragments in the supernatant, clarify and filter it with a 0.8μm filter membrane.

[0055] (3) Adjust the pH of the supernatant to neutral using sodium hydroxide or potassium hydroxide. Then, add Na2HPO4 with a final concentration of 0.01 mol / L and NaH2PO4 with a final concentration of 0.01 mol / L to the obtained solution as a buffer system. After complete dissolution, add sodium acetate with a final concentration of 0.6 mol / L and CaCl2 with a final concentration of 0.25 mol / L. Stir until dissolved. Add glacial acetic acid dropwise to the solution to adjust the pH to 5.3-5.5. Stir thoroughly and let stand at 2-8℃ for 12-16 hours. After standing, centrifuge at 12000g for 30 minutes, collect the supernatant, and discard the precipitate. If there are small fragments in the supernatant, they can be clarified and filtered through a 0.8 μm filter membrane.

[0056] (4) The supernatant obtained in the previous step was ultrafiltered with purified water. When the conductivity at the permeate end was lower than 10 μs / cm, the polysaccharide solution was collected. After sterilization filtration, the polysaccharide solution was collected. The polysaccharide solution was then freeze-dried to obtain pneumococcal capsular polysaccharide.

[0057] Example 2

[0058] (1) After sterilizing the type 2 pneumococcal fermentation broth, let it stand at 2-8℃ for 6-12 hours, then centrifuge at 12000g for 30-60min and collect the supernatant. Use a 100KD membrane to ultrafilter the supernatant to obtain an ultrafiltration concentrate. The solution used for ultrafiltration is purified water, and its volume is 5 times that of the supernatant.

[0059] (2) Add NaCl to the ultrafiltration concentrate until the concentration is 0.5 mol / L. After it is completely dissolved, add 0.7% (w / v) sodium deoxycholate. While stirring, add glacial acetic acid dropwise to adjust the pH to 5.0. After stirring thoroughly, let it stand at 2-8℃ for 3-16 hours. Then centrifuge at 12000g for 30min, collect the supernatant, discard the precipitate, and if there are small fragments in the supernatant, clarify and filter it with a 0.8μm filter membrane.

[0060] (3) Adjust the pH of the supernatant to neutral using sodium hydroxide or potassium hydroxide. Then, add Na2HPO4 with a final concentration of 0.01 mol / L and NaH2PO4 with a final concentration of 0.01 mol / L to the obtained solution as a buffer system. After complete dissolution, add sodium acetate with a final concentration of 0.6 mol / L and CaCl2 with a final concentration of 0.25 mol / L. Stir until dissolved. Add glacial acetic acid dropwise to the solution to adjust the pH to 5.3-5.5. Stir thoroughly and let stand at 2-8℃ for 12-16 hours. After standing, centrifuge at 12000g for 30 minutes, collect the supernatant, and discard the precipitate. If there are small fragments in the supernatant, they can be clarified and filtered through a 0.8 μm filter membrane.

[0061] (4) The supernatant obtained in the previous step was ultrafiltered with purified water. When the conductivity at the permeate end was lower than 10 μs / cm, the polysaccharide solution was collected. After sterilization filtration, the polysaccharide solution was collected. The polysaccharide solution was then freeze-dried to obtain pneumococcal capsular polysaccharide.

[0062] Example 3

[0063] (1) After sterilization, the fermentation broth of type 7F pneumococcus was allowed to stand at 2-8℃ for 6-12 hours, and then centrifuged at 12000g for 30-60min to collect the supernatant. The supernatant was ultrafiltered with a membrane with a pore size of 100KD to obtain an ultrafiltration concentrate. The solution used for ultrafiltration was purified water, and its volume was 5 times that of the supernatant.

[0064] (2) Add NaCl to the ultrafiltration concentrate to a concentration of 0.3 mol / L. After it is completely dissolved, add 0.7% (w / v) sodium deoxycholate. While stirring, add glacial acetic acid dropwise to adjust the pH to 4.5. After stirring thoroughly, let it stand at 2-8℃ for 3-16 hours. Then centrifuge at 12000g for 30 min, collect the supernatant, discard the precipitate, and if there are small fragments in the supernatant, clarify and filter it with a 0.8 μm filter membrane.

[0065] (3) Adjust the pH of the supernatant to neutral using sodium hydroxide or potassium hydroxide. Then, add Na2HPO4 with a final concentration of 0.01 mol / L and NaH2PO4 with a final concentration of 0.01 mol / L to the obtained solution as a buffer system. After complete dissolution, add sodium acetate with a final concentration of 0.6 mol / L and CaCl2 with a final concentration of 0.35 mol / L. Stir until dissolved. Add glacial acetic acid dropwise to the solution to adjust the pH to 5.3-5.5. Stir thoroughly and let stand at 2-8℃ for 12-16 hours. After standing, centrifuge at 12000g for 30 minutes, collect the supernatant, and discard the precipitate. If there are small fragments in the supernatant, they can be clarified and filtered through a 0.8 μm filter membrane.

[0066] (4) The supernatant obtained in the previous step was ultrafiltered with purified water. When the conductivity at the permeate end was lower than 10 μs / cm, the polysaccharide solution was collected. After sterilization filtration, the polysaccharide solution was collected. The polysaccharide solution was then freeze-dried to obtain pneumococcal capsular polysaccharide.

[0067] Example 4

[0068] (1) After sterilization, the fermentation broth of type 12F pneumococcus was allowed to stand at 2-8℃ for 6-12 hours, and then centrifuged at 12000g for 30-60min to collect the supernatant. The supernatant was ultrafiltered with a membrane with a pore size of 100KD to obtain an ultrafiltration concentrate. The solution used for ultrafiltration was purified water, and its volume was 5 times that of the supernatant.

[0069] (2) Add NaCl to the ultrafiltration concentrate until the concentration is 0.3 mol / L. After it is completely dissolved, add 0.5% (w / v) sodium deoxycholate. While stirring, add glacial acetic acid dropwise to adjust the pH to 5.0. After stirring thoroughly, let it stand at 2-8℃ for 12-16 hours. Then centrifuge at 12000g for 30min, collect the supernatant, discard the precipitate, and if there are small fragments in the supernatant, clarify and filter it with a 0.8μm filter membrane.

[0070] (3) Adjust the pH of the supernatant to neutral using sodium hydroxide or potassium hydroxide. Then, add Na2HPO4 with a final concentration of 0.01 mol / L and NaH2PO4 with a final concentration of 0.01 mol / L to the obtained solution as a buffer system. After complete dissolution, add sodium acetate with a final concentration of 0.4 mol / L and CaCl2 with a final concentration of 0.35 mol / L. Stir until dissolved. Add glacial acetic acid dropwise to the solution to adjust the pH to 5.3-5.5. Stir thoroughly and let stand at 2-8℃ for 12-16 hours. After standing, centrifuge at 12000g for 30 minutes, collect the supernatant, and discard the precipitate. If there are small fragments in the supernatant, they can be clarified and filtered through a 0.8 μm filter membrane.

[0071] (4) The supernatant obtained in the previous step was ultrafiltered with purified water. When the conductivity at the permeate end was lower than 10 μs / cm, the polysaccharide solution was collected. After sterilization filtration, the polysaccharide solution was collected. The polysaccharide solution was then freeze-dried to obtain pneumococcal capsular polysaccharide.

[0072] Example 5

[0073] (1) After sterilization, the fermentation broth of type 18C pneumococcus was allowed to stand at 2-8℃ for 6-12 hours, and then centrifuged at 12000g for 30-60min to collect the supernatant. The supernatant was ultrafiltered with a membrane with a pore size of 100KD to obtain an ultrafiltration concentrate. The solution used for ultrafiltration was purified water, and its volume was 5 times that of the supernatant.

[0074] (2) Add NaCl to the ultrafiltration concentrate to a final concentration of 0.3 mol / L. After it is completely dissolved, add 0.7% (w / v) sodium deoxycholate. While stirring, add glacial acetic acid dropwise to adjust the pH to 4.5. After stirring thoroughly, let it stand at 2-8℃ for 12-16 hours. Then centrifuge at 12000g for 30 min, collect the supernatant, discard the precipitate, and if there are small fragments in the supernatant, clarify and filter it with a 0.8 μm filter membrane.

[0075] (3) Adjust the pH of the supernatant to neutral using sodium hydroxide or potassium hydroxide. Then, add Na2HPO4 with a final concentration of 0.01 mol / L and NaH2PO4 with a final concentration of 0.01 mol / L to the obtained solution as a buffer system. After complete dissolution, add sodium acetate with a final concentration of 0.4 mol / L and CaCl2 with a final concentration of 0.35 mol / L. Stir until dissolved. Add glacial acetic acid dropwise to the solution to adjust the pH to 5.3-5.5. Stir thoroughly and let stand at 2-8℃ for 12-16 hours. After standing, centrifuge at 12000g for 30 minutes, collect the supernatant, and discard the precipitate. If there are small fragments in the supernatant, they can be clarified and filtered through a 0.8 μm filter membrane.

[0076] (4) The supernatant obtained in the previous step was ultrafiltered with purified water. When the conductivity at the permeate end was lower than 10 μs / cm, the polysaccharide solution was collected. After sterilization filtration, the polysaccharide solution was collected. The polysaccharide solution was then freeze-dried to obtain pneumococcal capsular polysaccharide.

[0077] Example 6

[0078] (1) After sterilizing the fermentation broth of type 20 pneumococcus, let it stand at 2-8℃ for 6-12 hours, then centrifuge at 12000g for 30-60min and collect the supernatant. Use a 100KD membrane to ultrafilter the supernatant to obtain an ultrafiltration concentrate. The solution used for ultrafiltration is purified water, and its volume is 5 times that of the supernatant.

[0079] (2) Add NaCl to the ultrafiltration concentrate to a final concentration of 0.7 mol / L. After it is completely dissolved, add 0.5% (w / v) sodium deoxycholate. While stirring, add glacial acetic acid dropwise to adjust the pH to 4.5. After stirring thoroughly, let it stand at 2-8℃ for 12-16 hours. Then centrifuge at 12000g for 30 minutes, collect the supernatant, and discard the precipitate. If there are small fragments in the supernatant, clarify and filter it with a 0.8μm filter membrane.

[0080] (3) Adjust the pH of the supernatant to neutral using sodium hydroxide or potassium hydroxide. Then, add Na2HPO4 with a final concentration of 0.01 mol / L and NaH2PO4 with a final concentration of 0.01 mol / L to the obtained solution as a buffer system. After complete dissolution, add sodium acetate with a final concentration of 0.9 mol / L and CaCl2 with a final concentration of 0.15 mol / L. Stir until dissolved. Add glacial acetic acid dropwise to the solution to adjust the pH to 5.3-5.5. Stir thoroughly and let stand at 2-8℃ for 12-16 hours. After standing, centrifuge at 12000g for 30 minutes, collect the supernatant, and discard the precipitate. If there are small fragments in the supernatant, they can be clarified and filtered through a 0.8 μm filter membrane.

[0081] (4) The supernatant obtained in the previous step was ultrafiltered with purified water. When the conductivity at the permeate end was lower than 10 μs / cm, the polysaccharide solution was collected. After sterilization filtration, the polysaccharide solution was collected. The polysaccharide solution was then freeze-dried to obtain pneumococcal capsular polysaccharide.

[0082] Example 7

[0083] This embodiment provides a method for preparing pneumococcal capsular polysaccharide. The only difference between this embodiment and Example 2 is that the final concentration of CaCl2 added in step (3) is 0.05 mol / L.

[0084] Example 8

[0085] This embodiment provides a method for preparing pneumococcal capsular polysaccharide. The only difference from Example 5 is that the final concentration of CaCl2 added in step (3) is 0.10 mol / L.

[0086] Example 9

[0087] This embodiment provides a method for preparing pneumococcal capsular polysaccharide. The only difference from Example 5 is that in step (2), the concentration of sodium deoxycholate is 0.4%.

[0088] Comparative Example 1

[0089] This comparative example provides a method for preparing pneumococcal capsular polysaccharide. The only difference from Example 2 is that in step (2), the concentration of sodium deoxycholate is 0.2%.

[0090] Comparative Example 2

[0091] This comparative example provides a method for preparing pneumococcal capsular polysaccharide. The only difference between this method and Example 2 is that in step (2), glacial acetic acid solution is used to adjust the pH to 5.7.

[0092] Comparative Example 3

[0093] This comparative example provides a method for preparing pneumococcal capsular polysaccharide. The only difference between this method and Example 5 is that in step (2), glacial acetic acid solution is used to adjust the pH to 4.0.

[0094] Test case

[0095] The pneumococcal capsular polysaccharides prepared in the examples and comparative examples were tested.

[0096] The recovery rate is calculated as: total sugar content of refined polysaccharides / total sugar content of polysaccharides in fermentation broth.

[0097] The determination of capsular polysaccharide content was performed according to the rate turbidimetric method (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 was performed according to General Chapter 0731 in Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; the determination of nucleic acid content was performed according to General Chapter 0401 in Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; the determination of total nitrogen content was performed according to General Chapter 0704 in Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; the determination of phosphorus content was performed according to General Chapter 3103 in Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; uronic acid... The content was determined according to the General Rule 0401 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; the O-acetyl content was determined according to the General Rule 3117 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; the methylpentose content was determined according to the General Rule 0401 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; the aminohexose content was determined according to the General Rule 0401 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China; and the size of the capsular polysaccharide molecule was determined according to Method I in Section 3.1.2.10 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China. The test results are shown in Tables 1 to 12.

[0098] Table 1 Recovery rate and quality control indicators of Example 1

[0099] Test items (Chinese Pharmacopoeia Standard) 71-1-22105001 71-1-22106012 71-1-22107021 Recovery rate % ( / ) 68.91 67.96 65.03 protein (≤2％) 0.25 0.32 0.29 Nucleic acid (≤2％) 0.03 0.05 0.03 Total nitrogen (3.5～6％) 4.72 4.67 4.83 Phosphorus content (0～1.5％) 0.46 0.42 0.48 Molecular weight (KD) CL-4B (≤0.15) 0.06 0.05 0.06 Glucuronic acid (≥45％) 50.9 51.5 51.3 O-acetyl (≥1.8) 5.35 5.47 5.62

[0100] Table 2 Recovery rate and quality control indicators of Example 2

[0101]

[0102]

[0103] Table 3 Recovery rate and quality control indicators of Example 3

[0104] Test items (Chinese Pharmacopoeia Standard) 71-7F-22107004 71-7F-22107006 71-7F-22107007 Recovery rate % ( / ) 65.83 69.25 67.86 protein (≤5％) 0.75 0.82 0.87 Nucleic acid (≤2％) 0.09 0.08 0.10 Total nitrogen (1.5～4.0％) 2.47 2.83 2.68 Phosphorus content (0～1.0％) 0.38 0.40 0.38 <![CDATA[Molecular weight size (K D )]]> CL-4B (≤0.20) 0.03 0.04 0.04 Methylpentose (≥13％) 18.6 17.3 17.8

[0105] Table 4 Recovery rate and quality control indicators of Example 4

[0106] Test items (Chinese Pharmacopoeia Standard) 71-12F-22109003 71-12F-22109004 71-12F-22109005 Recovery rate % ( / ) 61.31 58.46 60.32 protein (≤3％) 0.28 0.33 0.31 Nucleic acid (≤2％) 0.06 0.05 0.05 Total nitrogen (3.0～5.0％) 3.72 3.89 3.83 Phosphorus content (0～1.0％) 0.19 0.18 0.19 <![CDATA[Molecular weight size (K D )]]> CL-4B (≤0.25) 0.08 0.05 0.09 Aminohexose (≥25％) 28.5 27.8 28.9

[0107] Table 5 Recovery rate and quality control indicators of Example 5

[0108]

[0109]

[0110] Table 6 Recovery rate and quality control indicators of Example 6

[0111] Test items (Chinese Pharmacopoeia Standard) 71-20-22110010 71-20-22110012 71-20-22110013 Recovery rate % ( / ) 65.21 64.67 66.38 protein (≤2％) 0.51 0.43 0.47 Nucleic acid (≤2％) 0.02 0.03 0.01 Total nitrogen (0.5～2.5％) 1.08 1.01 1.02 Phosphorus content (1.5～4.0％) 2.43 2.51 2.58 <![CDATA[Molecular weight size (K D )]]> CL-2B (≤0.60) 0.38 0.40 0.39 Aminohexose (≥12％) 13.2 13.6 13.5

[0112] Table 7 Recovery rate and quality control indicators of Example 7

[0113] Test items (Chinese Pharmacopoeia Standard) 71-2-22109013 71-2-22109014 Recovery rate % ( / ) 63.42 65.21 protein (≤2％) 1.87 1.73 Nucleic acid (≤2％) 1.62 1.45 Total nitrogen (0～1％) 0.92 0.85 Phosphorus content (0～1.0％) 0.76 0.69 <![CDATA[Molecular weight size (K D )]]> CL-4B (≤0.15) 0.05 0.08 Glucuronic acid (≥15％) 17.6 16.8 Methylpentose (≥38％) 39.3 39.8

[0114] Table 8 Recovery rate and quality control indicators of Example 8

[0115] Test items (Chinese Pharmacopoeia Standard) 71-18C-22110018 71-18C-22110020 Recovery rate % ( / ) 58.98 60.32 protein (≤3％) 1.86 2.05 Nucleic acid (≤2％) 0.89 0.97 Total nitrogen (0～1.0％) 0.73 0.89 Phosphorus content (2.4～4.5％) 4.01 3.93 <![CDATA[Molecular weight size (K D )]]> CL-4B (≤0.15) 0.05 0.04 Methylpentose (≥14％) 16.1 15.6

[0116] Table 9 Recovery rate and quality control indicators of Example 9

[0117] Test items (Chinese Pharmacopoeia Standard) 71-18C-22109010 71-18C-22109013 Recovery rate % ( / ) 59.85 62.87 protein (≤3％) 2.76 2.83 Nucleic acid (≤2％) 1.48 1.56 Total nitrogen (0～1.0％) 0.58 0.69 Phosphorus content (2.4～4.5％) 3.97 4.18 <![CDATA[Molecular weight size (K D )]]> CL-4B (≤0.15) 0.06 0.04 Methylpentose (≥14％) 16.8 17.1

[0118] Table 10 Recovery rate and quality control indicators of Comparative Example 1

[0119] Test items (Chinese Pharmacopoeia Standard) 71-2-22108007 71-2-22108008 Recovery rate % ( / ) 69.87 65.36 protein (≤2％) 1.90 2.01 Nucleic acid (≤2％) 1.11 1.23 Total nitrogen (0～1％) 1.96 1.89 Phosphorus content (0～1.0％) 0.65 0.72 <![CDATA[Molecular weight size (K D )]]> CL-4B (≤0.15) 0.07 0.08 Glucuronic acid (≥15％) 17.4 16.1 Methylpentose (≥38％) 40.8 39.7

[0120] Table 11 Recovery rate and quality control indicators of Comparative Example 2

[0121] Test items (Chinese Pharmacopoeia Standard) 71-2-22108010 71-2-22109012 Recovery rate % ( / ) 60.13 59.84 protein (≤2％) 2.23 2.15 Nucleic acid (≤2％) 1.03 1.67 Total nitrogen (0～1％) 1.26 1.19 Phosphorus content (0～1.0％) 0.71 0.88 <![CDATA[Molecular weight size (K D )]]> CL-4B (≤0.15) 0.06 0.05 Glucuronic acid (≥15％) 17.4 16.1 Methylpentose (≥38％) 39.5 38.9

[0122] Table 12 Recovery rate and quality control indicators of Comparative Example 3

[0123] Test items (Chinese Pharmacopoeia Standard) 71-18C-22109014 71-18C-22110017 Recovery rate % ( / ) 52.45 51.16 protein (≤3％) 2.11 2.97 Nucleic acid (≤2％) 1.02 0.91 Total nitrogen (0～1.0％) 0.57 1.01 Phosphorus content (2.4～4.5％) 3.15 2.89 <![CDATA[Molecular weight size (K D )]]> CL-4B (≤0.15) 0.04 0.07 Methylpentose (≥14％) 15.3 16.1

[0124] The test results show that the preparation method of the present invention yields a high recovery rate of pneumococcal capsular polysaccharide, and all quality indicators meet the requirements of the Chinese Pharmacopoeia.

[0125] The pneumococcal capsular polysaccharide prepared in the comparative example did not meet the Chinese Pharmacopoeia standards for protein and total nitrogen content, or had a low recovery rate.

[0126] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A process for the preparation of pneumococcal capsular polysaccharide, characterized in that, The method comprises the following steps: (1) providing a pneumococcus fermentation liquor, sterilizing and centrifuging the pneumococcus fermentation liquor, collecting a first supernatant and ultrafiltering to obtain a first ultrafiltrate; (2) adding a neutral salt to the first ultrafiltrate, mixing sodium deoxycholate to obtain a mixed solution, controlling the chloride ion concentration of the mixed solution to be 0.3-0.7 mol / L, and adjusting the pH to 4.5-5.0; and then centrifuging to collect a second supernatant; (3) adjusting the pH of the second supernatant to neutral, precipitating impurities by a calcium salt, and centrifuging to collect a third supernatant; Then, pneumococcal capsular polysaccharide is prepared by drying the third supernatant. In step (2), the neutral salt is NaCl or KCl; the ratio of sodium deoxycholate to the first ultrafiltrate is 0.5-0.7 g:100 mL; In step (3), the calcium salt comprises CaCl2 and sodium acetate; the final concentration of CaCl2 is 0.15-0.35 mol / L, and the final concentration of sodium acetate is 0.4-0.9 mol / L, based on the volume of the second supernatant.

2. The production method according to claim 1, characterized by, No toxic and harmful reagents are used throughout the process, and the toxic and harmful reagents include one or more of phenol, acetone, and ethanol.

3. The preparation method according to claim 1, characterized in that, No exogenous macromolecular substances are used throughout the process, and the exogenous macromolecular substances include one or more of nucleases and proteases.

4. The method of claim 1, wherein, No chromatography filler and chromatography equipment are used throughout the process.

5. A method of preparing a multivalent pneumococcal vaccine, characterized in that, The method comprises: The pneumococcal capsular polysaccharide prepared by the preparation method of any one of claims 1-4 is used to prepare a multivalent pneumococcal vaccine.

6. The method of claim 5, wherein, The multivalent pneumococcal vaccine is a pneumococcal polysaccharide vaccine or a pneumococcal polysaccharide conjugate vaccine.

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