Purification methods and applications of pneumococcal capsular polysaccharides

Abstract

This invention discloses a method for purifying pneumococcal capsular polysaccharide, comprising the following steps: (1) treating pneumococcus to obtain a solution containing pneumococcal capsular polysaccharide; and (2) removing C-polysaccharide from the solution containing pneumococcal capsular polysaccharide. The purification method of this invention avoids the use of toxic or dangerous reagents such as phenol and ethanol, and can further reduce the C-polysaccharide impurity content in pneumococcal capsular polysaccharide after removing impurities such as proteins and nucleic acids, thus obtaining high-quality pneumococcal capsular polysaccharide that meets the requirements. It has the advantages of being safe and environmentally friendly, simple to operate, low in cost, and having a good impurity removal effect.

Description

Technical Field

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

[0002] Pneumococcus is a Gram-positive bacterium that commonly infects infants, the elderly, and individuals with underlying medical conditions. It is a major cause of pneumonia, meningitis, bacteremia, and other diseases. Pneumococcus can cause infection alone and frequently co-infects with other viruses and bacteria, such as influenza virus, respiratory syncytial virus, Haemophilus influenzae, Mycoplasma pneumoniae, and Mycobacterium tuberculosis, endangering the patient's life. Pneumococcal vaccines are the most effective means of preventing pneumococcal infection, and the WHO has listed pneumococcal disease as a "very high priority" disease for vaccine prevention.

[0003] Capsular polysaccharides are the most important virulence factor of pneumococcus. Currently marketed pneumococcal polysaccharide vaccines (PPV) and pneumococcal conjugate vaccines (PCV) are both developed and designed based on pneumococcal capsular polysaccharides. Extracting high-quality capsular polysaccharides has always been a challenge. Now, with the increasing potency of pneumococcal vaccines, residual impurities in the capsular polysaccharides continue to accumulate, posing even greater challenges to the quality control of pneumococcal vaccines.

[0004] Researchers have developed various purification methods for capsular polysaccharides, such as ethanol fractionation precipitation, phenol extraction, proteolysis, acid precipitation combined with activated carbon adsorption, hexadecyl trimethylammonium bromide (CTAB) precipitation, and diethylaminoethyl cellulose (DEAE) resin method. Other methods include ethanol fractionation combined with CTAB precipitation and a combination of multiple purification methods with chromatography. However, traditional polysaccharide purification methods focus on reducing protein and nucleic acid contamination, and are not very effective at removing heteropolysaccharides such as C-polysaccharides. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a purification method for pneumococcal capsular polysaccharides. This purification method avoids the use of toxic or hazardous reagents such as phenol and ethanol, and further reduces the C-polysaccharide impurity content in pneumococcal capsular polysaccharides after removing impurities such as proteins and nucleic acids, resulting in high-quality pneumococcal capsular polysaccharides that meet the requirements. It has advantages such as safety and environmental friendliness, simple operation, low cost, and good impurity removal effect.

[0006] According to one aspect of the present invention, a method for purifying pneumococcal capsular polysaccharide is provided, the method comprising the following steps:

[0007] (1) Treating pneumococcus to obtain a solution containing pneumococcal capsular polysaccharide; and

[0008] (2) The solution containing pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment.

[0009] In some embodiments, step (2) of the C polysaccharide removal treatment includes treating a solution containing pneumococcal capsular polysaccharide with a combination of reagents, the combination of reagents including acid and nitrite.

[0010] In some embodiments, the mass ratio of the acid to the nitrite is 1:(0.01-100), preferably 1:(0.1-20). In some embodiments, the mass ratio of the acid to the nitrite is 1:0.01, 1:0.05, 1:0.1, 1:0.5, 1:1, 1:2, 1:5, 1:10, 1:15, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, or any value between them.

[0011] In some embodiments, the acid includes at least one of acetic acid, phosphoric acid, carbonic acid, citric acid, hydrochloric acid, sulfuric acid, perchloric acid, or tetrafluoroboric acid.

[0012] In some embodiments, the mass concentration of the acid is 0.1%-20%, preferably 1%-10%, and more preferably 1%-5%. In some embodiments, the mass concentration of the acid is 0.1%, 0.2%, 0.5%, 0.8%, 1%, 2%, 3%, 4%, 5%, 8%, 10%, 12%, 14%, 15%, 16%, 18%, 20%, or any value between them.

[0013] In some embodiments, the nitrite includes at least one of sodium nitrite, potassium nitrite, calcium nitrite, or ammonium nitrite.

[0014] In some embodiments, the mass concentration of the nitrite is 0.1%-20%, preferably 1%-10%, and more preferably 1%-5%. In some embodiments, the mass concentration of the nitrite is 0.1%, 0.2%, 0.5%, 0.8%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 10%, 12%, 14%, 15%, 16%, 18%, 20%, or any value between them.

[0015] In some embodiments, the temperature for the C-polysaccharide removal treatment is 0℃-50℃, preferably 25℃-45℃, and more preferably 25℃-37℃. In some embodiments, the temperature for the C-polysaccharide removal treatment is 0℃, 5℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 37℃, 40℃, 45℃, 50℃, or any value between them.

[0016] In some embodiments, the C-polysaccharide removal treatment time is 0.1 h-24 h, preferably 1-20 h, and more preferably 1-15 h. In some embodiments, the C-polysaccharide removal treatment time is 0.1 h, 0.5 h, 1 h, 2 h, 4 h, 5 h, 6 h, 8 h, 10 h, 12 h, 15 h, 18 h, 20 h, 24 h, or any value between them.

[0017] In some implementations, step (1) includes the following steps:

[0018] (1-1) The bacterial suspension containing pneumococcus was lysed to obtain bacterial lysate;

[0019] (1-2) The lysis solution is subjected to acid precipitation treatment, and the first supernatant is collected;

[0020] (1-3) Add CTAB to the first supernatant and collect the second supernatant, or add CTAB to the first supernatant, collect the precipitate, dissolve the precipitate with sodium chloride, and then collect the second supernatant;

[0021] (1-4) Iodine precipitation is performed on the second supernatant, and the third supernatant is collected to obtain the solution containing pneumococcal capsular polysaccharide.

[0022] In some embodiments, in step (1-1), the pyrolysis process includes at least one of chemical pyrolysis, physical pyrolysis, or biological pyrolysis.

[0023] In some embodiments, the chemical pyrolysis includes pyrolysis with a pyrolysis agent and / or alkaline pyrolysis.

[0024] In some embodiments, the physical pyrolysis includes high-pressure pyrolysis and / or thermal pyrolysis.

[0025] In some embodiments, the biolysis includes enzymatic lysis.

[0026] In some embodiments, in steps (1-2), the acid precipitation treatment includes adding an acidic reagent to the lysis solution, adjusting the pH to acidic, and then collecting the supernatant.

[0027] In some embodiments, the acidic reagent includes at least one selected from acetic acid, phosphoric acid, carbonic acid, citric acid, hydrochloric acid, sulfuric acid, perchloric acid, and tetrafluoroboric acid.

[0028] In some embodiments, the pH value is in the range of 2-6, preferably 3-5. In some embodiments, the pH value is 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, or any value between them.

[0029] In some embodiments, steps (1-2) further include concentrating the supernatant after acid precipitation treatment, changing the liquid, and collecting the first supernatant.

[0030] In some embodiments, in steps (1-3), the final mass concentration of CTAB is 0.1%-5%, preferably 1%-3%. In some embodiments, in steps (1-3), the final mass concentration of CTAB is 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, or any value between them.

[0031] In some embodiments, the pneumococcal capsular polysaccharide includes acidic pneumococcal capsular polysaccharide and / or neutral pneumococcal capsular polysaccharide.

[0032] In some embodiments, the acidic pneumococcal capsular polysaccharide is harvested and precipitated, dissolved in sodium chloride solution, and the second supernatant is collected. In some embodiments, the acidic pneumococcus includes, but is not limited to, pneumococci types 2, 3, 4, and 5.

[0033] In some embodiments, the neutral pneumococcal capsular polysaccharide is directly harvested from the second supernatant. In some embodiments, the acidic pneumococcus includes, but is not limited to, pneumococci such as 7A, 7F, 14, 33F, 33A, and 37.

[0034] In some embodiments, the final concentration of sodium chloride is 0.01 mol / L to 5 mol / L, preferably 0.1 mol / L to 1 mol / L. In some embodiments, the final concentration of sodium chloride is 0.01 mol / L, 0.05 mol / L, 0.1 mol / L, 0.5 mol / L, 1 mol / L, 2 mol / L, 3 mol / L, 4 mol / L, 5 mol / L, or any value between them.

[0035] In some embodiments, in steps (1-4), the iodized salt comprises sodium iodide and / or potassium iodide.

[0036] In some embodiments, the final mass concentration of the iodized salt is 0.01%-5%, preferably 0.2%-1.5%. In some embodiments, the final mass concentration of the iodized salt is 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, or any value between them.

[0037] In some embodiments, the method further includes purification and drying processes after step (2).

[0038] In some embodiments, the purification process includes at least one of membrane filtration, depth filtration, and chromatography. In some embodiments, the chromatography includes, but is not limited to, hydroxyapatite chromatography columns and / or ion exchange chromatography columns and / or gel filtration chromatography.

[0039] In some embodiments, the purification process includes sequentially performing a first ultrafiltration concentration treatment, a chromatography treatment, and a second ultrafiltration concentration treatment on the solution after the removal of the C polysaccharide.

[0040] In some embodiments, the first ultrafiltration concentration treatment and / or the second ultrafiltration concentration treatment are performed using a membrane pack with a diameter of 30-300 kDa.

[0041] In some embodiments, the drying process employs freeze drying.

[0042] In some embodiments, the supernatant or precipitate is obtained by solid-liquid separation. Preferably, the solid-liquid separation includes, but is not limited to, at least one of centrifugation, clarification, and filtration.

[0043] In some embodiments, the concentration process is performed using ultrafiltration. Preferably, the ultrafiltration uses a membrane pack with a density of 30 kD to 300 kD.

[0044] In some embodiments, the serotype of the pneumococcus includes at least one of the following: type 2, type 3, type 4, type 5, type 6A, type 6B, type 6C, type 6D, type 7A, type 7C, type 7F, type 8, type 9N, type 9V, type 10A, type 10B, type 11A, type 11E, type 12F, type 13, type 14, type 15A, type 15B, type 15C, type 15F, type 16F, type 17F, type 18C, type 19A, type 19F, type 20, type 21, type 22F, type 23A, type 23B, type 23F, type 24A, type 24B, type 24F, type 25A, type 25F, type 27, type 28F, type 31, type 33A, type 33F, type 34, type 35B, type 37, and type 38.

[0045] In some embodiments, the solution containing pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the mass concentration of the acid is 1%-8%, preferably 4.5%-5.5%, and more preferably 5%.

[0046] In some embodiments, the solution containing pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the mass concentration of nitrite is 1%-5%, preferably 2.5%-3.5%, and more preferably 3%.

[0047] In some embodiments, the solution containing pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the temperature of the C polysaccharide removal treatment is 4℃-50℃, preferably 35℃-40℃, and more preferably 37℃.

[0048] In some embodiments, the solution containing pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the C polysaccharide removal treatment time is 5h-20h, preferably 14-16h, and more preferably 15h.

[0049] In some embodiments, the solution containing pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the mass concentration of the acid is 1%-4%, preferably 2.5%-3.5%, and more preferably 3%.

[0050] In some embodiments, the solution containing pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the mass concentration of nitrite is 0.5%-2%, preferably 0.8%-1.2%, and more preferably 1%.

[0051] In some embodiments, the solution containing pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the temperature of the C polysaccharide removal treatment is 4℃-40℃, preferably 22℃-30℃, and more preferably 25℃.

[0052] In some embodiments, the solution containing pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the C polysaccharide removal treatment time is 0.5h-2h, preferably 0.8h-1.2h, and more preferably 1.0h.

[0053] In some embodiments, the solution containing 7F type pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the mass concentration of the acid is 0.5%-2%, preferably 0.8%-1.2%, and more preferably 1%.

[0054] In some embodiments, the solution containing 7F type pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the mass concentration of nitrite is 1%-4%, preferably 2.5%-3.5%, and more preferably 3%.

[0055] In some embodiments, the solution containing 7F type pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the temperature of the C polysaccharide removal treatment is 4℃-40℃, preferably 22℃-30℃, and more preferably 25℃.

[0056] In some embodiments, the solution containing 7F type pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the C polysaccharide removal treatment time is 4h-10h, preferably 7h-9h, and more preferably 8.0h.

[0057] In some embodiments, the solution containing 12F type pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the mass concentration of the acid is 1%-4%, preferably 2.5%-3.5%, and more preferably 3%.

[0058] In some embodiments, the solution containing 12F type pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the mass concentration of nitrite is 1%-8%, preferably 4.5%-5.5%, and more preferably 5%.

[0059] In some embodiments, the solution containing 12F type pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the temperature of the C polysaccharide removal treatment is 4℃-40℃, preferably 22℃-30℃, and more preferably 25℃.

[0060] In some embodiments, the solution containing 12F type pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the C polysaccharide removal treatment time is 6h-12h, preferably 9h-11h, and more preferably 10h.

[0061] In some specific embodiments, the purification method of the pneumococcal capsular polysaccharide includes the following steps:

[0062] (1) The bacterial suspension containing pneumococcus was lysed to obtain bacterial lysate;

[0063] (2) The lysis solution is subjected to acid precipitation treatment, and the first supernatant is collected;

[0064] (3) Perform CTAB precipitation on the first supernatant and collect the second supernatant directly, or dissolve the precipitate with sodium chloride and collect the second supernatant;

[0065] (4) Iodine precipitation is performed on the second supernatant, and the third supernatant is collected to obtain the solution containing pneumococcal capsular polysaccharide;

[0066] (5) The solution containing pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment;

[0067] (6) Refining and purification;

[0068] (7) Drying.

[0069] In some embodiments, in step (1), a fermentation culture containing pneumococcus that can produce a selected serotype is provided, and a bacterial lysate containing cell debris, proteins, nucleic acids and polysaccharides is obtained by one or more of the following methods: chemical lysis (lysis agent lysis, alkaline lysis), physical lysis (high pressure lysis, thermal lysis), and biological lysis (enzymatic lysis).

[0070] Preferably, a fermentation broth containing pneumococcus that can produce a selected serotype is provided, and sodium deoxycholate is added to obtain the bacterial lysate.

[0071] Preferably, a fermentation broth containing pneumococcus that can produce a selected serotype is provided, and the mixture is treated with sodium deoxycholate at a mass concentration of 0.5-0.5% for more than 1 hour to obtain the bacterial lysate.

[0072] In some embodiments, in step (2), an acidic reagent (including but not limited to acetic acid, phosphoric acid, carbonic acid, citric acid, hydrochloric acid, sulfuric acid, perchloric acid, tetrafluoroboric acid, etc.) is added to the bacterial lysate to adjust the pH to acidic and precipitate impurities. The supernatant is harvested by centrifugation and / or clarification and / or filtration. The supernatant is concentrated and replaced to remove low molecular weight impurities, yielding a first clarified bacterial lysate (i.e., the first supernatant).

[0073] Preferably, acetic acid is added to the bacterial lysate to adjust the pH to 2-6 to precipitate impurities. The supernatant is harvested by centrifugation, filtered through a membrane to remove residual large particles. The supernatant is then concentrated and replaced using ultrafiltration to remove low-molecular-weight impurities, yielding a first clarified bacterial lysate.

[0074] Preferably, acetic acid is added to the bacterial lysate to adjust the pH to 3-5, and the mixture is allowed to stand at room temperature for at least 4 hours to precipitate impurities. The supernatant is collected by centrifugation and filtered through a 0.45 μm membrane to remove residual large particles. The supernatant is then ultrafiltered using a 30 kD-300 kD membrane to concentrate and remove low molecular weight impurities, yielding the first clarified bacterial lysate.

[0075] In some embodiments, in step (3), CTAB is added to the first clarified bacterial lysate to precipitate impurities. Solid-liquid separation is performed by centrifugation and / or clarification and / or filtration. In some embodiments, the supernatant is harvested to obtain a second clarified bacterial lysate (i.e., the second supernatant). In other embodiments, the precipitate is harvested and sodium chloride is added for depolymerization. The supernatant is harvested by centrifugation and / or clarification and / or filtration to obtain a second clarified bacterial lysate.

[0076] Preferably, CTAB at a final mass concentration of 0.1%-5% is added to the first clarified bacterial lysate to precipitate impurities. Solid-liquid separation is then performed by centrifugation. In some embodiments, the supernatant is harvested for neutral pneumococcal capsular polysaccharides to obtain a second clarified bacterial lysate, which is then added with sodium chloride. In other embodiments, the precipitate for acidic pneumococcal capsular polysaccharides is harvested and dissolved with sodium chloride. The supernatant is harvested by centrifugation to obtain the second clarified bacterial lysate.

[0077] Preferably, CTAB at a final mass concentration of 1%-3% is added to the first clarified bacterial lysate, and the mixture is allowed to stand overnight to precipitate impurities. Solid-liquid separation is then performed by centrifugation. In some embodiments, the supernatant of capsular polysaccharides from neutral pneumococcal types 7A, 7F, 14, 33F, 33A, and 37 is harvested to obtain a second clarified bacterial lysate, which is then dissolved in sodium chloride at a final concentration of 0.01 mol / L-5 mol / L. In other embodiments, the precipitate of capsular polysaccharides from acidic pneumococcal types 2, 3, 4, and 5 is harvested and dissolved in sodium chloride at a final concentration of 0.01 mol / L-5 mol / L. The supernatant is then harvested by centrifugation to obtain the second clarified bacterial lysate.

[0078] In some embodiments, in step (4), iodized salt is added to the second clarified bacterial lysate, and precipitates and large particles are removed by centrifugation and / or clarification and / or filtration. The supernatant is then concentrated and replaced to remove low molecular weight impurities, yielding a third clarified bacterial lysate (i.e., the third supernatant).

[0079] Preferably, sodium iodide with a final mass concentration of 0.01%-5% is added to the second clarified bacterial lysate, the precipitate is removed by centrifugation, and residual large particles are removed by membrane filtration. The harvested liquid is then concentrated by ultrafiltration to remove low molecular weight impurities, yielding the third clarified bacterial lysate.

[0080] Preferably, sodium iodide with a final mass concentration of 0.2%-1.5% is added to the second clarified bacterial lysate, the mixture is allowed to stand and precipitate, the supernatant is collected by centrifugation, and filtered using a 0.45 μm membrane to remove residual large particles. The harvested liquid is then ultrafiltered using a 30 kD-300 kD membrane to concentrate and replace the liquid, removing low molecular weight impurities to obtain the third clarified bacterial lysate.

[0081] In some embodiments, in step (5), acid and nitrite are added to the third clarified bacterial lysate, and the reaction is carried out under mild conditions. Solution replacement is performed by one-step or multi-step membrane filtration or deep filtration to obtain the fourth clarified bacterial lysate.

[0082] Preferably, acetic acid and sodium nitrite with a final mass concentration of 0.1%-20% are added to the third clarified bacterial lysate, and the reaction is carried out at 0℃-50℃ for 0.1 h–24 h. The solution is then replaced by membrane filtration to obtain the fourth clarified bacterial lysate.

[0083] Preferably, acetic acid and sodium nitrite with a final mass concentration of 1%–5% are added to the third clarified bacterial lysate, and the reaction is carried out at 25°C–37°C for 1 h–15 h. The solution is then replaced and concentrated using ultrafiltration with a 30 kD–300 kD membrane to obtain the fourth clarified bacterial lysate.

[0084] In some embodiments, in step (6), the fourth clarified bacterial lysate is purified (including but not limited to membrane filtration, deep filtration, and chromatography) to collect the target polysaccharide component and further concentrate and change the solution to obtain a purified capsular polysaccharide solution.

[0085] Preferably, the fourth clarified bacterial lysate is purified using a hydroxyapatite chromatography column and / or an ion exchange chromatography column and / or gel filtration chromatography, the target polysaccharide flow-through peak is collected, and ultrafiltration concentration and solution displacement are performed using 30 kD–300 kD to obtain a purified capsular polysaccharide solution.

[0086] Preferably, the fourth clarified bacterial lysate is purified using a hydroxyapatite chromatography column, the target polysaccharide flow-through peak is collected, and ultrafiltration concentration and solution replacement are performed using a 30 kD–300 kD membrane pack. The replacement solution is water for injection. The final product is a purified capsular polysaccharide solution.

[0087] In some embodiments, in step (7), the refined capsular polysaccharide is dried to remove moisture and obtain a capsular polysaccharide sample.

[0088] Preferably, the drying process is freeze drying.

[0089] According to another aspect of the present invention, a pneumococcal capsular polysaccharide prepared by the method according to the present invention is provided.

[0090] In some embodiments, the content of C polysaccharide in the pneumococcal capsular polysaccharide is less than 5%, preferably less than 1%, and more preferably less than 0.2%.

[0091] Preferably, the content of C-polysaccharide impurities in the pneumococcal capsular polysaccharide is significantly lower than that in the control serological capsular polysaccharide.

[0092] According to another aspect of the present invention, the use of the aforementioned pneumococcal capsular polysaccharide in the preparation of products containing pneumococcal capsular polysaccharide is provided.

[0093] In some embodiments, the pneumococcal capsular polysaccharide-containing product includes a pneumococcal vaccine.

[0094] In some embodiments, the pneumococcal vaccine includes a pneumococcal capsular polysaccharide vaccine or a pneumococcal capsular polysaccharide conjugate vaccine.

[0095] In some embodiments, the pneumococcal vaccine includes a monovalent vaccine or a multivalent vaccine.

[0096] According to another aspect of the present invention, a vaccine composition is provided comprising the pneumococcal capsular polysaccharide prepared by the method described in the present invention.

[0097] In some embodiments, the pneumococcal vaccine includes a pneumococcal capsular polysaccharide vaccine or a pneumococcal capsular polysaccharide conjugate vaccine.

[0098] In some embodiments, the pneumococcal vaccine includes a monovalent vaccine or a multivalent vaccine.

[0099] The method for purifying pneumococcal capsular polysaccharides of the present invention adds a step for removing C polysaccharides, which significantly improves the purity of capsular polysaccharides and is beneficial for quality control. At the same time, it avoids the use of toxic and harmful organic reagents such as phenol and acetone, and flammable and explosive reagents such as ethanol, making it safer and more environmentally friendly. Moreover, all reagents used are conventional reagents, which have the advantages of low cost, simple operation, and suitability for large-scale production. Attached Figure Description

[0100] Figure 1 The images show the control type 4 pneumococcal capsular polysaccharide Y4CH02C (A) and the type 4 pneumococcal capsular polysaccharide Y4CH23N purified by the method of this invention (B). 1 H NMR spectrum.

[0101] Figure 2 The images show the control pneumococcal type 5 capsular polysaccharide Y5CI25C (A) and the pneumococcal type 5 capsular polysaccharide Y5CG12N purified by the method of this invention (B). 1 H NMR spectrum.

[0102] Figure 3 The images show the control 7F type pneumococcal capsular polysaccharide Y7FCK22C (A) and the 7F type pneumococcal capsular polysaccharide Y7FCL15N purified by the method of this invention (B). 1 H NMR spectrum.

[0103] Figure 4 The images show the control 12F type pneumococcal capsular polysaccharide Y12FDA28C (A) and the purified 12F type pneumococcal capsular polysaccharide Y12FDC26N (B) by the method of this invention. 1 H NMR spectrum. Detailed Implementation

[0104] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0105] Cell wall polysaccharides (C-Ps), also known as C-polysaccharides, are species-specific polysaccharides found in the cell walls of pneumococci and are common to all serotypes. C-Ps exhibit high immunogenicity, but the antibodies produced by the host as an antigen do not provide protection; instead, they can trigger adverse reactions such as inflammation. Furthermore, antibodies produced by C-Ps can interfere with the quantification of capsular polysaccharide antibodies, affecting the detection of capsular polysaccharides. The varying levels of C-Ps residues in pneumococcal capsular polysaccharides pose significant challenges to the research and production of PPV and PCV. Currently, the WHO has listed C-Ps as an impurity and recommends controlling its content. The United States Pharmacopeia (USP) has included C-Ps content in its vaccine quality control criteria. Chinese vaccine review agencies have also begun to pay attention to and restrict the C-Ps impurity content in pneumococcal capsular polysaccharides. On July 25, 2024, the Center for Drug Evaluation of the National Medical Products Administration released the "Technical Guidelines for Quality Control of Polysaccharide Conjugate Vaccines (Draft for Comments)," which pointed out that the sterilization process may produce product-related impurities such as C polysaccharide. It suggested that research be conducted on the impact of the sterilization process on the quality and structure of polysaccharides, and recommended that appropriate methods such as quantitative nuclear magnetic resonance be used to determine C polysaccharide.

[0106] This invention provides a method for purifying pneumococcal capsular polysaccharides, creatively adding a step to remove C-polysaccharides to the existing precipitation method for impurity removal. Subsequent methods such as membrane filtration, depth filtration, or chromatography are used to separate the C-polysaccharides and capsular polysaccharides, effectively removing C-polysaccharide contamination. This invention can obtain high-purity, high-quality pneumococcal capsular polysaccharides, which is of great significance for the development of pneumococcal polysaccharide vaccines and polysaccharide conjugate vaccines.

[0107] The reagents and / or kits used in the following examples are commercially available or can be synthesized by known methods.

[0108] Unless otherwise specified, the methods described in the following embodiments are conventional. Unless otherwise specified, the materials used in the following embodiments are all available from publicly available commercial sources.

[0109] Main materials: Streptococcus pneumoniae strains were obtained from the China Medical Bacterial Culture Collection Center of the China National Institutes for Food and Drug Control; sodium deoxycholate, acetic acid, sodium hydroxide, sodium dihydrogen phosphate, disodium hydrogen phosphate, CTAB, sodium chloride, sodium iodide, and sodium nitrite were obtained from Sinopharm Chemical Reagent Co., Ltd.; heavy water was obtained from Shanghai Aladdin Biochemical Technology Co., Ltd.; and specific sera for Streptococcus pneumoniae types 4, 5, 7F, and 12F were obtained from the Danish Serum Institute.

[0110] Main instruments: Tangential flow system (Sartorius, Sartoflow Advanced); Centrifuge (Thermo, Sorvall LYNX 6000); Freeze dryer (CHRIST, Epsilon 2-4 Lscplus); 600 MHz ultra-low temperature nuclear magnetic resonance spectrometer (BRUKER, AV-HD-600X); Ultraviolet spectrophotometer (Shimadzu, UV-1900i); Protein purification system (Cytiva, AKTAPURE).

[0111] Example 1: Purification of capsular polysaccharides from pneumococcus type 4

[0112] Take 10 L of Pneumococcus type 4 fermentation broth and add sodium deoxycholate to a final concentration of 0.1% to obtain a lysate. Add acetic acid to the lysate to adjust the pH to 5, centrifuge to collect the supernatant, and adjust the pH of the supernatant to 7 using sodium hydroxide. Filter through a 0.45 μm membrane, concentrate using a 100 kD membrane pack, and replace the solution with phosphate buffer (pH 7.0). Add CTAB to the feed solution to a final concentration of 1%, centrifuge to collect the precipitate. Dissolve the precipitate with 1 L of 0.25 mol / L sodium chloride, centrifuge to collect the supernatant. Add sodium iodide to a final concentration of 0.5%, centrifuge to collect the supernatant. Filter through a 0.45 μm membrane, concentrate using a 100 kD membrane pack, and replace the solution with phosphate buffer (pH 7.0). Acetic acid (5% by mass) and sodium nitrite (3% by mass) were added to the feed solution, and the reaction was carried out at 37°C for 15 h. The solution was then concentrated by ultrafiltration using a 100 kD membrane, and the solution was replaced with phosphate buffer (pH 7.0). Purification was performed using a hydroxyapatite column, and the target flow-through peak was collected. The flow-through was then concentrated by ultrafiltration using a 100 kD membrane, and the solution was replaced with water for injection. The final product was freeze-dried to obtain capsular polysaccharide with batch number Y4CH23N for subsequent analysis.

[0113] Comparative Example 1: Purification of capsular polysaccharide from control type 4 pneumococcus

[0114] Take 10 L of Pneumococcus type 4 fermentation broth and add sodium deoxycholate to a final concentration of 0.1% to obtain the lysate. Add acetic acid to the lysate to adjust the pH to 5, centrifuge to collect the supernatant, and adjust the pH of the supernatant to 7 using sodium hydroxide. Filter through a 0.45 μm membrane, concentrate using a 100 kD membrane pack, and replace the solution with phosphate buffer (pH 7.0). Add 1% CTAB to the feed solution and centrifuge to collect the precipitate. Dissolve the precipitate with 1 L of 0.25 mol / L sodium chloride, centrifuge to collect the supernatant. Add sodium iodide to a final concentration of 0.5%, centrifuge to collect the supernatant. Filter through a 0.45 μm membrane, concentrate using a 100 kD membrane pack, and replace the solution with phosphate buffer (pH 7.0). Purify using a hydroxyapatite column and collect the target flow-through peak. Concentrate the flow-through using a 100 kD membrane pack and replace the solution with water for injection. The final product was freeze-dried to obtain capsular polysaccharide with batch number Y4CH02C for subsequent testing.

[0115] Example 2: Detection of capsular polysaccharides from pneumococcus type 4

[0116] 2.1 1 H NMR structural analysis

[0117] The control type 4 pneumococcal capsular polysaccharide purified in Comparative Example 1 and the type 4 pneumococcal capsular polysaccharide purified in Example 1 were dissolved in heavy water and then lyophilized. This process was repeated three times. The capsular polysaccharides were dissolved in heavy water to a final concentration of 5 mg / ml, allowed to dissolve completely at room temperature, centrifuged for 10 min, and the sample solutions were collected in NMR tubes. One-dimensional NMR spectra of each type of capsular polysaccharide were acquired using a 600 MHz NMR spectrometer, and their spectral characteristics were analyzed using Bruker TopSpin 4.2.0 software.

[0118] Quantitative analysis was performed using one-dimensional 1H NMR spectroscopy. The relative content of each substance was determined by measuring the integral of the peak area of ​​each component in the sample. Normalization was performed using a factor N, which divides the integral of each peak by the number of equivalent nuclei represented by that peak, thus obtaining the relative molar concentration of each component. The C-polysaccharide repeating unit has two phosphocholine substituents, and each phosphocholine contains three methyl groups. Therefore, the C-polysaccharide contains a total of 18 protons. The formula for calculating the C-polysaccharide content is as follows:

[0119]

[0120] Where: I is the peak area integral; N is the normalization factor; and the subscripts C and CPS represent C polysaccharide and capsular polysaccharide, respectively.

[0121] Pneumococcal capsular polysaccharide 1 The H NMR spectrum results are shown in Figure 1. 1 The 1H NMR spectrum shows a characteristic peak of C polysaccharide (phosphocholine peak) at 3.22 ppm. Figure 1 In the middle, A represents the control group, Y4CH02C, which is a capsular polysaccharide from pneumococcus type 4. Figure 1 B represents the capsular polysaccharide Y4CH23N purified by the method of this invention. Compared with the capsular polysaccharide spectrum of the control type 4 pneumococcus, the peak area of ​​the characteristic peak of C polysaccharide in the capsular polysaccharide purified by the method of this invention is reduced, while the other structural characteristic peaks are consistent. According to the formula for calculating C polysaccharide content, the C polysaccharide content of the control group is 5.67%, while the C polysaccharide content of the capsular polysaccharide purified by the method of this invention is 0.17%. In summary, this indicates that the method of this invention can specifically reduce the C polysaccharide impurity content.

[0122] 2.2 Immunomodulation

[0123] Melt 1% agar and dispense it into glass tubes. Incubate at 56°C in a water bath. Add type 4 pneumococcal capsular polysaccharide-specific serum and mix thoroughly. Spread 4 ml of agar solution onto a clean glass slide. After solidification, punch holes with a diameter of 3 mm and a spacing of 10 mm. Place 10 μl of the control capsular polysaccharide purified in Comparative Example 1 and the capsular polysaccharide purified in Example 1 into the holes respectively. Place the agar plate in a humidified chamber and react at room temperature for 12 h. Measure the diameter of the precipitate ring. Repeat three times and take the average value. The change in polysaccharide antigenicity is determined by measuring the diameter of the diffusion ring.

[0124] The results of immunodiffusion of Pneumococcal serotype 4 capsular polysaccharide are shown in Table 1. The diffusion loop diameter of the purified Pneumococcal serotype 4 capsular polysaccharide of this invention is comparable to that of the control Pneumococcal serotype 4 capsular polysaccharide, and slightly superior to that of the control capsular polysaccharide. This indicates that the purification method of this invention has no negative impact on the antigenicity of the capsular polysaccharide, and its effect is slightly better than that of the control group.

[0125] Table 1: Results of Immunosuppressive Monodiffusion of Pneumococcal Capsular Polysaccharide in Type 4 Pneumococcus

[0126] ;

[0127] 2.3 Physicochemical Testing

[0128] Physicochemical tests were performed on the type 4 pneumococcal capsular polysaccharide purified in Example 1 and the control type 4 capsular polysaccharide purified in Comparative Example 1.

[0129] 2.3.1 Protein content detection

[0130] The Lowry method was used: 1 mg / ml polysaccharide solution, gradient concentration control solutions, and alkaline copper solution were prepared, reacted, and the absorbance of each sample at 650 nm was measured. A linear regression equation was calculated using the concentrations of the control solutions and their corresponding absorbances, and the protein concentration in the polysaccharide solution was calculated from the linear regression equation.

[0131] 2.3.2 Nucleic acid content detection

[0132] Ultraviolet spectrophotometry was used: purified polysaccharide was dissolved in distilled water to obtain a 1 mg / ml polysaccharide solution. Distilled water was used as a blank control, and the absorbance at a wavelength of 260 nm was measured. The absorption coefficient of nucleic acid at a wavelength of 260 nm is 200. The nucleic acid content was calculated according to the absorption coefficient formula.

[0133] 2.3.3 Total Nitrogen Content Detection

[0134] Based on the Kjeldahl method, a fully automated nitrogen analyzer was used to determine the total nitrogen content. An appropriate amount of polysaccharide was weighed into a digestion tube, and potassium sulfate, copper sulfate, and sulfuric acid were added sequentially. The digestion tube was placed in the digester, and the temperature was set to 150℃ for 5 minutes to remove moisture; 350℃ for 5 minutes, close to the boiling point of sulfuric acid; and 400℃ for 60-80 minutes for digestion. After digestion, the tube was removed and cooled. The digestion tube was then placed in an automated distillation apparatus for distillation and titration to determine the total nitrogen content.

[0135] 2.3.4 Phosphorus content detection

[0136] Prepare a 1 mg / ml polysaccharide solution and a gradient concentration standard phosphorus solution using potassium dihydrogen phosphate. Accurately measure 1 ml of the sample solution into a test tube, add 4 drops of sulfuric acid, and heat until carbonized. Add 2 drops of perchloric acid and digest until colorless and clear. Let stand for a moment, add 2 ml of water and 0.4 ml of 0.04 M ammonium molybdate solution, mix well, add 0.2 ml of reducing agent, and mix well. Add water to 6 ml, and after 15 minutes, measure the absorbance at 820 nm using a UV spectrophotometer. Perform linear regression on the absorbance of the gradient concentration standard phosphorus solutions with the corresponding absorbance, substitute the absorbance of the polysaccharide sample solution into the linear regression equation, and calculate the phosphorus content.

[0137] 2.3.5 Molecular weight determination

[0138] An agarose gel chromatography column was prepared using CL-4B gel. Blue dextran 2000 solution and vitamin B12 solution were prepared for column calibration. Elution was performed with the mobile phase at a flow rate of 15 mL / h, and detection was performed at a wavelength of 206 nm. The eluent was collected using a fraction collector, and the chromatogram was recorded. In the chromatogram, the first peak represents blue dextran 2000, and the eluent volume at the peak is the empty flow volume V. o The second peak is the vitamin B12 peak, and the eluent volume at the peak is the column bed volume V. i Prepare a 4 mg / ml polysaccharide solution using the mobile phase. Add 1 ml of the polysaccharide solution to the calibrated column and elute with the mobile phase at a flow rate of 15 ml per hour. Detect at a wavelength of 206 nm. Collect the eluent using a fraction collector and record the chromatogram.

[0139] Calculate K using the following formula: D = (V e -V o ) / (V i -V o )

[0140] Where: K D V is the sample partition coefficient; e V represents the volume of the sample elution buffer, in ml; o V represents the volume of the airflow, in ml; i The volume of the column bed is in ml.

[0141] 2.3.6 Detection of aminohexose content

[0142] Prepare 3 ml of an 800 μg / ml polysaccharide solution and 10 ml of a 500 μg / ml D-glucosamine hydrochloride reference standard stock solution using purified water, and then serially dilute with purified water. Take 1 ml of each of the gradient concentration reference solutions and the prepared polysaccharide sample solutions into 20 ml stoppered test tubes. Add 1 ml of an appropriate concentration of HCl solution, stopper the tubes, and heat in a 100°C water bath. Cool to room temperature, add 0.5% phenolphthalein ethanol solution, mix thoroughly, neutralize the hydrolysate with 4 mol / L NaOH solution until color changes, add 1 mol / L HCl solution dropwise until the solution is colorless, and add purified water to 10 ml to obtain a neutral hydrolysate.

[0143] Accurately measure 1 ml of neutral hydrolysate into a glass-stopped test tube. Perform parallel measurements in two tubes. Add 1 ml of acetylacetone reagent to each tube, stopper the tubes, and heat in a 90°C water bath for 45 min. Cool to room temperature, add 2.5 ml of anhydrous ethanol to each tube, mix well, and slowly add 1 ml of p-dimethylaminobenzaldehyde solution to each tube, mixing well. Add anhydrous ethanol to a final volume of 10 ml, mix well, stopper the tubes, and incubate at room temperature in the dark for 1.5 h. Measure the absorbance of each tube at a wavelength of 530 nm.

[0144] A linear regression was performed on the absorbance values ​​of a series of D-glucosamine hydrochloride reference solutions at their corresponding concentrations to obtain the regression equation. The absorbance of the polysaccharide samples was then substituted into the regression equation, and the aminohexose content was calculated based on the dilution factor. Finally, the percentage of aminohexose content was calculated based on the dry weight of the polysaccharides.

[0145] The test results are shown in Table 2. All indicators of the purified pneumococcal capsular polysaccharide samples obtained in Example 1 and Comparative Example 1 meet the relevant standards stipulated in the pharmacopoeia.

[0146] Table 2: Physicochemical Detection Results of Polysaccharides from Pneumococcal Capsule Type 4

[0147] ;

[0148] Example 3: Optimization of purification process for polysaccharides from pneumococcal capsularis type 4

[0149] 3.1 Acetic acid concentration

[0150] The method of this invention was used to separate and purify capsular polysaccharides from Streptococcus pneumoniae type 4. In the C-polysaccharide removal step, the final mass concentrations of acetic acid added in each group were 1.0%, 2.5%, 5.0%, and 7.5%, respectively. The remaining steps were the same as in Example 1. The lyophilized polysaccharides were harvested and then subjected to... 1 H NMR detection. 1 The characteristic peak of C polysaccharide (phosphocholine peak) was observed at 3.22 ppm in the 1H NMR spectrum. The content of C polysaccharide was quantified based on the characteristic peak of type 4 capsular polysaccharide, and the results are shown in Table 3.

[0151] Table 3: Effect of acetic acid concentration on the C-polysaccharide content in purified Streptococcus pneumoniae capsular polysaccharide.

[0152] ;

[0153] The test results show that the C-polysaccharide content in the obtained capsular polysaccharide decreases with increasing acetic acid concentration. Purification using 5.0% and 7.5% acetic acid yielded capsular polysaccharides with comparable C-polysaccharide content. Considering both purification efficiency and economic cost, the preferred acetic acid concentration for the C-polysaccharide removal step in the purification process of Streptococcus pneumoniae type 4 capsular polysaccharide is 5.0%.

[0154] 3.2 Sodium nitrite concentration

[0155] The method of this invention was used to isolate and purify capsular polysaccharides from Streptococcus pneumoniae type 4. In the C polysaccharide removal step, the final mass concentrations of sodium nitrite added to each group were 1.0%, 2.0%, 3.0%, and 4.0%, respectively. The remaining steps were the same as in Example 1. The lyophilized polysaccharides were harvested and then subjected to… 1 H NMR detection. 1 The characteristic peak of C polysaccharide (phosphocholine peak) was observed at 3.22 ppm in the 1H NMR spectrum. The content of C polysaccharide was quantified based on the characteristic peak of type 4 capsular polysaccharide, and the results are shown in Table 4.

[0156] Table 4: Effect of sodium nitrite concentration on the C-polysaccharide content in purified Streptococcus pneumoniae capsular polysaccharide.

[0157] ;

[0158] The test results show that the C-polysaccharide content in the obtained capsular polysaccharide decreases with increasing sodium nitrite concentration. Purification using 3.0% and 4.0% sodium nitrite yielded similar C-polysaccharide contents in the obtained capsular polysaccharides. Considering both purification efficiency and economic cost, the preferred sodium nitrite concentration for the C-polysaccharide removal step in the purification process of Streptococcus pneumoniae capsular polysaccharide is 3.0%.

[0159] 3.3 Reaction Time

[0160] The method of this invention was used to isolate and purify capsular polysaccharides from Streptococcus pneumoniae type 4. The reaction times for the C polysaccharide removal step in each group were 5.0 h, 10.0 h, 15.0 h, and 20.0 h, respectively. The remaining steps were the same as in Example 1. The lyophilized polysaccharides were harvested and then subjected to... 1 H NMR detection. 1 The characteristic peak of C polysaccharide (phosphocholine peak) was observed at 3.22 ppm in the 1H NMR spectrum. The content of C polysaccharide was quantified based on the characteristic peak of type 4 capsular polysaccharide, and the results are shown in Table 5.

[0161] Table 5: Effect of reaction time on the C-polysaccharide content in the purified Streptococcus pneumoniae capsular polysaccharide.

[0162] ;

[0163] The test results show that the C-polysaccharide content in the obtained capsular polysaccharide decreases with increasing reaction time. The C-polysaccharide content in the obtained capsular polysaccharide is comparable between reactions of 15.0 h and 20.0 h. Considering both purification efficiency and economic cost, the optimal reaction time for the C-polysaccharide removal step in the purification process of Streptococcus pneumoniae capsular polysaccharide is 15.0 h.

[0164] 3.4 Reaction Temperature

[0165] The method of this invention was used to isolate and purify capsular polysaccharides from Streptococcus pneumoniae type 4. The reaction temperatures for the C polysaccharide removal step in each group were 4.0 ℃, 25.0 ℃, 37.0 ℃, and 45.0 ℃, respectively. The remaining steps were the same as in Example 1. The lyophilized polysaccharides were harvested and then subjected to… 1 H NMR detection. 1 The characteristic peak of C polysaccharide (phosphocholine peak) was observed at 3.22 ppm in the 1H NMR spectrum. The content of C polysaccharide was quantified based on the characteristic peak of type 4 capsular polysaccharide, and the results are shown in Table 6.

[0166] Table 6: Effect of reaction temperature on the C-polysaccharide content in the purified Streptococcus pneumoniae capsular polysaccharide.

[0167] ;

[0168] The test results show that the C-polysaccharide content in the obtained capsular polysaccharide decreases with increasing reaction temperature. The C-polysaccharide content is comparable between 37.0 ℃ and 45.0 ℃. Considering both purification efficiency and economic cost, the optimal reaction temperature for the C-polysaccharide removal step in the purification process of Streptococcus pneumoniae capsular polysaccharide is 37.0 ℃.

[0169] Example 4: Purification of capsular polysaccharides from pneumococcus type 5

[0170] Take 10 L of Pneumococcus type 5 fermentation broth and add sodium deoxycholate to a final concentration of 0.2% to obtain a lysate. Add acetic acid to the lysate to adjust the pH to 3.2, centrifuge to collect the supernatant, and adjust the pH of the supernatant to 7 using sodium hydroxide. Filter through a 0.45 μm membrane, perform ultrafiltration concentration using a 100 kD membrane pack, and replace the solution with phosphate buffer (pH 7.0). Add CTAB to the feed solution to a final concentration of 2%, centrifuge to collect the precipitate. Dissolve the precipitate with 1 L of 0.20 mol / L sodium chloride, centrifuge to collect the supernatant. Add sodium iodide to a final concentration of 0.5%, centrifuge to collect the supernatant. Filter through a 0.45 μm membrane, perform ultrafiltration concentration using a 100 kD membrane pack, and replace the solution with phosphate buffer (pH 7.0). Acetic acid (3.0% by mass) and sodium nitrite (1.0% by mass) were added to the feed solution, and the reaction was carried out at 25°C for 1.0 h. The solution was then concentrated by ultrafiltration using a 100 kD membrane, and the solution was replaced with phosphate buffer (pH 7.0). Purification was performed using a hydroxyapatite column, and the target flow-through peak was collected. The flow-through was then concentrated by ultrafiltration using a 100 kD membrane, and the solution was replaced with water for injection. The final product was freeze-dried to obtain capsular polysaccharide with batch number Y5CG12N for subsequent analysis.

[0171] Comparative Example 2: Purification of capsular polysaccharide from control type 5 pneumococcus

[0172] Take 10 L of Pneumococcus type 5 fermentation broth and add sodium deoxycholate to a final concentration of 0.2% to obtain the lysis buffer. Add acetic acid to the lysis buffer to adjust the pH to 3.2, centrifuge to collect the supernatant, and adjust the pH of the supernatant to 7 using sodium hydroxide. Filter through a 0.45 μm membrane, concentrate using a 100 kD membrane pack, and replace the solution with phosphate buffer (pH 7.0). Add CTAB to the feed solution to a final concentration of 2%, centrifuge to collect the precipitate. Dissolve the precipitate with 1 L of 0.20 mol / L sodium chloride, centrifuge to collect the supernatant. Add sodium iodide to a final concentration of 0.5%, centrifuge to collect the supernatant. Filter through a 0.45 μm membrane, concentrate using a 100 kD membrane pack, and replace the solution with phosphate buffer (pH 7.0). Purify using a hydroxyapatite column and collect the target flow-through peak. Concentrate the flow-through using a 100 kD membrane pack, and replace the solution with water for injection. The final product was freeze-dried to obtain capsular polysaccharide with batch number Y5CI25C for subsequent testing.

[0173] Example 5: Detection of capsular polysaccharides from pneumococcus type 5

[0174] 5.1 1 H NMR structural analysis

[0175] The control type 5 pneumococcal capsular polysaccharide purified in Comparative Example 2 and the type 5 pneumococcal capsular polysaccharide purified in Example 4 were dissolved in heavy water and then lyophilized. This process was repeated three times. Nuclear magnetic resonance (NMR) spectroscopy analysis was performed according to the method described in section 2.1 of Example 2.

[0176] Pneumococcal capsular polysaccharide 1 The H NMR spectrum results are shown in Figure 2 . 1 The characteristic peak of C polysaccharide is shown at 3.22 ppm in the 1H NMR spectrum. Figure 2 In section A, the control polysaccharide Y5CI25C from pneumococcus type 5 is shown. Figure 2 In the diagram, B represents the purified capsular polysaccharide Y5CG12N. Compared to the control pneumococcal capsular polysaccharide chromatogram, the characteristic peak area of ​​the C polysaccharide in the purified capsular polysaccharide obtained by the method of this invention is reduced, while the other structural characteristic peaks remain consistent. According to the C polysaccharide content calculation formula, the C polysaccharide content of the control capsular polysaccharide is 1.33%, while the C polysaccharide content of the capsular polysaccharide purified by the method of this invention is 0.15%. In summary, this indicates that the method of this invention can specifically reduce the C polysaccharide impurity content.

[0177] 5.2 Immunomodulation

[0178] Referring to the method in 2.2 of Example 2, an agar plate was prepared using sera containing polysaccharide specific to type 5 pneumococcus capsularis for an immunodiffusion experiment. The change in polysaccharide antigenicity was determined by measuring the diameter of the diffusion ring.

[0179] The results of immunodiffusion of Pneumococcal serotype 5 capsular polysaccharide are shown in Table 7. The diffusion ring diameter of the purified Pneumococcal serotype 5 capsular polysaccharide of this invention is comparable to that of the control Pneumococcal serotype 5 capsular polysaccharide, and slightly superior to that of the control capsular polysaccharide. This indicates that the purification method of this invention has no negative impact on the antigenicity of the capsular polysaccharide, and its effect is slightly better than that of the control group.

[0180] Table 7: Results of Immunosuppressive Monodiffusion of Pneumococcal Capsular Polysaccharide in Type 5 Pneumococcus

[0181] ;

[0182] 5.3 Physicochemical Testing

[0183] 5.3.1 Referring to the method in 2.3 of Example 2, the protein content, nucleic acid content, total nitrogen content, phosphorus content, molecular weight determination (using a CL-2B agarose gel chromatography column), and aminohexose content of the pneumococcal polysaccharide purified in Example 4 and the control pneumococcal polysaccharide purified in Comparative Example 2 were determined.

[0184] 5.3.2 Detection of uronic acid content

[0185] Prepare 3 ml of a 0.25 mg / ml pneumococcal capsular polysaccharide solution using purified water, and 200 ml of a 100 μg / ml D-uronic acid reference standard stock solution, using purified water for serial dilution. Take 1 ml of each concentration of the reference standard solution and the prepared polysaccharide sample solution into glass-stopped test tubes, with two tubes for parallel determination of the polysaccharide sample. While stirring, add 5 ml of 0.955% borate-sulfuric acid solution to each tube, stopper, and incubate at 100°C for 15 min. After cooling to room temperature, add 0.2 ml of 0.125% carbazole-ethanol solution, incubate at 100°C for 15 min, and after cooling to room temperature, read the absorbance value of each tube at 530 nm. Perform linear regression on the corresponding absorbance values ​​of the D-uronic acid reference standard solution series concentrations to obtain the regression equation. Substitute the absorbance of the polysaccharide sample into the regression equation, and calculate the uronic acid content based on the dilution factor. The percentage of uronic acid content is then calculated based on the dry weight of the polysaccharide.

[0186] The test results are shown in Table 8. All indicators of the purified pneumococcal capsular polysaccharide samples obtained in Example 4 and Comparative Example 2 met the relevant standards stipulated in the pharmacopoeia.

[0187] Table 8: Physicochemical Detection Results of Polysaccharides from Pneumococcal Capsule Type 5

[0188] ;

[0189] Example 6: Optimization of purification process for pneumococcal capsular polysaccharides of type 5

[0190] 6.1 Acetic acid concentration

[0191] The method of this invention was used to isolate and purify capsular polysaccharides from Streptococcus pneumoniae type 5. In the C-polysaccharide removal step, the final mass concentration of acetic acid added to each group was 1.0%, 2.0%, 3.0%, and 4.0%, respectively. The remaining steps were the same as in Example 4. The lyophilized polysaccharides were harvested and then subjected to… 1 H NMR detection. 1 The characteristic peak of C polysaccharide (phosphocholine peak) was observed at 3.22 ppm in the 1H NMR spectrum. The content of C polysaccharide was quantified based on the characteristic peak of type 5 capsular polysaccharide, and the results are shown in Table 9.

[0192] Table 9: Effect of acetic acid concentration on the C-polysaccharide content in purified Streptococcus pneumoniae capsular polysaccharide.

[0193] ;

[0194] The test results show that the C-polysaccharide content in the obtained capsular polysaccharide decreases with increasing acetic acid concentration. Purification using 3.0% and 4.0% acetic acid yielded capsular polysaccharides with comparable C-polysaccharide content. Considering both purification efficiency and economic cost, the preferred acetic acid concentration for the C-polysaccharide removal step in the purification process of Streptococcus pneumoniae capsular polysaccharide is 3.0%.

[0195] 6.2 Sodium nitrite concentration

[0196] The method of this invention was used to isolate and purify capsular polysaccharides from Streptococcus pneumoniae type 5. In the C polysaccharide removal step, the final mass concentrations of sodium nitrite added to each group were 0.5%, 1.0%, 1.5%, and 2.0%, respectively. The remaining steps were the same as in Example 4. The lyophilized polysaccharides were harvested and then subjected to… 1 H NMR detection. 1 The characteristic peak of C polysaccharide (phosphocholine peak) was observed at 3.22 ppm in the 1H NMR spectrum. The content of C polysaccharide was quantified based on the characteristic peak of type 5 capsular polysaccharide, and the results are shown in Table 10.

[0197] Table 10: Effect of sodium nitrite concentration on the C-polysaccharide content in purified Streptococcus pneumoniae capsular polysaccharide.

[0198] ;

[0199] The test results show that the C-polysaccharide content in the obtained capsular polysaccharide decreases with increasing sodium nitrite concentration. Purification using 1.0%, 1.5%, and 2.0% sodium nitrite yielded similar C-polysaccharide contents in the obtained capsular polysaccharides. Considering both purification efficiency and economic cost, the preferred sodium nitrite concentration for the C-polysaccharide removal step in the purification process of Streptococcus pneumoniae capsular polysaccharide is 1.0%.

[0200] 6.3 Reaction Time

[0201] The method of this invention was used to isolate and purify capsular polysaccharides from Streptococcus pneumoniae type 5. The reaction times for the C polysaccharide removal step in each group were 0.5 h, 1.0 h, 1.5 h, and 2.0 h, respectively. The remaining steps were the same as in Example 4. The lyophilized polysaccharides were harvested and then subjected to... 1 HNMR detection. 1 The characteristic peak of C polysaccharide (phosphocholine peak) was observed at 3.22 ppm in the 1H NMR spectrum. The content of C polysaccharide was quantified based on the characteristic peak of type 5 capsular polysaccharide, and the results are shown in Table 11.

[0202] Table 11: Effect of reaction time on the C-polysaccharide content in the purified Streptococcus pneumoniae capsular polysaccharide.

[0203] ;

[0204] The test results show that the C-polysaccharide content in the obtained capsular polysaccharide decreases with increasing reaction time. The C-polysaccharide content in the obtained capsular polysaccharide is comparable between reactions of 1.0 h, 1.5 h, and 2.0 h. Considering both purification efficiency and economic cost, the optimal reaction time for the C-polysaccharide removal step in the purification process of Streptococcus pneumoniae capsular polysaccharide is 1.0 h.

[0205] 6.4 Reaction Temperature

[0206] The method of this invention was used to isolate and purify capsular polysaccharides from Streptococcus pneumoniae type 5. The reaction temperatures for the C polysaccharide removal step in each group were 4.0℃, 20.0℃, 25.0℃, and 37.0℃, respectively. The remaining steps were the same as in Example 4. The lyophilized polysaccharides were harvested and then subjected to... 1 H NMR detection. 1 The characteristic peak of C polysaccharide (phosphocholine peak) was observed at 3.22 ppm in the 1H NMR spectrum. The content of C polysaccharide was quantified based on the characteristic peak of type 5 capsular polysaccharide, and the results are shown in Table 12.

[0207] Table 12: Effect of reaction temperature on the C polysaccharide content in the purified Streptococcus pneumoniae capsular polysaccharide.

[0208] ;

[0209] The test results show that the C-polysaccharide content in the obtained capsular polysaccharide decreases with increasing reaction temperature. The C-polysaccharide content in the obtained capsular polysaccharide is comparable between 25.0℃ and 37.0℃. Considering both purification efficiency and economic cost, the optimal reaction temperature for the C-polysaccharide removal step in the purification process of Streptococcus pneumoniae capsular polysaccharide is 25.0℃.

[0210] Example 7: Purification of capsular polysaccharides from type 7F pneumococcus

[0211] Take 10 L of 7F pneumococcal fermentation broth and add sodium deoxycholate to a final concentration of 0.2% to obtain a lysate. Add acetic acid to the lysate to adjust the pH to 5, centrifuge to collect the supernatant, and adjust the pH of the supernatant to 7 using sodium hydroxide. Filter through a 0.45 μm membrane and concentrate using ultrafiltration with a 100 kD membrane pack, then replace the solution with phosphate buffer (pH 7.0). Add CTAB to the feed solution to a final concentration of 1.5%, centrifuge to collect the supernatant, add sodium chloride to a final concentration of 0.20 mol / L, centrifuge to collect the supernatant. Add sodium iodide to a final concentration of 0.5%, centrifuge to collect the supernatant. Filter through a 0.45 μm membrane and concentrate using ultrafiltration with a 100 kD membrane pack, then replace the solution with phosphate buffer (pH 7.0). Acetic acid (1.0% by mass) and sodium nitrite (3.0% by mass) were added to the feed solution, and the reaction was carried out at 25.0℃ for 8.0 h. The solution was then concentrated by ultrafiltration using a 100 kD membrane, and the solution was replaced with phosphate buffer (pH 7.0). Purification was performed using a hydroxyapatite column, and the target flow-through peak was collected. The flow-through was then concentrated by ultrafiltration using a 100 kD membrane, and the solution was replaced with water for injection. The final product was freeze-dried to obtain capsular polysaccharide with batch number Y7FCL15N for subsequent analysis.

[0212] Comparative Example 3: Purification of capsular polysaccharide from control type 7F pneumococcus

[0213] Take 10 L of 7F pneumococcal fermentation broth and add sodium deoxycholate to a final concentration of 0.2% to obtain the lysate. Add acetic acid to the lysate to adjust the pH to 5, centrifuge to collect the supernatant, and adjust the pH of the supernatant to 7 using sodium hydroxide. Filter through a 0.45 μm membrane, concentrate using a 100 kD membrane pack, and replace the solution with phosphate buffer (pH 7.0). Add CTAB to the feed solution to a final concentration of 1.5%, centrifuge to collect the supernatant. Add sodium iodide to a final concentration of 0.5%, centrifuge to collect the supernatant. Filter through a 0.45 μm membrane, concentrate using a 100 kD membrane pack, and replace the solution with phosphate buffer (pH 7.0). Purify using a hydroxyapatite column and collect the target flow-through peak. Concentrate the flow-through using a 100 kD membrane pack and replace the solution with water for injection. The final product was freeze-dried to obtain capsular polysaccharide with batch number Y7FCK22C for subsequent testing.

[0214] Example 8: Detection of capsular polysaccharides from type 7F pneumococcus

[0215] 8.1 1 H NMR structural analysis

[0216] The control 7F type pneumococcal capsular polysaccharide purified in Comparative Example 3 and the 7F type pneumococcal capsular polysaccharide purified in Example 7 were dissolved in heavy water and then lyophilized, repeated three times. Nuclear magnetic resonance spectroscopy analysis was performed according to the method in 2.1 of Example 2.

[0217] 7F type pneumococcal capsular polysaccharide 1 The H NMR spectrum results are shown in Figure 3 . 1 The characteristic peak of C polysaccharide is shown at 3.22 ppm in the 1H NMR spectrum. Figure 3 In section A, the control polysaccharide Y7FCK22C is from pneumococcal capsular polysaccharide type 7F. Figure 3 In the diagram, B represents the purified capsular polysaccharide Y7FCL15N. Compared to the control 7F type pneumococcal capsular polysaccharide chromatogram, the characteristic peak area of ​​the C polysaccharide in the purified capsular polysaccharide obtained by the method of this invention is reduced, while the other structural characteristic peaks remain consistent. According to the C polysaccharide content calculation formula, the C polysaccharide content of the control capsular polysaccharide is 2.33%, while the C polysaccharide content of the purified capsular polysaccharide obtained by the method of this invention is 0.17%. In summary, this indicates that the method of this invention can specifically reduce the C polysaccharide impurity content.

[0218] 8.2 Immunomodulation

[0219] Referring to the method in 2.2 of Example 2, an agar plate was prepared using 7F type pneumococcal capsular polysaccharide-specific serum for an immunodiffusion experiment. The change in polysaccharide antigenicity was determined by measuring the diameter of the diffusion ring.

[0220] The results of immunodiffusion of 7F pneumococcal capsular polysaccharide are shown in Table 13. The diffusion ring diameter of the 7F pneumococcal capsular polysaccharide purified by this invention is comparable to that of the control 7F pneumococcal capsular polysaccharide, and slightly superior. This indicates that the purification method of this invention has no negative impact on the antigenicity of the capsular polysaccharide, and the effect is slightly better than that of the control group.

[0221] Table 13: Results of Immunosuppuration of Pneumococcal Capsular Polysaccharide in Type 7F Pneumococcus

[0222] ;

[0223] 8.3 Physicochemical tests

[0224] 8.3.1 Referring to the method in 2.3 of Example 2, the protein content, nucleic acid content, total nitrogen content, phosphorus content, and molecular weight of the 7F type pneumococcal capsular polysaccharide purified in Example 7 and the control 7F type capsular polysaccharide purified in Comparative Example 3 were determined.

[0225] 8.3.2 Detection of Methylpentose Content

[0226] Prepare 3 ml of a 50 μg / ml solution of 7F pneumococcal capsular polysaccharide using purified water, and 10 ml of a 20 μg / ml stock solution of rhamnose reference standard, both serially diluted with purified water. Take 1 ml of each concentration of reference standard solution and the prepared polysaccharide sample solution into glass-stopped test tubes, with the polysaccharide sample measured in duplicate. In an ice-water bath, with stirring, add 4.5 ml of pre-cooled sulfuric acid solution to each tube, stopper, warm to room temperature, and incubate at 100℃ for 5 min. After cooling to room temperature, add 0.1 ml of 3% mercaptoalanine hydrochloric acid solution, mix well, stopper, and let stand at room temperature for 1.5 h. Read the absorbance (A value) of each tube at wavelengths of 396 nm and 430 nm. A linear regression was performed on the corresponding corrected absorbance values ​​(A396~A430nm) of a series of rhamnose reference solution concentrations to obtain the regression equation. The corrected absorbance of the polysaccharide sample was then substituted into the regression equation, and the methylpentose content was calculated based on the dilution factor. Finally, the percentage content of methylpentose was calculated based on the dry weight of the polysaccharide.

[0227] The test results are shown in Table 14. All indicators of the purified 7F type pneumococcal capsular polysaccharide samples obtained in Example 7 and Comparative Example 3 met the relevant standards stipulated in the pharmacopoeia.

[0228] Table 14: Physicochemical Detection Results of Polysaccharides from the Capsular Polysaccharide Form of Pneumococcus 7F

[0229] ;

[0230] Example 9: Optimization of purification process for 7F pneumococcal capsular polysaccharides

[0231] 9.1 Acetic acid concentration

[0232] The method of this invention was used to separate and purify the capsular polysaccharide of Streptococcus pneumoniae type 7F. In the C polysaccharide removal step, the final mass concentration of acetic acid added to each group was 0.5%, 1.0%, 1.5%, and 2.0%, respectively. The remaining steps were the same as in Example 7. The lyophilized polysaccharides were harvested and then subjected to… 1 H NMR detection. 1 The characteristic peak of C polysaccharide (phosphocholine peak) was observed at 3.22 ppm in the 1H NMR spectrum. The content of C polysaccharide was quantified based on the characteristic peak of 7F capsular polysaccharide, and the results are shown in Table 15.

[0233] Table 15: Effect of acetic acid concentration on the C polysaccharide content in purified 7F pneumococcal capsular polysaccharide

[0234] ;

[0235] The test results show that the C-polysaccharide content in the obtained capsular polysaccharide decreases with increasing acetic acid concentration. Purification using 1.0%, 1.5%, and 2.0% acetic acid yielded similar C-polysaccharide contents in the obtained capsular polysaccharides. Considering both purification efficiency and economic cost, the preferred acetic acid concentration for the C-polysaccharide removal step in the purification process of Streptococcus pneumoniae type 7F capsular polysaccharide is 1.0%.

[0236] 9.2 Sodium nitrite concentration

[0237] The method of this invention was used to separate and purify the capsular polysaccharide of Streptococcus pneumoniae type 7F. In the C polysaccharide removal step, the final mass concentrations of sodium nitrite added to each group were 1.0%, 2.0%, 3.0%, and 4.0%, respectively. The remaining steps were the same as in Example 7. The lyophilized polysaccharides were harvested and then subjected to… 1 H NMR detection. 1 The characteristic peak of C polysaccharide (phosphocholine peak) was observed at 3.22 ppm in the 1H NMR spectrum. The content of C polysaccharide was quantified based on the characteristic peak of 7F capsular polysaccharide, and the results are shown in Table 16.

[0238] Table 16: Effect of sodium nitrite concentration on the C polysaccharide content in purified 7F pneumococcal capsular polysaccharide

[0239] ;

[0240] The test results show that the C-polysaccharide content in the obtained capsular polysaccharide decreases with increasing sodium nitrite concentration. Purification using 3.0% and 4.0% sodium nitrite yielded capsular polysaccharides with comparable C-polysaccharide content. Considering both purification efficiency and economic cost, the preferred sodium nitrite concentration for the C-polysaccharide removal step in the purification process of Streptococcus pneumoniae type 7F capsular polysaccharide is 3.0%.

[0241] 9.3 Reaction Time

[0242] The method of this invention was used to isolate and purify the capsular polysaccharide of Streptococcus pneumoniae type 7F. The reaction times for the C polysaccharide removal step in each group were 4.0 h, 6.0 h, 8.0 h, and 10.0 h, respectively. The remaining steps were the same as in Example 7. The lyophilized polysaccharide was harvested and subjected to... 1 H NMR detection. 1 The characteristic peak of C polysaccharide (phosphocholine peak) was observed at 3.22 ppm in the 1H NMR spectrum. The content of C polysaccharide was quantified based on the characteristic peak of 7F capsular polysaccharide, and the results are shown in Table 17.

[0243] Table 17: Effect of reaction time on the C polysaccharide content in the purified 7F type Streptococcus pneumoniae capsular polysaccharide

[0244] ;

[0245] The test results show that the C-polysaccharide content in the obtained capsular polysaccharide decreases with increasing reaction time. The C-polysaccharide content in the obtained capsular polysaccharide is comparable between reactions of 8.0 h and 10.0 h. Considering both purification efficiency and economic cost, the optimal reaction time for the C-polysaccharide removal step in the purification process of Streptococcus pneumoniae type 7F capsular polysaccharide is 8.0 h.

[0246] 9.4 Reaction Temperature

[0247] The method of this invention was used to separate and purify the capsular polysaccharide of Streptococcus pneumoniae type 7F. The reaction temperatures for the C polysaccharide removal step in each group were 4.0℃, 20.0℃, 25.0℃, and 37.0℃, respectively. The remaining steps were the same as in Example 7. The lyophilized polysaccharide was harvested and then subjected to… 1 H NMR detection. 1 The characteristic peak of C polysaccharide (phosphocholine peak) was observed at 3.22 ppm in the 1H NMR spectrum. The content of C polysaccharide was quantified based on the characteristic peak of 7F capsular polysaccharide, and the results are shown in Table 18.

[0248] Table 18: Effect of reaction temperature on the C polysaccharide content in the purified 7F type Streptococcus pneumoniae capsular polysaccharide

[0249] ;

[0250] The test results show that the C-polysaccharide content in the obtained capsular polysaccharide decreases with increasing reaction temperature. The C-polysaccharide content is comparable between 25.0℃ and 37.0℃. Considering both purification efficiency and economic cost, the optimal reaction temperature for the C-polysaccharide removal step in the purification process of Streptococcus pneumoniae type 7F capsular polysaccharide is 25.0℃.

[0251] Example 10: Purification of capsular polysaccharides from type 12F pneumococcus

[0252] Take 10 L of *Streptococcus pneumoniae* fermentation broth of type 12F and add sodium deoxycholate to a final concentration of 0.15% to obtain a lysate. Add acetic acid to the lysate to adjust the pH to 4.5, centrifuge to collect the supernatant, and adjust the pH of the supernatant to 7 using sodium hydroxide. Filter through a 0.45 μm membrane, concentrate using a 100 kD membrane pack, and replace the solution with phosphate buffer (pH 7.0). Add CTAB to the feed solution to a final concentration of 1.2%, centrifuge to collect the precipitate. Dissolve the precipitate with 1 L of 0.20 mol / L sodium chloride, centrifuge to collect the supernatant. Add sodium iodide to a final concentration of 0.5%, centrifuge to collect the supernatant. Filter through a 0.45 μm membrane, concentrate using a 100 kD membrane pack, and replace the solution with phosphate buffer (pH 7.0). Acetic acid (3.0% by mass) and sodium nitrite (5.0% by mass) were added to the feed solution, and the reaction was carried out at 25°C for 10 h. The solution was then concentrated by ultrafiltration using a 100 kD membrane, and the solution was replaced with phosphate buffer (pH 7.0). Purification was performed using a hydroxyapatite column, and the target flow-through peak was collected. The flow-through was then concentrated by ultrafiltration using a 100 kD membrane, and the solution was replaced with water for injection. The final product was freeze-dried to obtain capsular polysaccharide with batch number Y12FDC26N for subsequent analysis.

[0253] Comparative Example 4: Purification of capsular polysaccharide from control type 12F pneumococcus

[0254] Take 10 L of *Streptococcus pneumoniae* fermentation broth of type 12F and add sodium deoxycholate to a final concentration of 0.15%. Obtain the lysate. Add acetic acid to the lysate to adjust the pH to 4.5, centrifuge to collect the supernatant, and adjust the pH of the supernatant to 7 using sodium hydroxide. Filter through a 0.45 μm membrane, concentrate using a 100 kD membrane pack, and replace the solution with phosphate buffer (pH 7.0). Add CTAB to the feed solution to a final concentration of 1.2%, centrifuge to collect the precipitate. Dissolve the precipitate with 1 L of 0.20 mol / L sodium chloride, centrifuge to collect the supernatant. Add sodium iodide to a final concentration of 0.5%, centrifuge to collect the supernatant. Filter through a 0.45 μm membrane, concentrate using a 100 kD membrane pack, and replace the solution with phosphate buffer (pH 7.0). Purify using a hydroxyapatite column and collect the target flow-through peak. Concentrate the flow-through using a 100 kD membrane pack, and replace the solution with water for injection. The final product was freeze-dried to obtain capsular polysaccharide with batch number Y12FDA28C for subsequent testing.

[0255] Example 11: Detection of capsular polysaccharides from type 12F pneumococcus

[0256] 11.1 1 H NMR structural analysis

[0257] The control 12F type pneumococcal capsular polysaccharide purified in Comparative Example 4 and the 12F type pneumococcal capsular polysaccharide purified in Example 10 were dissolved in heavy water and then lyophilized, repeated three times. Nuclear magnetic resonance spectroscopy analysis was performed according to the method in 2.1 of Example 2.

[0258] 12F type pneumococcal capsular polysaccharide 1 The H NMR spectrum results are shown in Figure 4 . 1 The characteristic peak of C polysaccharide is shown at 3.22 ppm in the 1H NMR spectrum. Figure 4 In section A, the control polysaccharide Y12FDA28C is from the capsular polysaccharide of type 12F pneumococcus. Figure 4 In the diagram, B represents the purified capsular polysaccharide Y12FDC26N. Compared to the control 12F type pneumococcal capsular polysaccharide spectrum, the characteristic peak area of ​​the C polysaccharide in the purified capsular polysaccharide obtained by the method of this invention is reduced, while the other structural characteristic peaks remain consistent. According to the C polysaccharide content calculation formula, the C polysaccharide content of the control capsular polysaccharide is 3.83%, while the C polysaccharide content of the purified capsular polysaccharide obtained by the method of this invention is 0.33%. In summary, this indicates that the method of this invention can specifically reduce the C polysaccharide impurity content.

[0259] 11.2 Immunomodulation

[0260] Referring to the method in 2.2 of Example 2, an agar plate was prepared using 12F type pneumococcal capsular polysaccharide-specific serum for immunodiffusion experiment. The change in polysaccharide antigenicity was determined by measuring the diameter of the diffusion ring.

[0261] The results of immunodiffusion of the 12F pneumococcal capsular polysaccharide are shown in Table 19. The diffusion ring diameter of the 12F pneumococcal capsular polysaccharide purified by this invention is comparable to that of the control 12F pneumococcal capsular polysaccharide, and slightly superior. This indicates that the purification method of this invention has no negative impact on the antigenicity of the capsular polysaccharide, and the effect is slightly better than that of the control group.

[0262] Table 19: Results of Immunosuppuration of Pneumococcal Capsular Polysaccharide in Type 12F Pneumococcus

[0263] ;

[0264] 11.3 Physicochemical Testing:

[0265] Referring to the method in 2.3 of Example 2, the protein content, nucleic acid content, total nitrogen content, phosphorus content, molecular weight, and aminohexose content of the 12F type Streptococcus pneumoniae capsular polysaccharide purified in Example 10 and the control 12F type capsular polysaccharide purified in Comparative Example 4 were determined.

[0266] The test results are shown in Table 20. All indicators of the purified 12F type pneumococcal capsular polysaccharide samples obtained in Example 10 and Comparative Example 4 met the relevant standards stipulated in the pharmacopoeia.

[0267] Table 20: Physicochemical Detection Results of Polysaccharides from the Capsular Polysaccharide Form of Pneumococcus 12F

[0268] ;

[0269] Example 12: Optimization of purification process for 12F pneumococcal capsular polysaccharides

[0270] 12.1 Acetic acid concentration

[0271] The method of this invention was used to separate and purify the capsular polysaccharide of Streptococcus pneumoniae type 12F. In the C polysaccharide removal step, the final mass concentration of acetic acid added to each group was 1.0%, 2.0%, 3.0%, and 4.0%, respectively. The remaining steps were the same as in Example 10. The lyophilized polysaccharides were harvested and then subjected to… 1 H NMR detection. 1 The characteristic peak of C polysaccharide (phosphocholine peak) was observed at 3.22 ppm in the 1H NMR spectrum. The content of C polysaccharide was quantified based on the characteristic peak of 12F capsular polysaccharide, and the results are shown in Table 21.

[0272] Table 21: Effect of acetic acid concentration on the C-polysaccharide content in purified 12F pneumococcal capsular polysaccharide

[0273] ;

[0274] The test results show that the C-polysaccharide content in the obtained capsular polysaccharide decreases with increasing acetic acid concentration. Purification using 3.0% and 4.0% acetic acid yielded capsular polysaccharides with comparable C-polysaccharide content. Considering both purification efficiency and economic cost, the preferred acetic acid concentration for the C-polysaccharide removal step in the purification process of Streptococcus pneumoniae type 12F capsular polysaccharide is 3.0%.

[0275] 12.2 Sodium nitrite concentration

[0276] The method of this invention was used to separate and purify the capsular polysaccharide of Streptococcus pneumoniae type 12F. In the C polysaccharide removal step, the final mass concentrations of sodium nitrite added to each group were 1.0%, 2.5%, 5.0%, and 7.5%, respectively. The remaining steps were the same as in Example 10. The lyophilized polysaccharides were harvested and subjected to... 1 H NMR detection. 1 The characteristic peak of C polysaccharide (phosphocholine peak) was observed at 3.22 ppm in the 1H NMR spectrum. The content of C polysaccharide was quantified based on the characteristic peak of 12F capsular polysaccharide, and the results are shown in Table 22.

[0277] Table 22: Effect of sodium nitrite concentration on the C-polysaccharide content in purified 12F pneumococcal capsular polysaccharide

[0278] ;

[0279] The test results show that the C-polysaccharide content in the obtained capsular polysaccharide decreases with increasing sodium nitrite concentration. Purification using 5.0% and 7.5% sodium nitrite yielded capsular polysaccharides with comparable C-polysaccharide content. Considering both purification efficiency and economic cost, the preferred sodium nitrite concentration for the C-polysaccharide removal step in the purification process of Streptococcus pneumoniae type 12F capsular polysaccharide is 5.0%.

[0280] 12.3 Reaction Time

[0281] The method of this invention was used to isolate and purify the capsular polysaccharide of Streptococcus pneumoniae type 12F. The reaction times for the C polysaccharide removal step in each group were 6.0 h, 8.0 h, 10.0 h, and 12.0 h, respectively. The remaining steps were the same as in Example 10. The lyophilized polysaccharide was harvested and subjected to... 1 H NMR detection. 1 The characteristic peak of C polysaccharide (phosphocholine peak) was observed at 3.22 ppm in the 1H NMR spectrum. The content of C polysaccharide was quantified based on the characteristic peak of 12F capsular polysaccharide, and the results are shown in Table 23.

[0282] Table 23: Effect of reaction time on the C polysaccharide content in the purified 12F type Streptococcus pneumoniae capsular polysaccharide

[0283] ;

[0284] The test results show that the C-polysaccharide content in the obtained capsular polysaccharide decreases with increasing reaction time. The C-polysaccharide content in the obtained capsular polysaccharide is comparable between reactions of 10.0 h and 12.0 h. Considering both purification efficiency and economic cost, the optimal reaction time for the C-polysaccharide removal step in the purification process of Streptococcus pneumoniae capsular polysaccharide is 10.0 h.

[0285] 12.4 Reaction Temperature

[0286] The method of this invention was used to separate and purify the capsular polysaccharide of Streptococcus pneumoniae type 12F. The reaction temperatures for the C polysaccharide removal step in each group were 4.0℃, 20.0℃, 25.0℃, and 37.0℃, respectively. The remaining steps were the same as in Example 10. The lyophilized polysaccharide was harvested and then subjected to… 1 H NMR detection. 1 The characteristic peak of C polysaccharide (phosphocholine peak) was observed at 3.22 ppm in the 1H NMR spectrum. The content of C polysaccharide was quantified based on the characteristic peak of 12F capsular polysaccharide, and the results are shown in Table 24.

[0287] Table 24: Effect of reaction temperature on the C-polysaccharide content in the purified Streptococcus pneumoniae capsular polysaccharide.

[0288] ;

[0289] The test results show that the C-polysaccharide content in the obtained capsular polysaccharide decreases with increasing reaction temperature. The C-polysaccharide content is comparable between 25.0℃ and 37.0℃. Considering both purification efficiency and economic cost, the optimal reaction temperature for the C-polysaccharide removal step in the purification process of Streptococcus pneumoniae type 12F capsular polysaccharide is 25.0℃.

[0290] The technical solutions of the present invention are not limited to the specific embodiments described above. All technical modifications made according to the technical solutions of the present invention fall within the protection scope of the present invention, and the various technical features of the specific embodiments described in the present invention can be appropriately combined as needed.

Claims

1. A method for purifying pneumococcal capsular polysaccharide, the method comprising the following steps: (1) Treating pneumococcus to obtain a solution containing pneumococcal capsular polysaccharide; and (2) The solution containing pneumococcal capsular polysaccharide is subjected to C-polysaccharide removal treatment. in, In step (2), the C polysaccharide removal treatment includes treating the solution containing pneumococcal capsular polysaccharide with a combination reagent, wherein the combination reagent consists of an acid and a nitrite, wherein the mass concentration of the acid is 0.1%-20% and the mass concentration of the nitrite is 0.1%-20%; The serotype of the pneumococcus is at least one of the following: type 2, type 3, type 4, type 5, type 6A, type 6B, type 6C, type 6D, type 7A, type 7C, type 7F, type 8, type 9N, type 9V, type 10A, type 10B, type 11A, type 11E, type 12F, type 13, type 14, type 15A, type 15B, type 15C, type 15F, type 16F, type 17F, type 18C, type 19A, type 19F, type 20, type 21, type 22F, type 23A, type 23B, type 23F, type 24A, type 24B, type 24F, type 25A, type 25F, type 27, type 28F, type 31, type 33A, type 33F, type 34, type 35B, type 37, and type 38.

2. The purification method according to claim 1, characterized in that, The mass ratio of the acid to the nitrite is 1: (0.01-100)； The acid includes at least one of acetic acid, phosphoric acid, carbonic acid, citric acid, hydrochloric acid, sulfuric acid, perchloric acid, or tetrafluoroboric acid; The nitrite includes at least one of sodium nitrite, potassium nitrite, calcium nitrite, or ammonium nitrite.

3. The purification method according to claim 1, characterized in that, The mass ratio of the acid to the nitrite is 1: (0.1-20)； The mass concentration of the acid is 1%-10%; The mass concentration of the nitrite is 1%-10%.

4. The purification method according to claim 1, characterized in that, The temperature for the C polysaccharide removal treatment is 0℃-50℃; The removal time for the C polysaccharide is 0.1h-24h.

5. The purification method according to any one of claims 1-4, characterized in that, Step (1) includes the following steps: (1-1) The bacterial suspension containing pneumococcus was lysed to obtain bacterial lysate; (1-2) The lysis solution is subjected to acid precipitation treatment, and the first supernatant is collected; (1-3) Add CTAB to the first supernatant and collect the second supernatant, or add CTAB to the first supernatant, collect the precipitate, dissolve the precipitate with sodium chloride, and then collect the second supernatant. (1-4) Iodine precipitation is performed on the second supernatant, and the third supernatant is collected to obtain the solution containing pneumococcal capsular polysaccharide.

6. The purification method according to claim 5, characterized in that, In step (1-1), the pyrolysis treatment method includes at least one of chemical pyrolysis, physical pyrolysis, or biological pyrolysis; and / or In steps (1-2), the acid precipitation treatment includes adding an acidic reagent to the lysis buffer, adjusting the pH to acidic, and then collecting the supernatant; and / or Steps (1-2) further include concentrating the supernatant after acid precipitation treatment, changing the solution, and collecting the first supernatant; and / or In steps (1-3), the final mass concentration of CTAB is 0.1%-5%; and / or In steps (1-3), the acidic pneumococcal capsular polysaccharide is harvested and precipitated, dissolved in sodium chloride solution, and the second supernatant is collected; the neutral pneumococcal capsular polysaccharide is directly harvested from the second supernatant; and / or In steps (1-3), the final concentration of sodium chloride is 0.01 mol / L-5 mol / L; and / or In steps (1-4), the iodized salt comprises sodium iodide and / or potassium iodide; and / or In steps (1-4), the final concentration of the iodized salt is 0.01%-5%.

7. The purification method according to any one of claims 1-4, characterized in that, The method further includes purification and drying processes after step (2).

8. The purification method according to claim 7, characterized in that, The purification process includes at least one of membrane filtration, depth filtration, and chromatography. The drying process employs freeze drying.

9. The purification method according to claim 8, characterized in that, The purification process includes sequentially performing a first ultrafiltration concentration treatment, a chromatography treatment, and a second ultrafiltration concentration treatment on the solution after the removal of the C polysaccharide; the first ultrafiltration concentration treatment and / or the second ultrafiltration concentration treatment are performed using a 30-300KD membrane pack.

10. The purification method according to any one of claims 1-4, characterized in that, A solution containing capsular polysaccharide of type 4 pneumococcus is subjected to C-polysaccharide removal treatment, wherein the mass concentration of the acid is 1%-8%; and / or the mass concentration of the nitrite is 1%-5%; and / or the temperature of the C-polysaccharide removal treatment is 4℃-50℃; and / or the time of the C-polysaccharide removal treatment is 5h-20h; A solution containing capsular polysaccharide of type 5 pneumococcus is subjected to C-polysaccharide removal treatment, wherein the mass concentration of the acid is 1%-4%; and / or the mass concentration of the nitrite is 0.5%-2%; and / or the temperature of the C-polysaccharide removal treatment is 4℃-40℃; and / or the time of the C-polysaccharide removal treatment is 0.5h-2h. A solution containing 7F type pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the mass concentration of the acid is 0.5%-2%; and / or the mass concentration of the nitrite is 1%-4%; and / or the temperature of the C polysaccharide removal treatment is 4℃-40℃; and / or the time of the C polysaccharide removal treatment is 4h-10h. A solution containing 12F type pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the mass concentration of the acid is 1%-4%; and / or the mass concentration of the nitrite is 1%-8%; and / or the temperature of the C polysaccharide removal treatment is 4℃-40℃; and / or the time of the C polysaccharide removal treatment is 6h-12h.

11. The purification method according to any one of claims 1-4, characterized in that, A solution containing capsular polysaccharide of type 4 pneumococcus is subjected to C-polysaccharide removal treatment, wherein the mass concentration of the acid is 4.5%-5.5%; and / or the mass concentration of the nitrite is 2.5%-3.5%; and / or the temperature of the C-polysaccharide removal treatment is 35℃-40℃; and / or the time of the C-polysaccharide removal treatment is 14h-16h. A solution containing capsular polysaccharide of type 5 pneumococcus is subjected to C-polysaccharide removal treatment, wherein the mass concentration of the acid is 2.5%-3.5%; and / or the mass concentration of the nitrite is 0.8%-1.2%; and / or the temperature of the C-polysaccharide removal treatment is 22℃-30℃; and / or the time of the C-polysaccharide removal treatment is 0.8h-1.2h. A solution containing 7F type pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the mass concentration of the acid is 0.8%-1.2%; and / or the mass concentration of the nitrite is 2.5%-3.5%; and / or the temperature of the C polysaccharide removal treatment is 22℃-30℃; and / or the time of the C polysaccharide removal treatment is 7h-9h. A solution containing 12F pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the mass concentration of the acid is 2.5%-3.5%; and / or the mass concentration of the nitrite is 4.5%-5.5%; and / or the temperature of the C polysaccharide removal treatment is 22℃-30℃; and / or the time of the C polysaccharide removal treatment is 9h-11h.

12. The pneumococcal capsular polysaccharide prepared by any one of claims 1-11.

13. The pneumococcal capsular polysaccharide according to claim 12, characterized in that, The content of C-polysaccharide in the pneumococcal capsular polysaccharide is less than 5%.

14. The use of the pneumococcal capsular polysaccharide according to claim 12 or 13 in the preparation of products containing pneumococcal capsular polysaccharide.

15. The application according to claim 14, characterized in that, The product containing pneumococcal capsular polysaccharide includes pneumococcal vaccine.

16. The application according to claim 15, characterized in that, The pneumococcal vaccine includes pneumococcal capsular polysaccharide vaccine or pneumococcal capsular polysaccharide conjugate vaccine.

17. The application according to claim 15, characterized in that, The pneumococcal vaccine includes monovalent or multivalent vaccines.

18. A vaccine composition comprising pneumococcal capsular polysaccharide prepared by any one of claims 1-11.

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