Purification method and application of pneumococcal capsular polysaccharide

Abstract

The invention discloses a method for purifying pneumococcus capsular polysaccharide, which comprises the following steps: (1) treating pneumococcus to obtain a solution containing the pneumococcus capsular polysaccharide; and (2) carrying out C polysaccharide removal treatment on the solution containing the pneumococcus capsular polysaccharide. According to the purification method disclosed by the invention, the use of toxic or dangerous reagents such as phenol and ethanol is avoided, and the content of C polysaccharide impurities in the pneumococcal capsular polysaccharide can be further reduced on the basis of removing impurities such as protein and nucleic acid, so that the high-quality pneumococcal capsular polysaccharide meeting the requirements is obtained. The method has the advantages of safety, environmental protection, simplicity in operation, low cost, good impurity removal effect and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a method for purifying pneumococcal capsular polysaccharide. Background Art

[0002] Pneumococcus is a Gram-positive bacterium. Its infections are common in infants, the elderly, and people with underlying diseases, and it is the main cause of diseases such as pneumonia, meningitis, and bacteremia. Pneumococcus can not only cause disease alone, but also often cause mixed infections with other viruses and bacteria, such as influenza virus, respiratory syncytial virus, Haemophilus influenzae, Mycoplasma pneumoniae, Mycobacterium tuberculosis, etc., endangering the lives of patients. Pneumococcal vaccine is the most effective means to prevent pneumococcal infections. The WHO has listed pneumococcal diseases as diseases "of very high priority" (Very high priorities) to be prevented by vaccines.

[0003] Capsular polysaccharide is the most important virulence factor of pneumococcus. The currently marketed pneumococcal polysaccharide vaccine (PPV) and pneumococcal conjugate vaccine (PCV) are both designed and developed based on pneumococcal capsular polysaccharide. Extracting high-quality capsular polysaccharide has always been a challenge. Now, with the continuous increase in the number of valences of pneumococcal vaccines, the residual impurities in the capsular polysaccharide will also continue to accumulate, which brings more challenges to the quality control of pneumococcal vaccines.

[0004] Currently, researchers have developed various methods for purifying capsular polysaccharide, such as ethanol fractional precipitation method, phenol extraction method, protease digestion method, acid precipitation combined with activated carbon adsorption method, cetyltrimethylammonium bromide (CTAB) precipitation method, and diethylaminoethyl cellulose (DEAE) resin method, etc. In addition, there are also ethanol fractional precipitation combined with CTAB precipitation method, and various purification methods combined with chromatography method, etc. However, traditional polysaccharide purification methods focus on reducing protein and nucleic acid contamination, and have poor removal effects on heteropolysaccharides such as C polysaccharide. Summary of the Invention

[0005] Aiming at the deficiencies in the prior art, the present invention provides a method for purifying pneumococcal capsular polysaccharide. This purification method avoids the use of toxic or dangerous reagents such as phenol and ethanol, and can further reduce the content of C polysaccharide impurities in pneumococcal capsular polysaccharide on the basis of removing impurities such as protein and nucleic acid, so as to obtain high-quality pneumococcal capsular polysaccharide that meets the requirements. It has the advantages of safety, environmental protection, simple operation, low cost, and good impurity removal effect.

[0006] According to one aspect of the present invention, there is provided 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) performing C polysaccharide removal treatment on the solution containing pneumococcal capsular polysaccharide.

[0007] In some embodiments, in step (2), the C polysaccharide removal treatment comprises treating the solution containing pneumococcal capsular polysaccharide with a combined reagent, the combined reagent comprising an acid and a nitrite.

[0008] 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 therebetween.

[0009] In some embodiments, the acid comprises at least one of acetic acid, phosphoric acid, carbonic acid, citric acid, hydrochloric acid, sulfuric acid, perchloric acid or tetrafluoroboric acid.

[0010] In some embodiments, the mass concentration of the acid is 0.1% - 20%, preferably 1% - 10%, 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 therebetween.

[0011] In some embodiments, the nitrite comprises at least one of sodium nitrite, potassium nitrite, calcium nitrite or ammonium nitrite.

[0012] In some embodiments, the mass concentration of the nitrite is 0.1% - 20%, preferably 1% - 10%, 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 therebetween.

[0013] In some embodiments, the temperature of the C polysaccharide removal treatment is 0°C - 50°C, preferably 25°C - 45°C, more preferably 25°C - 37°C. In some embodiments, the temperature of the C polysaccharide removal treatment is 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C or any value therebetween.

[0014] In some embodiments, the time of the C polysaccharide removal treatment is 0.1 h - 24 h, preferably 1 - 20 h, more preferably 1 - 15 h. In some embodiments, the time of the C polysaccharide removal treatment 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 therebetween.

[0015] In some embodiments, step (1) includes the following steps: (1-1) Subjecting a bacterial solution containing pneumococcus to lysis treatment to obtain a bacterial lysate; (1-2) Subjecting the lysate to acid precipitation treatment and collecting a first supernatant; (1-3) Adding CTAB to the first supernatant and collecting a second supernatant, or adding CTAB to the first supernatant, collecting the precipitate, dissolving the precipitate with sodium chloride, and then collecting the second supernatant; (1-4) Subjecting the second supernatant to iodide salt precipitation treatment and collecting a third supernatant to obtain the solution containing pneumococcal capsular polysaccharide.

[0016] In some embodiments, in step (1-1), the lysis treatment method includes at least one of chemical lysis, physical lysis, or biological lysis.

[0017] In some embodiments, the chemical lysis includes lysis agent lysis and / or alkali lysis.

[0018] In some embodiments, the physical lysis includes high-pressure lysis and / or thermal lysis.

[0019] In some embodiments, the biological lysis includes enzyme lysis.

[0020] In some embodiments, in step (1-2), the acid precipitation treatment includes adding an acidic reagent to the lysate, adjusting the pH value to acidic, and then collecting the supernatant.

[0021] In some embodiments, the acidic reagent includes at least one of acetic acid, phosphoric acid, carbonic acid, citric acid, hydrochloric acid, sulfuric acid, perchloric acid, and tetrafluoroboric acid.

[0022] In some embodiments, the pH value ranges from 2 to 6, preferably from 3 to 5. In some embodiments, the pH value is 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6 or any value therebetween.

[0023] In some embodiments, step (1-2) further includes concentrating and changing the liquid of the supernatant after the acid precipitation treatment, and then collecting the first supernatant.

[0024] In some embodiments, in step (1-3), the final mass concentration of CTAB is 0.1% - 5%, preferably 1% - 3%. In some embodiments, in step (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 therebetween.

[0025] In some embodiments, the pneumococcal capsular polysaccharide includes acidic pneumococcal capsular polysaccharide and / or neutral pneumococcal capsular polysaccharide.

[0026] In some embodiments, the acidic pneumococcal capsular polysaccharide harvests the precipitate, adds a sodium chloride solution to dissolve it, and collects the second supernatant. In some embodiments, the acidic pneumococcus includes, but is not limited to, pneumococcus types 2, 3, 4, 5, etc.

[0027] In some embodiments, the neutral pneumococcal capsular polysaccharide directly harvests the second supernatant. In some embodiments, the acidic pneumococcus includes, but is not limited to, pneumococcus types 7A, 7F, 14, 33F, 33A, 37, etc.

[0028] In some embodiments, the final concentration of sodium chloride is 0.01 mol / L - 5 mol / L, preferably 0.1 mol / L - 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 therebetween.

[0029] In some embodiments, in step (1-4), the iodized salt includes sodium iodide and / or potassium iodide.

[0030] 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 therebetween.

[0031] In some embodiments, the method further includes performing a refining and purification treatment and a drying treatment after step (2).

[0032] In some embodiments, the refining and purification treatment 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, etc.

[0033] In some embodiments, the refining and purification treatment includes sequentially performing a first ultrafiltration concentration treatment, a chromatography treatment, and a second ultrafiltration concentration treatment on the solution after the C polysaccharide removal treatment.

[0034] In some embodiments, the first ultrafiltration concentration treatment and / or the second ultrafiltration concentration treatment is carried out using a membrane package with a molecular weight cut-off of 30 - 300 KD.

[0035] In some embodiments, the drying treatment is freeze-drying.

[0036] In some embodiments, in the method, the supernatant or precipitate is harvested by solid-liquid separation. Preferably, the solid-liquid separation includes at least one of, but is not limited to, centrifugation, clarification, and filtration.

[0037] In some embodiments, in the method, the concentration treatment is carried out by ultrafiltration. Preferably, the ultrafiltration uses a membrane package with a molecular weight cut-off of 30 kD - 300 kD.

[0038] In some embodiments, the serotypes of the pneumococcus include at least one of 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.

[0039] In some embodiments, a C polysaccharide removal treatment is performed on a solution containing type 4 pneumococcal capsular polysaccharide, wherein the mass concentration of the acid is 1% - 8%, preferably 4.5% - 5.5%, and more preferably 5%.

[0040] In some embodiments, a C polysaccharide removal treatment is performed on a solution containing type 4 pneumococcal capsular polysaccharide, wherein the mass concentration of the nitrite is 1% - 5%, preferably 2.5% - 3.5%, and more preferably 3%.

[0041] In some embodiments, the solution containing type 4 pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the temperature of the C polysaccharide removal treatment is 4°C - 50°C, preferably 35°C - 40°C, and more preferably 37°C.

[0042] In some embodiments, the solution containing type 4 pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the time of the C polysaccharide removal treatment is 5h - 20h, preferably 14 - 16h, and more preferably 15h.

[0043] In some embodiments, the solution containing type 5 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%.

[0044] In some embodiments, the solution containing type 5 pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the mass concentration of the nitrite is 0.5% - 2%, preferably 0.8% - 1.2%, and more preferably 1%.

[0045] In some embodiments, the solution containing type 5 pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the temperature of the C polysaccharide removal treatment is 4°C - 40°C, preferably 22°C - 30°C, and more preferably 25°C.

[0046] In some embodiments, the solution containing type 5 pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the time of the C polysaccharide removal treatment is 0.5h - 2h, preferably 0.8h - 1.2h, and more preferably 1.0 h.

[0047] In some embodiments, the solution containing type 7F 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%.

[0048] In some embodiments, the solution containing type 7F pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the mass concentration of the nitrite is 1% - 4%, preferably 2.5% - 3.5%, and more preferably 3%.

[0049] In some embodiments, the solution containing type 7F pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the temperature of the C polysaccharide removal treatment is 4°C - 40°C, preferably 22°C - 30°C, and more preferably 25°C.

[0050] In some embodiments, a solution containing type 7F pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the time of the C polysaccharide removal treatment is 4 h - 10 h, preferably 7 h - 9 h, and more preferably 8.0 h.

[0051] In some embodiments, a solution containing type 12F 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%.

[0052] In some embodiments, a solution containing type 12F pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the mass concentration of the nitrite is 1% - 8%, preferably 4.5% - 5.5%, and more preferably 5%.

[0053] In some embodiments, a solution containing type 12F pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the temperature of the C polysaccharide removal treatment is 4°C - 40°C, preferably 22°C - 30°C, and more preferably 25°C.

[0054] In some embodiments, a solution containing type 12F pneumococcal capsular polysaccharide is subjected to C polysaccharide removal treatment, wherein the time of the C polysaccharide removal treatment is 6 h - 12 h, preferably 9 h - 11 h, and more preferably 10 h.

[0055] In some specific embodiments, the method for purifying pneumococcal capsular polysaccharide comprises the following steps: (1) Subjecting a bacterial solution containing pneumococcus to lysis treatment to obtain a bacterial lysate; (2) Subjecting the lysate to acid precipitation treatment and collecting the first supernatant; (3) Subjecting the first supernatant to CTAB precipitation treatment, directly collecting the second supernatant, or dissolving the precipitate with sodium chloride and then collecting the second supernatant; (4) Subjecting the second supernatant to iodide salt precipitation treatment and collecting the third supernatant to obtain the solution containing pneumococcal capsular polysaccharide; (5) Subjecting the solution containing pneumococcal capsular polysaccharide to C polysaccharide removal treatment; (6) Refining and purifying; (7) Drying.

[0056] In some embodiments, in step (1), a fermentation culture solution containing pneumococcus capable of producing a selected serotype is provided, and a bacterial lysate containing substances such as cell debris, proteins, nucleic acids, and polysaccharides is obtained by one or more of chemical lysis (lysis agent lysis, alkali lysis), physical lysis (high-pressure lysis, thermal lysis), and biological lysis (enzyme lysis).

[0057] Preferably, a fermentation culture solution containing pneumococcus capable of producing a selected serotype is provided, and sodium deoxycholate is added to obtain the bacterial lysate.

[0058] Preferably, a fermentation culture solution containing pneumococcus capable of producing a selected serotype is provided, and sodium deoxycholate with a mass concentration of 0.5 - 0.5% is added and treated for more than 1 h to obtain the bacterial lysate.

[0059] 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 to precipitate impurities. Through centrifugation and / or clarification and / or filtration, the supernatant is harvested. The supernatant is concentrated and buffer exchanged to remove low molecular weight impurities to obtain a first clarified bacterial lysate (i.e., the first supernatant).

[0060] 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, and membrane filtration is performed to remove residual large particulate matter. The supernatant is concentrated and buffer exchanged by ultrafiltration to remove low molecular weight impurities to obtain a first clarified bacterial lysate.

[0061] Preferably, acetic acid is added to the bacterial lysate to adjust the pH to 3 - 5, and it is left standing at room temperature for more than 4 h to precipitate impurities. The supernatant is harvested by centrifugation, and 0.45 μm membrane filtration is performed to remove residual large particulate matter. The supernatant is ultrafiltered using a 30 kD - 300 kD membrane package to concentrate and buffer exchange to remove low molecular weight impurities to obtain a first clarified bacterial lysate.

[0062] In some embodiments, in step (3), CTAB is added to the first clarified bacterial lysate to precipitate impurities. Through centrifugation and / or clarification and / or filtration, solid - liquid separation is performed. In some examples, the supernatant is harvested to obtain a second clarified bacterial lysate (i.e., the second supernatant). In other examples, the precipitate is harvested and sodium chloride is added for depolymerization. Through centrifugation and / or clarification and / or filtration, the supernatant is harvested to obtain a second clarified bacterial lysate.

[0063] Preferably, CTAB with a final mass concentration of 0.1% - 5% is added to the first clarified bacterial lysate to precipitate impurities. Solid - liquid separation is performed by centrifugation. In some embodiments, for neutral pneumococcal capsular polysaccharide, the supernatant is harvested to obtain a second clarified bacterial lysate, and sodium chloride is added. In other embodiments, for acidic pneumococcal capsular polysaccharide, the precipitate is harvested and sodium chloride is added for dissolution. Through centrifugation, the supernatant is harvested to obtain a second clarified bacterial lysate.

[0064] Preferably, CTAB with a final mass concentration of 1% - 3% is added to the first clarified bacterial lysate, and it is left standing overnight to precipitate impurities. Solid-liquid separation is carried out by centrifugation. In some embodiments, for the capsular polysaccharides of Streptococcus pneumoniae types 7A, 7F, 14, 33F, 33A, 37, etc. (neutral), the supernatant is harvested to obtain the second clarified bacterial lysate, and sodium chloride with a final concentration of 0.01 mol / L - 5 mol / L is added. In other embodiments, for the capsular polysaccharides of Streptococcus pneumoniae types 2, 3, 4, 5, etc. (acidic), the precipitate is harvested, and sodium chloride with a final concentration of 0.01 mol / L - 5 mol / L is added for dissolution. The supernatant is harvested by centrifugation to obtain the second clarified bacterial lysate.

[0065] In some embodiments, in step (4), an iodized salt is added to the second clarified bacterial lysate, and the precipitate and large particulate matter are removed by centrifugation and / or clarification and / or filtration. The supernatant is concentrated and buffer exchanged to remove low molecular weight impurities, obtaining the third clarified bacterial lysate (i.e., the third supernatant).

[0066] 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 the remaining large particulate matter is removed by membrane filtration. The harvested solution is ultrafiltered, concentrated, and buffer exchanged to remove low molecular weight impurities, obtaining the third clarified bacterial lysate.

[0067] Preferably, sodium iodide with a final mass concentration of 0.2% - 1.5% is added to the second clarified bacterial lysate, left standing for precipitation, the supernatant is harvested by centrifugation, and filtered through a 0.45 μm membrane to remove the remaining large particulate matter. The harvested solution is ultrafiltered using a 30 kD - 300 kD membrane package for concentration and buffer exchange to remove low molecular weight impurities, obtaining the third clarified bacterial lysate.

[0068] In some embodiments, in step (5), an acid and nitrite are added to the third clarified bacterial lysate, and the reaction is carried out under mild conditions. One-step or multi-step membrane filtration or depth filtration is used for solution replacement, obtaining the fourth clarified bacterial lysate.

[0069] Preferably, acetic acid with a final mass concentration of 0.1% - 20% 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°C - 50°C for 0.1 h - 24 h. Membrane filtration is used for solution replacement, obtaining the fourth clarified bacterial lysate.

[0070] Preferably, acetic acid with a final mass concentration of 1% - 5% 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. Ultrafiltration using a 30 kD - 300 kD membrane package is used for solution replacement and concentration, obtaining the fourth clarified bacterial lysate.

[0071] In some embodiments, in step (6), the fourth clarified bacterial lysate is subjected to refining and purification treatment (including but not limited to membrane filtration, depth filtration, chromatography), the target polysaccharide component is collected, and the solution is further concentrated and exchanged to obtain a refined capsular polysaccharide solution.

[0072] Preferably, hydroxyapatite chromatography column and / or ion exchange chromatography column and / or gel filtration chromatography is used to purify the fourth clarified bacterial lysate, the target polysaccharide flow-through peak is collected, and ultrafiltration concentration and solution replacement are performed using 30 kD - 300 kD to obtain a refined capsular polysaccharide solution.

[0073] Preferably, a hydroxyapatite chromatography column is used to purify the fourth clarified bacterial lysate, the target polysaccharide flow-through peak is collected, and ultrafiltration concentration and solution replacement are performed using a 30 kD - 300 kD membrane package, and the replacement solution is water for injection. Finally, a refined capsular polysaccharide solution is obtained.

[0074] In some embodiments, in step (7), the refined capsular polysaccharide is subjected to drying treatment to remove moisture to obtain a capsular polysaccharide sample.

[0075] Preferably, the drying treatment is freeze-drying.

[0076] According to another aspect of the present invention, there is provided a pneumococcal capsular polysaccharide prepared by the method according to the present invention.

[0077] In some embodiments, the content of C polysaccharide in the pneumococcal capsular polysaccharide is less than 5%, preferably less than 1%, more preferably less than 0.2%.

[0078] Preferably, the content of C polysaccharide impurity in the pneumococcal capsular polysaccharide is significantly lower than that in the control capsular polysaccharide of the same serotype.

[0079] According to yet another aspect of the present invention, there is provided the use of the pneumococcal capsular polysaccharide in the preparation of a product containing pneumococcal capsular polysaccharide.

[0080] In some embodiments, the product containing pneumococcal capsular polysaccharide includes a pneumococcal vaccine.

[0081] In some embodiments, the pneumococcal vaccine includes a pneumococcal capsular polysaccharide vaccine or a pneumococcal conjugate vaccine.

[0082] In some embodiments, the pneumococcal vaccine includes a monovalent vaccine or a multivalent vaccine.

[0083] According to yet another aspect of the present invention, there is provided a vaccine composition, which includes the pneumococcal capsular polysaccharide prepared by the method of the present invention.

[0084] In some embodiments, the pneumococcal vaccine includes a pneumococcal capsular polysaccharide vaccine or a pneumococcal capsular polysaccharide conjugate vaccine.

[0085] In some embodiments, the pneumococcal vaccine includes a monovalent vaccine or a multivalent vaccine.

[0086] The method for purifying pneumococcal capsular polysaccharide of the present invention adds a step for removing C polysaccharide, significantly improves the purity of the capsular polysaccharide, and is conducive to 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, and is safer and more environmentally friendly; moreover, the reagents used are all conventional reagents, which have the advantages of low cost, simple operation, and suitability for scale-up. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 Shows the control type 4 pneumococcal capsular polysaccharide Y4CH02C (A) and the type 4 pneumococcal capsular polysaccharide Y4CH23N purified by the method of the present invention (B) 1 1H NMR spectra.

[0088] Figure 2 Shows the control type 5 pneumococcal capsular polysaccharide Y5CI25C (A) and the type 5 pneumococcal capsular polysaccharide Y5CG12N purified by the method of the present invention (B) 1 1H NMR spectra.

[0089] Figure 3 Shows the control type 7F pneumococcal capsular polysaccharide Y7FCK22C (A) and the type 7F pneumococcal capsular polysaccharide Y7FCL15N purified by the method of the present invention (B) 1 1H NMR spectra.

[0090] Figure 4 Shows the control type 12F pneumococcal capsular polysaccharide Y12FDA28C (A) and the type 12F pneumococcal capsular polysaccharide Y12FDC26N purified by the method of the present invention (B) 1 1H NMR spectra. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0091] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The specific embodiments described herein are only used to explain the present invention and do not constitute any limitation to the present invention. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessarily confusing the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0092] Cell wall polysaccharide (C-Ps), also known as C polysaccharide, is a species-specific polysaccharide present in the cell wall of pneumococcus and common to strains of each serotype. C polysaccharide has high immunogenicity, but the antibodies produced by stimulating the host as an antigen cannot play a protective role, but instead cause adverse reactions such as inflammation. In addition, the antibodies produced by C polysaccharide will also interfere with the quantification of capsular polysaccharide antibodies, affecting the detection of capsular polysaccharide. Different degrees of C polysaccharide residues in pneumococcal capsular polysaccharide bring many problems to the research and production of PPV and PCV. At present, WHO has listed C polysaccharide as an impurity and recommends controlling the content of C polysaccharide. The United States Pharmacopeia includes the content of C polysaccharide in the vaccine quality inspection items. The Chinese vaccine review agency has also begun to pay attention to and limit the content of C polysaccharide impurities in pneumococcal capsular polysaccharide. On July 25, 2024, the Center for Drug Evaluation of the National Medical Products Administration issued the "Technical Guidelines for Quality Control of Polysaccharide Conjugate Vaccines (Draft for Comment)", which proposed that product-related impurities such as C polysaccharide may be generated during the sterilization process. It is recommended to study the effects of the sterilization process on the quality and structure of polysaccharides, and it is recommended to use appropriate methods such as quantitative nuclear magnetic resonance spectroscopy to determine C polysaccharide.

[0093] The present invention provides a method for purifying pneumococcal capsular polysaccharide, and creatively adds a step of removing C polysaccharide on the basis of removing impurities by precipitation method. Subsequently, methods such as membrane filtration, depth filtration or chromatography are used to separate C polysaccharide and capsular polysaccharide to effectively remove C polysaccharide contamination. The present invention can obtain pneumococcal capsular polysaccharide with high purity and high quality, which is of great significance for the development of pneumococcal polysaccharide vaccine and polysaccharide conjugate vaccine.

[0094] The reagents and / or kits used in the following examples were all commercially obtained or could be synthesized by known methods.

[0095] In the following examples, unless otherwise specified, they are all conventional methods. The materials used in the following examples, unless otherwise specified, can be obtained from public commercial channels.

[0096] Main materials: The pneumococcus strain was derived from the China National Center for Medical Bacterial Culture Collection, National Institutes for Food and Drug Control; sodium deoxycholate, acetic acid, sodium hydroxide, sodium dihydrogen phosphate, disodium hydrogen phosphate, CTAB, sodium chloride, sodium iodide, sodium nitrite were from Sinopharm Chemical Reagent Co., Ltd.; heavy water was from Shanghai Aladdin Biochemical Technology Co., Ltd.; type 4, type 5, type 7F, type 12F pneumococcal specific sera were from Statens Serum Institut, Denmark.

[0097] Main instruments: tangential flow system (Sartorius, Sartoflow Advanced); centrifuge (Thermo, Sorvall LYNX 6000); freeze dryer (CHRIST, Epsilon2-4 Lscplus); 600 MHz ultra-low temperature nuclear magnetic resonance spectrometer (BRUKER, AV-HD-600X); ultraviolet spectrophotometer (Shimadzu, UV-1900i); protein purifier (Cytiva, AKTAPURE).

[0098] Example 1: Purification of type 4 pneumococcal capsular polysaccharide Take 10 L of type 4 pneumococcal fermentation broth, add sodium deoxycholate to make its final mass concentration 0.1% to obtain a lysate. Add acetic acid to the lysate to adjust the pH to 5, centrifuge to collect the supernatant, and use sodium hydroxide to adjust the pH of the supernatant to 7. Filter with a 0.45 μm filter membrane, ultrafiltrate and concentrate using a 100 kD membrane package, and replace the solution with phosphate buffer (pH 7.0). Add CTAB with a final mass concentration of 1% to the feed solution, 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 make its final mass concentration 0.5%, centrifuge to harvest the supernatant. Filter with a 0.45 μm filter membrane, ultrafiltrate and concentrate using a 100 kD membrane package, and replace the solution with phosphate buffer (pH 7.0). Add acetic acid with a final mass concentration of 5% and sodium nitrite with a final mass concentration of 3% to the feed solution, react at 37 °C for 15 h, ultrafiltrate and concentrate using a 100 kD membrane package, and replace the solution with phosphate buffer (pH 7.0). Refine using a hydroxyapatite chromatography column and collect the target flow-through peak. Ultrafiltrate and concentrate the flow-through solution using a 100 kD membrane package, and replace the solution with water for injection. Freeze-dry the final product to obtain capsular polysaccharide with batch number Y4CH23N for subsequent detection.

[0099] Comparative Example 1: Purification of control type 4 pneumococcal capsular polysaccharide Take 10 L of the fermentation culture solution of Streptococcus pneumoniae type 4, add sodium deoxycholate to make its final mass concentration 0.1% to obtain a lysate. Add acetic acid to the lysate to adjust the pH to 5, centrifuge to collect the supernatant, and use sodium hydroxide to adjust the pH of the supernatant to 7. Filter through a 0.45 μm filter membrane, perform ultrafiltration concentration using a 100 kD membrane package, and replace the solution with phosphate buffer (pH 7.0). Add CTAB with a final mass concentration of 1% to the feed solution, 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 make its final mass concentration 0.5%, centrifuge to harvest the supernatant. Filter through a 0.45 μm filter membrane, perform ultrafiltration concentration using a 100 kD membrane package, and replace the solution with phosphate buffer (pH 7.0). Refine using a hydroxyapatite chromatography column and collect the target flow-through peak. Perform ultrafiltration concentration on the flow-through solution using a 100 kD membrane package and replace the solution with water for injection. Lyophilize the final product to obtain the capsular polysaccharide with batch number Y4CH02C for subsequent detection.

[0100] Example 2: Detection of Streptococcus pneumoniae type 4 capsular polysaccharide 2.1 1 1H NMR structural analysis Dissolve the control Streptococcus pneumoniae type 4 capsular polysaccharide purified in Comparative Example 1 and the Streptococcus pneumoniae type 4 capsular polysaccharide purified in Example 1 with heavy water respectively and lyophilize, repeating 3 times. Dissolve the capsular polysaccharide with heavy water to make its final concentration 5 mg / ml, fully dissolve at room temperature, centrifuge for 10 min, and take the sample solution into a nuclear magnetic tube. Collect the one-dimensional nuclear magnetic spectrum of each type of capsular polysaccharide through a 600 MHz nuclear magnetic resonance instrument and analyze its spectral characteristics through Bruker TopSpin 4.2.0 software.

[0101] Perform quantitative analysis using one-dimensional nuclear magnetic hydrogen spectrum. Determine the relative content of each substance by measuring the integral of the peak area of each component in the sample. Perform normalization treatment through factor N, that is, divide the integral of each peak by the number of equivalent nuclei represented by this peak, so as to obtain the relative molar concentration of each component. The C polysaccharide repeating unit has two phosphorylcholine substituents, and each phosphorylcholine contains three methyl groups. Therefore, the C polysaccharide contains a total of 18 protons. The calculation formula for the C polysaccharide content is as follows:

[0102] Where: I is the integral of the peak area; N is the normalization factor; the subscripts C and CPS represent C polysaccharide and capsular polysaccharide respectively.

[0103] The 1H NMR spectrum results of Streptococcus pneumoniae type 4 capsular polysaccharide are shown in 1 Figure 1 . 1 ​The characteristic peak of C polysaccharide (phosphocholine peak) was shown at 3.22 ppm in the ¹H NMR spectrum. Figure 1 In it, A was the capsular polysaccharide Y4CH02C of Streptococcus pneumoniae type 4 as a control, Figure 1 In it, B was the capsular polysaccharide Y4CH23N purified by the method of the present invention. Compared with the spectrum of the capsular polysaccharide of Streptococcus pneumoniae type 4 as a control, the peak area of the C polysaccharide characteristic peak of the capsular polysaccharide obtained by the purification method of the present invention decreased, and the rest of the structural characteristic peaks were consistent. According to the calculation formula of the C polysaccharide content, the C polysaccharide content of the capsular polysaccharide in the control group was 5.67%, and the C polysaccharide content of the capsular polysaccharide purified by the method of the present invention was 0.17%. In summary, it shows that the method of the present invention can specifically reduce the content of C polysaccharide impurities.

[0104] 2.2 Immunodiffusion Melt 1% agar and dispense it into glass tubes, keep it warm in a water bath at 56 °C, add the specific serum of the capsular polysaccharide of Streptococcus pneumoniae type 4, and mix well. Take 4 ml of the agar solution and spread it on a clean glass slide. After solidification, punch holes with a diameter of 3 mm and a hole spacing of 10 mm. Respectively pipette 10 μl of the control capsular polysaccharide purified in Comparative Example 1 and the capsular polysaccharide purified in Example 1 into the holes. Place the agar plate in a wet box and react at room temperature for 12 h, and measure the diameter of the precipitation ring. Repeat three times and take the average value. By measuring the diameter of the diffusion ring, the change in the antigenicity of the polysaccharide was judged.

[0105] The immunodiffusion results of the capsular polysaccharide of Streptococcus pneumoniae type 4 are shown in Table 1. The diffusion ring diameter of the capsular polysaccharide of Streptococcus pneumoniae type 4 purified by the present invention is equivalent to that of the capsular polysaccharide of Streptococcus pneumoniae type 4 as a control, and is slightly better than that of the control capsular polysaccharide. It shows that the purification method of the present invention has no negative impact on the antigenicity of the capsular polysaccharide, and the effect is slightly better than that of the control group.

[0106] Table 1: Table of immunodiffusion results of the capsular polysaccharide of Streptococcus pneumoniae type 4 ; 2.3 Physicochemical detection Perform physicochemical detection on the capsular polysaccharide of Streptococcus pneumoniae type 4 purified in Example 1 and the control capsular polysaccharide of type 4 purified in Comparative Example 1.

[0107] 2.3.1 Protein content detection Adopt the Lowry method: Prepare a 1 mg / ml polysaccharide solution, a gradient concentration reference solution and an alkaline copper test solution, carry out the reaction, and detect the absorbance of each sample at a wavelength of 650 nm. Calculate the linear regression equation with the concentration of the reference solution and its corresponding absorbance, and calculate the protein concentration in the polysaccharide solution from the linear regression equation.

[0108] 2.3.2 Nucleic acid content detection Using ultraviolet spectrophotometry: Dissolve the purified polysaccharide in distilled water to obtain a 1 mg / ml polysaccharide solution. Use distilled water as a blank control and measure the absorbance at a wavelength of 260 nm. The absorption coefficient of nucleic acid at 260 nm is 200. Calculate the nucleic acid content according to the absorption coefficient formula.

[0109] 2.3.3 Detection of total nitrogen content According to the principle of the Kjeldahl method, use an automatic nitrogen analyzer to detect the total nitrogen content. Weigh an appropriate amount of polysaccharide into a digestion tube, and successively add potassium sulfate, copper sulfate, and sulfuric acid. Place the digestion tube in a digestion instrument, set it at 150 °C for 5 minutes to remove moisture; 350 °C for 5 minutes, close to the boiling point of sulfuric acid; 400 °C for 60 - 80 minutes for digestion. After digestion is completed, take it out and cool. Place the digestion tube into an automatic distillation device for distillation and titration to measure the total nitrogen content.

[0110] 2.3.4 Detection of phosphorus content Prepare a 1 mg / ml polysaccharide solution, and use potassium dihydrogen phosphate to prepare a gradient concentration standard phosphorus solution. 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 it stand for a while, add 2 ml of water, and 0.4 ml of ammonium molybdate solution with a concentration of 0.04 M, mix well, add 0.2 ml of reducing agent, and mix well. Add water to 6 ml, and after 15 minutes, use an ultraviolet spectrophotometer to measure the absorbance at a wavelength of 820 nm. Make a linear regression of the corresponding absorbance with the gradient concentration standard phosphorus solution, and substitute the absorbance of the polysaccharide sample solution into the linear regression equation to calculate the phosphorus content.

[0111] 2.3.5 Molecular weight determination Use CL-4B gel to prepare an agarose gel chromatography column. Prepare a blue dextran 2000 solution and a vitamin B12 solution for column calibration. Elute with the mobile phase at a flow rate of 15 ml per hour, detect at a wavelength of 206 nm, use a fraction collector to collect the eluate and record the chromatogram. In the chromatogram, the first peak is blue dextran 2000, and the eluate volume at the peak top is the void volume V o , the second peak is the vitamin B12 peak, and the eluate volume at the peak top is the column bed volume V i . Prepare a 4 mg / ml polysaccharide solution with the mobile phase, aspirate 1 ml of the polysaccharide solution and add it to the calibrated chromatography column, elute with the mobile phase at a flow rate of 15 ml per hour, detect at a wavelength of 206 nm, use a fraction collector to collect the eluate and record the chromatogram.

[0112] Calculate according to the following formula: K D = (V e - V o ) / (V i - V o ) Where: K D is the sample distribution coefficient; V e is the volume of the sample eluent, ml; V o is the void volume, ml; V i is the column bed volume, ml.

[0113] 2.3.6 Detection of hexosamine content Prepare 3 ml of a polysaccharide solution at 800 μg / ml with purified water, and prepare 10 ml of a stock solution of D-glucosamine hydrochloride reference substance at 500 μg / ml, and dilute it stepwise with purified water. Take 1 ml of the stepwise concentration reference substance solution and the prepared polysaccharide sample solution in 20-ml glass-stoppered test tubes. Add 1 ml of HCl solution with an appropriate concentration, stopper, and heat in a water bath at 100 °C. Cool to room temperature, add 0.5% phenolphthalein ethanol solution, mix well, neutralize the hydrolysis solution to change color with 4 mol / L NaOH solution, add 1 mol / L HCl solution dropwise until the solution is colorless, and add purified water to 10 ml to obtain a neutral hydrolysis solution.

[0114] Precisely measure 1 ml of the neutral hydrolysis solution into a glass-stoppered test tube, and determine it in duplicate. Add 1 ml of acetylacetone reagent to each tube, stopper, and heat in a water bath at 90 °C for 45 min. Cool to room temperature, add 2.5 ml of absolute ethanol to each tube, mix well, slowly add 1 ml of p-dimethylaminobenzaldehyde solution to each tube, and mix well. Make up the volume to 10 ml with absolute ethanol, mix well, stopper, and place in the dark at room temperature for 1.5 h. Measure the absorbance value of each tube at a wavelength of 530 nm.

[0115] Use the series of concentrations of the D-glucosamine hydrochloride reference substance solution to perform a linear regression on their corresponding absorbance values to obtain the regression equation. Substitute the absorbance of the polysaccharide sample into the regression equation, and calculate the hexosamine content according to the dilution factor. Then calculate the percentage content of hexosamine based on the dry weight of the polysaccharide.

[0116] The test results are shown in Table 2. All the indexes of the purified type 4 pneumococcal capsular polysaccharide samples in Example 1 and Comparative Example 1 meet the relevant standards specified in the pharmacopoeia.

[0117] Table 2: Physical and chemical test results of type 4 pneumococcal capsular polysaccharide ;

[0118] Example 3: Optimization of the purification process of type 4 pneumococcal capsular polysaccharide 3.1 Acetic acid concentration Using the method of the present invention to separate and purify type 4 Streptococcus pneumoniae capsular polysaccharide, the final mass concentrations of acetic acid added in each group in the C polysaccharide removal step are 1.0%, 2.5%, 5.0%, and 7.5% respectively, and the remaining steps are the same as in Example 1. Harvest the freeze-dried polysaccharide and perform 1 1H NMR detection.1 The characteristic peak of C polysaccharide (phosphocholine peak) appears at 3.22 ppm in the 1H NMR spectrum. Based on the characteristic peak of type 4 capsular polysaccharide, the content of C polysaccharide was quantified, and the results are shown in Table 3.

[0119] Table 3: Effect of acetic acid concentration on the content of C polysaccharide in purified type 4 Streptococcus pneumoniae capsular polysaccharide ; It can be seen from the test results that as the acetic acid concentration increases, the content of C polysaccharide in the obtained capsular polysaccharide shows a decreasing trend. When purified with 5.0% acetic acid and 7.5% acetic acid, the content of C polysaccharide in the obtained capsular polysaccharide is quite similar. Considering the purification effect and economic cost comprehensively, in the purification process of type 4 Streptococcus pneumoniae capsular polysaccharide, the mass concentration of acetic acid used in the C polysaccharide removal step is preferably 5.0%.

[0120] 3.2 Sodium nitrite concentration Using the method of the present invention to separate and purify type 4 Streptococcus pneumoniae capsular polysaccharide, the final mass concentrations of sodium nitrite added in each group in the C polysaccharide removal step were 1.0%, 2.0%, 3.0%, and 4.0% respectively, and the other steps were the same as those in Example 1. The freeze-dried polysaccharide was harvested and subjected to 1 1H NMR detection. 1 The characteristic peak of C polysaccharide (phosphocholine peak) appears at 3.22 ppm in the 1H NMR spectrum. Based on the characteristic peak of type 4 capsular polysaccharide, the content of C polysaccharide was quantified, and the results are shown in Table 4.

[0121] Table 4: Effect of sodium nitrite concentration on the content of C polysaccharide in purified type 4 Streptococcus pneumoniae capsular polysaccharide ; It can be seen from the test results that as the sodium nitrite concentration increases, the content of C polysaccharide in the obtained capsular polysaccharide shows a decreasing trend. When purified with 3.0% sodium nitrite and 4.0% sodium nitrite, the content of C polysaccharide in the obtained capsular polysaccharide is quite similar. Considering the purification effect and economic cost comprehensively, in the purification process of type 4 Streptococcus pneumoniae capsular polysaccharide, the mass concentration of sodium nitrite used in the C polysaccharide removal step is preferably 3.0%.

[0122] 3.3 Reaction time Using the method of the present invention to separate and purify type 4 Streptococcus pneumoniae capsular polysaccharide, the reaction time in the C polysaccharide removal step in each group was 5.0 h, 10.0 h, 15.0 h, and 20.0 h respectively, and the other steps were the same as those in Example 1. The freeze-dried polysaccharide was harvested and subjected to 1 1H NMR detection. 1The characteristic peak of C polysaccharide (phosphocholine peak) was shown at 3.22 ppm in the ¹H NMR spectrum. Based on the characteristic peak of type 4 capsular polysaccharide, the content of C polysaccharide was quantified, and the results are shown in Table 5.

[0123] Table 5: Effect of reaction time on the content of C polysaccharide in purified type 4 Streptococcus pneumoniae capsular polysaccharide ; It can be seen from the test results that with the extension of the reaction time, the content of C polysaccharide in the obtained capsular polysaccharide showed a decreasing trend. The content of C polysaccharide in the obtained capsular polysaccharide was comparable between the reaction for 15.0 h and the reaction for 20.0 h. Considering the purification effect and economic cost comprehensively, in the purification process of type 4 Streptococcus pneumoniae capsular polysaccharide, the preferred reaction time for the C polysaccharide removal step was 15.0 h.

[0124] 3.4 Reaction temperature Using the method of the present invention to isolate and purify type 4 Streptococcus pneumoniae capsular polysaccharide, the reaction temperatures in the C polysaccharide removal step for each group were 4.0 °C, 25.0 °C, 37.0 °C, and 45.0 °C respectively, and the remaining steps were the same as in Example 1. The harvested freeze-dried polysaccharide was subjected to 1 ¹H NMR detection. 1 The characteristic peak of C polysaccharide (phosphocholine peak) was shown at 3.22 ppm in the ¹H NMR spectrum. Based on the characteristic peak of type 4 capsular polysaccharide, the content of C polysaccharide was quantified, and the results are shown in Table 6.

[0125] Table 6: Effect of reaction temperature on the content of C polysaccharide in purified type 4 Streptococcus pneumoniae capsular polysaccharide ; It can be seen from the test results that with the increase of the reaction temperature, the content of C polysaccharide in the obtained capsular polysaccharide showed a decreasing trend. The content of C polysaccharide in the obtained capsular polysaccharide was comparable between 37.0 °C and 45.0 °C. Considering the purification effect and economic cost comprehensively, in the purification process of type 4 Streptococcus pneumoniae capsular polysaccharide, the preferred reaction temperature for the C polysaccharide removal step was 37.0 °C.

[0126] Example 4: Purification of type 5 pneumococcal capsular polysaccharide Take 10 L of the fermentation broth of Streptococcus pneumoniae type 5, add sodium deoxycholate to make its final mass concentration 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 use sodium hydroxide to adjust the pH of the supernatant to 7. Filter with a 0.45 μm filter membrane, perform ultrafiltration concentration using a 100 kD membrane package, and replace the solution with phosphate buffer (pH 7.0). Add CTAB with a final mass concentration of 2% to the feed solution, 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 with a final mass concentration of 0.5%, centrifuge to harvest the supernatant. Filter with a 0.45 μm filter membrane, perform ultrafiltration concentration using a 100 kD membrane package, and replace the solution with phosphate buffer (pH 7.0). Add acetic acid with a final mass concentration of 3.0% and sodium nitrite with a final mass concentration of 1.0% to the feed solution, react at 25 °C for 1.0 h, perform ultrafiltration concentration using a 100 kD membrane package, and replace the solution with phosphate buffer (pH 7.0). Refine using a hydroxyapatite chromatography column and collect the target flow-through peak. Perform ultrafiltration concentration on the flow-through solution using a 100 kD membrane package and replace the solution with water for injection. Lyophilize the final product to obtain the capsular polysaccharide with batch number Y5CG12N for subsequent detection.

[0127] Comparative Example 2: Purification of the capsular polysaccharide of Streptococcus pneumoniae type 5 Take 10 L of the fermentation broth of Streptococcus pneumoniae type 5, add sodium deoxycholate to make its final mass concentration 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 use sodium hydroxide to adjust the pH of the supernatant to 7. Filter with a 0.45 μm filter membrane, perform ultrafiltration concentration using a 100 kD membrane package, and replace the solution with phosphate buffer (pH 7.0). Add CTAB with a final mass concentration of 2% to the feed solution, 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 with a final mass concentration of 0.5%, centrifuge to harvest the supernatant. Filter with a 0.45 μm filter membrane, perform ultrafiltration concentration using a 100 kD membrane package, and replace the solution with phosphate buffer (pH 7.0). Refine using a hydroxyapatite chromatography column and collect the target flow-through peak. Perform ultrafiltration concentration on the flow-through solution using a 100 kD membrane package and replace the solution with water for injection. Lyophilize the final product to obtain the capsular polysaccharide with batch number Y5CI25C for subsequent detection.

[0128] Example 5: Detection of the capsular polysaccharide of Streptococcus pneumoniae type 5 5.1 1 1H NMR structural analysis Dissolve the control type 5 pneumococcal capsular polysaccharide purified in Comparative Example 2 and the type 5 pneumococcal capsular polysaccharide purified in Example 4 with heavy water respectively and then lyophilize, repeating 3 times. Perform nuclear magnetic spectroscopy analysis according to the method 2.1 in Example 2.

[0129] The 1 The results of the 1H NMR spectrum of the type 5 pneumococcal capsular polysaccharide are shown in Figure 2 . 1 A characteristic peak of C polysaccharide is shown at 3.22 ppm in the 1H NMR spectrum. Figure 2 In [reference], A is the control type 5 pneumococcal capsular polysaccharide Y5CI25C, Figure 2 In [reference], B is the purified capsular polysaccharide Y5CG12N. Compared with the spectrum of the control type 5 pneumococcal capsular polysaccharide, the peak area of the C polysaccharide characteristic peak of the capsular polysaccharide obtained by the purification method of the present invention decreases, and the rest of the structural characteristic peaks are the same. According to the calculation formula of the C polysaccharide content, the C polysaccharide content of the control capsular polysaccharide is 1.33%, and the C polysaccharide content of the capsular polysaccharide purified by the method of the present invention is 0.15%. In summary, it shows that the method of the present invention can specifically reduce the content of C polysaccharide impurities.

[0130] 5.2 Immunodiffusion Refer to the method 2.2 in Example 2, and use the type 5 pneumococcal capsular polysaccharide-specific serum to prepare an agar plate for immunodiffusion experiment. By measuring the diameter of the diffusion ring, the change in polysaccharide antigenicity is judged.

[0131] The immunodiffusion results of the type 5 pneumococcal capsular polysaccharide are shown in Table 7. The diffusion ring diameter of the type 5 pneumococcal capsular polysaccharide purified by the present invention is equivalent to that of the control type 5 pneumococcal capsular polysaccharide, and is slightly better than that of the control capsular polysaccharide. It shows that the purification method of the present invention has no negative impact on the antigenicity of the capsular polysaccharide, and the effect is slightly better than that of the control group.

[0132] Table 7: Table of immunodiffusion results of type 5 pneumococcal capsular polysaccharide ; 5.3 Physical and chemical detection 5.3.1 Refer to the method 2.3 in Example 2 to detect 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 type 5 Streptococcus pneumoniae capsular polysaccharide purified in Example 4 and the control type 5 capsular polysaccharide purified in Comparative Example 2.

[0133] 5.3.2 Detection of uronic acid content Prepare 3 ml of a 0.25 mg / ml solution of type 5 pneumococcal capsular polysaccharide using purified water, and prepare 200 ml of a mother solution of D-glucuronic acid reference substance at 100 μg / ml, and dilute it stepwise with purified water. Take 1 ml of the stepwise concentration reference substance solution and the prepared polysaccharide sample solution in a stoppered test tube, and measure the polysaccharide sample in duplicate. While stirring, add 5 ml of 0.955% borate-sulfuric acid solution dropwise to each tube, stopper, and place in a water bath at 100 °C for 15 min. After cooling to room temperature, add 0.2 ml of 0.125% carbazole-ethanol solution, place in a water bath at 100 °C for 15 min, and after cooling to room temperature, read the absorbance value of each tube at a wavelength of 530 nm. Make a linear regression of the series of concentrations of the D-glucuronic acid reference substance solution against their corresponding absorbance values to obtain the regression equation. Substitute the absorbance of the polysaccharide sample into the regression equation, and calculate the glucuronic acid content according to the dilution factor. Then calculate the percentage content of glucuronic acid based on the dry weight of the polysaccharide.

[0134] The test results are shown in Table 8. All the indicators of the type 5 pneumococcal capsular polysaccharide samples purified in Example 4 and Comparative Example 2 meet the relevant standards specified in the pharmacopoeia.

[0135] Table 8: Physical and chemical test results of type 5 pneumococcal capsular polysaccharide ;

[0136] Example 6: Optimization of the purification process of type 5 pneumococcal capsular polysaccharide

[0137] 6.1 Acetic acid concentration Using the method of the present invention to isolate and purify type 5 Streptococcus pneumoniae capsular polysaccharide, the final mass concentrations of acetic acid added in each group in the C polysaccharide removal step are 1.0%, 2.0%, 3.0%, and 4.0% respectively, and the remaining steps are the same as in Example 4. Harvest the freeze-dried polysaccharide and perform 1 1H NMR detection. 1 In the 1H NMR spectrum, a characteristic peak of C polysaccharide (phosphocholine peak) is shown at 3.22 ppm. Based on the characteristic peak of type 5 capsular polysaccharide, the content of C polysaccharide is quantified, and the results are shown in Table 9.

[0138] Table 9: Effect of acetic acid concentration on the content of C polysaccharide in the type 5 Streptococcus pneumoniae capsular polysaccharide purified ; It can be seen from the test results that as the acetic acid concentration increases, the content of C polysaccharide in the obtained capsular polysaccharide shows a decreasing trend. When purified with 3.0% acetic acid and 4.0% acetic acid, the content of C polysaccharide in the obtained capsular polysaccharide is quite the same. Considering the purification effect and economic cost comprehensively, in the purification process of type 5 Streptococcus pneumoniae capsular polysaccharide, the mass concentration of acetic acid used in the C polysaccharide removal step is preferably 3.0%.

[0139] 6.2 Sodium nitrite concentration When separating and purifying the capsular polysaccharide of Streptococcus pneumoniae type 5 by the method of the present invention, the final mass concentrations of sodium nitrite added in each group in the C polysaccharide removal step were 0.5%, 1.0%, 1.5%, and 2.0% respectively, and the remaining steps were the same as those in Example 4. The freeze-dried polysaccharide was harvested and subjected to 1 1H NMR detection. 1 The characteristic peak of C polysaccharide (phosphocholine peak) was shown at 3.22 ppm in the 1H NMR spectrum. Based on the characteristic peak of type 5 capsular polysaccharide, the content of C polysaccharide was quantified, and the results are shown in Table 10.

[0140] Table 10: Effect of sodium nitrite concentration on the content of C polysaccharide in the purified capsular polysaccharide of Streptococcus pneumoniae type 5 ; It can be seen from the detection results that as the sodium nitrite concentration increases, the content of C polysaccharide in the obtained capsular polysaccharide shows a decreasing trend. When using 1.0% sodium nitrite and 1.5% and 2.0% sodium nitrite for purification, the content of C polysaccharide in the obtained capsular polysaccharide is quite the same. Considering the purification effect and economic cost comprehensively, in the purification process of the capsular polysaccharide of Streptococcus pneumoniae type 5, the mass concentration of sodium nitrite used in the C polysaccharide removal step is preferably 1.0%.

[0141] 6.3 Reaction time When separating and purifying the capsular polysaccharide of Streptococcus pneumoniae type 5 by the method of the present invention, the reaction time in each group in the C polysaccharide removal step was 0.5 h, 1.0 h, 1.5 h, and 2.0 h respectively, and the remaining steps were the same as those in Example 4. The freeze-dried polysaccharide was harvested and subjected to 1 1HNMR detection. 1 The characteristic peak of C polysaccharide (phosphocholine peak) was shown at 3.22 ppm in the 1H NMR spectrum. Based on the characteristic peak of type 5 capsular polysaccharide, the content of C polysaccharide was quantified, and the results are shown in Table 11.

[0142] Table 11: Effect of reaction time on the content of C polysaccharide in the purified capsular polysaccharide of Streptococcus pneumoniae type 5 ; It can be seen from the detection results that as the reaction time prolongs, the content of C polysaccharide in the obtained capsular polysaccharide shows a decreasing trend. When comparing the reaction for 1.0 h with the reaction for 1.5 h and 2.0 h, the content of C polysaccharide in the obtained capsular polysaccharide is quite the same. Considering the purification effect and economic cost comprehensively, in the purification process of the capsular polysaccharide of Streptococcus pneumoniae type 5, the reaction time in the C polysaccharide removal step is preferably 1.0 h.

[0143] 6.4 Reaction temperature The method of the present invention is used to isolate and purify the capsular polysaccharide of Streptococcus pneumoniae type 5. The reaction temperatures in each group during the C polysaccharide removal step are 4.0°C, 20.0°C, 25.0°C, and 37.0°C respectively, and the remaining steps are the same as those in Example 4. The freeze-dried polysaccharide is harvested and subjected to 1 1H NMR detection. 1 In the 1H NMR spectrum, a characteristic peak of C polysaccharide (phosphocholine peak) appears at 3.22 ppm. Based on the characteristic peak of type 5 capsular polysaccharide, the content of C polysaccharide is quantified, and the results are shown in Table 12.

[0144] Table 12: Effect of reaction temperature on the content of C polysaccharide in the purified capsular polysaccharide of Streptococcus pneumoniae type 5 ; It can be seen from the detection results that as the reaction temperature increases, the content of C polysaccharide in the obtained capsular polysaccharide shows a decreasing trend. The content of C polysaccharide in the obtained capsular polysaccharide is quite the same at 25.0°C and 37.0°C. Considering the purification effect and economic cost comprehensively, in the purification process of the capsular polysaccharide of Streptococcus pneumoniae type 5, the reaction temperature in the C polysaccharide removal step is preferably 25.0°C.

[0145] Example 7: Purification of the capsular polysaccharide of Streptococcus pneumoniae type 7F Take 10 L of the fermentation culture solution of Streptococcus pneumoniae type 7F, add sodium deoxycholate to make its final mass concentration 0.2% to obtain a lysate. Add acetic acid to the lysate to adjust the pH to 5, centrifuge to collect the supernatant, and use sodium hydroxide to adjust the pH of the supernatant to 7. Filter with a 0.45 μm filter membrane, perform ultrafiltration and concentration using a 100 kD membrane package, and replace the solution with phosphate buffer (pH 7.0). Add CTAB with a final mass concentration of 1.5% to the feed solution, centrifuge to collect the supernatant, add sodium chloride with a final concentration of 0.20 mol / L, and centrifuge to collect the supernatant. Add sodium iodide with a final mass concentration of 0.5%, centrifuge to harvest the supernatant. Filter with a 0.45 μm filter membrane, perform ultrafiltration and concentration using a 100 kD membrane package, and replace the solution with phosphate buffer (pH 7.0). Add acetic acid with a final mass concentration of 1.0% and sodium nitrite with a final mass concentration of 3.0% to the feed solution, react at 25.0°C for 8.0 h, perform ultrafiltration and concentration using a 100 kD membrane package, and replace the solution with phosphate buffer (pH 7.0). Refine using a hydroxyapatite chromatography column, and collect the target flow-through peak. Perform ultrafiltration and concentration on the flow-through solution using a 100 kD membrane package, and replace the solution with injection water. Freeze-dry the final product to obtain the capsular polysaccharide with batch number Y7FCL15N for subsequent detection.

[0146] Comparative Example 3: Purification of the control capsular polysaccharide of Streptococcus pneumoniae type 7F Take 10 L of the fermentation culture solution of Streptococcus pneumoniae type 7F, add sodium deoxycholate to make its final mass concentration 0.2% to obtain a lysate. Add acetic acid to the lysate to adjust the pH to 5, centrifuge to collect the supernatant, and use sodium hydroxide to adjust the pH of the supernatant to 7. Filter with a 0.45 μm filter membrane, perform ultrafiltration concentration using a 100 kD membrane package, and replace the solution with phosphate buffer (pH 7.0). Add CTAB with a final mass concentration of 1.5% to the feed solution, centrifuge to collect the supernatant. Add sodium iodide with a final mass concentration of 0.5%, centrifuge to harvest the supernatant. Filter with a 0.45 μm filter membrane, perform ultrafiltration concentration using a 100 kD membrane package, and replace the solution with phosphate buffer (pH 7.0). Refine using a hydroxyapatite chromatography column and collect the target flow-through peak. Perform ultrafiltration concentration on the flow-through solution using a 100 kD membrane package and replace the solution with water for injection. Lyophilize the final product to obtain the capsular polysaccharide with batch number Y7FCK22C for subsequent detection.

[0147] Example 8: Detection of Streptococcus pneumoniae type 7F capsular polysaccharide 8.1 1 1H NMR structural analysis Dissolve the control Streptococcus pneumoniae type 7F capsular polysaccharide purified in Comparative Example 3 and the Streptococcus pneumoniae type 7F capsular polysaccharide purified in Example 7 with heavy water respectively and lyophilize, repeating 3 times. Refer to the method in 2.1 of Example 2 for nuclear magnetic spectroscopy analysis.

[0148] For the Streptococcus pneumoniae type 7F capsular polysaccharide 1 The results of the 1H NMR spectrum are shown in Figure 3 . 1 A characteristic peak of C polysaccharide is shown at 3.22 ppm in the 1H NMR spectrum. Figure 3 In [diagram], A is the control Streptococcus pneumoniae type 7F capsular polysaccharide Y7FCK22C, Figure 3 in [diagram], B is the purified capsular polysaccharide Y7FCL15N. Compared with the spectrum of the control Streptococcus pneumoniae type 7F capsular polysaccharide, the peak area of the C polysaccharide characteristic peak of the capsular polysaccharide obtained by the purification method of the present invention decreases, and the other structural characteristic peaks are the same. According to the calculation formula of the C polysaccharide content, the C polysaccharide content of the control capsular polysaccharide is 2.33%, and the C polysaccharide content of the capsular polysaccharide purified by the method of the present invention is 0.17%. In summary, it shows that the method of the present invention can specifically reduce the content of C polysaccharide impurities.

[0149] 8.2 Immunodiffusion Refer to the method in 2.2 of Example 2, use the specific serum of Streptococcus pneumoniae type 7F capsular polysaccharide to prepare an agar plate for immunodiffusion experiment, and judge the change of polysaccharide antigenicity by measuring the diameter of the diffusion ring.

[0150] The immunodiffusion results of the capsular polysaccharide of Streptococcus pneumoniae serotype 7F are shown in Table 13. The purified capsular polysaccharide of Streptococcus pneumoniae serotype 7F obtained in the present invention has a diffusion ring diameter equivalent to that of the control capsular polysaccharide of Streptococcus pneumoniae serotype 7F, and is slightly superior to the control capsular polysaccharide. This indicates that the purification method of the present invention has no negative impact on the antigenicity of the capsular polysaccharide and the effect is slightly better than that of the control group.

[0151] Table 13: Table of immunodiffusion results of the capsular polysaccharide of Streptococcus pneumoniae serotype 7F ; 8.3 Physical and chemical tests 8.3.1 Referring to the method 2.3 in Example 2, the protein content, nucleic acid content, total nitrogen content, phosphorus content, and molecular weight of the purified capsular polysaccharide of Streptococcus pneumoniae serotype 7F obtained in Example 7 and the control capsular polysaccharide of Streptococcus pneumoniae serotype 7F obtained in Comparative Example 3 were detected.

[0152] 8.3.2 Detection of methyl pentose content Prepare 3 ml of a 50 μg / ml solution of the capsular polysaccharide of Streptococcus pneumoniae serotype 7F using purified water, and prepare 10 ml of a stock solution of a rhamnose reference substance at 20 μg / ml, and dilute it stepwise with purified water. Take 1 ml of the reference substance solution at gradient concentrations and the prepared polysaccharide sample solution in stoppered test tubes, and measure the polysaccharide sample in duplicate. In an ice-water bath, with stirring, add 4.5 ml of pre-cooled sulfuric acid solution dropwise to each tube, stopper the tube, warm the tube to room temperature, and place it in a water bath at 100 °C for 5 min. After cooling to room temperature, add 0.1 ml of a 3% mercaptoalanine hydrochloride solution, mix well, stopper the tube, and let it stand at room temperature for 1.5 h. Read the absorbance A value of each tube at wavelengths of 396 nm and 430 nm. Use the series of concentrations of the rhamnose reference substance solution to perform a linear regression on its corresponding corrected absorbance values (A396 - A430 nm) to obtain the regression equation. Substitute the corrected absorbance of the polysaccharide sample into the regression equation, and calculate the methyl pentose content according to the dilution factor. Then calculate the percentage content of methyl pentose based on the dry weight of the polysaccharide.

[0153] The test results are shown in Table 14. The various indicators of the capsular polysaccharide samples of Streptococcus pneumoniae serotype 7F obtained in Example 7 and Comparative Example 3 all meet the relevant standards specified in the pharmacopoeia.

[0154] Table 14: Table of physical and chemical test results of the capsular polysaccharide of Streptococcus pneumoniae serotype 7F ;

[0155] Example 9: Optimization of the purification process of the capsular polysaccharide of Streptococcus pneumoniae serotype 7F 9.1 Acetic acid concentration Using the method of the present invention to isolate and purify the capsular polysaccharide of Streptococcus pneumoniae type 7F, the final mass concentration of acetic acid added in each group in the C polysaccharide removal step was 0.5%, 1.0%, 1.5%, and 2.0% respectively, and the remaining steps were the same as in Example 7. The freeze-dried polysaccharide was harvested and subjected to 1 1H NMR detection. 1 The C polysaccharide characteristic peak (phosphocholine peak) was shown at 3.22 ppm in the 1H NMR spectrum. Based on the characteristic peak of the type 7F capsular polysaccharide, the content of C polysaccharide was quantified, and the results are shown in Table 15.

[0156] Table 15: Effect of acetic acid concentration on the content of C polysaccharide in the purified capsular polysaccharide of Streptococcus pneumoniae type 7F ; It can be seen from the detection results that as the acetic acid concentration increases, the content of C polysaccharide in the obtained capsular polysaccharide shows a decreasing trend. When using 1.0% acetic acid and 1.5% and 2.0% acetic acid for purification, the content of C polysaccharide in the obtained capsular polysaccharide is quite the same. Considering the purification effect and economic cost comprehensively, in the purification process of the capsular polysaccharide of Streptococcus pneumoniae type 7F, the preferred mass concentration of acetic acid used in the C polysaccharide removal step is 1.0%.

[0157] 9.2 Sodium nitrite concentration Using the method of the present invention to isolate and purify the capsular polysaccharide of Streptococcus pneumoniae type 7F, the final mass concentration of sodium nitrite added in each group in the C polysaccharide removal step was 1.0%, 2.0%, 3.0%, and 4.0% respectively, and the remaining steps were the same as in Example 7. The freeze-dried polysaccharide was harvested and subjected to 1 1H NMR detection. 1 The C polysaccharide characteristic peak (phosphocholine peak) was shown at 3.22 ppm in the 1H NMR spectrum. Based on the characteristic peak of the type 7F capsular polysaccharide, the content of C polysaccharide was quantified, and the results are shown in Table 16.

[0158] Table 16: Effect of sodium nitrite concentration on the content of C polysaccharide in the purified capsular polysaccharide of Streptococcus pneumoniae type 7F ; It can be seen from the detection results that as the sodium nitrite concentration increases, the content of C polysaccharide in the obtained capsular polysaccharide shows a decreasing trend. When using 3.0% sodium nitrite and 4.0% sodium nitrite for purification, the content of C polysaccharide in the obtained capsular polysaccharide is quite the same. Considering the purification effect and economic cost comprehensively, in the purification process of the capsular polysaccharide of Streptococcus pneumoniae type 7F, the preferred mass concentration of sodium nitrite used in the C polysaccharide removal step is 3.0%.

[0159] 9.3 Reaction time Using the method of the present invention to isolate and purify the capsular polysaccharide of Streptococcus pneumoniae serotype 7F, the reaction times of each group in the C polysaccharide removal step were 4.0 h, 6.0 h, 8.0 h, and 10.0 h respectively, and the remaining steps were the same as those in Example 7. The freeze-dried polysaccharide was harvested and subjected to 1 1H NMR detection. 1 The 1H NMR spectrum showed a characteristic peak of C polysaccharide (phosphocholine peak) at 3.22 ppm. Based on the characteristic peak of the capsular polysaccharide of serotype 7F, the content of C polysaccharide was quantified, and the results are shown in Table 17.

[0160] Table 17: Effect of reaction time on the content of C polysaccharide in the purified capsular polysaccharide of Streptococcus pneumoniae serotype 7F ; It can be seen from the detection results that as the reaction time prolonged, the content of C polysaccharide in the obtained capsular polysaccharide showed a decreasing trend. The content of C polysaccharide in the obtained capsular polysaccharide was comparable between the reaction times of 8.0 h and 10.0 h. Considering the purification effect and economic cost comprehensively, in the purification process of the capsular polysaccharide of Streptococcus pneumoniae serotype 7F, the preferred reaction time for the C polysaccharide removal step is 8.0 h.

[0161] 9.4 Reaction temperature Using the method of the present invention to isolate and purify the capsular polysaccharide of Streptococcus pneumoniae serotype 7F, the reaction temperatures of each group in the C polysaccharide removal step were 4.0 °C, 20.0 °C, 25.0 °C, and 37.0 °C respectively, and the remaining steps were the same as those in Example 7. The freeze-dried polysaccharide was harvested and subjected to 1 1H NMR detection. 1 The 1H NMR spectrum showed a characteristic peak of C polysaccharide (phosphocholine peak) at 3.22 ppm. Based on the characteristic peak of the capsular polysaccharide of serotype 7F, the content of C polysaccharide was quantified, and the results are shown in Table 18.

[0162] Table 18: Effect of reaction temperature on the content of C polysaccharide in the purified capsular polysaccharide of Streptococcus pneumoniae serotype 7F ; It can be seen from the detection results that as the reaction temperature increased, the content of C polysaccharide in the obtained capsular polysaccharide showed a decreasing trend. The content of C polysaccharide in the obtained capsular polysaccharide was comparable between 25.0 °C and 37.0 °C. Considering the purification effect and economic cost comprehensively, in the purification process of the capsular polysaccharide of Streptococcus pneumoniae serotype 7F, the preferred reaction temperature for the C polysaccharide removal step is 25.0 °C.

[0163] Example 10: Purification of the capsular polysaccharide of Streptococcus pneumoniae serotype 12F Take 10 L of the fermentation broth of Streptococcus pneumoniae type 12F, add sodium deoxycholate to make its final mass concentration 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 use sodium hydroxide to adjust the pH of the supernatant to 7. Filter through a 0.45 μm filter membrane, perform ultrafiltration concentration using a 100 kD membrane package, and replace the solution with phosphate buffer (pH 7.0). Add CTAB with a final mass concentration of 1.2% to the feed solution, 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 with a final mass concentration of 0.5%, centrifuge to harvest the supernatant. Filter through a 0.45 μm filter membrane, perform ultrafiltration concentration using a 100 kD membrane package, and replace the solution with phosphate buffer (pH 7.0). Add acetic acid with a final mass concentration of 3.0% and sodium nitrite with a final mass concentration of 5.0% to the feed solution, react at 25 °C for 10 h, perform ultrafiltration concentration using a 100 kD membrane package, and replace the solution with phosphate buffer (pH 7.0). Refine using a hydroxyapatite chromatography column and collect the target flow-through peak. Perform ultrafiltration concentration on the flow-through solution using a 100 kD membrane package and replace the solution with water for injection. Lyophilize the final product to obtain the capsular polysaccharide with batch number Y12FDC26N for subsequent detection.

[0164] Comparative Example 4: Purification of the capsular polysaccharide of Streptococcus pneumoniae type 12F control Take 10 L of the fermentation broth of Streptococcus pneumoniae type 12F, add sodium deoxycholate to make its final mass concentration 0.15%. Obtain a lysate. Add acetic acid to the lysate to adjust the pH to 4.5, centrifuge to collect the supernatant, and use sodium hydroxide to adjust the pH of the supernatant to 7. Filter through a 0.45 μm filter membrane, perform ultrafiltration concentration using a 100 kD membrane package, and replace the solution with phosphate buffer (pH 7.0). Add CTAB with a final mass concentration of 1.2% to the feed solution, 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 with a final mass concentration of 0.5%, centrifuge to harvest the supernatant. Filter through a 0.45 μm filter membrane, perform ultrafiltration concentration using a 100 kD membrane package, and replace the solution with phosphate buffer (pH 7.0). Refine using a hydroxyapatite chromatography column and collect the target flow-through peak. Perform ultrafiltration concentration on the flow-through solution using a 100 kD membrane package and replace the solution with water for injection. Lyophilize the final product to obtain the capsular polysaccharide with batch number Y12FDA28C for subsequent detection.

[0165] Example 11: Detection of the capsular polysaccharide of Streptococcus pneumoniae type 12F 11.1 1 1H NMR structural analysis Dissolve the control 12F type pneumococcal capsular polysaccharide obtained by purification in Comparative Example 4 and the 12F type pneumococcal capsular polysaccharide obtained by purification in Example 10 with heavy water respectively and freeze-dry them, repeating 3 times. Refer to the method in 2.1 of Example 2 for nuclear magnetic spectroscopy analysis.

[0166] The 1 H NMR spectrum results of the 12F type pneumococcal capsular polysaccharide are shown in Figure 4 . 1 A characteristic peak of C polysaccharide is shown at 3.22 ppm in the H NMR spectrum. Figure 4 In it, A is the control 12F type pneumococcal capsular polysaccharide Y12FDA28C, Figure 4 In it, B is the purified capsular polysaccharide Y12FDC26N. Compared with the spectrum of the control 12F type pneumococcal capsular polysaccharide, the peak area of the C polysaccharide characteristic peak of the capsular polysaccharide obtained by the purification method of the present invention decreases, and the rest of the structural characteristic peaks are the same. According to the calculation formula of the C polysaccharide content, the C polysaccharide content of the control capsular polysaccharide is 3.83%, and the C polysaccharide content of the capsular polysaccharide purified by the method of the present invention is 0.33%. In summary, it shows that the method of the present invention can specifically reduce the content of C polysaccharide impurities.

[0167] 11.2 Immunodiffusion Referring to the method in 2.2 of Example 2, use the specific serum of 12F type pneumococcal capsular polysaccharide to prepare an agar plate for immunodiffusion experiment, and judge the change of polysaccharide antigenicity by measuring the diameter of the diffusion ring.

[0168] The immunodiffusion results of the 12F type pneumococcal capsular polysaccharide are shown in Table 19. The diffusion ring diameter of the 12F type pneumococcal capsular polysaccharide purified by the present invention is equivalent to that of the control 12F type pneumococcal capsular polysaccharide, and is slightly better than that of the control capsular polysaccharide. It shows that the purification method of the present invention has no negative impact on the antigenicity of the capsular polysaccharide, and the effect is slightly better than that of the control group.

[0169] Table 19: Immunodiffusion result table of 12F type pneumococcal capsular polysaccharide ; 11.3 Physical and chemical detection: Referring to the method in 2.3 of Example 2, detect the protein content, nucleic acid content, total nitrogen content, phosphorus content, molecular weight determination, 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.

[0170] The detection results are shown in Table 20. The various indicators of the 12F type pneumococcal capsular polysaccharide samples purified in Example 10 and Comparative Example 4 all meet the relevant standards specified in the pharmacopoeia.

[0171] Table 20: Table of Physicochemical Detection Results of 12F-Type Pneumococcal Capsular Polysaccharide ;

[0172] Example 12: Optimization of the Purification Process of 12F-Type Pneumococcal Capsular Polysaccharide 12.1 Acetic Acid Concentration Using the method of the present invention to isolate and purify 12F-type Streptococcus pneumoniae capsular polysaccharide, the final mass concentrations of acetic acid added in each group in the C polysaccharide removal step were 1.0%, 2.0%, 3.0%, and 4.0% respectively, and the remaining steps were the same as those in Example 10. The harvested freeze-dried polysaccharide was subjected to 1 1H NMR detection. 1 The 1H NMR spectrum showed a characteristic peak of C polysaccharide (phosphocholine peak) at 3.22 ppm. Based on the characteristic peak of 12F-type capsular polysaccharide, the content of C polysaccharide was quantified, and the results are shown in Table 21.

[0173] Table 21: Effect of Acetic Acid Concentration on the Content of C Polysaccharide in the Purified 12F-Type Streptococcus pneumoniae Capsular Polysaccharide ; It can be seen from the detection results that as the acetic acid concentration increases, the content of C polysaccharide in the obtained capsular polysaccharide shows a decreasing trend. When purified with 3.0% acetic acid and 4.0% acetic acid, the content of C polysaccharide in the obtained capsular polysaccharide is quite similar. Considering the purification effect and economic cost comprehensively, in the purification process of 12F-type Streptococcus pneumoniae capsular polysaccharide, the mass concentration of acetic acid used in the C polysaccharide removal step is preferably 3.0%.

[0174] 12.2 Sodium Nitrite Concentration Using the method of the present invention to isolate and purify 12F-type Streptococcus pneumoniae capsular polysaccharide, the final mass concentrations of sodium nitrite added in each group in the C polysaccharide removal step were 1.0%, 2.5%, 5.0%, and 7.5% respectively, and the remaining steps were the same as those in Example 10. The harvested freeze-dried polysaccharide was subjected to 1 1H NMR detection. 1 The 1H NMR spectrum showed a characteristic peak of C polysaccharide (phosphocholine peak) at 3.22 ppm. Based on the characteristic peak of 12F-type capsular polysaccharide, the content of C polysaccharide was quantified, and the results are shown in Table 22.

[0175] Table 22: Effect of Sodium Nitrite Concentration on the Content of C Polysaccharide in the Purified 12F-Type Streptococcus pneumoniae Capsular Polysaccharide ; It can be seen from the detection results that as the concentration of sodium nitrite increases, the content of C polysaccharide in the obtained capsular polysaccharide shows a decreasing trend. When purified with 5.0% sodium nitrite and 7.5% sodium nitrite, the content of C polysaccharide in the obtained capsular polysaccharide is quite the same. Considering the purification effect and economic cost comprehensively, in the purification process of Streptococcus pneumoniae type 12F capsular polysaccharide, the mass concentration of sodium nitrite used in the C polysaccharide removal step is preferably 5.0%.

[0176] 12.3 Reaction time Using the method of the present invention to isolate and purify Streptococcus pneumoniae type 12F capsular polysaccharide, the reaction time of each group in the C polysaccharide removal step is 6.0 h, 8.0 h, 10.0 h, and 12.0 h respectively, and the remaining steps are the same as those in Example 10. The freeze-dried polysaccharide was harvested and subjected to 1 1H NMR detection. 1 The characteristic peak of C polysaccharide (phosphocholine peak) is shown at 3.22 ppm in the 1H NMR spectrum. Based on the characteristic peak of type 12F capsular polysaccharide, the content of C polysaccharide was quantified, and the results are shown in Table 23.

[0177] Table 23: Effect of reaction time on the content of C polysaccharide in the purified Streptococcus pneumoniae type 12F capsular polysaccharide ; It can be seen from the detection results that as the reaction time prolongs, the content of C polysaccharide in the obtained capsular polysaccharide shows a decreasing trend. The content of C polysaccharide in the obtained capsular polysaccharide is quite the same when reacting for 10.0 h and 12.0 h. Considering the purification effect and economic cost comprehensively, in the purification process of Streptococcus pneumoniae type 12F capsular polysaccharide, the reaction time in the C polysaccharide removal step is preferably 10.0 h.

[0178] 12.4 Reaction temperature Using the method of the present invention to isolate and purify Streptococcus pneumoniae type 12F capsular polysaccharide, the reaction temperature of each group in the C polysaccharide removal step is 4.0 °C, 20.0 °C, 25.0 °C, and 37.0 °C respectively, and the remaining steps are the same as those in Example 10. The freeze-dried polysaccharide was harvested and subjected to 1 1H NMR detection. 1 The characteristic peak of C polysaccharide (phosphocholine peak) is shown at 3.22 ppm in the 1H NMR spectrum. Based on the characteristic peak of type 12F capsular polysaccharide, the content of C polysaccharide was quantified, and the results are shown in Table 24.

[0179] Table 24: Effect of reaction temperature on the content of C polysaccharide in the purified Streptococcus pneumoniae type 4 capsular polysaccharide ; It can be seen from the detection results that as the reaction temperature increases, the content of C polysaccharide in the obtained capsular polysaccharide shows a decreasing trend. Compared with 25.0 °C and 37.0 °C, the content of C polysaccharide in the obtained capsular polysaccharide is quite the same. Considering the purification effect and economic cost comprehensively, in the purification process of the capsular polysaccharide of Streptococcus pneumoniae type 12F, the reaction temperature of the C polysaccharide removal step is preferably 25.0 °C.

[0180] The technical solution of the present invention is not limited to the limitations of the above specific embodiments. Any technical deformation made according to the technical solution of the present invention falls within the protection scope of the present invention, and the various technical features of the specific embodiments described in the present invention can be combined as needed.

Claims

1. A method for purifying pneumococcal capsular polysaccharide, the method comprising the following steps: (1) Treat pneumococcus to obtain a solution containing pneumococcal capsular polysaccharide; and (2) Perform C polysaccharide removal treatment on the solution containing pneumococcal capsular polysaccharide.

2. The purification method according to claim 1, wherein In step (2), the C polysaccharide removal treatment includes treating the solution containing pneumococcal capsular polysaccharide with a combined reagent, and the combined reagent includes an acid and a nitrite.

3. The purification method according to claim 2, wherein 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 mass concentration of the acid is 0.1% - 20%; The nitrite includes at least one of sodium nitrite, potassium nitrite, calcium nitrite or ammonium nitrite; The mass concentration of the nitrite is 0.1% - 20%.

4. The purification method according to claim 2, wherein 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%.

5. The purification method according to claim 1, wherein The temperature of the C polysaccharide removal treatment is 0°C - 50°C; The time of the C polysaccharide removal treatment is 0.1 h - 24 h.

6. The purification method according to any one of claims 1-5, characterized in that, Step (1) includes the following steps: (1 - 1) Perform lysis treatment on the bacterial solution containing pneumococcus to obtain a bacterial lysate; (1 - 2) Perform acid precipitation treatment on the lysate, and collect the first supernatant; (1 - 3) Add CTAB to the first supernatant, 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) Perform iodide salt precipitation treatment on the second supernatant, collect the third supernatant, and obtain the solution containing pneumococcal capsular polysaccharide.

7. The purification method according to claim 6, characterized in that, In step (1 - 1), the lysis treatment method includes at least one of chemical lysis, physical lysis or biological lysis; and / or In step (1 - 2), the acid precipitation treatment includes adding an acidic reagent to the lysate, adjusting the pH value to acidic, and then collecting the supernatant; and / or Step (1 - 2) further includes concentrating and changing the solution of the supernatant after the acid precipitation treatment and then collecting the first supernatant; and / or In step (1 - 3), the final mass concentration of CTAB is 0.1% - 5%; and / or In step (1 - 3), acidic pneumococcal capsular polysaccharide harvests the precipitate, adds sodium chloride solution to dissolve it, and collects the second supernatant; neutral pneumococcal capsular polysaccharide directly harvests the second supernatant; and / or In step (1 - 3), the final concentration of sodium chloride is 0.01 mol / L - 5 mol / L; and / or In step (1 - 4), the iodide salt includes sodium iodide and / or potassium iodide; and / or In step (1 - 4), the final concentration of the iodide salt is 0.01% - 5%.

8. The purification method according to any one of claims 1-5, characterized in that, The method further includes performing refining and purification treatment and drying treatment after step (2).

9. The purification method according to claim 8, wherein The refining and purification treatment includes at least one of membrane filtration, depth filtration, chromatography; The drying treatment uses freeze-drying.

10. The purification method according to claim 9, characterized in that, The refining and purification treatment includes successively performing a first ultrafiltration concentration treatment, a chromatography treatment, and a second ultrafiltration concentration treatment on the solution after the C polysaccharide removal treatment; the first ultrafiltration concentration treatment and / or the second ultrafiltration concentration treatment is carried out using a membrane package with a molecular weight cut-off of 30 - 300 KD.

11. The purification method according to any one of claims 1-5, characterized in that, The serotypes of the pneumococcus include at least one of 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, type 38.

12. The purification method according to claim 11, wherein Perform C polysaccharide removal treatment on the solution containing type 4 pneumococcal capsular polysaccharide, 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°C - 50°C; and / or the time of the C polysaccharide removal treatment is 5 h - 20 h; Perform C polysaccharide removal treatment on the solution containing type 5 pneumococcal capsular polysaccharide, 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°C - 40°C; and / or the time of the C polysaccharide removal treatment is 0.5 h - 2 h; Perform C polysaccharide removal treatment on the solution containing type 7F pneumococcal capsular polysaccharide, 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°C - 40°C; and / or the time of the C polysaccharide removal treatment is 4 h - 10 h; Perform C polysaccharide removal treatment on the solution containing type 12F pneumococcal capsular polysaccharide, 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°C - 40°C; and / or the time of the C polysaccharide removal treatment is 6 h - 12 h.

13. The purification method according to claim 11, wherein, Perform C polysaccharide removal treatment on the solution containing type 4 pneumococcal capsular polysaccharide, 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°C - 40°C; and / or the time of the C polysaccharide removal treatment is 14 h - 16 h; Perform C polysaccharide removal treatment on the solution containing type 5 pneumococcal capsular polysaccharide, 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°C - 30°C; and / or the time of the C polysaccharide removal treatment is 0.8 h - 1.2 h; Perform C polysaccharide removal treatment on a solution containing type 7F pneumococcal capsular polysaccharide, 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°C - 30°C; and / or the time of the C polysaccharide removal treatment is 7 h - 9 h; Perform C polysaccharide removal treatment on a solution containing type 12F pneumococcal capsular polysaccharide, 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°C - 30°C; and / or the time of the C polysaccharide removal treatment is 9 h - 11 h.

14. Pneumococcal capsular polysaccharide prepared by the method according to any one of claims 1 - 13.

15. The pneumococcal capsular polysaccharide according to claim 14, characterized in that, The content of C polysaccharide in the pneumococcal capsular polysaccharide is less than 5%.

16. Use of the pneumococcal capsular polysaccharide according to claim 14 or 15 in the preparation of a product containing pneumococcal capsular polysaccharide.

17. The application according to claim 16, wherein The product containing pneumococcal capsular polysaccharide includes pneumococcal vaccines.

18. The application according to claim 17, characterized in that, The pneumococcal vaccines include pneumococcal capsular polysaccharide vaccines or pneumococcal conjugate vaccines.

19. The application according to claim 17, wherein The pneumococcal vaccines include monovalent vaccines or multivalent vaccines.

20. A vaccine composition comprising the pneumococcal capsular polysaccharide prepared by the method according to any one of claims 1 - 13.

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