CN116693706A - Purification method of streptococcus pneumoniae capsular polysaccharide - Google Patents

Abstract

The invention provides a purification method of streptococcus pneumoniae capsular polysaccharide, which comprises the following steps: adding sodium deoxycholate into streptococcus pneumoniae bacterial liquid, sterilizing, regulating pH to acidic precipitation to obtain supernatant, ultrafiltering, adding CTAB precipitation, collecting supernatant or collecting precipitation, adding salt solution to obtain heavy suspension, adding sodium iodide precipitation to remove residual CTAB, and ultrafiltering to obtain purified polysaccharide. The method has the advantages of reduced steps, high polysaccharide recovery rate, low impurity content, excellent purification effect, environmental friendliness, suitability for industrialization and good popularization and application value.

Description

Purification method of streptococcus pneumoniae capsular polysaccharide

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a purification method of streptococcus pneumoniae capsular polysaccharide.

Background

Streptococcus pneumoniae (Streptococcus pneumococcus) is a main pathogenic bacterium for causing diseases such as pneumonia, otitis media, meningitis, bacteremia and the like, is one of the important causes of death of infants and the elderly, and serious threat to the health of people is caused by Streptococcus pneumoniae infection. However, the abuse of antibiotics can cause pneumococci to develop resistance, which presents difficulties in clinical treatment, and vaccination has become the most effective means for preventing pneumococcal related diseases.

Bacterial polysaccharide is widely distributed in the interior and the surface of cells, has rich structure and biological functions, and has important value in the fields of food, medicine and industry. Currently, macromolecular polysaccharides are classified into three types according to their distribution positions: bacterial surface polysaccharides, cell wall polysaccharides, and extracellular polysaccharides. Wherein, the bacterial surface polysaccharide is also called capsular polysaccharide (capsular polysaccharides, CPS) in part of pathogenic bacteria, can be covalently connected with a peptidoglycan layer to cover the outer layer of bacterial cell walls, forms a capsular protection barrier, and the specific polysaccharide layer can act as an antigen substance. CPS of Streptococcus pneumoniae is the major virulence factor and can divide the capsular polysaccharide of Streptococcus pneumoniae into 48 serotypes, 93 serotypes, depending on the antigenic nature. Of these, 23 serotypes cause about 90% of invasive diseases, including 1,3,4,5,6A,6B,7F,9V,14, 18C,19A,19F,23F, and so forth, and vaccines that use the streptococcus pneumoniae capsular polysaccharides of the above serotypes as antigens are now in widespread use.

However, as the capsular polysaccharide used as the vaccine needs to be obtained by further separating and purifying after various serotypes of streptococcus pneumoniae are grown and cultured in a culture medium, how to realize the efficient purification and preparation of the streptococcus pneumoniae capsular polysaccharide, shorten the process steps, reduce the cost and simultaneously keep environment-friendly is a key in the industrialized production process of the vaccine.

At present, in the prior art for producing and purifying streptococcus pneumoniae capsular polysaccharide, phenol is partially adopted for sterilization (Wang Jianhong et al, streptococcus pneumoniae culture and preparation of capsular polysaccharide thereof [ J ]. Gansu science journal, 2004 (03): 40-44.), and although the process is relatively simple, the phenol used by the method is not friendly to human health and environment, and has a large improvement space.

Chinese patent CN101663327B discloses a shortened purification method for producing streptococcus pneumoniae capsular polysaccharide, which uses sodium Deoxycholate (DOC) to sterilize, filters and ultrafilters supernatant, uses protein to reduce solubility at isoelectric point to adjust acid condition, uses protein precipitation and then centrifugally removes, and further uses active carbon adsorption method to prepare 1,4,5,6A,6B,7F,9V,14, 18C,19A,19F,23F type streptococcus pneumoniae capsular polysaccharide of 12 serotypes, but the preparation steps of the method are still longer, the operation is complicated, the coverage of serotypes is not wide enough, and the used active carbon adsorption method does not have specificity for adsorption of impurities such as protein, so that polysaccharide loss of part of serotypes (such as type 4) is larger, recovery rate is low, batch-to-batch variation is larger, and quality is unstable.

Chinese patent CN101180079B discloses a streptococcus pneumoniae polysaccharide-protein conjugate composition and discloses in the description section a method of streptococcus pneumoniae purification. Comprises the steps of continuously stirring and lysing cells by using sodium deoxycholate at a specific temperature (7-13 ℃), regulating the pH to be 4.5-6.8, standing (15-25 ℃) for precipitation, and adding CTAB for precipitation; however, the purification process disclosed in the patent further requires multiple steps such as ultrafiltration with phosphate buffer or sodium chloride solution, filtration with 0.2 μm, adsorption with activated carbon, and column chromatography with hydroxyapatite, which not only has long and complicated process steps, but also has high ultrafiltration cost with phosphate buffer or sodium chloride solution, which is unfavorable for amplification into process production.

Therefore, the method for efficiently purifying the streptococcus pneumoniae capsular polysaccharide, which has simple process steps, low cost and environmental friendliness, has very important industrial significance.

Disclosure of Invention

The invention aims to provide a method for efficiently purifying streptococcus pneumoniae capsular polysaccharide, which has the advantages of reduced steps, low cost and environmental friendliness.

The invention provides a method for purifying streptococcus pneumoniae capsular polysaccharide, which comprises the following steps:

(1) Adding sodium deoxycholate into streptococcus pneumoniae bacterial liquid for sterilization, cooling and regulating the pH value to be not higher than 4.5, standing and precipitating at 2-8 ℃ to remove precipitate to obtain supernatant, and ultrafiltering to obtain ultrafiltrate; the final concentration of the added deoxycholate sodium is 0.1-0.5%w/v;

(2) Adding neutral phosphate buffer solution and CTAB (CTAB) into the ultrafiltrate to react to generate precipitate, and collecting supernatant to obtain mixed solution to be treated, or collecting precipitate and adding salt solution to resuspension to obtain supernatant to obtain mixed solution to be treated;

(3) Adding sodium iodide into the mixed solution to be treated to generate precipitate, removing the precipitate, and ultrafiltering to obtain purified Streptococcus pneumoniae capsular polysaccharide solution.

Further, the ultrafiltration in the step (1) and the step (3) is ultrafiltration with water for injection.

Further, when the streptococcus pneumoniae is a type 7F, type 14 or type 23F streptococcus pneumoniae, the mixed solution to be treated in the step (2) is a collected supernatant;

or, when the streptococcus pneumoniae is type 1, type 3, type 4, type 5, type 6A, type 6B, type 9V, type 18C, type 19A or type 19F, the mixed solution to be treated in the step (2) is: the precipitate was added to the supernatant obtained after resuspension of the salt solution.

Further, the sterilization by adding sodium deoxycholate in the step (1) is that: standing for 1 to 24 hours at a temperature of between 20 and 30 ℃, preferably for 1 to 15 hours;

The temperature is reduced to be not more than 15 ℃, preferably not more than 12 ℃;

the standing and precipitating time is 1-24 h;

the pH is adjusted to 3.5-4.5.

Further, when the streptococcus pneumoniae is type 1, type 3, type 5, type 6A, type 6B, type 9V, type 19A, type 19F or type 23F, the final concentration of the sodium deoxycholate added in the step (1) is 0.1-0.2%w/V;

or, the streptococcus pneumoniae is 4-type, 7F-type, 14-type or 18C-type, and the final concentration of the added deoxycholate sodium in the step (1) is 0.3-0.5% w/v.

Further, when the streptococcus pneumoniae is type 1, type 3 or type 6A, the pH is adjusted to 4.5;

or, when the streptococcus pneumoniae is type 6B, 7F, 9V, 14, 18C, 19A, 19F or 23F, the pH is adjusted to pH 4.4;

or, when the streptococcus pneumoniae is type 4 or type 5, the pH adjustment is to adjust the pH to 4.2.

Further, when the streptococcus pneumoniae is type 1, type 5, type 6A, type 6B, type 7F, type 9V, type 14, type 19A, type 19F or type 23F, the standing precipitation is carried out at 2-8 ℃ for 1-5 hours;

or when the streptococcus pneumoniae is 3 type, 4 type and 18C type, the standing precipitation is carried out for 5-24 hours at the temperature of 2-8 ℃.

Further, the neutral phosphate buffer solution in the step (2) is: phosphate buffer with pH of 6.0-8.0; the final concentration of the phosphate buffer solution is 0.01-0.10 mol/L; the final concentration of the added CTAB is 0.2-2.0%w/v; the reaction time is 60-180 minutes; the salt solution is sodium chloride aqueous solution with the concentration of 0.1-0.5 mol/L.

Further, when the streptococcus pneumoniae is type 1, type 3, type 6A, type 7F, type 9V, type 14 or type 18C, the neutral phosphate buffer solution in the step (2) is: phosphate buffer with pH of 6.0-7.0;

or, when the streptococcus pneumoniae is type 4, type 5, type 6B, type 19A, type 23F, the neutral phosphate buffer solution in step (2) is: phosphate buffer with pH 7.0-8.0;

or, when the streptococcus pneumoniae is 19F type, the neutral phosphate buffer solution in step (2) is: phosphate buffer with pH of 6.6-7.4.

Further, the streptococcus pneumoniae is 1 type, 4 type, 5 type, 6A type, 6B type, 9V type, 18C type, 19A type or 19F type, and the salt solution in the step (2) is sodium chloride aqueous solution with the concentration of 0.1-0.3 mol/L;

or the streptococcus pneumoniae is of type 3, and the salt solution in the step (2) is sodium chloride aqueous solution with the concentration of 0.3-0.5 mol/L.

Further, the final concentration of the added sodium iodide in the step (3) is 0.1-1.0% w/v.

The invention has the beneficial effects that:

in many existing purification processes, polysaccharide directly obtained through the process of precipitating impurities such as nucleic acid is difficult to reach quality standard required by regulatory authorities, and more process steps (such as ultrafiltration, adsorption, column chromatography and the like) are added to make up for the defect, but the increase of process steps tends to reduce polysaccharide yield, greatly improve various costs, process stability and the like are also challenged. Furthermore, introducing more purification steps may introduce more exogenous substances, increasing the risk of the product, and detecting the residual amount of exogenous substances also increases a lot of costs. The method directly adjusts the pH of the sterilizing liquid to a specific acidic pH range after the DOC is used for sterilization, and the principle of combining and precipitating the DOC and the protein under the acidic condition is utilized to precipitate and remove the protein, so that the method successfully avoids the large-scale loss of polysaccharide caused by precipitation of polysaccharide while precipitating the protein under the condition of complex components of the sterilizing liquid while reducing the steps, and ensures the yield; meanwhile, the method obviously reduces the content of impurities such as protein, nucleic acid and the like in the feed liquid through the DOC and CTAB precipitation treatment by reasonable preparation method and parameter design optimization, so that the polysaccharide meeting the quality standard is obtained, and purification processes such as adsorption, chromatography and the like are not needed to be further carried out, so that the purification steps are greatly reduced; in addition, the ultrafiltration step of the invention adopts ultrafiltration with water for injection, and phosphate buffer solution or salt solution is not needed, so that the cost is low.

Therefore, the method for purifying streptococcus pneumoniae capsular polysaccharide has the advantages of reduced steps, short flow and working hours, low cost and strong process controllability and stability; the obtained polysaccharide has high recovery rate and excellent quality, is suitable for large-scale production, and has very good industrial application prospect.

CTAB refers to cetyltrimethylammonium bromide in the present invention.

The method for removing or collecting the sediment is centrifugation and filtration.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

FIG. 1 shows the recovery of polysaccharide protein and nucleic acid from pneumococcal capsular polysaccharide type 1 20181207 batch purification process.

FIG. 2 shows the recovery of polysaccharide protein and nucleic acid from pneumococcal capsular polysaccharide 3 during batch purification 20201001.

FIG. 3 shows the recovery of polysaccharide protein and nucleic acid from pneumococcal capsular polysaccharide type 4 20190903 batch purification.

FIG. 4 shows polysaccharide protein and nucleic acid recovery from pneumococcal capsular polysaccharide type 5 20200801 batch purification.

FIG. 5 shows the recovery of polysaccharide protein and nucleic acid from a batch purification process of pneumococcal capsular polysaccharide 20190101 of 6A.

FIG. 6 shows the recovery of polysaccharide protein and nucleic acid from a batch purification process of pneumococcal capsular polysaccharide 20190101 of FIG. 6B.

FIG. 7 shows the recovery of polysaccharide protein and nucleic acid from 7F pneumococcal capsular polysaccharide 20190903 batch purification.

FIG. 8 shows the recovery of polysaccharide protein and nucleic acid from a batch purification process of pneumococcal capsular polysaccharide type 9V 20190501.

FIG. 9 shows polysaccharide protein and nucleic acid recovery from a batch purification process of pneumococcal capsular polysaccharide 20190201 of 14 type.

FIG. 10 shows the recovery of polysaccharide protein and nucleic acid from a batch purification process of 18C pneumococcal capsular polysaccharide 20190904.

FIG. 11 shows the recovery of polysaccharide protein and nucleic acid from a 19A pneumococcal capsular polysaccharide 20190301 batch purification process.

FIG. 12 shows the recovery of polysaccharide protein and nucleic acid from a 19F pneumococcal capsular polysaccharide 20190301 batch purification process.

FIG. 13 shows the recovery of polysaccharide protein and nucleic acid from a 23F pneumococcal capsular polysaccharide 20190701 batch purification process.

Detailed Description

1. The strain used in the invention is from China Medical Collection Center (CMCC), and the strain numbers are as follows:

2. seed culture

Starting working seed batch strain, inoculating to pneumococcus animal source-free culture medium, at 35-38deg.C, 5-15% CO ₂ Culturing at 100-250 rpm to logarithmic phase, which is the first seed liquid. Transferring the first-level seed liquid to a pneumococcus animal source-free culture medium, culturing under the same condition to obtain a second-level seed liquid, repeating the passage method, and culturing to obtain third-level and fourth-level seed liquids for fermentation culture.

3. Fermentation

Monitoring culture temperature, pH, stirring speed, ventilation, material supplementing and dissolved oxygen in the culture process; sampling and detecting the concentration of the bacterial liquid and the concentration of glucose.

Culture temperature: 35-38 ℃; pH: maintaining the pH of the fermentation broth with 5mol/L sodium hydroxide solution; stirring speed: dynamically adjusting according to the concentration of the bacterial liquid; dissolved oxygen: introducing nitrogen into the surface layer for control; and (3) material supplementing: in the fermentation process, the material is continuously fed according to the glucose concentration in the fermentation liquid, and the glucose concentration is preferably 50-90 mmol/L. Fermentation is ended when the optical density value is not obviously increased or begins to be reduced, and fermentation liquid is obtained.

4. Purification

The purification process of the Streptococcus pneumoniae capsular polysaccharide of the invention will be described by way of examples

EXAMPLE 1 purification of Streptococcus pneumoniae capsular polysaccharide

The purification of streptococcus pneumoniae capsular polysaccharides consists essentially of precipitation, centrifugation/filtration, ultrafiltration concentration, dissociation steps, all performed at room temperature unless otherwise indicated.

1.1 obtaining a sterilizing liquid

Adding deoxysodium cholate with a final concentration of 0.2% (w/v) into the streptococcus pneumoniae fermentation broth type 1, uniformly mixing, standing at room temperature for 15 hours, and lysing cells to release capsular polysaccharide, wherein the capsular polysaccharide is the sterilizing solution.

1.2 preparation of sterilizing solution clarifying and acid regulating ultrafiltrate

Cooling the sterilizing liquid to 5-15 ℃. Adding glacial acetic acid while stirring to regulate the pH value to 4.5, and standing for 4 hours at the temperature of 2-8 ℃. After the standing is finished, centrifuging to collect supernatant, and ultrafiltering with water for injection to obtain acid regulating ultrafiltrate. The step uses sodium deoxycholate to precipitate proteins under acidic conditions, ultrafiltering and other means to remove most of the proteins, nucleic acids, thalli and low molecular weight culture medium components.

1.3CTAB treatment

Phosphate buffer with pH of 7.0 and final concentration of 0.02mol/L was added, CTAB solution was slowly added and final concentration of 1.0% (w/v), and the reaction was stirred at room temperature for about 180 minutes, and the precipitate was collected by centrifugation. The precipitate was resuspended in 0.3mol/L aqueous sodium chloride solution, the precipitate was dissociated by stirring at room temperature, after dissociation was completed, the sodium chloride dissociation solution was centrifuged and the supernatant was harvested, which was sodium chloride dissociation supernatant. In this step nucleic acid impurities are significantly removed.

1.4 preparation of NaI ultrafiltrate

Sodium iodide solid was added to the sodium chloride dissociation supernatant to a final concentration of 1.0% (w/v) NaI, and the mixture was centrifuged and filtered after stirring to collect a filtrate. Ultrafiltering with injectable water, and concentrating to obtain NaI ultrafiltrate. At this time, the purified polysaccharide solution meeting the quality requirement is obtained, and the polysaccharide solution can be stored for a long time after freeze-drying.

The recovery results of polysaccharide, protein and nucleic acid in each step of the purification process are shown in table 1 and fig. 1:

table 1 1 results of 20181207 batch purification process

The polysaccharide recovery and quality analysis results are shown in Table 2, and the type 1 polysaccharide purification process has good stability.

TABLE 2 results of three batch 1 Streptococcus pneumoniae capsular polysaccharide assays

EXAMPLE 2 purification of Streptococcus pneumoniae capsular polysaccharide 3

The purification of streptococcus pneumoniae capsular polysaccharides consists essentially of precipitation, centrifugation/filtration, ultrafiltration concentration, dissociation steps, all performed at room temperature unless otherwise indicated.

1.1 obtaining a sterilizing liquid

Adding deoxysodium cholate with a final concentration of 0.2% (w/v) into the streptococcus pneumoniae fermentation broth 3, uniformly mixing, standing at room temperature for 15 hours, and cracking cells to release capsular polysaccharide, wherein the capsular polysaccharide is the sterilizing liquid.

1.2 preparation of sterilizing solution clarifying and acid regulating ultrafiltrate

Cooling the sterilizing liquid to 5-15 ℃. Adding glacial acetic acid while stirring to regulate the pH value to 4.5, and standing for 14 hours at the temperature of 2-8 ℃. After the standing is finished, centrifuging to collect supernatant, and ultrafiltering with water for injection to obtain acid regulating ultrafiltrate. The step uses sodium deoxycholate to precipitate proteins under acidic conditions, ultrafiltering and other means to remove most of the proteins, nucleic acids, thalli and low molecular weight culture medium components.

1.3CTAB treatment

Phosphate buffer with pH of 7.0 and final concentration of 0.02mol/L was added, CTAB solution was slowly added and final concentration of 1.0% (w/v), and the reaction was stirred at room temperature for about 180 minutes, and the precipitate was collected by centrifugation. The precipitate was resuspended in 0.4mol/L aqueous sodium chloride solution, the precipitate was dissociated by stirring at room temperature, and after dissociation, the sodium chloride dissociation solution was centrifuged and the supernatant was harvested, which was the sodium chloride dissociation supernatant. In this step nucleic acid impurities are significantly removed.

1.4 preparation of NaI ultrafiltrate

Sodium iodide solid was added to the sodium chloride dissociation supernatant to a final concentration of 1.0% (w/v) NaI, and the mixture was centrifuged and filtered after stirring to collect a filtrate. Ultrafiltering with injectable water, and concentrating to obtain NaI ultrafiltrate. At this time, the purified polysaccharide solution meeting the quality requirement is obtained, and the polysaccharide solution can be stored for a long time after freeze-drying.

The recovery results of polysaccharide, protein and nucleic acid in each step of the purification process are shown in table 3 and fig. 2:

table 3 3 results of 20201001 batch purification process

The polysaccharide recovery and quality analysis results are shown in Table 4, and the type 3 polysaccharide purification process has good stability.

TABLE 4 results of three batches of Streptococcus pneumoniae capsular polysaccharide assay 3

EXAMPLE 3 purification of Streptococcus pneumoniae capsular polysaccharide

The purification of streptococcus pneumoniae capsular polysaccharides consists essentially of precipitation, centrifugation/filtration, ultrafiltration concentration, dissociation steps, all performed at room temperature unless otherwise indicated.

1.1 obtaining a sterilizing liquid

Adding deoxysodium cholate with a final concentration of 0.4% (w/v) into the streptococcus pneumoniae fermentation broth, uniformly mixing, standing at room temperature for 15 hours, and lysing cells to release capsular polysaccharide, wherein the capsular polysaccharide is the sterilizing liquid.

1.2 preparation of sterilizing solution clarifying and acid regulating ultrafiltrate

Cooling the sterilizing liquid to 5-15 ℃. Adding glacial acetic acid while stirring to regulate the pH value to 4.2, and standing for 20 hours at the temperature of 2-8 ℃. After the standing is finished, centrifuging to collect supernatant, and ultrafiltering with water for injection to obtain acid regulating ultrafiltrate. The step uses sodium deoxycholate to precipitate proteins under acidic conditions, ultrafiltering and other means to remove most of the proteins, nucleic acids, thalli and low molecular weight culture medium components.

1.3CTAB treatment

Phosphate buffer with pH of 7.8 and final concentration of 0.02mol/L was added, CTAB solution was slowly added and final concentration of 1.0% (w/v), and the reaction was stirred at room temperature for about 180 minutes, and the precipitate was collected by centrifugation. The precipitate was resuspended in 0.3mol/L aqueous sodium chloride solution, the precipitate was dissociated by stirring at room temperature, after dissociation was completed, the sodium chloride dissociation solution was centrifuged and the supernatant was harvested, which was sodium chloride dissociation supernatant. In this step nucleic acid impurities are significantly removed.

1.4 preparation of NaI ultrafiltrate

Sodium iodide solid was added to the sodium chloride dissociation supernatant to a final concentration of 1.0% (w/v) NaI, and the mixture was centrifuged and filtered after stirring to collect a filtrate. Ultrafiltering with injectable water, and concentrating to obtain NaI ultrafiltrate. At this time, the purified polysaccharide solution meeting the quality requirement is obtained, and the polysaccharide solution can be stored for a long time after freeze-drying.

The recovery results of polysaccharide, protein and nucleic acid in each step of the purification process are shown in table 5 and fig. 3:

table 5 4 results of 20190903 batch purification process

The polysaccharide recovery and quality analysis results are shown in Table 6, and the type 4 polysaccharide purification process has good stability.

TABLE 6 results of three batches of 4 Streptococcus pneumoniae capsular polysaccharide assays

EXAMPLE 4 purification of Streptococcus pneumoniae capsular polysaccharide

The purification of streptococcus pneumoniae capsular polysaccharides consists essentially of precipitation, centrifugation/filtration, ultrafiltration concentration, dissociation steps, all performed at room temperature unless otherwise indicated.

1.1 obtaining a sterilizing liquid

Adding deoxysodium cholate with a final concentration of 0.2% (w/v) into the streptococcus pneumoniae fermentation broth, uniformly mixing, standing at room temperature for 15 hours, and cracking cells to release capsular polysaccharide, wherein the capsular polysaccharide is the sterilizing liquid.

1.2 preparation of sterilizing solution clarifying and acid regulating ultrafiltrate

Cooling the sterilizing liquid to 5-15 ℃. Adding glacial acetic acid while stirring to regulate the pH value to 4.2, and standing for 4 hours at the temperature of 2-8 ℃. After the standing is finished, centrifuging to collect supernatant, and ultrafiltering with water for injection to obtain acid regulating ultrafiltrate. The step uses sodium deoxycholate to precipitate proteins under acidic conditions, ultrafiltering and other means to remove most of the proteins, nucleic acids, thalli and low molecular weight culture medium components.

1.3CTAB treatment

Phosphate buffer with pH of 7.8 and final concentration of 0.02mol/L was added, CTAB solution was slowly added and final concentration of 1.0% (w/v), and the reaction was stirred at room temperature for about 180 minutes, and the precipitate was collected by centrifugation. The precipitate was resuspended in 0.3mol/L aqueous sodium chloride solution, the precipitate was dissociated by stirring at room temperature, after dissociation was completed, the sodium chloride dissociation solution was centrifuged and the supernatant was harvested, which was sodium chloride dissociation supernatant. In this step nucleic acid impurities are significantly removed.

1.4 preparation of NaI ultrafiltrate

Sodium iodide solid was added to the sodium chloride dissociation supernatant to a final concentration of 1.0% (w/v) NaI, and the mixture was centrifuged and filtered after stirring to collect a filtrate. Ultrafiltering with injectable water, and concentrating to obtain NaI ultrafiltrate. At this time, the purified polysaccharide solution meeting the quality requirement is obtained, and the polysaccharide solution can be stored for a long time after freeze-drying.

The recovery results of polysaccharide, protein and nucleic acid at each step of the purification process are shown in table 7, fig. 4:

table 7 5 results of 20200801 batch purification process

The polysaccharide recovery and quality analysis results are shown in Table 8, and the type 5 polysaccharide purification process has good stability

Table 8 results of three batches of Streptococcus pneumoniae capsular polysaccharide assay

EXAMPLE 5 purification of 6A Streptococcus pneumoniae capsular polysaccharide

The purification of streptococcus pneumoniae capsular polysaccharides consists essentially of precipitation, centrifugation/filtration, ultrafiltration concentration, dissociation steps, all performed at room temperature unless otherwise indicated.

1.1 obtaining a sterilizing liquid

Adding deoxysodium cholate with a final concentration of 0.2% (w/v) into the 6A streptococcus pneumoniae fermentation broth, uniformly mixing, standing at room temperature for 15 hours, and lysing cells to release capsular polysaccharide, wherein the capsular polysaccharide is the sterilizing liquid.

1.2 preparation of sterilizing solution clarifying and acid regulating ultrafiltrate

Cooling the sterilizing liquid to 5-15 ℃. Adding glacial acetic acid while stirring to regulate the pH value to 4.5, and standing for 4 hours at the temperature of 2-8 ℃. After the standing is finished, centrifuging to collect supernatant, and ultrafiltering with water for injection to obtain acid regulating ultrafiltrate. The step uses sodium deoxycholate to precipitate proteins under acidic conditions, ultrafiltering and other means to remove most of the proteins, nucleic acids, thalli and low molecular weight culture medium components.

1.3CTAB treatment

Phosphate buffer with pH of 7.0 and final concentration of 0.02mol/L was added, CTAB solution was slowly added and final concentration of 1.0% (w/v), and the reaction was stirred at room temperature for about 180 minutes, and the precipitate was collected by centrifugation. The precipitate was resuspended in 0.3mol/L aqueous sodium chloride solution, the precipitate was dissociated by stirring at room temperature, after dissociation was completed, the sodium chloride dissociation solution was centrifuged and the supernatant was harvested, which was sodium chloride dissociation supernatant. In this step nucleic acid impurities are significantly removed.

1.4 preparation of NaI ultrafiltrate

Sodium iodide solid was added to the sodium chloride dissociation supernatant to a final concentration of 1.0% (w/v) NaI, and the mixture was centrifuged and filtered after stirring to collect a filtrate. Ultrafiltering with injectable water, and concentrating to obtain NaI ultrafiltrate. At this time, the purified polysaccharide solution meeting the quality requirement is obtained, and the polysaccharide solution can be stored for a long time after freeze-drying.

The recovery results of polysaccharide, protein and nucleic acid at each step of the purification process are shown in table 9, fig. 5:

TABLE 9 detection results for model 6A 20190101 batch purification process

The polysaccharide recovery and quality analysis results are shown in Table 10, and the 6A polysaccharide purification process has good stability

Table 10 results of three batches of 6A Streptococcus pneumoniae capsular polysaccharide assays

EXAMPLE 6 purification of 6B Streptococcus pneumoniae capsular polysaccharide

The purification of streptococcus pneumoniae capsular polysaccharides consists essentially of precipitation, centrifugation/filtration, ultrafiltration concentration, dissociation steps, all performed at room temperature unless otherwise indicated.

1.1 obtaining a sterilizing liquid

Adding deoxysodium cholate with a final concentration of 0.2% (w/v) into the 6B streptococcus pneumoniae fermentation broth, uniformly mixing, standing at room temperature for 15 hours, and lysing cells to release capsular polysaccharide, wherein the capsular polysaccharide is the sterilizing liquid.

1.2 preparation of sterilizing solution clarifying and acid regulating ultrafiltrate

Cooling the sterilizing liquid to 5-15 ℃. Adding glacial acetic acid while stirring to regulate the pH value to 4.4, and standing for 4 hours at the temperature of 2-8 ℃. After the standing is finished, centrifuging to collect supernatant, and ultrafiltering with water for injection to obtain acid regulating ultrafiltrate. The step uses sodium deoxycholate to precipitate proteins under acidic conditions, ultrafiltering and other means to remove most of the proteins, nucleic acids, thalli and low molecular weight culture medium components.

1.3CTAB treatment

Phosphate buffer with pH of 7.8 and final concentration of 0.02mol/L was added, CTAB solution was slowly added and final concentration of 1.0% (w/v), and the reaction was stirred at room temperature for about 180 minutes, and the precipitate was collected by centrifugation. The precipitate was resuspended in 0.3mol/L aqueous sodium chloride solution, the precipitate was dissociated by stirring at room temperature, after dissociation was completed, the sodium chloride dissociation solution was centrifuged and the supernatant was harvested, which was sodium chloride dissociation supernatant. In this step nucleic acid impurities are significantly removed.

1.4 preparation of NaI ultrafiltrate

Sodium iodide solid was added to the sodium chloride dissociation supernatant to a final concentration of 1.0% (w/v) NaI, and the mixture was centrifuged and filtered after stirring to collect a filtrate. Ultrafiltering with injectable water, and concentrating to obtain NaI ultrafiltrate. At this time, the purified polysaccharide solution meeting the quality requirement is obtained, and the polysaccharide solution can be stored for a long time after freeze-drying.

The recovery results of polysaccharide, protein and nucleic acid at each step of the purification process are shown in table 11, fig. 6:

TABLE 11 detection results for 6B type 20190101 batch purification process

The polysaccharide recovery and quality analysis results are shown in Table 12, and the 6B type polysaccharide purification process has good stability.

Table 12 results of three batches of 6B Streptococcus pneumoniae capsular polysaccharide assays

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EXAMPLE 7 purification of 7F Streptococcus pneumoniae capsular polysaccharide

The purification of 7F pneumococcal capsular polysaccharide consisted mainly of precipitation, centrifugation/filtration, ultrafiltration concentration, all steps being performed at room temperature unless otherwise noted.

1.1 obtaining a sterilizing liquid

Adding deoxysodium cholate with a final concentration of 0.4% (w/v) into the 7F streptococcus pneumoniae fermentation broth, uniformly mixing, standing at room temperature for 15 hours, and lysing cells to release capsular polysaccharide, wherein the capsular polysaccharide is the sterilizing liquid.

1.2 preparation of sterilizing solution clarifying and acid regulating ultrafiltrate

Cooling the sterilizing liquid to 5-15 ℃. Adding glacial acetic acid while stirring to regulate the pH value to 4.4, and standing for 4 hours at the temperature of 2-8 ℃. After the standing is finished, centrifuging to collect supernatant, and ultrafiltering with water for injection to obtain acid regulating ultrafiltrate. The step uses sodium deoxycholate to precipitate proteins under acidic conditions, ultrafiltering and other means to remove most of the proteins, nucleic acids, thalli and low molecular weight culture medium components.

1.3CTAB treatment

Phosphate buffer solution with pH of 7.0 is added, the final concentration is about 0.05mol/L, CTAB solution is slowly added, the final concentration is about 1.0% (w/v), the mixture is stirred at room temperature for 180 minutes, and the supernatant is collected by centrifugation, and is CTAB supernatant. In this step nucleic acid impurities are significantly removed.

1.4 preparation of NaI ultrafiltrate

Sodium iodide solid was added to CTAB supernatant to a final NaI concentration of about 1.0% (w/v), and the mixture was centrifuged and filtered after stirring to collect a filtrate. Ultrafiltering with injectable water, and concentrating to obtain NaI ultrafiltrate. At this time, the purified polysaccharide solution meeting the quality requirement is obtained, and the polysaccharide solution can be stored for a long time after freeze-drying.

The recovery results of polysaccharide, protein and nucleic acid at each step of the purification process are shown in table 13, fig. 7:

TABLE 13 detection results of 7F type 20190903 batch purification process

The polysaccharide recovery and quality analysis results are shown in Table 14, and the 7F type polysaccharide purification process has good stability.

Table 14 results of three batches of 7F Streptococcus pneumoniae capsular polysaccharide assays

EXAMPLE 8 purification of the capsular polysaccharide of Streptococcus pneumoniae type 9V

The purification of streptococcus pneumoniae capsular polysaccharides consists essentially of precipitation, centrifugation/filtration, ultrafiltration concentration, dissociation steps, all performed at room temperature unless otherwise indicated.

1.1 obtaining a sterilizing liquid

Adding deoxysodium cholate with a final concentration of 0.2% (w/V) into the 9V streptococcus pneumoniae fermentation broth, uniformly mixing, standing at room temperature for 15 hours, and lysing cells to release capsular polysaccharide, wherein the capsular polysaccharide is the sterilizing liquid.

1.2 preparation of sterilizing solution clarifying and acid regulating ultrafiltrate

Cooling the sterilizing liquid to 5-15 ℃. Adding glacial acetic acid while stirring to regulate the pH value to 4.4, and standing for 4 hours at the temperature of 2-8 ℃. After the standing is finished, centrifuging to collect supernatant, and ultrafiltering with water for injection to obtain acid regulating ultrafiltrate. The step uses sodium deoxycholate to precipitate proteins under acidic conditions, ultrafiltering and other means to remove most of the proteins, nucleic acids, thalli and low molecular weight culture medium components.

1.3CTAB treatment

Phosphate buffer with pH of 7.0 and final concentration of 0.02mol/L was added, CTAB solution was slowly added and final concentration of 1.0% (w/v), and the reaction was stirred at room temperature for about 180 minutes, and the precipitate was collected by centrifugation. The precipitate was resuspended in 0.3mol/L aqueous sodium chloride solution, the precipitate was dissociated by stirring at room temperature, after dissociation was completed, the sodium chloride dissociation solution was centrifuged and the supernatant was harvested, which was sodium chloride dissociation supernatant. In this step nucleic acid impurities are significantly removed.

1.4 preparation of NaI ultrafiltrate

Sodium iodide solid was added to the sodium chloride dissociation supernatant to a final concentration of 1.0% (w/v) NaI, and the mixture was centrifuged and filtered after stirring to collect a filtrate. Ultrafiltering with injectable water, and concentrating to obtain NaI ultrafiltrate. At this time, the purified polysaccharide solution meeting the quality requirement is obtained, and the polysaccharide solution can be stored for a long time after freeze-drying.

The recovery results of polysaccharide, protein and nucleic acid at each step of the purification process are shown in table 15, fig. 8:

TABLE 15 detection results for 9V type 20190501 batch purification process

The polysaccharide recovery and quality analysis results are shown in Table 16, and the 9V type polysaccharide purification process has good stability.

Table 16 results of three batches of 9V Streptococcus pneumoniae capsular polysaccharide assay

EXAMPLE 9 purification of Streptococcus pneumoniae capsular polysaccharide 14

The purification of the capsular polysaccharide from pneumonia consisted mainly of precipitation, centrifugation/filtration, ultrafiltration concentration, all steps being performed at room temperature unless otherwise indicated.

1.1 obtaining a sterilizing liquid

Adding deoxysodium cholate with a final concentration of 0.4% (w/v) into the fermentation liquor of the streptococcus pneumoniae 14, uniformly mixing, standing at room temperature for 15 hours, and cracking cells to release capsular polysaccharide, wherein the capsular polysaccharide is the sterilizing liquor.

1.2 preparation of sterilizing solution clarifying and acid regulating ultrafiltrate

Cooling the sterilizing liquid to 5-15 ℃. Adding glacial acetic acid while stirring to regulate the pH value to 4.4, and standing for 4 hours at the temperature of 2-8 ℃. After the standing is finished, centrifuging to collect supernatant, and ultrafiltering with water for injection to obtain acid regulating ultrafiltrate. The step uses sodium deoxycholate to precipitate proteins under acidic conditions, ultrafiltering and other means to remove most of the proteins, nucleic acids, thalli and low molecular weight culture medium components.

1.3CTAB treatment

Phosphate buffer solution with pH of 7.0 is added, the final concentration is about 0.05mol/L, CTAB solution is slowly added, the final concentration is about 1.0% (w/v), the mixture is stirred at room temperature for 180 minutes, and the supernatant is collected by centrifugation, and is CTAB supernatant. In this step nucleic acid impurities are significantly removed.

1.4 preparation of NaI ultrafiltrate

Sodium iodide solid was added to CTAB supernatant to a final NaI concentration of about 1.0% (w/v), and the mixture was centrifuged and filtered after stirring to collect a filtrate. Ultrafiltering with injectable water, and concentrating to obtain NaI ultrafiltrate. At this time, the purified polysaccharide solution meeting the quality requirement is obtained, and the polysaccharide solution can be stored for a long time after freeze-drying.

The recovery results of polysaccharide, protein and nucleic acid at each step of the purification process are shown in table 17, fig. 9:

table 17 results of batch 20190201 purification procedure

The polysaccharide recovery and quality analysis results are shown in Table 18, and the type 14 polysaccharide purification process has good stability.

Table 18 results of three batches of 14-type Streptococcus pneumoniae capsular polysaccharide assays

EXAMPLE 10 purification of 18C Streptococcus pneumoniae capsular polysaccharide

The purification of streptococcus pneumoniae capsular polysaccharides consists essentially of precipitation, centrifugation/filtration, ultrafiltration concentration, dissociation steps, all performed at room temperature unless otherwise indicated.

1.1 obtaining a sterilizing liquid

Adding deoxysodium cholate with a final concentration of 0.4% (w/v) into the 18C streptococcus pneumoniae fermentation broth, uniformly mixing, standing at room temperature for 15 hours, and lysing cells to release capsular polysaccharide, wherein the capsular polysaccharide is the sterilizing liquid.

1.2 preparation of sterilizing solution clarifying and acid regulating ultrafiltrate

Cooling the sterilizing liquid to 5-15 ℃. Adding glacial acetic acid while stirring to regulate the pH value to 4.4, and standing for 14 hours at the temperature of 2-8 ℃. After the standing is finished, centrifuging to collect supernatant, and ultrafiltering with water for injection to obtain acid regulating ultrafiltrate. The step uses sodium deoxycholate to precipitate proteins under acidic conditions, ultrafiltering and other means to remove most of the proteins, nucleic acids, thalli and low molecular weight culture medium components.

1.3CTAB treatment

Phosphate buffer with pH of 7.0 and final concentration of 0.02mol/L was added, CTAB solution was slowly added and final concentration of 1.0% (w/v), and the reaction was stirred at room temperature for about 180 minutes, and the precipitate was collected by centrifugation. The precipitate was resuspended in 0.3mol/L aqueous sodium chloride solution, the precipitate was dissociated by stirring at room temperature, after dissociation was completed, the sodium chloride dissociation solution was centrifuged and the supernatant was harvested, which was sodium chloride dissociation supernatant. In this step nucleic acid impurities are significantly removed.

1.4 preparation of NaI ultrafiltrate

Sodium iodide solid was added to the sodium chloride dissociation supernatant to a final concentration of 1.0% (w/v) NaI, and the mixture was centrifuged and filtered after stirring to collect a filtrate. Ultrafiltering with injectable water, and concentrating to obtain NaI ultrafiltrate. At this time, the purified polysaccharide solution meeting the quality requirement is obtained, and the polysaccharide solution can be stored for a long time after freeze-drying.

The recovery results of polysaccharide, protein and nucleic acid at each step of the purification process are shown in table 19, fig. 10:

TABLE 19 detection results for 18C type 20190904 batch purification process

The polysaccharide recovery and quality analysis results are shown in Table 20, and the 18C type polysaccharide purification process has good stability.

Table 20 results of three batches of 18C Streptococcus pneumoniae capsular polysaccharide assays

EXAMPLE 11, 19A purification of Streptococcus pneumoniae capsular polysaccharide

The purification of streptococcus pneumoniae capsular polysaccharides consists essentially of precipitation, centrifugation/filtration, ultrafiltration concentration, dissociation steps, all performed at room temperature unless otherwise indicated.

1.1 obtaining a sterilizing liquid

Adding deoxysodium cholate with a final concentration of 0.2% (w/v) into the fermentation liquor of the 19A streptococcus pneumoniae, uniformly mixing, standing at room temperature for 15 hours, and cracking cells to release capsular polysaccharide, wherein the capsular polysaccharide is the sterilizing liquor.

1.2 preparation of sterilizing solution clarifying and acid regulating ultrafiltrate

Cooling the sterilizing liquid to 5-15 ℃. Adding glacial acetic acid while stirring to regulate the pH value to 4.4, and standing for 4 hours at the temperature of 2-8 ℃. After the standing is finished, centrifuging to collect supernatant, and ultrafiltering with water for injection to obtain acid regulating ultrafiltrate. The step uses sodium deoxycholate to precipitate proteins under acidic conditions, ultrafiltering and other means to remove most of the proteins, nucleic acids, thalli and low molecular weight culture medium components.

1.3CTAB treatment

Phosphate buffer with pH of 7.8 and final concentration of 0.02mol/L was added, CTAB solution was slowly added and final concentration of 1.0% (w/v), and the reaction was stirred at room temperature for about 180 minutes, and the precipitate was collected by centrifugation. The precipitate was resuspended in 0.3mol/L aqueous sodium chloride solution, the precipitate was dissociated by stirring at room temperature, after dissociation was completed, the sodium chloride dissociation solution was centrifuged and the supernatant was harvested, which was sodium chloride dissociation supernatant. In this step nucleic acid impurities are significantly removed.

1.4 preparation of NaI ultrafiltrate

Sodium iodide solid was added to the sodium chloride dissociation supernatant to a final concentration of 1.0% (w/v) NaI, and the mixture was centrifuged and filtered after stirring to collect a filtrate. Ultrafiltering with injectable water, and concentrating to obtain NaI ultrafiltrate. At this time, the purified polysaccharide solution meeting the quality requirement is obtained, and the polysaccharide solution can be stored for a long time after freeze-drying.

The recovery results of polysaccharide, protein and nucleic acid at each step of the purification process are shown in table 21, fig. 11:

TABLE 21 19A type 20190301 batch purification procedure assay results

The polysaccharide recovery and quality analysis results are shown in Table 22, and the type 19A polysaccharide purification process has good stability.

Table 22 results of three 19A Streptococcus pneumoniae capsular polysaccharide assays

EXAMPLE 12 purification of 19F Streptococcus pneumoniae capsular polysaccharide

The purification of streptococcus pneumoniae capsular polysaccharides consists essentially of precipitation, centrifugation/filtration, ultrafiltration concentration, dissociation steps, all performed at room temperature unless otherwise indicated.

1.1 obtaining a sterilizing liquid

Adding deoxysodium cholate with a final concentration of 0.2% (w/v) into the 19F streptococcus pneumoniae fermentation broth, uniformly mixing, standing at room temperature for 15 hours, and lysing cells to release capsular polysaccharide, wherein the capsular polysaccharide is the sterilizing liquid.

1.2 preparation of sterilizing solution clarifying and acid regulating ultrafiltrate

Cooling the sterilizing liquid to 5-15 ℃. Adding glacial acetic acid while stirring to regulate the pH value to 4.4, and standing for 4 hours at the temperature of 2-8 ℃. After the standing is finished, centrifuging to collect supernatant, and ultrafiltering with water for injection to obtain acid regulating ultrafiltrate. The step uses sodium deoxycholate to precipitate proteins under acidic conditions, ultrafiltering and other means to remove most of the proteins, nucleic acids, thalli and low molecular weight culture medium components.

1.3CTAB treatment

Phosphate buffer with pH of 7.4 and final concentration of 0.02mol/L was added, CTAB solution was slowly added and final concentration of 1.0% (w/v), and the reaction was stirred at room temperature for about 180 minutes, and the precipitate was collected by centrifugation. The precipitate was resuspended in 0.3mol/L aqueous sodium chloride solution, the precipitate was dissociated by stirring at room temperature, after dissociation was completed, the sodium chloride dissociation solution was centrifuged and the supernatant was harvested, which was sodium chloride dissociation supernatant. In this step nucleic acid impurities are significantly removed.

1.4 preparation of NaI ultrafiltrate

Sodium iodide solid was added to the sodium chloride dissociation supernatant to a final concentration of 1.0% (w/v) NaI, and the mixture was centrifuged and filtered after stirring to collect a filtrate. Ultrafiltering with injectable water, and concentrating to obtain NaI ultrafiltrate. At this time, the purified polysaccharide solution meeting the quality requirement is obtained, and the polysaccharide solution can be stored for a long time after freeze-drying.

The recovery results of polysaccharide, protein and nucleic acid at each step of the purification process are shown in table 23, fig. 12:

TABLE 23 19F type 20190301 batch purification procedure assay results

The polysaccharide recovery and quality analysis results are shown in Table 24, and the 19F type polysaccharide purification process has good stability.

Table 24 results of three 19F Streptococcus pneumoniae capsular polysaccharide assays

Examples 13, 23F purification of Streptococcus pneumoniae capsular polysaccharide

The purification of the 23F pneumococcal capsular polysaccharide consisted mainly of precipitation, centrifugation/filtration, ultrafiltration concentration, all steps being performed at room temperature unless otherwise noted.

1.1 obtaining a sterilizing liquid

Adding deoxysodium cholate with a final concentration of 0.2% (w/v) into 23F streptococcus pneumoniae fermentation broth, uniformly mixing, standing at room temperature for 15 hours, and lysing cells to release capsular polysaccharide, which is a sterilizing solution.

1.2 preparation of sterilizing solution clarifying and acid regulating ultrafiltrate

Cooling the sterilizing liquid to 5-15 ℃. Adding glacial acetic acid while stirring to regulate the pH value to 4.4, and standing for 4 hours at the temperature of 2-8 ℃. After the standing is finished, centrifuging to collect supernatant, and ultrafiltering with water for injection to obtain acid regulating ultrafiltrate. The step uses sodium deoxycholate to precipitate proteins under acidic conditions, ultrafiltering and other means to remove most of the proteins, nucleic acids, thalli and low molecular weight culture medium components.

1.3CTAB treatment

Phosphate buffer solution with pH of 7.8, with a final concentration of about 0.05mol/L, CTAB solution with a final concentration of about 1.0% (w/v) was added slowly, and the reaction was stirred at room temperature for 180 minutes, and the supernatant was collected by centrifugation, which was CTAB supernatant. In this step nucleic acid impurities are significantly removed.

1.4 preparation of NaI ultrafiltrate

Sodium iodide solid was added to CTAB supernatant to a final NaI concentration of about 1.0% (w/v), and the mixture was centrifuged and filtered after stirring to collect a filtrate. Ultrafiltering with injectable water, and concentrating to obtain NaI ultrafiltrate. At this time, the purified polysaccharide solution meeting the quality requirement is obtained, and the polysaccharide solution can be stored for a long time after freeze-drying.

The recovery results of polysaccharide, protein and nucleic acid at each step of the purification process are shown in table 25, fig. 13:

table 25 23F type 20190701 batch purification procedure test results

The polysaccharide recovery and quality analysis results are shown in Table 26, and the 23F type polysaccharide purification process has good stability.

Table 26 results of three 23F Streptococcus pneumoniae capsular polysaccharide assays

The following experiments prove the beneficial effects of the invention.

Experimental example 1 optimization of sodium deoxycholate dosage

The inventor screens the preferential dosage of sodium deoxycholate for streptococcus pneumoniae of different serotypes, and discovers that the dosage of sodium deoxycholate has obvious influence on the purified result, and takes the purification of 7F type and 14 type streptococcus pneumoniae capsular polysaccharide as an example, as shown in table 27, when the DOC with the final concentration of 0.2% is adopted for sterilization, the protein content of the purified polysaccharide is relatively high, and after the DOC final concentration is increased to 0.4%, the protein content of the purified polysaccharide can be further obviously reduced.

Table 27 results after sterilization and purification of Streptococcus pneumoniae capsular polysaccharides of type 7F and 14 using different concentrations of sodium deoxycholate

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Experimental example 2, acid-regulating precipitation process parameter optimization

The inventor screens the technological parameters of the DOC sterilization and acid regulation precipitation process aiming at streptococcus pneumoniae with different serotypes, and discovers that the combination of the DOC sterilization and acid regulation precipitation process with parameters such as pH value, standing precipitation time and the like has obvious influence on the purification result besides the DOC dosage. Taking streptococcus pneumoniae capsular polysaccharides type 4 as an example, the purification effect of different parameter combinations in the DOC sterilization and acid precipitation-modulating process is demonstrated, as shown in table 28. It can be seen that for the streptococcus pneumoniae capsular polysaccharide type 4, a higher recovery of polysaccharide and a significantly reduced impurity content can be achieved at a pH of 4.2 and a settling time of 20 hours on the basis of a DOC usage of 0.4% w/v final concentration. Therefore, in the parameter optimization of the acid adjusting precipitation process step, the adjustment of a single factor sometimes cannot obtain an ideal effect, and a plurality of factors are required to be simultaneously adjusted to form an optimized parameter combination so as to obtain a good purification effect. Correspondingly, the inventor performs screening optimization on the purification parameters of streptococcus pneumoniae capsular polysaccharides of other serotypes, and finally obtains the optimal scheme (high polysaccharide recovery rate, low impurity content and excellent quality) in examples.

Table 28 comparison of preparation parameters of acid-regulating ultrafiltrate of streptococcus pneumoniae capsular polysaccharide

It is noted that, in contrast to example 13 described in the comparative document CN101180079B (which is also a method for purifying streptococcus pneumoniae capsular polysaccharide 4), the corresponding parameters of the process (DOC final concentration 0.12%, ph 6.4-6.8) and the scheme of the present invention are completely different, which results in insufficient purification effect in the step of treatment with DOC, and thus require further purification in the subsequent steps of activated carbon adsorption filtration and hydroxyapatite column chromatography.

Experimental examples 3, 23F optimization of CTAB treatment parameters for Streptococcus pneumoniae capsular polysaccharide

The process of the present invention has been developed to find that a lower CTAB concentration does not provide an ideal precipitation of polysaccharide, and that increasing the concentration can relatively increase the precipitation efficiency, for example to 3% in the CTAB treatment step disclosed in example 13 of patent CN101180079B for purification of 23F Streptococcus pneumoniae capsular polysaccharide, where the final concentration of CTAB is different from other serotypes.

However, the high concentration of CTAB is unfavorable for the stability of the reaction system, firstly, the solubility of CTAB is lower, the concentration is increased, the mother liquor consumption is increased to increase the whole reaction volume, in addition, the CTAB is easy to separate out in a precipitation form under high concentration, and is unfavorable for separating polysaccharide precipitation, so that the invention forms another path, impurities are precipitated in the final concentration of 1.0% of CTAB, as can be seen from table 29, 23F type polysaccharide exists in the supernatant of the reaction solution, polysaccharide precipitation is not formed with CTAB, and further, the steps of re-dissolution precipitation of sodium chloride solution, centrifugation and the like are omitted, and the excellent purification effect is obtained unexpectedly. Compared with the purification method disclosed in the patent CN101180079B, the method shortens the process flow, saves a large amount of labor and material cost and has remarkable advantages as a whole.

Table 29 23F CTAB treatment results

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Experimental example 4, optimization of type 3 NaCl dissociation parameters

After CTAB precipitation, the concentration of NaCl solution used for resuspension precipitation is adjusted, and different substances can be dissociated from the precipitation by using NaCl with different concentrations, so that the separation effect of impurities and target polysaccharide is affected, as shown in table 30, the concentration of nucleic acid in supernatant is relatively high after suspension centrifugation, the product has the risk of disqualification, and the nucleic acid content is obviously reduced after the concentration of NaCl solution is finely adjusted.

TABLE 30 results of NaCl dissociation supernatant

Experimental example 5, process stability verification of the invention

Table 31 shows the results of a 19A type 3 batch of purified polysaccharide assay on the same scale as the purification method disclosed in patent CN101663327B, in comparison with the data obtained from the purification methods disclosed in patent CN 29276-116, L29276-143.

Table 31 confirmation of preparation Process of 19A pneumococcal polysaccharide

The above table shows that the process disclosed in patent CN101663327B is not strong in stability and large in product quality lot-to-lot variation. The invention has small difference among batches and strong process controllability and stability.

In summary, the invention provides a method for purifying streptococcus pneumoniae capsular polysaccharide, which has the advantages of reduced steps, short flow and working hours, low cost, strong process controllability and stability, high recovery rate and excellent quality of the obtained polysaccharide, suitability for amplified production and good industrial application prospect.

Claims (10)

1. A method for purifying streptococcus pneumoniae capsular polysaccharides, comprising the steps of:

(1) Adding sodium deoxycholate into streptococcus pneumoniae bacterial liquid for sterilization, cooling and regulating the pH value to be not higher than 4.5, standing and precipitating at 2-8 ℃ to remove precipitate to obtain supernatant, and ultrafiltering to obtain ultrafiltrate; the final concentration of the added deoxycholate sodium is 0.1-0.5%w/v;

(2) Adding neutral phosphate buffer solution and CTAB (CTAB) into the ultrafiltrate to react to generate precipitate, and collecting supernatant to obtain mixed solution to be treated, or collecting precipitate and adding salt solution to resuspension to obtain supernatant to obtain mixed solution to be treated;

(3) Adding sodium iodide into the mixed solution to be treated to generate precipitate, removing the precipitate, and ultrafiltering to obtain purified Streptococcus pneumoniae capsular polysaccharide solution.

2. The method of claim 1, wherein said streptococcus pneumoniae is type 7F, type 14 or type 23F streptococcus pneumoniae and said mixture to be treated in step (2) is a collected supernatant;

or, the streptococcus pneumoniae is type 1, type 3, type 4, type 5, type 6A, type 6B, type 9V, type 18C, type 19A or type 19F, and the mixed solution to be treated in the step (2) is: the precipitate was added to the supernatant obtained after resuspension of the salt solution.

3. The method of claim 1, wherein the sterilizing with sodium deoxycholate in step (1) is performed by: standing for 1 to 24 hours at a temperature of between 20 and 30 ℃, preferably for 1 to 15 hours;

the temperature is reduced to be not more than 15 ℃, preferably not more than 12 ℃;

the standing and precipitating time is 1-24 h;

the pH is adjusted to 3.5-4.5;

and/or the ultrafiltration is ultrafiltration with water for injection.

4. The method of any one of claims 1 to 3, wherein streptococcus pneumoniae is type 1, type 3, type 5, type 6A, type 6B, type 9V, type 19A, type 19F or type 23F, and wherein sodium deoxycholate is added in step (1) at a final concentration of 0.1 to 0.2% w/V;

or, the streptococcus pneumoniae is 4-type, 7F-type, 14-type or 18C-type, and the final concentration of the added deoxycholate sodium in the step (1) is 0.3-0.5% w/v.

5. The method of any one of claims 1 to 3, wherein the streptococcus pneumoniae is type 1, type 3 or type 6A and the pH is adjusted to pH 4.5;

or, the streptococcus pneumoniae is type 6B, 7F, 9V, 14, 18C, 19A, 19F or 23F, and the pH is adjusted to pH 4.4;

or, the streptococcus pneumoniae is type 4 or type 5, and the pH adjustment is to adjust the pH to 4.2.

6. The method according to any one of claims 1 to 3, wherein the streptococcus pneumoniae is type 1, type 5, type 6A, type 6B, type 7F, type 9V, type 14, type 19A, type 19F or type 23F and the stationary precipitation is stationary at 2 to 8 ℃ for 1 to 5 hours;

or the streptococcus pneumoniae is of type 3, type 4 and type 18C, and the standing precipitation is carried out for 5-24 hours at the temperature of 2-8 ℃.

7. A method according to any one of claims 1 to 3, wherein the neutral phosphate buffer of step (2) is: phosphate buffer with pH of 6.0-8.0; the final concentration of the phosphate buffer solution is 0.01-0.10 mol/L; the final concentration of the added CTAB is 0.2-2.0%w/v; the reaction time is 60-180 minutes; the salt solution is sodium chloride aqueous solution with the concentration of 0.1-0.5 mol/L.

8. The method of claim 7, wherein the streptococcus pneumoniae is type 1, type 3, type 6A, type 7F, type 9V, type 14 or type 18C and the neutral phosphate buffer of step (2) is: phosphate buffer with pH of 6.0-7.0;

or, the streptococcus pneumoniae is type 4, type 5, type 6B, type 19A, type 23F, and the neutral phosphate buffer solution in the step (2) is: phosphate buffer with pH 7.0-8.0;

Or, the streptococcus pneumoniae is 19F type, and the neutral phosphate buffer solution in the step (2) is: phosphate buffer with pH of 6.6-7.4.

9. The method of any one of claims 1 to 3, wherein the streptococcus pneumoniae is type 1, type 4, type 5, type 6A, type 6B, type 9V, type 18C, type 19A or type 19F, and the salt solution in step (2) is an aqueous sodium chloride solution having a concentration of 0.1 to 0.3 mol/L;

or the streptococcus pneumoniae is of type 3, and the salt solution in the step (2) is sodium chloride aqueous solution with the concentration of 0.3-0.5 mol/L.

10. A method according to any one of claims 1 to 3 wherein the final concentration of sodium iodide added in step (3) is from 0.1 to 1.0% w/v; and/or the ultrafiltration is ultrafiltration with water for injection.