TW201522622A - Method for preparing capsular polysaccharides of streptococcus pneumoniae serotype - Google Patents

Abstract

The present invention provides an improved process for preparing capsular polysaccharides of Streptococcus pneumoniae serotypes. The process according to the invention includes further culturing the S. pneumoniae serotype without pH adjustment, thereby removing the protein precipitation via acidification by use of a pH adjuster.

Description

Method for preparing capsular polysaccharide of S. pneumoniae serotype Field of invention

The present invention relates to a method for preparing a capsular polysaccharide of a S. pneumoniae serotype. In particular, the present invention relates to the preparation of a capsular polysaccharide of a pneumococcal serotype by removing impurities such as proteins and nucleic acids from a serotype culture of a Streptococcus pneumoniae (S. pneumoniae) serotype capable of producing a pneumococcal polysaccharide. Methods.

Background of the invention

Streptococcus pneumoniae is a Gram-positive bacterium belonging to the genus Streptococcus faecalis and causes human diseases such as pneumonia, bacteremia, otitis media, and meningitis. The cells of the S. pneumoniae serotype have a capsule which is a polysaccharide shell surrounding each cell. This capsule interferes with phagocytosis by preventing antibodies from attaching to bacterial cells. Depending on the immunological properties, about 90 or more serotypes have been identified, some of which are reported to cause invasive diseases. A total of 37 serotypes were collected from invasive pneumococcal disease identification between 1996 and 2008, with serotype prevalence of 19F>23F>19A>6A>3>9V>6B. These major serotypes account for the blood that causes invasive pneumococcal disease The majority of the clear type, up to 53.4%. A capsular polysaccharide development vaccine from such S. pneumoniae serotypes, such as a polysaccharide vaccine and a polysaccharide-protein binding vaccine, has been used.

The capsular polysaccharide used to manufacture the polysaccharide vaccine and the polysaccharide-protein-binding vaccine is obtained from a culture of a pneumococcal strain capable of producing each serotype polysaccharide. Each strain was incubated at the optimum conditions of temperature, pH, and shaking until the growth reached a maximum. To obtain a non-secreting cell-derived S. pneumonia capsular polysaccharide, the cell line is subjected to a cell lysis method using sodium deoxycholate (DOC). Subsequently, in addition to the capsular polysaccharide, numerous proteins and nucleic acids are released from the cells, and such numerous proteins and nucleic acids should be removed in a purification process after culture. In order to minimize adverse events caused by substances other than the subject antigen after vaccination, the protein content and nucleic acid content must meet strict specifications. For example, in the case of Prevenar 7 ^(TM ), the specification of 2-5% or less protein and 1-2% or less of nucleic acid by dry weight should be met.

International Publication No. WO 2006/110352 discloses a method for preparing a capsular polysaccharide comprising culturing S. pneumoniae, lysing cells, precipitating proteins by acidification to pH 5.5 or below, culturing without oscillation, and by centrifugation and/or filtration purification. International Publication No. WO 2008/118752 discloses a method for preparing a capsular polysaccharide by filtering a cell lysate by ultrafiltration/dialysis and precipitating the protein by acidification. Conventional preparation methods essentially involve precipitating proteins via acidification using a pH adjusting agent, which complicates the overall process and also causes the capsular polysaccharide to be modified and produce harmful substances.

Detailed description of the invention

The inventors have conducted studies to improve the preparation of capsular polysaccharides of the S. pneumoniae serotype. Unexpectedly, it was found that further cultivation under the same conditions, without pH adjustment, caused the pH to fall to a range suitable for protein precipitation, i.e., pH 5.5 or below, due to its product, especially lactic acid. In other words, the inventors found that further culture, without pH adjustment, followed by cell lysis and protein precipitation can remove the step of precipitating the protein via acidification using a pH adjuster.

Accordingly, it is an object of the present invention to provide an improved method for preparing a capsular polysaccharide of a S. pneumoniae serotype.

In an aspect of the invention, there is provided a method for preparing a capsular polysaccharide of a S. pneumoniae serotype comprising the steps of: (a) cultivating a S. pneumoniae serotype while maintaining a pH in the culture solution at 7.0 (b) between the time when the absorbance of the culture solution reaches a level and when the absorbance begins to decrease, the culture in the step (a) is terminated; (c) the culture liquid obtained from the step (b) is further cultured, and has no pH. Adjust until the pH of the culture solution drops to 5.5 or below; (d) add a lysing agent to the culture solution obtained from step (c) to lyse the cells, precipitate the protein and remove the precipitated proteins and cell debris; and (e) The capsular polysaccharide is isolated and purified from the solution obtained in step (d).

In the method of the invention, the S. pneumoniae serotype can be 1, 2, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F, 22F, 23F or 33F.

In the method of the present invention, the culture in the step (a) can be carried out at 34-38 ° C The oscillation was carried out at 50-150 rpm.

In a specific example of the present invention, the step (b) can be carried out by terminating the culture in the step (a) within 1 to 3 hours from the time when the absorbance of the culture liquid reaches a level.

Further cultivation in step (c) was carried out at 34-38 ° C with shaking at 50-150 rpm without pH adjustment.

The lysing agent in step (d) may be sodium deoxycholate.

In still another embodiment of the present invention, the step (d) can be carried out by adding a lysing agent to the culture solution obtained from the step (c) to lyse the cells, and maintaining the resulting solution at 10-20 ° C for 3-24 After hours, there was no oscillation to precipitate the protein and the precipitated protein and cell debris were removed by centrifuging the solution.

In still another embodiment of the present invention, the separating and purifying in the step (e) comprises the steps of: (i) filtering the solution obtained from the step (d) via a depth filter to obtain a filtrate; (ii) concentrating From the filtrate of step (i), followed by ultrafiltration and centrifugation to obtain a supernatant; (iii) adding a cationic detergent to the supernatant obtained from step (ii) for cultivation, and centrifuging the resulting mixture to obtain a sedimentation granule or supernatant comprising a capsular polysaccharide; (iv) reacting the capsular polysaccharide obtained from step (iii) with sodium iodide, followed by centrifuging the resulting reaction mixture to obtain a supernatant; (v) adding activated carbon To the solution obtained from step (iv) and filtering the resulting mixture to obtain a filtrate; (vi) Concentrate the filtrate obtained from step (v) and perform ultrafiltration and centrifugation to obtain a capsular polysaccharide.

In this specific example, the concentration in step (ii) can be carried out using a 100 kDa membrane and the concentration in step (vi) is carried out using a 30 kDa membrane. Further, the cationic detergent used in the step (iii) may be cetyl cetyltrimethylammonium, and the brominated cetyltrimethylammonium is used in a concentration of 0.5 to 3.0%. The activated carbon in the step (v) may be used in an amount of from 1 to 5% (w/v).

The method of the invention does not use any pH adjusting agent. The present invention is based on the discovery that further culturing of serotype S. pneumoniae under the same conditions without any pH adjustment may result in a pH drop to a range suitable for protein precipitation, ie pH 5.5 or below, due to the culture product, especially lactic acid. . Thus, the method of the present invention does not require the use of any pH adjusting agent for protein precipitation, thereby minimizing the production of any modification and hazardous substances of the capsular polysaccharide and simplifying the preparation process. The S. pneumoniae serotype capsular polysaccharide obtained by the method of the present invention can be advantageously used as a polysaccharide vaccine and a polysaccharide-protein-binding vaccine.

Figure 1 shows the pH change of S. pneumoniae serotype 3 culture and its conditions.

Figure 2 shows pH changes and conditions of S. pneumoniae serotype 6B culture.

Figure 3 shows pH changes and conditions of S. pneumoniae serotype 7F culture.

Figure 4 shows the pH change of S. pneumoniae serotype 14 culture and Its conditions.

Figure 5 shows pH changes and conditions of S. pneumoniae serotype 19A culture.

Figure 6 shows pH changes and conditions of S. pneumoniae serotype 23F culture.

Invention mode

The present invention provides a method for preparing a capsular polysaccharide of a S. pneumoniae serotype comprising the steps of: (a) cultivating a S. pneumoniae serotype while maintaining a pH in the culture solution in the range of 7.0 to 9.4; (b) terminating the culture in the step (a) between when the absorbance of the culture solution reaches a level and when the absorbance starts to decrease; (c) further incubating the culture solution obtained from the step (b) without pH adjustment until the culture solution The pH is lowered to 5.5 or below; (d) a lysing agent is added to the culture solution obtained from step (c) to lyse the cells to precipitate the protein and remove the precipitated protein and cell debris; and (e) from the step (d) The solution separates and purifies the capsular polysaccharide.

In step (a) of the method of the invention, the S. pneumoniae serotype may be any serotype used to prepare a pneumococcal vaccine, including but not limited to serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F, 22F, 23F, 33F and the like. S. pneumoniae serotype strains are well known in the art and such strains can be used without limitation (for example, see WO2006/110381). The culture in step (a) of the method of the invention is based on general culture. The nutrient is carried out, for example, in a soybean-based medium while maintaining the pH in the culture solution in the range of 7.0 to 9.4, preferably 7.2 to 8.2, by using a pH adjusting agent such as sodium hydroxide. The culture can be carried out under generally known conditions, for example, at a temperature of 34-38 ° C, with an oscillation of 50-150 rpm. The pH conditions which can be used for individual S. pneumoniae serotype inoculum culture and main culture are shown in Table 1 below.

The method of the invention does not use any pH adjusting agent. Unexpectedly, the inventors have found that further cultivation under the same conditions, without pH adjustment, causes the pH to fall to the protein for its product, especially lactic acid. The range of precipitation is pH 5.5 or below. Thus, the method of the invention comprises culturing a S. pneumoniae serotype, preferably until the growth reaches its maximum, and further culturing without any pH adjustment, followed by cell lysis and protein precipitation. Thus, the method of the present invention does not require the use of any pH adjusting agent for protein precipitation, thereby minimizing the production of any modification and hazardous substances of the capsular polysaccharide and simplifying the preparation process.

The method of the present invention comprises, between the point when the absorbance of the culture solution reaches a level and the point at which the absorbance begins to decrease, terminating the culture in the step (a) [ie, the step (b)] and further comprising the culture liquid obtained from the step (b), Until the pH drops to 5.5 or below [ie step (c)].

In the culture of step (a) of the method of the present invention, as the cells grow, the absorbance of the culture solution - for example, the absorbance at 590 nm (OD ₅₉₀ ) - is gradually increased to a maximum value (about 7-24 hours after the start of the culture). And then maintain that value. After one to three hours, the absorbance begins to drop. Thus, in a specific example of the present invention, the step (b) can be carried out by terminating the culture in the step (a) within one to three hours from the time when the absorbance of the culture liquid reaches a level. Further culturing in step (c) can be carried out under the same conditions as in step (a), i.e., at a temperature of 34-38 ° C, with shaking at 50-150 rpm, without pH adjustment. This further culture naturally causes the pH of the culture to fall to 5.5 or below, thereby providing a pH suitable for protein precipitation.

The method of the invention comprises the addition of a lysing agent to the culture fluid obtained from step (c) to lyse the cells, precipitate the protein, and remove the precipitated proteins and cell debris [ie, step (d)].

Cell lysis can be carried out by methods known in the art, such as Shown in WO2006/110381 and WO2008/118752. For example, cell lysis can be carried out using sodium deoxycholate as a lysing agent. Sodium deoxycholate can be used in a concentration of 0.10 to 0.15% (concentration when sodium deoxycholate is added), but is not limited thereto. As the cells lyse, the capsular polysaccharide of the S. pneumoniae serotype is released from the cytoplasm. In addition, precipitation of the protein can be carried out by maintaining the reaction mixture at a temperature of 10 to 20 ° C and the precipitated protein and cell debris can be removed by a general method such as centrifugation. In a specific embodiment of the present invention, the step (d) can be carried out by adding a lysing agent to the culture solution obtained from the step (c), and maintaining the resulting solution at 10-20 ° C for 3-24 hours without oscillation. To precipitate the protein and remove precipitated proteins and cell debris by centrifugation.

The method of the invention comprises the removal of impurities, such as proteins and nucleic acids present in cells, from the solution obtained in step (d) to separate and purify the capsular polysaccharide [ie step (e)]. Isolation and purification of the capsular polysaccharide can be carried out by conventional purification methods, such as those disclosed in WO2006/110381 and WO2008/118752.

In one embodiment, the separation and purification of step (e) comprises the steps of: (i) filtering the solution obtained from step (d) via a depth filter to obtain a filtrate; (ii) concentrating from step (i) a filtrate, followed by ultrafiltration and centrifugation to obtain a supernatant; (iii) adding a cationic detergent to the supernatant obtained from step (ii) for cultivation, and centrifuging the resulting mixture to obtain a capsular polysaccharide Settling pellet or supernatant; (iv) reacting the capsular polysaccharide obtained from step (iii) with sodium iodide, followed by centrifuging the resulting reaction mixture to obtain a supernatant; (v) adding activated carbon to the solution obtained from step (iv) and filtering The mixture is obtained to obtain a filtrate; and (vi) the filtrate obtained from the step (v) is concentrated and subjected to ultrafiltration and centrifugation to obtain a capsular polysaccharide.

In this specific example, the lysate remaining after centrifugation to remove precipitated proteins and cell debris is removed by filtration using a depth filter.

In addition, two concentrations of concentration/ultrafiltration can be performed to remove proteins and nucleic acids. In one embodiment, the concentration in step (ii) can be carried out using a 100 kDa membrane and the concentration in step (iv) can be carried out using a 30 kDa membrane. The ultrafiltration used in this case is also referred to as "diafiltration".

The cationic detergent used in step (iii) may be cetyl cetyltrimethylammonium bromide. Cetyl trimethylammonium bromide [Cetyl cetyltrimethylammonium bromide, cetyltrimethylammonium bromide (HB)] can be 0.5-3% (when cetyltrimethylammonium bromide is added) The concentration of the concentration is used. A cationic detergent, such as HB, can be removed by precipitation using sodium iodide. If centrifugation is carried out in step (iii), the capsular polysaccharide may be a sedimentary granule (for example, serotypes 1, 2, 3, 4, 5, 6A, 6B, 9N, 9V, 18C, 19A, 19F, 22F, It exists in the form of 23F), or a supernatant (for example, 7F, 14, and 33F). The capsular polysaccharide obtained in the form of settled granules can be dissolved in a suitable solvent (for example, an aqueous solution of sodium chloride) for use in the subsequent step (i.e., reaction with sodium iodide). The capsular polysaccharide present in the supernatant can be used in the subsequent step (i.e., reaction with sodium iodide) without further isolation.

Further, the activated carbon in the step (v) may preferably be 1-5% (w/v). The amount used.

Hereinafter, the present invention is further exemplified with reference to the following examples. However, the examples provided are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any way.

Example

The compositions of the soy-based medium used in the following examples are shown in Table 2.

Example 1. Preparation of S. pneumoniae capsular polysaccharide serotypes 3, 6B, and 19A

Preparation of cell bank

The strains of S. pneumoniae serotypes 3, 6B, and 19A were obtained from the American Center for Strain (ATCC). The strains used for inoculum culture and main culture are shown in Table 3.

Create several generations of preserved strains (F1, F2, and F3 generations). Create two additional generations of preserved strains. The first extra generation was made from F3 vials, and the subsequent generations were made from the first extra generation vials. The vials of the strains were stored frozen (<-70 ° C) with synthetic glycerol as a cryoprotectant. To prepare a cell bank, all cultures were grown in soy-based medium. Prior to freezing, the cells are concentrated by centrifugation, the spent medium is removed, and the cell pellet is resuspended in fresh medium containing a cryoprotectant, such as synthetic glycerol.

Cultivation and collection

A strain containing a soybean-based medium is inoculated using a culture from a working cell bank and the bottle is incubated. After the target optical density (absorbance) is reached, the strain cell containing the soybean-based medium is inoculated with the strain bottle. The fermentation tank was incubated at 34-38 ° C with shaking at 120 rpm while maintaining the pH at about 7.2 or above with 3N NaOH. Samples were taken every 2-3 hours and their optical density was measured. When the optical density begins to grow substantially, sampling is performed every 30 minutes to 1 hour to measure the optical density. The culture was terminated after the optical density measurement reached a certain value and was maintained constant for 1 hour. Then, without any pH adjustment, further cultivation was continued at the same temperature and the same rpm oscillation until the pH reached 5.5 or less. After the further culture was terminated, 0.12% sodium deoxycholate was added to the culture to lyse the bacterial cells. The contents of the resulting fermenter were cooled to 10-15 ° C and maintained at this temperature without shaking for about 3 hours to induce protein precipitation. The mixture is then centrifuged to remove precipitated proteins and cell debris.

purification

The solution obtained from centrifugation is filtered with a depth filter to remove Protein and cell debris that were not precipitated during centrifugation. The filtrate was concentrated on a 100 kDa MW membrane and the concentrate was dialyzed against 25 mM sodium phosphate buffer (pH 7.2). The diafiltration system is carried out until the conductivity of the filtrate reaches about 3-4 mS/cm, and the TMP is set to 0.5 to 1.5 bar or less. After the impurities were removed from the sample, brominated cetyltrimethylammonium bromide (methyl cetyl bromide, cetyl bromide) was added from the stock solution to provide a final concentration of about 1.0% HB(w/v). Trimethylammonium (HB)] precipitated the polysaccharide from the concentrated diafiltration solution while shaking for about 1 hour, followed by centrifugation. The resulting polysaccharide pellet was dissolved in about 0.25 M aqueous sodium chloride solution and sodium iodide (NaI) from the stock NaI solution to provide a final concentration of about 0.5% (w/v) was added to the polysaccharide solution. The solution was centrifuged to obtain a supernatant and activated carbon was slowly added to the obtained solution to about 2.0 w/v% while shaking for about 1 hour, and then the solution was filtered. The filtrate was concentrated on a 30 kDa membrane and the concentrate was dialyzed against about 10 volumes of three distilled water. The diafiltration system was carried out until the conductivity of the filtrate reached about 10 μS/cm, and the TMP was set to 0.5 to 1.5 bar or less. The concentrate was sterile filtered and stored at -20 °C.

The total amount of protein, nucleic acid and polysaccharide obtained in each purification step and the purified yield thereof are shown in Tables 4 to 6.

As shown in Table 4-6, the capsular polysaccharide obtained by the method of the present invention conforms to the specification of residual protein content, and does not use any individual acidification method, and the recovered yield is 49% or more (serotype 3 capsular polysaccharide). , 65% or more (serotype 6B capsular polysaccharide), and 60% or more (serotype 19A capsular polysaccharide). Thus, the capsular polysaccharide can be efficiently produced in a simplified manner according to the method of the present invention.

Example 2. Preparation of S. pneumoniae capsular polysaccharide serotype 7F and 14 preparation cell bank

Preservative strains of S. pneumoniae serotypes 7F and 14 were obtained from the American Center for Strain (ATCC). The strains used for inoculum culture and main culture are shown in Table 7.

Create several generations of preserved strains (F1, F2, and F3 generations). Create two additional generations of preserved strains. The first extra generation was made from F3 vials, and the subsequent generations were made from the first extra generation vials. The vials of the strains were stored frozen (<-70 ° C) with synthetic glycerol as a cryoprotectant. To prepare a cell bank, all cultures were grown in soy-based medium. Prior to freezing, the cells are concentrated by centrifugation, the spent medium is removed, and the cell pellet is resuspended in fresh medium containing a cryoprotectant, such as synthetic glycerol.

Cultivation and collection

A strain containing a soybean-based medium is inoculated using a culture from a working cell bank and the bottle is incubated. After the target optical density (absorbance) is reached, the strain cell containing the soybean-based medium is inoculated with the strain bottle. The fermentation tank was incubated at 34-38 ° C with shaking at 120 rpm while maintaining the pH at about 7.2 or above with 3N NaOH. Samples were taken every 2-3 hours and their optical density was measured. When the optical density begins to grow substantially, sampling is performed every 30 minutes to 1 hour to measure the optical density. The culture was terminated after the optical density measurement reached a certain value and was maintained constant for 1 hour. Then, without any pH adjustment, further cultivation was continued at the same temperature and the same rpm oscillation until the pH reached 5.5 or less. After the further culture was terminated, 0.12% sodium deoxycholate was added to the culture to lyse the bacterial cells. The contents of the resulting fermenter were cooled to 10-15 ° C and maintained at this temperature without shaking for about 3 hours to induce protein precipitation. The mixture is then centrifuged to remove precipitated proteins and cell debris.

purification

The solution obtained from centrifugation is filtered with a depth filter to remove Protein and cell debris that were not precipitated during centrifugation. The concentrate was dialyzed against 25 mM sodium phosphate buffer (pH 7.2). The diafiltration system is carried out until the conductivity of the filtrate reaches about 3-4 mS/cm, and the TMP is set to 0.5 to 1.5 bar or less. After the impurities were removed from the sample, brominated cetyltrimethylammonium bromide (methyl cetyl bromide, cetyl bromide) was added from the stock solution to provide a final concentration of about 1.0% HB(w/v). Trimethylammonium (HB)] to the concentrated diafiltration solution while shaking for about 1 hour, followed by centrifugation to obtain a supernatant. Sodium iodide (NaI) from the stock NaI solution to a final concentration of about 0.5% (w/v) was added to the resulting supernatant. The solution was centrifuged to obtain a supernatant and activated carbon was slowly added to the obtained solution to about 2.0 w/v% while shaking for about 1 hour, and then the solution was filtered. The filtrate was concentrated on a 30 kDa membrane and the concentrate was dialyzed against about 10 volumes of three distilled water. The diafiltration system was carried out until the conductivity of the filtrate reached about 10 μS/cm, and the TMP was set to 0.5 to 1.5 bar or less. The concentrate was sterile filtered and stored at -20 °C.

The total amount of protein, nucleic acid and polysaccharide obtained in each purification step and its purified yield are shown in Tables 8 and 9.

As shown in Tables 8 and 9, the capsular polysaccharide obtained by the method of the present invention conforms to the specification of residual protein content, and does not use any individual acidification method, and the recovered yield is 60% or more (serotype 7F capsular polysaccharide). With 55% or more (serotype 14 capsular polysaccharide). Thus, the capsular polysaccharide can be efficiently produced in a simplified manner according to the method of the present invention.

Example 3. Preparation of Streptococcus pneumoniae capsular polysaccharide serotype 23F

Preparation of cell bank

The preserved strain of Streptococcus pneumoniae serotype 23F was obtained from the American Center for Strain, ATCC No. 6323.

Create several generations of preserved strains (F1, F2, and F3 generations). Create two additional generations of preserved strains. The first extra generation was made from F3 vials, and the subsequent generations were made from the first extra generation vials. The vials of the strains were stored frozen (<-70 ° C) with synthetic glycerol as a cryoprotectant. To prepare a cell bank, all cultures were grown in soy-based medium. Prior to freezing, the cells are concentrated by centrifugation, the spent medium is removed, and the cell pellet is resuspended in fresh medium containing a cryoprotectant, such as synthetic glycerol.

Cultivation and collection

A strain containing a soybean-based medium is inoculated using a culture from a working cell bank and the bottle is incubated. After the target optical density (absorbance) is reached, the strain cell containing the soybean-based medium is inoculated with the strain bottle. The fermentation tank was incubated at 34-38 ° C with shaking at 120 rpm while maintaining the pH at about 7.2 or above with 3N NaOH. Samples were taken every 2-3 hours and their optical density was measured. When the optical density begins to grow substantially, sampling is performed every 30 minutes to 1 hour to measure the optical density. The culture was terminated after the optical density measurement reached a certain value and was maintained constant for 1 hour. Then, without any pH adjustment, further cultivation was continued at the same temperature and the same rpm oscillation until the pH reached 5.5 or less. After the further culture was terminated, 0.12% sodium deoxycholate was added to the culture to lyse the bacterial cells. The contents of the resulting fermenter were cooled to 10-15 ° C and maintained at this temperature without shaking for about 3 hours to induce protein precipitation. Then, the mixture is centrifuged to remove precipitated proteins. Quality and cell debris.

purification

The solution obtained from centrifugation was filtered through a depth filter to remove proteins and cell debris that were not precipitated during centrifugation. The filtrate was concentrated on a 100 kDa MW membrane and the concentrate was dialyzed against 25 mM sodium phosphate buffer (pH 7.2). The diafiltration system is carried out until the conductivity of the filtrate reaches about 3-4 mS/cm, and the TMP is set to 0.5 to 1.5 bar or less. After the impurities were removed from the sample, brominated cetyltrimethylammonium bromide (methyl cetyl bromide, cetyl bromide) was added from the stock solution to provide a final concentration of about 2.5% HB(w/v). Trimethylammonium (HB)] precipitated the polysaccharide from the concentrated diafiltration solution while shaking for about 1 hour, followed by centrifugation. The resulting polysaccharide pellet was dissolved in about 0.25 M aqueous sodium chloride solution and sodium iodide (NaI) from the stock NaI solution to provide a final concentration of about 0.5% (w/v) was added to the polysaccharide solution. The solution was centrifuged to obtain a supernatant and activated carbon was slowly added to the supernatant to about 2.0 w/v% while shaking for about 1 hour, and then the solution was filtered. The filtrate was concentrated on a 30 kDa membrane and the concentrate was dialyzed against about 10 volumes of three distilled water. The diafiltration system was carried out until the conductivity of the filtrate reached about 10 μS/cm, and the TMP was set to 0.5 to 1.5 bar or less. The concentrate was sterile filtered and stored at -20 °C.

The total amount of protein, nucleic acid and polysaccharide obtained in each purification step and the purified yield thereof are shown in Table 10.

As shown in Table 10, the capsular polysaccharide serotype 23F obtained by the method of the present invention conformed to the specification of the residual protein content, and did not use any individual acidification method, and the recovery yield was 60% or more. Thus, the capsular polysaccharide can be efficiently produced in a simplified manner according to the method of the present invention.

Claims (12)

A method for preparing a capsular polysaccharide of a S. pneumoniae serotype, comprising: (a) cultivating a S. pneumoniae serotype while maintaining a pH in the culture solution in a range of 7.0 to 9.4; (b) in culturing The liquid in the step (a) is terminated between when the liquid absorbance reaches a level and when the absorbance starts to decrease; (c) the culture liquid obtained in the step (b) is further cultured without pH adjustment until the pH of the culture solution drops to 5.5 or Following; (d) adding a lysing agent to the culture broth obtained from step (c) to lyse the cells, precipitating the protein and removing the precipitated protein and cell debris; and (e) separating from the solution obtained from step (d) The capsular polysaccharide is purified. The method of claim 1, wherein the S. pneumoniae serotype is 1, 2, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F, 22F, 23F or 33F. The method of claim 1, wherein the culturing in step (a) is carried out at 34-38 ° C with shaking at 50-150 rpm. The method of claim 1, wherein the step (b) is carried out by terminating the culture in the step (a) within 1 to 3 hours from the time when the absorbance of the culture solution reaches a level. The method of claim 1, wherein the further culturing in step (c) is carried out at 34-38 ° C with shaking at 50-150 rpm without pH adjustment. The method of claim 1, wherein the lysing agent in step (d) is sodium deoxycholate. The method of claim 1, wherein the step (d) is carried out by adding a lysing agent to the culture solution obtained from the step (c) to lyse the cells, and maintaining the resulting solution at 10-20 ° C for 3 to 24 hours. There is no oscillation to precipitate the protein and the precipitated protein and cell debris are removed by centrifugation of the resulting solution. The method of claim 1, wherein the separating and purifying in the step (e) comprises the steps of: (i) filtering the solution obtained from the step (d) via a depth filter (Depth filter) to obtain a filtrate; (ii) Concentrating the filtrate obtained from step (i), followed by ultrafiltration and centrifugation to obtain a supernatant; (iii) adding a cationic detergent to the supernatant obtained from step (ii) for cultivation, and centrifuging the resulting mixture Obtaining a pellet or supernatant comprising a capsular polysaccharide; (iv) reacting the capsular polysaccharide obtained from step (iii) with sodium iodide, and then centrifuging the resulting reaction mixture to obtain a supernatant; (v) Activated carbon is added to the solution obtained from step (iv) and the resulting mixture is filtered to obtain a filtrate; and (vi) the filtrate obtained from step (v) is concentrated and subjected to ultrafiltration and centrifugation to obtain a capsular polysaccharide. The method of claim 8, wherein the concentration in step (ii) is carried out using a 100 kDa membrane. The method of claim 8, wherein the concentration in step (vi) is carried out using a 30 kDa film. The method of claim 8, wherein the cationic detergent used in the step (iii) is cetyltrimethylammonium bromide. The method of claim 8, wherein the activated carbon in the step (v) is used in an amount of from 1 to 5% (w/v).