STREPTOCOCCUS PNEUMONIAE CAPSULAR POLYSACCHARIDES AND IMMUNOGENIC CONJUGATE THEREOF

Abstract

The present invention provides an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, comprising a capsular polysaccharide derived from one or more selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F, derived from Streptococcus pneumoniae; and one or 2 or more of carrier proteins conjugated to the respective capsular polysaccharide, and method of preparation thereof. Through one example of the present invention, an immunogenic composition for preventing or treating pneumococcal infection can be provided.

Claims

1. An immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, comprising a capsular polysaccharide derived from one or more selected from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F, derived from Streptococcus pneumoniae; and one or 2 or more of carrier proteins conjugated to the respective capsular polysaccharide.

2. The immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate according to claim 1, wherein the polysaccharide is activated and binds to the carrier protein at a molecular weight of 100 to 400 kDa to form a conjugate, when the immunogenic composition comprises a polysaccharide derived from serotype 2, or the polysaccharide is activated and binds to the carrier protein at a molecular weight of 400 to 900 kDa to form a conjugate, when the immunogenic composition comprises a polysaccharide derived from serotype 17F, or the polysaccharide is activated and binds to the carrier protein at a molecular weight of 400 to 800 kDa to form a conjugate, when the immunogenic composition comprises a polysaccharide derived from serotype 20.

3. The immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate according to claim 1, wherein an immunogenic conjugate comprising the polysaccharide derived from serotype 2 has a molecular weight of 1,000 to 16,000 kDa, when the immunogenic composition comprises a polysaccharide derived from serotype 2, or an immunogenic conjugate comprising the polysaccharide derived from serotype 17F has a molecular weight of 300 to 4,500 kDa, when the immunogenic composition comprises a polysaccharide derived from serotype 17F, or an immunogenic conjugate comprising the polysaccharide derived from serotype 20 has a molecular weight of 1,000 to 4,000 kDa, when the immunogenic composition comprises a polysaccharide derived from serotype 20.

4. The immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate according to claim 1, wherein the carrier protein is TT (Tetanus toxoid) or CRM197.

5. The immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate according to claim 1, wherein the ratio of the serotype 2 capsular polysaccharide to the carrier protein in the immunogenic conjugate (polysaccharide/protein, W/W) when the immunogenic composition comprises a polysaccharide derived from serotype 2 is 0.5 to 2.0, or the ratio of the serotype 2 capsular polysaccharide to the carrier protein in the immunogenic conjugate (polysaccharide/protein, W/W) when the immunogenic composition comprises a polysaccharide derived from serotype 17F is 0.5 to 18, or the ratio of the serotype 20 capsular polysaccharide to the carrier protein in the immunogenic conjugate (polysaccharide/protein, W/W) when the immunogenic composition comprises a polysaccharide derived from serotype 20 is 1 to 5.

6. The immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate according to claim 1, wherein the immunogenic composition is that 20 to 60% of the total molecular weight is present within 0.3 Kd in a CL-4B column, in case of the immunogenic conjugate comprising a polysaccharide derived from serotype 2, or 15 to 60% of the total molecular weight is present within 0.3 Kd in a CL-4B column, in case of the immunogenic conjugate comprising a polysaccharide derived from serotype 17F, or 70 to 90% of the total molecular weight is present within 0.3 Kd in a CL-4B column, in case of the immunogenic conjugate comprising a polysaccharide derived from serotype 20.

7. The immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate according to claim 1, wherein the immunogenic composition is that the degree of oxidation of the polysaccharide conjugated to the conjugate is 2 to 18, in case of the immunogenic conjugate comprising a polysaccharide derived from serotype 2, or the degree of oxidation of the polysaccharide conjugated to the conjugate is 1 to 22, in case of the immunogenic conjugate comprising a polysaccharide derived from serotype 17F, or the degree of oxidation of the polysaccharide conjugated to the conjugate is 4 to 16, in case of the immunogenic conjugate comprising a polysaccharide derived from serotype 20.

8. The immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate according to claim 1, wherein the immunogenic composition is that polysaccharides derived from 15 serotypes different each other are conjugated to respective carrier proteins, and the serotypes are 1, 2, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F, and 23F, and the serotypes are conjugated to CRM197, respectively.

9. The immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate according to claim 1, wherein the immunogenic composition is that polysaccharides derived from 23 serotypes different each other are conjugated to respective carrier proteins, and the serotypes are 1, 2, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F, and among the serotypes, capsular polysaccharides derived from serotypes 3 and 5 are conjugated to carrier protein TT and capsular polysaccharides derived from serotypes 1, 2, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F are conjugated to carrier protein CRM197, respectively.

10. The immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate according to claim 1, wherein the immunogenic composition is that polysaccharides derived from 24 serotypes different each other are conjugated to respective carrier proteins, and the serotypes are 2, 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F, and capsular polysaccharides derived from serotypes 1 and 5 are conjugated to carrier protein TT and capsular polysaccharides derived from serotypes 2, 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F are conjugated to carrier protein CRM197, respectively.

11. The immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate according to claim 1, wherein the immunogenic composition comprises a physiologically acceptable vehicle.

12. The immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate according to claim 1, wherein the immunogenic composition is a vaccine.

13. A preparation method of an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate comprising (a) a step of fermenting and dissolving a bacterial cell which produces a capsular polysaccharide derived from one or more serotypes selected from the group consisting of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F; (b) a step of purifying a capsular polysaccharide derived from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F in the dissolved cell; (c) a step of reacting the purified polysaccharide with an oxidizing agent to activate it; and (d) a step of combining the activated polysaccharide with a carrier protein to form a Streptococcus pneumoniae polysaccharide-protein conjugate bound to the carrier protein.

14. The preparation method of an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate according to claim 13, wherein the preparation method further comprises a step of hydrolyzing the purified Streptococcus pneumoniae capsular polysaccharide to size it, before the (c) step, in case of the capsular polysaccharides derived from serotypes 2 and 17F.

15. The preparation method of an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate according to claim 13, wherein the combined carrier protein of the (d) step forms a conjugate with the polysaccharide activated by reacting with one or more reducing agents selected from the group consisting of cyanoborohydride, borane-pyridine and borohydride exchange resin.

16. The preparation method of an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate according to claim 13, wherein the (c) step is reacting 0.01˜0.22 μg of periodate per 1 μg polysaccharide at a temperature of 20 to 25° C. for 15 to 20 hours.

17. (canceled)

18. A method for preventing or treating infection of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and/or 33F in a subject, by administering an immunogenic composition comprising a Streptococcus pneumoniae polysaccharide-protein conjugate, comprising a capsular polysaccharide derived from one or more selected from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F, derived from Streptococcus pneumoniae; and one or 2 or more of carrier proteins conjugated to the respective capsular polysaccharide, into a subject.

19. (canceled)

Description

MODE FOR INVENTION

[0393] Hereinafter, the present invention will be described with reference to the following examples and the like in order to describe it more specifically. However, the examples according to the present invention may be modified into various other forms, and the scope of the present invention should not be construed as being limited to the examples described below. The examples of the present invention are provided to illustrate the present invention in order to facilitate a specific understanding of the present invention.

EXAMPLE 1. PREPARATION OF SEROTYPE 2, 9N, 17F or 20-DERIVED POLYSACCHARIDE-PROTEIN CONJUGATE VACCINE

[0394] [1. Streptococcus pneumoniae Serotype 2-Derived Polysaccharide-Protein Conjugate]

[0395] Preparation of Polysaccharide Protein Conjugate from Streptococcus pneumoniae Serotype 2

[0396] Preparation of Cell Bank for Master and Preparation

[0397] Streptococcus pneumoniae serotype 2 was acquired from American Type Culture Collection (ATCC) (strain ATCC 6302). In order to enhance the strain and remove components of animal origin, a seed stock was cultured for several generations. The seed vial was frozen with synthetic glycerol as a cryopreservative (<−70r). For preparation of cell bank, all cultures were proliferated in a soybean-based medium. Before freezing, cells were concentrated by centrifugation and the used medium was removed, and then a cell pellet was resuspended in a new medium containing a cryopreservative (e.g.: synthetic glycerol).

[0398] Fermentation

[0399] The cultures derived from the cell bank for preparation was used and inoculated into a seed bottle containing a soybean-based medium. Before satisfying the growth requirements, it was cultured at a certain temperature without stirring. Using the seed bottle, it was inoculated to a seed fermenter containing the soybean-based medium in which the temperature, pH and stirring speed were controlled. After the growth was stopped, or at the time of reaching the work capacity of the fermenter, fermentation was terminated. After terminating the fermentation process by adding an inactivator, cell residuals were removed using the combination of continuous flow centrifugation and filtration.

[0400] Purification

[0401] The purification of the pneumococcal polysaccharide was composed of multiple media filtration, several times of concentration/diafiltration work and precipitation/elution steps.

[0402] Activation

[0403] The final polysaccharide concentration was adjusted to be about 2.0 g/L in 0.01N hydrochloric acid solution by adding a calculated amount of 0.1N hydrochloric acid solution and WFI in order. For hydrolysis of the polysaccharide, the hydrolysis reaction was carried out at 60° C. for 60 minutes. After lowering the temperature of the reaction solution to the room temperature, the reaction pH was adjusted to approximately 6.0 by adding 0.1M sodium monohydrogen phosphate solution. After adjusting the pH, the temperature was adjusted to 23° C. The oxidation was initiated by adding sodium periodic acid of approximately 0.023˜0.114 mg per 1 mg sugar. The oxidation reaction was carried out at 23° C. for 18 hours.

[0404] The concentration and diafiltration of the activated polysaccharide were performed using 100 kDa MWCO ultrafiltration membrane. Diafiltration was performed on WFI of 10-fold diafiltration volume. Then, the purified activated polysaccharide was stored at 2˜8° C. The purified activated polysaccharide was characterized by in particular, (i) polysaccharide concentration by colorimetric determination, (ii) aldehyde concentration by colorimetric determination, (iii) degree of oxidation and (iv) molecular weight by SEC-MALLS.

[0405] SEC-MALLS is used for determining the molecular weights of the polysaccharide and polysaccharide-protein conjugate. SEC is used for separating the polysaccharide by fluid dynamical volume. The refractive index (RI) and multi-angle laser light scattering detector are used for molecular weight determination. When light interacts with a material, light is scattering and the amount of scattered light is related to concentration, square of do/dc (unique refractive index increase) and molar mass of a material. The molecular weight measured value is calculated on the basis of the reading value from scattered light signal from MALLS detector and the concentration signal from RI detector.

[0406] The degree of oxidation (DO) of the activated polysaccharide was determined by ‘mole of sugar repeating unit mole of aldehyde’. By various colorimetry methods, for example, using Anthrone method, the mole of sugar repeating unit was determined. In addition, at the same time, using Park-Johnson colorimetry method, the mole of aldehyde was determined.

[0407] Preferably, the activated Streptococcus pneumoniae serotype 2 capsular polysaccharide obtained by the method has a degree of oxidation of 2 to 18 and a molecular weight of about 100 kDa to 400 kDa.

[0408] Conjugation Process

[0409] The activated polysaccharide was combined with sucrose at a ratio of sucrose of 2 to 8 g per the activated polysaccharide gram. Subsequently, the bottle of the combined mixture was lyophilized. Following lyophilization, the bottle containing the lyophilized activated polysaccharide was stored at −20 to −30° C. The calculated amount of CRM197 protein was separately lyophilized. The lyophilized CRM197 was stored at −20 to −30° C.

[0410] The lyophilized activated polysaccharide was recomposed in anhydrous dimethyl sulfoxide solution (DMSO). When completing dissolution of the polysaccharide, for recomposing anhydrous DMSO, it was added to lyophilized CRM197. The activated polysaccharide recomposed in the reaction container was combined with the recomposed CRM197 (input ratio 0.5 to 2:1) and then it was mixed thoroughly. The conjugation reaction was initiated by adding sodium cyanoborohydride (NaBH.sub.3CN) of 1.0 mole equivalent to the reaction mixture. WFI was added to the reaction mixture at a target concentration of 1% (v/v) and it was reacted at 23° C. for 22 to 26 hours. The conjugation reaction was terminated by adding sodium borohydride (NaBH.sub.4) of 2.0 mole equivalent and adding WFI at a target concentration of 5%(v/v) to the reaction mixture, thereby capping unreacted aldehyde. The capping reaction was conducted at 23° C. for 4.5 hours.

[0411] The conjugate solution was diluted with 0.9% sodium chloride solution during the preparation for purification by concentration and diafiltration using 100 kDa MWCO membrane. The diluted conjugate solution was passed through a 0.8 μm filter and diafiltration was carried out using 0.9% sodium chloride at a 15-fold to 40-fold diafiltration volume. After completing the diafiltration, the residual solution was filtrated through a 0.2 μm filter. The conjugate solution was diluted with 0.9% sodium chloride solution so as to be less than approximately 0.55 mg/mL concentration and was under sterile filtration and was stored at 2 to 8° C.

[0412] The purified serotype 2 conjugate was characterized by (i) polysaccharide concentration by colorimetric determination, (ii) protein concentration by colorimetric determination (Lowry), (iii) ratio of polysaccharides to protein, (iv) molecular size distribution by size exclusion chromatography (CL-4B), (iv) content of free sugar and (v) molecular weight by SEC-MALLS.

[0413] The characteristic change of the serotype 2 conjugate was observed by controlling the degree of oxidation (DO) based on the preparation method. The result was summarized in Table 1.

TABLE-US-00001 TABLE 1 Conjugate number 1-1 1-2 1-3 1-4 Activated 365 313 300 231 polysaccharide molecular weight, kDa D0 14.2 8.6 6.7 2.7 Input ratio 1:1 (P:S) % Conjugate 72 47 57 67 yield Ratio of 1.2 1.1 1.2 1.0 saccharides to protein % Free 27 13 8 1 polysaccharide % Molecular 93 92 88 64 weight distribution Conjugate 4,918 3,845 3,485 1,988 molecular weight, kDa

[0414] The characteristic change of the serotype 2 conjugate was observed by controlling the mixing ratio of the activated polysaccharides and CRM197 during the lyophilization on the basis of the preparation method. The result was summarized in Table 2.

TABLE-US-00002 TABLE 2 Conjugate number 1-5 1-6 1-7 1-8 1-9 1-10 1-11 1-12 1-13 1-14 Activated 260 147 polysaccharide molecular weight, kDa D0 9.4 3.0 Input ratio 2:1 1.5:1 1:1 0.7:1 0.5:1 2:1 1.5:1 1:1 0.7:1 0.5:1 (P:S) % Conjugate 47 53 57 58 58 64 70 70 73 68 yield Ratio of 0.53 0.69 0.99 1.44 1.81 0.58 0.69 1.04 1.41 1.86 saccharides to protein % Free 14 15 18 26 27 1 0 1 9 14 polysaccharide % Molecular 94 96 97 96 94 76 71 67 65 63 weight distribution Conjugate 15,819 11,389 5,532 3,544 2,629 8,255 4,070 2,185 1,226 1,050 molecular weight, kDa

[0415] Research on Immunogenicity of Serotype 2 Polysaccharide-Protein Conjugate

[0416] A monovalent conjugate composition comprising a polysaccharide-protein conjugate from Streptococcus pneumoniae serotype 2 all individually conjugated to CRM197 was formulated.

[0417] The immunogenicity of the monovalent immunogenic composition of the Table 1 and Table 2 was analyzed using ELISA in a rabbit, thereby measuring a serotype-specific IgG concentration in serum.

[0418] 5 female New Zealand white rabbit group of 2.5 kg to 3.5 kg was immunized via intramuscular route at the 0th week with the proposed human clinical dose (conjugate 2.2 μg; +aluminum 0.25 mg/10 as AlPO.sub.4). The rabbit was further immunized at the 2nd week with the same dose of conjugate vaccine, and subsequently blood-gathering was carried out at the 4th week. The serotype-specific ELISA was performed in the 0th and 4th serum samples.

[0419] The analysis result was shown in Table 3. The rabbit immunized with the monovalent conjugate composition (conjugate number 1-6) exhibited a significant increase of the total IgG titer against serotype 2. In the rabbit immunized with other conjugate, a significant increase of the total IgG titer was shown.

[0420] The values of the following Table 3 are the result of showing the measured IgG concentration after immunizing with the conjugate number 1-6 of the Table 1.

TABLE-US-00003 TABLE 3 IgG concentration (U/mL) Serotype Pre-immunization Post-immunization 2 130.0 62,164.7

[0421] [2. Streptococcus pneumoniae Serotype 9N-Derived Polysaccharide-Protein Conjugate]

[0422] Preparation of Streptococcus pneumoniae Serotype 9N-Derived Polysaccharide-Protein Conjugate

[0423] Preparation of Cell Band for Master and Preparation

[0424] Streptococcus pneumoniae serotype 9N was obtained from American Type Culture Collection (ATCC) (strain ATCC 6309). It was progressed in the same manner as serotype 2.

[0425] Fermentation

[0426] It was progressed in the same manner as serotype 2.

[0427] Purification

[0428] It was progressed in the same manner as serotype 2.

[0429] Activation

[0430] The final polysaccharide concentration of about 2.0 g/L was provided by adding a calculated amount of WFI in order. If needed, the reaction pH was adjusted to approximately 6.0. After adjusting the pH, the reaction temperature was adjusted to 23° C. The oxidation was initiated by adding sodium periodic acid of 0.024˜0.189 mg per approximately 1 mg sugar. The oxidation reaction was carried out at 23° C. for 18 hours.

[0431] The concentration and diafiltration of the activated polysaccharide were performed using 100 kDa MWCO ultrafiltration membrane. Diafiltration was performed on WFI of 10-fold diafiltration volume. Then, the purified activated polysaccharide was stored at 2˜8° C. The purified activated polysaccharide was characterized by in particular, (i) polysaccharide concentration by colorimetric determination, (ii) aldehyde concentration by colorimetric determination, (iii) degree of oxidation and (iv) molecular weight by SEC-MALLS.

[0432] SEC-MALLS is used for determining the molecular weights of the polysaccharide and polysaccharide-protein conjugate. SEC is used for separating the polysaccharide by fluid dynamical volume. The refractive index (RI) and multi-angle laser light scattering detector are used for molecular weight determination. When light interacts with a material, light is scattering and the amount of scattered light is related to concentration, square of do/dc (unique refractive index increase) and molar mass of a material. The molecular weight measured value is calculated on the basis of the reading value from scattered light signal from MALLS detector and the concentration signal from RI detector.

[0433] The degree of oxidation (DO) of the activated polysaccharide was determined by ‘mole of sugar repeating unit mole of aldehyde’. By various colorimetry methods, for example, using Anthrone method, the mole of sugar repeating unit was determined. In addition, at the same time, using Park-Johnson colorimetry method, the mole of aldehyde was determined.

[0434] Preferably, the activated Streptococcus pneumoniae serotype 9N capsular polysaccharide obtained by the method has a degree of oxidation of 2 to 19 and a molecular weight of about 200 kDa to 700 kDa.

[0435] Conjugation Process

[0436] The activated polysaccharide was combined with the carrier protein, CRM197 at a ratio of CRM197 of 0.5 to 2 grams per the activated polysaccharide gram. Subsequently, the combined mixture was lyophilized. Following lyophilization, the lyophilized mixture of the activated polysaccharide and CRM197 was stored at −20° C.

[0437] The lyophilized mixture of the activated polysaccharide and CRM197 was recomposed in 0.1M sodium phosphate solution and then was mixed sufficiently. The final polysaccharide concentration in the reaction solution is about 10 to 20 g/L. The conjugation was initiated by adding sodium cyanoborohydride (NaBH.sub.3CN) of 1.2 mole equivalent to the mixture, and it was reacted at 37° C. for 48 hours. The conjugation reaction was terminated by adding 0.9% sodium chloride solution at the same volume as the conjugation reaction solution and then adding sodium borohydride (NaBH.sub.4) of 2.0 mole equivalent, thereby capping unreacted aldehyde. The capping reaction was conducted at 23° C. for 4.5 hours.

[0438] The conjugate solution was diluted with 0.9% sodium chloride solution during the preparation for purification by concentration and diafiltration using 100 kDa MWCO membrane. The diluted conjugate solution was passed through a 0.45 μm filter and purification by concentration and diafiltration was carried out. Diafiltration using 100 kDa MWCO membrane was carried out using 0.9% sodium chloride solution at a 15-fold to 40-fold diafiltration volume. After completing the diafiltration, the residual solution was filtrated through a 0.2 μm filter. The conjugate solution was diluted so as to be less than approximately 0.55 mg/mL concentration and was under sterile filtration, and was stored at 2 to 8° C.

[0439] The purified serotype 9N conjugate was particularly characterized by (i) polysaccharide concentration by colorimetric determination, (ii) protein concentration by colorimetric determination (Lowry), (iii) ratio of polysaccharides to protein, (iv) molecular size distribution by size exclusion chromatography (CL-4B), (iv) content of free sugar and (v) molecular weight by SEC-MALLS.

[0440] The characteristic change of the serotype 9N conjugate was observed by controlling the degree of oxidation (DO) based on the preparation method. The result was summarized in Table 4.

TABLE-US-00004 TABLE 4 Conjugate number 2-1 2-2 2-3 2-4 2-5 2-6 Activated 582 619 459 563 490 427 polysaccharide molecular weight, kDa D0 18.2 9.4 7.4 6.7 4.3 2.3 Input ratio (P:S) 0.8:1 Polysaccharide 20.0 concentration in conjugation reaction solution, g/L % Conjugate 53 43 39 32 33 39 yield Ratio of 2.1 1.5 1.3 1.1 1.0 0.78 saccharides to protein % Free 44 28 22 20 21 31 polysaccharide % Molecular 52 49 50 55 44 31 weight distribution Conjugate 860 1,110 1,912 1,168 1,189 1,160 molecular weight, kDa

[0441] The characteristic change of the serotype 9N conjugate was observed by controlling the mixing ratio of the activated polysaccharides and CRM197 during the lyophilization on the basis of the preparation method. The result was summarized in Table 5.

TABLE-US-00005 TABLE 5 Conjugate number 2-7 2-8 2-9 2-10 2-11 Activated 287 polysaccharide molecular weight, kDa D0 5.6 Input ratio (P:S) 2:1 1.5:1 1:1 0.67:1 0.5:1 Polysaccharide 20.0 concentration in conjugation reaction solution, g/L % Conjugate yield 25 50 43 41 66 Ratio of saccharides 0.71 0.85 1.0 1.2 1.8 to protein % Free polysaccharide 5 6 15 27 62 % Molecular weight 52 58 50 40 22 distribution Conjugate molecular 3,720 3,713 1,327 1,016 545 weight, kDa

[0442] The characteristic change of the serotype 9N conjugate was observed by controlling the polysaccharide concentration in the conjugation reaction solution on the basis of the preparation method. The result was summarized in Table 6.

TABLE-US-00006 TABLE 6 Conjugate number 2-12 2-13 2-14 2-15 2-16 Activated 560 polysaccharide molecular weight, kDa D0 6.1 Input ratio (P:S) 0.8:1 Polysaccharide 10.0 12.5 15.0 17.5 20.0 concentration in conjugation reaction solution, g/L % Conjugate yield 20 31 28 40 42 Ratio of saccharides 1.0 1.0 0.93 0.99 0.97 to protein % Free polysaccharide 32 30 22 21 18 % Molecular weight 17 27 40 47 54 distribution Conjugate molecular 560 546 845 932 1,438 weight, kDa

[0443] Research on Immunogenicity of Serotype 9N Polysaccharide-Protein Conjugate

[0444] A monovalent conjugate composition comprising a polysaccharide-protein conjugate from Streptococcus pneumoniae serotype 9N all individually conjugated to CRM197 was formulated.

[0445] The immunogenicity of the monovalent immunogenic composition of the Table 4 to Table 6 was analyzed using ELISA in a rabbit, thereby measuring a serotype-specific IgG concentration in serum.

[0446] The female New Zealand white rabbit group was immunized via intramuscular route in the same manner as serotype 2.

[0447] The analysis result was shown in Table 7. The rabbit immunized with the monovalent conjugate composition (conjugate number 2-8) exhibited a significant increase of the total IgG titer against serotype 9N. In the rabbit immunized with other conjugate, a significant increase of the total IgG titer was shown.

[0448] The values of the following Table 7 are the result of showing the measured IgG concentration after immunizing with the conjugate number 2-8 of the Table 5.

TABLE-US-00007 TABLE 7 IgG concentration (U/mL) Serotype Pre-immunization Post-immunization 9N 130.0 656,345.3

[0449] [3. Streptococcus pneumoniae Serotype 17F-Derived Polysaccharide-Protein Conjugate]

[0450] Preparation of Streptococcus pneumoniae Serotype 17F-Derived Polysaccharide-Protein Conjugate

[0451] Preparation of Cell Band for Master and Preparation

[0452] Streptococcus pneumoniae serotype 17F was obtained from American Type Culture Collection (ATCC) (strain ATCC 6317). In order to enhance the strain and remove components of animal origin, a seed stock was cultured for several generations. It was progressed in the same manner as serotype 2.

[0453] Fermentation

[0454] It was progressed in the same manner as serotype 2.

[0455] Purification

[0456] It was progressed in the same manner as serotype 2.

[0457] Activation

[0458] The final polysaccharide concentration was adjusted so as to be about 2.0 g/L in 0.01N hydrochloric acid solution by adding a calculated amount of 0.1N hydrochloric acid solution and WFI in order. For hydrolysis of the polysaccharide, the hydrolysis reaction was carried out at about 60° C. for 60 minutes. After lowering the temperature of the reaction solution to a room temperature, the reaction pH was adjusted to approximately 6.0 by adding 0.1M sodium monohydrogen phosphate solution. After adjusting the pH, the temperature was adjusted to 23° C. The oxidation was initiated by adding sodium periodic acid of approximately 0.008˜0.219 mg per 1 mg sugar. The oxidation reaction was carried out at 23° C. for 18 hours.

[0459] The concentration and diafiltration of the activated polysaccharide were performed using 100 kDa MWCO ultrafiltration membrane. Diafiltration was performed on WFI of 10-fold diafiltration volume. Then, the purified activated polysaccharide was stored at 2˜8° C. The purified activated polysaccharide was characterized by in particular, (i) polysaccharide concentration by colorimetric determination, (ii) aldehyde concentration by colorimetric determination, (iii) degree of oxidation and (iv) molecular weight by SEC-MALLS.

[0460] SEC-MALLS is used for determining the molecular weights of the polysaccharide and polysaccharide-protein conjugate. SEC is used for separating the polysaccharide by fluid dynamical volume. The refractive index (RI) and multi-angle laser light scattering detector are used for molecular weight determination. When light interacts with a material, light is scattering and the amount of scattered light is related to concentration, square of do/dc (unique refractive index increase) and molar mass of a material. The molecular weight measured value is calculated on the basis of the reading value from scattered light signal from MALLS detector and the concentration signal from RI detector.

[0461] The degree of oxidation (DO) of the activated polysaccharide was determined by ‘mole of sugar repeating unit mole of aldehyde’. By various colorimetry methods, for example, using Anthrone method, the mole of sugar repeating unit was determined. In addition, at the same time, using Park-Johnson colorimetry method, the mole of aldehyde was determined.

[0462] Preferably, the activated Streptococcus pneumoniae serotype 17F capsular polysaccharide obtained by the method has a degree of oxidation of 1 to 22 and a molecular weight of about 400 kDa to 900 kDa.

[0463] Conjugation Process

[0464] The activated polysaccharide was combined with the carrier protein, CRM197 at a ratio of CRM197 of 1.0 gram per the activated polysaccharide gram. Subsequently, the combined mixture was lyophilized. Following lyophilization, the lyophilized mixture of the activated polysaccharide and CRM197 was stored at −20° C.

[0465] The lyophilized mixture of the activated polysaccharide and CRM197 was recomposed in 0.1M sodium phosphate solution (pH 7.2±0.1). The final polysaccharide concentration in the reaction solution is 15.0 to 25.0 g/L. The conjugation was initiated by adding sodium cyanoborohydride (NaBH.sub.3CN) of 1.2 mole equivalent to the mixture, and it was reacted at 37° C. for 48 hours. The conjugation reaction was terminated by adding 0.9% sodium chloride solution at the same volume as the conjugation reaction solution and then adding sodium borohydride (NaBH.sub.4) of 2.0 mole equivalent, thereby capping unreacted aldehyde. The capping reaction was conducted at 23° C. for 4.5 hours.

[0466] The conjugate solution was diluted with 0.9% sodium chloride solution during the preparation for purification by concentration and diafiltration using 100 kDa MWCO membrane. The diluted conjugate solution was passed through a 0.45 μm filter and purification by concentration and diafiltration was carried out. Diafiltration using 100 kDa MWCO membrane was carried out using 0.9% sodium chloride solution at a 15-fold to 40-fold diafiltration volume. After completing primary diafiltration, the residual solution was filtrated through a 0.2 μm filter and was stored at 2 to 8° C.

[0467] The purified serotype 17F conjugate was particularly characterized by (i) polysaccharide concentration by colorimetric determination, (ii) protein concentration by colorimetric determination (Lowry), (iii) ratio of polysaccharides to protein, (iv) molecular size distribution by size exclusion chromatography (CL-4B), (iv) content of free sugar and (v) molecular weight by SEC-MALLS.

[0468] The characteristic change of the serotype 17F conjugate was observed by controlling the degree of oxidation (DO) and the polysaccharide concentration in the reaction solution based on the preparation method. The result was summarized in Table 8.

TABLE-US-00008 TABLE 8 Conjugate number 3-1 3-2 3-3 3-4 3-5 3-6 3-7 3-8 3-9 Activated 551 560 577 628 801 polysaccharide molecular weight, kDa D0 21.3 9.4 7.5 3.8 1.3 Input ratio 1:1 (P:S) Polysaccharide 20.0 15.0 17.5 20.0 22.5 25.0 concentration in conjugation reaction solution, g/L % Conjugate 31 48 58 35 28 25 28 37 37 yield Ratio of 14.9 5.3 4.9 2.8 0.68 0.65 0.67 0.70 0.71 saccharides to protein % Free 84 82 78 60 8 4 4 6 4 polysaccharide % Molecular — 18 38 48 49 58 weight distribution Conjugate 372 706 456 1,064 1,346 2,115 2,531 3,150 4,423 molecular weight, kDa

[0469] Research on Immunogenicity of Serotype 17F Polysaccharide-Protein Conjugate

[0470] A monovalent conjugate composition comprising a polysaccharide-protein conjugate from Streptococcus pneumoniae serotype 17F all individually conjugated to CRM197 was formulated.

[0471] The immunogenicity of the monovalent immunogenic composition of the Table 8 to Table 6 was analyzed using ELISA in a rabbit, thereby measuring a serotype-specific IgG concentration in serum.

[0472] The female New Zealand white rabbit group was immunized via intramuscular route in the same manner as serotype 2.

[0473] The analysis result was shown in Table 9. The rabbit immunized with the monovalent conjugate composition (conjugate number 3-8) exhibited a significant increase of the total IgG titer against serotype 17F. In the rabbit immunized with other conjugate, a significant increase of the total IgG titer was shown.

[0474] The values of the following Table 9 are the result of showing the measured IgG concentration after immunizing with the conjugate number 3-8 of the Table 8.

TABLE-US-00009 TABLE 9 IgG Concentration (U/mL) Serotype Pre-immunization Post-immunization 17F 130.0 227,590.3

[0475] [4. Streptococcus pneumoniae Serotype 20-Derived Polysaccharide-Protein Conjugate]

[0476] Preparation of Streptococcus pneumoniae Serotype 20-Derived Polysaccharide-Protein Conjugate

[0477] Preparation of Cell Band for Master and Preparation

[0478] Streptococcus pneumoniae serotype 20 was obtained from American Type Culture Collection (ATCC) (strain ATCC 6320). It was progressed in the same manner as serotype 2.

[0479] Fermentation

[0480] It was progressed in the same manner as serotype 2.

[0481] Purification

[0482] It was progressed in the same manner as serotype 2.

[0483] Activation

[0484] The final polysaccharide concentration was adjusted so as to be about 2.0 g/L in 0.01N hydrochloric acid solution by adding a calculated amount of 0.1N hydrochloric acid solution and WFI in order. The oxidation was initiated by adding sodium periodic acid of approximately 0.010˜0.038 mg per 1 mg sugar. The oxidation reaction was carried out at 23° C. for 18 hours.

[0485] The concentration and diafiltration of the activated polysaccharide were performed using 100 kDa MWCO ultrafiltration membrane. Diafiltration was performed on WFI of 10-fold diafiltration volume. Then, the purified activated polysaccharide was stored at 2˜8° C. The purified activated polysaccharide was characterized by in particular, (i) polysaccharide concentration by colorimetric determination, (ii) aldehyde concentration by colorimetric determination, (iii) degree of oxidation and (iv) molecular weight by SEC-MALLS.

[0486] SEC-MALLS is used for determining the molecular weights of the polysaccharide and polysaccharide-protein conjugate. SEC is used for separating the polysaccharide by fluid dynamical volume. The refractive index (RI) and multi-angle laser light scattering detector are used for molecular weight determination. When light interacts with a material, light is scattering and the amount of scattered light is related to concentration, square of do/dc (unique refractive index increase) and molar mass of a material. The molecular weight measured value is calculated on the basis of the reading value from scattered light signal from MALLS detector and the concentration signal from RI detector.

[0487] The degree of oxidation (DO) of the activated polysaccharide was determined by ‘mole of sugar repeating unit÷mole of aldehyde’. By various colorimetry methods, for example, using Anthrone method, the mole of sugar repeating unit was determined. In addition, at the same time, using Park-Johnson colorimetry method, the mole of aldehyde was determined.

[0488] Preferably, the activated Streptococcus pneumoniae serotype 20 capsular polysaccharide obtained by the method has a degree of oxidation of 4 to 16 and a molecular weight of about 400 kDa to 800 kDa.

[0489] Conjugation Process

[0490] The activated polysaccharide was combined with the carrier protein, CRM197 at a ratio of CRM197 of 1.0 gram per the activated polysaccharide gram. Subsequently, the combined mixture was lyophilized. Following lyophilization, the lyophilized mixture of the activated polysaccharide and CRM197 was stored at −20° C.

[0491] The lyophilized mixture of the activated polysaccharide and CRM197 was recomposed in 0.1M sodium phosphate solution (pH 7.2±0.1). The final polysaccharide concentration in the reaction solution is 15.0 g/L. The conjugation was initiated by adding sodium cyanoborohydride (NaBH.sub.3CN) of 1.2 mole equivalent to the mixture, and it was reacted at 37° C. for 48 hours. The conjugation reaction was terminated by adding 0.9% sodium chloride solution at the same volume as the conjugation reaction solution and then adding sodium borohydride (NaBH.sub.4) of 2.0 mole equivalent, thereby capping unreacted aldehyde. The capping reaction was conducted at 23° C. for 4.5 hours.

[0492] The conjugate solution was diluted with 0.9% sodium chloride solution during the preparation for purification by concentration and diafiltration using 100 kDa MWCO membrane. The diluted conjugate solution was passed through a 0.45 μm filter and 2-step purification by concentration and diafiltration was carried out. Diafiltration using 100 kDa MWCO membrane was carried out using 0.9% sodium chloride solution at a 20-fold diafiltration volume. After completing primary diafiltration, the residual solution was filtrated through a 0.2 μm filter and was stored at 2 to 8° C. The conjugate solution was diluted so as to be less than approximately 0.55 mg/mL concentration and was under sterile filtration, and was stored at 2 to 8° C.

[0493] The purified serotype 20 conjugate was particularly characterized by (i) polysaccharide concentration by colorimetric determination, (ii) protein concentration by colorimetric determination (Lowry), (iii) ratio of polysaccharides to protein, (iv) molecular size distribution by size exclusion chromatography (CL-4B), (iv) content of free sugar and (v) molecular weight by SEC-MALLS.

[0494] The characteristic change of the serotype 20 conjugate was observed by controlling the degree of oxidation (DO) based on the preparation method. The result was summarized in Table 10.

TABLE-US-00010 TABLE 10 Conjugate number 4-1 4-2 4-3 4-4 4-5 4-6 4-7 Activated 651 749 675 463 613 444 712 polysaccharide molecular weight, kDa D0 15.7 15.4 8.9 7.3 6.7 4.8 4.6 Input ratio (P:S) 1:1 % Conjugate yield 65 57 56 49 45 24 20 Ratio of 3.7 2.9 2.5 2.7 2.2 2.1 1.5 saccharides to protein % Free 29 28 16 15 16 11 8 polysaccharide % Molecular weight 84 — 79 78 82 78 — distribution Conjugate 1,968 1,271 3,349 2,458 3,645 2,123 2,563 molecular weight, kDa

[0495] Research on Immunogenicity of Streptococcus pneumoniae Serotype 20 Polysaccharide-Protein Conjugate

[0496] A monovalent conjugate composition comprising a polysaccharide-protein conjugate from Streptococcus pneumoniae serotype 20 all individually conjugated to CRM197 was formulated.

[0497] The immunogenicity of the monovalent immunogenic composition of the Table 10 was analyzed using ELISA in a rabbit, thereby measuring a serotype-specific IgG concentration in serum.

[0498] The female New Zealand white rabbit group was immunized via intramuscular route in the same manner as serotype 2.

[0499] The analysis result was shown in Table 11. The rabbit immunized with the monovalent conjugate composition (conjugate number 4-7) exhibited a significant increase of the total IgG titer against serotype 20. In the rabbit immunized with other conjugate, a significant increase of the total IgG titer was shown.

[0500] The values of the following Table 11 is the result of showing the measured IgG concentration after immunizing with the conjugate number 4-7 of the Table 10.

TABLE-US-00011 TABLE 11 IgG concentration (U/mL) Serotype Pre-immunization Post-immunization 20 166.2 277,210.1

EXAMPLE 2. PREPARATION OF MULTIVALENT STREPTOCOCCUS PNEUMONIAE POLYSACCHARIDE-PROTEIN CONJUGATE

[0501] [5. Streptococcus pneumoniae 15-Valent Polysaccharide-Protein Conjugate]

[0502] Preparation of Streptococcus pneumoniae 15-Valent Polysaccharide-Protein Conjugate

[0503] A 15-valent conjugate composition comprising the polysaccharide-protein conjugate derived from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F, and 23F all individually conjugated to CRM197 (15vPnC) was formulated.

[0504] For serotypes 2 and 9N, the conjugate was prepared by the afore-mentioned method, and for other serotypes, the conjugate was prepared according to the method disclosed in Korean Patent Application 2012-0065893.

[0505] The required volume of the final bulk concentrate was calculated based on batch volume and bulk saccharide concentration. A required amount of 0.85% sodium chloride, polysorbate 80 and succinate buffer were added to a pre-labeled formulation container, and then the bulk concentrate was added. It was sufficiently mixed and was filtrated through a 0.22 μm filter. During and after addition of bulk aluminum phosphate, the formulated bulk solution was slowly mixed. pH was checked and was adjusted if necessary. The formulated bulk product was stored at 2 to 8° C. The obtained vaccine composition contained each saccharide of 2.2 μg, but 6B of 4.4 μg; CRM 197 carrier protein of about 32 μg; adjuvant of aluminum element of 0.125 mg (0.5 mg aluminum phosphate); sodium chloride about 4.25 mg; succinate buffer about 295 μg; and polysorbate 80 about 100 μg in the total 0.5 mL.

[0506] Research on Immunogenicity of Streptococcus pneumoniae 15-Valent Polysaccharide-Protein Conjugate

[0507] IgG Concentration Measurement

[0508] The immunogenicity of the 15-valent immunogenic composition was analyzed in a rabbit using ELISA, thereby measuring the serotype-specific IgG concentration in serum.

[0509] 6 female New Zealand white rabbit group of 2.5 kg to 3.5 kg was immunized via intramuscular route at the 0th week with the proposed human clinical dose (conjugate 2.2 μg, except for serotype 6b determined as 4.4 μg; +aluminum 0.25 mg/ml as AlPO.sub.4). The rabbit was further immunized at the 3rd week with the same dose of conjugate vaccine, and subsequently blood-gathering was carried out at an interval of 3 weeks. The serotype-specific ELISA was performed in serum samples of each week.

[0510] The serotype specific immune response for the vaccine formulation according to the present invention and the vaccine formulation of the comparative example was evaluated by IgG ELISA. The analysis result was summarized in Table 12. It shows IgG concentrations (U/mL) as time passes after inoculation. It was shown that the rabbit immunized by the 15vPnC produced antibodies against serotypes 2 and 9N which could not be obtained with Prevnar13, and particularly, it could induce an equivalent or excellent serum IgG titer compared to Prevnar13, even though the valence number increased by serotype addition.

TABLE-US-00012 TABLE 12 Prevnar 13 SK-15 ELISA Day Day Day Day Type 0 21 Day 42 Day 63 0 21 Day 42 Day 63 1 0 8522 14187 10207 0 2053 17110 7752 2 — — — — 134 47864 36482 23790 3 0 968 7229 5330 0 2084 11069 10947 4 0 3831 23100 16654 0 2592 15000 10179 5 97 5597 13083 14819 111 4866 19615 16062  6A 0 15810 28609 20581 0 3634 20313 10141  6B 0 12920 43575 34932 0 4296 29763 14204  7F 0 48129 26694 18014 0 43979 21590 10878  9N — — — — 0 9197 17085 13021  9V 436 17281 35392 9442 373 15128 12143 12304 14 194 8365 10403 10786 416 7066 13160 16043 18C 0 19101 23989 24862 0 19385 14192 14565 19A 0 71160 136042 139273 0 24713 64138 59135 19F 0 40695 53165 56443 0 8382 38304 30852 23F 0 11171 62331 47522 0 7116 65352 67085

[0511] OPA Analysis Result

[0512] In order to confirm whether the 15-valent polysaccharide-protein conjugate induced a functional antibody reaction, multiplexed opsonophagocytic killing assay (MOPA) was carried out.

[0513] By collecting the same amount of serum by each subject, serum was pooled between the same groups. Streptococcus pneumoniae was cultured in a THY medium by each serum and was diluted to be 1000 CFU/10 uL. Opsonization buffer 200 uL, diluted serum 10 uL, and diluted Streptococcus pneumoniae 10 uL were mixed and it was reacted at a room temperature for 1 hour. The mixed solution of a pre-differentiated HL-60 cell and a complement was added and it was reacted in a CO.sub.2 incubator (37° C.) for 1 hour. The phagocytosis was stopped by lowering the temperature and the reaction solution 5 uL was painted out in an agar medium dried for 30 to 60 minutes in advance. It was cultured in the CO.sub.2 incubator (37° C.) for 12 to 18 hours and the number of colonies was counted. The OPA titer was represented by the dilution rate in which 50% death was observed. As a comparative example, a 13-valent vaccine (Prevnar13, Pfizer) was used to evaluate in the same manner, and the result was summarized in Table 13.

TABLE-US-00013 TABLE 13 OPA Prevnar 13 SK-15 Type Day 21 Day 42 Day 63 Day 21 Day 42 Day 63 1 16 64 64 4 64 64 2 — — — 128 512 512 3 1 2 4 1 4 4 4 128 1024 1024 128 1024 1024 5 64 256 512 32 256 512 6A 512 2048 2048 256 2048 2048 6B 256 2048 2048 128 2048 2048 7F 1024 2048 2048 1024 2048 2048 9N — — — 512 2048 2048 9V 256 512 512 256 512 512 14 256 1024 1024 256 1024 1024 18C  1024 1024 2048 1024 512 2048 19A  512 1024 2048 256 1024 1024 19F  256 1024 1024 128 512 512 23F  256 2048 2048 256 2048 2048

[0514] [6. Streptococcus pneumoniae 23-Valent Polysaccharide-Protein Conjugate]

[0515] Preparation of Streptococcus pneumoniae 23-Valent Polysaccharide-Protein Conjugate

[0516] A 23-valent conjugate composition comprising the polysaccharide-protein conjugate in which polysaccharides derived from Streptococcus pneumoniae serotypes 1, 2, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F were conjugated to CRM197 and the polysaccharide-protein conjugate in which Streptococcus pneumoniae serotypes 3 and 5 were conjugated to TT (Tenus toxoid) (23vPnC) was formulated.

[0517] For serotypes 2, 9N, 17F and 20, the conjugate was prepared by the afore-mentioned method, and for other serotypes, the conjugate conjugated to CRM197 or TT was prepared according to the methods disclosed in Korean Patent Application 2012-0065893, U.S. Patent Applications 62/371,529, 62/371,553 and 62/626,482.

[0518] The required volume of the final bulk concentrate was calculated based on batch volume and bulk saccharide concentration. A required amount of 0.85% sodium chloride, polysorbate 80 and succinate buffer were added to a pre-labeled formulation container, and then the bulk concentrate was added. It was sufficiently mixed and was filtrated through a 0.22 μm filter. During and after addition of bulk aluminum phosphate, the formulated bulk solution was slowly mixed. pH was checked and was adjusted if necessary. The formulated bulk product was stored at 2 to 8° C. The obtained vaccine composition contained each saccharide of 2.2 μg, but 6B of 4.4 μg; CRM 197 carrier protein of about 50 to 85 μg; an experimental amount of aluminum adjuvant (for example, in case of Group 4 in Table 14, aluminum element of 0.125 mg, that is, 0.5 mg aluminum phosphate); sodium chloride about 4.25 mg; succinate buffer about 295 μg; and polysorbate 80 about 100 μg in the total 0.5 mL.

[0519] Research on Immunogenicity of Streptococcus pneumoniae 23-Valent Polysaccharide-Protein Conjugate

[0520] IgG Concentration Measurement

[0521] The immunogenicity of the 23-valent immunogenic composition was analyzed in a rabbit using ELISA, thereby measuring the serotype-specific IgG concentration in serum. The ability of inducing a serotype-specific immune response of the 23vPnC vaccine containing an adjuvant was investigated.

[0522] 5 female New Zealand white rabbit group of 2.5 kg to 3.5 kg was immunized via intramuscular route at the 0th week with the proposed human clinical dose (conjugate 2.2 μg, except for serotype 6b determined as 4.4 μg) in which aluminum of 0.0625 mg/ml, 0.125 mg/ml, 0.25 mg/ml, 0.5 mg/ml and 1 mg/ml, respectively was comprised as AlPO4. The rabbit was further immunized at the 2nd week with the same dose of conjugate vaccine, and subsequently blood-gathering was carried out at the 4th week. The serotype-specific ELISA was performed in the 0th and 4th serum samples.

[0523] As a comparative example, a 13-valent vaccine (Prevnar13, Pfizer) was used to evaluate in the same manner, and the analysis result was summarized in Table 14. It shows IgG concentrations (U/mL) as 4th week passes after inoculation.

[0524] The geometric mean titer (GMT) measured in the pooled serum sample after administering the 23vPnV vaccine and Prevnar13 twice was proposed. These data demonstrate that a higher level of IgG antibody is induced compared to the same vaccine which does not contain an adjuvant, when an adjuvant is comprised in the 23vPnV formulation. In particular, it was confirmed that a 23-valent immunogenic vaccine comprising all serotypes 2, 9N, 17F and 20 could be obtained. As could be seen in the following result, in particular, an antibody against serotype 2 and the like, which could not be obtained by Prevnar13, could be produced. In addition, it was confirmed that it could induce an immune response against all the comprised serotype without greatly affecting production of an antibody against an antigen of other serotypes, despite greatly increased valence number.

TABLE-US-00014 TABLE 14 Group 1: Group 7 PCV24 I (Prevnar ® alum 0 Group 2: Group 3: Group 4: Group 5: Group 6: 13) Control PCV24 I PCV24 I PCV24 I PCV24 I PCV24 I Reference group alum 62.5 alum 125 alum 250 alum 500 alum 1000 group Type 4421 10951 11205 9595 12821.5 12038.5 9131.0 1 Type 4850.7 7347 7358.2 6833.1 9909.4 7875.3 — 2 Type 15552.6 17362.7 23574.8 11718 24425.5 18313.2 4265.9 3 Type 5676.1 11391.8 10274.5 10699.7 13622.7 15469 5930.1 5 Type 15265.3 13034.2 30631.9 17203.6 21335.1 21011.1 5697.2  6A Type 3566.3 8567.3 17964.5 8101.7 21985.6 19993.7 4136.6  6B Type 10474.8 32904.5 36935.5 28650.3 45878.8 48559 31991.5  7F Type 18684.5 26486.5 39449.2 26369.3 43372.9 56812.6 — 8 Type 29350.5 52915.1 61918.7 25465.9 73406.3 95465.2 — 9N Type 9041 19108 24479.9 24602.9 30087.5 36732.8 23053.6 9V Type 19833.3 38644.7 39959.5 73948.6 40524.8 60464.4 — 10A Type 893.6 3845.9 4447.6 2474.3 5683.2 8358.5 — 11A Type 4785.2 10497.8 7381.7 8444.8 8113.6 14211.8 — 12F Type 9177.7 17574 12811.4 15834.6 13543.4 23876.5 11178.8 14 Type 4908.7 17293.7 18345.2 4936.3 19436.1 28797.9 — 15B Type 7441.1 9373.4 14848.8 10186.3 14319.7 32906.8 — 17F Type 16747.2 27864.6 44605.6 29416.3 37658 38860.5 34163.2 18C Type 781.9 4088.9 5846.5 4114.6 8824.5 12216.5 14381.8 19A Type 4501.9 32543.6 27502.3 28629.2 36290.5 68218.5 15315.5 19F Type 18252.8 32553.5 34663.5 21760.3 37978.2 44630 — 20 Type 6790.8 24687.1 24800.1 19953 42634.5 55543.4 — 22F Type 823.6 3859.8 7938 6634 9468.7 10348.8 9440.5 23F Type 7261.6 23864.8 24843.6 20105.5 22586.9 28958 — 33F

[0525] [7. Streptococcus pneumoniae 24-Valent Polysaccharide-Protein Conjugate]

[0526] Preparation of Streptococcus pneumoniae 24-Valent Polysaccharide-Protein Conjugate

[0527] A 24-valent conjugate composition comprising the polysaccharide-protein conjugate in which polysaccharides derived from Streptococcus pneumoniae serotypes 2, 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F were conjugated to CRM197 and the polysaccharide-protein conjugate in which Streptococcus pneumoniae serotypes 1 and 5 were conjugated to TT (Tenus toxoid) (24vPnC) was formulated. For serotypes 2, 9N, 17F and 20, the conjugate was prepared by the afore-mentioned method, and for other serotypes, the conjugate conjugated to CRM197 or TT was prepared according to the methods disclosed in Korean Patent Application 2012-0065893, U.S. Patent Applications 62/371,529, 62/371,553 and 62/626,482.

[0528] The required volume of the final bulk concentrate was calculated based on batch volume and bulk saccharide concentration. A required amount of 0.85% sodium chloride, polysorbate 80 and succinate buffer were added to a pre-labeled formulation container, and then the bulk concentrate was added. It was sufficiently mixed and was filtrated through a 0.22 μm filter. During and after addition of bulk aluminum phosphate, the formulated bulk solution was slowly mixed. pH was checked and was adjusted if necessary. The formulated bulk product was stored at 2 to 8° C. The obtained vaccine composition contained each saccharide of 2.2 μg, but 6B of 4.4 μg; CRM 197 carrier protein of about 50 to 90 μg; adjuvant of aluminum element of 0.125 mg (0.5 mg aluminum phosphate); sodium chloride about 4.25 mg; succinate buffer about 295 μg; and polysorbate 80 about 100 μg in the total 0.5 mL.

[0529] Research on Immunogenicity of Streptococcus pneumoniae 24-Valent Polysaccharide-Protein Conjugate

[0530] OPA Analysis Result

[0531] In order to confirm whether the 24-valent polysaccharide-protein conjugate induced a functional antibody reaction, opsonophagocytic killing assay (OPA) was carried out in three rabbits in the same manner as the 15-valent vaccine.

TABLE-US-00015 TABLE 15 Serotype PCV24 Prevnar 13  1 309  54  2 490 —  3 407 393  4 1290 2072   5 1516 306  6A 1817 2355   6B 2888 1614   7F 1277 952  8 279 —  9N 653  52  9V 178 324 10A 658 — 11A 675 — 12F 471 — 14 959 539 15B 371 — 17F 348 — 18C 1357 1996  19A 642 1870  19F 1521 1516  20 306 — 22F 1703 — 23F 1531 928 33F 428 —

[0532] Through the result, it was confirmed that a 24-valent vaccine comprising all the serotypes 2, 9N, 17F and 20 could be obtained. As could be seen in the result, in particular, an antibody against serotypes 2, 17F and the like, which could not be obtained by Prevnar13, could be produced. In addition, it was confirmed that the immune response against all comprised serotypes could be induced without greatly affecting production of an antibody against an antigen of other serotypes, although 11 serotypes were added to Prevnar13.

INDUSTRIAL APPLICABILITY

[0533] The immunogenic composition of one example of the present invention may be used as medicament.

[0534] The immunogenic composition of one example of the present invention may be used as various therapeutic or prophylactic methods for prevention, treatment or improvement of bacterial infection, diseases or conditions. In particular, the immunogenic composition disclosed in the present invention may be used for prevention, treatment or improvement of Streptococcus pneumoniae infection, diseases or conditions in a subject.

[0535] In one embodiment, a method for inducing an immune response against Streptococcus pneumoniae in a subject, comprising administering an effective dose of the immunogenic composition of the present invention into the subject may be provided.