Compositions comprising streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof

Abstract

To provide additional pneumococcal vaccine compositions that can provide complementary protection against pneumococcal serotypes that are not present in currently available vaccines.SOLUTION: Provided is an immunogenic conjugate comprising Streptococcus pneumoniae (S. pneumoniae) serotype 9N polysaccharide conjugated to a CRM197 carrier protein, in which a) the polysaccharide has a molecular weight of 100 kDa to 500 kDa, b) the conjugate has a molecular weight of 1,000 to 10,000 kDa, and c) a ratio of the polysaccharide to carrier protein is 0.8 to 2.0, the conjugate being capable of inducing an immune response against 9N pneumococcal bacteria in a human patient. Also provided is an immunogenic composition comprising the conjugate and a pharmaceutically acceptable carrier.SELECTED DRAWING: Figure 31A

Description

[Technical Field]

[0001] The present invention provides multivalent immunogenic compositions having distinct polysaccharide-protein conjugates. Each conjugate is conjugated to a carrier protein, preferably CRM197. Different serotypes of Streptococcus pneumoniae The immunogenic composition comprises capsular polysaccharides prepared from the pneumococcal strains of Streptococcus pneumoniae. Provide coverage. [Background technology]

[0002] Streptococcus pneumoniae is a gram-positive bacterium. It is the most common cause of invasive bacterial diseases in infants and young children (such as pneumonia, bacteremia, meningitis, and otitis media). Pneumococci are encapsulated with chemically bound polysaccharides that confer serotype specificity. There are over 90 known serotypes of pneumococcus, and the capsule is a protective barrier against the bacterial interior. Not only does it protect the body, but it is also a major virulence determinant of pneumococci because it is poorly immunogenic in itself. Polysaccharides are T-cell independent antigens and interact mostly with T cells. However, they cannot be processed or presented by MHC molecules. The immune system can be stimulated through alternative mechanisms, including cross-linking of surface receptors on the immune system.

[0003] The multivalent pneumococcal polysaccharide vaccine, which has been licensed for many years, is used in adults, especially the elderly and have proven beneficial in preventing pneumococcal disease in people at high risk. However, infants do not respond well to unconjugated pneumococcal polysaccharides. The seven most frequently isolated serotypes (4, 6B, 9V, 1) causing invasive pneumococcal disease Pneumococcal conjugate vaccine containing 4, 18C, 19F and 23F Revnar® was first approved in the United States in February 2000. Serum present in Prevnar® after universal use of Prevnar® The study found that invasive pneumococcal disease in children was significantly reduced by 100%. sease Control and Prevention,MMWR Morb M See Oral Wkly Rep 2005,54(36):893-7. However, serotype coverage by Prevnar® in certain regions of the world is limited. There is some evidence that certain serotypes are emerging in the United States. (e.g., 19A). O'Brien et al., 2004, Am J Epi demiol 159:634-44;Whitney et al.,2003,N Engl J Med 348:1737-46;Kyaw et al.,2006, N Engl J Med 354:1455-63;Hicks et al.,20 07,J Infect Dis 196:1346-54;Traore et al .,2009,Clin Infect Dis 48:S181-S189 sea ​​bream.

[0004] U.S. Patent Application Publication No. 2006 / 0228380 describes serotypes 1, 3, 4, 5, and 6 13-valent pneumocytes containing A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F It describes a bacterial polysaccharide-protein conjugate vaccine. No. 1590224 covers serotypes 1, 2, 4, 5, 6A, 6B, 7F, 9N, 9V, 1 14-valent pneumococcal polysaccharide-protein complex containing 4, 18C, 19A, 19F, and 23F The adjugate vaccine is described.

[0005] Other PCVs cover serotypes 7, 10, 11, or 13, which are included in PCV-15. (US Patent Application Publication No. 2011 / 0195086), but immunity to some serotypes Interference (e.g., low protection against serotype 3 of GSK's PCV-11) and Pfiz The response rate of PCV-13 (PREVNAR® 13) to serotype 6B in er It has been observed that the incidence of HIV infection is low. Prymula et al., 2006, Lancet 367:740-48 and Kieninger et al., Safety and Immunologic Non-inferiority of 13-valen t Pneumococcal Conjugate Vaccine Compare d to 7-valent Pneumococcal Conjugate Vac cine Given as a 4-Dose Series in Healthy Infants and Toddlers,presented at the 4 8 th Annual ICAAC / ISDA 46 th Annual Meeting g, Washington DC, October 25-28, 2008 stomach.

[0006] Current multivalent pneumococcal vaccines limit the risk of pneumococcal disease associated with the serotypes present in the vaccine. However, there are currently no vaccines available that are effective in reducing the incidence of The prevalence of pneumococci expressing serotypes that are not pneumococcal is increasing. provides protection against a different set of pneumonia types, which are not present in currently available vaccines There is a need for additional pneumococcal vaccine compositions that can provide complementary protection against pneumococcal serotypes. It is considered essential. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] US Patent Application Publication No. 2006 / 0228380 [Patent Document 2] Chinese Patent Application Publication No. 101590224 [Patent Document 3] US Patent Application Publication No. 2011 / 0195086 [Non-patent literature]

[0008] [Non-Patent Document 1] Centers for Disease Control and Prevention, MMWR Morb Mortal Wkly Rep 2005,54(36):893-7 [Non-patent document 2] O'Brien et al., 2004, Am J Epidemiol 159:634-44 [Non-patent document 3] Whitney et al.,2003,N Engl J Med 348:1737-46 [Non-patent document 4] Kyaw et al.,2006,N Engl J Med 354:1455-63 [Non-patent document 5] Hicks et al.,2007,J Infect Dis 196:1346-54 [Non-patent document 6] Traore et al., 2009, Clin Infect Dis 48:S181-S189 [Non-Patent Document 7] Prymula et al.,2006,Lancet 367:740-48 [Non-patent document 8] Kieninger et al.,Safety and Immunologic Non-inferiority of 13-valent Pneumococcal Conjugate Vaccine Compared to 7-valent Pneumococcal Conjugate Vaccine Given as a 4-Dose Series in Healthy Infants and Toddlers,presented at the 48th Annual ICAAC / ISDA 46th Annual Meeting,Washington DC,October 25-28,2008 Summary of the Invention [Means for solving the problem]

[0009] The present invention provides a pneumococcal (S. pneumoniae) polysaccharide-protein conjugate. a multivalent immunogenic composition comprising: Conjugated polysaccharides of Streptococcus pneumoniae serotypes, and the serotypes of S. pneumoniae are as defined herein. A composition is provided.

[0010] In a specific embodiment of the invention, the serotype of S. pneumoniae is a) 15A, 16F, 23A, 23B, 24F, 31 and 35B; b)15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 1 1A, 12F, 15C, 17F and 20; and c)3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20 The set of serotypes selected from the group consisting of:

[0011] In a further particular embodiment of the present invention, serum of S. pneumoniae The type is a) 15A, 16F, 23A, 23B, 24F, 31 and 35B; b)15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 1 1A, 12F, 15C, 17F and 20A; and c)3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20A The set of serotypes selected from the group consisting of:

[0012] In a further particular embodiment of the present invention, serum of S. pneumoniae The type is a) 15A, 16F, 23A, 23B, 24F, 31 and 35B; b)15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 1 1A, 12F, 15C, 17F and 20B; and c)3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20B The set of serotypes selected from the group consisting of:

[0013] In some embodiments, the pneumococcus (S. pneumoniae) strain is selected from the group consisting of Streptococcus pneumoniae (S. pneumoniae) strains listed in a), b), or c). moniae) serotype set is serotype 6C, serotype 6A, or serotype 6A and Further contains 6B.

[0014] In some embodiments, at least one of the polysaccharide protein conjugates is a non-protein Conjugation reactions involving protic solvents, such as dimethyl sulfoxide (DMSO), In a specific embodiment, each of the polysaccharide protein conjugates is formed by a conjugation reaction involving an aprotic solvent. DM as a solvent during the reductive amination of polysaccharide-protein conjugates, as described Using SO, these blood samples were significantly reduced compared to the same conjugates prepared under aqueous conditions. The unexpectedly superior stability and enhanced immunogenicity of the purified form are obtained.

[0015] A method for inducing a protective immune response in a human patient, comprising administering a multivalent immunogenic composition of the invention to the patient. In some embodiments of the methods of the present invention, a method is also provided, comprising administering to The patient had previously been treated with a multivalent pneumococcal vaccine.

[0016] The multivalent immunogenic compositions of the invention are useful in treating regimens with different complementary pneumococcal vaccines. Thus, the present invention provides a method for inducing a protective immune response in a human patient. A method comprising the steps of administering, in any order, a multivalent immunogenic composition of the invention to a patient. and further comprising the step of administering a multivalent pneumococcal vaccine to the patient. In certain embodiments, the multivalent pneumococcal vaccine comprises multiple S. pneumoniae e) polysaccharide-protein conjugates, wherein each of the conjugates is a carrier Protein-conjugated polysaccharides of S. pneumoniae serotypes In other embodiments, the multivalent pneumococcal vaccine comprises a polysaccharide comprising more than one non-conjugated capsular polysaccharide. Rinaru.

[0017] Conjugation of serotype 8 pneumococcus (Streptococcus pneumoniae) using a conjugation reaction in aprotic solvents Peptococcus pneumoniae polysaccharide-protein conjugates were prepared. wherein the conjugation reaction does not involve the use of cyanoborohydride. A method for doing this is also provided.

[0018] The present invention also provides S. pneumoniae polysaccharide-protein conjugates. and a multivalent immunogenic composition comprising a conjugate of a carrier protein, wherein each of the conjugates is linked to a carrier protein. a polysaccharide of a serovar S. pneumoniae conjugated to Here, the selected serotype of Streptococcus pneumoniae is cross-reactive with other selected serotypes. Provides responsiveness. [Brief explanation of the drawings]

[0019] [Figure 1] Figure 1 shows a 600 MHz one-dimensional H NMR spectrum of the capsular polysaccharide of S. pneumoniae serotype 6C in heavy water (DO) at 50 °C. Signals arising from internal standards (DMSO and DSS-d6) and residual water (HOD) are shown. Minor signals, indicated by *, are due to S. pneumoniae cell wall residues such as C-polysaccharide and / or peptidoglycan. [Figure 2]Figure 1 shows a 600 MHz one-dimensional H NMR spectrum of the capsular polysaccharide of S. pneumoniae serotype 15A in heavy water (DO) at 50 °C. Signals arising from internal standards (DMSO and DSS-d6) and residual water (HOD) are shown. Minor signals, indicated with *, are due to S. pneumoniae cell wall residues such as C-polysaccharide and / or peptidoglycan. [Figure 3] Figure 1 shows a 600 MHz one-dimensional H NMR spectrum of the capsular polysaccharide of S. pneumoniae serovar 15B de-O-acetylated in deuterium oxide (DO) at 50 °C. Signals arising from internal standards (DMSO and DSS-d6) and residual water (HOD) are shown. Minor signals indicated with * are due to S. pneumoniae cell wall residues such as C-polysaccharide and / or peptidoglycan. [Figure 4] Figure 1 shows a 600 MHz one-dimensional H NMR spectrum of the capsular polysaccharide of S. pneumoniae serotype 35B in DO at 50 °C. Signals arising from internal standards (DMSO and DSS-d6) and residual water (HOD) are shown. Minor signals, indicated by *, are due to S. pneumoniae cell wall residues such as C-polysaccharide and / or peptidoglycan. [Figure 5] 1 shows the H NMR identification regions useful for serotype identification of S. pneumoniae serotype 6C. The signal positions of each anomeric proton of the repeating unit from each monosaccharide residue are indicated. [Figure 6] 1 shows the H NMR identification regions useful for serotype identification of S. pneumoniae serotype 15A. The signal positions of each anomeric proton of the repeating unit from each monosaccharide residue are indicated. [Figure 7] 1 shows the H NMR identification regions useful for serotype identification of S. pneumoniae serotype de-O-acetylated 15B. The signal positions of each anomeric proton from each monosaccharide residue are indicated. [Figure 8]1 shows the H NMR identification regions useful for serotype identification of S. pneumoniae serotype 35B. The signal positions of each anomeric proton of the repeating unit from each monosaccharide residue are indicated. [Figure 9A] FIG. 1 shows a 600 MHz one-dimensional 1H NMR spectrum of native capsular polysaccharide of S. pneumoniae serotype 15B. [Figure 9B] FIG. 1 shows a 600 MHz one-dimensional 1H NMR spectrum of de-O-acetylated capsular polysaccharide of S. pneumoniae serotype 15B. [Figure 9C] Figure 1 shows a 600 MHz one-dimensional H NMR spectrum of the capsular polysaccharide of S. pneumoniae serotype 15C. The spectrum was acquired at 50 °C in heavy water (DO). Signals arising from internal standards (DMSO and DSS-d6) and residual water (HOD) are shown. Signals indicated with an * are due to S. pneumoniae cell wall residues such as C-polysaccharide and / or peptidoglycan. [Figure 10A] FIG. 1 shows the anomeric region of the 600 MHz one-dimensional 1H NMR spectrum of the native capsular polysaccharide of S. pneumoniae serotype 15B. [Figure 10B] FIG. 1 shows the anomeric region of the 600 MHz one-dimensional 1H NMR spectrum of de-O-acetylated capsular polysaccharide of S. pneumoniae serotype 15B. [Figure 10C] FIG. 1 shows the anomeric region of the 600 MHz one-dimensional 1H NMR spectrum of the capsular polysaccharide of S. pneumoniae serotype 15C. [Figure 10D] FIG. 1 shows the spectral region of the O-acetyl and N-acetylmethyl signals of native capsular polysaccharide of S. pneumoniae serotype 15B. [Figure 10E] FIG. 1 shows the spectral region of the O-acetyl and N-acetylmethyl signals of de-O-acetylated capsular polysaccharide of S. pneumoniae serotype 15B. [Figure 10F]FIG. 1 shows the spectral region of the O-acetyl and N-acetylmethyl signals of the capsular polysaccharide of S. pneumoniae serotype 15C. [Figure 11] Figure 3 shows the effect of time and temperature (4°C for up to 12 weeks - diamonds, 25°C for up to 4 weeks - squares, 37°C for up to 4 weeks - triangles) on polysaccharide concentration of PCV16 (0.128 mg / mL) or PCV21 (0.084 mg / mL or 0.169 mg / mL) formulations using HPSEC-UV / MALS / RI (see Example 39). [Figure 12] 1 shows the effect of horizontal rotational agitation and temperature (at either 4°C, 25°C, or 37°C for 1 week) on the polysaccharide concentration of PCV16 (0.128 mg / mL) or PCV21 (0.084 mg / mL or 0.169 mg / mL) formulations dispensed into prefilled syringes (see Example 39). [Figure 13] Figure 1 shows the effect of PS concentration over time and temperature (up to 4 weeks at either 4°C, 25°C, or 37°C) on the average molecular weight (Mw and Mn) of PCV16 (0.128 mg / mL) or PCV21 (0.084 mg / mL or 0.169 mg / mL) formulations using HPSEC-UV / MALS / RI. [Figure 14A] FIG. 1 shows the effect of time and temperature (up to 1 week at 4° C. or 37° C.) on the stability of pneumococcal conjugate vaccines (PCV15 formulated using all-aqueous conjugation) prepared with drug substances conjugated in protic solvents using intrinsic protein fluorescence spectroscopy at an excitation wavelength of 280 nm (see Example 40). [Figure 14B] FIG. 1 shows the effect of time and temperature (up to 1 week at 4° C. or 37° C.) on the stability of pneumococcal conjugate vaccines (PCV16 formulated using all-DMSO conjugation at 0.064 mg / mL PnPs) prepared with drug substances conjugated in aprotic solvents using intrinsic protein fluorescence spectroscopy at an excitation wavelength of 280 nm (see Example 40). [Figure 15]Figure 1 shows ELISA IgG antibody dilution titers (post-dose 2) of rabbits immunized with S. pneumoniae monovalent serotypes conjugated to CRM197 and formulated in aluminum phosphate adjuvant (APA). Symbols indicate individual antibody titers, and error bars represent 95% confidence intervals (CI) of the geometric mean titers (GMT). [Figure 16] Figure 1 shows serotype-specific OPA-dilution antibody titers (post-dose 2) of rabbits immunized with S. pneumoniae monovalent serotypes conjugated to CRM197 and formulated in aluminum phosphate adjuvant (APA). Symbols indicate individual antibody titers, and error bars represent the 95% confidence interval (CI) of the geometric mean titer (GMT). [Figure 17] Figure 1 shows ELISA IgG antibody dilution titers (post-dose 2) of rabbits immunized with S. pneumoniae serotype 15 monovalent conjugate. The x-axis indicates the vaccine used to immunize the rabbits. Dashed lines separate three separate ELISA assays using different pneumococcal polysaccharides as coating antigens. Symbols indicate individual antibody titers, and error bars represent the 95% confidence interval (CI) of the geometric mean titer (GMT). [Figure 18] Figure 1 shows serotype-specific OPA dilution antibody titers (pre-immunization, post-dose 1 (PD1, pooled), and post-dose 2 (PD2)) of rabbits immunized with S. pneumoniae serotype 15 monovalent conjugates. The X-axis indicates the vaccine used to immunize the rabbits. Dashed lines separate three OPA assays using distinct S. pneumoniae strains. Symbols indicate individual antibody titers, and error bars represent the 95% confidence interval (CI) of the geometric mean antibody titer (GMT). [Figure 19A] Figure 1 shows a comparison of PD1 antibody responses in NZWR (5 animals per group) after vaccination with 4, 2, 1, 0.4, 0.08, or 0.016 μg / dose of PCV21. Symbols indicate PD1 geometric mean titer (GMT) ratios (2 μg / dose group vs. other dose groups), and error bars represent 95% confidence intervals (CI). [Figure 19B]Figure 19B shows PCV21 dose comparison PD1 GMT ratios (95% CI) corresponding to the GMT ratios in Figure 19A, where GMT ratios whose lower 95% confidence limit is greater than 1.0 are light gray and GMT ratios whose upper 95% confidence limit is less than 1.0 are dark gray. Serotype 15B data are included to assess cross-protection. [Figure 20A] Figure 1 shows a comparison of PD2 antibody responses in NZWR (5 animals per group) after vaccination with 4, 2, 1, 0.4, 0.08, or 0.016 μg / dose of PCV21. Symbols indicate PD2 GMT ratios (2 μg / dose group vs. other dose groups), and error bars represent 95% CI. [Figure 20B] Figure 20B shows PCV21 dose comparison PD2 GMT ratios (95% confidence intervals) corresponding to the GMT ratios in Figure 20A. GMT ratios whose lower 95% confidence limit is greater than 1.0 are light gray. Serotype 15B data are included to assess cross-protection. [Figure 21A] Figure 1 shows serotype-specific PD1 OPA dilution antibody titers in rabbits immunized with PCV21 (2 μg / PnPs). Symbols indicate individual antibody titers, and error bars represent the 95% confidence interval (CI) of the geometric mean titer (GMT). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 21B] Figure 1 shows serotype-specific PD2 OPA dilution antibody titers for rabbits immunized with PCV21 (2 μg / PnPs). Symbols indicate individual antibody titers, and error bars represent the 95% confidence interval (CI) of the geometric mean titer (GMT). ****p<0.0001. Serotype 15B data are included to assess cross-protection. [Figure 22A] FIG. 1 shows the effect of time and temperature (i.e., 4° C. for 7 days, 37° C. for 1 day, or 37° C. for 7 days) on the stability of pneumococcal conjugate vaccine (PCV1) prepared with all drug substances conjugated in aqueous or DMSO solvents using intrinsic protein fluorescence spectroscopy at an excitation wavelength of 280 nm. [Figure 22B]FIG. 1 shows the effect of time and temperature (i.e., 4° C. for 7 days, 37° C. for 1 day, or 37° C. for 7 days) on the stability of pneumococcal conjugate vaccine (PCV7) prepared with all drug substances conjugated in aqueous or DMSO solvents using intrinsic protein fluorescence spectroscopy at an excitation wavelength of 280 nm. [Figure 22C] FIG. 1 shows the effect of time and temperature (i.e., 4° C. for 7 days, 37° C. for 1 day, or 37° C. for 7 days) on the stability of pneumococcal conjugate vaccine (PCV14) prepared with all drug substances conjugated in aqueous or DMSO solvents using intrinsic protein fluorescence spectroscopy at an excitation wavelength of 280 nm. [Figure 22D] FIG. 1 shows the effect of time and temperature (i.e., 7 days at 4° C. or 37° C.) on the stability of a 21-valent pneumococcal conjugate vaccine (PCV21 at 0.084 mg / mL PnPs) prepared with all drug substances conjugated in DMSO solvent using intrinsic protein fluorescence spectroscopy at an excitation wavelength of 280 nm. [Figure 23] FIG. 1 shows the effect of temperature (4° C. or 37° C. for 7 days) and agitation at 4° C. on particle size distribution as analyzed by nanoparticle tracking analysis (NTA) of six PCV21 pneumococcal conjugate vaccine formulations formulated with PS-20 (0%, 0.025%, 0.05%, 0.1%, 0.15%, or 0.2%; all w / v PS-20) at 0.084 mg / mL PnPs. [Figure 24] Figure 1 shows the effect of temperature (4°C and 37°C) and agitation on the average molecular weight of three PCV21 formulations analyzed by HPSEC / UV / MALS / RI assay. Three PCV21 pneumococcal conjugate vaccine formulations were formulated with different concentrations of PS-20 (0.05%, 0.1%, and 0.15% w / v) at 0.084 mg / mL PnPs. [Figure 25] FIG. 1 shows that PCV21-immunized mice are protected from intratracheal challenge with S. pneumoniae 24F. [Figure 26A]Figure 1 shows pre(pool), PD1(pool) and PD2 IgG antibody dilution titers determined by ECL for rabbits immunized with non-adjuvanted PCV21. [Figure 26B] Figure 1 shows pre(pool), PD1(pool), and PD2 IgG antibody dilution titers as determined by ECL for rabbits immunized with PCV21 formulated with APA. Error bars represent the 95% confidence interval (CI) of the geometric mean titer (GMT). Serotype 15B data are included to assess cross-protection. [Figure 27A] Figure 1 shows pre(pooled), PD1(pooled) and PD2 IgG antibody dilution titers determined by ECL for rabbits immunized with non-adjuvanted PCV8. [Figure 27B] Figure 1 shows pre(pool), PD1(pool) and PD2 IgG antibody dilution titers determined by ECL for rabbits immunized with non-adjuvanted PCV16. [Figure 27C] Figure 1 shows pre-(pooled), PD1(pooled), and PD2 IgG antibody dilution titers determined by ECL for rabbits immunized with PCV31 using APA. Error bars represent the 95% confidence interval (CI) of the geometric mean titer (GMT). Serotype 15B data are included to assess cross-protection. [Figure 28] Figure 1 shows a comparison of PD2 ECL antibody responses in NZWR (5 animals per group) after vaccination with PCV21 with or without APA. Symbols indicate PD2 GMT ratios, and error bars represent 95% CI. Serotype 15B data are included to assess cross-protection. [Figure 29] Figure 1 shows a comparison of PD2 ECL antibody responses in NZWR (5 per group) for common and cross-protective serotype 15B after vaccination with PCV21, PCV8, or PCV16. Symbols indicate PD2 ECL GMT ratios, and error bars represent 95% CI. [Figure 30]Figure 1 shows a comparison of PD2 ECL antibody responses in NZWR (5 animals per group) for common and cross-protective serotype 15B after vaccination with PCV21 / APA or PCV31 / APA. Symbols indicate PD2 ECL GMT ratios, and error bars represent 95% CI. [Figure 31A] FIG. 1 shows serotype-specific pre-(pooled) OPA dilution antibody titers for rabbits immunized with PCV. [Figure 31B] FIG. 1 shows serotype-specific pre-(pooled) OPA dilution antibody titers for rabbits immunized with PCV. [Figure 31C] FIG. 1 shows serotype-specific PD2 OPA dilution antibody titers for rabbits immunized with PCV. [Figure 31D] Figure 1 shows serotype-specific PD2 OPA dilution antibody titers for rabbits immunized with PCV. Error bars represent the 95% CI of the GMT. PCV21 was used as the benchmark for statistical comparison. *p<0.05. Figures A and C show data for eight common serotypes of all PCVs evaluated (6C, 15A, 16F, 23A, 23B, 24F, 31, 35B) and eight additional common serotypes of PCV16, PCV21, and PCV31 (8, 9N, 10A, 11A, 12F, 15C, 17F, 20B). Figures B and D show data for an additional 16 serotypes not included in Figures A and C. Five of these serotypes are also common in PCV21 and PCV31 (3, 7F, 19A, 22F, and 33F), and 10 serotypes are found only in PCV31 (1, 4, 5, 6A, 6B, 9V, 14, 18C, 19F, and 23F). Data for serotype 15B are included to assess cross-protection. [Figure 32] Figure 1 shows pre-, PD1, PD2, and PD3 IgG antibody dilution titers determined by ECL for adult rhesus macaques (n=8) immunized with PCV21. Error bars represent the 95% CI of the GMT. Serotype 15B data are included to assess cross-protection. [Figure 33]Pre, PD1 (pooled), and PD3 OPA dilution antibody titers of adult rhesus macaques (n=8) immunized with PCV21. Error bars represent the 95% CI of the GMT. Serotype 15B data are included to assess cross-protection. [Figure 34] FIG. 1 shows pre- and PD3 (day 70) OPA antibody titers against four serotypes not included in the PCV21 vaccine for adult rhesus macaques immunized with PCV21. [Figure 35] Figure 1 shows a comparison of PD1 ECL antibody responses in adult rhesus macaques (5 per group) after vaccination with PCV21 with or without APA. Symbols indicate PD1 ECL GMT ratios (PCV21 vs. PCV21 / APA), and error bars represent 95% CI. Serotype 15B data are included to assess cross-protection. [Figure 36] Figure 1 shows PD1 IgG antibody responses to serotypes 3, 7F, 19A, 22F, and 33F in adult rhesus macaques (2-5 per group) after vaccination with PCV21 compared to PCV15 or Prevnar 13. Symbols indicate PD1 ECL GMT ratios, and error bars represent 95% CI. [Figure 37A] FIG. 10 shows ELISA IgG antibody dilution titers against 6A, 6B and 6C ((pre-immune and PD1, pooled) and PD2) for rabbits immunized with 6A-CRM197 monovalent formulation. [Figure 37B] Figure 1 shows ELISA IgG antibody dilution titers against 6A, 6B, and 6C (pre-immune and PD1, pooled) and PD2 for rabbits immunized with the 6B-CRM197 monovalent formulation. Bars indicate geometric mean titers (GMTs), and error bars indicate 95% confidence intervals (CIs) of the GMTs. [Figure 38A] FIG. 1 shows serotype-specific OPA dilution antibody titers against 6A, 6B and 6C ((pre-immune and PD1, pooled) and PD2) for rabbits immunized with the 6A-CRM197 monovalent formulation. [Figure 38B]Figure 1 shows serotype-specific OPA-dilution antibody titers against 6A, 6B, and 6C (pre-immune and PD1, pooled) and PD2 for rabbits immunized with the 6B-CRM197 monovalent formulation. Bars indicate geometric mean titers (GMTs), and error bars indicate 95% confidence intervals (CIs) of the GMTs. [Figure 39A] 20A-Serotype-specific OPA dilution antibody titers against 20A and 20B (pre-immune (pooled), PD1 and PD2) for rabbits immunized with CRM197 / APA. [Figure 39B] Figure 1 shows serotype-specific OPA dilution antibody titers for rabbits immunized with PCV21 against 20A and 20B (pre-immune (pooled), PD1, and PD2). Bars indicate geometric mean antibody titers (GMTs), and error bars indicate 95% confidence intervals (CIs) of GMTs. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention is a multivalent immunogenic composition comprising a pneumococcal polysaccharide-protein conjugate. wherein each of the conjugates is a pneumococcal antibody conjugated to a carrier protein. (S. pneumoniae) serotype polysaccharides, where pneumococcus (S. pneu moniae) serotypes as defined herein. In embodiments, the immunogenic composition comprises: (a) 15A, 16F, 23A, 23B, 24F, 31 and 35B; (b) 15A, 16F, 23A, 23B, 24F, 31, 35B, 8 , 9N, 10A, 11A, 12F, 15C, 17F and 20; and (c) 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8 , 9N, 10A, 11A, 12F, 15C, 17F and 20 In some embodiments, the immunogenic composition comprises a set of pneumococcal serotypes. 5A, 16F, 23A, 23B, 24F, 31 and 35B; (b) 15A, 16F, 2 3A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 1 7F and 20B; and (c) 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, The set of pneumococcal serotypes selected from the group consisting of 17F and 20B. In one embodiment, the immunogenic composition comprises: (a) 15A, 16F, 23A, 23B, 24F, 31, and and 35B; (b) 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N , 10A, 11A, 12F, 15C, 17F and 20A; and (c) 3, 7F, 19 A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9 Lungs selected from the group consisting of N, 10A, 11A, 12F, 15C, 17F and 20A In a further embodiment, the immunogenic composition comprises a set of Streptococcus pneumoniae serotypes. (ii) serotypes 6A and 6B; or (iii) serotype 6C. In one embodiment, the present invention provides a polysaccharide template containing multiple pneumococcal S. pneumoniae polysaccharides. and protein conjugates, wherein each of the conjugates is conjugated to a carrier protein. Glutamate polysaccharides of Streptococcus pneumoniae serovars, The serotypes of Streptococcus pneumoniae (S. pneumoniae) are serotypes 3, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 20A, 22F, The compositions contain 23A, 23B, 24F, 31, 33F and 35B. It was immunogenic against the vaccine-type bacterial strain and produced functional antibodies that killed the vaccine-type bacterial strain at all doses tested. In another specific embodiment, the present invention provides a method for treating multiple pneumonia. containing a cocci Streptococcus pneumoniae (S. pneumoniae) polysaccharide-protein conjugate, Here, each of the conjugates is a pneumococcus (S. pneumoniae serotype polysaccharides, where S. pneumoniae iae) serotypes are serotypes 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, and 1 1A, 12F, 15C, 17F, 20, 6A, 15A, 15C, 16F, 23A, 23B In another specific embodiment, the present invention provides a method for the treatment of pneumonia comprising administering to a patient a therapeutically effective amount of pneumonia using a method ... The compound contains a polysaccharide-protein conjugate of the bacterium Streptococcus pneumoniae (S. pneumoniae), Here, each of the conjugates is a pneumococcus (S. pneumoniae serotype polysaccharides, where S. pneumoniae iae) serotypes are serotypes 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, and 1 1A, 12F, 15C, 17F, 20B, 6A, 15A, 15C, 16F, 23A, 23 In another specific embodiment, the present invention provides a method for treating multiple pneumonia. containing a cocci Streptococcus pneumoniae (S. pneumoniae) polysaccharide-protein conjugate, wherein each of the conjugates comprises Streptococcus pneumoniae (S) conjugated to a carrier protein. pneumoniae serotype polysaccharides, where pneumococcus (S. pneumoniae) niae) serotypes are serotypes 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15C, 17F, 20A, 6A, 15A, 15C, 16F, 23A, 2 Includes 3B, 24F, 31 and 35B.

[0021] The multivalent immunogenic compositions of the present invention are directed to vaccine-type S. pneumoniae serogroups. and different complementary pneumococcal vaccine(s) were used to immunize patients against serotypes. Thus, the present invention is useful as part of a treatment regimen to induce a protective immune response in a human patient. a method for inducing a HIV-1-associated ... and further comprising the step of administering a multivalent pneumococcal vaccine to the patient. In another embodiment, the multivalent immunogenic compositions of the invention are prepared by combining different multivalent immunogens. It is administered to patients who have been immunized with the pneumococcal vaccine.

[0022] In an embodiment of the invention, the conjugate from at least one pneumococcal serotype is D It is prepared using reductive amination in an aprotic solvent such as MSO. In embodiments, the multivalent immunogenic compositions each comprise a multivalent immunogen comprising a nucleotide sequence selected from the group consisting of nucleotides obtained by reductive amination in an aprotic solvent. Each of the pneumococcal conjugates was prepared by reduction in an aprotic solvent. PCV1, PCV7, PCV14, PCV8, PCV9, PCV10, PCV11, PCV12, PCV13, PCV14, PCV14, PCV15, PCV16, PCV17, PCV18, PCV19, PCV20, PCV21, PCV22, PCV23, PCV24, PCV25, PCV30, PCV31, PCV32, PCV33, PC V16 and PCV21 (defined below) formulations are formulated to depolymerize or chemically decompose carbohydrates. and are shown herein to be stable to protein aggregation in formulations. (See Examples 40 and 45.) When DMSO solvent is used, the carrier Covalent association of polysaccharides with proteins through the direct consumption of lysine residues on the protein surface The increased covalent association is observed for polysaccharide antigens conjugated in DMSO. Increasing the stability of a polysaccharide-protein conjugate of a multivalent immunogenic composition comprising directly benefit the

[0023] I. Definitions and Abbreviations As used throughout this specification and the appended claims, the following abbreviations apply: R: APA Aluminum phosphate adjuvant APC antigen presenting cells CI confidence interval DMSO dimethyl sulfoxide DS Polysaccharide-Protein Drug Substance GMC geometric mean concentration GMT Geometric Mean Titer HPSEC High-Performance Size Exclusion Chromatography IM intramuscular or intramuscularly LOS Lipooligosaccharide LPS lipopolysaccharide MALS multi-angle light scattering MBC Monovalent Bulk Conjugate MOPA Multiplex Opsonophagocytic Assay MW molecular weight NMWCO Nominal Molecular Weight Cutoff NZWR New Zealand White Rabbit OPA Opsonophagocytosis Assay PCV Pneumococcal Conjugate Vaccine PD1 administration 1 day later PD2 after 2 doses PnPs Pneumococcus polysaccharide Ps polysaccharide PS-20 Polysorbate-20 RI refractive index UV ultraviolet light w / v Weight per volume.

[0024] In order that the present invention may be more readily understood, certain technical and scientific terms will be specifically defined below. Unless specifically defined elsewhere in this document, all techniques used herein Technical and scientific terms have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs. It has.

[0025] As used throughout this specification and in the appended claims, the singular forms "a," "a" and "b" are used interchangeably. "n" and "the" refer to plurals unless the context clearly indicates otherwise. Includes the target.

[0026] A reference to "or" means either or, unless the context clearly indicates one of the possibilities indicated. Indicate either or both possibilities. In some cases, emphasize either or both possibilities. I used "and / or" to

[0027] The terms "aqueous solvent" or "aqueous conditions" refer to the use of conjugation methods such as reductive amination. When used in this context, it refers to the use of water as the solvent for the conjugation reaction. may contain buffers and other ingredients except that organic solvents are absent.

[0028] The terms "aprotic solvent," "DMSO solvent," or "DMSO conditions" refer to the reduction When used in conjugation such as selective amination, aprotic solvents, or combinations of aprotic solvents (or This refers to the use of aprotic solvents, e.g., up to 1%, 2%, 5% It may contain some water present, 10% or 20%.

[0029] The term "comprises" when used in reference to the immunogenic compositions of the present invention includes adjuvants and and the inclusion of other optional ingredients such as excipients, or one or more polysaccharides not specifically listed. The term "consisting of" refers to the addition of a polysaccharide-protein conjugate. - When used in protein conjugate mixtures, certain S. pneumoniae oniae) polysaccharide-protein conjugates and other pneumococci of different serotypes (S pneumoniae) refers to a mixture that does not have a polysaccharide-protein conjugate. "consists essentially of" and " "consist essentially of" or "consist essentially of" "consisting essentially of" Such variations include the inclusion of a listed element or elements and the administration regimen, method, or or compositions similar to the recited elements that do not materially change the basic or novel characteristics of the composition. indicates the optional inclusion of other elements of a different nature.

[0030] An "effective amount" of a composition of the present invention is an amount that will kill or inhibit a microorganism, such as Streptococcus pneumoniae (S. pneumoniae), during subsequent exposure. significantly increase the likelihood or severity of infection with cecumonia It refers to the dose required to induce antibodies that reduce the level of vasopressin.

[0031] As used herein, the term "required to prevent pneumococcal disease" means The phrase refers to a vaccine or immunogenic composition that has been approved by one or more regulatory authorities, such as the U.S. Food and Drug Administration. These include, but are not limited to, pneumococcal disease in general, pneumococcal pneumonia, pneumococcal meningitis, pneumococcal bacteremia, caused by Streptococcus pneumoniae (S. pneumoniae) Invasive disease and middle ear infection caused by S. pneumoniae caused by any serotype of Streptococcus pneumoniae, including pneumonia This means that the drug is approved to prevent one or more of the diseases listed in the "Diseases and Conditions" section.

[0032] "Multivalent pneumococcal vaccine" is a vaccine that contains two or more strains of pneumococcus (S. pneumoniae). Two or more that provide active immunity against disease or pathological symptoms caused by the serotype active agent (e.g., pneumococcal capsular polysaccharide or pneumococcal polysaccharide protein conjugate) It is a medicine containing (

[0033] The term "polysaccharide" refers to any of the antibodies commonly used in immunization and bacterial vaccine technology. "sugar" is meant to include, but is not limited to, raw sugar elements (or antigenic units); "Oligosaccharides", "polysaccharides" or "lipopolysaccharides", "lipooligosaccharides (LOS)", "lipopolysaccharides" (LPS), "glycosylates", "glycoconjugates" ate)" etc.

[0034] "PCV1," as used herein, refers to a PCV1 conjugated to a carrier protein. One pneumococcus (S) containing capsular polysaccharide of the S. pneumoniae serotype Monovalent pneumococcal conjugates containing a (.pneumoniae) polysaccharide-protein conjugate A gate vaccine, wherein the serotype of pneumococcus (S. pneumoniae) is 3 In certain embodiments, the vaccine is The body protein is CRM197.

[0035] "PCV7," as used herein, refers to a virus conjugated to a carrier protein. Seven pneumococci containing capsular polysaccharides of selected S. pneumoniae serotypes Heptavalent pneumococcal vaccine containing S. pneumoniae polysaccharide protein conjugate A conjugate vaccine, comprising a serogroup containing Streptococcus pneumoniae (S. pneumoniae) The types are 3, 8, 9N, 11A, 19A, 15A and 10A. In a specific embodiment , carrying one or more of the pneumococcal (S. pneumoniae) polysaccharide-protein conjugates In a further embodiment, the body protein is CRM197. The carrier protein of each of the (niae) polysaccharide-protein conjugates is CRM197. be.

[0036] "PCV8," as used herein, refers to a virus conjugated to a carrier protein. Eight pneumococci containing capsular polysaccharides of selected S. pneumoniae serotypes Octavalent pneumococcal vaccine containing S. pneumoniae polysaccharide protein conjugate A conjugate vaccine, comprising a serogroup containing Streptococcus pneumoniae (S. pneumoniae) The types are 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B. In one embodiment, a S. pneumoniae polysaccharide protein conjugate is In a further embodiment, the one or more carrier proteins of the antibody is CRM197. Each carrier protein of the S. pneumoniae polysaccharide-protein conjugate It's CRM197.

[0037] "PCV14," as used herein, refers to a virus conjugated to a carrier protein. Fourteen pneumonia samples containing capsular polysaccharides of the identified S. pneumoniae serotypes were 14-valent pneumococci containing S. pneumoniae polysaccharide-protein conjugates A bacterial conjugate vaccine, wherein the pneumococcus (S. pneumoniae) Serotypes are 3, 7F, 8, 9N, 10A, 11A, 12F, 15A, 16F, 17F, and 19 A, 20, 22F and 33F. In a specific embodiment, Streptococcus pneumoniae (S. pneumoniae) moniae) polysaccharide-protein conjugates, wherein one or more carrier proteins are CRM19 7. In a further embodiment, the S. pneumoniae polysaccharide protein The carrier protein for each of the protein conjugates is CRM197.

[0038] "PCV15," as used herein, refers to a virus conjugated to a carrier protein. 15 pneumonia samples containing capsular polysaccharides of the identified S. pneumoniae serotypes 15-valent pneumococci containing S. pneumoniae polysaccharide-protein conjugate A bacterial conjugate vaccine, wherein the pneumococcus (S. pneumoniae) Serotypes are 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 2 2F, 23F and 33F. In a specific embodiment, the antibodies against Streptococcus pneumoniae (S. pneumoniae) niae) one or more carrier proteins of the polysaccharide-protein conjugate is CRM197 In a further embodiment, a S. pneumoniae polysaccharide protein The carrier protein in each of the conjugates is CRM197.

[0039] "PCV16," as used herein, refers to a virus conjugated to a carrier protein. Sixteen pneumonia samples containing capsular polysaccharides of the identified S. pneumoniae serotypes were 16-valent pneumococci containing S. pneumoniae polysaccharide-protein conjugates A bacterial conjugate vaccine, wherein the pneumococcus (S. pneumoniae) Serotypes are 6C, 8, 9N, 10A, 11A, 12F, 15A, 16F, 17F, 20A, 23A, 23B, 24F, 31, and 35B, as well as the following serotypes of serogroup 15: 15B , 15C, or de-O-acetylated 15B. In certain embodiments, The serotype of serogroup 15 is serotype 15C or de-O-acetylated 15B. As shown (see Example 3 below), de-O-acetylated serotype 15B pneumococcal polysaccharides is equivalent to the serotype 15C pneumococcal polysaccharide and has the same NMR spectrum. In a preferred embodiment, a S. pneumoniae polysaccharide protein conjugate In a further embodiment, one or more carrier proteins of the Streptococcus pneumoniae antibody is CRM197. Each carrier protein of the S. pneumoniae polysaccharide-protein conjugate is CRM197.

[0040] "PCV21," as used herein, refers to a virus conjugated to a carrier protein. Twenty-one pneumonia samples containing capsular polysaccharides of the identified S. pneumoniae serotypes were 21-valent pneumococci containing S. pneumoniae polysaccharide-protein conjugates A bacterial conjugate vaccine, wherein the pneumococcus (S. pneumoniae) Serotypes are 3, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 16F, and 17F. , 19A, 20A, 22F, 23A, 23B, 24F, 31, 33F and 35B and and at least one of the following serogroup 15 serotypes: 15B, 15C, or de-O-acetylated 15B. In a specific embodiment, the serotype of serogroup 15 is serotype 15C or de-O- acetylated 15B. In a specific embodiment, the antibody against Streptococcus pneumoniae (S. pneumoniae ) One or more carrier proteins of the polysaccharide-protein conjugate is CRM197. In a further embodiment, the S. pneumoniae polysaccharide protein conjugate The carrier protein for each of the gates is CRM197.

[0041] Streptococcus pneumoniae (S. pneumoniae) serotypes 20A and 20B were identified by genetic analysis. It was identified within serogroup 20 in 2012 (Calix, JJ et al., JB iol.Chem.(2012)287(33):27885-27894). Cali As discussed by JJ et al., traditional serotyping methods and commercially available antibodies These two serotypes could not be distinguished previously, and serotyping of serotypes 20A and 20B was performed. Information on the efforts to combat this disease is currently limited. The prevalence of 20A or 20B disorders within the identified disorders is not fully understood. Therefore, those skilled in the art will be able to incorporate this into S. pneumoniae vaccine compositions. The inclusion of either or both of these two serotypes (20A and / or 20B) On the other hand, Streptococcus pneumoniae (S. pneumococcal) containing serotype 20 polysaccharide antigens may be selected. niae) vaccine may be cross-protective against other serotypes ( For example, the polysaccharide protein conjugate of S. pneumoniae serotype 20A A vaccine containing serotype 20B polysaccharide-protein conjugate was developed. , cross-protection against infection with S. pneumoniae serotype 20B Therefore, as used herein, "serotype 20" includes serotypes 20A and 20B. The term "polysaccharide-protein conjugate" may refer to a composition comprising a polysaccharide-protein conjugate of serotype 20B and / or serotype 20C.

[0042] "CpG-containing nucleotides," "CpG-containing oligonucleotides," "CpG oligonucleotides" "Nucleotide" and similar terms refer to a nucleotide sequence of 6 to 50 nucleotides containing an unmethylated CpG moiety. For example, Wang et al., 2003, Vaccine See e 21:4297. CpG-containing oligonucleotides may be synthetic nucleosides. These include modified oligonucleotides using inter-chain linkages, modified bases and / or modified sugars.

[0043] As defined herein, an "adjuvant" is an agent that enhances the immunogenicity of the immunogenic composition of the invention. Immune adjuvants are substances that are weakly immunogenic when administered alone. , e.g., immunity to an antigen that induces no or weak antibody titers or cell-mediated immune responses enhance the response, increase antibody titers to an antigen, and / or achieve an immune response in an individual Adjuvants can reduce the effective dose of antigen required to produce a It is typically given to enhance the immune response and is well known to those skilled in the art.

[0044] A "patient" (also referred to herein as a "subject") is a person infected with Streptococcus pneumoniae (S. pneumoniae). In a preferred embodiment, the patient is a human. Patients can be treated prophylactically or therapeutically. Prophylactic treatment is directed against pneumococcus. sufficient to reduce the likelihood or severity of an infection or its effects, e.g., pneumococcal pneumonia Therapeutic treatment provides sufficient protective immunity against S. pneumoniae infections. Or it may be performed to reduce the severity of its clinical effects or prevent recurrence. As described herein, the multivalent immunogenic compositions of the invention are used to perform prophylactic treatments. The compositions of the present invention can be administered to the general population or to individuals at high risk for pneumococcal infection. people, such as older people or those living with or caring for older people It can be administered individually.

[0045] Those who "need treatment" include those who have had previous exposure to or have had S. pneumoniae or infected with pneumococcus (S. pneumoniae) or have previously been vaccinated against it. those who have been infected, as well as those who are prone to infection or for whom it is desirable to reduce the possibility of infection, such as Examples include immunocompromised, elderly, children, adults or healthy individuals.

[0046] A "stable" multivalent immunogenic composition is one that remains stable at least at refrigerated temperatures (e.g., 2-8°C or 4°C). Significant changes were observed at 1 month, 2 months, 3 months, 6 months, 12 months, and / or 24 months. Furthermore, a "stable" composition is one in which the temperature is maintained at temperatures including 25°C and 37°C. temperature over a period including 1 month, 3 months, 6 months, 12 months and / or 24 months. Typical acceptance criteria for stability are as follows: The above variation is about 5%, about 10%, about 15%, or about 20% or less: (a) in the composition Number-average molecular weight of pneumococcal (S. pneumoniae) polysaccharide-protein conjugates (Mn), (b) the amount of pneumococcal (S. pneumoniae) polysaccharide protein in the composition (c) the weight average molecular weight (Mw) of the conjugate; (d) the total polysaccharide concentration in the composition; Compositions measured using intrinsic protein fluorescence spectroscopy at an excitation wavelength, e.g., 280 nm emission maxima, and (e) measured using intrinsic protein fluorescence spectroscopy at specific excitation wavelengths. The term "stable" also refers to the fluorescence intensity of a particular immunogenic composition within a multivalent immunogenic composition. It may also be used to refer to pneumococcal conjugates. The term refers to a conjugate that exhibits a desired property over time at a particular temperature, such property being , about 5%, about 10%, about 15%, or about 20% or less over the times and temperatures recited Only it changes.

[0047] II. Multivalent Immunogenic Compositions The present invention provides a method for producing multiple S. pneumoniae polysaccharide-protein conjugates. a multivalent immunogenic composition comprising a conjugate of a medicament for use in a pharmaceutical composition comprising a carrier protein and a medicament for use in a pharmaceutical composition comprising ... Contains polysaccharides of Streptococcus pneumoniae serovars conjugated to a substrate Different aspects and embodiments of the multivalent immunogenic compositions of the present invention are described below. Posted.

[0048] In one embodiment (embodiment E1), the present invention provides a method for the preparation of medicaments comprising administering to a subject a medicament ..., each of which is conjugated to a carrier protein. Multiple pneumococci containing capsular polysaccharides of selected S. pneumoniae serotypes Polyvalent immunogenic compositions containing S. pneumoniae polysaccharide-protein conjugates The composition, wherein the serotype of Streptococcus pneumoniae is (1) 15A, (2) 16F, (3) (a) 23A and 23B, (b) 23A and 23F, (c) 23B and 2 One or more serogroups selected from (d) 3F, (d) 23A, (e) 23B, and (f) 23F 23 serotypes, (4) 24F, (5)31, and (6) 35B The present invention provides a composition comprising, consisting of, or consisting essentially of:

[0049] In a subembodiment of embodiment E1, the serotypes of one or more of serogroup 23 are 23A and 23B and 23B (i.e., no other serotypes of serogroup 23 are present). In one subembodiment, the one or more serotypes of serogroup 23 are 23A and 23F. In another subembodiment of Form E1, one or more of the serotypes of serogroup 23 are 23B and 23F. In yet another subembodiment of embodiment E1, 23A is the only serotype of serogroup 23. In a further subembodiment of embodiment E1, 23B is the only serotype of serogroup 23. In yet a further subembodiment of embodiment E1, 23F is the only serogroup 23 serogroup It is a Qing type.

[0050] In a second embodiment (embodiment E2), the present invention provides a method for producing a medicament for the treatment of rhesus malabsorption, comprising administering to a mammalian subject the medicament for the treatment of rhesus malabsorption, ... Multiple pneumococci, including capsular polysaccharides of selected S. pneumoniae serotypes Multivalent immunogenic compositions comprising S. pneumoniae polysaccharide-protein conjugates The product of embodiment E1, wherein the serotype of S. pneumoniae is or any subembodiment of embodiment E1, including a serotype: (1) (1) a composition further comprising 6C, (2) 6A, or (3) 6A and 6B.

[0051] In a subembodiment of embodiment E2, the composition comprises serotype 6C. In a subembodiment, the composition comprises serotypes 6A and 6B, and does not comprise serotype 6C. In another subembodiment of embodiment E2, the composition comprises serotype 6A.

[0052] In a third embodiment (embodiment E3), the present invention provides a method for producing a medicament for the treatment of rhesus malabsorption, comprising administering to a mammalian subject the medicament for the treatment of rhesus malabsorption, ... Multiple pneumococci, including capsular polysaccharides of selected S. pneumoniae serotypes Multivalent immunogenic compositions comprising S. pneumoniae polysaccharide-protein conjugates The product of embodiment E1, wherein the serotype of S. pneumoniae is or any subembodiment of embodiment E1, or embodiment E2, or embodiment E2, including a serotype as set forth in any subembodiment thereof, and including a serotype: (1)8, (2) 9N, (3)11A (4) 12F, (5) 15B or 15C, (6) 17F, and (7) 20A and / or 20B The composition further comprises:

[0053] In a fourth embodiment (embodiment E4), the present invention provides a method for producing a medicament for the treatment of rhesus malabsorption, comprising administering to said patient a therapeutically effective amount of rhesus malabsorption, ... Multiple pneumococci, including capsular polysaccharides of selected S. pneumoniae serotypes Multivalent immunogenic compositions comprising S. pneumoniae polysaccharide-protein conjugates The product of embodiment E1, wherein the serotype of S. pneumoniae is to E3 or any subembodiment thereof, and , serotype: 10A or 39.

[0054] In a fifth embodiment (Embodiment E5), the present invention provides a method for the preparation of a vaccine against a plurality of pneumococci (S. pneumoniae) strains as described above. 1. A multivalent immunogenic composition comprising a B. umoniae polysaccharide-protein conjugate, Here, the serotypes of pneumococcus (S. pneumoniae) are 8, 9N, 10A, 11A, 12F, 15C, 17F, 20A, 6C, 15A, 16F, 23A, 23B, 24F, 3 The present invention provides a composition comprising, consisting of, or consisting essentially of 1 and 35B.

[0055] In a sixth embodiment (Embodiment E6), the present invention provides a method for the preparation of a vaccine against a plurality of pneumococci (S. pneumoniae) strains as described above. 1. A multivalent immunogenic composition comprising a B. umoniae polysaccharide-protein conjugate, Here, the serotypes of pneumococcus (S. pneumoniae) are 8, 9N, 39, 11A, 1 2F, 15C, 17F, 20A, 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B.

[0056] In a seventh embodiment (Embodiment E7), the present invention provides a method for the preparation of a pneumococcus strain (S. pneumoniae) as described above. 1. A multivalent immunogenic composition comprising a B. umoniae polysaccharide-protein conjugate, Here, the serotypes of pneumococcus (S. pneumoniae) are 8, 9N, 10A, 11A, 12F, 15B, 17F, 20A, 6C, 15A, 16F, 23A, 23B, 24F, 3 The present invention provides a composition comprising, consisting of, or consisting essentially of 1 and 35B.

[0057] In an eighth embodiment (Embodiment E8), the present invention provides a method for the preparation of a pneumococcus strain (S. pneumoniae) as described above. 1. A multivalent immunogenic composition comprising a B. umoniae polysaccharide-protein conjugate, Here, the serotypes of pneumococcus (S. pneumoniae) are 8, 9N, 39, 11A, 1 2F, 15B, 17F, 20A, 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B.

[0058] In a ninth embodiment (Embodiment E9), the present invention provides a method for the preparation of a Streptococcus pneumoniae (S. pneumoniae) strain as described above. 1. A multivalent immunogenic composition comprising a B. umoniae polysaccharide-protein conjugate, wherein the S. pneumoniae serotype is any of the embodiments E1 to E8 (or any subembodiment thereof), and serotype: 3 , 7F and 19A.

[0059] In a tenth embodiment (Embodiment E10), the present invention provides a method for the preparation of a plurality of pneumococci (Sp and a multivalent immunogenic composition comprising a polysaccharide-protein conjugate of B. neumoniae. Wherein the S. pneumoniae serotype is any of the embodiments E1 to E9 (or or any subembodiment thereof), and The composition further comprises 22F.

[0060] In an eleventh embodiment (Embodiment E11), the present invention provides a method for the preparation of a plurality of pneumococci (Sp and a multivalent immunogenic composition comprising a polysaccharide-protein conjugate of B. neumoniae. Wherein the pneumococcus (S. pneumoniae) serotype is any of the embodiments E1 to E10 ( or any subembodiment thereof), and A composition is provided which further comprises Form 33F.

[0061] In a twelfth embodiment (Embodiment E12), the present invention provides a method for the preparation of a plurality of pneumococci (Sp and a multivalent immunogenic composition comprising a polysaccharide-protein conjugate of B. neumoniae. Here, the serotype of pneumococcus (S. pneumoniae) is (1) 15A, 16F, 23A, 23B, 24F, 31 and 35B, (2) 15A, 16F, 23F, 23B, 24F, 31 and 35B, (3) 15A, 16F, 23A, 23F, 24F, 31 and 35B, (4) 15A, 16F, 23A, 24F, 31 and 35B, (5) 15A, 16F, 23B, 24F, 31 and 35B, (6) 15A, 16F, 23F, 24F, 31 and 35B, (7) 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B, (8) 6C, 15A, 16F, 23F, 23B, 24F, 31 and 35B, (9) 6C, 15A, 16F, 23A, 23F, 24F, 31 and 35B, (10) 6C, 15A, 16F, 23A, 24F, 31 and 35B, (11) 6C, 15A, 16F, 23B, 24F, 31 and 35B, (12) 6C, 15A, 16F, 23F, 24F, 31 and 35B, (13) 6A, 15A, 16F, 23A, 23B, 24F, 31 and 35B, (14) 6A, 15A, 16F, 23F, 23B, 24F, 31 and 35B, (15) 6A, 15A, 16F, 23A, 23F, 24F, 31 and 35B, (16) 6A, 15A, 16F, 23A, 24F, 31 and 35B, (17) 6A, 15A, 16F, 23B, 24F, 31 and 35B, (18) 6A, 15A, 16F, 23F, 24F, 31 and 35B, (19) 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31 and 35B, (20) 6A, 6B, 15A, 16F, 23F, 23B, 24F, 31 and 35B, (21) 6A, 6B, 15A, 16F, 23A, 23F, 24F, 31 and 35B, (22) 6A, 6B, 15A, 16F, 23A, 24F, 31, and 35B, (23) 6A, 6B, 15A, 16F, 23B, 24F, 31, and 35B, and (24) 6A, 6B, 15A, 16F, 23F, 24F, 31, and 35B A set of S. pneumoniae serotypes selected from the group consisting of The present invention provides a composition comprising, consisting of, or consisting essentially of:

[0062] In a thirteenth embodiment (Embodiment E13), the present invention provides a method for the preparation of a plurality of pneumococci (Sp and a multivalent immunogenic composition comprising a polysaccharide-protein conjugate of B. neumoniae. Here, the serotype of pneumococcus (S. pneumoniae) is (1)8, 9N, 10A, 11A, 12F, 15C, 17F, 20A, 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B, (2)8, 9N, 39, 11A, 12F, 15C, 17F, 20A, 6C, 15A, 1 6F, 23A, 23B, 24F, 31 and 35B, (3)8, 9N, 10A, 11A, 12F, 15B, 17F, 20A, 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B, (4)8, 9N, 39, 11A, 12F, 15B, 17F, 20A, 6C, 15A, 1 6F, 23A, 23B, 24F, 31 and 35B, (5)8, 9N, 10A, 11A, 12F, 15C, 17F, 20A, 15A, 16F , 23A, 23B, 24F, 31 and 35B, (6)8, 9N, 10A, 11A, 12F, 15C, 17F, 20A, 6A, 6B, 1 5A, 16F, 23A, 23B, 24F, 31 and 35B, (7)8, 9N, 10A, 11A, 12F, 15C, 17F, 20A, 6A, 15A, 16F, 23A, 23B, 24F, 31 and 35B, (8)8, 9N, 39, 11A, 12F, 15C, 17F, 20A, 15A, 16F, 23A, 23B, 24F, 31 and 35B, (9)8, 9N, 39, 11A, 12F, 15C, 17F, 20A, 6A, 6B, 15 A, 16F, 23A, 23B, 24F, 31 and 35B, (10)8, 9N, 39, 11A, 12F, 15C, 17F, 20A, 6A, 15A, 16F, 23A, 23B, 24F, 31 and 35B, (11)8, 9N, 10A, 11A, 12F, 15B, 17F, 20A, 15A, 16 F, 23A, 23B, 24F, 31 and 35B, (12)8, 9N, 10A, 11A, 12F, 15B, 17F, 20A, 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31 and 35B, (13)8, 9N, 10A, 11A, 12F, 15B, 17F, 20A, 6A, 15A , 16F, 23A, 23B, 24F, 31 and 35B, (14)8, 9N, 39, 11A, 12F, 15B, 17F, 20A, 15A, 16F , 23A, 23B, 24F, 31 and 35B, (15)8, 9N, 39, 11A, 12F, 15B, 17F, 20A, 6A, 6B, 1 5A, 16F, 23A, 23B, 24F, 31 and 35B, (16)8, 9N, 39, 11A, 12F, 15B, 17F, 20A, 6A, 15A, 16F, 23A, 23B, 24F, 31 and 35B, (17)8, 9N, 10A, 11A, 12F, 15C, 17F, 20A, 6C, 15A , 16F, 23F, 24F, 31 and 35B, (18)8, 9N, 39, 11A, 12F, 15C, 17F, 20A, 6C, 15A, 16F, 23F, 24F, 31 and 35B, (19)8, 9N, 10A, 11A, 12F, 15B, 17F, 20A, 6C, 15A , 16F, 23F, 24F, 31 and 35B, (20)8, 9N, 39, 11A, 12F, 15B, 17F, 20A, 6C, 15A, 16F, 23F, 24F, 31 and 35B, (21)8, 9N, 10A, 11A, 12F, 15C, 17F, 20A, 15A, 16 F, 23F, 24F, 31 and 35B, (22)8, 9N, 10A, 11A, 12F, 15C, 17F, 20A, 6A, 6B, 15A, 16F, 23F, 24F, 31 and 35B, (23)8, 9N, 10A, 11A, 12F, 15C, 17F, 20A, 6A, 15A , 16F, 23F, 24F, 31 and 35B, (24)8, 9N, 39, 11A, 12F, 15C, 17F, 20A, 15A, 16F , 23F, 24F, 31 and 35B, (25)8, 9N, 39, 11A, 12F, 15C, 17F, 20A, 6A, 6B, 1 5A, 16F, 23F, 24F, 31 and 35B, (26)8, 9N, 39, 11A, 12F, 15C, 17F, 20A, 6A, 15A, 16F, 23F, 24F, 31 and 35B, (27)8, 9N, 10A, 11A, 12F, 15B, 17F, 20A, 15A, 16 F, 23F, 24F, 31 and 35B, (28)8, 9N, 10A, 11A, 12F, 15B, 17F, 20A, 6A, 6B, 15A, 16F, 23F, 24F, 31 and 35B, (29)8, 9N, 10A, 11A, 12F, 15B, 17F, 20A, 6A, 15A , 16F, 23F, 24F, 31 and 35B, (30)8, 9N, 39, 11A, 12F, 15B, 17F, 20A, 15A, 16F , 23F, 24F, 31 and 35B, (31)8, 9N, 39, 11A, 12F, 15B, 17F, 20A, 6A, 6B, 1 5A, 16F, 23F, 24F, 31 and 35B, and (32)8, 9N, 39, 11A, 12F, 15B, 17F, 20A, 6A, 15A, 16F, 23F, 24F, 31 and 35B A set of S. pneumoniae serotypes selected from the group consisting of The present invention provides a composition comprising, consisting of, or consisting essentially of:

[0063] A thirteenth embodiment (Embodiment E13) provides a S. pneumoniae serum The set of serotypes in rows (1) to (32) further includes serotype 20A in each set. It is replaced by either serotype 20 or serotype 20B.

[0064] In a fourteenth embodiment (embodiment E14) of the invention, the present invention provides a method for producing a plurality of pneumococcal bacteria as described above. Multivalent immunogenic compositions comprising S. pneumoniae polysaccharide-protein conjugates The product of embodiment E13, wherein the S. pneumoniae serotype is Streptococcus pneumoniae ( comprising, consisting of, or consisting essentially of a set of S. pneumoniae serovars The present invention provides a composition further comprising serotypes 23A and / or 23B.

[0065] In a fifteenth embodiment (Embodiment E15), the present invention provides a method for the preparation of a plurality of pneumococci (Sp and a multivalent immunogenic composition comprising a polysaccharide-protein conjugate of B. neumoniae. Here, the serotype of pneumococcus (S. pneumoniae) is (1)3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15 C, 17F, 20A, 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35 B. (2)3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15C , 17F, 20A, 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B , (3)3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15 B, 17F, 20A, 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35 B. (4)3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15B , 17F, 20A, 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B , (5)3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15 C, 17F, 20A, 15A, 16F, 23A, 23B, 24F, 31 and 35B, (6)3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15 C, 17F, 20A, 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31 and and 35B, (7)3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15 C, 17F, 20A, 6A, 15A, 16F, 23A, 23B, 24F, 31 and 35 B. (8)3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15C , 17F, 20A15A, 16F, 23A, 23B, 24F, 31 and 35B, (9)3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15C , 17F, 20A, 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31 and 35B, (10)3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15 C, 17F, 20A, 6A, 15A, 16F, 23A, 23B, 24F, 31 and 35 B. (11)3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 1 5B, 17F, 20A, 15A, 16F, 23A, 23B, 24F, 31 and 35B, (12)3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 1 5B, 17F, 20A, 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31 and 35B, (13)3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 1 5B, 17F, 20A, 6A, 15A, 16F, 23A, 23B, 24F, 31 and 3 5B, (14)3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15 B, 17F, 20A, 15A, 16F, 23A, 23B, 24F, 31 and 35B, (15)3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15 B, 17F, 20A, 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31 and and 35B, (16)3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15 B, 17F, 20A, 6A, 15A, 16F, 23A, 23B, 24F, 31 and 35 B. (17)3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 1 5C, 17F, 20A, 6C, 15A, 16F, 23F, 24F, 31 and 35B, (18)3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15 C, 17F, 20A, 6C, 15A, 16F, 23F, 24F, 31 and 35B, (19)3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 1 5B, 17F, 20A, 6C, 15A, 16F, 23F, 24F, 31 and 35B, (20)3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15 B, 17F, 20A, 6C, 15A, 16F, 23F, 24F, 31 and 35B, (21)3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 1 5C, 17F, 20A, 15A, 16F, 23F, 24F, 31 and 35B, (22)3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 1 5C, 17F, 20A, 6A, 6B, 15A, 16F, 23F, 24F, 31 and 35 B. (23)3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 1 5C, 17F, 20A, 6A, 15A, 16F, 23F, 24F, 31 and 35B, (24)3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15 C, 17F, 20A, 15A, 16F, 23F, 24F, 31 and 35B, (25)3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15 C, 17F, 20A, 6A, 6B, 15A, 16F, 23F, 24F, 31 and 35B , (26)3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15 C, 17F, 20A, 6A, 15A, 16F, 23F, 24F, 31 and 35B, (27)3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 1 5B, 17F, 20A, 15A, 16F, 23F, 24F, 31 and 35B, (28)3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 1 5B, 17F, 20A, 6A, 6B, 15A, 16F, 23F, 24F, 31 and 35 B. (29)3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 1 5B, 17F, 20A, 6A, 15A, 16F, 23F, 24F, 31 and 35B, (30)3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15 B, 17F, 20A, 15A, 16F, 23F, 24F, 31 and 35B, (31)3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15 B, 17F, 20A, 6A, 6B, 15A, 16F, 23F, 24F, 31 and 35B , (32)3, 7F, 19A, 22F, 33F, 8, 9N, 39, 11A, 12F, 15 B, 17F, 20A, 6A, 15A, 16F, 23F, 24F, 31 and 35B, (33)3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B , 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 2 0B, (34)3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B , 24B, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 2 0A, (35)3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B , 24B, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 2 0B, (36)3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B , 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 2 0B, (37)3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B , 24B, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 2 0A, (38)3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B , 24B, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 2 0B, (39)3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B , 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 2 0 (40)3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B , 24B, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 2 0 (41)3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B , 24B, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 2 0 (42)3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B , 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 2 0 (43)3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B , 24B, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 2 0, and (44)3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B , 24B, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 2 0 A set of S. pneumoniae serotypes selected from the group consisting of The present invention provides a composition comprising, consisting of, or consisting essentially of:

[0066] In a sixteenth embodiment (embodiment E16) of the invention, the present invention provides a method for producing a plurality of pneumococcal strains as described above. Multivalent immunogenic compositions comprising S. pneumoniae polysaccharide-protein conjugates The product of embodiment E15, wherein the S. pneumoniae serotype is Streptococcus pneumoniae ( comprising, consisting of, or consisting essentially of a set of S. pneumoniae serovars The present invention provides a composition further comprising serotypes 23A and / or 23B.

[0067] In a seventeenth embodiment (embodiment E17), the present invention provides a method for conjugating a medicament to a carrier protein. Multiple pneumonia cases, including capsular polysaccharides of selected S. pneumoniae serotypes Polyvalent immunogenicity containing S. pneumoniae polysaccharide-protein conjugates A composition, wherein the serotype of S. pneumoniae is i.6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B, ii.6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 1 0A, 11A, 12F, 15C, 17F and 20A, and iii.3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B , 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 2 0A The present invention provides a composition comprising a set of serotypes selected from the group consisting of:

[0068] In a subembodiment of embodiment E17, the immunogenic composition comprises a further Streptococcus pneumoniae (S. pn eumoniae) polysaccharide-protein conjugates.

[0069] In an eighteenth embodiment (embodiment E18), the present invention provides a method for the treatment of Streptococcus pneumoniae (S. pneumoniae) iae) pods of serotypes 6C, 15A, 16F, 23A, 23B, 24F, 31, and 35B Multiple pneumococcal (S. pneumoniae) polysaccharide-protein conjugates, including membrane polysaccharides In a subembodiment of embodiment E18, a multivalent immunogenic composition is provided, comprising a gate. The probiotic composition contains additional S. pneumoniae polysaccharide protein conjugates. Does not include duguate.

[0070] In a nineteenth embodiment (Embodiment E19), the present invention provides a method for the treatment of Streptococcus pneumoniae (S. pneumoniae) iae) serotypes 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9 Multiple lung cancers containing capsular polysaccharides N, 10A, 11A, 12F, 15C, 17F, and 20A Polyvalent immunogens containing S. pneumoniae polysaccharide-protein conjugates In a subembodiment of embodiment E19, the immunogenic composition further comprises Does not contain S. pneumoniae polysaccharide-protein conjugates.

[0071] In a twentieth embodiment (Embodiment E20), the present invention provides a method for the treatment of Streptococcus pneumoniae (S. pneumoniae) iae) serotypes 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 2 3B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and Multiple S. pneumoniae polysaccharide proteins, including capsular polysaccharides of 20A and 20B A multivalent immunogenic composition comprising the protein conjugate is provided. In some embodiments, the immunogenic composition may further comprise a pneumococcal (S. pneumoniae) polysaccharide protein. Contains no protein conjugates.

[0072] In another embodiment, the present invention provides a method for the treatment of S. pneumoniae serotypes 3 and 7F. , 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 3 Capsular polysaccharides 5B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20A Contains multiple S. pneumoniae polysaccharide-protein conjugates, including Subembodiments of this embodiment (i.e., 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35 B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20A) are immunogenic. The composition may further comprise a S. pneumoniae polysaccharide-protein conjugate. Does not include port.

[0073] In another embodiment, the present invention provides a method for the treatment of S. pneumoniae serotypes 3 and 7F. , 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 3 Contains capsular polysaccharides 5B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20 Contains multiple pneumococcal (S. pneumoniae) polysaccharide-protein conjugates Multivalent immunogenic compositions are provided. Subembodiments of this embodiment (i.e., 3, 7F, 1 9A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B , 8, 9N, 10A, 11A, 12F, 15C, 17F and 20) immunogenic composition The subject matter also includes a further S. pneumoniae polysaccharide-protein conjugate. Does not include.

[0074] In another embodiment, the present invention provides a method for the treatment of S. pneumoniae serotypes 3 and 7F. , 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 3 Capsular polysaccharides of 5B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20B Contains multiple S. pneumoniae polysaccharide-protein conjugates, including Subembodiments of this embodiment (i.e., 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35 B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20B) are immunogenic. The composition may further comprise a S. pneumoniae polysaccharide-protein conjugate. Does not include port.

[0075] As shown herein (see Example 3 below), de-O-acetylated serotype 15B lung The pneumococcal polysaccharide is equivalent to the serotype 15C pneumococcal polysaccharide and has an identical NMR spectrum. The de-O-acetylated serotype 15B pneumococcal polysaccharides described and disclosed herein is equivalent to serotype 15C pneumococcal polysaccharides, both in the range of 0-5% or 0-4%. % range, or 0-3% range, or 0-2% range, or 0-1% range, or O-acetyl content per repeat unit in the range of 0-0.5% or 0-0.1% In a report by Spencer BL et al. may be slightly O-acetylated at 15C (Spencer, BLett. al., Clin.Vac.Immuno.(2017)24(8):1-13). death Therefore, in any of the embodiments of the multivalent immunogenic compositions herein, instead of serotype 15C, Alternatively, de-O-acetylated serotype 15B can be used. The de-O-acetylation method is ,e.g., Rajam et al., Clinical and Vaccine I As described in Immunology, 2007, 14(9):1223-1227 are known in the art.

[0076] Multivalent immunogens of the invention, including embodiments E1 to E20 and any subembodiments thereof In certain embodiments of any of the compositions, the composition further comprises a pharmaceutically acceptable carrier. include.

[0077] Cross-reactivity In one embodiment, the present invention provides a S. pneumoniae polysaccharide protein. A multivalent immunogenic composition comprising conjugates, wherein each of the conjugates is Streptococcus pneumoniae (S. pneumoniae) containing serotype 6C conjugated to a carrier protein iae serotype polysaccharides, where the serotype of Streptococcus pneumoniae (S. pneumoniae) Type 6C is cross-reactive with serotypes 6A and 6B of S. pneumoniae. Compositions that provide protection are provided.

[0078] In another embodiment, the present invention relates to a method for treating Streptococcus pneumoniae (Sp) selected from embodiments E1 to E20. and a multivalent immunogenic composition comprising a polysaccharide-protein conjugate of B. neumoniae. wherein each of the conjugates is a serotype 6C conjugated to a carrier protein. The polysaccharides of S. pneumoniae serotypes include: Serotype 6C of S. pneumoniae is a type of Streptococcus pneumoniae The present invention provides compositions that provide cross-protection against serotypes 6A and 6B of H. pneumoniae.

[0079] In one embodiment, the present invention provides a S. pneumoniae polysaccharide protein. A multivalent immunogenic composition comprising conjugates, wherein each of the conjugates is a carrier. Streptococcus pneumoniae (S. pneumoniae) containing serotype 19A conjugated to body proteins iae serotype polysaccharides, where the serotype of Streptococcus pneumoniae (S. pneumoniae) Serotype 19A provides cross-protection against serotype 19F of S. pneumoniae. A composition is provided.

[0080] In another embodiment, the present invention relates to a method for treating Streptococcus pneumoniae (Sp) selected from embodiments E1 to E20. and a multivalent immunogenic composition comprising a polysaccharide-protein conjugate of B. neumoniae. wherein each of the conjugates is a serotype 19 conjugated to a carrier protein. A polysaccharide of S. pneumoniae serotype A, Serotype 19A of S. pneumoniae is a type of Streptococcus pneumoniae e) provides a composition that provides cross-protection against serotype 19F.

[0081] In one embodiment, the present invention provides a S. pneumoniae polysaccharide protein. A multivalent immunogenic composition comprising conjugates, wherein each of the conjugates is a carrier. Streptococcus pneumoniae containing serotypes 23A and / or 23B conjugated to body proteins (S. pneumoniae) serotype polysaccharides, where pneumococcus (S. pneu moniae serotypes 23A and / or 23B are associated with Streptococcus pneumoniae (S. pneumoniae The present invention provides compositions that provide cross-protection against serotype 23F of S. iae.

[0082] In another embodiment, the present invention relates to a method for treating Streptococcus pneumoniae (Sp) selected from embodiments E1 to E20. and a multivalent immunogenic composition comprising a polysaccharide-protein conjugate of B. neumoniae. wherein each of the conjugates is a serotype 23 conjugated to a carrier protein. Polysaccharides of S. pneumoniae serotypes including A and / or 23B wherein the serotypes 23A and / or 23B of Streptococcus pneumoniae are 3B provides cross-protection against S. pneumoniae serotype 23F The present invention provides a composition for

[0083] In one embodiment, the present invention provides a S. pneumoniae polysaccharide protein. A multivalent immunogenic composition comprising conjugates, wherein each of the conjugates is a carrier. Streptococcus pneumoniae (S. pneumoniae) containing serotype 6A conjugated to body proteins ae) serotype polysaccharides, where serotypes of Streptococcus pneumoniae (S. pneumoniae) 6A is directed against serotypes 6B and / or 6C of Streptococcus pneumoniae. The present invention provides compositions that provide cross-protection against

[0084] In another embodiment, the present invention relates to a method for treating Streptococcus pneumoniae (Sp) selected from embodiments E1 to E20. and a multivalent immunogenic composition comprising a polysaccharide-protein conjugate of B. neumoniae. wherein each of the conjugates is a serotype 6A conjugated to a carrier protein. The polysaccharides of S. pneumoniae serotypes include: Serotype 6A of S. pneumoniae is a type of Streptococcus pneumoniae The present invention provides compositions that provide cross-protection against serotypes 6B and / or 6C of H. pneumoniae.

[0085] In one embodiment, the present invention provides a S. pneumoniae polysaccharide protein. A multivalent immunogenic composition comprising conjugates, wherein each of the conjugates is a carrier. Streptococcus pneumoniae (S. pneumoniae) containing serotype 20A conjugated to body proteins iae serotype polysaccharides, where the serotype of Streptococcus pneumoniae (S. pneumoniae) Serotype 20A provides cross-protection against serotype 20B of S. pneumoniae. A composition is provided.

[0086] In another embodiment, the present invention relates to a method for treating Streptococcus pneumoniae (Sp) selected from embodiments E1 to E20. and a multivalent immunogenic composition comprising a polysaccharide-protein conjugate of B. neumoniae. wherein each of the conjugates is a serotype 20 conjugated to a carrier protein. A polysaccharide of S. pneumoniae serotype A, Serotype 20A of S. pneumoniae is a type of Streptococcus pneumoniae e) provides a composition that provides cross-protection against serotype 20B.

[0087] In one embodiment, the present invention provides a S. pneumoniae polysaccharide protein. A multivalent immunogenic composition comprising conjugates, wherein each of the conjugates is a carrier. Streptococcus pneumoniae (S. pneumoniae) containing serotype 20B conjugated to body proteins iae serotype polysaccharides, where the serotype of Streptococcus pneumoniae (S. pneumoniae) Serotype 20B provides cross-protection against serotype 20A of S. pneumoniae. A composition is provided.

[0088] In another embodiment, the present invention relates to a method for treating Streptococcus pneumoniae (Sp) selected from embodiments E1 to E20. and a multivalent immunogenic composition comprising a polysaccharide-protein conjugate of B. neumoniae. wherein each of the conjugates is a serotype 20 conjugated to a carrier protein. Polysaccharides of S. pneumoniae serotypes, including B, wherein pneumococcus Serotype 20B of S. pneumoniae is a type of Streptococcus pneumoniae e) provides a composition that provides cross-protection against serotype 20A.

[0089] In one embodiment, the present invention provides a S. pneumoniae polysaccharide protein. A multivalent immunogenic composition comprising conjugates, wherein each of the conjugates is a carrier. Streptococcus pneumoniae (S. pneumoniae) containing serotype 15C conjugated to body proteins iae serotype polysaccharides, where the serotype of Streptococcus pneumoniae (S. pneumoniae) Serotype 15C provides cross-protection against serotype 15B of S. pneumoniae. A composition is provided.

[0090] In another embodiment, the present invention relates to a method for treating Streptococcus pneumoniae (Sp) selected from embodiments E1 to E20. and a multivalent immunogenic composition comprising a polysaccharide-protein conjugate of B. neumoniae. wherein each of the conjugates comprises serotype 15 conjugated to a carrier protein. C, wherein the polysaccharides of S. pneumoniae serotypes Serotype 15C of S. pneumoniae is a type of Streptococcus pneumoniae e) provides a composition that provides cross-protection against serotype 15B.

[0091] Carrier proteins In a particular embodiment of the invention, CRM197 is used as the carrier protein. M197 has the following amino acid sequence: GADDVVDSSK SFVMENFSSY HGTKPGYVDS IQKGIQ KPKS GTQGNYDDDW KEFYSTDNKY DAAGYSVDNE NP LSGKAGGV VKVTYPGLTK VLALKVDNAE TIKKELGLS L TEPLMEQVGT EEFIKRFGDG ASRVVLSLPF AEGSS SVEYI NNWEQAKALS VELEINFETR GKRGQDAMYE Y MAQACAGNR VRRSVGSSLS CINLDWDVIR DKTKTKIE SL KEHGPIKNKM SESPNKTVSE EKAKQYLEEF HQTA LEHPEL SELKTVTGTN PVFAGANYAA WAVNVAQVID SETADNLEKT TAALSILPGI GSVMGIADGA VHHNTEE IVA QSIALSSLMV AQAIPLVGEL VDIGFAAYNF VES IINLFQV VHNSYNRPAY SPGHKTQPFL HDGYAVSWNT VEDSIIRTGF QGESGHDIKI TAENTPLPIA GVLLPT IPGK LDVNKSKTHI SVNGRKIRMR CRAIDGDVTF CR PKSPVYVG NGVHANLHVA FHRSSSEKIH SNEISSDSI G VLGYQKTVDH TKVNSKLSLF FEIKS (SEQ ID NO: 1) It is a non-toxic variant (i.e., toxoid) of diphtheria toxin having the formula:

[0092] In one embodiment, CRM197 is a recombinant human leukocyte grown in a casamino acid and yeast extract-based medium. Corynebacterium diphtheria strain C7(β19 7). In another embodiment, CRM197 is isolated from the culture of U.S. Pat. No. 5,616,616. Typically, the recombinant protein is prepared recombinantly according to the methods described in CR4,382. M197 is a combination of ultrafiltration, ammonium sulfate precipitation, and ion exchange chromatography. In some embodiments, CRM197 is purified through a combination of Pfenex Expression Technology™ (Pfenex Inc., Pseudomonas fluorescens (Pseudomonas fluo) was grown using a 200-well platelet-free platelet culture system (San Diego, California). It is prepared in the genus cepa (Cornus rescens).

[0093] Other suitable carrier proteins include DT (diphtheria toxoid) or fragments of DT. Tetanus toxoid B (DTFB), TT (tetanus toxoid) or TT fragment C, pertussis toxoid toxoid, cholera toxoid (e.g., as described in WO 2004 / 083251), ), Escherichia coli (E. coli) LT, Escherichia coli (E. coli) ST, and Pseudomonas aeruginosa (Ps Additional inactivating bacterial toxins, such as exotoxin A of Eudomonas aeruginosa, Bacterial outer membrane proteins, such as outer membrane complex c (OMPC), porins, trans Feline-binding protein, pneumococcal surface protein A (PspA; WO 02 / 09 1998), pneumococcal adhesin protein (PsaA), group A or group B linkage C5a peptidase of cocci, or Haemophilus influenzae luenzae protein D, pneumococcal pneumolysin (Kuo et al., 1999 95, Infect Immun 63;2706-13) (in some ways detoxified pl y, such as dPLY-GMBS (see WO 04 / 081515) or dPLY -including formol), PhtX (PhtA, PhtB, PhtD, PhtE and Ph t protein fusions, such as PhtDE fusions, PhtBE fusions (WO 01 / 024666), 98334 and WO 03 / 54007) can also be used. Ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin BSA or purified protein derivative of tuberculin (PPD), PorB (meningitis N. meningitidis), PD (Haemophilus influenzae Illus influenzae protein D; e.g., EP 0594 610 see specification) or immunologically functional equivalents thereof, synthetic peptides (European Patent No. 0378 881 and EP 0 427 347), heat shock proteins (See WO 93 / 17712 and WO 94 / 03208), pertussis Proteins (see WO 98 / 58668 and EP 0471177) ), cytokines, lymphokines, growth factors or hormones (WO 91 / 0114 6), artificial tags containing multiple human CD4+ T cell epitopes of various pathogen-derived antigens. Protein (Falugi et al., 2001, Eur J Immunol 31 :3816-3824), e.g., N19 protein (Baraldoi et al. ., 2004, Infect Immun 72:4884-7), iron uptake protein proteins (see WO 01 / 72337), Clostridium difficile (C. difficile toxin A or B (see WO 00 / 61761), and Flagellin (Ben-Yedidia et al., 1998, Immunol L Other proteins, such as ribosomal proteins (see RIBOFLAVORS 64:9), can also be used as carrier proteins. Cut.

[0094] CRM 176 , CRM 228 , CRM 45 (Uchida et al., 1973, J Biol Chem 218:3838-3844);CRM9, CRM 45 , C.R. M 102 , CRM 103 and CRM 107 and Nicholls and You le in Genetically Engineered Toxins ,Ed:F Others described by Rankel, Maecel Dekker Inc., 1992 Mutation; Glu-148 to Asp, Gln or Ser and / or Ala 15 8 to Gly deletion or mutation and the or other mutations disclosed in U.S. Pat. No. 4,950,740; at least one or more Residues Lys 516, Lys 526, Phe 530 and / or Lys 53 on 4 mutation and the method described in U.S. Pat. No. 5,917,017 or U.S. Pat. No. 6,455,455 5,673; or other mutations disclosed in U.S. Pat. No. 5,843,711. Other DT mutants, such as the fragments disclosed herein, can be used as carrier proteins. Such DT mutants can be used to generate B fragments containing the epitope region. It is also possible to generate DTFB mutants containing the nucleotides.

[0095] In certain embodiments, the carrier protein is an outer membrane protein complex (OMPC), tetanus toxoid, diphtheria toxoid, protein D and CRM197 It is selected.

[0096] In some embodiments of the invention, the second carrier is a polysaccharide protein in a multivalent immunogenic composition. A second carrier protein may be used in one or more of the protein conjugates. or non-toxic, non-reactogenic, in sufficient quantities and The second carrier protein also enhances the immunogenicity of the antigen. Conjugated or linked to pneumococcal (S. pneumoniae) polysaccharides to The carrier protein should be amenable to standard conjugation procedures. In an embodiment, each capsular polysaccharide that is not conjugated to a first carrier protein is conjugated to a second carrier protein (e.g., each capsular polysaccharide molecule is a single In another embodiment, the first carrier protein The capsular polysaccharides that are not conjugated to a protein are conjugated to two or more carrier proteins. Each capsular polysaccharide molecule is conjugated to a single carrier protein. In such embodiments, each capsular polysaccharide of the same serotype is typically carried by the same carrier protein. It is conjugated to protein.

[0097] The embodiments of the present invention include any of embodiments E1 to E20 and any subembodiments thereof. In embodiments, one or more of the polysaccharide serotypes (where applicable, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) Conjugated to CRM197. In further embodiments of the invention, including any of the subembodiments, each of the polysaccharide serotypes is C Conjugated to RM197.

[0098] Formulation of the polysaccharide-protein conjugates of the present invention is art-recognized. For example, individual pneumococcal conjugates can be prepared using methods such as It can be formulated using a physiologically acceptable vehicle to prepare the product. Examples of such vehicles include, but are not limited to, water, buffered saline, polio alcohols (e.g., glycerol, propylene glycol, liquid polyethylene glycol) and and dextrose solution.

[0099] In a preferred embodiment, the vaccine composition is an L-histidine buffer containing sodium chloride. It is formulated in a formulation.

[0100] In some embodiments of the invention, the multivalent immunogenic composition comprises a medicament conjugated to a carrier protein. Multiple lung tumors containing capsular polysaccharides of gated S. pneumoniae serotypes S. pneumoniae polysaccharide protein conjugate and adjuvant wherein the S. pneumoniae serotype is as described herein. Suitable adjuvants for enhancing the effectiveness of the composition include, but are not limited to: These include, but are not limited to: (1) Aluminum salts (alum), such as aluminum hydroxide and aluminum phosphate aluminum, aluminum sulfate, etc.; (2) Oil-in-water emulsion formulations (other specific immunostimulants, e.g., muramyl peptides) (as defined below) or bacterial cell wall components), such as (a) Mo del 110Y Microfluidizer (Microfluidics, Newton , MA) into submicron particles. % squalene, 0.5% Tween 80, and 0.5% Span 85 (various amounts MF59 (International Patent Application Publication No. WO 90 / 09999) containing MTP-PE / 14837), (b) microfluidized or vortexed into submicron emulsions Mixed to produce a larger particle size emulsion, 10% squalene, 0.4% Tw een 80, containing 5% Pluronic block polymer L121 and thr-MDP SAF, (c) 2% squalene, 0.2% Tween 80, and U.S. Pat. 3-O-deacylated monophospholipid A (MPL™) as described in US Pat. No. 12,094 ), trehalose dimycolate (TDM) and cell wall skeleton (CWS), preferably MP L+CWS (Ribi ( (Trademark) Adjuvant System (RAS) (Corixa, Hamilton, MT); and (d) Montanide ISA; (3) Saponin adjuvants, such as Quil A or STIMULON™ Q S-21 (Antigenics, Framingham, MA) (U.S. Patent No. 5,055, 7,540) may be used, or particles made therefrom, e.g., I SCOM (combination of cholesterol, saponin, phospholipids and amphiphilic proteins) Immunostimulating complex formed by Iscomatrix® (ISC has essentially the same structure as OM but does not contain proteins); (4) bacterial lipopolysaccharide, a synthetic lipid A analogue, available, for example, from Corixa; Aminoalkylglucosamine phosphatase as described in U.S. Pat. No. 6,113,918 phosphate compounds (AGP) or derivatives or analogs thereof; one such AGP 529 (formerly known as RC529), formulated in aqueous form or as a stable emulsion. 2-[(R)-3-tetradecanoyloxyte (formerly known as 2-deoxy-4-O-phosphono-3-O-[(R)-3 -tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoyl] oxytetradecanoylamino]-bD-glucopyranoside, (5) Synthetic polynucleotides, e.g., oligonucleotides containing one or more CpG motifs Otide (U.S. Pat. No. 6,207,646); and (6) Cytokines, such as interleukins (e.g., IL-1, IL-2, IL- 4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc.), Inter -ferons (e.g., gamma interferon), granulocyte-macrophage colony-stimulating factor ( GM-CSF), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TN F), costimulatory molecules such as B7-1 and B7-2; and (7) Complement, e.g., the trimer of complement component C3d.

[0101] In another embodiment, the adjuvant is a mixture of two, three or more of the above adjuvants. compounds, such as SBAS2 (also containing 3-deacylated monophosphoryl lipid A and QS21) oil-in-water emulsion).

[0102] Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-succinate. Leonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L- Alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryl) methylparaben-2-yl methylparaben (MTP-PE) and the like.

[0103] In certain embodiments, the adjuvant is an aluminum salt. The vaccine may be an alum-precipitated or alum-adsorbed vaccine. Mu salt adjuvants are well known in the art and are described, for example, in Harlow, E. and D. Lane(1988;Antibodies:A Laboratory Manual Cold Spring Harbor Laboratory) and Nickla s,W.(1992;Aluminum salts.Research in Imm (Unology 143:489-493). Aluminum salts have limited Examples include, but are not limited to, hydrated alumina, alumina hydrate, and alumina trihydrate (ATH). , aluminum hydrate, aluminum trihydrate, alhydrogel, Superfo s, Amphogel, Aluminum(III) Hydroxyphosphate, Aluminum Sulfate aluminum hydroxyphosphate sulfate, phosphorus Acid aluminum adjuvant (APA), amorphous alumina, alumina trihydrate, or trihydrate Contains hydroxyaluminum.

[0104] APA is an aqueous suspension of aluminum hydroxyphosphate. APA is also known as aluminum chloride. Aluminum hydroxyphosphate is prepared by blending aluminum and sodium phosphate in a 1:1 volume ratio. After the mixing process, the material is sized in a high shear mixer. The product is then diafiltered against saline to achieve a monodisperse particle size distribution. , steam sterilize.

[0105] In certain embodiments, commercially available Al(OH)3 (e.g., Denmark / Accura te Chemical and Scientific Co., Westbury; 50-200μg of TA using NY Alhydrogel or Superfos In another embodiment, the protein is adsorbed at a ratio of mg of protein / mg of aluminum hydroxide. Protein adsorption depends on the pI (isoelectric pH) of the protein and the pH of the medium. Proteins with low pIs are more strongly attracted to positively charged aluminum ions than proteins with high pIs. The aluminum salts act as depots for antigens that are slowly released over a period of 2-3 weeks. establish a cellular network that is involved in the nonspecific activation of macrophages and complement activation, and / or can stimulate the innate immune system (probably through stimulation by uric acid). For example, Lambrech See t et al., 2009, Curr Opin Immunol 21:23 I want to be done that.

[0106] Monovalent bulk aqueous conjugates are typically blended together to a target concentration of 8 μg / mL. Dilute to a target of 4 μg / mL for all serotypes except for dilution 6B. Once complete, filter sterilize the batch and add an equal volume of aluminum phosphate adjuvant, 250 μg Add aluminum aseptically to achieve a final aluminum concentration of 1 / mL. The drug will be filled into single-use 0.5 mL / dose vials.

[0107] In certain embodiments, the adjuvant comprises a CpG-containing nucleotide sequence, e.g., CpG containing oligonucleotides, particularly CpG-containing oligodeoxynucleotides (CpG ODs) In another embodiment, the adjuvant is ODN 1826, which is Col It is available from ey Pharmaceutical Group.

[0108] Methods for using CpG oligonucleotides are well known in the art, e.g., Sur et al.,1999,J Immunol.162:6284-93;Verth elyi,2006,Methods Mol Med.127:139-58;and Yasuda et al.,2006,Crit Rev Ther Drug C Arrier Syst.23:89-110.

[0109] In alternative embodiments, the immunogenic compositions are those described herein, for example, in embodiments E1-E20 or E21. any of the subembodiments thereof. oniae) polysaccharide-protein conjugate and no adjuvant .

[0110] formulation The compositions of the present invention may be presented in single dose vials, multi-dose vials, or pre-filled glasses. It may be formulated as a syringe or plastic syringe.

[0111] In another embodiment, the compositions of the present invention are administered orally and therefore may be in a form suitable for oral administration. Solid oral formulations include tablets, These include capsules, pills, granules, pellets, etc. Liquid oral preparations include liquids, suspensions, etc. , dispersants, emulsions, oils, etc.

[0112] Pharmaceutically acceptable carriers for liquid formulations include aqueous or non-aqueous solutions, suspensions, emulsions, and the like. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Includes water, alcohol / aqueous solutions, emulsions or suspensions, saline and buffered media Examples of oils include those of animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil. , olive oil, sunflower oil, fish liver oil, another marine oil, or milk or egg lipids.

[0113] Pharmaceutical compositions may be isotonic, hypotonic or hypertonic. It is generally preferred that pharmaceutical compositions be essentially isotonic when administered. For this reason, the pharmaceutical composition may preferably be isotonic or hypertonic. If the solution is hypertonic, it can be diluted to an isotonic solution before administration.

[0114] The isotonic agent may be an ionic isotonic agent, such as a salt, or a non-ionic isotonic agent, such as a carbohydrate. Examples of ionic isotonic agents include, but are not limited to, NaCl, HCl, KCl, Examples of non-ionic isotonic agents include, but are not limited to, HCl and MgCl. includes mannitol, sorbitol and glycerol.

[0115] It is also preferred that the at least one pharmaceutically acceptable excipient is a buffering agent. For example, if the pharmaceutical composition is intended for infusion or injection, the composition may be A buffer capable of buffering a solution to a pH in the range of 4 to 10, for example 5 to 9, for example 6 to 8. It is usually desirable to include

[0116] Buffers include, for example, TRIS, acetate, glutamate, lactate, maleate, tartar, etc. Acid salts, phosphates, citrates, carbonates, glycinates, histidine, glycine, succinic acid The buffer may be selected from the group consisting of salts and triethanolamine buffers.

[0117] Buffering agents may be used in combination with U.S. Pat. No. 4,999,499 for parenteral use, especially when the pharmaceutical preparation is for parenteral use. The buffer may be selected from SP-compatible buffers. For example, the buffer may be acetic acid, benzoic acid, gluconic acid, Monobasic acids such as glyceric and lactic acid; aconitic acid, adipic acid, ascorbic acid , carbonic acid, dibasic acids such as glutamic acid, malic acid, succinic acid and tartaric acid, citric acid and and polybasic acids such as phosphoric acid; ammonia, diethanolamine, glycine, triethanolamine The base may be selected from the group consisting of bases such as dimethylamine and TRIS.

[0118] Parenteral vehicles (for subcutaneous, intravenous, intraarterial, or intramuscular injection) include sodium chloride solution. solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, Examples include surfactants and and other pharmaceutically acceptable adjuvants. There are various sterile liquids available. Commonly, these include water, saline, aqueous dextrose, and related sugar solutions. glycols such as propylene glycol or polyethylene glycol, polysols Polysorbate 80 (PS-80), Polysorbate 20 (PS-20), and Poloxamer 1 88 (P188) is a preferred liquid carrier, particularly for injectable solutions. Examples of oils include: of animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, olive oil, sunflower oil , fish liver oil, another marine oil, or milk or egg lipids.

[0119] The formulations of the present invention may also include a surfactant. Preferred surfactants include, but are not limited to: Polyoxyethylene sorbitan ester surfactants (commonly known as Tween s), especially PS-20 and PS-80; sold under the trade name DOWFAX™ Commercially available ethylene oxide (EO), propylene oxide (PO) and / or Copolymers of ethylene oxide (BO), such as linear EO / PO block copolymers; repeating Octoxynol (octoxy) with variable number of ethoxy (oxy-1,2-ethanediyl) groups Toxinol-9 (Triton X-100, or t-octylphenoxypolyether (Octylphenoxy)polyethoxyethanol is particularly important); (Octylphenoxy)polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids, e.g., phosphatidylcholine (lecithin); nonylphenol ethoxylates, such as Tergitol™ NP Series: Poly(ethylene glycol) derived from lauryl, cetyl, stearyl, and oleyl alcohols Oxyethylene fatty ethers (known as Brij surfactants), e.g., triethylene ethylene glycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as SPAN), e.g., sorbitan trioleate (Span) n 85) and sorbitan monolaurate. Preferred for inclusion in emulsions are A suitable surfactant is PS-80.

[0120] Mixtures of surfactants can be used, for example PS-80 / Span 85 mixtures. Polyoxyethylene such as polyoxyethylene sorbitan monooleate (PS-80) Triethylene sorbitan ester and t-octylphenoxypolyethoxyethanol (Trit Combinations of octoxynol compounds such as octoxynol (on X-100) are also suitable. The combination is laureth 9 with polyoxyethylene sorbitan ester and / or octanoic acid. Contains xynol.

[0121] The preferred amount (wt%) of surfactant is polyoxyethylene sorbitan ester (PS -80, etc.) 0.01 to 1%, especially about 0.1%; octyl- or nonylphenoxypoly Oxyethanol (Triton X-100, or other interfaces in the Triton series) Active ingredients) 0.001-0.1%, especially 0.005-0.02%; Polyoxyethylene ether (such as Laureth 9) 0.1 to 20%, preferably 0.1 to 10%, particularly 0.1 to 1% or about 0.5%.

[0122] In certain embodiments, the composition contains 250 μg / mL of APA (aluminum azide phosphate). Histidine (20 mM), saline (150 mM), pH 5.8 and 0.2% PS-20 or 0.04% PS-80. can range from 0.005% to 0.3% (w / v) to prevent aggregation during simulated manufacturing. For controlled formulations and shipments using primary packaging, PS-20 or PS-80 is present. In another embodiment, PS-20 is in the range of 0.025% to 0.8% (w / v). In another embodiment, PS-20 may be in the range of 0.05% to 0.8% (w / v). In another embodiment, PS-20 may be in the range of 0.05% to 0.2% (w / v). The method involves up to 24 h in histidine, saline, and PS-20 or PS-80. A blend of serotypes is combined and the blended material is then incubated with an antimicrobial preservative. It consists of combining with or without APA and saline.

[0123] In certain embodiments, the multivalent immunogenic composition is directed against S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates is linked to a carrier protein. Contains polysaccharides of Streptococcus pneumoniae serovars conjugated to a protein , where S. pneumoniae in a polysaccharide-protein conjugate The serotypes include any of the sets of serotypes set forth herein, and 20 to 80 min Further containing 100 mM histidine pH 5.8 and 150 mM NaCl. In some embodiments, the multivalent immunogenic composition further comprises 0.2% to 0.8% w / v polysorbate 20. Included.

[0124] The choice of surfactant may need to be optimized for different formulations and drug substances. For multivalent vaccines with the above serotypes, PS-20 and P188 are preferred. The choice of chemistry used to make the conjugate also plays an important role in the stabilization of the formulation. In particular, it can be used to prepare different polysaccharide-protein conjugates into multivalent compositions. If the conjugation reaction involves both aqueous and DMSO solvents, The surfactant system makes a significant difference in stability. Improved stability of polysaccharide-protein conjugates The effect was observed with polysorbate 20 alone or with poloxamer 188 in combination with polyols. It was.

[0125] The exact mechanism of how specific surfactants protect biopharmaceuticals is not well understood. Possible stabilization mechanisms include priority selective hydration, preferential exclusion, air / liquid interfacial competition between biopharmaceuticals and surfaces, surface tension, and Surfactants and biopharmaceuticals mask hydrophobic patches that act as aggregation seeds and / or This includes in-person meetings.

[0126] Poloxamers may also be used in the compositions of the present invention. Poloxamers are polyoxyethylene Two hydrophilic chains of poly(ethylene oxide) are adjacent to each other. Nonionic triblock copolymers composed of central hydrophobic chains of poly(propylene oxide) Poloxamers are also known by the trade name Pluronic®. The length of the polymer blocks can be customized, resulting in slightly different There are many different poloxamers with different properties. These copolymers are generally designated by the letter "P" (for poloxamer) followed by a three-digit number. The first two digits x 100 represent the approximate molecular mass of the polyoxypropylene core. The last digit x 10 indicates the polyoxyethylene content (e.g., P407 = 4,000 of polyoxypropylene molecular mass of 0 g / mol and 70% polyoxyethylene content Poloxamers). The Pluronic® brand name refers to these copolymers. The coding is a letter that defines the physical form at room temperature (L=liquid, P=paste, F=flake). The first digit of the numerical display (a three-digit number) is followed by two or three numbers. The molecular weight of the hydrophobic substance is calculated by multiplying the molecular weight (two digits) by 300; the last digit x 10 indicates the polyoxyethylene content (for example, L61 = 1,800 g / mol of polyoxyethylene) Pluronic (registered trademark) with a polypropylene molecular mass and 10% polyoxyethylene content (Registered trademark). See U.S. Patent No. 3,740,421.

[0127] Examples of poloxamers have the general formula: HO(C2H4O) a (C3H6O) b (C2H4O) a H (wherein a and b blocks The block has the following values: Having: [Table 1]

[0128] Preferably, the poloxamer generally has a molecular weight of 1100 to 17,400 Da, a molecular weight of 7,500 to 1 5,000 Da, or in the range of 7,500 to 10,000 Da. The poloxamer can be selected from poloxamer 188 or poloxamer 407. The final concentration of poloxamer in the formulation is 0.001% to 5% weight / volume, or 0.025% In certain embodiments, the polyol is propylene glycol; The final concentration is 1% to 20% weight / volume. In certain embodiments, the polyol is polyethylene. Glycol 400, at a final concentration of 1% to 20% weight / volume.

[0129] Suitable polyols for the formulation of the present invention include, but are not limited to, polymer polyols, particularly No, but propylene glycol and polyethylene glycol, polyethylene glycol It is a polyether diol containing monomethyl ether. Propylene glycol is a monomethyl ether. Polyethylene glycols with molecular weights ranging from about 425 to about 2700 are available. Polyethylene glycol monomethyl ether also has a molecular weight ranging from about 200 to about 35,000. Available in a range of molecular weights, including but not limited to PEG200, PEG300, 00, PEG400, PEG1000, PEG MME 550, PEG MME 60 0, PEG MME 2000, PEG MME 3350 and PEG MME 40 00. A preferred polyethylene glycol is polyethylene glycol 400. The final concentration of polyol in the formulation of the present invention is 1% to 20% weight / volume or 6% to 20% weight / volume. It may be % weight / volume.

[0130] The formulation also includes a pH buffered saline solution. Buffering agents include, for example, TRIS, acetate, glutathione, and glutathione. Salt, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycerin Phosphate, Histidine, Glycine, Succinate, HEPES (4-(2-hydroxyethyl) )-1-piperazineethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid) sulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid) and triethanolamine The buffer may be selected from the group consisting of amine buffers. Alternatively, the pH may be buffered to a range of 5.8 to 7.0. Buffers include phosphate, succinate, histidine, MES, MOPS, HEPES, and acetate. or citrate. Buffering agents may also be used, for example, in particular pharmaceutical formulations. If the formulation is for parenteral use, select from USP compatible buffers for parenteral use. The concentration of the buffering agent may be in the range of 1 mM to 100 mM. The buffer concentration ranges from 1mM to 50mM or 5mM to 50mM. In certain embodiments, the buffering agent is histidine at a final concentration of 5 mM to 50 mM, or In certain embodiments, the final concentration of histidine is 20 mM to 10 mM succinate. The final concentration is 2 mM.

[0131] A saline solution (i.e., a solution containing NaCl) is preferred, but other salts suitable for the formulation may be used. These include, but are not limited to, CaCl2, KCl, and MgCl2, and their salts. Combinations of sucrose, trehalose, mannose, and maltodextrin are included, but are not limited to. Non-ionic isotonic agents, including ethanol, sorbitol, and glycerol, can be used in place of salt. Suitable salts may be used in a range of, but not limited to, 25 mM to 500 mM. In one embodiment, the physiological saline is NaCl, and the physiological saline is 20 mM to 170 mM. It may be present at a concentration of from 1 mM to 170 mM.

[0132] In a preferred embodiment, the formulation comprises an L-histidine buffer containing sodium chloride.

[0133] In another embodiment, the pharmaceutical composition is delivered in a controlled release system. It can be administered using intravenous infusion, transdermal patch, liposomes, or other modes of administration. In another embodiment, the polymeric material is used, for example, in microspheres or implants. It is used.

[0134] The amount of conjugate in each vaccine dose induced an immune protective response without significant adverse effects. Such an amount may vary depending on the serotype of the pneumococcus. In the case of polysaccharide-based conjugates, each dose contains 0.08 to 100 μg of each polysaccharide. In some embodiments of the invention, the dose of each polysaccharide conjugate is between 0.08 and 10 μg. In further embodiments, the dose is 1-5 μg, 0.4-4 μg, 0.4-3 μg. g, 0.4 to 2 μg, or 0.4 to 1 μg. In some embodiments, one or more The doses of saccharide conjugates were 100, 150, 200, 250, 300, 400, 50 0 or 750ng or 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0. 9, 1, 1.5, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 25, 30, 40, 50, 60, 70, 80, 9 It is either 0 or 100 μg.

[0135] In some embodiments of the compositions of the present invention, all of the polysaccharide conjugates are present in the composition. In a further embodiment, the polysaccharide conjugates are present in different amounts in the composition. amount (i.e., at least one polysaccharide conjugate is present in a proportion not greater than the other polysaccharides of the composition) (These conjugates are present in different amounts than one or more of the conjugates.)

[0136] The optimal amount of a particular vaccine component can be determined through standard clinical trials involving observation of an appropriate immune response in a subject. For example, in another embodiment, the method may be used for human vaccination. Dosages for the treatment of rheumatoid arthritis are determined by extrapolation from animal studies to human data. The dosage is determined empirically.

[0137] In one embodiment, the dose of aluminum salt is 10, 15, 20, 25, 30, 50, 7 0, 100, 125, 150, 200, 300, 500 or 700 μg, or 1, In yet another embodiment, the dose is 1.2, 1.5, 2, 3, 5 mg or more. of alum salt per μg of recombinant protein.

[0138] The compositions of the present invention also contain one or more proteins of S. pneumoniae. Examples of S. pneumoniae proteins suitable for inclusion include: The international patent application publication numbers are WO 02 / 083855 and WO 02 Examples include those identified in issue 053761.

[0139] In certain embodiments, the compositions of the present invention are administered to a subject by one or more methods known to those of skill in the art. by, for example, parenteral, transmucosal, transdermal, intramuscular, intravenous, intradermal, intranasal, subcutaneous, intraperitoneal In one embodiment, the compositions of the present invention are administered intravenously and formulated accordingly. via epidermal, intramuscular, intravenous, intraarterial, subcutaneous, or intramucosal injection of the substance Liquid preparations for injection include solutions.

[0140] III. Preparation method The capsular polysaccharides of Streptococcus pneumoniae are Polysaccharides can be prepared by standard techniques known to those skilled in the art. and can be isolated by known methods (e.g., EP 497524 and EP 497525), preferably using a homogenizer. achieved by microfluidisation or chemical hydrolysis In one embodiment, each pneumococcal polysaccharide serotype The strains are grown in soy-based media and then subjected to standard filtration techniques including centrifugation, sedimentation, and ultrafiltration. Through this step, individual polysaccharides are purified. See U.S. Pat. Nos. 286,838 and 5,847,112. To reduce the viscosity of the sugar sample and / or to reduce the viscosity of the sugar sample, such as by mechanical or chemical sizing, To improve the filterability of conjugated products using such techniques, polysaccharides are used. Chemical hydrolysis can be carried out using acetic acid. Ising can be carried out using high pressure homogenizing shear.

[0141] The purified polysaccharides are chemically activated to make the saccharides reactive with the carrier protein. The purified polysaccharide can be linked to a linker. Once attached to the linker, each capsular polysaccharide can be individually conjugated to a carrier protein. The polysaccharide conjugates are formed by coupling with the saccharides of known coupling agents. They may be prepared by gelling techniques.

[0142] The polysaccharide is coupled to a linker, and the free end of the linker is an ester group. Thus, the linker can be formed by forming a linker-linker intermediate. The other end reacts with the polysaccharide to form a polysaccharide-linker. The copolymer is selected so as to be capable of forming an anchor intermediate.

[0143] The polysaccharide can be coupled to the linker using primary amine groups in the polysaccharide. In this case, the linker typically has ester groups at both ends. by reacting one of the terephthalate groups with a primary amine group in the polysaccharide by nucleophilic acyl substitution. The reaction involves attaching the polysaccharide to the linker via an amide bond. This results in a coupled polysaccharide-linker intermediate.

[0144] Thus, the linker contains a first ester group for reaction with the primary amine groups of the polysaccharide. A bifunctional linker that provides a second ester group for reaction with a primary amine group on the carrier molecule. A typical linker is adipic acid N-hydroxysuccinimide diester (S IDEA).

[0145] Coupling can also be done indirectly, i.e., by derivatizing the polysaccharide prior to coupling to the linker. This can also be done with an additional linker used for derivatization.

[0146] Polysaccharides are coupled to additional linkers using the carbonyl group at the reducing end of the polysaccharide. This coupling is carried out in two steps: (a1) reacting the carbonyl group with an additional linker; and (a2) reacting the free end of the additional linker with the linker. In these embodiments, the additional linker typically comprises a primary amine at each end. group, which allows one of the primary amine groups to be attached to the carbonyl group of the polysaccharide by reductive amination. Step (a1) can be carried out by reacting the carbonyl group of the polysaccharide with the A primary amine group reactive with the hydroxyl group is used. A hydrazide or hydroxylamino group is suitable. Typically, the additional linker has the same primary amine group at both ends. Polysaccharide-additional linker, in which the saccharide is coupled to the additional linker via a C-N bond -leading to intermediates.

[0147] The polysaccharides can be coupled to additional linkers using different groups in the polysaccharide, particularly the carboxyl groups. This coupling can be accomplished in two steps: (a1) coupling the group to an additional linker; and (a2) reacting the free end of the additional linker with the linker. In this case, the additional linker typically has primary amine groups at both ends. whereby EDAC activation converts one of the primary amine groups to the carboxyl group of the polysaccharide. Step (a1) can be carried out by reacting the EDAC activity of the polysaccharide with the hydroxyl group. A primary amine group reactive with the functionalized carboxyl group is used. A hydrazide group is suitable. Typically, the additional linker has the same primary amine group at both ends. A polysaccharide-additional linker intermediate coupled to an additional linker via an amide bond. results.

[0148] In one embodiment, the carrier protein is prepared by chemical activation of the polysaccharide followed by reductive amination. Conjugation with proteins is described in U.S. Pat. Nos. 4,365,170 and 4,666,670. 73,574 and 4,902,506, U.S. Patent Application Publication No. 20 Specification No. 06 / 0228380, Specification No. 2007 / 184072, Specification No. 2007 / 0231340 and 2007 / 0184071, as well as International Publication Nos. WO 2006 / 110381, WO 2008 / 079653 and WO 2008 / 079653 This can be achieved by the means described in JP 2008 / 143709. salts (containing sodium periodate, potassium periodate, or periodic acid), of pneumococcal polysaccharides by reaction with any oxidizing agent that oxidizes the terminal hydroxyl groups to aldehydes. This reaction may involve random oxidative activation of adjacent hydroxyl groups on carbohydrates. It is cleaved to form a reactive aldehyde group.

[0149] Coupling to carrier proteins is via direct amination of the protein's lysyl groups. For example, conjugation can be achieved by the ligation of activated polysaccharides with carrier proteins. The mixture is reacted with a reducing agent such as sodium cyanoborohydride in the presence of nickel. The conjugation reaction can be carried out in aqueous solution or in DMSO. It can be carried out in the presence of SO. See, for example, U.S. Patent Application Publication No. 2015 / 023127 0, U.S. Patent Application Publication No. 2011 / 0195086 and European Patent No. See US Pat. No. 4,711,777. Then, a strong ion exchanger such as sodium borohydride is used. A reducing agent is added to cap any unreacted aldehydes.

[0150] Reductive amination involves (1) oxidation of polysaccharides to form reactive aldehydes, and (2) activation Stable amides are formed by reduction of imines (Schiff bases) formed between polysaccharides and carrier proteins. The oxidation process involves two steps: the formation of a hydroxyl group and the formation of a hydroxyl group. The grain size may be reduced by using mechanical methods (e.g., homogenization) or chemical hydrolysis. The chemical hydrolysis can be carried out using acetic acid. The oxidation step can be carried out using periodic acid. For the purposes of the present invention, the term "periodate" refers to Includes both periodate and periodic acid; the term also includes metaperiodate (IO4) and orthoperiodate (IO6), and various periodates (e.g., periodic acid In one embodiment, the capsular polysaccharide comprises meta- Acid in the presence of periodate, preferably sodium periodate (NaIO4) In another embodiment, the capsular polysaccharide is converted to a soluble form, preferably in the presence of orthoperiodate. is oxidized in the presence of periodate.

[0151] In one embodiment, the oxidizing agent is: Stable nitroxy compounds such as piperidine-N-oxy or pyrrolidine-N-oxy compounds nitroxide radical compounds (as described in WO 2014 / 097099). In the reaction, the actual oxidant is N-oxoammonium in the catalytic cycle. In one embodiment, the stable nitroxyl or nitroxide radical compound is a piperidine-N-oxy or pyrrolidine-N-oxy compound. The stable nitroxyl or nitroxide radical compound is TEMPO(2,2,6 ,6-tetramethyl-1-piperidinyloxy) or PROXYL(2,2,5,5- In one embodiment, the stable nitro The xyl radical compound is TEMPO or a derivative thereof. is a molecule having an N-halo moiety. In one embodiment, the oxidizing agent is N-chlorosuccinic acid. Imide, N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid Acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione, dib Bromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6 -trione, diiodoisocyanuric acid and 1,3,5-triiodo-1,3,5-tri Preferably, the oxidizing agent is selected from the group consisting of azinane-2,4,6-trione. N-chlorosuccinimide.

[0152] In certain embodiments, the oxidizing agent is 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) free radical and N-chlorosuccinimide (NC) as a co-oxidant S) (as described in WO 2014 / 097099). Thus, in one embodiment So, glycoconjugates of S. pneumoniae are: a) aqueous solvent; In this study, sugars were treated with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and and N-chlorosuccinimide (NCS) to obtain an activated sugar; and b) reacting the activated sugar with a carrier protein containing one or more amine groups. (The method was subsequently referred to as "TEMPO / NCS reductive amination") (This is the case.)

[0153] A quenching agent may be added to quench the oxidation reaction. , 1-, 2-amino alcohols, amino acids, glutathione, sulfites, bisulfites, dithionite Sulfates, metabisulfites, thiosulfates, phosphites, hypophosphites or phosphorous acids (e.g. Glycerol, ethylene glycol, propane-1,2-diol, butane-1,2-diol or butane-2,3-diol, ascorbic acid, etc.

[0154] In certain embodiments, the present invention utilizes conjugation reactions in aprotic solvents. Serotype 8 Streptococcus pneumoniae polysaccharides for use - a method for preparing a protein conjugate, wherein the conjugation reaction In a further embodiment, the conjugate is free of cyanoborohydride. The oxidative reaction is a Schiff base reduction or a reductive amination. The protein is tetanus toxoid, diphtheria toxoid, or CRM197. In another embodiment, the protein is CRM197. The conjugation reaction is a reductive amination. In a further embodiment, the reductive amination is , performed in dimethyl sulfoxide (DMSO).

[0155] In some embodiments, the oxidized polysaccharide prior to conjugation is between 30 kDa and 1,0 The molecular weight was determined by size exclusion chromatography (SEC) and multi- Calculations using a combined angular light scattering detector (MALS) and a refractive index detector (RI) In some embodiments, the polysaccharide has a molecular weight of 50 kDa to 300 kDa. In some embodiments, the polysaccharide has a molecular weight of 50 kDa to 1,000 kDa. In additional embodiments, the polysaccharide has a molecular weight of between 70 kDa and 900 kDa. In one embodiment, the polysaccharide has a molecular weight of between 100 kDa and 800 kDa. In a further embodiment, the polysaccharide has a molecular weight of 200 kDa to 600 kDa. Polysaccharides range from 100kDa to 1,000kDa; a~800kDa;100kDa~700kDa;100kDa~600kDa;100 kDa~500kDa;100kDa~400kDa;100kDa~300kDa;1 50kDa~1,000kDa;150kDa~900kDa;150kDa~800k Da;150kDa~700kDa;150kDa~600kDa;150kDa~50 0kDa;150kDa~400kDa;150kDa~300kDa;200kDa~ 1,000kDa;200kDa~900kDa;200kDa~800kDa;200 kDa~700kDa;200kDa~600kDa;200kDa~500kDa;2 00kDa~400kDa;200kDa~300;250kDa~1,000kDa; 250kDa~900kDa;250kDa~800kDa;250kDa~700kDa a;250kDa~600kDa;250kDa~500kDa;250kDa~400 kDa;250kDa~350kDa;300kDa~1,000kDa;300kDa ~900kDa;300kDa~800kDa;300kDa~700kDa;300k Da~600kDa;300kDa~500kDa;300kDa~400kDa;40 0kDa~1,000kDa;400kDa~900kDa;400kDa~800kDa a;400kDa~700kDa;400kDa~600kDa;500kDa~600 It has a molecular weight of 100 kDa.

[0156] The second step of the conjugation method is to activate the polysaccharide and carrier using a reducing agent. Reduces the imine (Schiff base) bond between the protein and the carboxyl group to form a stable conjugate. The formation of a hydroxybenzoate (so-called reductive amination) is carried out by the formation of a hydroxybenzoate. Suitable reducing agents include cyanoborohydride, These include sodium cyanoborohydride or sodium borohydride. In embodiments, the reducing agent is sodium cyanoborohydride.

[0157] In certain embodiments, the reductive amination reaction is carried out in an aprotic solvent (or In one embodiment, the reduction reaction is carried out in DMSO or DMF (a mixture of solvents). If lyophilized, the solvent is DMSO or DMSO. The F solvent may be used to reconstitute the activated polysaccharide and carrier protein. In the example, the aprotic solvent is DMSO.

[0158] At the end of the reduction reaction, unreacted aldehyde groups may remain in the conjugate. , which can be capped using a suitable capping agent. In the example, this capping agent is sodium borohydride (NaBH4). The compounds include sodium triacetoxyborohydride in the presence of Bronsted or Lewis acids. hydride or sodium or zinc borohydride, amine boranes, e.g., pyridine borane Borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine-borane, t -BuMe'PrN-BH3, benzylamine-BH3 or 5-ethyl-2-methylpi Conjugation includes lysine borane (PEMB) or borohydride exchange resin. After the reduction reaction and optional capping, the glycoconjugate can be prepared by any method known to those skilled in the art. Purification (enrichment in terms of the amount of polysaccharide-protein conjugate) can be achieved by various techniques that have been developed. These techniques include dialysis, concentration / diafiltration, tandem Fenton flow filtration, precipitation / elution, column chromatography (ion exchange chromatography) , multimodal ion exchange chromatography, DEAE or hydrophobic interaction chromatography In one embodiment, glycoconjugates are used in a variety of methods, including filtration, depth filtration, and microfiltration. The resulting product is purified by diafiltration or ion exchange chromatography or size exclusion chromatography. Purify by chromatography.

[0159] Glycoconjugates prepared using reductive amination in aprotic solvents Commonly used in multivalent pneumococcal conjugate vaccines. In certain embodiments of the multivalent composition that is not prepared in a protic solvent, the remaining blood The reduction reaction of the purified form is carried out in an aqueous solvent (e.g., PBS (phosphate buffered saline), MES (2- (N-morpholino)ethanesulfonic acid), HEPES(4-(2-hydroxyethyl)- 1-Piperazineethanesulfonic acid), Bis-Tris, ADA (N-(2-acetamide) iminodiacetic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)) , MOPSO (3-morpholino-2-hydroxypropanesulfonic acid), BES (N,N -bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS(3-(N -morpholino)propanesulfonic acid), DIPSO(3-bis(2-hydroxyethyl) Amino-2-hydroxypropane-1-sulfonic acid), MOBS (4-(N-morpholino) ) butanesulfonic acid), HEPPSO (N-(2-hydroxyethyl)piperazine-N- (2-hydroxypropanesulfonic acid)), POPSO (piperazine-1,4-bis(2 -hydroxy-3-propanesulfonic acid), TEA (triethanolamine), EPP S(4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid), pH 6.0 -8.5, 7.0-8.0 or 7.0-7.5 Bicine It can be done.

[0160] Streptococcus pneumoniae (S) can be prepared using reductive amination in aprotic solvents. pneumonia) capsular polysaccharide-protein conjugates include, but are not limited to: No, but serotypes: 3, 6A, 6B, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 19A, 20A, 22F, 23A, 23B, 23F, 24F, 31, 33F, 35B and 39. S. pneumoniae capsules can be prepared using reductive amination Membrane polysaccharide-protein conjugates include, but are not limited to, serotypes: 3, 6 A, 6B, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 19A, 20, 22F, 23A, 23B, 23F, 24F, 31, 33 These include 35F, 35B, and 39. These compounds are prepared using reductive amination in aprotic solvents. Pneumococcal (S. pneumoniae) capsular polysaccharide-protein conjugates that can be produced The serotypes include, but are not limited to: serotypes 3, 6A, 6B, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 19A, 2 Includes 0B, 22F, 23A, 23B, 23F, 24F, 31, 33F, 35B and 39 Polysaccharides can be used in the form of oligosaccharides. These can be used in the form of oligosaccharides (e.g., hydrolyzed They are conveniently formed by fragmentation of purified polysaccharides (by digestion), usually followed by the production of the desired saccharides. Fragments of the size are purified.

[0161] In certain embodiments, serotypes 3, 6A, 6B, 6C, 7F, 8, 9N, 10A, 11A , 12F, 15A, 15B, 15C, 16F, 17F, 19A, 20A, 22F, 23A , 23B, 23F, 24F, 31, 33F, 35B, and one or more of the pneumococcal polysaccharides 39 -Protein conjugates prepared using reductive amination in aprotic solvents In certain embodiments, serotypes 3, 6A, 6B, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 19A, 20, 22F, 2 Pneumococcal polypeptic ulcers: 3A, 23B, 23F, 24F, 31, 33F, 35B, and 39 Sugar-protein conjugates are synthesized using reductive amination in aprotic solvents. In certain embodiments, serotypes 3, 6A, 6B, 6C, 7F, 8, 9N, 10 A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 19A, 20B, 22 Pneumonia with one or more of F, 23A, 23B, 23F, 24F, 31, 33F, 35B, and 39 Coccal polysaccharide-protein conjugates were synthesized using reductive amination in aprotic solvents. In certain embodiments, each of the serotypes in the multivalent immunogenic composition is prepared using a non-promoter serotype. In certain embodiments, the multivalent groups are prepared using reductive amination in a rotonic solvent. Polysaccharides of one or more serotypes in the composition can be purified using reductive amination in an aprotic solvent. The conjugated and one or more serotype polysaccharides are subjected to reductive amino acid synthesis in an aqueous medium. In certain embodiments, two or more antibodies in a multivalent composition are conjugated using a fusion. The purified polysaccharides were conjugated using reductive amination in aprotic solvents. In other embodiments, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more in a multivalent composition. Above, 9 and above, 10 and above, 11 and above, 12 and above, 13 and above, 14 and above, 15 and above, 16 and above Polysaccharides of serotypes 17 or more, 18 or more, 19 or more, 20 or more, or 21 or more are non-proteobacterial. The conjugation is carried out using reductive amination in a rotonic solvent. The polysaccharides of one or more serotypes in the multivalent composition are dissolved in an aprotic or aqueous solvent. Conjugated using other chemicals possible.

[0162] Thus, the present invention provides a method for the production of Streptococcus pneumoniae (S. pneumoniae) antibodies, each conjugated to a carrier protein. Multiple pneumococci (S. pneumoniae) containing capsular polysaccharides of the serovar eumoniae ae) a multivalent immunogenic composition comprising a polysaccharide-protein conjugate, wherein the pulmonary Serotypes of S. pneumoniae are described herein (i.e., in Section II, "Multivalent Immunogenic Compositions"), wherein the polysaccharide-protein conjugate The one or more polysaccharides are conjugated to a carrier protein. In certain embodiments, the reaction is in an aprotic solvent. At least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% are prepared in aprotic solvents. The remainder of the serotypes are prepared in alternative solvents. They are prepared using chemicals and / or in aqueous solvents.

[0163] DMSO was used as a solvent in the reductive amination of polysaccharide-protein conjugates. When used, the predicted serotypes were significantly higher than those of the same conjugates prepared under aqueous conditions. It has been determined that unexpectedly superior stability and enhanced immunogenicity are obtained (U.S. Application No. 6 See US Pat. Nos. 2 / 463,216 and 62 / 555,444. As shown in Example 40, reduction in an aprotic solvent (e.g., DMSO) Formulations containing the pneumococcal conjugates of the invention prepared using reactive amination are Using a protic (i.e., aqueous) solvent during reductive amination in a jugation method Superior physical and chemical stability compared to vaccines using formulated drug substances Thus, in some embodiments, the pneumococcal polysaccharide conjugates in the multivalent compositions All of the gates are prepared in aprotic solvents.

[0164] In certain embodiments of the present invention, the total polysaccharide concentration in the composition is from about 0.02 to about 0.175 In certain embodiments of the present invention, the total polysaccharide concentration in the composition is about 0.0 mg / mL. In certain embodiments of the present invention, the total polysaccharides in the composition are from 3 to about 0.175 mg / mL. In another embodiment, the total amount of the hydroxybenzoates in the composition is about 0.04 to about 0.175 mg / mL. The polysaccharide concentration is about 0.065 to about 0.085 mg / mL, about 0.070 to about 0.080 mg / mL. g / mL, about 0.065 to about 0.080 mg / mL, about 0.070 to about 0.085 mg / mL, about 0.110 to about 0.128 mg / mL, about 0.110 to about 0.175 mg / mL , about 0.10 to about 0.175mg / mL, about 0.110 to about 0.170mg / mL, about 0 .115~approx. 0.15mg / mL, approx. 0.110~approx. 0.15mg / mL, approx. 0.110 ~0.125mg / mL, approx. 0.150~0.170mg / mL, approx. 0.150~approx. 0.165mg / mL, about 0.140 to about 0.170mg / mL, about 0.130 to about 0. 170mg / mL, about 0.150 to about 0.175mg / mL, about 0.070 to about 0.17 0 mg / mL, about 0.065 to about 0.175 mg / mL, or about 0.065 to about 0.18 0 mg / mL.

[0165] One or more or all of the polysaccharide-protein conjugates in the multivalent immunogenic composition may be non-protein. In embodiments of the invention prepared in a rotonic solvent, the total polysaccharide concentration in the composition is at 37°C. It is stable for at least 4 weeks at room temperature, at least 4 weeks at 25°C, and at least 12 weeks at 4°C.

[0166] One or more or all of the polysaccharide-protein conjugates in the multivalent immunogenic composition may be non-protein. In certain embodiments of the present invention, the composition is prepared in a rotonic solvent. Weight average molecular weight (Mw) of all polysaccharide-protein conjugates of C. neumoniae (average of all conjugates in the composition) is about 3,500 to about 4,700 kDa, 3,500 to approximately 4,600 kDa, approximately 3,500 to approximately 4,500 kDa, approximately 3,500 to Approximately 4,400 kDa, approximately 3,500 to approximately 4,300 kDa, approximately 3,500 to approximately 4,200 kDa, about 3,600 to about 4,700kDa, about 3,600 to about 4,600kDa, about 3 ,600 to approximately 4,500 kDa, approximately 3,600 to approximately 4,400 kDa, approximately 3,600 to approximately 4,300kDa, approximately 3,600~4,200kDa, approximately 3,700~4,700k Da, about 3,700 to about 4,600kDa, about 3,700 to about 4,500kDa, about 3, 700 to approximately 4,400 kDa, approximately 3,700 to approximately 4,300 kDa, approximately 3,700 to approximately 4 ,200kDa, about 3,800 to about 4,700kDa, about 3,800 to about 4,600kD a, about 3,800 to about 4,500kDa, about 3,800 to about 4,400kDa, about 3,8 00~about 4,300kDa, about 3,800~about 4,200kDa, about 3,900~about 4, 700kDa, approximately 3,900 to approximately 4,600kDa, approximately 3,900 to approximately 4,500kDa , about 3,900 to about 4,400 kDa, about 3,900 to about 4,300 kDa, or about 3, 900 to approximately 4,200 kDa.

[0167] The polysaccharide-protein conjugates in the multivalent immunogenic composition are prepared in an aprotic solvent. In certain embodiments of the present invention, the pneumococcus (S. pneumoniae) in the composition The Mw of each of the polysaccharide-protein conjugates (for a single serotype) is approximately 1.00 0 to approximately 10,000 kDa, approximately 1,500 to approximately 5,500 kDa, approximately 1,500 to approximately 5, 600kDa, approximately 1,500 to approximately 5,700kDa, approximately 1,500 to approximately 5,800kDa , about 1,500 to about 5,900 kDa, about 1,500 to about 6,000 kDa, about 1,00 0 to approximately 5,500 kDa, approximately 1,000 to approximately 5,000 kDa, approximately 1,000 to approximately 4,0 00kDa, approximately 1,000 to approximately 4,500kDa, approximately 1,000 to approximately 4,000kDa or about 1,000 to about 3,500 kDa. The Mw of the purified conjugates is approximately 1,000 kDa, approximately 1,100 kDa, and approximately 1,200 kDa. 0kDa, approx. 1,300kDa, approx. 1,400kDa, approx. 1,500kDa, approx. 1,60 0kDa, approx. 1,700kDa, approx. 1,800kDa, approx. 1,900kDa, approx. 2,00 0kDa, approx. 2,100kDa, approx. 2,200kDa, approx. 2,300kDa, approx. 2,40 0kDa, approx. 2,500kDa, approx. 2,600kDa, approx. 2,700kDa, approx. 2,80 0kDa, approx. 2,900kDa, approx. 3,000kDa, approx. 3,100kDa, approx. 3,20 0kDa, approx. 3,300kDa, approx. 3,400kDa, approx. 3,500kDa, approx. 3,60 0kDa, approx. 3,700kDa, approx. 3,800kDa, approx. 3,900kDa, approx. 4,00 0kDa, approx. 4,100kDa, approx. 4,200kDa, approx. 4,300kDa, approx. 4,40 0kDa, approx. 4,500kDa, approx. 4,600kDa, approx. 4,700kDa, approx. 4,80 0kDa, approx. 4,900kDa, approx. 5,000kDa, approx. 5,100kDa, approx. 5,20 0 kDa, about 5,300 kDa, about 5,400 kDa or about 5,500 kDa.

[0168] In certain embodiments of the invention, the polysaccharide-protein conjugate in the multivalent immunogenic composition The esters are prepared in aprotic solvents (as opposed to protic solvents). ) Streptococcus pneumoniae (S. pneumoniae) conjugated to a carrier protein in an aprotic solvent Compositions with a high proportion of (moniae) polysaccharides may be preferred. In certain embodiments, Serotype-specific coagulation of Streptococcus pneumoniae (S. pneumoniae) prepared in aprotic solvents. The percentage of conjugates (the number of polysaccharide serotypes prepared in aprotic solvents) The total number of solvents is calculated by dividing by the total number of solvents in aprotic or protic solvents. (including those prepared) is more than 50%, or more than 60%, or more than 70%, or more than 80% It may be greater than, or greater than 90%, or is 100%.

[0169] In certain embodiments of the invention, the serotype 3 polysaccharide-protein conjugate in the composition is prepared in an aprotic solvent, and the Mw of the conjugate is about 1,000 to about 5 ,000 kDa, or about 1,000 to about 4,000 kDa, or about 1,000 to about 3 ,000 kDa, or about 1,000 to about 2,500 kDa, or about 1,000 to about 2 ,000kDa.

[0170] One or more or all of the polysaccharide-protein conjugates in the multivalent immunogenic composition may be non-protein. In certain embodiments of the present invention, the composition is prepared in a rotonic solvent. neumoniae) polysaccharide-protein conjugate number average molecular weight (Mn) (composition The average of all conjugates in the 000kDa, about 1000 to about 2500kDa, about 1500 to about 2500kDa, about 18 00 to about 2500 kDa, about 1900 to about 2500 kDa, or about 2000 to about 2500 kDa.

[0171] One or more or all of the polysaccharide-protein conjugates in the multivalent immunogenic composition may be non-protein. In certain embodiments of the present invention, the composition is prepared in a rotonic solvent. neumoniae polysaccharide-protein conjugates (for a single serotype) (approximately 700 to 7000 kDa, approximately 1000 to 6000 kDa, approximately 1000 to approx. 5000kDa, approx. 1000 to approx. 4000kDa, approx. 1000 to approx. 3000kDa, approx. 9 00 to approximately 5500 kDa, approximately 900 to approximately 5000 kDa, approximately 900 to approximately 4500 kDa, About 900 to about 4000 kDa, about 900 to about 3500 kDa, or about 900 to about 3000 kDa.

[0172] In an embodiment of the present invention, the S. pneumoniae polysaccharide protein in the composition The Mw and / or Mn of the protein conjugates were measured at 37°C for more than 4 weeks and at 25°C for 4 weeks. Stable for at least 12 weeks at 4°C and / or at 4°C.

[0173] In an embodiment of the invention, the polysaccharide concentration, Mw and / or Mn can be determined by HPSEC UV / Determined using MALS / RI.

[0174] One or more or all of the polysaccharide-protein conjugates in the multivalent immunogenic composition may be non-protein. Some embodiments of the present invention prepared in rotic solvents have a molecular weight of 280 nanometers (n The emission maximum of the composition measured using intrinsic protein fluorescence spectroscopy at an excitation wavelength of m is In some embodiments, the emission maximum is about 335 nm to about 342 nm. The fluorescence intensity remains stable at 37°C for at least one week. In some embodiments, the emission maximum is maintained at about 335 nm to about 342 nm, and the fluorescence intensity is maintained at 37°C. It is stable for one week.

[0175] In some embodiments, all of the pneumococcal polysaccharide conjugates in the multivalent composition are D In certain subembodiments, all are prepared using reductive amination in MSO. Multivalent compositions containing polysaccharide conjugates prepared using MSO contain adjuvants. Does not include.

[0176] Without being bound by any theory, glycoconjugates prepared in DMSO One possible mechanism for the enhanced immunogenicity observed in mice is the interaction between the protein and polysaccharide. Provides additional attachment points to confer stability and prevents chemical depolymerization or depolymerization of carbohydrate bonds in peptides. The linkage between carbohydrates (capsular polysaccharides) and lysine residues on the surface of the carrier protein resists degradation. This involves increasing the number of bonds between the n of Saccharide-CRM197 Conjugate Vaccine s in Brown F,Corbel M,Griffiths E(eds):P hysico-Chemical Procedures for the Chara cterization of Vaccines.Dev.Biol.Basel,K See Arger, 2000, Vol. 103, pp. 93-104. DMSO The additional benefit of the increased polysaccharide-protein bonds created during conjugation in solvent This may provide additional opportunities for successful peptide-carbohydrate presentation to T cells. Loading with specific peptide sequences conjugated to carbohydrate antigens or Genetic diversity in human populations, resulting in different capacities and susceptibilities to association with carrier proteins, Additional attachment points on the protein increase the chances of successful antigen presentation on the surface of antigen-presenting cells (APCs). This allows for increased T cell response and allows for T cell-dependent responses to other T cell-independent antigens. It can be seen that the immunogenicity observed by conjugation in DMSO solvent Another possible mechanism of enhancement is the ability to bind different peptides, exposing additional lysines for polysaccharide binding. Mechanism of glycopeptide presentation on the surface of APCs for T cell-dependent responses to glycopeptide epitopes This may be due to the denaturation of CRM 197 in organic solvents, which leads to an increase in the activity of the i et al.,2011,Nature Medicine 17:1602-16 Please refer to 10.

[0177] Conjugation in organic solvents yields denatured CRM197 in the conjugate. A further advantage is the immunological impact of reduced antibodies against the native CRM 197 epitope. Polysaccharide-protein complexes produced during conjugation in DMSO solvent may be An additional benefit of increased protein binding is the larger size of the polysaccharides, resulting in improved immunogenicity. The compositions of the present invention may be capable of eliciting a human response, such as the formation of a protein conjugate. It is believed to offer important advantages in

[0178] In certain embodiments, the conjugation reaction is carried out by reductive amination, To increase the efficiency of the conjugation reaction and to aid in the removal of free cyanide Nickel is used. Transition metals are known to form stable complexes with cyanides. The reduction of protein amino groups with formaldehyde by sodium cyanoborohydride was It is known to improve basal methylation (S Gidley et al., Biol. chem J.1982,203:331-334;Jentoft et al.An al Biochem. 1980, 106:186-190). By complexing, the addition of nickel reduces the consumption of protein during conjugation. This increases the binding capacity of the antibody, leading to the formation of larger and potentially more immunogenic conjugates.

[0179] Differences in starting cyanide levels between sodium cyanoborohydride reagent lots also contribute to consistency. Conjugate size and conjugate size are optimized to provide conjugation performance without The addition of nickel alters product attributes such as the Ps to CRM197 ratio. Complexation of the substance reduces conjugation mismatch, and sodium cyanoborohydride Lot differences were eliminated.

[0180] Suitable alternative chemistries include 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, The process involves activating sugars with cyanate esters (CDAP). Thus, the activated sugar is attached to the carrier protein directly or via a spacer (linker) group. For example, the spacer can be coupled to an amino group on a protein. The thiolated polysaccharides may be cystamine or cysteamine, which give thiolated polysaccharides, and maleimides. Deactivated carrier proteins (e.g., using GMBS) or haloacetylated carrier proteins Substrates (e.g., iodoacetimide [e.g., ethyl iodoacetimide HCl] or N Succinimidyl bromoacetate or SIAB, or SIA, or SBAP It can be coupled to the support via a thioether bond obtained after reaction with Preferably, cyanate esters (which may be produced by CDAP chemistry) is coupled with hexanediamine or adipic dihydrazide (ADH) to give the amine The derivatized sugar is coupled to a carbodiimide (e.g., Conjugation to a carrier protein is performed using EDAC or EDC chemistry. Suitable conjugates are described in International Patent Application Publication No. WO 93 / 15760, ... No. 95 / 08348 and WO 96 / 29094; and Chu et al. l., 1983, Infect. Immunity 40:245-256 There are.

[0181] Other suitable techniques include carbodiimides, hydrazides, active esters, norborane, p-dinitrobenzoates, Uses benzoic acid, N-hydroxysuccinimide, S-NHS, EDC, and TSTU. Many of these are described in International Patent Application Publication No. WO 98 / 42721. Conjugation is the formation of carbohydrates that can be formed by the reaction of free hydroxyl groups of sugars with CDI. bonyl linker (Bethell et al., 1979, J. Biol. Chem. 254:2572-4;Hearn et al., 1981, J. Chromatog 218:509-18), which subsequently reacts with proteins to form carbamates. This is achieved by reduction of the anomeric terminal to the primary hydroxyl group, optional protection / deprotection of the hydroxyl group, and CDI capping by reaction of the primary hydroxyl group with CDI. Formation of carbamate intermediates, and the cleavage of CDI carbamate intermediates with amino groups on proteins Coupling may be included.

[0182] After conjugation of the capsular polysaccharide to the carrier protein, the polysaccharide-protein conjugate The gates are purified by one or more of a variety of techniques (e.g., purification of polysaccharide-protein conjugates). Examples of these techniques are well known to those skilled in the art and include concentration / diafiltration filtration, ultrafiltration, precipitation / elution, column chromatography and depth filtration. See, for example, U.S. Patent No. 6,146,902.

[0183] After the individual glycoconjugates are purified, they are combined to form the immunogenic compositions of the invention. These pneumococcal conjugates are prepared by separate methods and formulated into single-dose The formulation is bulk formulated.

[0184] An alternative method for characterizing the glycoconjugates of the present invention is to characterize the conjugated lysine Conjugated to sugars can be characterized as the extent of conjugation This is due to the number of lysine residues in the carrier protein (e.g. CRM197) used. Evidence for lysine modification of carrier proteins by covalent attachment to polysaccharides is known to those skilled in the art. This can be obtained by amino acid analysis using routine methods. compared to the carrier protein starting material used to generate the conjugate. In a preferred embodiment, the glycoconjugates of the present invention are The degree of conjugation of the jugates was 2–15, 2–13, 2–10, 2–8, and 2 ~6, 2~5, 2~4, 3~15, 3~13, 3~10, 3~8, 3~6, ​​3~5, 3~ 4, 5-15, 5-10, 8-15, 8-12, 10-15 or 10-12. In embodiments, the degree of conjugation of the glycoconjugates of the present invention is about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14 or about 15. In a preferred embodiment, the conjugates of the glycoconjugates of the present invention In some such embodiments, the carrier protein has a degree of affinity of 4 to 7. It's CRM197.

[0185] The glycoconjugates of the compositions of the present invention may also be prepared by adjusting the ratio of sugar to carrier protein (weight / weight) )(Ps:Pr). In some embodiments, the amount of glycol in the composition may be The polysaccharide to carrier protein ratio (w / w) of the co-conjugate is 0.5–3.0 (e.g., , about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2 , about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0 , approx. 2.1, approx. 2.2, approx. 2.3, approx. 2.4, approx. 2.5, approx. 2.6, approx. 2.7, approx. 2.8 , about 2.9 or about 3.0). In other embodiments, the ratio of sugar to carrier protein (w / w) is 0.5~2.5, 0.5~1.5, 0.8~2.5, 0.5~1.0, 1.0~ 1.5, 1.0~2.0, 0.8~2.4, 0.8~2.3, 0.8~2.2, 0.8~ 2.1, 0.8~2.0, 0.8~1.9, 0.8~1.8, 0.8~1.7, 0.8~ 1.6, 0.8~1.5, 0.8~1.4, 0.8~1.3, 0.9~2.4, 0.9~ 2.3, 0.9~2.2, 0.9~2.1, 0.9~2.0, 0.9~1.9, 0.9~ 1.8, 0.9~1.7, 0.9~1.6, 0.9~1.5, 0.9~1.4, 0.9~ 1.3, 0.9~1.2, 1.0~2.4, 1.0~2.3, 1.0~2.2, 1.0~ 2.1, 1.0~2.0, 1.0~1.9, 1.0~1.8, 1.0~1.7, 1.0~ 1.6, 1.0-1.5, 1.0-1.4, 1.0-1.3 or 1.0-1.2 In a further embodiment, the sugar to carrier protein ratio (w / w) is between 0.8 and 1.2. In some such embodiments, the carrier protein is CRM197. The glycoconjugates and immunogenic compositions are covalently conjugated to a carrier protein. Although not specifically incorporated, they are nonetheless present in the glycoconjugate composition. The free sugar may be non-covalently associated with the glycoconjugate. The molecule may be non-covalently bound, adsorbed, or entrapped.

[0186] In a specific embodiment, the saccharide and carrier protein for serotype 15A conjugates The ratio (w / w) is about 1.0 to about 2.0, about 1.25 to about 1.75, or about 1.3 to about 1. In another embodiment, the saccharide to carrier protein ratio (w / w) for serotype 15A is 7. ) is approximately 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, or 1.8 .

[0187] In a specific embodiment, the saccharide and carrier protein for a serotype 15C conjugate are The ratio (w / w) is about 1.0 to about 2.0, about 1.25 to about 1.75, or about 1.3 to about 1. In another embodiment, the saccharide to carrier protein ratio (w / w) for serotype 15C is 7. ) is approximately 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, or 1.8 .

[0188] In a specific embodiment, the sugar and carrier protein for the serotype 33F conjugate are The ratio (w / w) is about 1.0 to about 2.0, about 1.25 to about 1.75, or about 1.3 to about 1. In another embodiment, the saccharide to carrier protein ratio (w / w) for serotype 33F is 7. ) is approximately 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, or 1.8 .

[0189] In a specific embodiment, the saccharide and carrier protein for serotype 35B conjugates The ratio (w / w) is about 1.25 to about 2.25, about 1.25 to about 2.0, or about 1.3 to about 1. In another embodiment, the saccharide to carrier protein ratio (w / w) is about 1.2, 1.3, 1.3, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 .0.

[0190] In a specific embodiment, the sugar and carrier protein for the serotype 24F conjugate are The ratio (w / w) is about 0.5 to about 1.5, about 0.75 to about 1.25, or about 0.8 to about 1. In another embodiment, the saccharide to carrier protein ratio (w / w) for serotype 24F is 0. ) is about 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0.

[0191] In a preferred embodiment, the glycoconjugates comprise about 50% of the total amount of polysaccharides, Contains less than 45%, 40%, 35%, 30%, 25%, 20% or 15% free polysaccharides In a preferred embodiment, the glycoconjugates comprise less than about 25% of the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate contains less than 100% of the total amount of free polysaccharides. In a preferred embodiment, the glycoconjugates contain less than about 20% free polysaccharides compared to the glycoconjugates. The syrup contains less than about 15% free polysaccharides compared to the total amount of polysaccharides.

[0192] IV.How to use Embodiments of the present invention also include: (a) therapeutic (e.g., human body); (b) pharmaceutical; (c) pulmonary Inhibition of infection by Streptococcus pneumoniae; (d ) Induction of an immune response or a protective immune response against Streptococcus pneumoniae (S. pneumoniae); (e) Prevention of infection with Streptococcus pneumoniae (S. pneumoniae); (f) Prevention of infection with Streptococcus pneumoniae (S. (g) preventing recurrence of E. pneumoniae infections; (g) preventing related diseases such as brain damage, hearing loss, and seizures Prevention of pathological conditions associated with S. pneumoniae infection, including prevention of complications (h) reduction in progression, onset, or severity of pneumococcal (S. pneumoniae) infection or (i) a reduction in the likelihood of, but not limited to, pneumococcal pneumonia, pneumococcal pneumococcal disease(s), including bacteremia, pneumococcal meningitis, otitis media, and sinusitis (i) for use in treating, preventing, or delaying the onset, severity, or progression of or (iii) for use in the preparation of a medicament, These uses also include one or more of the multivalent immunogenic compositions described herein. The serovalent pneumococcal polysaccharide-conjugate compositions may be combined with or with one or more adjuvants. It may be used with or without adjuvant.

[0193] Thus, the present invention provides a multivalent immunogenic pneumococcal polysaccharide-protein conjugate of the invention. and administering to a patient in need thereof one or more of the following compositions: pneumoniae infection or pneumococcal disease prophylactic treatment (i.e., protection) This provides a method for:

[0194] The compositions and formulations of the present invention can be used via systemic or mucosal routes to administer the compositions of the present invention. administration of the compound to protect or treat people susceptible to infectious diseases, such as pneumococcal infections. can be treated.

[0195] In one embodiment, the present invention provides an immune response to S. pneumoniae. A method for inducing a leukemia comprising administering to a patient an immunologically effective amount of a multivalent immunogenic composition of the present invention. In another embodiment, the present invention provides a method for treating pneumococcal infections, comprising the steps of: a method of vaccinating a human by administering an immunologically effective amount of a multivalent immunogenic composition of the present invention to a subject; to a human.

[0196] Thus, in one aspect, the present invention provides a method for (1) inducing an immune response in a human patient; (2) administering a therapeutic agent to a human patient; (3) induce a protective immune response in individuals infected with Streptococcus pneumoniae (S. pneumoniae). or (4) vaccinating a human patient against S. pneumoniae in a human patient. moniae infection, comprising administering to a subject a multivalent immunogenic composition of the present invention ( That is, any of the multivalent immunogenic compositions described herein, e.g., the "multivalent" immunogenic compositions described above, administering to the patient a multivalent immunogenic composition (a multivalent immunogenic composition described in Section II entitled "Immunogenic Composition"); The present invention provides a method comprising the steps of:

[0197] In one embodiment, the present invention provides a method for the treatment of pneumococcal pneumonia and invasive disease in adults 18 years of age and older. In another embodiment, the present invention provides a method for preventing infection with 24 species of Streptococcus pneumoniae (Streptococcus pneumoniae). eptococcus pneumoniae) strains (3, 6A, 6C, 7F, 8, 9N , 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 19A, 20A , 20B, 22F, 23A, 23B, 24F, 31, 33F and 35B) The present invention provides a method for preventing pneumococcal pneumonia and invasive disease caused by the pneumococcal pneumonia.

[0198] In one embodiment of the above method, the composition comprises a plurality of S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates is linked to a carrier protein. Contains polysaccharides of Streptococcus pneumoniae serovars conjugated to a protein Here, the serotypes of Streptococcus pneumoniae (S. pneumoniae) are serotypes: 3, 7F, 19 A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9 Sets of N, 10A, 11A, 12F, 15C, 17F and 20A are included. In one embodiment, the composition comprises a plurality of S. pneumoniae polysaccharide proteins. and protein conjugates, wherein each of the conjugates is conjugated to a carrier protein. Polysaccharides of the gated S. pneumoniae serotype, where pneumonia The serotypes of S. pneumoniae are: serotypes 3, 7F, 19A, 22F, 3 3F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 1 In one embodiment of the above method, the set includes: 1A, 12F, 15C, 17F, and 20. The composition comprises a plurality of S. pneumoniae polysaccharide-protein conjugates. wherein each of the conjugates is a pneumonia conjugated to a carrier protein. The polysaccharides of the serotype S. pneumoniae, wherein S. pneumoniae eumoniae) serotypes are serotypes: 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, In another embodiment of the above method, the composition comprises the set of: 15C, 17F and 20B. Serotype 6A or 6C S. pneumoniae polysaccharide protein conjugates It further includes a ducate.

[0199] Pneumococcal conjugate vaccines containing serotype 6A show some cross-reactivity to serotype 6C. It has been shown that vaccines can provide differential protection (Cooper et al., Vaccine 29(2011)7207-7211). Thus, some embodiments of the above method Therefore, the present invention also does not include serotype 6C, but instead serotype 6A or serotype 6A and In another embodiment, the immunogenic composition comprises: , serotype 6A, 6B and 6C pneumococcal conjugates. In terms of morphology, the serotypes of Streptococcus pneumoniae (S. pneumoniae) are: Ia)3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24 F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20A; Ib)3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B , 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 2 0A; Ic)3, 7F, 19A, 22F, 33F, 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and and 20A; and Id)3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B , 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 2 0A The set of serotypes selected from the group consisting of:

[0200] In a particular embodiment of the set of serotypes (Ia-Id) above, serotype 20 or 20 B can substitute for serotype 20A.

[0201] In a further embodiment of the above method, the serotype of S. pneumoniae is , II-a) 15A, 16F, 23A, 23B, 24F, 31 and 35B; II-b) 6A, 15A, 16F, 23A, 23B, 24F, 31 and 35B; II-c) 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31 and 35B ; II-d) 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B; II-e) 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10 A, 11A, 12F, 15C, 17F and 20A; II-f)6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N , 10A, 11A, 12F, 15C, 17F, and 20A; II-g)6A, 6B, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8 , 9N, 10A, 11A, 12F, 15C, 17F and 20A; and II-h)6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N , 10A, 11A, 12F, 15C, 17F and 20A The set of serotypes selected from the group consisting of:

[0202] In a particular embodiment of the set of serotypes (II-e to II-h) above, serotype 20 or 2 0B can be substituted for serotype 20A.

[0203] In a further embodiment of the above method, the serotype of S. pneumoniae is , a set of serotypes as shown in any of embodiments Ia), II-a) or II-e). and further includes serotypes 6A, 6B and 6C.

[0204] Pneumococcal conjugate vaccines containing serotype 10A have some efficacy against serotype 39. It has also been shown to be capable of providing cross-protection (see WO 2017 / 085586). Thus, in some embodiments of the above methods, the present invention also includes serotype 10A. In another embodiment, the present invention provides the use of a multivalent immunogenic composition that does not contain serotype 39 but instead contains serotype 39. The immunogenic composition comprises serotype 10A and 39 pneumococcal conjugates. In certain embodiments of the method, the serotype of S. pneumoniae is III-a)3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 39, 11A, 12F, 15C, 17F and 20A ; III-b)3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 2 3B, 24F, 31, 35B, 8, 9N, 39, 11A, 12F, 15C, 17F and 20A; III-c)3, 7F, 19A, 22F, 33F, 6A, 6B, 15A, 16F, 23 A, 23B, 24F, 31, 35B, 8, 9N, 39, 11A, 12F, 15C, 17F and 20A; III-d)3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 2 3B, 24F, 31, 35B, 8, 9N, 39, 11A, 12F, 15C, 17F and 20A; III-e) 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 3 9, 11A, 12F, 15C, 17F and 20A; III-f)6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9 N, 39, 11A, 12F, 15C, 17F and 20A; III-g)6A, 6B, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 39, 11A, 12F, 15C, 17F and 20A; and III-h)6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9 N, 39, 11A, 12F, 15C, 17F and 20A The set of serotypes selected from the group consisting of:

[0205] In a particular embodiment of the set of serotypes (II-a to III-h) above, serotype 20 or 20B can be substituted for serotype 20A.

[0206] In a further embodiment of the above method, the serotype of S. pneumoniae is , comprising a set of serotypes as set forth in any of embodiments III-a) to III-h), and further includes serotype 10A.

[0207] S. pneumoniae serotypes covalently bound to carrier proteins Immunogenic conjugates containing 15B capsular polysaccharides are also available for use with serotype 15C and / or serotype 15D. It has also been shown that IFN-γ may provide some cross-protection against IFN-γ (International Publication No. WO 2014 / 013998). (See, e.g., US Pat. No. 5,110,942.) Thus, in some embodiments of the above method, the present invention provides It also does not contain serotype 15C (or de-O-acetylated 15B), but instead contains serotype 15B (i.e., the serotype 15B polysaccharide is not de-O-acetylated) In another embodiment, the immunogenic composition comprises a serotype 15B and a serotype 15C. The pneumococcal conjugates of 5C (or de-O-acetylated 15B) are also included. In certain embodiments, the serotype of S. pneumoniae is IV-a)3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 2 4F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15B, 17F and 20A ; IV-b)3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23 B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15B, 17F and 20A; IV-c)3, 7F, 19A, 22F, 33F, 6A, 6B, 15A, 16F, 23A , 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15B, 17F and 20A; IV-d)3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23 B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15B, 17F and 20A; IV-e) 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10 A, 11A, 12F, 15B, 17F and 20A; IV-f)6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N , 10A, 11A, 12F, 15B, 17F and 20A; IV-g)6A, 6B, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8 , 9N, 10A, 11A, 12F, 15B, 17F and 20A; and IV-h)6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N , 10A, 11A, 12F, 15B, 17F and 20A The set of serotypes selected from the group consisting of:

[0208] In a particular embodiment of the set of serotypes (IVa-IVh) above, serotype 20 or 20 B can be substituted for serotype 20A.

[0209] In a further embodiment of the above method, the serotype of S. pneumoniae is , comprising a set of serotypes as set forth in any of embodiments IV-a) to IV-h), and , further including serotype 15C (and / or de-O-acetylated 15B).

[0210] The compositions of the present invention are effective in preventing the development of Streptococcus pneumoniae (S. This use is useful in methods of providing complementary protection against S. pneumoniae. The compositions of the present invention are effective against specific pneumococci (S. pneumoniae) against which patients have not previously been vaccinated. eumoniae serotypes, and if the patient has not been previously vaccinated Provides additional protection against selected S. pneumoniae serotypes or if the patient has not previously been vaccinated against S. pneumoniae iae serotype and the pneumococcus (S. pneumoniae) against which the patient had previously been vaccinated ) serotypes.

[0211] Thus, the present invention provides a method for enhancing an immune response to S. pneumoniae in a patient. A method of inducing, vaccinating or inducing a protective immune response comprising administering a multivalent immunogen The method includes administering to a patient a composition containing a plurality of Streptococcus pneumoniae (S. pneumoniae) strains, wherein the composition contains a plurality of Streptococcus pneumoniae strains, eumoniae polysaccharide-protein conjugates, wherein the polysaccharide-protein The conjugate is a pneumococcal (S. pneumoniae) antibody conjugated to a carrier protein. iae serotype, where the patient has previously been infected with S. pneumoniae The present invention provides a method for vaccinating against Vaccinia oniae. In this context, the multivalent immunogenic composition is any multivalent immunogenic composition described herein. In certain embodiments of the methods of the present invention, the multivalent immunogenic composition may be a multivalent pneumocystis The multivalent immunogenic vaccine is administered to patients who have been treated with a pneumococcal (Sp) vaccine. Prevent pneumococcal disease caused by two or more serotypes of Streptococcus pneumoniae The vaccine may be any vaccine required to

[0212] In a specific embodiment of the above method, the patient has previously a) 4, 6B, 9V, 14, 18C, 19F and 23F; b) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F and 19A; c) 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F; d)4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19 A, 22F and 33F; e)4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 2 2F, 33F, 2, 8, 9N, 10A, 11A, 12F, 15B, 17F and 20; and Call f)4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19 A, 22F, 33F, 8, 10A, 11A, 12F and 15B Streptococcus pneumoniae (S. pneumoniae) serotype selected from the group consisting of Treated with a multivalent pneumococcal vaccine required to prevent pneumococcal disease caused by Ta.

[0213] In a specific embodiment of the above method, the multivalent pneumococcal vaccine is a multivalent pneumococcal vaccine containing S. pneumoniae moniae) serotypes 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7 F, 19A, 22F, 33F, 2, 8, 9N, 10A, 11A, 12F, 15B, 17F and 20A capsular polysaccharides. In a specific embodiment of the above method, the multivalent pneumococcal vaccine Chin is effective against Streptococcus pneumoniae (S. pneumoniae) serotypes 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 22F, 33F, 2, 8, 9N, 10A, The capsular polysaccharides of the above-mentioned methods include 11A, 12F, 15B, 17F and 20. In embodiments, the multivalent pneumococcal vaccine is a multivalent vaccine for S. pneumoniae serotype 4. , 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 22F, 3 Capsular polysaccharides 3F, 2, 8, 9N, 10A, 11A, 12F, 15B, 17F and 20B Includes types.

[0214] In a specific embodiment of the above method, the multivalent pneumococcal vaccine comprises a plurality of polysaccharide protein complexes. and a polysaccharide-protein conjugate, wherein the polysaccharide-protein conjugate is conjugated to a carrier protein. Contains polysaccharides of conjugated S. pneumoniae serotypes. In embodiments, the multivalent pneumococcal vaccine is not conjugated to a carrier protein. Contains multiple S. pneumoniae capsular polysaccharides.

[0215] In an additional embodiment of the above method, the patient has previously received PREVNAR® 13 (pulmonary Streptococcus pneumoniae 13-valent conjugate vaccine [diphtheria CRM197 protein], Pfiz er, Inc., Philadelphia, PA, USA).

[0216] In a further embodiment of the above method, the patient has previously received PNEUMOVAX® 2 3 (Pneumococcal Vaccine Multivalent, Merck & Co., Inc., Kenilworth, NJ, USA) ) was treated.

[0217] In yet a further embodiment of the above method, the patient has previously received SYNFLORIX™ ( Pneumococcal polysaccharide conjugate vaccine (adsorbed), GlaxoSmithKline B The patients were treated at the National Institute of Neurology, Rixensart, Belgium.

[0218] In an embodiment of the above method, the multivalent immunogenic composition of the invention is administered to a medical professional, e.g., a physician. Optional treatment after a patient receives a multivalent pneumococcal vaccine according to the treatment regimen provided by In certain embodiments, the multivalent immunogenic compositions of the invention are administered to a patient at a time point. 1 month to 5 years after receiving the multivalent pneumococcal vaccine, or 1 month to 1 year after receiving the test, 1 month to 2 years after receiving the test, 1 month to 3 years after receiving the test, 1 month to 4 years after receiving the test, 6 months later, 2 to 6 months later, 2 months to 1 year later, 1 to 5 years later, 6 months to 5 years later, 6 months ~4 years later, 6 months to 3 years later, 6 months to 2 years later, 6 months to 1 year later, 1 to 4 years later, 1 year to 3 years later In a further embodiment, the multivalent immunogenic composition is administered to the patient after one year or one to two years. The results were obtained approximately 1 month, 2 months, 3 months, and 4 months after the patient received the multivalent pneumococcal vaccine. months later, about 5 months later, about 6 months later, about 7 months later, about 8 months later, about 9 months later, about 10 months later , about 11 months later, about 1 year later, about 1.25 years later, about 1.5 years later, about 1.75 years later, about 2 years later , about 2.25 years later, about 2.5 years later, about 2.75 years later, about 3 years later, about 3.25 years later, about 3. 5 years later, approximately 3.75 years later, approximately 4 years later, approximately 4.25 years later, approximately 4.5 years later, approximately 4.75 years later The drug is administered to patients approximately five years later.

[0219] In further embodiments, the present invention provides a method for (1) inducing an immune response in a human patient; (2) inducing an immune response in a human patient; (3) induce protective immune responses in patients; and (4) prevent infection caused by Streptococcus pneumoniae (S. pneumoniae). (4) vaccinating a human patient against S. pneumoniae infection; or immunizations of the present invention in any order. administering an immunogenic composition to a patient and administering a multivalent pneumococcal vaccine to a patient. The multivalent pneumococcal vaccine is a vaccine containing Streptococcus pneumoniae (S. pneumoniae) e) required to prevent pneumococcal disease caused by two or more serotypes The vaccine may be any vaccine that is suitable for use in the prevention of HIV infection.

[0220] In certain embodiments of the above methods, the patient: a) 4, 6B, 9V, 14, 18C, 19F and 23F; b) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F and 19A; c) 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F; d)4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19 A, 22F and 33F; e)4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 2 2F, 33F, 2, 8, 9N, 10A, 11A, 12F, 15B, 17F and 20; and Call f)4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19 A, 22F, 33F, 8, 10A, 11A, 12F and 15B Streptococcus pneumoniae (S. pneumoniae) serotype selected from the group consisting of The multivalent immunogenic compositions of the present invention are required to prevent pneumococcal disease caused by and polyvalent pneumococcal vaccine.

[0221] In a specific embodiment of the above method, the multivalent pneumococcal vaccine is a multivalent pneumococcal vaccine containing S. pneumoniae moniae) serotypes 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7 F, 19A, 22F, 33F, 2, 8, 9N, 10A, 11A, 12F, 15B, 17F and 20A capsular polysaccharide.

[0222] In a specific embodiment of the above method, the multivalent pneumococcal vaccine comprises a plurality of polysaccharide protein complexes. and a polysaccharide-protein conjugate, wherein the polysaccharide-protein conjugate is conjugated to a carrier protein. Contains polysaccharides of conjugated S. pneumoniae serotypes. In embodiments, the multivalent pneumococcal vaccine is not conjugated to a carrier protein. Contains multiple S. pneumoniae capsular polysaccharides.

[0223] In an additional embodiment of the above method, the patient receives, in any order, a multivalent immunogenic composition of the invention. and PREVNAR® 13 (pneumococcal 13-valent conjugate vaccine) [Diphtheria CRM197 protein], Pfizer, Inc., Philadelphia, In one embodiment, patients are first treated with PREVNAR®. 13 and the patient is secondly administered a multivalent immunogenic composition of the invention. In this study, patients were first administered the multivalent immunogenic composition of the present invention and secondly, PREVNAR. The patient was administered 13mg of steroids (registered trademark).

[0224] In a further embodiment of the above method, the patient receives, in any order, a multivalent immunogenic composition of the invention. and PNEUMOVAX® 23 (Pneumococcal Vaccine Multivalent, Merc In one embodiment, the patient is treated with steroids (e.g., steroids ... Patients were first administered PNEUMOVAX® 23 and patients were second administered the polyvalent In an alternative embodiment, the patient is first administered the multivalent immunogenic composition of the present invention. First, patients will receive the composition and second, they will receive PNEUMOVAX® 23.

[0225] In still further embodiments of the above methods, the patient receives, in any order, a multivalent immunogenic antibody of the invention. and treated with the composition and administered SYNFLORIX™ (pneumococcal polysaccharide conjugate vaccine). (adsorption), GlaxoSmithKline Biologicals sa, Li In one embodiment, patients are first treated with SYNFLOR. IX™, and the patient is secondarily administered a multivalent immunogenic composition of the invention. In an embodiment, a patient is first administered a multivalent immunogenic composition of the invention and second administered SYNFL Administered ORIX™.

[0226] In some embodiments of the above methods, the multivalent immunogenic compositions and multivalent pneumococcal vaccines As used herein, "co-administration" refers to administering two compositions at the same time. The present invention is not limited to administering the compounds in a sequential order, but includes administering them one after the other in any order. In some embodiments, the multivalent immunogenic composition and the multivalent pneumococcal vaccine are administered intramuscularly or subcutaneously. The vaccine is administered via a bolus of 100 mg / kg to separate anatomical sites, for example, two different arms.

[0227] In some embodiments of the above methods, the multivalent immunogenic composition of the invention and the multivalent pneumococcal vaccine are administered. In an alternative embodiment, the amount of time between administrations of the steroid is from about 4 weeks to about 1 year. It ranges from 1 month to about 5 years.

[0228] In one embodiment, the patient is first administered a multivalent pneumococcal vaccine and second administered a multivalent pneumococcal vaccine of the invention. In an alternative embodiment, the patient is first administered the multivalent immunogenic composition of the present invention. First, they receive a polyvalent pneumococcal vaccine, and second, they receive a polyvalent pneumococcal vaccine.

[0229] Inducing an immune response against Streptococcus pneumoniae in patients 1. A method of inoculating or inducing a protective immune response in a mammal, comprising: (1) A multivalent immunogenic composition is administered to a patient, the composition comprising multiple S. pneumoniae antibodies. moniae) polysaccharide-protein conjugates, wherein the polysaccharide-protein conjugate Each of the conjugates contains a pneumococcal antibody (S. pneumoniae antibody) conjugated to a carrier protein. oniae serovar capsular polysaccharides; (2) waiting a predetermined amount of time to elapse; (3) administering a multivalent pneumococcal vaccine to the patient; Also provided is a method comprising:

[0230] In this method, the multivalent immunogenic composition comprises a multivalent immunogen comprising ... oniae) polysaccharide-protein conjugates, Multivalent pneumococcal vaccines are vaccines that contain two or more strains of pneumococcus (S. pneumoniae). Any vaccine required to prevent disease caused by a strain of do.

[0231] Inducing an immune response against Streptococcus pneumoniae in patients 1. A method of inoculating or inducing a protective immune response in a mammal, comprising: (1) administering a multivalent pneumococcal vaccine to a patient; (2) waiting a predetermined amount of time to elapse; (3) administering to a patient a multivalent immunogenic composition, the composition comprising multiple S. pneumoniae antibodies; oniae) polysaccharide-protein conjugates, including polysaccharide-protein conjugates Each of the following is a pneumococcus (S. pneumoniae) conjugated to a carrier protein: containing the capsular polysaccharide of the serotype; Also provided by the present invention is a method comprising:

[0232] In this method, the multivalent immunogenic composition comprises a multivalent immunogen comprising ... oniae) polysaccharide-protein conjugates, Multivalent pneumococcal vaccines are vaccines that contain two or more strains of pneumococcus (S. pneumoniae). Any vaccine required to prevent disease caused by a strain of do.

[0233] In some embodiments of the above methods, the multivalent pneumococcal vaccine comprises multiple pneumococcal (Sp neumoniae) polysaccharide-protein conjugates, wherein the polysaccharide-protein The protein conjugate is a S. pneumoniae antibody conjugated to a carrier protein. In an alternative embodiment, the multivalent pneumococcal vaccine comprises capsular polysaccharides of the P. oniae serotype. S. pneumoniae capsules not conjugated to carrier proteins Contains membrane polysaccharides.

[0234] In any embodiment of the method of the present invention (i.e., any of the methods described herein), The method further comprises administering to the patient one or more additional doses of the multivalent immunogenic composition of the invention. In such a method, the patient may receive a multivalent immunogenic composition of the invention as described above. Patients may have already received a multivalent pneumococcal vaccine before receiving the first dose of this vaccine, or Vaccination against Streptococcus pneumoniae (S. pneumoniae) before receiving the polyvalent immunogenic composition Thus, in one embodiment, the bacteria may be inoculated with Streptococcus pneumoniae (S. pneumoniae). Multivalent pneumococcal vaccines required to prevent pneumococcal disease caused by Streptococcus pneumoniae Patients who have received a pneumococcal vaccine may be administered two or more doses of a multivalent immunogenic composition of the invention. In an alternative embodiment, any vaccine previously required to prevent pneumococcal disease is administered. Patients not treated with IFN-γ are administered two or more doses of the multivalent immunogenic composition of the present invention. can be.

[0235] In an embodiment of the above method, two or more doses are the same multivalent immunogenic composition of the invention. In an alternative embodiment, two or more doses are different multivalent immunogenic compositions of the invention.

[0236] In specific embodiments of any of these methods, the patient receives two, three, or four doses. In certain embodiments, the patient is administered a multivalent immunogenic composition of the invention (e.g., stem cell transplant). (Immunosuppressive regimen after cell transplantation) immunocompromised, with three doses.

[0237] In some embodiments, the amount of time between administration of each dose of the multivalent immunogenic composition of the invention is: In an alternative embodiment, the administration of each dose of the multivalent immunogenic composition of the present invention is from about 4 weeks to about 1 year. The duration of the training period ranges from approximately one month to approximately five years.

[0238] In any embodiment of the method of the present invention, the patient treated with the composition(s) of the present invention The patient is a human. In certain embodiments, the human patient is a young child (approximately 12 to 24 months of age). The compositions of the present invention may also be used in older children, adolescents, or children (approximately 2-5 years of age). and adults (e.g., 18–45 years, 18–50 years, 18–55 years, 18–60 years, or In other embodiments of any of the methods of the present invention, The patient is between about 2 and about 18 years of age. In a further embodiment of any of the methods of the present invention, the patient is 18 years of age or older.

[0239] In a further embodiment of the method of the invention, the human patient is elderly. In some embodiments of the present invention, the patient is 50 years of age or older. In some embodiments, the patient is 55 years of age or older. In an embodiment, the patient is 60 years of age or older. In some embodiments, the patient is 65 years of age or older. In additional embodiments of any of the methods of the invention, the patient is over 70 years old.

[0240] In some embodiments of any of the methods of the present invention, the subject is treated with an immunogenic composition of the present invention. Patients affected are immunocompromised.

[0241] In some embodiments of any of the methods of the invention, the multivalent immunogenic composition is In certain embodiments, the vaccine is administered simultaneously with a vaccine against influenza. Chin is a "senior flu vaccine," a vaccine needed for older people, such as those aged 65 and over. It is a high-dose influenza vaccine.

[0242] The present invention provides a method for inducing a protective immune response in a patient against pneumococcal infection, comprising administering to the patient an immunization a therapeutically effective amount of the multivalent immunogenic pneumococcal polysaccharide-protein conjugate described herein. The present invention provides a method for administering to a patient a vaccine comprising administering to the patient any of the vaccine compositions. The optimal amount of the components (i.e., the multivalent immunogenic composition) is determined in view of an appropriate immune response in the subject. This can be confirmed by standard tests, including observations. For example, in another embodiment, Dosages for vaccination are determined by extrapolation from animal studies to human data. In another embodiment, dosages are empirically determined.

[0243] The method of the present invention can be applied to invasive infections (meningitis, pneumonia, and bacteremia) and non-invasive infections (acute Microorganisms, including both otitis media and sinusitis, such as Streptococcus pneumoniae (S. pneumoniae) ) can be used to prevent and / or reduce the primary clinical syndrome caused by Cut.

[0244] Administration of the compositions of the present invention may be by injection via intramuscular, intraperitoneal, intradermal or subcutaneous routes; may include one or more of oral / dietary, mucosal administration to the respiratory or genitourinary tract. In one embodiment, intranasal administration is used to treat pneumonia or otitis media (e.g., pneumococcal nasal to more effectively prevent pharyngeal carriage, thereby attenuating the infection at its early stage In a specific embodiment, the compositions of the invention are administered to a patient via intramuscular or subcutaneous administration. It is given.

[0245] All publications mentioned herein are incorporated by reference in their entirety for purposes of illustration only and are not intended to be limiting unless otherwise specified. ... No. 6,399,423, filed on Oct. 1, 2007, and incorporated by reference for purposes of describing and disclosing the fees charged thereto.

[0246] Although different embodiments of the present invention have been described herein with reference to the accompanying drawings, the present invention It is understood that the scope of the invention is not limited to these precise embodiments, but is instead defined by the appended claims. Various changes and modifications may be made therein by those skilled in the art without departing from the spirit or scope of the present invention. It should be understood that this may be the case.

[0247] The following examples illustrate, but do not limit, the present invention.

[0248] [Example 1] Preparation of Pneumococcal (S. Pneumoniae) Capsular Polysaccharides Methods for culturing pneumococci are well known in the art. See, e.g., Chase, 1967 ,Methods of Immunology and Immunochemist See, e.g., J. Med. Chem. Soc. 1:52. Methods for preparing pneumococcal capsular polysaccharides are also well known in the art. See, for example, European Patent No. 0 497 524. The method generally follows that described in EP 0 497 524, Generally applicable to all pneumococcal serotypes.

[0249] Isolates of pneumococcal strains of serotypes 6C, 23B, and 31 were identified by the U.S. Department of Disease Control and Prevention. Serotypes 3, 8, 10A, 11A, and 12 were obtained from the Center for Strain Research (Atlanta, GA). Strains for F, 15B, 22F, and 33F were obtained from the University of Pennsylvania (Robert Strains for serotypes 17F and 19A were obtained from The serotype 7F was obtained from the FDA Office of Biologics (Dr. John Robbins). New York State University, Downstate Medical Center (Gerald Schmidt) Isolates of pneumococcal serotypes not listed above were obtained from the American Cell lineages were obtained from the Italian Type Culture Collection (Manassas, VA). were distinguished based on the Querung reaction using specific antisera. See, e.g., 847,112. The resulting isolates were grown on soybean peptides without hemin. Agar plates consisting of an animal component-free medium containing tonsils, yeast extract, and glucose Further clonal isolation was performed by successively plating on serotype 7F in two steps. The agar plates used also contained hemin. Clonal isolates for each serotype were Soybean peptone, yeast extract, HEPES, sodium chloride, sodium bicarbonate, potassium phosphate Liquid culture using an animal component-free medium containing sodium, glucose, and glycerol The cells were further expanded in culture to prepare a pre-master cell bank.

[0250] The production of each serotype of pneumococcal polysaccharide involves cell expansion and batch production fermentation followed by downstream The purification consisted of chemical inactivation prior to soy peptone or soy peptone ultrafiltrate, Yeast extract or yeast extract ultrafiltrate, HEPES, sodium chloride, sodium bicarbonate Contains a sterile, animal component-free growth medium containing cereals, potassium phosphate, and glucose. Expand thawed cell bank vials of each serotype using shake flasks or culture bottles. Cell expansion cultures were grown in sealed shake flasks or bottles to control temperature and agitation. Gas exchange was minimized by controlling serotypes 3, 7F, 8, 9N, 10A, 11A, and 12F. For 15B, 17F, 19A, 20, 22F and 33F, fermenters containing the same medium were used. During cell expansion of these serotypes, Temperature, pH, pressure and agitation were controlled. Sparging was not used, so aeration over The incubation period was also controlled. A specific culture density was reached, as measured by optical density at 600 nm. Afterwards, a portion of the cell expansion culture was cultured in a medium containing soybean peptone or soybean peptone ultrafiltrate, yeast extract, or yeast extract ultrafiltrate, containing sodium chloride, potassium phosphate, and glucose The culture was transferred to a production fermenter containing sterile, animal-component-free growth medium. Temperature, pH, pressure and The aeration overlay was also controlled since no sparging was used. .

[0251] Once glucose is almost depleted, the batch is started by adding phenol, a chemical deactivator. Fermentation was terminated. Cells were inactivated by adding pure phenol to a final concentration of 0.8–1.2%. The primary inactivation was performed in the fermenter for the specified time. After primary inactivation, the batch is transferred to a separate container. This is then held at a controlled temperature for a further specified time and agitated to ensure complete inactivation. This was confirmed by microbial plating techniques or by verification of phenol concentrations and specified times. The inactivated broth was then purified.

[0252] [Example 2] Purification of pneumococcal polysaccharides Several methods for the purification of pneumococcal polysaccharides include centrifugation, depth filtration, concentration / diafiltration, and All procedures consisted of a treatment and precipitation step. was carried out at room temperature.

[0253] Inactivated broth from fermenter cultures of S. pneumoniae was cationized. Resistant polymer (BPA-1000, TRETOLITE® (Baker Hull) Hes Inc., Houston, TX), Spectrum 8160, Poly(ethylene Cationic surfactants were used to aggregate the cellulose with methylaminoimine and Millipore pDADMAC. The polymer bound to impurity proteins, nucleic acids, and cell debris. After this period, the flocculated solids were removed via centrifugation and multiple depth filtration steps. Concentrate the clarified broth to a MWCO (molecular weight cut-off) of 100 kDa to 500 kDa. The solution was diafiltered using a filter. Diafiltration was achieved using sodium phosphate buffer. Residual nucleic acids and proteins were removed by filtration.

[0254] Sodium acetate and phenacetin with denatured alcohol and / or isopropanol Further removal of impurities was achieved by reprecipitation of the polysaccharides in ethanol. In the cup, sodium acetate and phenol (liquefied phenol) in sodium phosphate saline buffer The diafiltration retentate was then charged with phenol (pure phenol or solid phenol). The alcohol fractionation of polysaccharides was carried out in two stages. In the first stage, low-concentration alcohol was prepared. was added to precipitate cell debris and other unwanted impurities, while the crude polysaccharides remained in solution. Impurities were removed via centrifugation followed by a depth filtration step. Adding additional isopropanol or denatured alcohol to the batch will remove the The precipitated polysaccharide pellet was collected by centrifugation, triturated, and powdered. The mixture was dried and stored frozen at -70°C.

[0255] [Example 3] Structural identity analysis of certain pneumococcal serotypes by NMR studies. 0.01% dimethyl sulfoxide (DMSO) and 0.01% 2,2-dimethyl-2 -Silapentane-5-sulfonate-d6 sodium salt (DSS-d6) containing heavy water (D Prepare the polysaccharide powder for NMR analysis by dissolving it in 20 mL of HCl at 5 mg powder / mL of solution. DMSO was the internal standard used for quantitative analysis, and DSS-d6 was the chemical standard. The NMR data set was used to set the soft scale to 0 ppm. was acquired at 50°C, and the portion of the spectrum containing the anomeric resonances was then analyzed using a Microsof t Save x,y coordinates to an ASCII file for analysis using an Excel workbook The Y coordinate (i.e., the spectral profile) was then written selectively as a target. The spectral profiles of capsular bacterial polysaccharides were compared with those in a reference database. The file is then created for the selected preparation of each serotype, which is designated as the reference lot. The y values ​​from the sample spectrum and the reference spectrum were compared pairwise to determine the The correlation coefficient (ρ) is used as a measure of similarity between xy ) was created. A ρ value of .95 or greater was considered a positive identification of the polysaccharide structure.

[0256] Figures 1 to 4 show the results of S. pneumoniae serotypes 6C, 15A, and 16C, respectively. 600MHz one-dimensional analysis of de-O-acetylated 15B and 35B capsular polysaccharides 1 H NMR Provides a spectrum. Streptococcus pneumoniae (S. pneumonia) serotype 6C, 15A, deoxyribonucleic acid Used for acetylated 15B and 35B serotyping 1 The H NMR identification region is These are provided in FIGS. 5 to 8, respectively.

[0257] De-O-acetylated capsules of Streptococcus pneumoniae (S. pneumoniae) serotypes 15B and 15C Membrane polysaccharide structure An immunogenicity study was conducted using PCV21 in New Zealand White rabbits (see below). (See Example 43 below.) In these studies, de-O-acetylated polysaccharides were used instead of serotype 15C. Saccharide serotype 15B was used in the polyvalent composition. NMR studies were performed to confirm the de-O-acetylation of 15B. It was confirmed that the B polysaccharide is equivalent to the serotype 15C polysaccharide.

[0258] Structural differences between capsular polysaccharide serotypes are manifested as differences in chemical shifts in NMR spectra. The anomeric region (approximately 4.4 ppm to 6.0 ppm) contains all the structures of the repeating units of polysaccharides. It is sensitive to structural features, such as differences in stereochemistry, monosaccharide composition, O-acetylation, and glycosidic linkages. However, leaving this region of the spectrum unique to each serotype, the chemical shifts of the anomeric signals In the case of de-O-acetylated serotype 15B polysaccharide, the complete 1 H NMR spectrum Toru is a serotype 15C polysaccharide 1 The H NMR spectra are identical, and the repeating units of both polysaccharides are These results show that the saccharides are composed of the same monosaccharide substituents and glycosidic binding sites (Figures 9B-9C 10B-10C). The de-O-acetylation step removes the 15B polysaccharide. It was confirmed that the O-acetate groups present in the hydroxyl group were removed and that the O-acetate groups observed were essentially It is also clearly shown that no sebum remains in the blood (see FIGS. 10A to 10F).

[0259] [Example 4] Preparation of serotype 3 conjugates for multivalent testing using DMSO conjugation Polysaccharides are dissolved, sized to a target molecular mass, chemically activated, and purified by ultrafiltration. The activated polysaccharide and purified CRM 197 were lyophilized separately. The polysaccharide solution was then redissolved in dimethyl sulfoxide (DMSO). The and CRM197 solutions were combined and conjugated as follows. The gate was purified by ultrafiltration before a final 0.2 micron filtration. Several process parameters within each step, such as temperature and time, can be controlled to achieve the desired A conjugate with the desired properties was obtained.

[0260] Size reduction and oxidation of polysaccharides Purified pneumococcal Ps capsule powder was dissolved in water and filtered through a 0.45 micron filter. The polysaccharides were homogenized to reduce the molecular mass of Ps. The homogenization pressure and homogenizer The number of passes was controlled at 380 bar / 5 passes.

[0261] Size-reduced polysaccharides were concentrated and filtered through a 10 kDa NMWCO tangential flow ultrafiltration membrane. The column was diafiltered against water using a filtration filter.

[0262] The polysaccharide solution was then heated to 22°C and adjusted to pH 5 with sodium acetate buffer to activate Polysaccharide activation was performed with 100 mM sodium metaperiodate. The amount of sodium metaperiodate added was determined by the amount of polysaccharide activity. To achieve the target level of polymerization, 0.25 moles of metaperiodic acid were added per mole of polysaccharide repeating unit. Sodium urate (moles of aldehyde per mole of polysaccharide repeating unit). Oxidation reaction The reaction proceeded for 12 hours at 22°C.

[0263] The activated product was diafiltrated against 10 mM potassium phosphate, pH 6.4. followed by filtration using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Diafiltration was performed against water at 2-8°C.

[0264] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876), Pseudomonas fluorescens (Pseudomonas fluorescens) Purified CRM197 obtained through expression in C. fluorescens , filtered through a 5 kDa NMWCO tangential flow ultrafiltration membrane in 2 mM phosphate, pH 7. The solution was diafiltered against 0.2 buffer and 0.2 micron filtered.

[0265] The activated polysaccharide was lyophilized at 2 mg Ps / mL with a sucrose concentration of 10% w / v. CRM197 was formulated for drying at 6 mg Pr / mL with a sucrose concentration of 1% w / v. It was formulated for lyophilization.

[0266] The formulated Ps and CRM197 solutions were lyophilized separately. The polysaccharide solution and CRM197 materials were separately redissolved in an equal volume of DMSO. The 197 solutions were blended to give polysaccharide concentrations of 1.0 g Ps / L and polysaccharide and CRM197. The mass ratio of the resulting conjugate polysaccharide to CRM197 was 1.3. The ratio of sodium cyanoborohydride (1 mole of polysaccharide repeating unit) was selected to control the (1 mole per 1000 mg of ATP) was added and conjugation was allowed to proceed for 1 hour at 22°C.

[0267] Reduction with sodium borohydride Conjugation with sodium borohydride (2 moles per mole of polysaccharide repeating unit) After the reaction, the mixture was added and incubated at 22°C for 1.6 hours. The batch was then cooled to approximately 4°C and It was diluted in 150 mM sodium chloride containing 0.025% (w / v) polysorbate 20. The pH was then neutralized by adding potassium phosphate buffer. The batch was concentrated and Using a Da NMWCO tangential flow ultrafiltration membrane, 150 mM sodium chloride, 2 Diafiltered against 5 mM potassium phosphate pH 7 at approximately 4°C.

[0268] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The solution was then incubated at 4°C in 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20. The solution was diafiltered against 10 mM histidine in 10 ml of ethanol, pH 7.0.

[0269] The retentate batch was filtered to 0.2 microns and then diluted with 0.015% (w / v) Polysorbate Dilute with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 containing 20 mg of PEG-20. The solution was diluted, divided into aliquots, and frozen at -60°C or below. [Table 2]

[0270] [Example 5] Preparation of serotype 6C for conjugate multivalent testing using DMSO conjugation The polysaccharides were dissolved, chemically activated, and buffer exchanged by ultrafiltration. The polysaccharide and purified CRM197 were separately lyophilized and redissolved in DMSO. The reconstituted polysaccharide solution and CRM197 solution were combined and conjugated as described below. The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration. Several process parameters within each step such as pH, temperature, concentration and time were adjusted. The parameters were controlled to obtain conjugates with desired attributes.

[0271] Polysaccharide oxidation The purified pneumococcal Ps powder was dissolved in water and filtered through a 0.45 micron filter. The digested polysaccharides were concentrated and filtered using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The solution was diafiltered against

[0272] The polysaccharide solution was then heated to 22°C and adjusted to pH 5 with sodium acetate buffer to activate Polysaccharide activation was performed with 100 mM sodium metaperiodate. The amount of sodium metaperiodate added was determined by the amount of polysaccharide activity. To achieve the target level of polymerization, 0.10 moles of metaperiodic acid were added per mole of polysaccharide repeating unit. Sodium urate (moles of aldehyde per mole of polysaccharide repeating unit). Oxidation reaction The reaction proceeded for 2 hours at 22°C.

[0273] The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4. and subsequently purified using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was carried out at 2-8°C.

[0274] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876), Pseudomonas fluorescens (Pseudomonas fluorescens) Purified CRM197 obtained through expression in C. fluorescens , filtered through a 5 kDa NMWCO tangential flow ultrafiltration membrane in 2 mM phosphate, pH 7. The solution was diafiltered against 0.2 buffer and 0.2 micron filtered.

[0275] The activated polysaccharide was lyophilized at 6 mg Ps / mL in 5% w / v sucrose concentration. CRM197 was formulated at 6 mg Pr / mL with a sucrose concentration of 1% w / v. Formulated for lyophilization.

[0276] The formulated Ps and CRM197 solutions were lyophilized separately. The polysaccharide solution and CRM197 materials were separately redissolved in an equal volume of DMSO. The 197 solution was blended to give a polysaccharide concentration of 2.2 g Ps / L and a polysaccharide and CRM197 The mass ratio of the resulting conjugate polysaccharide to CRM197 was 1.4. The ratio of sodium cyanoborohydride (1 mole of polysaccharide repeating unit) was selected to control the (1 mole per 1000 mg of ATP) was added and the conjugation was allowed to proceed for 15 hours at 22°C.

[0277] Reduction with sodium borohydride Conjugation with sodium borohydride (2 moles per mole of polysaccharide repeating unit) After the reaction, the mixture was added and incubated at 22°C for 3 hours. The batch was then heated at approximately 4°C and Diluted in 150 mM sodium chloride containing 0.025% (w / v) polysorbate 20. The pH was then neutralized by adding potassium phosphate buffer. NMWCO tangential flow ultrafiltration membranes were used to filter 150 mM sodium chloride, 25 mM The solution was diafiltered against 1 M potassium phosphate pH 7 at approximately 4°C.

[0278] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The solution was then incubated at 4°C in 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20. The solution was diafiltered against 10 mM histidine in 10 ml of ethanol, pH 7.0.

[0279] The retentate batch was filtered to 0.2 microns and then diluted with 0.015% (w / v) polysorbate. Dilute with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 containing 20 mg of PEG-20. The solution was diluted, divided into aliquots, and frozen at -60°C or below. [Table 3]

[0280] [Example 6] Preparation of serotype 6C conjugates for multivalent testing using DMSO conjugation The polysaccharides were dissolved, chemically activated, and buffer exchanged by ultrafiltration. The polysaccharide and purified CRM197 were separately lyophilized and redissolved in DMSO. The reconstituted polysaccharide solution and CRM197 solution were combined and conjugated as described below. The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration. Several process parameters within each step such as pH, temperature, concentration and time were adjusted. The parameters were controlled to obtain conjugates with desired attributes.

[0281] Polysaccharide oxidation The purified pneumococcal Ps powder was dissolved in water and filtered through a 0.45 micron filter. The digested polysaccharides were concentrated and filtered using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The solution was diafiltered against

[0282] The polysaccharide solution was then heated to 22°C and adjusted to pH 5 with sodium acetate buffer to activate Polysaccharide activation was performed with 100 mM sodium metaperiodate. The amount of sodium metaperiodate added was determined by the amount of polysaccharide activity. To achieve the target level of polymerization, 0.10 moles of metaperiodic acid were added per mole of polysaccharide repeating unit. Sodium urate (moles of aldehyde per mole of polysaccharide repeating unit). Oxidation reaction The reaction proceeded for 2 hours at 22°C.

[0283] The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4. and subsequently purified using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was carried out at 2-8°C.

[0284] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876), Pseudomonas fluorescens (Pseudomonas fluorescens) Purified CRM197 obtained through expression in C. fluorescens , filtered through a 5 kDa NMWCO tangential flow ultrafiltration membrane in 2 mM phosphate, pH 7. The solution was diafiltered against 0.2 buffer and 0.2 micron filtered.

[0285] The activated polysaccharide was lyophilized at 6 mg Ps / mL in 5% w / v sucrose concentration. CRM197 was formulated at 6 mg Pr / mL with a sucrose concentration of 1% w / v. Formulated for lyophilization.

[0286] The formulated Ps and CRM197 solutions were lyophilized separately. The polysaccharide solution and CRM197 materials were separately redissolved in an equal volume of DMSO. The 197 solution was blended to give a polysaccharide concentration of 1.9 g Ps / L and a polysaccharide and CRM197 The mass ratio of the resulting conjugate polysaccharide to CRM197 was 1.4. The ratio of sodium cyanoborohydride (1 mole of polysaccharide repeating unit) was selected to control the (1 mole per 1000 mg of ATP) was added and the conjugation was allowed to proceed for 15 hours at 22°C.

[0287] Reduction with sodium borohydride Conjugation with sodium borohydride (2 moles per mole of polysaccharide repeating unit) After the reaction, the mixture was added and incubated at 22°C for 3 hours. The batch was then heated at approximately 4°C and Diluted in 150 mM sodium chloride containing 0.025% (w / v) polysorbate 20. The pH was then neutralized by adding potassium phosphate buffer. NMWCO tangential flow ultrafiltration membranes were used to filter 150 mM sodium chloride, 25 mM The solution was diafiltered against 1 M potassium phosphate pH 7 at approximately 4°C.

[0288] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The solution was then incubated at 4°C in 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20. The solution was diafiltered against 10 mM histidine in 10 ml of ethanol, pH 7.0.

[0289] The retentate batch was filtered to 0.2 microns and then diluted with 0.015% (w / v) Polysorbate Dilute with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 containing 20 mg of PEG-20. The solution was diluted, divided into aliquots, and frozen at -60°C or below. [Table 4]

[0290] Preparation of serotype 6A conjugates for monovalent testing using DMSO conjugation The polysaccharides were dissolved, chemically activated, and buffer exchanged by ultrafiltration. The polysaccharide and purified CRM197 were separately lyophilized and redissolved in DMSO. The reconstituted polysaccharide solution and CRM197 solution were combined and conjugated as described below. The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration. Several process parameters within each step such as pH, temperature, concentration and time were adjusted. The parameters were controlled to obtain conjugates with desired attributes.

[0291] Size reduction and oxidation of polysaccharides Purified pneumococcal Ps capsule powder was dissolved in water and filtered through a 0.45 micron filter. The polysaccharides were homogenized to reduce the molecular mass of Ps. The homogenization pressure and homogenizer The number of passes was controlled at 200 bar / 5 passes. The size-reduced polysaccharides were concentrated and Diafiltrate against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane It was rationed.

[0292] The polysaccharide solution was then heated to 22°C and adjusted to pH 5 with sodium acetate buffer to activate Polysaccharide activation was performed with 100 mM sodium metaperiodate. The amount of sodium metaperiodate added was determined by the amount of polysaccharide activity. To achieve the target level of polymerization, 0.10 moles of metaperiodic acid were added per mole of polysaccharide repeating unit. Sodium urate (moles of aldehyde per mole of polysaccharide repeating unit). Oxidation reaction The reaction proceeded for 2 hours at 22°C.

[0293] The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4. and subsequently purified using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was carried out at 2-8°C.

[0294] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876), Pseudomonas fluorescens (Pseudomonas fluorescens) Purified CRM197 obtained through expression in C. fluorescens , filtered through a 5 kDa NMWCO tangential flow ultrafiltration membrane in 2 mM phosphate, pH 7. The solution was diafiltered against 0.2 buffer and 0.2 micron filtered.

[0295] The activated polysaccharide was lyophilized at 6 mg Ps / mL in 5% w / v sucrose concentration. CRM197 was formulated at 6 mg Pr / mL with a sucrose concentration of 1% w / v. Formulated for lyophilization.

[0296] The formulated Ps and CRM197 solutions were lyophilized separately. The polysaccharide solution and CRM197 materials were separately redissolved in an equal volume of DMSO. The 197 solution was blended to give a polysaccharide concentration of 1.5 g Ps / L and a polysaccharide and CRM197 The mass ratio of the resulting conjugate polysaccharide to CRM197 was 1.4. The ratio of sodium cyanoborohydride (1 mole of polysaccharide repeating unit) was selected to control the (1 mole per 1000 mg of ATP) was added and the conjugation was allowed to proceed for 15 hours at 22°C.

[0297] Reduction with sodium borohydride Conjugation with sodium borohydride (2 moles per mole of polysaccharide repeating unit) After the reaction, the mixture was added and incubated at 22°C for 3 hours. The batch was then heated at approximately 4°C and Diluted in 150 mM sodium chloride containing 0.025% (w / v) polysorbate 20. The pH was then neutralized by adding potassium phosphate buffer. NMWCO tangential flow ultrafiltration membranes were used to filter 150 mM sodium chloride, 25 mM The solution was diafiltered against 1 M potassium phosphate pH 7 at approximately 4°C.

[0298] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The solution was then incubated at 4°C in 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20. The solution was diafiltered against 10 mM histidine in 10 ml of ethanol, pH 7.0.

[0299] The retentate batch was filtered to 0.2 microns and then diluted with 0.015% (w / v) Polysorbate Dilute with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 containing 20 mg of PEG-20. The solution was diluted, divided into aliquots, and frozen at -60°C or below. [Table 5]

[0300] [Example 7] Preparation of serotype 7F conjugates for multivalent testing using DMSO conjugation Polysaccharides are dissolved, sized to a target molecular mass, chemically activated, and purified by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately. The polysaccharide solution was then redissolved in DMSO. The redissolved polysaccharide solution and CRM197 solution were then mixed together. The resulting conjugate was added to the final 0.2 microgram of The mixture was purified by ultrafiltration before filtration. Each step, such as pH, temperature, concentration and time, was Several process parameters within the process can be controlled to produce conjugates with desired attributes. obtained.

[0301] Size reduction and oxidation of polysaccharides Purified pneumococcal Ps capsule powder was dissolved in water and filtered through a 0.45 micron filter. The polysaccharides were homogenized to reduce the molecular mass of Ps. The homogenization pressure and homogenizer The number of passes was controlled at 150 bar / 7 passes.

[0302] Size-reduced polysaccharides were concentrated and filtered through a 10 kDa NMWCO tangential flow ultrafiltration membrane. The column was diafiltered against water using a filtration filter.

[0303] The polysaccharide solution was then heated to 22°C and adjusted to pH 5 with sodium acetate buffer to activate Polysaccharide activation was performed with 100 mM sodium metaperiodate. The amount of sodium metaperiodate added was determined by the amount of polysaccharide activity. To achieve the target level of polymerization, 0.24 moles of metaperiodic acid were added per mole of polysaccharide repeating unit. Sodium urate (moles of aldehyde per mole of polysaccharide repeating unit). Oxidation reaction The reaction proceeded for 4 hours at 4°C.

[0304] The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4. and subsequently purified using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was carried out at 2-8°C.

[0305] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876), Pseudomonas fluorescens (Pseudomonas fluorescens) Purified CRM197 obtained through expression in C. fluorescens , filtered through a 5 kDa NMWCO tangential flow ultrafiltration membrane in 2 mM phosphate, pH 7. The solution was diafiltered against 0.2 buffer and 0.2 micron filtered.

[0306] The activated polysaccharide was lyophilized at 6 mg Ps / mL in 5% w / v sucrose concentration. CRM197 was formulated at 6 mg Pr / mL with a sucrose concentration of 1% w / v. Formulated for lyophilization.

[0307] The formulated Ps and CRM197 solutions were lyophilized separately. The polysaccharide solution and CRM197 materials were separately redissolved in an equal volume of DMSO. The 197 solution was blended to give a polysaccharide concentration of 2.6 g Ps / L and a polysaccharide and CRM197 The mass ratio of the resulting conjugate polysaccharide to CRM197 was 1.5. The ratio of sodium cyanoborohydride (1 mole of polysaccharide repeating unit) was selected to control the (1 mole per 1000 mg of ATP) was added and conjugation was allowed to proceed for 4 hours at 22°C.

[0308] Reduction with sodium borohydride Conjugation with sodium borohydride (2 moles per mole of polysaccharide repeating unit) After the reaction, the mixture was added and incubated at 22°C for 3 hours. The batch was then heated at approximately 4°C and Diluted in 150 mM sodium chloride containing 0.025% (w / v) polysorbate 20. The pH was then neutralized by adding potassium phosphate buffer. NMWCO tangential flow ultrafiltration membranes were used to filter 150 mM sodium chloride, 25 mM The solution was diafiltered against 1 M potassium phosphate pH 7 at approximately 4°C.

[0309] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The solution was then incubated at 4°C in 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20. The solution was diafiltered against 10 mM histidine in 10 ml of ethanol, pH 7.0.

[0310] The retentate batch was filtered to 0.2 microns and then diluted with 0.015% (w / v) Polysorbate Dilute with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 containing 20 mg of PEG-20. The solution was diluted, divided into aliquots, and frozen at -60°C or below. [Table 6]

[0311] [Example 8] Preparation of serotype 8 conjugates for multivalent testing using DMSO conjugation Polysaccharides are dissolved, sized to a target molecular mass, chemically activated, and purified by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately. The polysaccharide solution was then redissolved in DMSO. The redissolved polysaccharide solution and CRM197 solution were then mixed together. The resulting conjugate was added to the final 0.2 microgram of The mixture was purified by ultrafiltration before filtration. Each step, such as pH, temperature, concentration and time, was Several process parameters within the process can be controlled to produce conjugates with desired attributes. obtained.

[0312] Size reduction and oxidation of polysaccharides Purified pneumococcal Ps capsule powder was dissolved in water and filtered through a 0.45 micron filter. The polysaccharides were homogenized to reduce the molecular mass of Ps. The homogenization pressure and homogenizer The number of passes was controlled at 600 bar / 5 passes.

[0313] Size-reduced polysaccharides were concentrated and filtered through a 5 kDa NMWCO tangential flow ultrafiltration membrane. Diafiltration was performed against water using

[0314] The polysaccharide solution was then heated to 22°C and adjusted to pH 5 with sodium acetate buffer to activate Polysaccharide activation was performed with 100 mM sodium metaperiodate. The amount of sodium metaperiodate added was determined by the amount of polysaccharide activity. To achieve the target level of polymerization, 0.18 moles of metaperiodic acid were added per mole of polysaccharide repeating unit. Sodium urate (moles of aldehyde per mole of polysaccharide repeating unit). Oxidation reaction The reaction proceeded for 4 hours at 22°C.

[0315] The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4. and subsequently filtered into water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was carried out at 2-8°C.

[0316] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876), Pseudomonas fluorescens (Pseudomonas fluorescens) Purified CRM197 obtained through expression in C. fluorescens , filtered through a 5 kDa NMWCO tangential flow ultrafiltration membrane in 2 mM phosphate, pH 7. The solution was diafiltered against 0.2 buffer and 0.2 micron filtered.

[0317] The activated polysaccharide was lyophilized at 6 mg Ps / mL in 5% w / v sucrose concentration. CRM197 was formulated at 6 mg Pr / mL with a sucrose concentration of 1% w / v. Formulated for lyophilization.

[0318] The formulated Ps and CRM197 solutions were lyophilized separately. The polysaccharide solution and CRM197 materials were separately redissolved in an equal volume of DMSO. The 197 solution was blended to obtain a polysaccharide concentration of 4.0 g Ps / L and a polysaccharide and CRM197 The mass ratio of the resulting conjugate polysaccharide to CRM197 was 1.5. After blending, the conjugation reaction was carried out at 22°C for 2 hours. The addition of sodium cyanoborohydride accelerated the conjugation of serotype 8. It was observed that irreversible precipitation may occur during the reaction, so cyanoborohydride No sodium was added to the conjugation reaction. Omitting thorium avoids precipitation without significantly affecting the properties of the conjugate. was done.

[0319] Reduction with sodium borohydride Conjugation with sodium borohydride (2 moles per mole of polysaccharide repeating unit) After the reaction, the mixture was added and incubated at 22°C for 1 hour. The batch was then heated at approximately 4°C and Diluted in 150 mM sodium chloride containing 0.025% (w / v) polysorbate 20. The pH was then neutralized by adding potassium phosphate buffer. NMWCO tangential flow ultrafiltration membranes were used to filter 150 mM sodium chloride, 25 mM The solution was diafiltered against 1 M potassium phosphate pH 7 at approximately 4°C.

[0320] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The solution was then incubated at 4°C in 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20. The solution was diafiltered against 10 mM histidine in 10 ml of ethanol, pH 7.0.

[0321] The retentate batch was filtered to 0.2 microns (using a 0.5 micron prefilter) and then 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20, pH 7 Dilute with additional 10 mM histidine in 0.0 mL of PBS, dispense into aliquots, and freeze at -60°C or below. did. [Table 7]

[0322] [Example 9] Preparation of serotype 9N conjugates for multivalent testing using DMSO conjugation Polysaccharides are dissolved, sized to a target molecular mass, chemically activated, and purified by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately. The polysaccharide solution was then redissolved in DMSO. The redissolved polysaccharide solution and CRM197 solution were then mixed together. The resulting conjugate was added to the final 0.2 microgram of The mixture was purified by ultrafiltration before filtration. Each step, such as pH, temperature, concentration and time, was Several process parameters within the process can be controlled to produce conjugates with desired attributes. obtained.

[0323] Size reduction and oxidation of polysaccharides Purified pneumococcal Ps capsule powder was dissolved in water and filtered through a 0.45 micron filter. The polysaccharides were homogenized to reduce the molecular mass of Ps. The homogenization pressure and homogenizer The number of passes was controlled at 250 bar / 5 passes.

[0324] Size-reduced polysaccharides were concentrated and filtered through a 10 kDa NMWCO tangential flow ultrafiltration membrane. The column was diafiltered against water using a filtration filter.

[0325] The polysaccharide solution was then heated to 22°C and adjusted to pH 5 with sodium acetate buffer to activate Polysaccharide activation was performed with 100 mM sodium metaperiodate. The amount of sodium metaperiodate added was determined by the amount of polysaccharide activity. To achieve the target level of polymerization, 0.16 moles of metaperiodic acid were added per mole of polysaccharide repeating unit. Sodium urate (moles of aldehyde per mole of polysaccharide repeating unit). Oxidation reaction The reaction proceeded for 4 hours at 22°C.

[0326] The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4. and subsequently purified using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was carried out at 2-8°C.

[0327] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876), Pseudomonas fluorescens (Pseudomonas fluorescens) Purified CRM197 obtained through expression in C. fluorescens , filtered through a 5 kDa NMWCO tangential flow ultrafiltration membrane in 2 mM phosphate, pH 7. The solution was diafiltered against 0.2 buffer and 0.2 micron filtered.

[0328] The activated polysaccharide was lyophilized at 6 mg Ps / mL in 5% w / v sucrose concentration. CRM197 was formulated at 6 mg Pr / mL with a sucrose concentration of 1% w / v. Formulated for lyophilization.

[0329] The formulated Ps and CRM197 solutions were lyophilized separately. The polysaccharide solution and CRM197 materials were separately redissolved in an equal volume of DMSO. The 197 solutions were blended to obtain a polysaccharide concentration of 3.25 g Ps / L and a polysaccharide and CRM19 A mass ratio of 1.5 was achieved for the resulting conjugate polysaccharide to CRM19. The ratio of sodium cyanoborohydride (polysaccharide repeating unit 1) was selected to control the (1 mole per unit) was added and conjugation was allowed to proceed for 1 hour at 22°C.

[0330] Reduction with sodium borohydride Conjugation with sodium borohydride (2 moles per mole of polysaccharide repeating unit) After the reaction, the mixture was added and incubated at 22°C for 1 hour. The batch was then heated at approximately 4°C and Diluted in 150 mM sodium chloride containing 0.025% (w / v) polysorbate 20. The pH was then neutralized by adding potassium phosphate buffer. NMWCO tangential flow ultrafiltration membranes were used to filter 150 mM sodium chloride, 25 mM The solution was diafiltered against 1 M potassium phosphate pH 7 at approximately 4°C.

[0331] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The solution was then incubated at 4°C in 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20. The solution was diafiltered against 10 mM histidine in 10 ml of ethanol, pH 7.0.

[0332] The retentate batch was filtered to 0.2 microns and then diluted with 0.015% (w / v) polysorbate. Dilute with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 containing 20 mg of PEG-20. The solution was diluted, divided into aliquots, and frozen at -60°C or below. [Table 8]

[0333] [Example 10] Preparation of serotype 10A conjugates for monovalent testing using DMSO conjugation Made Polysaccharides are dissolved, sized to a target molecular mass, chemically activated, and purified by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately. The polysaccharide solution was then redissolved in DMSO. The redissolved polysaccharide solution and CRM197 solution were then mixed together. The resulting conjugate was added to the final 0.2 microgram of The mixture was purified by ultrafiltration before filtration. Each step, such as pH, temperature, concentration and time, was Several process parameters within the process can be controlled to produce conjugates with desired attributes. obtained.

[0334] Size reduction and oxidation of polysaccharides Purified pneumococcal Ps capsule powder was dissolved in water and filtered through a 0.45 micron filter. The polysaccharides were homogenized to reduce the molecular mass of Ps. The homogenization pressure and homogenizer The number of passes is controlled to 200 bar / 5 passes, followed by 600 bar / 5 passes. The target molecular mass was achieved.

[0335] Size-reduced polysaccharides were concentrated and filtered through a 10 kDa NMWCO tangential flow ultrafiltration membrane. The column was diafiltered against water using a filtration filter.

[0336] The polysaccharide solution was then heated to 22°C and adjusted to pH 5 with sodium acetate buffer to activate Polysaccharide activation was performed with 100 mM sodium metaperiodate. The amount of sodium metaperiodate added was determined by the amount of polysaccharide activity. To achieve the target level of polymerization, 0.15 moles of metaperiodic acid were added per mole of polysaccharide repeating unit. Sodium urate (moles of aldehyde per mole of polysaccharide repeating unit). Oxidation reaction The reaction proceeded for 2 hours at 22°C.

[0337] The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4. and subsequently purified using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was carried out at 2-8°C.

[0338] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876), Pseudomonas fluorescens (Pseudomonas fluorescens) Purified CRM197 obtained through expression in C. fluorescens , filtered through a 5 kDa NMWCO tangential flow ultrafiltration membrane in 2 mM phosphate, pH 7. The solution was diafiltered against 0.2 buffer and 0.2 micron filtered.

[0339] The activated polysaccharide was lyophilized at 6 mg Ps / mL in 5% w / v sucrose concentration. CRM197 was formulated at 6 mg Pr / mL with a sucrose concentration of 1% w / v. Formulated for lyophilization.

[0340] The formulated Ps and CRM197 solutions were lyophilized separately. The polysaccharide solution and CRM197 materials were separately redissolved in an equal volume of DMSO. The 197 solution was blended to obtain a polysaccharide concentration of 5.0 g Ps / L and a polysaccharide and CRM197 The mass ratio of the polysaccharide to CRM197 in the resulting conjugate was 2.0. The ratio of sodium cyanoborohydride (1 mole of polysaccharide repeating unit) was selected to control the (1 mole per 1000 mg of ATP) was added and conjugation was allowed to proceed for 4 hours at 22°C.

[0341] Reduction with sodium borohydride Conjugation with sodium borohydride (2 moles per mole of polysaccharide repeating unit) After the reaction, the mixture was added and incubated at 22°C for 1 hour. The batch was then heated at approximately 4°C and Diluted in 150 mM sodium chloride containing 0.025% (w / v) polysorbate 20. The pH was then neutralized by adding potassium phosphate buffer. NMWCO tangential flow ultrafiltration membranes were used to filter 150 mM sodium chloride, 25 mM The solution was diafiltered against 1 M potassium phosphate pH 7 at approximately 4°C.

[0342] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The solution was then incubated at 4°C in 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20. The solution was diafiltered against 10 mM histidine in 10 ml of ethanol, pH 7.0.

[0343] The retentate batch was filtered to 0.2 microns (using a 0.5 micron prefilter) and then 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20, pH 7 Dilute with additional 10 mM histidine in 0.0 mL of PBS, dispense into aliquots, and freeze at -60°C or below. did. [Table 9]

[0344] [Example 11] Preparation of serotype 10A conjugates for multivalent testing using DMSO conjugation Made Polysaccharides are dissolved, sized to a target molecular mass, chemically activated, and purified by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately. The polysaccharide solution was then redissolved in DMSO. The redissolved polysaccharide solution and CRM197 solution were then mixed together. The resulting conjugate was added to the final 0.2 microgram of The mixture was purified by ultrafiltration before filtration. Each step, such as pH, temperature, concentration and time, was Several process parameters within the process can be controlled to produce conjugates with desired attributes. obtained.

[0345] Size reduction and oxidation of polysaccharides Purified pneumococcal Ps capsule powder was dissolved in water and filtered through a 0.45 micron filter. The polysaccharides were homogenized to reduce the molecular mass of Ps. The homogenization pressure and homogenizer The number of passes was controlled at 600 bar / 5 passes.

[0346] Size-reduced polysaccharides were concentrated and filtered through a 10 kDa NMWCO tangential flow ultrafiltration membrane. The column was diafiltered against water using a filtration filter.

[0347] The polysaccharide solution was then heated to 22°C and adjusted to pH 5 with sodium acetate buffer to activate Polysaccharide activation was performed with 100 mM sodium metaperiodate. The amount of sodium metaperiodate added was determined by the amount of polysaccharide activity. To achieve the target level of polymerization, 0.16 moles of metaperiodic acid were added per mole of polysaccharide repeating unit. Sodium urate (moles of aldehyde per mole of polysaccharide repeating unit). Oxidation reaction The reaction proceeded for 2 hours at 22°C.

[0348] The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4. and subsequently purified using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was carried out at 2-8°C.

[0349] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876), Pseudomonas fluorescens (Pseudomonas fluorescens) Purified CRM197 obtained through expression in C. fluorescens , filtered through a 5 kDa NMWCO tangential flow ultrafiltration membrane in 2 mM phosphate, pH 7. The solution was diafiltered against 0.2 buffer and 0.2 micron filtered.

[0350] The activated polysaccharide was lyophilized at 6 mg Ps / mL in 5% w / v sucrose concentration. CRM197 was formulated at 6 mg Pr / mL with a sucrose concentration of 1% w / v. Formulated for lyophilization.

[0351] The formulated Ps and CRM197 solutions were lyophilized separately. The polysaccharide solution and CRM197 materials were separately redissolved in an equal volume of DMSO. The 197 solution was blended to obtain a polysaccharide concentration of 4.0 g Ps / L and a polysaccharide and CRM197 The mass ratio of the resulting conjugate polysaccharide to CRM19 was 1.75. The ratio of sodium cyanoborohydride (polysaccharide repeating unit 1) was selected to control the 1 mole per unit was added and conjugation was allowed to proceed for 4 hours at 22°C.

[0352] Reduction with sodium borohydride Conjugation with sodium borohydride (2 moles per mole of polysaccharide repeating unit) After the reaction, the mixture was added and incubated at 22°C for 1 hour. The batch was then heated at approximately 4°C and Diluted in 150 mM sodium chloride containing 0.025% (w / v) polysorbate 20. The pH was then neutralized by adding potassium phosphate buffer. NMWCO tangential flow ultrafiltration membranes were used to filter 150 mM sodium chloride, 25 mM The solution was diafiltered against 1 M potassium phosphate pH 7 at approximately 4°C.

[0353] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The solution was then incubated at 4°C in 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20. The solution was diafiltered against 10 mM histidine in 10 ml of ethanol, pH 7.0.

[0354] The retentate batch was filtered to 0.2 microns (using a 0.5 micron prefilter) and then 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20, pH 7 Dilute with additional 10 mM histidine in 0.0 mL of PBS, dispense into aliquots, and freeze at -60°C or below. did. [Table 10]

[0355] [Example 12] Preparation of serotype 11A conjugates for multivalent testing using DMSO conjugation Made Polysaccharides are dissolved, sized to a target molecular mass, chemically activated, and purified by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately. The polysaccharide solution was then redissolved in DMSO. The redissolved polysaccharide solution and CRM197 solution were then mixed together. The resulting conjugate was added to the final 0.2 microgram of The mixture was purified by ultrafiltration before filtration. Each step, such as pH, temperature, concentration and time, was Several process parameters within the process can be controlled to produce conjugates with desired attributes. obtained.

[0356] Size reduction and oxidation of polysaccharides Purified pneumococcal Ps capsule powder was dissolved in water and filtered through a 0.45 micron filter. The polysaccharides were size reduced by acid hydrolysis by adding acetic acid to 200 mM and then heated at 92°C. Incubate for 75 minutes, then add cold potassium phosphate pH 7 buffer to 400 mM and neutralized it.

[0357] Size-reduced polysaccharides were concentrated and filtered through a 5 kDa NMWCO tangential flow ultrafiltration membrane. Diafiltration was performed against water using

[0358] The polysaccharide solution was then heated to 22°C and adjusted to pH 5 with sodium acetate buffer to activate Polysaccharide activation was performed with 100 mM sodium metaperiodate. The amount of sodium metaperiodate added was determined by the amount of polysaccharide activity. To achieve the target level of polymerization, 0.13 moles of metaperiodic acid were used per mole of polysaccharide repeating unit. Sodium urate (moles of aldehyde per mole of polysaccharide repeating unit). Oxidation reaction The reaction proceeded for 2 hours at 22°C.

[0359] The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4. and subsequently filtered into water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was carried out at 2-8°C.

[0360] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876), Pseudomonas fluorescens (Pseudomonas fluorescens) Purified CRM197 obtained through expression in C. fluorescens , filtered through a 5 kDa NMWCO tangential flow ultrafiltration membrane in 2 mM phosphate, pH 7. The solution was diafiltered against 0.2 buffer and 0.2 micron filtered.

[0361] The activated polysaccharide was lyophilized at 6 mg Ps / mL in 5% w / v sucrose concentration. CRM197 was formulated at 6 mg Pr / mL with a sucrose concentration of 1% w / v. Formulated for lyophilization.

[0362] The formulated Ps and CRM197 solutions were lyophilized separately. and CRM197 materials were separately redissolved in an equal volume of DMSO containing 25 mM sodium chloride. The polysaccharide solution and CRM197 solution were blended to give a polysaccharide concentration of 3.5 g Ps / A mass ratio of 1.5 between L and polysaccharide and CRM197 was achieved. The ratio of the gate polysaccharide to CRM197 was selected to control. sodium (1 mole per mole of polysaccharide repeating unit) was added and the conjugation was carried out at 22°C. It advanced by 4 hours.

[0363] Reduction with sodium borohydride Conjugation with sodium borohydride (2 moles per mole of polysaccharide repeating unit) After the reaction, the mixture was added and incubated at 22°C for 1 hour. The batch was then heated at approximately 4°C and Diluted in 150 mM sodium chloride containing 0.025% (w / v) polysorbate 20. The pH was then neutralized by adding potassium phosphate buffer. NMWCO tangential flow ultrafiltration membranes were used to filter 150 mM sodium chloride, 25 mM The solution was diafiltered against 1 M potassium phosphate pH 7 at approximately 4°C.

[0364] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The solution was then incubated at 4°C in 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20. The solution was diafiltered against 10 mM histidine in 10 ml of ethanol, pH 7.0.

[0365] The retentate batch was filtered to 0.2 microns (using a 0.5 micron prefilter) and then 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20, pH 7 Dilute with additional 10 mM histidine in 0.0 mL of PBS, dispense into aliquots, and freeze at -60°C or below. did. [Table 11]

[0366] [Example 13] Preparation of serotype 12F conjugates for multivalent testing using DMSO conjugation Made Polysaccharides are dissolved, sized to a target molecular mass, chemically activated, and purified by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately. The polysaccharide solution was then redissolved in DMSO. The redissolved polysaccharide solution and CRM197 solution were then mixed together. The resulting conjugate was added to the final 0.2 microgram of The mixture was purified by ultrafiltration before filtration. Each step, such as pH, temperature, concentration and time, was Several process parameters within the process can be controlled to produce conjugates with desired attributes. obtained.

[0367] Size reduction and oxidation of polysaccharides Purified pneumococcal Ps capsule powder was dissolved in water and filtered through a 0.45 micron filter. The polysaccharides were size reduced by acid hydrolysis by adding acetic acid to 200 mM and then heated at 90°C. Incubate for 45 minutes, then add cold potassium phosphate pH 7 buffer to 400 mM and neutralized it.

[0368] Size-reduced polysaccharides were concentrated and filtered through a 5 kDa NMWCO tangential flow ultrafiltration membrane. Diafiltration was performed against water using

[0369] The polysaccharide solution was then heated to 22°C and adjusted to pH 5 with sodium acetate buffer to activate Polysaccharide activation was performed with 100 mM sodium metaperiodate. The amount of sodium metaperiodate added was determined by the amount of polysaccharide activity. To achieve the target level of polymerization, 0.26 moles of metaperiodic acid were used per mole of polysaccharide repeating unit. Sodium urate (moles of aldehyde per mole of polysaccharide repeating unit). Oxidation reaction The reaction proceeded for 2 hours at 22°C.

[0370] The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4. and subsequently filtered into water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was carried out at 2-8°C.

[0371] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876), Pseudomonas fluorescens (Pseudomonas fluorescens) Purified CRM197 obtained through expression in C. fluorescens , filtered through a 5 kDa NMWCO tangential flow ultrafiltration membrane in 2 mM phosphate, pH 7. The solution was diafiltered against 0.2 buffer and 0.2 micron filtered.

[0372] The activated polysaccharide was lyophilized at 6 mg Ps / mL in 5% w / v sucrose concentration. CRM197 was formulated at 6 mg Pr / mL with a sucrose concentration of 1% w / v. Formulated for lyophilization.

[0373] The formulated Ps and CRM197 solutions were lyophilized separately. and CRM197 materials were separately redissolved in an equal volume of DMSO containing 25 mM sodium chloride. The polysaccharide solution and CRM197 solution were blended to give a polysaccharide concentration of 3.0 g Ps / A mass ratio of 1.5 between L and polysaccharide and CRM197 was achieved. The ratio of the gate polysaccharide to CRM197 was selected to control. sodium (1 mole per mole of polysaccharide repeating unit) was added and the conjugation was carried out at 22°C. It advanced by 4 hours.

[0374] Reduction with sodium borohydride Conjugation with sodium borohydride (2 moles per mole of polysaccharide repeating unit) After the reaction, the mixture was added and incubated at 22°C for 3 hours. The batch was then heated at approximately 4°C and Diluted in 150 mM sodium chloride containing 0.025% (w / v) polysorbate 20. The pH was then neutralized by adding potassium phosphate buffer. NMWCO tangential flow ultrafiltration membranes were used to filter 150 mM sodium chloride, 25 mM The solution was diafiltered against 1 M potassium phosphate pH 7 at approximately 4°C.

[0375] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The solution was then incubated at 4°C in 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20. The solution was diafiltered against 10 mM histidine in 10 ml of ethanol, pH 7.0.

[0376] The retentate batch was filtered to 0.2 microns and then diluted with 0.015% (w / v) Polysorbate Dilute with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 containing 20 mg of PEG-20. The solution was diluted, divided into aliquots, and frozen at -60°C or below. [Table 12]

[0377] [Example 14] Preparation of serotype 15A conjugates for monovalent testing using DMSO conjugation Made Polysaccharides are dissolved, sized to a target molecular mass, chemically activated, and purified by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately. The polysaccharide solution was then redissolved in DMSO. The redissolved polysaccharide solution and CRM197 solution were then mixed together. The resulting conjugate was added to the final 0.2 microgram of The mixture was purified by ultrafiltration before filtration. Each step, such as pH, temperature, concentration and time, was Several process parameters within the process can be controlled to produce conjugates with desired attributes. obtained.

[0378] Size reduction and oxidation of polysaccharides Purified pneumococcal Ps capsule powder was dissolved in water and filtered through a 0.45 micron filter. The polysaccharides were homogenized to reduce the molecular mass of Ps. The homogenization pressure and homogenizer The number of passes was controlled at 200 bar / 5 passes.

[0379] Size-reduced polysaccharides were concentrated and filtered through a 10 kDa NMWCO tangential flow ultrafiltration membrane. The column was diafiltered against water using a filtration filter.

[0380] The polysaccharide solution was then heated to 22°C and adjusted to pH 5 with sodium acetate buffer to activate Polysaccharide activation was performed with 100 mM sodium metaperiodate. The amount of sodium metaperiodate added was determined by the amount of polysaccharide activity. To achieve the target level of polymerization, 1.75 moles of metaperiodic acid were added per mole of polysaccharide repeating unit. Sodium urate (moles of aldehyde per mole of polysaccharide repeating unit). Oxidation reaction The reaction proceeded for 20 hours at 22°C.

[0381] The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4. and subsequently purified using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was carried out at 2-8°C.

[0382] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876), Pseudomonas fluorescens (Pseudomonas fluorescens) Purified CRM197 obtained through expression in C. fluorescens , filtered through a 5 kDa NMWCO tangential flow ultrafiltration membrane in 2 mM phosphate, pH 7. The solution was diafiltered against 0.2 buffer and 0.2 micron filtered.

[0383] The activated polysaccharide was lyophilized at 6 mg Ps / mL in 5% w / v sucrose concentration. CRM197 was formulated at 6 mg Pr / mL with a sucrose concentration of 1% w / v. Formulated for lyophilization.

[0384] The formulated Ps and CRM197 solutions were lyophilized separately. and CRM197 materials were separately redissolved in an equal volume of DMSO containing 25 mM sodium chloride. The polysaccharide solution and CRM197 solution were blended to give a polysaccharide concentration of 6.0 g Ps / The mass ratio of polysaccharide to CRM197 was 2.0. The ratio of the gate polysaccharide to CRM197 was selected to control. sodium (1 mole per mole of polysaccharide repeating unit) was added and the conjugation was carried out at 22°C. I advanced 2.5 hours.

[0385] Reduction with sodium borohydride Conjugation with sodium borohydride (2 moles per mole of polysaccharide repeating unit) After the reaction, the mixture was added and incubated at 22°C for 1 hour. The batch was then heated at approximately 4°C and Diluted in 150 mM sodium chloride containing 0.025% (w / v) polysorbate 20. The pH was then neutralized by adding potassium phosphate buffer. NMWCO tangential flow ultrafiltration membranes were used to filter 150 mM sodium chloride, 25 mM The solution was diafiltered against 1 M potassium phosphate pH 7 at approximately 4°C.

[0386] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The solution was then incubated at 4°C in 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20. The solution was diafiltered against 10 mM histidine in 10 ml of ethanol, pH 7.0.

[0387] The retentate batch was filtered to 0.2 microns (using a 0.5 micron prefilter) and then 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20, pH 7 Dilute with additional 10 mM histidine in 0.0 mL of PBS, dispense into aliquots, and freeze at -60°C or below. did. [Table 13]

[0388] [Example 15] Serotype 15A conjugate for 15A / B / C cross-protection testing using aqueous conjugation Preparation of jugate Polysaccharides are dissolved, size reduced, chemically activated, and buffer exchanged by ultrafiltration The purified CRM197 was then activated using nickel chloride in an aqueous reaction mixture. The resulting conjugate was filtered through a final 0.2 micron filtration. The mixture was purified by ultrafiltration beforehand. Several process parameters were controlled to obtain conjugates with desired attributes.

[0389] Size reduction and oxidation of polysaccharides Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered through a 0.45 micron filter. The resulting polysaccharides were homogenized to reduce the molecular mass. The number of passes was controlled at 200 bar / 5 passes to achieve the target molecular mass. The reduced polysaccharides were concentrated and filtered using a 10kDa NMWCO tangential flow ultrafiltration membrane. and diafiltered against water.

[0390] The polysaccharide solution was then heated to 22°C and adjusted to pH 5 with sodium acetate buffer to activate Polysaccharide activation was performed with 100 mM sodium metaperiodate. The amount of sodium metaperiodate added was determined by the amount of polysaccharide repeating units. The amount of sodium metaperiodate was 0.45 moles per mole of unit. The oxidation reaction was carried out at 22°C. The reaction proceeded for 20 hours. The activated product was purified by filtration at a 10 kDa NMWCO tangential flow limit. Diafiltrate using a membrane filter against 10 mM potassium phosphate, pH 6.4. Ultrafiltration was carried out at 2-8°C. The ultrafiltration product was adjusted to 22°C and pH 5. Further activation was achieved by increasing the amount of polysaccharides in the saccharin-containing medium. 2.0 moles of sodium metaperiodate per mole of polysaccharide repeating unit ( (moles of aldehyde per mole of aldehyde) and 100 mM sodium metaperiodate The activation was carried out by adding the solution. The oxidation reaction proceeded for 20 hours at 22°C. The resulting product was filtered through a 10 kDa NMWCO tangential flow ultrafiltration membrane at 1000 rpm. Diafiltration was performed against potassium phosphate, pH 6.4. Ultrafiltration was performed in 2-8 The temperature was ℃.

[0391] Polysaccharide conjugation to CRM197 The oxidized polysaccharide solution was mixed with water and 1.5 M potassium phosphate pH 6.0. The buffer pH is used to improve the stability of the activated polysaccharide during the conjugation reaction. As previously described (WO 2012 / 173876), Pseudomonas fluorescens (P Purified CRM obtained through expression in Pseudomonas fluorescens 197, filtered to 0.2 microns, and buffered at a mass ratio of polysaccharide to CRM197 of 0.6. The mass ratio controls the ratio of polysaccharide to CRM197 in the resulting conjugate. The polysaccharide and phosphate concentrations were 9.75 g / L and 10.5 g / L, respectively. The polysaccharide concentration was adjusted to 100 mM to control the size of the resulting conjugate. Using a 100 mM nickel chloride solution, add nickel chloride to approximately 2 mM. Sodium cyanoborohydride (2 moles per mole of polysaccharide repeating unit) was added. The conjugate was incubated at 10°C for 148 hours to maximize the consumption of polysaccharides and proteins. We continued the project.

[0392] Reduction with sodium borohydride After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.0 g / L and The batch was cooled to 8°C and filtered through a 100 kDa NMWCO Tanger Using a fast-flow ultrafiltration membrane, the solution was filtered at 2-8°C in 100 mM potassium phosphate, pH 7.0. The batch recovered in the retentate was then diafiltered at approximately 2.0 g polysaccharide / L and adjust the pH by adding 1.2 M sodium bicarbonate, pH 9.4. Sodium borohydride (1 mole per mole of polysaccharide repeating unit) was added. M potassium phosphate, pH 6.0 was added later.

[0393] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane. and 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4°C. Polysorbate 20 was added to the retentate batch at 0.05% ( w / v) and the batch was then 0.2 micron filtered (0.5 micron with prefilter).

[0394] The batch was diluted with 150 mM sodium chloride containing 0.03% (w / v) polysorbate 20. , with additional 10 mM L-histidine in pH 7.0 buffer to a polysaccharide concentration of 1.0 g / L. The batch was divided into aliquots and frozen at -60°C or below. [Table 14]

[0395] [Example 16] Preparation of serotype 15A conjugates for multivalent testing using DMSO conjugation Made Polysaccharides are dissolved, sized to a target molecular mass, chemically activated, and purified by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately. The polysaccharide solution was then redissolved in DMSO. The redissolved polysaccharide solution and CRM197 solution were then mixed together. The resulting conjugate was added to the final 0.2 microgram of The mixture was purified by ultrafiltration before filtration. Each step, such as pH, temperature, concentration and time, was Several process parameters within the process can be controlled to produce conjugates with desired attributes. obtained.

[0396] Size reduction and oxidation of polysaccharides Purified pneumococcal Ps capsule powder was dissolved in water and filtered through a 0.45 micron filter. The polysaccharides were homogenized to reduce the molecular mass of Ps. The homogenization pressure and homogenizer The number of passes was controlled at 200 bar / 5 passes.

[0397] Size-reduced polysaccharides were concentrated and filtered through a 10 kDa NMWCO tangential flow ultrafiltration membrane. The column was diafiltered against water using a filtration filter.

[0398] The polysaccharide solution was then heated to 22°C and adjusted to pH 5 with sodium acetate buffer to activate Polysaccharide activation was performed with 100 mM sodium metaperiodate. The amount of sodium metaperiodate added was determined by the amount of polysaccharide activity. To achieve the target level of polymerization, 1.75 moles of metaperiodic acid were added per mole of polysaccharide repeating unit. Sodium urate (moles of aldehyde per mole of polysaccharide repeating unit). Oxidation reaction The reaction proceeded for 20 hours at 22°C.

[0399] The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4. and subsequently purified using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was carried out at 2-8°C.

[0400] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876), Pseudomonas fluorescens (Pseudomonas fluorescens) Purified CRM197 obtained through expression in C. fluorescens , filtered through a 5 kDa NMWCO tangential flow ultrafiltration membrane in 2 mM phosphate, pH 7. The solution was diafiltered against 0.2 buffer and 0.2 micron filtered.

[0401] The activated polysaccharide was lyophilized at 6 mg Ps / mL in 5% w / v sucrose concentration. CRM197 was formulated at 6 mg Pr / mL with a sucrose concentration of 1% w / v. Formulated for lyophilization.

[0402] The formulated Ps and CRM197 solutions were lyophilized separately. The polysaccharides and CRM197 materials were separately redissolved in an equal volume of DMSO preheated to 34°C. Sodium chloride was added to the solution to a concentration of 50 mM. The liquids were blended to a polysaccharide concentration of 5.0 g Ps / L and a mass ratio of polysaccharide to CRM197 of 1.0 g Ps / L. The mass ratio of the resulting conjugate was controlled by the ratio of polysaccharide to CRM197. Sodium cyanoborohydride (1 mole per mole of polysaccharide repeating unit) was selected to control the mol) was added and conjugation was allowed to proceed at 34°C for 2 hours.

[0403] Reduction with sodium borohydride Conjugation with sodium borohydride (2 moles per mole of polysaccharide repeating unit) After the reaction, the mixture was added and incubated at 22°C for 1 hour. The batch was then heated at approximately 4°C and Diluted in 150 mM sodium chloride containing 0.025% (w / v) polysorbate 20. The pH was then neutralized by adding potassium phosphate buffer. NMWCO tangential flow ultrafiltration membranes were used to filter 150 mM sodium chloride, 25 mM The solution was diafiltered against 1 M potassium phosphate pH 7 at approximately 4°C.

[0404] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The solution was then incubated at 4°C in 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20. The solution was diafiltered against 10 mM histidine in 10 ml of ethanol, pH 7.0.

[0405] The retentate batch was filtered to 0.2 microns and then diluted with 0.015% (w / v) polysorbate. Dilute with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 containing 20 mg of PEG-20. The solution was diluted, divided into aliquots, and frozen at -60°C or below. [Table 15]

[0406] [Example 17] Serotype 15B for 15A / B / C cross-protection testing using DMSO conjugation Preparation of conjugates Polysaccharides are dissolved, sized to a target molecular mass, chemically activated, and purified by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately. The polysaccharide solution was then redissolved in DMSO. The redissolved polysaccharide solution and CRM197 solution were then mixed together. The resulting conjugate was added to the final 0.2 microgram of The solution was purified by ultrafiltration before filtration. Each step, such as pH, temperature, concentration and time, was Several process parameters within the process can be controlled to produce conjugates with desired attributes. obtained.

[0407] Size reduction and oxidation of polysaccharides Purified pneumococcal Ps capsule powder was dissolved in water and filtered through a 0.45 micron filter. The polysaccharides were homogenized to reduce the molecular mass of Ps. The homogenization pressure and homogenizer The number of passes was controlled at 300 bar / 5 passes.

[0408] Size-reduced polysaccharides were concentrated and filtered through a 10 kDa NMWCO tangential flow ultrafiltration membrane. The column was diafiltered against water using a filtration filter.

[0409] The polysaccharide solution was then heated to 22°C and adjusted to pH 5 with sodium acetate buffer to activate Polysaccharide activation was performed with 100 mM sodium metaperiodate. The amount of sodium metaperiodate added was determined by the amount of polysaccharide activity. To achieve the target level of polymerization, 0.20 moles of metaperiodic acid were added per mole of polysaccharide repeating unit. Sodium urate (moles of aldehyde per mole of polysaccharide repeating unit). Oxidation reaction The reaction proceeded for 4 hours at 22°C.

[0410] The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4. and subsequently purified using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was carried out at 2-8°C.

[0411] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876), Pseudomonas fluorescens (Pseudomonas fluorescens) Purified CRM197 obtained through expression in C. fluorescens , filtered through a 5 kDa NMWCO tangential flow ultrafiltration membrane in 2 mM phosphate, pH 7. The solution was diafiltered against 0.2 buffer and 0.2 micron filtered.

[0412] The activated polysaccharide was lyophilized at 6 mg Ps / mL in 5% w / v sucrose concentration. CRM197 was formulated at 6 mg Pr / mL with a sucrose concentration of 1% w / v. Formulated for lyophilization.

[0413] The formulated Ps and CRM197 solutions were lyophilized separately. The polysaccharide solution and CRM197 materials were separately redissolved in an equal volume of DMSO. The 197 solution was blended to give a polysaccharide concentration of 3.0 g Ps / L and a polysaccharide and CRM197 The mass ratio of the polysaccharide to CRM197 in the resulting conjugate was 2.0. The ratio of sodium cyanoborohydride (1 mole of polysaccharide repeating unit) was selected to control the (1 mole per 1000 mg of ATP) was added and the conjugation was allowed to proceed for 5 hours at 22°C.

[0414] Reduction with sodium borohydride Conjugation with sodium borohydride (2 moles per mole of polysaccharide repeating unit) After the reaction, the mixture was added and incubated at 22°C for 1 hour. The batch was then heated at approximately 4°C and Diluted in 150 mM sodium chloride containing 0.025% (w / v) polysorbate 20. The pH was then neutralized by adding potassium phosphate buffer. NMWCO tangential flow ultrafiltration membranes were used to filter 150 mM sodium chloride, 25 mM The solution was diafiltered against 1 M potassium phosphate pH 7 at approximately 4°C.

[0415] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The solution was then incubated at 4°C in 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20. The solution was diafiltered against 10 mM histidine in 10 ml of ethanol, pH 7.0.

[0416] The retentate batch was filtered to 0.2 microns (using a 0.5 micron prefilter) and then 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20, pH 7 Dilute with additional 10 mM histidine in 0.0 mL of PBS, dispense into aliquots, and freeze at -60°C or below. did. [Table 16]

[0417] [Example 18] For monovalent and 15A / B / C cross-protection testing using DMSO conjugation Preparation of serotype 15C conjugates Derived from Streptococcus pneumoniae serotype 15B The polysaccharides are dissolved, sized to a target molecular mass, and subjected to mild base hydrolysis to obtain O-amino acids. The cetyl groups were released, chemically activated, and buffer exchanged by ultrafiltration. The polysaccharide and purified CRM197 were separately lyophilized and redissolved in DMSO. The reconstituted polysaccharide solution and CRM197 solution were combined and conjugated as described below. The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration. Several process parameters within each step such as pH, temperature, concentration and time were adjusted. The parameters were controlled to obtain conjugates with desired attributes.

[0418] Size reduction, base hydrolysis, and oxidation of polysaccharides Purified serotype 15B pneumococcal capsular Ps powder was dissolved in water and filtered to 0.45 microns. The dissolved polysaccharides were homogenized to reduce the molecular mass of Ps. The number of passes through the genizer was controlled at 300 bar / 5 passes.

[0419] Size-reduced polysaccharides were concentrated and filtered through a 10 kDa NMWCO tangential flow ultrafiltration membrane. The column was diafiltered against water using a filtration filter.

[0420] Heat the polysaccharide solution to 60 °C and add sodium bicarbonate pH 9 buffer to a final concentration of 50 mM. The batch was incubated at 60°C for 13 hours with mixing to remove the O-acetyl groups. The pH was neutralized by adding potassium phosphate pH 6 buffer to a final concentration of 136 mM. The solution was then concentrated to give a 10 kDa NMWCO tandem. The solution was diafiltered against water using a vent flow ultrafiltration membrane.

[0421] The polysaccharide solution was kept at 22°C and adjusted to pH 5 with sodium acetate buffer to determine the amount of polysaccharide produced by activation. Polysaccharide activation was performed with 100 mM sodium metaperiodate to minimize size reduction of the saccharides. The amount of sodium metaperiodate added was determined by the target amount of polysaccharide activation. To achieve this level, 0.20 moles of sodium metaperiodate per mole of polysaccharide repeating unit was used. The oxidation reaction was carried out at 22°C. The meeting lasted for two hours.

[0422] The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4. and subsequently purified using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was carried out at 2-8°C.

[0423] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876), Pseudomonas fluorescens (Pseudomonas fluorescens) Purified CRM197 obtained through expression in C. fluorescens , filtered through a 5 kDa NMWCO tangential flow ultrafiltration membrane in 2 mM phosphate, pH 7. The solution was diafiltered against 0.2 buffer and 0.2 micron filtered.

[0424] The activated polysaccharide was lyophilized at 6 mg Ps / mL in 5% w / v sucrose concentration. CRM197 was formulated at 6 mg Pr / mL with a sucrose concentration of 1% w / v. Formulated for lyophilization.

[0425] The formulated Ps and CRM197 solutions were lyophilized separately. The polysaccharide solution and CRM197 materials were separately redissolved in an equal volume of DMSO. The 197 solution was blended to give a polysaccharide concentration of 3.0 g Ps / L and a polysaccharide and CRM197 The mass ratio of the polysaccharide to CRM197 in the resulting conjugate was 2.0. The ratio of sodium cyanoborohydride (1 mole of polysaccharide repeating unit) was selected to control the (1 mole per 1000 mg of ATP) was added and conjugation was allowed to proceed for 4 hours at 22°C.

[0426] Reduction with sodium borohydride Conjugation with sodium borohydride (2 moles per mole of polysaccharide repeating unit) After the reaction, the mixture was added and incubated at 22°C for 1 hour. The batch was then heated at approximately 4°C and Diluted in 150 mM sodium chloride containing 0.025% (w / v) polysorbate 20. The pH was then neutralized by adding potassium phosphate buffer. NMWCO tangential flow ultrafiltration membranes were used to filter 150 mM sodium chloride, 25 mM The solution was diafiltered against 1 M potassium phosphate pH 7 at approximately 4°C.

[0427] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The solution was then incubated at 4°C in 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20. The solution was diafiltered against 10 mM histidine in 10 ml of ethanol, pH 7.0.

[0428] The retentate batch was filtered to 0.2 microns (using a 0.5 micron prefilter) and then 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20, pH 7 Dilute with additional 10 mM histidine in 0.0 mL of PBS, dispense into aliquots, and freeze at -60°C or below. did. [Table 17]

[0429] [Example 19] Preparation of serotype 15C conjugates for multivalent testing using DMSO conjugation Made Derived from Streptococcus pneumoniae serotype 15B The polysaccharides are dissolved, sized to a target molecular mass, and subjected to mild base hydrolysis to obtain O-amino acids. The cetyl groups were released, chemically activated, and buffer exchanged by ultrafiltration. The polysaccharide and purified CRM197 were separately lyophilized and redissolved in DMSO. The reconstituted polysaccharide solution and CRM197 solution were combined and conjugated as described below. The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration. Several process parameters within each step such as pH, temperature, concentration and time were adjusted. The parameters were controlled to obtain conjugates with desired attributes.

[0430] Size reduction, base hydrolysis, and oxidation of polysaccharides Purified serotype 15B pneumococcal capsular Ps powder was dissolved in water and filtered to 0.45 microns. The dissolved polysaccharides were homogenized to reduce the molecular mass of Ps. The number of passes through the genizer was controlled at 300 bar / 5 passes.

[0431] Size-reduced polysaccharides were concentrated and filtered through a 10 kDa NMWCO tangential flow ultrafiltration membrane. The column was diafiltered against water using a filtration filter.

[0432] Heat the polysaccharide solution to 60 °C and add sodium bicarbonate pH 9 buffer to a final concentration of 50 mM. The batch was incubated at 60°C for 13 hours with mixing to remove the O-acetyl groups. The pH was neutralized by adding potassium phosphate pH 6 buffer to a final concentration of 136 mM. and the solution was cooled to ambient temperature.

[0433] The solution was then concentrated and filtered using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Diafiltered against water.

[0434] The polysaccharide solution was kept at 22°C and adjusted to pH 5 with sodium acetate buffer to determine the amount of polysaccharide produced by activation. Polysaccharide activation was performed with 100 mM sodium metaperiodate to minimize size reduction of the saccharides. The amount of sodium metaperiodate added was determined by the target amount of polysaccharide activation. To achieve this level, 0.20 moles of sodium metaperiodate per mole of polysaccharide repeating unit was used. The oxidation reaction was carried out at 22°C. The meeting lasted for two hours.

[0435] The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4. and subsequently purified using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was carried out at 2-8°C.

[0436] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876), Pseudomonas fluorescens (Pseudomonas fluorescens) Purified CRM197 obtained through expression in C. fluorescens , filtered through a 5 kDa NMWCO tangential flow ultrafiltration membrane in 2 mM phosphate, pH 7. The solution was diafiltered against 0.2 buffer and 0.2 micron filtered.

[0437] The activated polysaccharide was lyophilized at 6 mg Ps / mL in 5% w / v sucrose concentration. CRM197 was formulated at 6 mg Pr / mL with a sucrose concentration of 1% w / v. Formulated for lyophilization.

[0438] The formulated Ps and CRM197 solutions were lyophilized separately. The polysaccharide solution and CRM197 materials were separately redissolved in an equal volume of DMSO. The 197 solution was blended to give a polysaccharide concentration of 3.0 g Ps / L and a polysaccharide and CRM197 The mass ratio of the resulting conjugate polysaccharide to CRM19 was 1.75. The ratio of sodium cyanoborohydride (polysaccharide repeating unit 1) was selected to control the 1 mole per unit was added and conjugation was allowed to proceed for 8 hours at 22°C.

[0439] Reduction with sodium borohydride Conjugation with sodium borohydride (2 moles per mole of polysaccharide repeating unit) After the reaction, the mixture was added and incubated at 22°C for 1 hour. The batch was then heated at approximately 4°C and Diluted in 150 mM sodium chloride containing 0.025% (w / v) polysorbate 20. The pH was then neutralized by adding potassium phosphate buffer. NMWCO tangential flow ultrafiltration membranes were used to filter 150 mM sodium chloride, 25 mM The solution was diafiltered against 1 M potassium phosphate pH 7 at approximately 4°C.

[0440] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The solution was then incubated at 4°C in 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20. The solution was diafiltered against 10 mM histidine in 10 ml of ethanol, pH 7.0.

[0441] The retentate batch was filtered to 0.2 microns (using a 0.5 micron prefilter) and then 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20, pH 7 Dilute with additional 10 mM histidine in 0.0 mL of PBS, dispense into aliquots, and freeze at -60°C or below. did. [Table 18]

[0442] [Example 20] Preparation of serotype 16F conjugate for monovalent testing using aqueous conjugation Polysaccharides are dissolved, size reduced, chemically activated, and buffer exchanged by ultrafiltration The purified CRM197 was then activated using nickel chloride in an aqueous reaction mixture. The resulting conjugate was filtered through a final 0.2 micron filtration. The mixture was purified by ultrafiltration beforehand. Several process parameters were controlled to obtain conjugates with desired attributes.

[0443] Size reduction and oxidation of polysaccharides Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered through a 0.45 micron filter. The resulting polysaccharides were homogenized to reduce the molecular mass. The number of passes is controlled to 200 bar / 5 passes, followed by 500 bar / 5 passes. The size-reduced polysaccharides were then concentrated to obtain the 10 kDa NMWC. The solution was diafiltered against water using a tangential flow ultrafiltration membrane.

[0444] The polysaccharide solution was then heated to 22°C and adjusted to pH 5 with sodium acetate buffer to activate Polysaccharide activation was performed with 100 mM sodium metaperiodate. The amount of sodium metaperiodate added was determined by the amount of polysaccharide activity. To achieve the target level of polymerization, 0.15 moles of metaperiodic acid were added per mole of polysaccharide repeating unit. Sodium urate (moles of aldehyde per mole of polysaccharide repeating unit). Oxidation reaction The reaction proceeded for 2 hours at 22°C.

[0445] The activated product was filtered using a 10 kDa NMWCO tangential flow ultrafiltration membrane. , and diafiltered against 10 mM potassium phosphate, pH 6.4. Filtration was carried out at 2 to 8°C.

[0446] Polysaccharide conjugation to CRM197 The oxidized polysaccharide solution was mixed with water and 1.5 M potassium phosphate pH 7.0. The buffer pH is used to improve the stability of the activated polysaccharide during the conjugation reaction. As previously described (WO 2012 / 173876), Pseudomonas fluorescens (P Purified CRM obtained through expression in Pseudomonas fluorescens 197, filtered to 0.2 microns, and buffered at a polysaccharide to CRM197 mass ratio of 0.7. The mass ratio controls the ratio of polysaccharide to CRM197 in the resulting conjugate. The polysaccharide and phosphate concentrations were selected to be 7.5 g / L and 1.0 g / L, respectively. The polysaccharide concentration was chosen to control the size of the resulting conjugate. The solution was then filtered through 0.2 microns. A 100 mM nickel chloride solution was used. Nickel chloride was added to approximately 2 mM. (2 moles per mole of repeating unit) was added to maximize polysaccharide and protein consumption. Conjugation was allowed to proceed for 122 hours at 22°C.

[0447] Reduction with sodium borohydride After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.0 g / L and The batch was cooled to 8°C and filtered through a 100 kDa NMWCO Tanger Using a fast-flow ultrafiltration membrane, the solution was filtered at 2-8°C in 100 mM potassium phosphate, pH 7.0. The batch recovered in the retentate was then diafiltered at approximately 2.0 g polysaccharide / L and adjust the pH by adding 1.2 M sodium bicarbonate, pH 9.4. Sodium borohydride (1 mole per mole of polysaccharide repeating unit) was added. M potassium phosphate, pH 6.0 was added later.

[0448] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane. and 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4°C. Polysorbate 20 was added to the retentate batch at 0.05% ( w / v) and the batch was then 0.2 micron filtered (0.5 micron with prefilter).

[0449] The batch was diluted with 150 mM sodium chloride containing 0.03% (w / v) polysorbate 20. , with additional 10 mM L-histidine in pH 7.0 buffer to a polysaccharide concentration of 1.0 g / L. The batch was divided into aliquots and frozen at -60°C or below. [Table 19]

[0450] [Example 21] Preparation of serotype 16F conjugates for multivalent testing using DMSO conjugation Made Polysaccharides are dissolved, sized to a target molecular mass, chemically activated, and purified by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately. The polysaccharide solution was then redissolved in DMSO. The redissolved polysaccharide solution and CRM197 solution were then mixed together. The resulting conjugate was added to the final 0.2 microgram of The solution was purified by ultrafiltration before filtration. Each step, such as pH, temperature, concentration and time, was Several process parameters within the process can be controlled to produce conjugates with desired attributes. obtained.

[0451] Size reduction and oxidation of polysaccharides Purified pneumococcal Ps capsule powder was dissolved in water and filtered through a 0.45 micron filter. The polysaccharides were homogenized to reduce the molecular mass of Ps. The homogenization pressure and homogenizer The number of passes was controlled at 1000 bar / 5 passes.

[0452] Size-reduced polysaccharides were concentrated and filtered through a 10 kDa NMWCO tangential flow ultrafiltration membrane. The column was diafiltered against water using a filtration filter.

[0453] The polysaccharide solution was then heated to 22°C and adjusted to pH 5 with sodium acetate buffer to activate Polysaccharide activation was performed with 100 mM sodium metaperiodate. The amount of sodium metaperiodate added was determined by the amount of polysaccharide activity. To achieve the target level of polymerization, 0.15 moles of metaperiodic acid were added per mole of polysaccharide repeating unit. Sodium urate (moles of aldehyde per mole of polysaccharide repeating unit). Oxidation reaction The reaction proceeded for 2 hours at 22°C.

[0454] The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4. and subsequently purified using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was carried out at 2-8°C.

[0455] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876), Pseudomonas fluorescens (Pseudomonas fluorescens) Purified CRM197 obtained through expression in C. fluorescens , filtered through a 5 kDa NMWCO tangential flow ultrafiltration membrane in 2 mM phosphate, pH 7. The solution was diafiltered against 0.2 buffer and 0.2 micron filtered.

[0456] The activated polysaccharide was lyophilized at 6 mg Ps / mL in 5% w / v sucrose concentration. CRM197 was formulated at 6 mg Pr / mL with a sucrose concentration of 1% w / v. Formulated for lyophilization.

[0457] The formulated Ps and CRM197 solutions were lyophilized separately. The polysaccharide solution was redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 solution were blended to a concentration of 50 mM. , a polysaccharide concentration of 2.0 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.5 were achieved. The mass ratio is chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mole per mole of polysaccharide repeating unit) was added, Conjugation was allowed to proceed for 2 hours at 22°C.

[0458] Reduction with sodium borohydride Conjugation with sodium borohydride (2 moles per mole of polysaccharide repeating unit) After the reaction, the mixture was added and incubated at 22°C for 1 hour. The batch was then heated at approximately 4°C and Diluted in 150 mM sodium chloride containing 0.025% (w / v) polysorbate 20. The pH was then neutralized by adding potassium phosphate buffer. The batch was concentrated to 30 kDa Using NMWCO tangential flow ultrafiltration membranes, 150 mM sodium chloride, 25 mM Diafiltered against potassium phosphate pH 7 at approximately 4°C.

[0459] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The solution was then incubated at 4°C in 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20. The solution was diafiltered against 10 mM histidine in 10 ml of ethanol, pH 7.0.

[0460] The retentate batch was filtered to 0.2 microns and then diluted with 0.015% (w / v) polysorbate. Dilute with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 containing 20 mg of PEG-20. The solution was diluted, divided into aliquots, and frozen at -60°C or below. [Table 20]

[0461] [Example 22] Preparation of serotype 17F conjugate for monovalent testing using DMSO conjugation Made Polysaccharides are dissolved, sized to a target molecular mass, chemically activated, and purified by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately. The polysaccharide solution was then redissolved in DMSO. The redissolved polysaccharide solution and CRM197 solution were then mixed together. The resulting conjugate was added to the final 0.2 microgram of The solution was purified by ultrafiltration before filtration. Each step, such as pH, temperature, concentration and time, was Several process parameters within the process can be controlled to produce conjugates with desired attributes. obtained.

[0462] Size reduction and oxidation of polysaccharides Purified pneumococcal Ps capsule powder was dissolved in water and filtered through a 0.45 micron filter. The polysaccharides were homogenized to reduce the molecular mass of Ps. The homogenization pressure and homogenizer The number of passes was controlled at 200 bar / 5 passes.

[0463] Size-reduced polysaccharides were concentrated and filtered through a 10 kDa NMWCO tangential flow ultrafiltration membrane. The column was diafiltered against water using a filtration filter.

[0464] The polysaccharide solution was then heated to 22°C and adjusted to pH 5 with sodium acetate buffer to activate Polysaccharide activation was performed with 100 mM sodium metaperiodate. The amount of sodium metaperiodate added was determined by the amount of polysaccharide activity. To achieve the target level of polymerization, 0.11 moles of metaperiodic acid were used per mole of polysaccharide repeating unit. Sodium urate (moles of aldehyde per mole of polysaccharide repeating unit). Oxidation reaction The reaction proceeded for 2 hours at 22°C.

[0465] The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4. and subsequently purified using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was carried out at 2-8°C.

[0466] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876), Pseudomonas fluorescens (Pseudomonas fluorescens) Purified CRM197 obtained through expression in C. fluorescens , filtered through a 5 kDa NMWCO tangential flow ultrafiltration membrane in 2 mM phosphate, pH 7. The solution was diafiltered against 0.2 buffer and 0.2 micron filtered.

[0467] The activated polysaccharide was lyophilized at 6 mg Ps / mL in 5% w / v sucrose concentration. CRM197 was formulated at 6 mg Pr / mL with a sucrose concentration of 1% w / v. Formulated for lyophilization.

[0468] The formulated Ps and CRM197 solutions were lyophilized separately. The polysaccharide solution was redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 solution were blended to a concentration of 50 mM. , a polysaccharide concentration of 2.0 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.5 were achieved. The mass ratio is chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mole per mole of polysaccharide repeating unit) was added, Conjugation was allowed to proceed for 2 hours at 22°C.

[0469] Reduction with sodium borohydride Conjugation with sodium borohydride (2 moles per mole of polysaccharide repeating unit) After the reaction, the mixture was added and incubated at 22°C for 1 hour. The batch was then heated at approximately 4°C and Diluted in 150 mM sodium chloride containing 0.025% (w / v) polysorbate 20. The pH was then neutralized by adding potassium phosphate buffer. NMWCO tangential flow ultrafiltration membranes were used to filter 150 mM sodium chloride, 25 mM The solution was diafiltered against 1 M potassium phosphate pH 7 at approximately 4°C.

[0470] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The solution was then incubated at 4°C in 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20. The solution was diafiltered against 10 mM histidine in 10 ml of ethanol, pH 7.0.

[0471] The retentate batch was filtered to 0.2 microns and then diluted with 0.015% (w / v) Polysorbate Dilute with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 containing 20 mg of PEG-20. The solution was diluted, divided into aliquots, and frozen at -60°C or below. [Table 21]

[0472] [Example 23] Preparation of serotype 17F conjugates for multivalent testing using DMSO conjugation Made Polysaccharides are dissolved, sized to a target molecular mass, chemically activated, and purified by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately. The polysaccharide solution was then redissolved in DMSO. The redissolved polysaccharide solution and CRM197 solution were then mixed together. The resulting conjugate was added to the final 0.2 microgram of The solution was purified by ultrafiltration before filtration. Each step, such as pH, temperature, concentration and time, was Several process parameters within the process can be controlled to produce conjugates with desired attributes. obtained.

[0473] Size reduction and oxidation of polysaccharides Purified pneumococcal Ps capsule powder was dissolved in water and filtered through a 0.45 micron filter. The polysaccharides were homogenized to reduce the molecular mass of Ps. The homogenization pressure and homogenizer The number of passes was controlled at 200 bar / 5 passes.

[0474] Size-reduced polysaccharides were concentrated and filtered through a 10 kDa NMWCO tangential flow ultrafiltration membrane. The column was diafiltered against water using a filtration filter.

[0475] The polysaccharide solution was then heated to 22°C and adjusted to pH 5 with sodium acetate buffer to activate Polysaccharide activation was performed with 100 mM sodium metaperiodate. The amount of sodium metaperiodate added was determined by the amount of polysaccharide activity. To achieve the target level of polymerization, 0.11 moles of metaperiodic acid were used per mole of polysaccharide repeating unit. Sodium urate (moles of aldehyde per mole of polysaccharide repeating unit). Oxidation reaction The reaction proceeded for 2 hours at 22°C.

[0476] The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4. and subsequently purified using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was carried out at 2-8°C.

[0477] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876), Pseudomonas fluorescens (Pseudomonas fluorescens) Purified CRM197 obtained through expression in C. fluorescens , filtered through a 5 kDa NMWCO tangential flow ultrafiltration membrane in 2 mM phosphate, pH 7. The solution was diafiltered against 0.2 buffer and 0.2 micron filtered.

[0478] The activated polysaccharide was lyophilized at 6 mg Ps / mL in 5% w / v sucrose concentration. CRM197 was formulated at 6 mg Pr / mL with a sucrose concentration of 1% w / v. Formulated for lyophilization.

[0479] The formulated Ps and CRM197 solutions were lyophilized separately. The polysaccharide solution was redissolved in an equal volume of DMSO. The polysaccharide solution and CRM197 solution were blended to a concentration of 20 mM. , a polysaccharide concentration of 2.1 g Ps / L and a polysaccharide to CRM197 mass ratio of 1.5 were achieved. The mass ratio is chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mole per mole of polysaccharide repeating unit) was added, Conjugation was allowed to proceed for 2 hours at 22°C.

[0480] Reduction with sodium borohydride Conjugation with sodium borohydride (2 moles per mole of polysaccharide repeating unit) After the reaction, the mixture was added and incubated at 22°C for 1 hour. The batch was then heated at approximately 4°C and Diluted in 150 mM sodium chloride containing 0.025% (w / v) polysorbate 20. The pH was then neutralized by adding potassium phosphate buffer. NMWCO tangential flow ultrafiltration membranes were used to filter 150 mM sodium chloride, 25 mM The solution was diafiltered against 1 M potassium phosphate pH 7 at approximately 4°C.

[0481] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The solution was then incubated at 4°C in 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20. The solution was diafiltered against 10 mM histidine in 10 ml of ethanol, pH 7.0.

[0482] The retentate batch was filtered to 0.2 microns (using a 0.5 micron prefilter) and then 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20, pH 7 Dilute with additional 10 mM histidine in 0.0 mL of PBS, dispense into aliquots, and freeze at -60°C or below. did. [Table 22]

[0483] [Example 24] Preparation of serotype 19A conjugates for multivalent testing using DMSO conjugation Made The polysaccharides were dissolved, chemically activated, and buffer exchanged by ultrafiltration. The polysaccharide and purified CRM197 were separately lyophilized and redissolved in DMSO. The reconstituted polysaccharide solution and CRM197 solution were combined and conjugated as described below. The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration. Several process parameters within each step such as pH, temperature, concentration and time were adjusted. The parameters were controlled to obtain conjugates with desired attributes.

[0484] Polysaccharide oxidation Purified pneumococcal Ps capsule powder was dissolved in water and filtered through a 0.45 micron filter. Concentrate and filter against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. It was filtered.

[0485] The polysaccharide solution was then heated to 22°C and adjusted to pH 5 with sodium acetate buffer to activate Polysaccharide activation was performed with 100 mM sodium metaperiodate. The amount of sodium metaperiodate added was determined by the amount of polysaccharide activity. To achieve the target level of polymerization, 0.26 moles of metaperiodic acid were used per mole of polysaccharide repeating unit. Sodium urate (moles of aldehyde per mole of polysaccharide repeating unit). Oxidation reaction The reaction proceeded for 20 hours at 22°C.

[0486] The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4. and subsequently purified using a 10 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was carried out at 2-8°C.

[0487] Polysaccharide conjugation to CRM197 As previously described (WO 2012 / 173876), Pseudomonas fluorescens (Pseudomonas fluorescens) Purified CRM197 obtained through expression in C. fluorescens , filtered through a 5 kDa NMWCO tangential flow ultrafiltration membrane in 2 mM phosphate, pH 7. The solution was diafiltered against 0.2 buffer and 0.2 micron filtered.

[0488] The activated polysaccharide was lyophilized at 6 mg Ps / mL in 5% w / v sucrose concentration. CRM197 was formulated at 6 mg Pr / mL with a sucrose concentration of 1% w / v. Formulated for lyophilization.

[0489] The formulated Ps and CRM197 solutions were lyophilized separately. The polysaccharide solution and CRM197 materials were separately redissolved in an equal volume of DMSO. The 197 solution was blended to give a polysaccharide concentration of 3.8 g Ps / L and a polysaccharide and CRM197 The mass ratio of the resulting conjugate polysaccharide to CRM19 was 1.33. The ratio of sodium cyanoborohydride (polysaccharide repeating unit 1) was selected to control the 1 mole per 100 ml of PEG-400 (1 mole per 100 ml of PEG-400) was added and the conjugation was allowed to proceed for 1.5 hours at 22°C.

[0490] Reduction with sodium borohydride Conjugation with sodium borohydride (2 moles per mole of polysaccharide repeating unit) After the reaction, the mixture was added and incubated at 22°C for 3 hours. The batch was then heated at approximately 4°C and ...

Claims

1. Multivalent Immunoglobulins Containing S. pneumoniae Polysaccharide-Protein Conjugates an immunogenic composition, wherein each of said conjugates is conjugated to a carrier protein; and a polysaccharide of a serovar S. pneumoniae that is induced by seroselective immunization, Serotype of S. pneumoniae of the polysaccharide-protein conjugate teeth, a) 15A, 16F, 23A, 23B, 24F, 31 and 35B; b) 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 1 1A, 12F, 15C, 17F and 20; and c) 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20 A multivalent immunogenic composition comprising a set of serotypes selected from the group consisting of:

2. Serotypes of S. pneumoniae listed in a), b) or c) The set includes: (i) serotype 6C, (ii) serotype 6A, or (iii) Serotypes 6A and 6B The multivalent immunogenic composition of claim 1, further comprising:

3. The serotype of S. pneumoniae is a) 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10 A, 11A, 12F, 15C, 17F and 20; b) 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10 A, 11A, 12F, 15C, 17F and 20; and c) 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N , 10A, 11A, 12F, 15C, 17F and 20 The set of serotypes selected from the group consisting of Here, the serotype 20 in the sets a) to c) is either serotype 20A or serotype 20B. The multivalent immunogenic composition of claim 1, optionally substituted with:

4. The serotype of S. pneumoniae is a) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 2 4F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20A ； b) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 2 4F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20A and c) 3, 7F, 19A, 22F, 33F, 6A, 6B, 15A, 16F, 23A, 23 B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20A The multivalent immunogenic composition of claim 1, comprising a set of serotypes selected from the group consisting of: thing.

5. The serotypes of S. pneumoniae are 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 1 A serotype selected from the group consisting of 0A, 11A, 12F, 15C, 17F and 20A. The multivalent immunogenic composition of claim 1 .

6. The serotypes of S. pneumoniae are 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 1 A serotype selected from the group consisting of 0A, 11A, 12F, 15C, 17F and 20A. The multivalent immunogenic composition of claim 1 .

7. The immunogenic composition further comprises a S. pneumoniae polysaccharide protein. The multivalent immunogenic composition according to any one of claims 1 to 6, which does not comprise a protein conjugate. Composition.

8. At least one of the polysaccharide-protein conjugates comprises an aprotic solvent. The multivalent ligand according to any one of claims 1 to 7, which is formed by a conjugation reaction. Immunogenic composition.

9. Each of the polysaccharide-protein conjugates comprises an aprotic solvent. The multivalent immunogenic composition according to any one of claims 1 to 8, which is formed by a covalent reaction. Composition.

10. 1 to 5, wherein the total polysaccharide concentration in the composition is about 0.02 to about 0.175 mg / mL.

10. The multivalent immunogenic composition according to any one of claims 9.

11. The S. pneumoniae polysaccharide protein conjugate in the composition The weight average molecular weight (Mw) of the copolymer is about 1,000 to about 6,000 kDa.

11. A multivalent immunogenic composition according to any one of claims 10.

12. The S. pneumoniae polysaccharide protein conjugate in the composition 12. The method according to claim 1, wherein the number average molecular weight (Mn) of the copolymer is about 500 to about 4,000 kDa. The multivalent immunogenic composition according to any one of claims 1 to 4.

13. The polyvalent immunoglobulin according to any one of claims 1 to 12, which is stable at 37°C for up to 4 weeks or more. Epidemiogenic composition.

14. The polyvalent immunoglobulin according to any one of claims 1 to 12, which is stable at 4°C for up to 12 weeks or more. Epidemiogenic composition.

15. 13. The stability is determined using HPSEC UV / MALS / RI.

15. The multivalent immunogenic composition according to 14.

16. Measured using intrinsic protein fluorescence spectroscopy at an excitation wavelength of 280 nanometers (nm) The composition according to any one of claims 1 to 15, wherein the composition has an emission maximum of about 335 nm to about 342 nm. A multivalent immunogenic composition according to any one of claims 1 to 4.

17. The emission maximum remains at about 335 nm to about 342 nm, and the fluorescence intensity is low at 37° C.

17. The multivalent immunogenic composition of claim 16, which is stable for at least one week.

18. 17. The method according to claim 8, wherein the aprotic solvent is dimethylsulfoxide (DMSO).

13. The multivalent immunogenic composition according to any one of claims 1 to 12.

19. The multivalent conjugation reaction of claim 8 or 9, wherein the conjugation reaction is a reductive amination. Immunogenic composition.

20. The carrier protein may be an outer membrane protein complex (OMPC), a tetanus toxoid, a diphtheria toxoid, teria toxoid, protein D and CRM197.

20. The multivalent immunogenic composition of any one of 1 to 19.

21. 21. The method of claim 1, wherein the carrier protein is CRM197. Multivalent immunogenic compositions.

22. The multivalent immunogenic composition according to any one of claims 1 to 21, further comprising an adjuvant. Composition.

23. 22. The multivalent immunogenic composition of any one of claims 1 to 21, which does not contain an adjuvant. thing.

24. The composition further comprises 10-80 mM histidine pH 5.8 and 150 mM NaCl.

24. The multivalent immunogenic composition of any one of claims 1 to 23.

25. 25. The method of claim 24, further comprising 0.025% to 0.8% w / v polysorbate 20. A multivalent immunogenic composition of claim 1.

26. Multivalent Immunoglobulins Containing S. pneumoniae Polysaccharide-Protein Conjugates an immunogenic composition, wherein each of said conjugates is conjugated to CRM197; The polysaccharides of the gated S. pneumoniae serotypes, The serotype of the pneumococcus (S. pneumoniae) of the polysaccharide-protein conjugate is 、 a) 15A, 16F, 23A, 23B, 24F, 31 and 35B; b) 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31 and 35B; c) 6A, 15A, 16F, 23A, 23B, 24F, 31 and 35B; d) 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B; e) 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 1 1A, 12F, 15C, 17F and 20A; f) 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10 A, 11A, 12F, 15C, 17F and 20A; g) 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N , 10A, 11A, 12F, 15C, 17F, and 20A; h) 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10 A, 11A, 12F, 15C, 17F and 20A; i) 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20A; j) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 2 4F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20A ； k) 3, 7F, 19A, 22F, 33F, 6A, 6B, 15A, 16F, 23A, 23 B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20A; and l) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 2 4F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20A A set of serotypes selected from the group consisting of: Here, serotype 20A in set e) to l) is either serotype 20 or serotype 20B. wherein each of said polysaccharide protein conjugates is substituted with a non- is formed by a conjugation reaction involving a protic solvent, and wherein the composition A multivalent immunogenic composition that does not include an adjuvant.

27. Claims including S. pneumoniae polysaccharide protein conjugates 27. The composition according to claim 26, wherein each of the conjugates is linked to CRM197. The polysaccharides of S. pneumoniae serotype conjugated thereto, The polysaccharide-protein conjugate is used to treat blood Streptococcus pneumoniae (S. pneumoniae). The types are 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20 A, wherein serotype 20A is selected from serotype 20 or serotype 2 0B; wherein the polysaccharide protein conjugate Each of the above is formed by a conjugation reaction involving an aprotic solvent, wherein the composition is a multivalent immunogenic composition that does not include an adjuvant.

28. Claims including S. pneumoniae polysaccharide protein conjugates 27. The composition according to claim 26, wherein each of the conjugates is linked to CRM197. The polysaccharides of S. pneumoniae serotype conjugated thereto, The polysaccharide-protein conjugate is used to treat blood Streptococcus pneumoniae (S. pneumoniae). The types are 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20 A, wherein serotype 20A is selected from serotype 20 or serotype 2 0B; wherein the polysaccharide protein conjugate Each of the above is formed by a conjugation reaction involving an aprotic solvent, wherein the composition is a multivalent immunogenic composition that does not include an adjuvant.

29. 29. A method of inducing a protective immune response in a human patient comprising administering to a subject any one of claims 1 to 28. Administering to said patient the multivalent immunogenic composition of claim 1.

30. The serotype of S. pneumoniae is a) 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20A; b) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 2 4F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20A ； c) 3, 7F, 19A, 22F, 33F, 6A, 6B, 15A, 16F, 23A, 23 B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20A; and d) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 2 4F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20A The set of serotypes selected from the group consisting of: Here, serotype 20A in sets a) to d) is either serotype 20 or serotype 20B.

30. The method of claim 29, wherein said amino acid is optionally substituted with

31. The serotype of S. pneumoniae is a) 15A, 16F, 23A, 23B, 24F, 31 and 35B; b) 6A, 15A, 16F, 23A, 23B, 24F, 31 and 35B 6A, 6B , 15A, 16F, 23A, 23B, 24F, 31 and 35B; c) 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B; d) 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 1 1A, 12F, 15C, 17F and 20A; e) 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10 A, 11A, 12F, 15C, 17F, and 20A; f) 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N , 10A, 11A, 12F, 15C, 17F and 20A; and g) 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10 A, 11A, 12F, 15C, 17F and 20A The set of serotypes selected from the group consisting of: Here, serotype 20A in set d) to g) is either serotype 20 or serotype 20B.

30. The method of claim 29, wherein said amino acid is optionally substituted with

32. The serotypes of S. pneumoniae are 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 1 The serotypes selected from 0A, 11A, 12F, 15C, 17F and 20A are included.

31. The method according to claim 30.

33. The serotypes of S. pneumoniae are 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 1 The serotypes selected from 0A, 11A, 12F, 15C, 17F and 20A are included.

31. The method according to claim 30.

34. 34. The method of claim 29, wherein the patient has previously been treated with a multivalent pneumococcal vaccine.

3. The method according to claim 1 .

35. The multivalent pneumococcal vaccine comprises: a) 4, 6B, 9V, 14, 18C, 19F and 23F; b) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F and 19A; c) 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F; d) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19 A, 22F and 33F; e) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 2 2F, 33F, 2, 8, 9N, 10A, 11A, 12F, 15B, 17F and 20; f) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19 A, 22F, 33F, 8, 10A, 11A, 12F and 15B; and g) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 6C, 7F , 19A, 22F, 33F, 8, 10A, 11A, 12F and 15B For preventing pneumococcal disease caused by a serotype selected from the group consisting of The method of claim 34, wherein

36. The multivalent pneumococcal vaccine comprises a plurality of polysaccharide-protein conjugates, wherein: Streptococcus pneumoniae, wherein the polysaccharide-protein conjugate is conjugated to a carrier protein The method according to claim 34 or 35, comprising a polysaccharide of the S. pneumoniae serotype. Law.

37. administering to said patient one or more additional doses of said multivalent immunogenic composition.

37. The method of any one of claims 31 to 36, comprising:

38. 38. The method of claim 37, wherein the amount of time between doses is from about 4 weeks to about 1 year.

39. 2. The method of claim 1, wherein two additional doses are administered to the patient and the patient is immunocompromised. 37 or 38. The method of claim 37 or 38.

40. further comprising administering to said patient a multivalent pneumococcal vaccine in any order, and wherein the multivalent immunogenic composition and the multivalent pneumococcal vaccine are not the same.

34. The method according to any one of claims 1 to 33.

41. The multivalent pneumococcal vaccine comprises: a) 4, 6B, 9V, 14, 18C, 19F and 23F; b) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F and 19A; c) 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F; d) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19 A, 22F and 33F; e) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 2 2F, 33F, 2, 8, 9N, 10A, 11A, 12F, 15B, 17F and 20; f) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19 A, 22F, 33F, 8, 10A, 11A, 12F and 15B; and g) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 6C, 7F , 19A, 22F, 33F, 8, 10A, 11A, 12F and 15B A pneumococcus (S. pneumoniae) serotype selected from the group consisting of 41. The method of claim 40, wherein said method is required to prevent pneumococcal disease from occurring.

42. The multivalent pneumococcal vaccine comprises a plurality of S. pneumoniae polysaccharide tags. and a protein conjugate, each of said conjugates being linked to a carrier protein. The present invention relates to a method for the preparation of a polysaccharide of a serotype of S. pneumoniae conjugated to a substrate.

42. The method of claim 41 .

43. 2. The method of claim 1, wherein the multivalent immunogenic composition and the multivalent pneumococcal vaccine are administered simultaneously.

43. The method according to any one of claims 40 to 42.

44. The amount of time between administration of the multivalent immunogenic composition and the multivalent pneumococcal vaccine is about 4 weeks.

43. The method of any one of claims 40 to 42, wherein the treatment is for up to about 1 year.

45. 41. The method of claim 40, wherein the multivalent immunogenic composition is administered prior to the multivalent pneumococcal vaccine. 42 or 44. The method according to any one of claims 42 to 44.

46. 41. The method of claim 40, wherein the multivalent pneumococcal vaccine is administered prior to the multivalent immunogenic composition. 42 or 44. The method according to any one of claims 42 to 44.

47. 47. The method of any one of claims 31 to 46, wherein the patient is 50 years of age or older.

48. 47. The method of any one of claims 31 to 46, wherein the patient is aged between 2 and 18 years.

49. 47. The method of any one of claims 31 to 46, wherein the patient is 18 years of age or older.

50. 47. The method of any one of claims 31 to 46, wherein the patient is 65 years of age or older.

51. 51. The method of any one of claims 31 to 50, wherein the patient is immunocompromised.

52. 51. The immunogenic composition of claim 31, wherein the immunogenic composition is administered by subcutaneous or intramuscular injection.

2. The method according to claim 1 .

53. 4. The method of claim 1, wherein the immunogenic composition is administered simultaneously with a vaccine against influenza.

53. A method according to any one of claims 31 to 52.

54. Conjugation reaction of serotype 8 pneumococcus (Streptococcus pneumoniae) in aprotic solvents eptococcus pneumoniae polysaccharide-protein conjugates wherein the conjugation reaction uses cyanoborohydride. No way.

55. 6. The method of claim 5, wherein the conjugation reaction is a Schiff base reduction or a reductive amination.

5. The method according to claim 4.

56. The protein is a tetanus toxoid, a diphtheria toxoid, or CRM197. The method according to claims 54 and 55.

57. 57. The method of claim 56, wherein the protein is CRM197.

58. 58. Any of claims 54 to 57, wherein the conjugation reaction is a reductive amination.

13. The method according to claim 1.

59. 5. The method of claim 4, wherein the reductive amination is carried out in dimethylsulfoxide (DMSO).

49. The method of any one of claims 4 to 48.

60. A method for inducing a protective immune response in a human patient comprising administering to a subject a multimeric antibody as claimed in any one of claims 1 to 28. administering to said human patient a multivalent immunogenic composition comprising: The method, wherein the saccharide dosage is from about 0.4 to about 4 μg.

61. Multivalent Immunoglobulins Containing S. pneumoniae Polysaccharide-Protein Conjugates an immunogenic composition, wherein each of said conjugates is conjugated to a carrier protein; The polysaccharides of the endonucleated serotypes of S. pneumoniae are The polysaccharide-protein conjugate comprises: I) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 2 4F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20; II) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20 A; III) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B , 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 2 0B; IV) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20 ； V) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 2 4F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20A and VI) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20 B The polysaccharides of S. pneumoniae serotypes selected from the group consisting of Multivalent immunogenic compositions.

62. The immunogenic composition further comprises a polysaccharide of a further S. pneumoniae serotype.

62. The multivalent immunogen of claim 61, which does not include a polysaccharide-protein conjugate having sexual composition.

63. At least one of the polysaccharide-protein conjugates comprises an aprotic solvent. The multivalent immunogenic agent of claim 61 or 62, which is formed by a conjugation reaction. composition.

64. Each of the polysaccharide-protein conjugates comprises an aprotic solvent.

64. The multivalent immunogen of any one of claims 61 to 63, formed by a cation reaction. sexual composition.

65. 6. The method according to claim 63, wherein the aprotic solvent is dimethylsulfoxide (DMSO).

5. A multivalent immunogenic composition according to any one of claims 4.

66. The carrier protein may be an outer membrane protein complex (OMPC), a tetanus toxoid, a diphtheria toxoid, teria toxoid, protein D and CRM197.

66. A multivalent immunogenic composition according to any one of claims 61 to 65.

67. 67. The method of any one of claims 61 to 66, wherein the carrier protein is CRM197. A multivalent immunogenic composition of claim 1.

68. 68. The multivalent composition of any one of claims 61 to 67, wherein the composition further comprises an adjuvant. Immunogenic composition.

69. 68. The multivalent immunization composition of any one of claims 61 to 67, wherein the composition does not include an adjuvant. Epidemiogenic composition.

70. A method of inducing a protective immune response in a human patient comprising administering to a subject a compound according to any one of claims 61 to 69.

2. A method comprising administering to a patient the multivalent immunogenic composition of claim 1.

71. 71. The method of claim 70, wherein the patient has previously been treated with a multivalent pneumococcal vaccine.

72. administering to said patient one or more additional doses of said multivalent immunogenic composition.

72. The method of any one of claims 70 to 71, comprising:

73. 73. The method according to any one of claims 70 to 72, wherein the amount of time between doses is from about 4 weeks to about 1 year. How to.

74. 71. The method of claim 70, wherein two additional doses are administered to the patient, and the patient is immunocompromised.

73. The method of any one of claims 73.

75. further comprising administering to said patient a multivalent pneumococcal vaccine in any order, and wherein the multivalent immunogenic composition and the multivalent pneumococcal vaccine are not the same.

75. The method of any one of claims 0 to 74.

76. The multivalent pneumococcal vaccine comprises a plurality of S. pneumoniae polysaccharide proteins. and protein conjugates, each of which is linked to a carrier protein. Conjugated S. pneumoniae serotype polysaccharides. The method of claim 75.

77. 2. The method of claim 1, wherein the multivalent immunogenic composition and the multivalent pneumococcal vaccine are administered simultaneously.

77. The method of any one of claims 75 to 76.

78. The amount of time between administration of the multivalent immunogenic composition and the multivalent pneumococcal vaccine is about 4 weeks.

78. The method of any one of claims 75 to 77, wherein the treatment is for up to about 1 year.

79. 76. The method of claim 75, wherein the multivalent immunogenic composition is administered prior to the multivalent pneumococcal vaccine.

79. The method according to any one of claims 78 to 78.

80. 76. The method of claim 75, wherein the multivalent pneumococcal vaccine is administered prior to the multivalent immunogenic composition.

79. The method according to any one of claims 78 to 78.

81. 81. The method of any one of claims 70 to 80, wherein the patient is 50 years of age or older.

82. 82. The method of any one of claims 70 to 81, wherein the patient is aged between 2 and 18 years.

83. 83. The method of any one of claims 70 to 82, wherein the patient is 18 years of age or older.

84. 84. The method of any one of claims 70 to 83, wherein the patient is 65 years of age or older.

85. 85. The method of any one of claims 70 to 84, wherein the patient is immunocompromised.

86. 85. The immunogenic composition of claim 70, wherein the immunogenic composition is administered by subcutaneous or intramuscular injection.

2. The method according to claim 1 .

87. 4. The method of claim 1, wherein the immunogenic composition is administered simultaneously with a vaccine against influenza.

87. The method of any one of claims 70 to 86.

88. 1. A method for preventing pneumococcal pneumonia and invasive disease in adults 18 years of age or older, comprising:

63. A method comprising administering to a patient the multivalent immunogenic composition of claim 62.

89. 24 strains of Streptococcus pneumoniae (3, 6A, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16 F, 17F, 19A, 20A, 20B, 22F, 23A, 23B, 24F, 31, 33F and 35B)と、55A、55B、55C ...

63. A method comprising administering to a patient the multivalent immunogenic composition of claim 62. 。

90. 1. A multivalent immunogenic composition comprising 21 distinct polysaccharide-protein conjugates, wherein each of the conjugates is a pneumococcus conjugated to a carrier protein ( and a capsular polysaccharide of the S. pneumoniae serotype, wherein the polysaccharide is S. pneumoniae serotypes 3, 7F, 19A, 22F, 33F, 6A, 15 A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12 F, 15C, 17F and 20A, and wherein the carrier protein is C A multivalent immunogenic composition that is RM197.

91. Immunogenic compositions have been prepared from other S. pneumoniae serotypes.

91. The multivalent immunogenic composition of claim 90, which does not comprise a polysaccharide protein conjugate.

92. Each of the polysaccharide-protein conjugates comprises an aprotic solvent. The aprotic solvent is dimethyl sulfoxide ( 91. The multivalent immunogenic composition of claim 90, wherein the immunogenic composition is DMSO.

93. 91. The multivalent immunogenic composition of claim 90, wherein the composition does not include an adjuvant.

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