EA043489B1 - COMPOSITIONS CONTAINING CONJUGATES OF STREPTOCOCCUS PNEUMONIAE POLYSACCHARIDE WITH PROTEIN AND METHODS OF THEIR APPLICATION - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to multivalent immunogenic compositions containing different polysaccharide-protein conjugates. Each conjugate consists of a capsular polysaccharide derived from a different (different) serotype of Streptococcus pneumoniae conjugated to a carrier protein, preferably CRM197. Immunogenic compositions provide a broad spectrum of action against pneumococcal disease.

State of the art

Streptococcus pneumoniae is a Gram-positive bacterium and the most common cause of invasive bacterial diseases (such as pneumonia, bacteremia, meningitis, and otitis media) in infants and young children. The pneumococcus is encapsulated by a chemically cross-linked polysaccharide, which imparts serotype specificity. There are more than 90 known serotypes of pneumococci, and the capsule is the main factor determining the virulence of pneumococci, since the capsule not only protects the inner surface of the bacteria from complement, but is itself weakly immunogenic. Polysaccharides are T-independent antigens and, in most cases, cannot be processed or presented by MHC molecules for interaction with T cells. However, they may stimulate the immune system through an alternative mechanism that involves cross-linking of surface receptors on B cells.

Polyvalent pneumococcal polysaccharide vaccines, licensed for many years, have proven value in the prevention of pneumococcal disease in adults, especially the elderly and those at high risk. However, infants and young children respond poorly to unconjugated pneumococcal polysaccharides. Prevnar® pneumococcal conjugate vaccine, containing the 7 most commonly isolated serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) that cause invasive pneumococcal disease in young children and infants, was first licensed in the United States in February 2000. Since the widespread use of Prevnar® in the United States, there has been a significant reduction in the incidence of invasive pneumococcal disease in children caused by serotypes present in Prevnar®. See Centers for Disease Control and Prevention, MMWR, Morb. Mortal. Wkly. Rep., 2005, 54(36):893-7. However, Prevnar® has limited serotype coverage in some regions of the world, and there is evidence of some new serotypes (eg, 19A and others) emerging in the United States.

See O'Brien et al., 2004, Am. J Epidemiol 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., 2007, J. Infect. Dis., 196:1346-54; Traore et al., 2009, Clin. Infect. Dis., 48:S181–S189.

US Patent Application Publication No. US 2006/0228380 describes a 13-valent pneumococcal polysaccharide-protein conjugate vaccine comprising serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. Chinese Patent Application Publication No. CN 101590224 A describes a 14-valent pneumococcal polysaccharide-protein conjugate vaccine including serotypes 1, 2, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F and 23F .

Other PCVs include the 7, 10, 11 or 13 serotypes contained in PCV-15 (US Patent Application Publication 2011/0195086), but immune interference has been observed with some serotypes (eg, lower protection for serotype 3 in GSK PCV-11 ) and lower response rates to serotype 6B in Pfizer's PCV-13 (PREVNAR® 13). See Prymula et al., 2006, Lancet, 367:740-48; and 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.

Current multivalent pneumococcal vaccines are effective in reducing the incidence of pneumococcal disease associated with the serotypes present in the vaccines. However, the prevalence of pneumococci expressing serotypes not present in currently available vaccines is increasing. Accordingly, there is a need for additional pneumococcal vaccine compositions that provide protection against different sets of pneumococcal serotypes and that can provide additional protection against pneumococcal serotypes not present in currently available vaccines.

The essence of the invention

The invention relates to polyvalent immunogenic compositions containing conjugates of S. pneumoniae polysaccharide with protein (S. pneumoniae polysaccharide-protein conjugates), where each of the conjugates contains a polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, and the serotypes of S. pneumoniae are as follows: as defined herein.

In specific embodiments, the S. pneumoniae serotypes comprise a set of serotypes selected from the group consisting of

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a) 15A, 16F, 23A, 23B, 24F, 31 and 35B;

b) 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11 A, 12F, 15C, 17F and 20; And

c) 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, PA, 12F, 15C, 17F and 20.

In further specific embodiments, the S. pneumoniae serotypes comprise a set of serotypes selected from the group consisting of

a) 15A, 16F, 23A, 23B, 24F, 31 and 35B;

b) 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, IA, 12F, 15C, 17F and 20A; And

c) 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, PA, 12F, 15C, 17F and 20A.

In further specific embodiments, the S. pneumoniae serotypes comprise a set of serotypes selected from the group consisting of

a) 15A, 16F, 23A, 23B, 24F, 31 and 35B;

b) 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, PA, 12F, 15C, 17F and 20B; And

c) 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, PA, 12F, 15C, 17F and 20B.

In some embodiments, the set of S. pneumoniae serotypes listed in a), b) or c) further comprises serotype 6C, serotype 6A, or serotypes 6A and 6B.

In some embodiments, at least one of the polysaccharide-protein conjugates is formed by a conjugation reaction containing an aprotic solvent, such as dimethyl sulfoxide (DMSO). In specific embodiments, each of the polysaccharide-protein conjugates is formed by a conjugation reaction containing an aprotic solvent. As shown herein, the use of DMSO as a solvent during the reductive amination of polysaccharide-protein conjugates provides unexpectedly high stability and increased immunogenicity of these serotypes compared to the same conjugates prepared under aqueous conditions.

The invention also relates to methods for inducing a protective immune response in a human patient, including administering to the patient the multivalent immunogenic compositions of the invention. In some embodiments of the methods of the invention, the patient has previously received treatment with a multivalent pneumococcal vaccine.

The multivalent immunogenic composition of the invention can be used as part of a treatment regimen with another complementary pneumococcal vaccine. Accordingly, the invention relates to a method of inducing a protective immune response in a human patient, comprising administering to the patient a multivalent immunogenic composition of the invention, further comprising administering to the patient a multivalent pneumococcal vaccine in any order. In specific embodiments, the multivalent pneumococcal vaccine consists of several S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates contains a S. pneumoniae serotype polysaccharide conjugated to a carrier protein. In other embodiments, the multivalent pneumococcal vaccine is composed of unconjugated capsular polysaccharides.

The invention also relates to methods for preparing a conjugate of a Streptococcus pneumoniae serotype 8 polysaccharide with a protein using a conjugation reaction in an aprotic solvent, where the conjugation reaction does not use cyanoborohydride.

The invention also relates to multivalent immunogenic compositions containing S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates contains a polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, wherein selected S. pneumoniae serotypes provide cross-reactivity with other selected serotypes.

Brief description of drawings

In fig. Figure 1 shows a 600 MHz one-dimensional 1H NMR spectrum of S. pneumonia serotype 6C capsular polysaccharide in deuterium oxide (D2O) at 50°C. Signals due to internal standards (DMSO and DSS-d ₆ ) and residual water (HOD) are noted. Minor signals marked * are due to S. pneumonia cell wall residues such as C-polysaccharide and/or peptidoglycans.

In fig. Figure 2 shows the 600 MHz one-dimensional 1H NMR spectrum of S. pneumonia serotype 15A capsular polysaccharide in deuterium oxide (D2O) at 50°C. Signals due to internal standards (DMSO and DSS-d ₆ ) and residual water (HOD) are noted. Minor signals marked * are due to S. pneumonia cell wall residues such as C-polysaccharide and/or peptidoglycans.

In fig. 3 shows a 600 MHz one-dimensional 1H NMR spectrum of de-O-acetylated S. pneumonia serotype 15B capsular polysaccharide in deuterium oxide (D ₂ O) at 50°C. Signals due to internal standards (DMSO and DSS-d6) and residual water (HOD) are noted. Minor signals marked * are due to S. pneumonia cell wall residues such as C-polysaccharide and/or peptidoglycans.

In fig. Figure 4 shows the 600 MHz one-dimensional 1H NMR spectrum of capsular polysaccharide serotype 35B

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S. pneumonia in D2O at 50°C. Signals due to internal standards (DMSO and

DSS-d ₆ ) and residual water (HOD). Minor signals marked * are due to S. pneumonia cell wall residues such as C-polysaccharide and/or peptidoglycans.

In fig. Figure 5 shows a 1H NMR identical region useful for identifying S. pneumonia serotype 6C. The signal positions of each anomeric proton of the repeating unit of each monosaccharide residue are marked.

In fig. Figure 6 shows an 1H NMR identical region useful for identifying S. pneumonia serotype 15A. The signal positions of each anomeric proton of the repeating unit of each monosaccharide residue are marked.

In fig. 7 shows a 1H NMR identical region useful for identifying de-O-acetylated S. pneumonia serotype 15B. The signal positions of each anomeric proton of each monosaccharide residue are indicated.

In fig. 8 shows a 1H NMR identical region useful for identifying S. pneumonia serotype 35B. The signal positions of each anomeric proton of the repeating unit of each monosaccharide residue are marked.

In fig. 9A-9C show the 600 MHz one-dimensional 1H NMR spectrum of native S. pneumonia serotype 15B capsular polysaccharide (Fig. 9A), de-O-acetylated S. pneumonia serotype 15B capsular polysaccharide (Fig. 9B), and S. pneumonia serotype 15C capsular polysaccharide (Fig. 9C). The spectra were obtained in deuterium oxide (D2O) at 50°C. Signals due to internal standards (DMSO and DSS-d ₆ ) and residual water (HOD) are noted. Signals marked * are due to S. pneumonia cell wall residues such as C-polysaccharide and/or peptidoglycans.

In fig. 10A-10C show the 600 MHz anomeric region of the one-dimensional 1H NMR spectrum of native S. pneumonia serotype 15B capsular polysaccharide (Fig. 10A), de-O-acetylated S. pneumonia serotype 15B capsular polysaccharide (Fig. 10B), and S. serotype 15C capsular polysaccharide. pneumonia (Fig. 10C). In fig. 10D-10F show the signal spectrum regions of O-acetyl- and N-acetylmethyl of native S. pneumonia serotype 15B capsular polysaccharide (Fig. 10D), de-O-acetylated S. pneumonia serotype 15B capsular polysaccharide (Fig. 10E), and serotype 10E capsular polysaccharide 15C S. pneumonia (Fig. 10F).

In fig. Figure 11 shows the effect of time and temperature (up to 12 weeks at 4°C (diamonds), up to 4 weeks at 25°C (squares), up to 4 weeks at 37°C (triangles)) on the concentration of polysaccharide in PCV16 drugs (0.128 mg /ml) or PCV21 (0.084 or 0.169 mg/ml) determined using HPSEC-UV/MALS/RI methods (see example 39).

In fig. Figure 12 shows the effect of horizontal agitation and temperature (1 week at 4°C, 25°C, or 37°C) on the polysaccharide concentration of PCV16 (0.128 mg/ml) or PCV21 (0.084 or 0.169 mg/ml) drug formulations in in pre-filled syringes (see example 39).

In fig. Figure 13 shows the effect of PS concentration as a function of time and temperature (up to 4 weeks at 4, 25 or 37°C) on the average molecular weight (Mw and Mn) of drugs PCV16 (0.128 mg/ml) or PCV21 (0.084 or 0.169 mg/ ml), determined using HPSECUV/MALS/RI methods.

In fig. 14A and 14B show the effect of time and temperature (up to 1 week at 4 or 37°C) on the stability of pneumococcal conjugate vaccine (PCV15 or PCV16) prepared with drugs conjugated in A) protic solvent (PCV15 obtained by conjugation in all-aqueous medium) or B) aprotic solvent (PCV16, obtained by conjugation in DMSO at 0.064 mg/ml PnPs) using internal protein fluorescence spectroscopy with an excitation wavelength of 280 nm (Example 40).

In fig. Figure 15 shows IgG antibody dilution titers by ELISA (after dose 2) in rabbits immunized with monovalent S. pneumoniae serotypes conjugated to CRM197 and formulated with aluminum phosphate adjuvant. Symbols indicate individual titers and error bars represent 95% confidence intervals (CI) of geometric mean titers (GMT).

In fig. Figure 16 shows serotype-specific OPA dilution titers (after dose 2) in rabbits immunized with monovalent S. pneumoniae serotypes conjugated to CRM197 and formulated with aluminum phosphate adjuvant (APA). Symbols indicate individual titers and error bars represent 95% confidence intervals (CI) of geometric mean titers (GMT).

In fig. Figure 17 shows IgG antibody dilution titers by ELISA (after dose 2) in rabbits immunized with monovalent S. pneumoniae serotype 15 conjugates. The x-axis shows the vaccine used to immunize rabbits. Dashed lines separate three separate ELISA assays using a separate pneumococcal polysaccharide as the coating antigen. Symbols indicate individual titers and error bars represent 95% confidence intervals (CI) of geometric mean titers (GMT).

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In fig. 18 shows serotype-specific OPA dilution titers (pre-immunization, post-dose 1 (PD1), combined) and post-dose 2 (PD2)) in rabbits immunized with monovalent S. pneumoniae serotype 15 conjugates. The x-axis shows the vaccine used to immunize rabbits. Dashed lines separate three OPA assays using individual S. pneumoniae bacterial strains. Symbols indicate individual titers and error bars represent 95% confidence intervals (CI) of geometric mean titers (GMT).

In fig. 19A shows a comparison of PD1 antibody responses in NZWR (5 per group) following vaccination with 4, 2, 1, 0.4, 0.08 or 0.016 μg/dose PCV21. Symbols indicate geometric mean titer (GMT) ratios of PD1 (2 μg/dose group versus other dose groups) and error bars represent 95% confidence intervals (CI). In fig. 19B shows the PD1 GMT ratios (95% CI) for the PCV21 doses being compared, corresponding to the GMT ratios in FIG. 19A, the lower 95% confidence limit of which is greater than 1.0, is shaded in light gray, and the GMT ratios, the upper 95% confidence limit of which is below 1.0, are shaded dark gray. Data for serotype 15B are included to assess cross-protection.

In fig. 20A shows a comparison of PD2 antibody responses in NZWR (5 per group) following vaccination with 4, 2, 1, 0.4, 0.08 or 0.016 μg/dose PCV21. Symbols indicate PD2 GMT ratios (2 μg/dose group versus other dose groups) and error bars represent 95% CIs. In fig. 20B shows PD2 GMT ratios (95% confidence interval) for the PCV21 doses being compared, corresponding to the GMT ratios in FIG. 20A, for which the lower 95% confidence interval greater than 1.0 is shaded in light gray. Data for serotype 15B are included to assess cross-protection.

In fig. 21A shows serotype-specific OPA PD1 dilution titers in rabbits immunized with PCV21 (2 μg/PnPs). Symbols indicate individual titers and error bars represent 95% confidence intervals (CI) of geometric mean titers (GMT). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. In fig. 21B shows the PD2 dilution titers as determined by ORA in rabbits immunized with PCV21 (2 μg/PnPs). Symbols indicate individual titers and error bars represent 95% confidence intervals (CI) of geometric mean titers (GMT), ****p<0.0001. Data from serotype 15B are included to assess cross-protection.

In fig. 22A-22C show the effect of time and temperature (i.e., 7 days at 4°C, 1 day at 37°C, or 7 days at 37°C) on the stability of three pneumococcal conjugate vaccines (PCV1, PCV7, and PCV14) prepared with all drugs conjugated in either aqueous or DMSO solvent by internal protein fluorescence spectroscopy with an excitation wavelength of 280 nm. In fig. 22D shows the effect of time and temperature (i.e., 7 days at 4 or 37°C) on the stability of a 21-valent pneumococcal conjugate vaccine (PCV21 at 0.084 mg/ml PnPs) prepared with all drugs conjugated in DMSO using the method fluorescence spectroscopy with internal protein with an excitation wavelength of 280 nm.

In fig. Figure 23 shows the effect of temperature (7 days at 4 or 37°C) and agitation at 4°C on particle size distribution as determined by nanoparticle trajectory analysis (NTA) for six (6) PCV21 pneumococcal conjugate vaccine formulations formulated with PS- 20 (0, 0.025, 0.05, 0.1, 0.15, or 0.2%; all in PS-20 w/v units) at 0.084 mg/mL PnPs.

In fig. Figure 24 shows the effect of temperature (4 and 37°C) and agitation on the average molecular weight of three PCV21 drugs as determined by HPSEC/UV/MALS/RI analysis. Three pneumococcal conjugate PCV21 vaccine formulations were prepared with different concentrations of PS-20 (0.05, 0.1, and 0.15% w/v) at 0.084 mg/mL PnPs.

In fig. 25 shows that mice immunized with PCV21 are protected from intratracheal challenge with 24F S. pneumoniae.

In fig. 26A and 26B show preimmunization IgG (pooled), PD1 (pooled), and PD2 antibody dilution titers determined by ECL in rabbits immunized with PCV21 formulated without adjuvants or with APA. Error bars represent 95% confidence intervals (CI) of the geometric mean titer (GMT). Data from serotype 15B are included to assess cross-protection.

In fig. 27A-27C show preimmunization IgG (pooled), PD1 (pooled), and PD2 antibody dilution titers determined by ECL in rabbits immunized with PCV8 without adjuvants, PCV16 without adjuvants, and PCV31 with APA. Error bars represent 95% confidence intervals (CI) of the geometric mean titer (GMT). Data from serotype 15B are included to assess cross-protection.

In fig. Figure 28 shows a comparison of PD2 levels of antibody synthesis as measured by ECL in NZWR (5 per group) following PCV21 vaccination with or without ARA. Symbols indicate GMT PD2 ratios with error bars representing 95% CI. Data from serotype 15B are included to assess cross-protection.

In fig. Figure 29 shows a comparison of PD2 levels of antibody synthesis based on ECL results in NZWR (5 per group) for common serotypes and cross-protected serotype 15B after vaccination with PCV21, PCV8

- 4 043489 or PCV16. Symbols indicate GMT PD2 ratios from ECL results with error bars representing 95% CI.

In fig. Figure 30 shows a comparison of PD2 levels of antibody synthesis by ECL in NZWR (5 per group) for common serotypes and cross-protected serotype 15B following vaccination with PCV21/APA or PCV31/APA. Symbols indicate GMT PD2 ratios from ECL results with error bars representing 95% CI.

In fig. 31A-31D show serotype-specific dilution titers of pre-immunization OPA (combined) (A, B) and PD2 (C, D) in rabbits immunized with PCV. Error bars represent 95% CI GMT. PCV21 was used as reference for statistical comparisons, *p < 0.05. In fig. 31A and 31C show data for 8 common serotypes in all PCVs evaluated (6C, 15A, 16F, 23A, 23B, 24F, 31, 35B) and an additional 8 common serotypes for PCV16, PCV21 and PCV31 (8, 9N, 10A, 11A, 12F, 15 C, 17F, 20B). In fig. 31B and 31D show data for an additional 16 serotypes not shown in FIG. 31A and 31C. Five of them are also common serotypes for PCV21 and PCV31 (3, 7F, 19A, 22F, 33F), ten serotypes are contained exclusively in PCV31 (1, 4, 5, 6A, 6B, 9B, 14, 18C, 19F, 23F) . Data for serotype 15B are included to assess cross-protection.

In fig. 32 shows pre-immunization IgG antibody dilution titers, PD1, PD2 and PD3, determined from ECL results in adult rhesus monkeys (n=8) immunized with PCV21. Error bars represent 95% CI GMT. Data from serotype 15B are included to assess cross-protection.

In fig. 33 shows pre-immunization dilution titers of OPA, PD1 (combined) and PD3 in macaques immunized with PCV21 (n=8). Error bars represent 95% CI GMT. Data from serotype 15B are included to assess cross-protection.

In fig. Figure 34 shows pre-immunization OPA and PD3 titers (day 70) for four non-PCV21 vaccine serotypes in adult rhesus monkeys immunized with PCV21.

In fig. Figure 35 shows a comparison of PD1 levels of antibody synthesis as measured by ECL in adult macaquesus (5 per group) after vaccination with PCV21 with or without ARA. Symbols indicate GMT PD2 ratios from ECL results (PCV21 vs. PCV21/APA) with error bars representing 95% CIs. Data from serotype 15B are included to assess cross-protection.

In fig. 36 shows PD1 levels of IgG antibody synthesis to serotypes 3, 7F, 19A, 22F and 33F in adult rhesus monkeys (2-5 per group) after vaccination with PCV21 compared with PCV15 or Prevnar13. Symbols indicate GMT PD2 ratios from ECL results with error bars representing 95% CI.

In fig. 37 shows the ELISA dilution titers of IgG antibodies for 6A, 6B and 6C ((pre-immunization and PD1 combined) and PD2) in rabbits immunized with a drug based on monovalent 6A-CRM197 (A) or 6B-CRM197 (B). Columns indicate geometric mean titers (GMT) and error bars indicate 95% confidence intervals (CI) of GMT.

In fig. Figure 38 shows serotype 6A, 6B and 6C-specific OPA dilution titers ((pre-immunization and PD1 combined) and PD2) in rabbits immunized with drugs based on monovalent 6A-CRM197 (A) or 6B-CRM197 (B). Columns indicate geometric mean titers (GMT) and error bars -95% confidence intervals (CI) GMT.

In fig. 39 shows serotype 20A and 20B specific OPA dilution titers (pre-immunization (combined), PD1 and PD2) in rabbits immunized with 2OA-CRM197/APA (A) or PCV21 (B). Columns indicate geometric mean titers (GMT) and error bars indicate 95% confidence intervals (CI) of GMT.

Detailed Description of the Invention

The present invention relates to multivalent immunogenic compositions containing pneumococcal polysaccharide-protein conjugates, wherein each of the conjugates contains a polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, the S. pneumoniae serotypes being as defined herein. In some embodiments, the immunogenic composition contains a set of pneumococcal serotypes selected from the group consisting of (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 contains a set of pneumococcal serotypes selected from the group consisting of (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 20B; and (c) 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20B.

In other embodiments, the immunogenic composition contains a set of pneumococcal serotypes selected from the group consisting of (a) 15A, 16F, 23A, 23B, 24F, 31 and 35B;

(b) 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9H, 10A, 11A, 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.

In other embodiments, the immunogenic composition comprises

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(ii) serotypes 6A and 6B; or (iii) serotype 6C.

In a specific embodiment, the invention comprises multiple pneumococcal S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates comprises a polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes include serotypes 3, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 20A, 22F, 23A, 23B, 24F, 31, 33F and 35B. The composition was found to be immunogenic in rabbits and generated a functional antibody that killed vaccine-type bacterial strains at all doses tested (Example 43). In another specific embodiment, the invention comprises multiple pneumococcal S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates comprises a S. pneumoniae serotype polysaccharide conjugated to a carrier protein, wherein the S. pneumoniae serotypes comprise serotypes 3, 7F, 19A, 22F, 33F , 8, 9N, 10A, 11A, 12F, 15C, 17F, 20, 6A, 15A, 15C, 16F, 23A, 23B, 24F, 31 and 35B. In another specific embodiment, the invention comprises multiple pneumococcal S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates comprises a S. pneumoniae serotype polysaccharide conjugated to a carrier protein, wherein the S. pneumoniae serotypes comprise serotypes 3, 7F, 19A, 22F, 33F , 8, 9N, 10A, 11A, 12F, 15C, 17F, 20B, 6A, 15A, 15C, 16F, 23A, 23B, 24F, 31 and 35B. In another specific embodiment, the invention comprises multiple pneumococcal S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates comprises a S. pneumoniae serotype polysaccharide conjugated to a carrier protein, wherein the S. pneumoniae serotypes comprise serotypes 3, 7F, 19A, 22F, 33F , 8, 9N, 10A, 11A, 12F, 15C, 17F, 20A, 6A, 15A, 15C, 16F, 23A, 23B,24F,31 and 35B.

The multivalent immunogenic compositions of the invention are useful for immunizing a patient against vaccine serotypes of S. pneumoniae and as a component of a treatment regimen with other complementary pneumococcal vaccine(s). Accordingly, the invention relates to a method of inducing a protective immune response in a human patient, comprising administering to the patient a multivalent immunogenic composition of the invention and further comprising administering to the patient a multivalent pneumococcal vaccine in any order. In other embodiments, the multivalent immunogenic compositions of the invention are administered to a patient who has previously been immunized with another multivalent pneumococcal vaccine.

In embodiments of the invention, conjugates of at least one pneumococcal serotype are prepared by a reductive amination mechanism in an aprotic solvent such as DMSO. In other embodiments, the multivalent immunogenic composition comprises pneumococcal conjugates, each of which was produced by a reductive amination mechanism in an aprotic solvent. The present disclosure demonstrates that drug formulations PCV1, PCV7, PCV14, PCV16 and PCV21 (defined below), containing drug substances each produced by a reductive amination mechanism in an aprotic solvent, are resistant to depolymerization or chemical degradation of carbohydrate, and are resistant to protein aggregation in the drug (see examples 40 and 45). The use of DMSO solvent strengthens the covalent bonds between the polysaccharide and the protein, formed with the direct participation of lysine residues on the surface of the carrier protein. Improved covalent bonds provide a direct benefit in terms of increasing the stability of the polysaccharide-protein conjugate of multivalent immunogenic compositions containing polysaccharide antigens conjugated in DMSO.

I. Definitions and abbreviations.

In the description and attached claims the following abbreviations are used:

APA - adjuvant based on aluminum phosphate;

APC - antigen presenting cell;

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;

LOS - lipooligosaccharide;

LPS - lipopolysaccharide;

MALS - multi-angle light scattering;

MBC - monovalent mixed (general) conjugate;

MORA - multiplex analysis of opsonophagocytic reactions;

MW - molecular weight;

NMWCO - nominal molecular weight cutoff;

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NZWR - New Zealand white rabbit;

OPA - opsonophagocytosis assay;

PCV - pneumococcal conjugate vaccine;

PD1 - after dose 1;

PD2 - after dose 2;

PnPs - pneumococcal polysaccharide;

Ps - polysaccharide;

PS-20 - polysorbate-20;

RI - refractive index;

UV - ultraviolet;

w/v - w/v.

To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used herein have the meaning commonly understood by one skilled in the art to which the present invention relates.

As used in the specification and the accompanying claims, the singular form includes a reference to the plural unless the context clearly indicates otherwise.

A reference to or indicates one or both possibilities unless one of the specified possibilities is clearly implied by the context. In some cases, and/or is used to highlight one or both possibilities.

The terms aqueous solvent or aqueous conditions used in connection with conjugation such as reductive amination refer to the use of water as a solvent in the conjugation reaction. Water may contain buffers and other components, with the exception of the organic solvent.

The terms aprotic solvent, DMSO solvent, or DMSO terms used in connection with conjugation such as reductive amination refer to the use of an aprotic solvent or combination of aprotic solvents (or DMSO, as appropriate) as the solvent in the conjugation reaction. The aprotic solvent may contain some amount of water, for example up to 1, 2, 5, 10 or 20%.

The term contains, as used in connection with an immunogenic composition of the invention, refers to the inclusion of any other components, such as adjuvants and excipients, or the addition of one or more polysaccharide-protein conjugates that are not specifically stated. The term "consisting of", as used in reference to a polyvalent polysaccharide-protein conjugate mixture, refers to a mixture containing those particular S. pneumoniae polysaccharide-protein conjugates and not containing any other polysaccharide-protein conjugates of another S. pneumoniae serotype. essentially consists of and variations such as essentially consist of or essentially consisting of indicate the inclusion of any of the listed elements or groups of elements and the optional inclusion of other elements similar to or different in nature from the listed elements that do not substantially alter the essential or new properties of the specified dosage regimen, method or composition.

An effective amount of a composition of the invention refers to the dose required to generate antibodies that significantly reduce the likelihood or severity of infectivity of a microbe, such as S. pneumonia, during subsequent infection.

As used herein, indicated for the prevention of pneumococcal disease means that the vaccine or immunogenic composition is approved by one or more regulatory authorities, such as the US Food and Drug Administration, for the prevention of one or more diseases caused by any serotype of S. pneumoniae , including but not limited to generally pneumococcal disease, pneumococcal pneumonia, pneumococcal meningitis, pneumococcal bacteremia, invasive disease caused by S. pneumoniae, and otitis media caused by S. pneumoniae.

A polyvalent pneumococcal vaccine is a pharmaceutical preparation containing more than one active agent (eg, pneumococcal capsular polysaccharide or pneumococcal polysaccharide-protein conjugate) that provides active immunity to a disease or condition caused by more than one serotype of S. pneumoniae.

The term polysaccharide means to include any antigenic saccharide element (or antigenic unit) commonly used in the field of immunological and bacterial vaccines, including, without limitation, a saccharide, oligosaccharide, polysaccharide, liposaccharide, lipooligosaccharide (LOS), lipopolysaccharide (LPS), glycosylate, glycoconjugate, etc. P.

PCV1 as used herein refers to a 1-valent pneumococcal conjugate vaccine containing one S. pneumoniae polysaccharide-protein conjugate comprising a capsular polysaccharide of the S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotype is serotype 3 (as shown in examples 38 and 45). In specific embodiments

- 7 043489 carrier protein is CRM197.

PCV7 as used herein refers to a 7-valent pneumococcal conjugate vaccine containing seven S. pneumoniae polysaccharide-protein conjugates, each containing capsular polysaccharides of S. pneumoniae serotypes conjugated to a carrier protein, wherein the S. pneumoniae serotypes are 3, 8, 9N, 11A, 19A, 15A and 10A. In specific embodiments, the carrier protein of the one or more S. pneumoniae polysaccharide-protein conjugates is CRM197. In other embodiments, the carrier protein of each of the S. pneumoniae polysaccharide-protein conjugates is CRM197.

PCV8 as used herein refers to an 8-valent pneumococcal conjugate vaccine containing eight S. pneumoniae polysaccharide-protein conjugates, each containing a capsular polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes are 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B. In specific embodiments, the carrier protein of the one or more S. pneumoniae polysaccharide-protein conjugates is CRM197. In other embodiments, the carrier protein of each of the S. pneumoniae polysaccharide-protein conjugates is CRM197.

PCV14 as used herein refers to a 14-valent pneumococcal conjugate vaccine containing fourteen S. pneumoniae polysaccharide-protein conjugates, each containing capsular polysaccharides of an S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes are 3, 7F, 8, 9N, 10A, 11A, 12F, 15A, 16F, 17F, 19A, 20, 22F and 33F. In specific embodiments, the carrier protein of the one or more S. pneumoniae polysaccharide-protein conjugates is CRM197. In other embodiments, the carrier protein of each of the S. pneumoniae polysaccharide-protein conjugates is CRM197.

PCV15 as used herein refers to a 15-valent pneumococcal conjugate vaccine containing fifteen S. pneumoniae polysaccharide-protein conjugates, each containing capsular polysaccharides of an S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes are 1, 3, 4, 5, 6A, 6B, 7F, 9B, 14, 18C, 19A, 19F, 22F, 23F and 33F. In specific embodiments, the carrier protein of the one or more S. pneumoniae polysaccharide-protein conjugates is CRM197. In other embodiments, the carrier protein of each of the S. pneumoniae polysaccharide-protein conjugates is CRM197.

PCV16 as used herein refers to a 16-valent pneumococcal conjugate vaccine containing sixteen S. pneumoniae polysaccharide-protein conjugates, each containing a capsular polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes are 6C, 8, 9N, 10A, 11A, 12F, 15A, 16F, 17F, 20A, 23A, 23B, 24F, 31 and 35B, and at least one of the following serotypes of serogroup 15: 15B, 15C or de-Oacetylated 15B. In specific embodiments, serotype 15 is serotype 15C or de-O-acetylated 15B. As shown herein (see Example 3 below), de-Oacetylated pneumococcal polysaccharide serotype 15B is equivalent to pneumococcal polysaccharide serotype 15C and has identical NMR spectra. In specific embodiments, the carrier protein of the one or more S. pneumoniae polysaccharide-protein conjugates is CRM197. In other embodiments, the carrier protein of each of the S. pneumoniae polysaccharide-protein conjugates is CRM197.

PCV21 as used herein refers to a 21-valent pneumococcal conjugate vaccine containing twenty-one S. pneumoniae polysaccharide-protein conjugates, each containing a capsular polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes are 3 , 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 16F, 17F, 19A, 20A, 22F, 23A, 23B, 24F, 31, 33F and 35B, and at least one of the following serotypes of serogroup 15: 15B, 15C or de-O-acetylated-15B. In specific embodiments, serotype 15 is serotype 15C or de-O-acetylated 15B. In specific embodiments, the carrier protein of the one or more S. pneumoniae polysaccharide-protein conjugates is CRM197. In other embodiments, the carrier protein of each of the S. pneumoniae polysaccharide-protein conjugates is CRM197.

S. pneumoniae serotypes 20A and 20B were identified (Calix, J.J. et al., J. Biol., Chemical (2012), 287(33):27885-27894) in serogroup 20 in 2012 using genetic analysis. As stated by Calix J.J. et al., these two serotypes have not previously been identified using conventional serotyping methods and commercially available antibodies, and limited information is currently available regarding attempts to serotype serotypes 20A and 20B. In addition, the prevalence of diseases caused by 20A or 20B among identified diseases caused by serogroup 20 remains unclear. As such, one or both of these serotypes (20A and/or 20B) can be selected by one of ordinary skill in the art for inclusion in the S. pneumoniae vaccine composition. It is also possible that an S. pneumoniae vaccine containing one serotype 20 polysaccharide antigen may provide cross-protection against another antigen (e.g., a vaccine containing conjugates of S. pneumoniae serotype 20A polysaccharide, rather than serotype 20B, with protein may provide cross-protection against infection caused by serotype 20B S. pneumoniae). Therefore, the term serotype 20 as used herein may refer to a composition containing serotype 20A and/or serotype 20B polysaccharide conjugates with protein.

CpG-containing nucleotide, CpG-containing oligonucleotide, CpG oligonucleotide and similar terms refer to a nucleotide molecule 6-50 nucleotides in length that contains an unmethylated CpG fragment. See, for example, Wang et al., 2003, Vaccine, 21:4297. CpG-containing oligonucleotides include modified oligonucleotides formed by any synthetic modified base and/or modified sugar internucleoside linkages.

An adjuvant, as used herein, is a substance that serves to enhance the immunogenicity of the immunogenic composition of the invention. An immune adjuvant can enhance the immune response to an antigen that, when administered alone, is weakly immunogenic, e.g., inducing no or weak antibody titers or a cellular immune response, can increase antibody titers to the antigen, and/or allows a reduction in the dose of antigen effective to achieve an immune response in person. Thus, adjuvants are often used to enhance the immune response and are well known to one skilled in the art.

A patient (alternatively referred to herein as a subject) refers to a mammal capable of being infected with S. pneumoniae. In preferred embodiments, the patient is human. The patient may receive preventive or therapeutic treatment. Prophylactic treatment provides sufficient protective immunity to reduce the likelihood or severity of pneumococcal infection or its consequences, such as pneumococcal pneumonia. Therapeutic treatment may be used to reduce the severity or prevent recurrence of S. pneumoniae infection or its clinical effects. Prophylactic treatment can be carried out using the multivalent immunogenic composition of the invention disclosed herein. The composition of the invention can be administered to the general population or to people at increased risk of pneumococcal infection, for example, elderly people or those who live with or care for elderly people.

The term in need of treatment includes those previously exposed to or infected with S. pneumoniae, previously vaccinated against S. pneumoniae, and susceptible to infection, or anyone for whom a reduction in the likelihood of infection is desired, for example, immunocompromised individuals, elderly individuals, children, adults or healthy individuals.

A stable multivalent immunogenic composition is a composition that does not exhibit significant changes observed at refrigeration temperature (eg, 2-8 or 4°C) for at least 1, 2, 3, 6, 12 and/or 24 months. In addition, a stable composition includes a composition that exhibits the desired properties at temperatures, including 25 and 37°C, for periods of time, including 1, 3, 6, 12 and/or 24 months. Typical acceptable criteria for stability are: variability of no more than about 5%, about 10%, about 15%, or about 20% in one or more of the following:

(a) number average molecular weight (Mn) of S. pneumoniae polysaccharide-protein conjugates in the composition;

(b) the weight average molecular weight (Mw) of the S. pneumoniae polysaccharide-protein conjugates in the composition;

(c) the total concentration of polysaccharide in the composition;

(d) the maximum emission of the composition, measured by fluorescence spectroscopy with the internal protein at a specific excitation wavelength, for example, 280 nanometers; and (e) the fluorescence intensity of the composition measured by internal protein fluorescence spectroscopy at a specific excitation wavelength.

The term stable may also be used to refer to a specific pneumococcal conjugate in a multivalent immunogenic composition. When used as such, the term refers to a conjugate that exhibits desired properties over time at a specified temperature, and such properties vary over time and at a specified temperature by no more than about 5%, about 10%, about 15%, or approximately 20%.

II. Polyvalent immunogenic compositions.

The invention relates to multivalent immunogenic compositions containing several S. pneumoniae polysaccharide-protein conjugates, where each of the conjugates contains a polysaccharide of the S. pneumoniae serotype conjugated to a carrier protein. Various aspects and variations of the multivalent immunogenic compositions of the invention are described below.

In one embodiment (Option E1), the invention relates to a multivalent immunogenic composition containing several S. pneumoniae polysaccharide-protein conjugates, each of which contains a capsular polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes consist of or essentially consist of

- 9 043489 (1) 15A, (2) 16F, Data</GipSegment>

(3) one or more of 23 serotypes selected from: (a) 23A and 23B, (b) 23A and 23F, (c) 23B and 23F, (d) 23A, (e) 23B and (f) 23F, Data</GipSegment>

(4) 24F, Data</GipSegment>

(5) 31, and (6) 35B.

In a sub-embodiment of embodiment E1, one or more serogroup 23 serotypes are 23A and 23B (ie, no other serogroup 23 serotypes are present). In a further sub-embodiment of embodiment E1, one or more serotypes of serogroup 23 are 23A and 23F. In another sub-embodiment of embodiment E1, one or more serogroup 23 serotypes are 23B and 23F. In yet another sub-embodiment of embodiment E1, 23A is the only serotype of serogroup 23. In a further sub-embodiment of embodiment, E1 23B is the only serotype of serogroup 23. In yet another further sub-embodiment of embodiment E1, 23F is the only serotype of serogroup 23.

In a second embodiment (Option E2), the invention relates to a multivalent immunogenic composition containing several S. pneumoniae polysaccharide-protein conjugates, comprising a capsular polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes comprise the set of serotypes presented in embodiment E1 or any sub-embodiment of embodiment E1, and further comprises serotype: (1) 6C, (2) 6A, or (3) 6A and 6B.

In a sub-embodiment of embodiment E2, the composition comprises serotype 6C. In a further sub-embodiment of embodiment E2, the composition contains serotypes 6A and 6B and does not contain serotype 6C. In another sub-embodiment of embodiment E2, the composition comprises serotype 6A.

In a third embodiment (Option E3), the invention relates to a multivalent immunogenic composition comprising multiple S. pneumoniae polysaccharide-protein conjugates comprising a capsular polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes comprise the set of serotypes given in embodiments E1, or any sub-embodiments of embodiment E1, or embodiment E2, or any sub-embodiment of embodiment E2, and further comprise serotypes (1)8, (2)9N, Data</GipSegment>

(3) PA (4) 12F, Data</GipSegment>

(5) 15V or 15C, (6) 17F and (7) 20A and/or 20V.

In a fourth embodiment (Option E4), the invention relates to a multivalent immunogenic composition containing multiple S. pneumoniae polysaccharide-protein conjugates comprising a capsular polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes comprise the set of serotypes given in any of embodiments E1-E3 or any sub-embodiments thereof, and further comprises serotypes 10A or 39.

In a fifth embodiment (Embodiment E5), the invention relates to a multivalent immunogenic composition comprising several S. pneumoniae polysaccharide-protein conjugates described above, wherein the S. pneumoniae serotypes contain, consist of, or consist essentially of 8, 9N, 10A, 11A, 12F , 15C, 17F, 20A, 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B.

In a sixth embodiment (Embodiment E6), the invention relates to a multivalent immunogenic composition comprising several S. pneumoniae polysaccharide-protein conjugates described above, wherein the S. pneumoniae serotypes contain, consist of, or consist essentially of 8, 9N, 39, 11A, 12F , 15C, 17F, 20A, 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B.

In a seventh embodiment (embodiment E7), the invention relates to a multivalent immunogenic composition comprising several S. pneumoniae polysaccharide-protein conjugates described above, wherein the S. pneumoniae serotypes contain, consist of, or consist essentially of 8, 9N, 10A, 11A, 12F, 15B, 17F, 20A, 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B.

In an eighth embodiment (embodiment E8), the invention relates to a multivalent immunogenic composition comprising several S. pneumoniae polysaccharide-protein conjugates described above, wherein the S. pneumoniae serotypes contain, consist of, or consist essentially of 8, 9N, 39, 11A, 12F, 15B, 17F, 20A, 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B.

In a ninth embodiment (embodiment E9), the invention relates to a multivalent immunogenic composition comprising several S. pneumoniae polysaccharide-protein conjugates described above, wherein the S. pneumoniae serotypes comprise the serotypes shown in any of embodiments E1 to E8 (or any subvariant thereof), and additionally contain serotypes 3, 7F and 19A.

In a tenth embodiment (embodiment E10), the invention relates to a multivalent immunogenic composition comprising several S. pneumoniae polysaccharide-protein conjugates described above, wherein the S. pneumoniae serotypes comprise the serotypes shown in any of embodiments E1-E9 (or any of them subvariant), and additionally contain serotype 22F.

In an eleventh embodiment (embodiment E11), the invention relates to a multivalent immunogenic composition comprising several S. pneumoniae polysaccharide-protein conjugates described above, wherein the S. pneumoniae serotypes comprise the serotypes shown in any of embodiments E1-E10 (or any of them subvariant), and additionally contain serotype 33F.

In a twelfth embodiment (embodiment E12), the invention relates to a multivalent immunogenic composition comprising several S. pneumoniae polysaccharide-protein conjugates described above, wherein the S. pneumoniae serotypes comprise, consist of, or are substantially composed of a set of S. pneumoniae serotypes selected from group consisting of (1) 15A, 16F, 23A, 23B, 24F, 31 and 35B, (2) 15A, 16F, 23F, 23B, 24F, 31 and 35B, Data</GipSegment>

(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, Data</ GipSegment>

(6) 15A, 16F, 23F, 24F, 31 and 35B, (7) 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</GipSegment>

(8) 6C, 15A, 16F, 23F, 23B, 24F, 31 and 35B, Data</GipSegment>

(9) 6C, 15A, 16F, 23A, 23F, 24F, 31 and 35B, Data</GipSegment>

(10) 6C, 15A, 16F, 23A, 24F, 31 and 35B, Data</GipSegment>

(11) 6С, 15А, 16F, 23В, 24F, 31 and 35В, Data</GipSegment>

(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, 23V, 24F, 31 and 35V, Data</GipSegment>

(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.

In a thirteenth embodiment (embodiment E13), the invention relates to a multivalent immunogenic composition comprising several S. pneumoniae polysaccharide-protein conjugates described above, wherein the S. pneumoniae serotypes comprise, consist of, or are substantially composed of a set of S. pneumoniae serotypes selected from group consisting of

- 11 043489 (1) 8, 9N, 10A, PA, 12F, 15C, 17F, 20A, 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</GipSegment>

(2) 8, 9N, 39, PA, 12F, 15С, 17F, 20А, 6С, 15А, 16F, 23А, 23В, 24F, 31 and 35В, Data</GipSegment>

(3) 8, 9N, 10A, PA, 12F, 15B, 17F, 20A, 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</GipSegment>

(4) 8, 9N, 39, PA, 12F, 15B, 17F, 20A, 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</GipSegment>

(5) 8, 9N, 10A, 11 A, 12F, 15C, 17F, 20A, 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</GipSegment>

(6) 8, 9N, 10A, PA, 12F, 15C, 17F, 20A, 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</GipSegment>

(7) 8, 9N, 10A, PA, 12F, 15C, 17F, 20A, 6A, 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</GipSegment>

(8) 8, 9N, 39, PA, 12F, 15С, 17F, 20А, 15А, 16F, 23А, 23В, 24F, 31 and 35В, Data</GipSegment>

(9) 8, 9N, 39, PA, 12F, 15С, 17F, 20А, 6А, 6В, 15А, 16F, 23А, 23В, 24F, 31 and 35В, Data</GipSegment>

(10) 8, 9N, 39, PA, 12F, 15C, 17F, 20A, 6A, 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</GipSegment>

(11) 8, 9N, 10A, PA, 12F, 15B, 17F, 20A, 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</GipSegment>

(12) 8, 9N, 10A, PA, 12F, 15B, 17F, 20A, 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</GipSegment>

(13) 8, 9N, 10A, PA, 12F, 15B, 17F, 20A, 6A, 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</GipSegment>

(14) 8, 9N, 39, PA, 12F, 15B, 17F, 20A, 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</GipSegment>

(15) 8, 9N, 39, PA, 12F, 15B, 17F, 20A, 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</GipSegment>

(16) 8, 9N, 39, PA, 12F, 15B, 17F, 20A, 6A, 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</GipSegment>

(17) 8, 9N, 10A, 11A, 12F, 15C, 17F, 20A, 6C, 15A, 16F, 23F, 24F, 31 and 35B, Data</GipSegment>

(18) 8, 9N, 39, 11A, 12F, 15C, 17F, 20A, 6C, 15A, 16F, 23F, 24F, 31 and 35B, Data</GipSegment>

(19) 8, 9N, 10A, 11A, 12F, 15B, 17F, 20A, 6C, 15A, 16F, 23F, 24F, 31 and 35B, Data</GipSegment>

(20) 8, 9N, 39, 11A, 12F, 15B, 17F, 20A, 6C, 15A, 16F, 23F, 24F, 31 and 35B,

- 12 043489

Data</GipSegment>

(21) 8, 9N, 1OA, 11 A, 12F, 15C, 17F, 20A, 15A, 16F, 23F, 24F, 31 and 35B,

Data</GipSegment>

(22) 8, 9N, 1OA, 11 A, 12F, 15C, 17F, 20A, 6A, 6B, 15A, 16F, 23F, 24F, 31 and 35B,

Data</GipSegment>

(23) 8, 9N, 10A, 11 A, 12F, 15C, 17F, 20A, 6A, 15A, 16F, 23F, 24F, 31 and 35B,

Data</GipSegment>

(24) 8, 9N, 39, 11 A, 12F, 15C, 17F, 20A, 15A, 16F, 23F, 24F, 31 and 35B,

Data</GipSegment>

(25) 8, 9N, 39, 11 A, 12F, 15C, 17F, 20A, 6A, 6B, 15A, 16F, 23F, 24F, 31 and 35B,

Data</GipSegment>

(26) 8, 9N, 39, 11 A, 12F, 15C, 17F, 20A, 6A, 15A, 16F, 23F, 24F, 31 and 35B,

Data</GipSegment>

(27) 8, 9N, 10A, 11 A, 12F, 15B, 17F, 20A, 15A, 16F, 23F, 24F, 31 and 35B,

Data</GipSegment>

(28) 8, 9N, 10A, 11 A, 12F, 15B, 17F, 20A, 6A, 6B, 15A, 16F, 23F, 24F, 31 and 35B,

Data</GipSegment>

(29) 8, 9N, 10A, 11 A, 12F, 15B, 17F, 20A, 6A, 15A, 16F, 23F, 24F, 31 and 35B,

Data</GipSegment>

(30) 8, 9N, 39, 11 A, 12F, 15B, 17F, 20A, 15A, 16F, 23F, 24F, 31 and 35B, Data</GipSegment>

(31) 8, 9N, 39, 11 A, 12F, 15B, 17F, 20A, 6A, 6B, 15A, 16F, 23F, 24F, 31 and 35B, and (32) 8, 9N, 39, PA, 12F, 15B, 17F, 20A, 6A, 15A, 16F, 23F, 24F, 31 and 35B.

The thirteenth embodiment (embodiment E13) further comprises a set of S. pneumoniae serotypes, lines (1)-(32), wherein serotype 20A in each set is replaced by either serotype 20 or serotype 20B.

In a fourteenth embodiment (embodiment E14), the invention relates to a multivalent immunogenic composition comprising several S. pneumoniae polysaccharide-protein conjugates described above, wherein the S. pneumoniae serotypes comprise, consist of, or are substantially composed of a set of S. pneumoniae serotypes selected from any set of serotypes shown in embodiment E13, lines (17)-(32), and additionally contain serotypes 23A and/or 23B.

In a fifteenth embodiment (embodiment E15), the invention relates to a multivalent immunogenic composition comprising several S. pneumoniae polysaccharide-protein conjugates described above, wherein the S. pneumoniae serotypes comprise, consist of, or are substantially composed of a set of S. pneumoniae serotypes selected from group consisting of (1) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15C, 17F, 20A, 6C, 15A, 16F, 23A,

23V, 24F, 31 and 35V, Data</GipSegment>

(2) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, PA, 12F, 15C, 17F, 20A, 6C, 15A, 16F, 23A, 23B,

- 13 043489

24F, 31 and 35V, Data</GipSegment>

(3) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, PA, 12F, 15B, 17F, 20A, 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</GipSegment>

(4) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, PA, 12F, 15B, 17F, 20A, 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</GipSegment>

(5) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15C, 17F, 20A 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</GipSegment>

(6) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, PA, 12F, 15C, 17F, 20A, 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</ GipSegment>

(7) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, PA, 12F, 15C, 17F, 20A, 6A, 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</GipSegment>

(8) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, PA, 12F, 15C, 17F, 20A 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</GipSegment>

(9) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, PA, 12F, 15C, 17F, 20A, 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</ GipSegment>

(10) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, PA, 12F, 15C, 17F, 20A, 6A, 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</GipSegment>

(11) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, PA, 12F, 15B, 17F, 20A 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</GipSegment>

(12) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, PA, 12F, 15B, 17F, 20A, 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</ GipSegment>

(13) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, PA, 12F, 15B, 17F, 20A, 6A, 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</GipSegment>

(14) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, PA, 12F, 15B, 17F, 20A 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</GipSegment>

(15) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, PA, 12F, 15B, 17F, 20A, 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</ GipSegment>

(16) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, PA, 12F, 15B, 17F, 20A, 6A, 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</GipSegment>

(17) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, PA, 12F, 15C, 17F, 20A, 6C, 15A, 16F, 23F, 24F, 31 and 35B, Data</GipSegment>

(18) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, PA, 12F, 15C, 17F, 20A, 6C, 15A, 16F, 23F, 24F, 31 and 35B, Data</GipSegment>

(19) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, PA, 12F, 15B, 17F, 20A, 6C, 15A, 16F, 23F, 24F, 31 and 35B, Data</GipSegment>

(20) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, PA, 12F, 15B, 17F, 20A, 6C, 15A, 16F, 23F, 24F, 31 and 35B, Data</GipSegment>

(21) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11 A, 12F, 15C, 17F, 20A, 15A, 16F, 23F, 24F, 31 and 35B, Data</GipSegment>

(22) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11A, 12F, 15C, 17F, 20A, 6A, 6B, 15A, 16F, 23F,

- 14 043489

24F, 31 and 35V, Data</GipSegment>

(23) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, PA, 12F, 15C, 17F, 20A, 6A, 15A, 16F, 23F,

24F, 31 and 35V, Data</GipSegment>

(24) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, PA, 12F, 15C, 17F, 20A, 15A, 16F, 23F, 24F, 31 and

35V, Data</GipSegment>

(25) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, PA, 12F, 15C, 17F, 20A, 6A, 6B, 15A, 16F, 23F,

24F, 31 and 35V, Data</GipSegment>

(26) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, PA, 12F, 15C, 17F, 20A, 6A, 15A, 16F, 23F, 24F, and 35B, Data</GipSegment>

(27) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, 11 A, 12F, 15B, 17F, 20A, 15A, 16F, 23F, 24F, 31 and 35B, Data</GipSegment>

(28) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, IA, 12F, 15B, 17F, 20A, 6A, 6B, 15A, 16F, 23F,

24F, 31 and 35V, Data</GipSegment>

(29) 3, 7F, 19A, 22F, 33F, 8, 9N, 10A, PA, 12F, 15B, 17F, 20A, 6A, 15A, 16F, 23F,

24F, 31 and 35V, Data</GipSegment>

(30) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, PA, 12F, 15B, 17F, 20A, 15A, 16F, 23F, 24F, 31 and

35V, Data</GipSegment>

(31) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, PA, 12F, 15B, 17F, 20A, 6A, 6B, 15A, 16F, 23F,

24F, 31 and 35V, Data</GipSegment>

(32) 3, 7F, 19A, 22F, 33F, 8, 9N, 39, PA, 12F, 15B, 17F, 20A, 6A, 15A, 16F, 23F, 24F, and 35B, Data</GipSegment>

(33) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, PA, 12F,

15С, 17F and 20В, Data</GipSegment>

(34) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24B, 31, 35B, 8, 9N, 10A, 11A, 12F,

15С, 17F and 20А, (35) 3, 7F, 19А, 22F, 33F, 6С, 15А, 16F, 23А, 23В, 24В, 31, 35В, 8, 9N, 10А, 11А, 12F,

15С, 17F and 20В, Data</GipSegment>

(36) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F,

15С, 17F and 20В, Data</GipSegment>

(37) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24B, 31, 35B, 8, 9N, 10A, PA,

12F, 15C, 17F and 20A, Data</GipSegment>

(38) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24B, 31, 35B, 8, 9N, 10A, PA,

12F, 15C, 17F and 20B, Data</GipSegment>

(39) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, PA, 12F,

15С, 17F and 20, (40) 3, 7F, 19А, 22F, 33F, 6С, 15А, 16F, 23А, 23В, 24В, 31, 35В, 8, 9N, 10А, 11А, 12F,

15С, 17F and 20, (41) 3, 7F, 19А, 22F, 33F, 6С, 15А, 16F, 23А, 23В, 24В, 31, 35В, 8, 9N, 10А, 11А, 12F,

15C, 17F, and 20, (42) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 1 ( 43) 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24B, 31, 35B, 8, 9N, 10A, PA, 12F,

15C, 17F, and 20, and (44) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24B, 31, 35B, 8, 9N, 10A, PA, 12F, 15C, 17F and 20.

In a sixteenth embodiment (embodiment E16), the invention relates to a multivalent immunogenic composition comprising several S. pneumoniae polysaccharide-protein conjugates described above, wherein the S. pneumoniae serotypes comprise, consist of, or are substantially composed of a set of S. pneumoniae serotypes selected from any set of serotypes shown in embodiment E15, lines (17)-(32), and additionally contain serotypes 23A and/or 23B.

In a seventeenth embodiment (embodiment E17), the invention relates to a multivalent immunogenic composition comprising multiple S. pneumoniae polysaccharide-protein conjugates comprising a capsular polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes comprise a set of serotypes selected from group consisting of

- 15 043489

i. 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B, Data</GipSegment>ii. 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 12F5 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 20A;

In sub-embodiments of embodiment E17, the immunogenic composition does not contain any additional S. pneumoniae polysaccharide-protein conjugates.

In an eighteenth embodiment (embodiment E18), the invention relates to a multivalent immunogenic composition comprising several S. pneumoniae polysaccharide-protein conjugates containing the capsular polysaccharide of S. pneumoniae serotypes 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B. In sub-embodiments of embodiment E18, the immunogenic composition does not contain any additional S. pneumoniae polysaccharide-protein conjugates.

In a nineteenth embodiment (embodiment E19), the invention relates to a multivalent immunogenic composition containing several S. pneumoniae polysaccharide-protein conjugates containing the capsular polysaccharide of S. pneumoniae serotypes 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8 , 9N, 10A, 11A, 12F, 15C, 17F and 20A. In sub-embodiments of embodiment E19, the immunogenic composition does not contain any additional S. pneumoniae polysaccharide-protein conjugates.

In the twentieth embodiment (embodiment E20), the invention relates to a multivalent immunogenic composition containing several S. pneumoniae polysaccharide-protein conjugates containing the capsular polysaccharide of S. pneumoniae serotypes 3, 7F, 19A, 22F, 33F, 6C, 25A, 15C, 15C . In sub-embodiments of embodiment E20, the immunogenic composition does not contain any additional S. pneumoniae polysaccharide-protein conjugates.

In another embodiment, the invention relates to a multivalent immunogenic composition containing several S. pneumoniae polysaccharide-protein conjugates containing the capsular polysaccharide of S. pneumoniae serotypes 3, 7F, 19A, 22F, 33F, 6A and 15F, 15C, 15C. In sub-embodiments of this embodiment (i.e., 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F, and 20A), the immunogenic composition does not contain any additional S. pneumoniae polysaccharide-protein conjugates.

In another embodiment, the invention relates to a multivalent immunogenic composition containing several S. pneumoniae polysaccharide-protein conjugates containing a capsular polysaccharide from S. pneumoniae serotypes 3, 7F, 19A, 22F, 33F, 6A, 15F, 15C, 1. In sub-embodiments of this embodiment (i.e., 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F, and 20), the immunogenic composition does not contain any additional S. pneumoniae polysaccharide-protein conjugates.

In another embodiment, the invention relates to a multivalent immunogenic composition containing several S. pneumoniae polysaccharide-protein conjugates containing capsular polysaccharide serotypes 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B , 8, 9H, 10A, 11A, 12F, 15C, 17F and 20B S. pneumoniae. In sub-embodiments of this embodiment (i.e., 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F, and 20B), the immunogenic composition does not contain any additional S. pneumoniae polysaccharide-protein conjugates.

As shown herein (see Example 3 below), de-O-acetylated pneumococcal polysaccharide serotype 15B is equivalent to pneumococcal polysaccharide serotype 15C and has identical NMR spectra. The serotype 15B de-O-acetylated pneumococcal polysaccharide described and disclosed herein is equivalent to the serotype 15C pneumococcal polysaccharide, and in both of these serotypes, the O-acetyl content of the repeat unit may be in the range of 0-5% or in the range of 0-4% , or in the range of 0-3%, or in the range of 0-2%, or in the range of 0-1%, or in the range of 0-0.5%, or in the range of 0-0.1%, or O-acetyl can absent. Spencer B.L. et al. report that the level of 15C O-acetylation may be low (Spencer, B.L. et al., Clin. Vac. Immuno. (2017), 24(8):1-13). Thus, in any of the embodiments of the multivalent immunogenic compositions provided herein, de-Oacetylated serotype 15B can be used instead of serotype 15C. Methods for de-O-acetylation are known in the art, for example, as described by Rajam et al., Clinical. and Vaccine Immunology, 2007, 14(9):1223-1227.

In some embodiments, any of the multivalent immunogenic compositions of the invention, including embodiments E1-E20 and any sub-embodiment thereof, further comprises a pharmaceutically acceptable carrier.

Cross reactivity.

In one embodiment, the invention relates to multivalent immunogenic compositions containing S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates contains a polysaccharide of a S. pneumoniae serotype, including serotype 6C, conjugated to a carrier protein, wherein S. pneumoniae serotype 6C provides cross-protection against S. pneumoniae serotypes 6A and 6B.

- 16 043489

In another embodiment, the invention relates to multivalent immunogenic compositions containing S. pneumoniae polysaccharide-protein conjugates selected from embodiments E1-E20, wherein each of the conjugates contains a polysaccharide of a S. pneumoniae serotype, including serotype 6C, conjugated to a carrier protein, wherein S. pneumoniae serotype 6C provides cross-protection against S. pneumoniae serotypes 6A and 6B.

In one embodiment, the invention relates to multivalent immunogenic compositions containing S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates contains a polysaccharide of a S. pneumoniae serotype, including serotype 19A, conjugated to a carrier protein, wherein S. pneumoniae serotype 19A provides cross-protection against serotype 19F S. pneumoniae.

In another embodiment, the invention relates to multivalent immunogenic compositions containing S. pneumoniae polysaccharide-protein conjugates selected from embodiments E1-E20, wherein each of the conjugates contains a polysaccharide of a S. pneumoniae serotype, including serotype 19A, conjugated to a carrier protein, wherein S. pneumoniae serotype 19A provides cross-protection against S. pneumoniae serotype 19F.

In one embodiment, the invention relates to multivalent immunogenic compositions containing S. pneumoniae polysaccharide-protein conjugates, where each of the conjugates contains a polysaccharide of a S. pneumoniae serotype, including serotypes 23A and/or 23B, conjugated to a carrier protein, where serotypes 23A and /or S. pneumoniae 23B provides cross-protection against S. pneumoniae serotype 23F.

In another embodiment, the invention relates to multivalent immunogenic compositions containing S. pneumoniae polysaccharide-protein conjugates selected from embodiments E1-E20, wherein each of the conjugates contains a polysaccharide of a S. pneumoniae serotype, including serotypes 23A and/or 23B, conjugated to a protein -carrier, where S. pneumoniae serotypes 23A and/or 23B provide cross-protection against S. pneumoniae serotype 23F.

In an embodiment, the invention relates to multivalent immunogenic compositions containing S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates contains a polysaccharide of a S. pneumoniae serotype, including serotype 6A, conjugated to a carrier protein, wherein S. pneumoniae serotype 6A provides cross-protection against serotypes 6B and/or 6C of S. pneumoniae.

In another embodiment, the invention relates to multivalent immunogenic compositions containing S. pneumoniae polysaccharide-protein conjugates selected from embodiments E1-E20, wherein each of the conjugates contains a polysaccharide of a S. pneumoniae serotype, including serotype 6A, conjugated to a carrier protein, wherein S. pneumoniae serotype 6A provides cross-protection against S. pneumoniae serotypes 6B and/or 6C.

In one embodiment, the invention relates to multivalent immunogenic compositions containing S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates contains a polysaccharide of a S. pneumoniae serotype, including serotype 20A, conjugated to a carrier protein, wherein S. pneumoniae serotype 20A provides cross-protection against serotype 20B S. pneumoniae.

In another embodiment, the invention relates to multivalent immunogenic compositions containing S. pneumoniae polysaccharide-protein conjugates selected from embodiments E1-E20, wherein each of the conjugates contains a polysaccharide of a S. pneumoniae serotype, including serotype 20A, conjugated to a carrier protein, wherein S. pneumoniae serotype 20A provides cross-protection against S. pneumoniae serotype 20B.

In one embodiment, the invention relates to multivalent immunogenic compositions containing S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates contains a polysaccharide of a S. pneumoniae serotype, including serotype 20B, conjugated to a carrier protein, wherein S. pneumoniae serotype 20B provides cross-protection against serotype 20A S. pneumoniae.

In another embodiment, the invention relates to multivalent immunogenic compositions containing S. pneumoniae polysaccharide-protein conjugates selected from embodiments E1-E20, wherein each of the conjugates contains a polysaccharide of a S. pneumoniae serotype, including serotype 20B, conjugated to a carrier protein, wherein S. pneumoniae serotype 20B provides cross-protection against S. pneumoniae serotype 20A.

In one embodiment, the invention relates to multivalent immunogenic compositions containing S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates contains a polysaccharide of a S. pneumoniae serotype, including serotype 15C, conjugated to a carrier protein, wherein S. pneumoniae serotype 15C provides cross-protection against serotype 15B S. pneumoniae.

In another embodiment, the invention relates to multivalent immunogenic compositions containing S. pneumoniae polysaccharide-protein conjugates selected from embodiments E1-E20, wherein each of the conjugates contains a polysaccharide of a S. pneumoniae serotype, including

- 17 043489 serotype 15C conjugated to a carrier protein, wherein S. pneumoniae serotype 15C provides cross-protection against S. pneumoniae serotype 15B.

Carrier protein.

In specific embodiments of the present invention, CRM197 is used as a carrier protein. CRM197 is a non-toxic variant (i.e. toxoid) of diphtheria toxin having the following amino acid sequence:

GADDVVDSSK SFVMENFSSY HGTKPGYVDS IQKGIQKPKS GTQGNYDDDW

KEFYSTDNKY DAAGYSVDNE NPLSGKAGGV VKVTYPGLTK VLALKVDNAE

TIKKELGLSL TEPLMEQVGT EEFIKRFGDG ASRVVLSLPF AEGSSSVEYI

NNWEQAKALS VELEINFETR GKRGQDAMYE YMAQACAGNR VRRSVGSSLS

CINLDWDVIR DKTKTKIESL KEHGPIKNKM SESPNKTVSE EKAKQYLEEF

HQTALEHPEL SELKTVTGTN PVFAGANYAA WAVNVAQVID SETADNLEKT

TAALSILPGI GSVMGIADGA VHHNTEEIVA QSIALSSLMV AQAIPLVGEL

VDIGFAAYNF VESIINLFQV VHNSYNRPAY SPGHKTQPFL HDGYAVSWNT

VEDSIIRTGF QGESGHDIKI TAENTPLPIA GVLLPTIPGK LDVNKSKTHI

SVNGRKIRMR CRAIDGDVTF CRPKSPVYVG NGVHANLHVA FHRSSSEKIH

SNEISSDSIG VLGYQKTVDH TKVNSKLSLF FEIKS (SEQIDNO:1)

In one embodiment, CRM197 is isolated from cultures of Corynebacterium diphtheria strain C7 (β 197) grown in the presence of casamino acids in yeast extract medium. In another embodiment, CRM197 is produced recombinantly in accordance with the methods described in US 5614382. Typically, CRM197 is purified using a combination of ultrafiltration, ammonium sulfate precipitation and ion exchange chromatography methods. In some embodiments, CRM197 is produced in Pseudomonas fluorescens using Pfenex Expression Technology™ (Pfenex Inc., San Diego, CA).

Other suitable carrier proteins include additional inactivated bacterial toxins such as DT (diphtheria toxoid) or DT fragment B (DTFB), TT (tetanus toxoid) or TT fragment C, pertussis toxoid, cholera toxoid (for example, as described in WO 2004/ 083251), E.coli LT, E.coli ST and Pseudomonas aeruginosa exotoxin A. Bacterial outer membrane proteins such as outer membrane protein complex (OMPC), porins, transferrin-binding proteins, pneumococcal surface protein A (PspA; see WO 02/091998), pneumococcal adhesin (PsaA), peptidase can also be used as protein carriers. C5a from group A or group B streptococci or protein D of Haemophilias influenzae, pneumococcal pneumolysin (Kuo et al., 1995, Infect. Immun., 63, 2706-13), including detoxified ply, for example dPLYGMBS fusion protein (see WO 04/ 081515) or dPLY-formol, PhtX, including PhtA, PhtB, PhtD, PhtE, and Pht fusion proteins, for example PhtDE fusion proteins, PhtBE fusion proteins can also be used (see WO 01/98334 and WO 03/54007). Other proteins may also be used, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tuberculin purified protein derivative (PPD), PorB (N. meningitidis), PD (Protein D of Haemophilus influenzae; see e.g. , EP 0594610 B) or their immunologically functional equivalents, synthetic peptides (see EP 0378881 and EP 0427347), 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 (see WO 91/01146), artificial proteins containing several human CD4+ T cell epitopes from various pathogen-derived antigens (see Falugi et al., 2001, Eur J. Immunol., 31:3816-3824), such as N19 protein (see Baraldoi et al., 2004, Infect. Immun., 72:4884-7), iron uptake proteins (see WO 01/72337) , C. difficile toxin A or B (see WO 00/61761) and flagellin (see Ben-Yedidia et al., 1998, Immunol. Lett., 64: 9).

Other DT mutants such as CRM ₁₇₆ , CRM ₂₂₈ , CRM45 can be used as carrier protein (Uchida et al., 1973, J. Biol. Chem., 218:3838-3844); CRM9, CRM45, CRM102, CRM103 and CRM107 and with other mutations described by Nicholls and Youle in Genetically Engineered Toxins, ed: Frankel, Maecel Dekker Inc, 1992; deletions or mutations of Glu-148 to Asp, Gln or Ser and/or Ala 158 to Gly and other mutations disclosed in US Pat. No. 4,709,017 or 4,950,740; mutations of at least one or more residues of Lys 516, Lys 526, Phe 530 and/or Lys 534 and other mutations disclosed in US Pat. No. 5,917,017 or 6,455,673; or a fragment disclosed in US Pat. No. 5,843,711. Such DT mutants can also be used to create DTFB variants, where the variants contain fragment B containing epitope regions.

In certain embodiments, the carrier protein is selected from the group consisting of outer membrane protein complex (OMPC), tetanus toxoid, diphtheria toxoid, protein D, and CRM197.

In some embodiments, a second carrier may be used in a multivalent immunogenic composition for one or more polysaccharide protein conjugates. The second carrier protein is preferably a protein that is non-toxic and non-reactogenic and can be obtained in sufficient quantity and purity. The second carrier protein is also conjugated or linked to the S. pneumoniae polysaccharide to enhance the immunogenicity of the antigen. Carrier proteins must be amenable to standard conjugation procedures. In one embodiment, each capsular polysaccharide not conjugated to a first carrier protein is conjugated to the same second carrier protein (eg, each capsular polysaccharide molecule is conjugated to one carrier protein). In another embodiment, capsular polysaccharides not conjugated to a first carrier protein are conjugated to two or more carrier proteins (each capsular polysaccharide molecule is conjugated to one carrier protein). In such embodiments, each capsular polysaccharide of the same serotype is typically conjugated to the same carrier protein.

In embodiments of the invention, including any of embodiments E1-E20 and any sub-embodiments thereof, one or more polysaccharide serotypes (including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 where applicable) are conjugated to CRM197. In additional embodiments of the invention, including any of embodiments E1-E20 and any sub-embodiments thereof, the polysaccharide of each of the serotypes is conjugated to CRM197.

The composition of the polysaccharide-protein conjugates of the present invention can be prepared using methods known in the art. For example, individual pneumococcal conjugates can be prepared using a physiologically acceptable carrier to obtain the composition. Examples of such carriers include, but are not limited to, water, buffered saline, polyols (eg, glycerin, propylene glycol, liquid polyethylene glycol), and dextrose solutions.

In a preferred embodiment, the vaccine composition is prepared in L-histidine buffer with sodium chloride.

In some embodiments, the multivalent immunogenic composition comprises multiple S. pneumoniae polysaccharide-protein conjugates comprising a capsular polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, and an adjuvant, wherein the S. pneumoniae serotypes are as disclosed herein. Suitable adjuvants for enhancing the effectiveness of the composition include, but are not limited to (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.;

(2) formulations based on oil-in-water emulsions (with or without other specific immunostimulating agents such as muramyl peptides (defined below) or bacterial cell wall components), such as, for example, (a) MF59 (publication of international patent application No. WO 90/14837) containing 5% squalene, 0.5% Tween 80 and 0.5% Span 85 (optionally containing varying amounts of MTP-PE), prepared into submicron particles using a microfluidizer such as the HOY model microfluidizer ( Microfluidics, Newton, MA);

(b) SAF containing 10% squalene, 0.4% Tween 80, 5% L121 pluronic-capped polymer, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to form a large particle size emulsion;

(c) Ribi™ Adjuvant System (RAS), (Corixa, Hamilton, MT) containing 2% squalene, 0.2% Tween 80 and one or more bacterial cell wall components from the group consisting of 3-O-deacylated monophosphorylipid A (MPL™), described in US patent 4,912,094, trehalose dimycolate (TDM) and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); and (d) Montanide ISA;

(3) saponin adjuvants, such as Quil A or STIMULON™ QS-21 (Antigenics, Framingham, MA) (see, for example, US Pat. No. 5,057,540) or particles derived from them, such as ISCOM (immune stimulatory complexes formed by a combination of cholesterol , saponin, phospholipid and amphipathic proteins) and Iskomatrix® (having essentially the same structure as ISCOM, but without the protein);

(4) bacterial lipopolysaccharides, synthetic analogs of lipid A, such as aminoalkylglucosamine phosphate compounds (AGP) or derivatives or analogs thereof, which are available from Corixa and which are described in US patent 6113918; one such AGP is 2-[(R)-3tetradecanoyloxytetradecanoylamino]ethyl 2-deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino] -b-O-glucopyranoside, which is also known as 529 (formerly known as RC529), prepared in the form of an aqueous solution or as a stable emulsion;

(5) synthetic polynucleotides, such as oligonucleotides containing CpG motif(s) (US 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.) etc.), interferons (eg, interferon gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), co-stimulatory molecules B7-1 and B7-2, and etc.; And

- 19 043489 (7) complement, such as trimer of complement component C3d.

In another embodiment, the adjuvant is a mixture of 2, 3 or more of the above adjuvants, for example, SBAS2 (an oil-in-water emulsion also containing 3-deacylated monophosphoryl lipid A and QS21).

Muramyl peptides include, but are not limited to, K-acetyl-muramyl-E-threonyl-O-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanine-2-(1'-2'dipalmitoyl-sn)-glycero-3hydroxyphosphoryloxy )ethylamine (MTP-PE), etc.

In certain embodiments, the adjuvant is an aluminum salt. The aluminum salt adjuvant may be an alum-precipitated vaccine or an alum-adsorbed vaccine. Aluminum salt adjuvants are well known in the art and are described, for example, in Harlow, E., D. Lane (1988, Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory) and Nicklas, W. (1992, Aluminum salts, Research in Immunology , 143:489-493). Aluminum salt includes, but is not limited to, hydrated alumina, alumina hydrate, alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate, alhydrogel, Superfos, amphogel, aluminum(III) hydroxide, hydroxyphosphate aluminum sulfate, aluminum phosphate adjuvant (APA). , amorphous alumina, trihydrated alumina or trihydroxyaluminum.

ARA is an aqueous suspension of aluminum hydroxyphosphate. APA is prepared by mixing aluminum chloride and sodium phosphate in a 1:1 volume ratio to precipitate aluminum hydroxyphosphate. After the mixing process, the material is ground using a high shear mixer to achieve a monodisperse particle size distribution. The product is then diafiltered against saline and steam sterilized.

In some embodiments, commercially available Al(OH) ₃ (eg, Alhydrogel or Superfos of Denmark/Accurate Chemical and Scientific Co., Westbury, NY) is used for protein adsorption at a ratio of 50-200 μg protein/mg aluminum hydroxide. Protein adsorption, in another embodiment, depends on the pI (isoelectric pH) value of the protein and the pH of the medium. A protein with a lower pI value is adsorbed onto the positively charged aluminum ion more strongly than a protein with a higher pI value. Aluminum salts may form an antigen depot that is slowly released over 2-3 weeks, participate in nonspecific macrophage activation and complement activation, and/or stimulate the innate immune mechanism (possibly through uric acid stimulation). See, for example, Lambrecht et al., 2009, Curr. Opin. Immunol., 21:23.

Stock aqueous solutions of monovalent conjugates are typically mixed together and diluted to the target value of 4 μg/ml for all serotypes except 6B, which is diluted to the target value of 8 μg/ml. After dilution, the batch is filter sterilized and an equal volume of aluminum phosphate adjuvant is added aseptically to achieve a final aluminum concentration of 250 µg/ml. The prepared batch with the adjuvant is poured into disposable vials of 0.5 ml per dose.

In some embodiments, the adjuvant is a CpG-containing nucleotide sequence, for example, a CpG-containing oligonucleotide, in particular a CpG-containing oligodeoxynucleotide (CpG ODN). In another embodiment, the adjuvant is ODN 1826, which is available from Coley Pharmaceutical Group.

Methods for using CpG oligonucleotides are well known in the art and are described, for example, in Sur et al., 1999, J. Immunol., 162:6284-93; Verthelyi, 2006, Methods Mol. Med., 127:139-58; and Yasuda et al., 2006, Crit. Rev. Ther. Drug. Carrier. Syst., 23:89-110.

In alternative embodiments, the immunogenic composition contains multiple S. pneumoniae polysaccharide-protein conjugates disclosed herein, for example, in any of embodiments E1-E20 or any sub-embodiment thereof, and does not contain an adjuvant.

Compositions.

The composition of the invention may be provided in single dose vials, multi-dose vials or pre-filled glass or plastic syringes.

In another embodiment, the compositions of the present invention are administered orally and are thus provided in a form suitable for oral administration, i.e. in the form of a solid or liquid preparation. Solid oral formulations include tablets, capsules, pills, granules, pellets, and the like. Liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils, and the like.

Pharmaceutically acceptable carriers for liquid formulations are aqueous or non-aqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol and injectable organic esters such as ethyl oleate. Aqueous vehicles include water, alcohol/aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils are oils of animal, vegetable or synthetic origin, such as peanut oil, soybean oil, olive oil, sunflower oil, fish oil, other marine oil, or lipid from milk or eggs.

The pharmaceutical composition may be isotonic, hypotonic or hypertonic. However, it is often preferable that upon administration the pharmaceutical composition for infusion or

- 20 043489 injection was essentially isotonic. Therefore, for storage, the pharmaceutical composition may preferably be isotonic or hypertonic. If a hypertonic pharmaceutical composition is used for storage, it may be diluted to an isotonic solution before administration.

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, CaCl2, KCl and MgCl2. Examples of nonionic isotonic agents include, but are not limited to, mannitol, sorbitol, and glycerin.

It is also preferred that the at least one pharmaceutically acceptable additive is a buffer. For some purposes, for example, when the pharmaceutical composition is intended for infusion or injection, it is often desirable that the composition contains a buffer that is capable of buffering the solution to a pH in the range of 4 to 10, such as 5 to 9, such as 6 to 8.

The buffer, for example, can be selected from the group consisting of TRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate and triethanolamine buffer.

The buffer may be selected from USP-compatible buffers for parenteral use, particularly when the pharmaceutical composition is intended for parenteral use. For example, the buffer may be selected from the group consisting of monobasic acids such as acetic, benzoic, gluconic, glyceric and lactic; dibasic acids such as aconitic, adipic, ascorbic, carbonic, glutamic, malic, succinic and tartaric acids, polybasic acids such as citric and phosphoric; and bases such as ammonia, diethanolamine, glycine, triethanolamine and TRIS.

Parenteral vehicles (for subcutaneous, intravenous, intra-arterial, or intramuscular injection) include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, and fatty oils. Carriers for intravenous administration include fluids and nutritional supplements, electrolyte supplements such as those based on Ringer's dextrose, and the like. Examples are sterile liquids such as water and oils, with or without the addition of surfactant and other pharmaceutically acceptable adjuvants. In general, preferred liquid carriers are water, saline, aqueous solutions of dextrose and related sugars, glycols such as propylene glycols or polyethylene glycol, polysorbate 80 (PS-80), polysorbate 20 (PS-20) and poloxamer 188 (P188), in particular for solutions for injections. Examples of oils are oils of animal, vegetable or synthetic origin, such as peanut oil, soybean oil, olive oil, sunflower oil, fish oil, other marine oil, or lipid from milk or eggs.

The compositions of the invention may also contain a surfactant. Preferred surfactants include, but are not limited to, polyoxyethylene sorbitan ester surfactants (commonly referred to as Tweens), especially PS-20 and PS-80; ethylene oxide (EO), propylene oxide (PO) and/or butylene oxide (BO) copolymers sold under the trade name DOWFAX™, such as linear EO/PO block copolymers; octoxynols, which may differ in the number of repeating ethoxy(oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100 or tert-octylphenoxypolyethoxyethanol) being of practical interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids such as phosphatidylcholine (lecithin); nonylphenol ethoxylates such as Tergitol™ NP series; polyoxyethylene fatty alcohol ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as SPAN), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. The preferred surfactant for inclusion in the emulsion is PS-80.

Mixtures of surfactants can be used, for example PS-80/Span 85 mixtures. A combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (PS-80) and an octoxynol such as tert-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable. . Another useful combination includes laureth 9 plus polyoxyethylene sorbitan ester and/or octoxynol.

Preferred surfactant amounts (% by weight) are: 0.01 to 1% for polyoxyethylene sorbitan esters (such as PS-80), particularly about 0.1%; 0.001 to 0.1% for octyl or nonylphenoxypolyoxyethanols (such as Triton X-100 or other Triton detergents), in particular 0.005 to 0.02%; from 0.1 to 20% for polyoxyethylene ethers (such as laureth 9), preferably from 0.1 to 10% and in particular from 0.1 to 1% or about 0.5%.

In some embodiments, the composition consists essentially of histidine (20 mM), saline (150 mM), and 0.2% PS-20 or 0.04% PS-80 pH 5.8 with 250 μg/mL ARA (adjuvant based on aluminum phosphate). PS-20 content can vary from 0.005 to 0.3% (w/v), where the presence of PS-20 or PS-80 in the composition allows control of aggregation during

- 21 043489 imitation of preparation and transportation using primary packaging. In another embodiment, PS-20 may be in the range of 0.025 to 0.8% (w/v). In another embodiment, PS-20 may be from 0.05 to 0.8% (w/v). In another embodiment, PS-20 may be from 0.05 to 0.2% (w/v). The process consists of combining up to 24 serotypes in histidine, saline and PS-20 or PS-80, and then combining this mixed material with APA and saline with or without antimicrobial preservatives.

In specific embodiments, the multivalent immunogenic composition comprises S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates contains a polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, wherein the serotypes in the S. pneumoniae polysaccharide-protein conjugates comprise any of the sets of serotypes given in the present description, and additionally contain 20-80 mm histidine, pH 5.8 and 150 mm NaCl. In some embodiments, the multivalent immunogenic composition further contains from 0.2 to 0.8% w/v. polysorbate 20.

Surfactant selection may be required to optimize different drugs and drug substances. For multivalent vaccines having 15 or more serotypes, PS-20 and P188 are preferred. The choice of chemical composition used to prepare the conjugate can also play an important role in stabilizing the composition. In particular, when the conjugation reactions used to produce the various polysaccharide-protein conjugates in a multivalent composition involve both an aqueous solvent and a DMSO solvent, the stability is highly dependent on the particular surfactant system. Increased stability of polysaccharide-protein conjugates was observed with polysorbate 20 alone or with poloxamer 188 in combination with a polyol.

The exact mechanism of how a particular detergent protects a biotherapeutic agent is poorly understood and cannot be predicted a priori. Possible stabilization mechanisms include preferential hydration, preferential non-incorporation, competition at the air/liquid interface between the biotherapeutic and the surface, surface tension, and/or direct association of the detergent with the biotherapeutic to mask hydrophobic patches that serve as aggregation seeds.

Poloxamer may also be used in the compositions of the invention. The poloxamer is a nonionic triblock copolymer consisting of a central hydrophobic polyoxypropylene (poly(propylene oxide)) chain flanked by two hydrophilic polyoxyethylene (poly(ethylene oxide)) chains. Poloxamers are also known under the trade name Pluronic®. Because the lengths of the polymer blocks can be adjusted, there are many different poloxamers that vary slightly in their properties. Copolymers under the general term poloxamer are usually designated by the letter P (for poloxamer) followed by three digits, the first two digits x100 giving the approximate molecular weight of the polyoxypropylene core and the last digit x10 giving the percentage of polyoxyethylene (e.g. P407=poloxamer with molecular weight polyoxypropylene 4000 g/mol and 70% polyoxyethylene content). For the Pluronic® brand, the coding for these copolymers begins with a letter identifying the physical form of the copolymer at room temperature (L=liquid, P=paste, F=flakes (solid)), followed by two or three digits. The first digit (two digits in a three-digit number) in the numerical designation, multiplied by 300, indicates the approximate molecular weight of the hydrophobe; and the last digit x10 indicates the percentage of polyoxyethylene (for example, L61=Pluronic® with a polyoxypropylene molecular weight of 1800 g/mol and 10% polyoxyethylene content). See US Patent 3740421.

Examples of poloxamers have the general formula

HO(C2H4O)a(C3H6O)b(C2H4O)aH, where blocks a and b have the following meanings.__________________________________________

Pluronic® Poloxamer A IN: Molecular mass L31 2 16 1100 (average) L35 1900 (average) L44NF 124 12 20 2090-2360 L64 2900 (average) L81 2800 (average) L121 4400 (average) P123 20 70 5750 (average) F68NF 188 80 27 7680-9510 F87NF 237 64 37 6840-8830 F108NF 338 141 44 12700-17400 F127NF 407 101 56 9840-14600

The units of molecular weight used herein are given in Daltons (Da)

- 22 043489 or g/mol.

Preferably, the poloxamer typically has a molecular weight in the range of 1100 to 17,400 Da, 7,500 to 15,000 Da, or 7,500 to 10,000 Da. The poloxamer may be selected from poloxamer 188 or poloxamer 407. The final concentration of poloxamer in the formulations ranges from 0.001 to 5% w/v. or from 0.025 to 1% w/v. In certain aspects, the polyol is propylene glycol and is present in a final concentration of 1 to 20% w/v. In certain aspects, the polyol is polyethylene glycol 400 and is in a final concentration of 1 to 20% w/v.

Suitable polyols for the compositions of the invention are polymeric polyols, in particular polyether diols, including, without limitation, propylene glycol and polyethylene glycol, polyethylene glycol monomethyl ethers.

Propylene glycol is available in a range of monomer molecular weights from ~425 to ~2700. Polyethylene glycol and polyethylene glycol monomethyl ether are also available in a molecular weight range of ~200 to ~35,000, including but not limited to PEG200, PEG300, PEG400, PEG1000, PEG MME 550, PEG MME 600, PEG MME 2000, PEG MME 3350 and PEG MME 4000. Preferred the polyethylene glycol is polyethylene glycol 400. The final concentration of polyol in the compositions of the invention can range from 1 to 20% w/v. or from 6 to 20% w/v.

The formulation also contains a pH-buffered saline solution. The buffer may be selected, for example, from the group consisting of tris, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate, HEPES (4-(2-hydroxyethyl)-1piperazinethanesulfonic acid ), MOPS (3-(N-morpholino)propanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid) and triethanolamine buffers. The buffer is capable of buffering a solution to a pH ranging from 4 to 10, 5.2 to 7.5, or 5.8 to 7.0. In a certain aspect of the invention, the buffer is selected from the group consisting of phosphate, succinate, histidine, MES, MOPS, HEPES, acetate or citrate. In addition, the buffer may be selected, for example, from USP-compatible buffers for parenteral use, in particular when the pharmaceutical composition is intended for parenteral use. Buffer concentrations can vary from 1 to 100 mM. Buffer concentrations can vary from 10 to 80 mM. Buffer concentrations can vary from 1 to 50 mM or from 5 to 50 mM. In certain aspects, the buffer is histidine at a final concentration of 5 to 50 mM or succinate at a final concentration of 1 to 10 mM. In certain aspects, histidine has a final concentration of 20±2 mM.

Although saline (ie, a solution containing NaCl) is preferred, other salts suitable for formulation include, but are not limited to, CaCl ₂ , KCl and MgCl ₂ and combinations thereof. Nonionic isotonic agents may be used in place of salt, including, but not limited to, sucrose, trehalose, mannitol, sorbitol, and glycerol. Suitable salt content ranges include, but are not limited to, 25 to 500 mM or 40 to 170 mM. In one aspect, the saline solution is NaCl, optionally present at a concentration of from 20 to 170 mM.

In a preferred embodiment, the compositions contain L-histidine buffer with sodium chloride.

In another embodiment, the pharmaceutical composition is delivered in a controlled release system. For example, the agent can be administered by intravenous infusion, transdermal patch, liposomes, or other routes of administration. Another embodiment uses polymeric materials; for example, in microspheres or implants.

The amount of conjugate in each vaccine dose is selected to produce an immunoprotective response without significant side effects. This amount may vary depending on the pneumococcal serotype. Typically for polysaccharide conjugates, each dose of each polysaccharide will range from 0.08 to 100 μg. In some embodiments, the dose of each polysaccharide conjugate is from 0.08 to 10 μg. In other embodiments, the dose is 1 to 5 μg, 0.4 to 4 μg, 0.4 to 3 μg, 0.4 to 2 μg, or 0.4 to 1 μg. In some embodiments, the dose of one or more polysaccharide conjugates is 100, 150, 200, 250, 300, 400, 500, or 750 ng, 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, 90 or 100 mcg.

In some embodiments of the compositions of the invention, all polysaccharide conjugates are present in the composition in equal amounts. In other embodiments, the polysaccharide conjugates are present in the composition in different amounts (ie, at least one polysaccharide conjugate is present in an amount that is different from the amount of one or more other polysaccharide conjugates in the composition).

The optimal amount of components for a particular vaccine can be determined through standard studies involving observation of appropriate immune responses in subjects. For example, in another embodiment, the dose for human vaccination is determined by extrapolating data obtained from animal studies to humans. In another embodiment, the dose is determined empirically.

In one embodiment, the dose of aluminum salt is 10, 15, 20, 25, 30, 50, 70, 100,

- 23 043489

125, 150, 200, 300, 500 or 700 mcg or 1, 1.2, 1.5, 2, 3.5 mg or more. In yet another embodiment, the dose of aluminum salt described above is per microgram of recombinant protein.

The compositions of the present invention may also include one or more S. pneumoniae proteins. Examples of S. pneumoniae proteins suitable for inclusion include those identified in International Patent Application Publications No. WO 02/083855 and WO 02/053761.

In certain embodiments, the compositions of the invention are administered to a subject by one or more routes known to one of ordinary skill in the art, for example, parenterally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, intranasally, subcutaneously, intraperitoneally, and are formulated accordingly. In one embodiment, the compositions of the present invention are administered by epidermal injection, intramuscular injection, intravenous, intra-arterial, subcutaneous injection, or intra-respiratory injection of a liquid preparation into the mucous membrane of the respiratory tract. Liquid injectable formulations include solutions and the like.

Cooking methods

Streptococcus pneumoniae capsular polysaccharides can be prepared by standard methods known to those skilled in the art. For example, polysaccharides can be isolated from bacteria and brought to a certain size by known methods (see, for example, European patents No. EP 497524 and EP 497525); and preferably microfluidization using a homogenizer or chemical hydrolysis. In one embodiment, each pneumococcal polysaccharide serotype is grown in a soy-based medium. The individual polysaccharides are then purified using standard steps including centrifugation, sedimentation and ultrafiltration. See, for example, US Patent Application Publication No. 2008/0286838 and US Patent No. 5847112. Polysaccharides can be sized to reduce viscosity in polysaccharide samples and/or to improve filterability of conjugated products by mechanical or chemical methods to obtain the desired size. Chemical hydrolysis can be performed using acetic acid. A mechanical method for obtaining the desired size can be achieved using high pressure homogenization.

Purified polysaccharides can be chemically activated to allow the saccharides to react with the carrier protein. Purified polysaccharides may be linked to a linker. Once activated or bound to a linker, each capsular polysaccharide is individually conjugated to a carrier protein to form a glycoconjugate. Polysaccharide conjugates can be prepared by known coupling methods.

The polysaccharide may be linked to a linker to form a polysaccharide-linker intermediate in which the free end of the linker is an ester group. Therefore, a linker is a linker in which at least one end is an ester group. The other end is chosen so that it can react with the polysaccharide to form a polysaccharide-linker intermediate.

The polysaccharide may be linked to a linker through a primary amino group on the polysaccharide. In this case, the linker usually has an ester group at both ends. This allows binding through the interaction of one of the ester groups with the primary amino group of the polysaccharide through the mechanism of nucleophilic substitution in the acyl group. As a result of the reaction, a polysaccharide-linker intermediate is formed, in which the polysaccharide is linked to the linker through an amide bond. Therefore, the linker is a bifunctional linker in which the first ester group reacts with the primary amino group of the polysaccharide and the second ester group reacts with the primary amino group of the carrier molecule. A typical linker is N-hydroxysuccinimide diester of adipic acid (SIDEA).

Binding can also occur indirectly, i.e. through an additional linker, which is used to derivatize the polysaccharide before binding to the linker.

The polysaccharide is linked to an additional linker involving a carbonyl group at the reducing end of the polysaccharide. This coupling reaction consists of two steps:

(a1) interaction of a carbonyl group with an additional linker; and (a2) interaction of the free end of the additional linker with the linker.

In these embodiments, the additional linker typically has a primary amino group at both ends, allowing step (a1) to occur through the interaction of one of the primary amino groups with the carbonyl group of the polysaccharide via a reductive amination mechanism. A primary amino group is used which reacts with the carbonyl group of the polysaccharide. Suitable ones are hydrazide groups or hydroxylamine groups. The same primary amino groups are usually present at both ends of the additional linker. The reaction produces a polysaccharide-additional linker intermediate in which the polysaccharide is linked to the additional linker via a C-N bond.

The polysaccharide may be linked to an additional linker by another group on the polysaccharide, in particular a carboxyl group. This coupling reaction consists of two steps:

(a1) interaction of the group with an additional linker; And

- 24 043489 (a2) interaction of the free end of the additional linker with the linker.

In this case, the additional linker usually has a primary amino group at both ends, which allows step (a1) to occur through the interaction of one of the primary amino groups with the carboxyl group of the polysaccharide by activating EDAC. A primary amino group is used which reacts with the EDAC-activated carboxyl group in the polysaccharide. A hydrazide group is suitable. The same primary amino groups are usually present at both ends of the additional linker. The reaction produces a polysaccharide-additional linker intermediate in which the polysaccharide is linked to the additional linker via an amide bond.

In one embodiment, chemical activation of polysaccharides and subsequent conjugation to a carrier protein through a reductive amination mechanism can be achieved using the means described in US Patent Nos. 4,365,170, 4,673,574 and 4,902,506, US Patent Application Publications No. 2006/0228380, 2007 /184072, 2007/0231340 and 2007/0184071 and WO 2006/110381, WO 2008/079653 and WO 2008/143709). The chemistry may involve activation of the pneumococcal polysaccharide through reaction with any oxidizing agent that oxidizes the terminal hydroxyl group to an aldehyde, such as a periodate (including sodium periodate, potassium periodate, or periodic acid). The reaction results in random oxidative cleavage of the vicinal hydroxyl groups of carbohydrates to form reactive aldehyde groups.

The coupling reaction with a carrier protein proceeds through a reductive amination mechanism through direct amination at the lysyl residues of the protein. For example, conjugation can be accomplished by reacting a mixture of activated polysaccharide and carrier protein with a reducing agent, such as sodium cyanoborohydride, in the presence of nickel. The conjugation reaction can occur in aqueous solution or in the presence of DMSO. See, for example, US 2015/0231270, US 2011/0195086 and EP 0471177 B1. The unreacted aldehydes are then capped by adding a strong reducing agent such as sodium borohydride.

Reductive amination involves two stages:

(1) oxidation of the polysaccharide to form reactive aldehydes;

(2) reduction of the imine (Schiff base) formed between the activated polysaccharide and the carrier protein to form a stable amine conjugate bond.

The size of the polysaccharide is not necessarily reduced before oxidation. Mechanical methods (eg homogenization) or chemical hydrolysis can be used. Chemical hydrolysis can be performed using acetic acid. The oxidation step may include reaction with a periodate. In the context of the present invention, the term periodate includes both periodate and periodic acid; the term also includes both metaperiodate (IO4 ^- ) and orthoperiodate (IO6 ^- ) and includes various periodate salts (for example, sodium periodate and potassium periodate). In one embodiment, the capsular polysaccharide is oxidized in the presence of metaperiodate, preferably in the presence of sodium periodate (NaIO ₄ ). In another embodiment, the capsular polysaccharide is oxidized in the presence of orthoperiodate, preferably in the presence of periodic acid.

In one embodiment, the oxidizing agent is a stable nitroxide or nitroxide radical compound, such as piperidine-N-oxy or pyrrolidine-Noxy compounds, in the presence of an oxidizing agent to selectively oxidize primary hydroxyls (as described in WO 2014/097099). In this reaction, the actual oxidizing agent in the catalytic cycle is the N-oxoammonium salt. In one aspect, said stable nitroxyl or nitroxide radical compounds are piperidine-N-oxy or pyrrolidine-M-oxy compounds. In one aspect, said stable nitroxyl or nitroxide radical compound has a TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) or PROXYL (2,2,5,5tetramethyl-1-pyrrolidinyloxy) moiety. In one aspect, said stable nitroxide radical compound is TEMPO or a derivative thereof. In one aspect, said oxidizing agent is a molecule containing an N-halogen-containing moiety. In one aspect, said oxidizing agent is selected from the group consisting of N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1,2,3-trichloro-1,2,5-triazinan-2,4,6-trione , dibromoisocyanuric acid, 1,5-tribromo-1,2,3-triazinan-2,4,6-trione, diiodisocyanuric acid and 3,5-triiodo-1,3,5-triazinan-2,4,6-trione .

Preferably, said oxidizing agent is N-chlorosuccinimide.

In certain aspects, the oxidizing agent is the free radical 2,2,6,6 tetramethyl-1-piperidinyloxy (TEMPO) and N-chlorosuccinimide (NCS) as a co-oxidant (as described in WO 2014/097099). Therefore, in one aspect, S. pneumoniae glycoconjugates can be prepared by a process comprising the steps

a) reacting the saccharide with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and N-chlorosuccinimide (NCS) in an aqueous solvent to obtain an activated saccharide; And

b) reacting the activated saccharide with a carrier protein containing one or more amino groups (hereinafter referred to as TEMPO/NCS-reductive amination).

Optionally, the oxidation reaction is stopped by adding a quenching agent. Quenching agent

- 25 043489 may be selected from vicinal diols, 1-, 2-amino alcohols, amino acids, glutathione, sulfite, bisulfate, dithionite, metabisulfite, thiosulfate, phosphites, hypophosphites or phosphorous acid (for example glycerol, ethylene glycol, propane-1-, 2 -diol, butane-1,2-diol or butane-2,3-diol, ascorbic acid).

In some embodiments, the present invention provides a method for producing a conjugate of a Streptococcus pneumoniae serotype 8 polysaccharide to a protein by a conjugation reaction in an aprotic solvent, wherein the conjugation reaction does not use cyanoborohydride. In other embodiments, the conjugation reaction is a Schiff reduction or reductive amination. In other embodiments, the protein is tetanus toxoid, diphtheria toxoid, or CRM197. In other embodiments, the protein is CRM197. In other embodiments, the conjugation reaction is a reductive amination. In other embodiments, the reductive amination is carried out in dimethyl sulfoxide (DMSO).

In some embodiments, the oxidized polysaccharides have a molecular weight of 30 kDa to 1000 kDa before conjugation. Molecular weight can be determined by size exclusion chromatography (SEC) in combination with a multi-angle light scattering (MALS) detector and a refractive index (RI) detector. In some embodiments, the polysaccharide has a molecular weight of 50 to 300 kDa. In some embodiments, the polysaccharide has a molecular weight of 50 kDa to 1000 kDa. In additional embodiments, the polysaccharide has a molecular weight of 70 to 900 kDa. In other embodiments, the polysaccharide has a molecular weight of 100 to 800 kDa. In other embodiments, the polysaccharide has a molecular weight of 200 to 600 kDa. In other embodiments, the polysaccharide has a molecular weight of 100 to 1000 kDa; from 100 to 900 kDa; from 100 to 800 kDa; from 100 to 700 kDa; from 100 to 600 kDa; from 100 to 500 kDa; from 100 to 400 kDa; from 100 to 300 kDa; from 150 to 1000 kDa; from 150 to 900 kDa; from 150 to 800 kDa; from 150 to 700 kDa; from

150 to 600 kDa; from 150 to 500 kDa; from 150 to 400 kDa; from 150 to 300 kDa; from 200 to 1000 kDa; from

200 to 900 kDa; from 200 to 800 kDa; from 200 to 700 kDa; from 200 to 600 kDa; from 200 to 500 kDa; from

200 to 400 kDa; from 200 to 300; from 250 to 1000 kDa; from 250 to 900 kDa; from 250 to 800 kDa; from 250 to 700 kDa; from 250 to 600 kDa; from 250 to 500 kDa; from 250 to 400 kDa; from 250 to 350 kDa; from

300 to 1000 kDa; from 300 to 900 kDa; from 300 to 800 kDa; from 300 to 700 kDa; from 300 to 600 kDa; from

300 to 500 kDa; from 300 to 400 kDa; from 400 to 1000 kDa; from 400 to 900 kDa; from 400 to 800 kDa; from

400 to 700 kDa; from 400 to 600 kDa; from 500 to 600 kDa.

The second step in the conjugation process is the reduction of the imine bond (Schiff base) between the activated polysaccharide and the carrier protein to form a stable conjugate bond (called reductive amination) using a reducing agent. Suitable reducing agents include cyanoborohydrides (such as sodium cyanoborohydride or sodium borohydride). In one embodiment, the reducing agent is sodium cyanoborohydride.

In certain embodiments, the reductive amination reaction is carried out in an aprotic solvent (or mixture of aprotic solvents). In one embodiment, the reduction reaction is carried out in a solvent of DMSO or DMF (dimethylformamide). The solvent DMSO or DMF can be used to reconstitute the activated polysaccharide and protein carrier if it is lyophilized. In one embodiment, the aprotic solvent is DMSO.

At the end of the reduction reaction, unreacted aldehyde groups may remain in the conjugates, which can be capped with a suitable capping agent. In one embodiment, the capping agent is sodium borohydride (NaBH ₄ ). Suitable alternatives include sodium triacetoxyborohydride or sodium or zinc borohydride in the presence of Bronsted or Lewis acids), amine boranes such as pyridine-borane, 2-picoline-borane, 2,6-diboranemethanol, dimethylamine-borane, tert-BuMe'PrN-BH ₃ , benzylamine-BH ₃ or 5-ethyl-2-methylpyridineborane (PEMB), or borohydride exchange resins. After conjugation (reduction reaction and, optionally, capping), the glycoconjugates can be purified (enriched in the amount of polysaccharide-protein conjugate) by various methods known to one skilled in the art. These methods include dialysis, concentration/diafiltration operations, tangential flow filtration, precipitation/elution, column chromatography (ion exchange chromatography, multimodal ion exchange chromatography, DEAE or hydrophobic interaction chromatography) and depth filtration. In one embodiment, the glycoconjugates are purified by diafiltration or ion exchange chromatography or size exclusion chromatography.

Glycoconjugates produced by a reductive amination mechanism in an aprotic solvent are commonly used in multivalent pneumococcal conjugate vaccines. Thus, in certain embodiments, for multivalent compositions in which not all serotypes are produced in an aprotic solvent, the reduction reaction of the remaining serotypes is carried out in an aqueous solvent (eg, selected from PBS, MES (2-(N-morpholino )ethanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1- 26 043489 piperazineethanesulfonic acid), bis-tris, ADA (N-(2-acetamido)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-(Nmorpholino)propanesulfonic acid), DIPSO (3-bis(2-hydroxyethyl)amino-2-hydroxypropane-1sulfonic acid), MOBS (4-(N-morpholino)butanesulfonic acid), HEPPSO (N-(2-hydroxyethyl)piperazineN-(2-hydroxypropanesulfonic acid)), POPSO (piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid)), TEA (triethanolamine), EPPS (4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid), bicina at pH 6.0 to 8.5, from 7.0 to 8.0 or from 7.0 to 7.5).

S. pneumonia capsular polysaccharide-protein conjugates that can be prepared by reductive amination in an aprotic solvent include, but are not limited to, 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. Conjugates of S. pneumonia capsular polysaccharide and protein, which can be obtained by the mechanism of reductive amination in an aprotic solvent, include without restrictions serotypes 3, 6A, 6B, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 19A, 20, 22F, 23A, 23B, 23F, 24F, 31, 33F, 35B and 39. Conjugates of S. pneumonia capsular polysaccharide and protein that can be prepared by a reductive amination mechanism in an aprotic solvent include, but are not limited to, serotypes 3, 6A, 6B, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A , 15B, 15C, 16F, 17F, 19A, 20B, 22F, 23a, 23B, 23F, 24F, 31, 33F, 35B and 39. Polysaccharides can be used in the form of oligosaccharides. They are conveniently prepared by fragmentation of the purified polysaccharide (eg by hydrolysis), usually followed by purification of the fragments to the desired size.

In some embodiments, pneumococcal polysaccharide-protein conjugates of one or more 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 are prepared by the reductive amination mechanism in an aprotic solvent. In some embodiments, pneumococcal polysaccharide-protein conjugates of one or more serotypes 3, 6A, 6B, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 19A, 20, 22F, 23A, 23B, 23F, 24F, 31, 33F, 35B and 39 are prepared by the reductive amination mechanism in an aprotic solvent. In some embodiments, pneumococcal polysaccharide protein conjugates of one or more serotypes 3, 6A, 6B, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 19A, 20B, 22F, 23A, 23B , 23F, 24F, 31, 33F, 35B and 39 are prepared by the reductive amination mechanism in an aprotic solvent. In certain embodiments, each of the serotypes in the multivalent immunogenic composition is produced by a reductive amination mechanism in an aprotic solvent. In some embodiments, the polysaccharides of one or more serotypes in the multivalent composition are conjugated by a reductive amination mechanism in an aprotic solvent, and the polysaccharides of one or more serotypes are conjugated by a reductive amination mechanism in an aqueous solvent. In some embodiments, the polysaccharides of two or more serotypes in a multivalent composition are conjugated by a reductive amination mechanism in an aprotic solvent. In other embodiments, the polysaccharides are three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, twenty or more, or twenty one or more serotypes in a polyvalent composition are conjugated by the mechanism of reductive amination in an aprotic solvent. In certain embodiments, the polysaccharides of one or more serotypes in the multivalent composition are conjugated with other chemical compounds, which may be in an aprotic solvent or an aqueous solvent.

Thus, the invention relates to a multivalent immunogenic composition containing several S. pneumoniae polysaccharide-protein conjugates, each of which contains a capsular polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes are as disclosed herein (i.e., Section II, Polyvalent Immunogenic Compositions), wherein the conjugation reaction resulting in the conjugation of the polysaccharide of one or more polysaccharide-protein conjugates to a carrier protein occurs in an aprotic solvent. In some embodiments, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% of the serotypes in the multivalent composition are prepared in an aprotic solvent. The remaining serotypes are produced using alternative chemistry and/or in an aqueous solvent.

Surprisingly, it has been found that the use of DMSO as a solvent during the reductive amination of polysaccharide-protein conjugates results in high stability and increased immunogenicity of these serotypes compared to the same conjugates prepared under aqueous conditions (see US Application Nos. 62/463216 and 62/ 555444). As shown herein (see Example 40), drug formulations containing the pneumococcal conjugates of the invention, obtained by the mechanism of reductive amination in an aprotic solvent (for example, DMSO), provided high physical and chemical stability compared to the vaccine, in

- 27 043489 which used medicinal substances obtained in protic (i.e. aqueous) solvents during reductive amination in the conjugation process. Thus, in some embodiments, all of the pneumococcal polysaccharide conjugates in the multivalent composition are prepared in an aprotic solvent.

In certain embodiments of the invention, the total concentration of polysaccharide in the composition is from about 0.02 mg/ml to about 0.175 mg/ml. In certain embodiments of the invention, the total concentration of polysaccharide in the composition is from about 0.03 mg/ml to about 0.175 mg/ml. In certain embodiments of the invention, the total concentration of polysaccharide in the composition is from about 0.04 mg/ml to about 0.175 mg/ml. In other embodiments, the total polysaccharide concentration in the composition is from about 0.065 mg/ml to about 0.085 mg/ml, from about 0.070 mg/ml to about 0.080 mg/ml, from about 0.065 mg/ml to about 0.080 mg/ml, from about 0.070 mg/ml to about 0.085 mg/ml, about 0.110 mg/ml to about 0.128 mg/ml, about 0.110 mg/ml to about 0.175 mg/ml, about 0.10 mg/ml to about 0.175 mg /ml, from about 0.110 mg/ml to about 0.170 mg/ml, from about 0.115 mg/ml to about 0.15 mg/ml, from about 0.110 mg/ml to about 0.15 mg/ml, from about 0.110 mg /ml to about 0.125 mg/ml, from about 0.150 mg/ml to about 0.170 mg/ml, from about 0.150 mg/ml to about 0.165 mg/ml, from about 0.140 mg/ml to about 0.170 mg/ml, from about 0.130 mg/ml to about 0.170 mg/ml, about 0.150 mg/ml to about 0.175 mg/ml, about 0.070 mg/ml to about 0.170 mg/ml, about 0.065 mg/ml to about 0.175 mg/ml, or from about 0.065 mg/ml to about 0.180 mg/ml.

In embodiments of the invention where one or more or all of the polysaccharide-protein conjugates in the multivalent immunogenic compositions are prepared in an aprotic solvent, the total polysaccharide concentration in the composition is stable for 4 weeks or more at 37°C, 4 weeks or more at 25°C, and 12 weeks or more at 4°C.

In certain embodiments of the invention, where one or more or all of the polysaccharide-protein conjugates in multivalent immunogenic compositions are prepared in an aprotic solvent, the weight average molecular weight (Mw) of all S. pneumoniae polysaccharide-protein conjugates in the composition (average of all conjugates in the composition) is between about 3500 kDa to about 4700 kDa, about 3500 kDa to about 4600 kDa, about 3500 kDa to about 4500 kDa, about 3500 kDa to about 4400 kDa, about 3500 kDa to about 4300 kDa, about 3500 kDa to about 4200 kDa, from about 3600 kDa to about 4700 kDa, from about 3600 kDa to about 4600 kDa, from about 3600 kDa to about 4500 kDa, from about 3600 kDa to about 4400 kDa, from about 3600 kDa to about 4300 kDa from about 3600 kDa to about 4200 kDa, from about 3700 kDa to about 4700 kDa, from about 3700 kDa to about 4600 kDa, from about 3700 kDa to about 4500 kDa, from about 3700 kDa to about 4400 kDa, from about 3700 kDa to about 4300 kDa , from about 3700 kDa to about 4200 kDa, from about 3800 kDa to about 4700 kDa, from about 3800 kDa to about 4600 kDa, from about 3800 kDa to about 4500 kDa, from about 3800 kDa to about 4400 kDa, from about 3800 kDa to about 4300 kDa, from about 3800 kDa to about 4200 kDa, from about 3900 kDa to about 4700 kDa, from about 3900 kDa to about 4600 kDa, from about 3900 kDa to about 4500 kDa, from about 3900 kDa to about 4400 kDa, from about 3900 kDa to about 4300 kDa or from about 3900 kDa to about 4200 kDa.

In some embodiments in which the polysaccharide-protein conjugates in multivalent immunogenic compositions are prepared in an aprotic solvent, the Mw of each of the S. pneumoniae polysaccharide-protein conjugates in the composition (for one serotype) is from about 1000 kDa to about 10,000 kDa, from about 1500 kDa to about 5500 kDa, about 1500 kDa to about 5600 kDa, about 1500 kDa to about 5700 kDa, about 1500 kDa to about 5800 kDa, about 1500 kDa to about 5900 kDa, about 1500 kDa to about 6000 kDa, from about 1000 kDa to about 5500 kDa, from about 1000 kDa to about 5000 kDa, from about 1000 kDa to about 4000 kDa, from about 1000 kDa to about 4500 kDa, from about 1000 kDa to about 4000 kDa, or from about 1000 kDa to about 3500 kDa. In other embodiments, the Mw of a single serotype conjugate in the composition is about 1000 kDa, about 1100 kDa, about 1200 kDa, about 1300 kDa, about 1400 kDa, about 1500 kDa, about

1600 kDa, about 1700 kDa, about 1800 kDa, about 1900 kDa, about 2000 kDa, about

2100 kDa, about 2200 kDa, about 2300 kDa, about 2400 kDa, about 2500 kDa, about

2600 kDa, about 2700 kDa, about 2800 kDa, about 2900 kDa, about 3000 kDa, about

3100 kDa, about 3200 kDa, about 3300 kDa, about 3400 kDa, about 3500 kDa, about

3600 kDa, about 3700 kDa, about 3800 kDa, about 3900 kDa, about 4000 kDa, about

4100 kDa, about 4200 kDa, about 4200 kDa, about 300 kDa, about 4400 kDa, about 4500 kDa, about 4600 kDa, about 4700 kDa, about 4800 kDa, about 4900 kDa, about 5000 kDa, about 5100 kDa, about 5200 kDa , about 5300 kDa, about 5400 kDa, or about 5500 kDa.

- 28 043489

In certain embodiments of the invention, the polysaccharide-protein conjugates in multivalent immunogenic compositions are prepared in an aprotic solvent. Compositions containing a higher percentage of S. pneumoniae polysaccharides conjugated to a carrier protein in an aprotic solvent (as opposed to being prepared in a protic solvent) may be preferred. In some embodiments, the percentage (calculated as the number of polysaccharide serotypes produced in an aprotic solvent divided by the total number of polysaccharide serotypes, where the total number includes serotypes produced in an aprotic solvent or protic solvent) of conjugates of certain S. pneumoniae serotypes produced in an aprotic solvent is may be more than 50%, or more than 60%, or more than 70%, or more than 80%, or more than 90%, or be 100%.

In some embodiments, the serotype 3 polysaccharide-protein conjugate of the composition is prepared in an aprotic solvent and the Mw of the conjugate is from about 1000 kDa to about 5000 kDa, or from about 1000 kDa to about 4000 kDa, or from about 1000 kDa to about 3000 kDa , or from about 1000 kDa to about 2500 kDa, or from about 1000 kDa to about 2000 kDa.

In certain embodiments of the invention in which one or more or all of the polysaccharide-protein conjugates in multivalent immunogenic compositions are prepared in an aprotic solvent, the number average molecular weight (Mn) of the S. pneumoniae polysaccharide-protein conjugates in the composition (average of all conjugates in the composition) is from about 900 kDa to about 3000 kDa, from about 1000 kDa to about 3000 kDa, from about 1000 kDa to about 2500 kDa, from about 1500 kDa to about 2500 kDa, from about 1800 kDa to about 2500 kDa from about 1900 kDa to about 2500 kDa or from about 2000 kDa to about 2500 kDa.

In certain embodiments of the invention in which one or more or all of the polysaccharide-protein conjugates in multivalent immunogenic compositions are prepared in an aprotic solvent, the Mn of each of the S. pneumoniae polysaccharide-protein conjugates in the composition (for one serotype) is from about 700 kDa to about 7000 kDa, from about 1000 kDa to about 6000 kDa, from about 1000 kDa to about 5000 kDa, from about 1000 kDa to about 4000 kDa, from about 1000 kDa to about 3000 kDa, from about 900 kDa to about 5500 kDa, from about 900 kDa to about 5000 kDa, about 900 kDa to about 4500 kDa, about 900 kDa to about 4000 kDa, about 900 kDa to about 3500 kDa, or about 900 kDa to about 3000 kDa.

In embodiments of the invention, the Mw and/or Mn of the S. pneumoniae polysaccharide-protein conjugates in the composition remains stable for 4 weeks or more at 37°C, 4 weeks or more at 25°C, and/or 12 weeks or more at 4°C .

In embodiments of the invention, the concentration of polysaccharide, Mw and/or Mn is determined using HP SEC UV/MALS/RI.

In some embodiment of the invention, in which one or more or all of the polysaccharide protein conjugates in the multivalent immunogenic compositions are prepared in an aprotic solvent, the maximum emission of the composition, as measured by internal protein fluorescence spectroscopy with an excitation wavelength of 280 nanometers (nm), is from about 335 nm to approximately 342 nm. In some embodiments, the emission maximum remains in the range from about 335 nm to about 342 nm, and the fluorescence intensity remains stable for at least 1 week at 37°C. In some embodiments, the emission maximum is maintained between about 335 nm and about 342 nm and the fluorescence intensity remains stable for 1 week at 37°C.

In some embodiments, all of the pneumococcal polysaccharide conjugates in the multivalent composition are produced by a reductive amination mechanism in DMSO. In some sub-embodiments, the polyvalent composition comprising polysaccharide conjugates, all of which were prepared in DMSO, does not contain an adjuvant.

Without being limited to any theory, one possible mechanism for the increased immunogenicity observed in glycoconjugates prepared in DMSO involves an increase in the number of bonds between the carbohydrate (capsular polysaccharide) and lysine residues on the surface of the carrier protein, which may lead to additional points of attachment of the protein to polysaccharide, providing stability and resistance to chemical depolymerization or cleavage of the peptide carbohydrate bond. See, for example, Hsieh, Characterization of Saccharide-CRM197 Conjugate Vaccines in Brown F., Corbel M., Griffiths E. (eds): Physico-Chemical Procedures for the Characterization of Vaccines, Dev. Biol. Basel, Karger, 2000, vol. 103, p. 93-104. An additional benefit of increasing the number of polysaccharide-protein bonds that are formed during conjugation in the solvent DMSO may provide additional opportunities for successful presentation of peptide-carbohydrates to T cells. It should be understood that due to genetic variability in the human population resulting in varying abilities and loading sensitivities or associations with specific peptide sequences conjugated to carbohydrate antigens, such additional attachment points to

- 29 043489 carrier protein will increase the chances of successful presentation of the antigen on the surface of the antigen-presenting cell (APC), providing a T-cell-dependent response to another T-cell-independent antigen. Another possible mechanism for enhanced immunogenicity observed with DMSO conjugation may be due to the denaturation of CRM197 in an organic solvent, which exposes additional lysine residues, leading to the formation of polysaccharide bonds, which increases the chances of presentation of the glycopeptide on the surface of the APC to T cells -dependent response to various peptide epitopes. See Avci et al., 2011, Nature Medicine, 17:1602–1610.

Another advantage of conjugation in an organic solvent generating denatured CRM197 in conjugates may be a reduction in immunological interference of antibodies to native CRM197 epitopes. An additional benefit of increasing the number of polysaccharide-protein bonds that are formed during conjugation in the DMSO solvent may be the formation of larger polysaccharide-protein conjugates, resulting in increased immunogenicity. It is believed that the compositions of the invention provide significant benefits in initiating a reaction in humans.

In some embodiments, the conjugation reaction is performed via a reductive amination mechanism, which uses nickel to provide a more efficient conjugation reaction and to remove free cyanide. Transition metals are known to form stable complexes with cyanide and are known to enhance reductive methylation of amino groups of protein and formaldehyde with sodium cyanoborohydride (S. Gidley et al., Biochem. J., 1982, 203:331-334; Jentoft et al. ., Anal. Biochem., 1980, 106:186-190). By forming a residual inhibitory cyanide complex, the addition of nickel increases protein consumption during conjugation and results in the formation of larger, potentially more immunogenic conjugates.

Differences in initial cyanide levels between sodium cyanoborohydride reagent batches also result in inconsistent conjugation characteristics, resulting in different product characteristics such as conjugate size and Ps-k-CRM197 ratio. The addition of nickel reduces the variability of conjugation reaction results due to cyanide complexation, eliminating batch variations of sodium cyanoborohydride.

Suitable alternative chemistries include activation of the saccharide with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form the cyanate ester. Thus, the activated saccharide can be linked directly or through a spacer (linker) group to the amino group on the carrier protein. For example, the spacer may be cystamine or cysteamine to produce a thiolated polysaccharide, which can be linked to a carrier via a thioester linkage obtained by reaction with a maleimide-activated carrier protein (eg, using GMBS) or a halogen-acetylated carrier protein ( for example, using iodoacetimide (eg ethyl-iodoacetimide HCl) or N-succinimidyl bromoacetate or SIAB or SIA or SBAP). Preferably, a cyanate ester (optionally prepared using CDAP chemistry) is coupled to a hexanediamine or adipic acid dihydrazide (ADH), and the amino derivative saccharide is conjugated to a carrier protein using carbodiimide chemistry (eg, EDAC or EDC) via the carboxyl group on the carrier protein. Such conjugates are described in International Patent Application Publications No. WO 93/15760, WO 95/08348 and WO 96/29094; and Chu et al., 1983, Infect. Immunity 40:245–256.

Other suitable methods use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU. Many of them are described in International Patent Application Publication No. WO 98/42721. The conjugation process may involve a carbonyl linker, which may be formed by reacting the free hydroxyl group of the saccharide with CDI (See Bethell et al., 1979, J. Biol. Chem., 254:2572-4; Hearn et al., 1981 , J. Chromatogr., 218:509-18) followed by reaction with the protein to form a carbamate bond. This may include reduction of the anomeric end to a primary hydroxyl group, optional protection/deprotection of the primary hydroxyl group, reaction of the primary hydroxyl group with CDI to form a CDI carbamate intermediate, and coupling of the CDI carbamate intermediate to the amino group of the protein.

After conjugation of the capsular polysaccharide to the carrier protein, the polysaccharide-protein conjugates are purified (enriched in the amount of polysaccharide-protein conjugate) by one or more of a variety of methods. Examples of these methods are well known to one skilled in the art and include concentration/diafiltration, ultrafiltration, precipitation/elution, column chromatography and depth filtration operations. See, for example, US Pat. No. 6,146,902.

After purification of the individual glycoconjugates, they are mixed to obtain the immunogenic composition of the present invention. These pneumococcal conjugates are each prepared separately and mixed into a unit dose formulation.

An alternative way to characterize the glycoconjugates of the invention is by the number of lysine residues in the carrier protein (e.g., CRM197) that become conjugated to calcium

- 30 043489 charide, and this number of conjugated lysines can be characterized as a range (degree of conjugation). Evidence of modification of lysine residues of the carrier protein by formation of covalent bonds with polysaccharides can be obtained by amino acid analysis using conventional methods known to those skilled in the art. Conjugation results in a decrease in the amount of reduced lysine residues compared to the starting carrier protein material used to prepare the conjugate materials. In a preferred embodiment, the degree of conjugation of the glycoconjugate of the invention is 2 to 15, 2 to 13, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 3 to 15, 3 to 13, from 3 to 10, from 3 to 8, from 3 to 6, from 3 to 5, from 3 to 4, from 5 to 15, from 5 to 10, from 8 to 15 from 8 to 12, from 10 to 15 or from 10 to 12. In one embodiment, the degree of conjugation of the glycoconjugate of the 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 degree of conjugation of the glycoconjugate of the invention is from 4 to 7. In some such embodiments, the carrier protein is CRM197.

Glyconoconjugates of the compositions of the invention can also be characterized by the ratio (w/w) of saccharide to carrier protein (Ps:Pr). In some embodiments, the ratio of polysaccharide to carrier protein in glycoconjugates (wt/wt) in the composition is from 0.5 to 3.0 (e.g., about 0.5, about 0.6, about 0.7, about 0 .8, approximately 0.9, approximately 1.0, approximately 1.1, approximately 1.2, approximately 1.3, approximately 1.4, approximately 1.5, approximately 1.6, approximately 1.7, approximately 1 ,8, approximately 1.9, approximately 2.0, approximately 2.1, approximately 2.2, approximately 2.3, approximately 2.4, approximately 2.5, approximately 2.6, approximately 2.7, approximately 2 .8, about 2.9 or about 3.0). In other embodiments, the ratio of saccharide to carrier protein (w/w) is 0.5 to 2.5, 0.5 to 1.5, 0.8 to 2.5, 0.5 to 1.0, from 1.0 to 1.5, from 1.0 to 2.0, from 0.8 to 2.4 from 0.8 to 2.3, from 0.8 to 2.2, from 0 ,8 to 2.1, from 0.8 to 2.0, from 0.8 to 1.9, from 0.8 to 1.8, from 0.8 to 1.7, from 0.8 to 1, 6, from 0.8 to 1.5, from 0.8 to 1.4 from 0.8 to 1.3, from 0.9 to 2.4, from 0.9 to 2.3, from 0.9 up to 2.2, from 0.9 to 2.1, from 0.9 to 2.0, from 0.9 to 1.9, from 0.9 to 1.8, from 0.9 to 1.7, from 0.9 to 1.6 from 0.9 to 1.5, from 0.9 to 1.4, from 0.9 to 1.3, from 0.9 to 1.2, from 1.0 to 2 ,4, from 1.0 to 2.3, from 1.0 to 2.2, from 1.0 to 2.1, from 1.0 to 2.0, from 1.0 to 1.9 from 1, 0 to 1.8, 1.0 to 1.7, 1.0 to 1.6, 1.0 to 1.5, 1.0 to 1.4, 1.0 to 1.3 or from 1.0 to 1.2. In other embodiments, the saccharide to carrier protein (w/w) ratio is from 0.8 to 1.2. In some such embodiments, the carrier protein is CRM197. The glycoconjugates and immunogenic compositions of the invention may contain a free saccharide that is not covalently conjugated to the carrier protein but is nonetheless present in the glycoconjugate composition. The free saccharide may be non-covalently bound (ie, non-covalently attached to, adsorbed or entrapped) to the glycoconjugate.

In specific embodiments, the saccharide to carrier protein (w/w) ratio for the serotype 15A conjugate is from about 1.0 to about 2.0, from about 1.25 to about 1.75, or from about 1.3 to approximately 1.7. In other embodiments, the saccharide to carrier protein (w/w) ratio for serotype 15A is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, or 1. 8.

In specific embodiments, the saccharide to carrier protein (w/w) ratio for the serotype 15C conjugate is from about 1.0 to about 2.0, from about 1.25 to about 1.75, or from about 1.3 to approximately 1.7. In other embodiments, the saccharide to carrier protein (w/w) ratio for serotype 15C is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, or 1. 8.

In specific embodiments, the saccharide to carrier protein (w/w) ratio for the serotype 33F conjugate is from about 1.0 to about 2.0, from about 1.25 to about 1.75, or from about 1.3 to approximately 1.7. In other embodiments, the saccharide to carrier protein (w/w) ratio for serotype 33F is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, or 1. 8.

In specific embodiments, the saccharide to carrier protein (w/w) ratio for the serotype 35B conjugate is from about 1.25 to about 2.25, from about 1.25 to about 2.0, or from about 1.3 to approximately 1.8. In other embodiments, the saccharide to carrier protein (w/w) ratio for serotype 33B is about 1.2, 1.3, 1.3, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0.

In specific embodiments, the saccharide to carrier protein (w/w) ratio for the serotype 24F conjugate is from about 0.5 to about 1.5, from about 0.75 to about 1.25, or from about 0.8 to approximately 1.0. In other embodiments, the saccharide to carrier protein (w/w) ratio for serotype 24F is about 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0.

In a preferred embodiment, the glycoconjugate contains less than about 50, 45, 40, 35, 30, 25, 20, or 15% free polysaccharide, based on the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate contains less than about 25% free polysaccharide, relative to the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate contains less than about 20% free polysaccharide, relative to the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate contains less than about 15% free polysaccharide, relative to the total amount of polysaccharide.

IV. Methods of application.

Embodiments of the invention also include one or more multivalent immunogens

- 31 043489 ny compositions disclosed herein, (i) for use, (ii) for use as a medicament or composition for, or (iii) for use in the preparation of a medicament (a) for therapy (for example, the human body );

(b) in medicine;

(c) to inhibit Streptococcus pneumoniae infection;

(d) to induce an immune response or a protective immune response against S. pneumoniae;

(e) for the prevention of S. pneumoniae infection;

(f) to prevent recurrence of S. pneumoniae infection;

(g) to reduce the progression, occurrence or severity of pathological symptoms associated with S. pneumoniae infection, including the prevention of associated complications such as brain damage, hearing loss and seizures;

(h) to reduce the likelihood of infection with S. Pneumoniae; or (i) to treat, prevent, or delay the onset, severity, or progression of pneumococcal disease(s), including, without limitation, pneumococcal pneumonia, pneumococcal bacteremia, pneumococcal meningitis, otitis media, and sinusitis.

In these applications, the multivalent pneumococcal polysaccharide conjugate compositions of the invention may optionally be used in combination with one or more adjuvants or without an adjuvant.

Accordingly, the invention relates to methods of prophylactically treating (ie, protecting against) S. pneumoniae infection or pneumococcal disease, comprising administering one or more multivalent immunogenic pneumococcal polysaccharide-protein conjugates of the invention to a patient in need of treatment.

The compositions and compositions of the present invention can be used to protect or treat a person susceptible to an infection, for example, pneumococcal infection, by administering a composition of the invention systemically or mucosally.

In one embodiment, the invention relates to a method of inducing an immune response to S. pneumoniae, comprising administering to a patient an immunologically effective amount of a multivalent immunogenic composition of the invention. In another embodiment, the invention relates to a method of vaccinating a person against pneumococcal infection, comprising the step of administering to the person an immunogenically effective amount of a multivalent immunogenic composition according to the invention.

Thus, in one aspect, the invention relates to a method of (1) inducing an immune response in a human patient;

(2) inducing a protective immune response in a human patient;

(3) vaccinating the human patient against S. pneumoniae infection; or (4) reducing the likelihood of infection of a human patient with S. pneumoniae, the method comprising administering to the patient a multivalent immunogenic composition of the invention (i.e., any multivalent immunogenic composition disclosed herein, such as the multivalent immunogenic compositions described in Section II, entitled Polyvalent immunogenic compositions, see above).

In one embodiment, the invention provides a method for preventing pneumococcal pneumonia and invasive disease in adults 18 years of age and older. In another embodiment, the invention relates to a method for the prevention of pneumococcal pneumonia and invasive disease caused by 24 strains of Streptococcus pneumoniae (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).

In one embodiment of the above methods, the composition contains multiple S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates contains a polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes comprise a set of serotypes 3, 7F, 19A, 22F , 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20A. In one embodiment of the above methods, the composition contains several S. pneumoniae polysaccharide-protein conjugates, where each of the conjugates contains a polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, and the S. pneumoniae serotypes contain a set of serotypes: 3, 7F, 19A , 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20. In one embodiment of the above methods, the composition contains several S. pneumoniae polysaccharide-protein conjugates, where each of the conjugates contains a polysaccharide of the S. pneumoniae serotype conjugated to a carrier protein, and the S. pneumoniae serotypes contain a set of serotypes 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B , 8, 9N, 10A, 11A, 12F, 15C, 17F and 20B. In another embodiment of the above methods, the composition further comprises a S. pneumoniae serotype 6A or 6C polysaccharide conjugate with a protein.

It has been shown that pneumococcal conjugate vaccine containing serotype 6A can provide

- 32 043489 to provide some cross-protection against serotype 6C (Cooper et al., Vaccine 29 (2011), 7207-7211). Therefore, in some embodiments of the above methods, the invention also relates to the use of multivalent immunogenic compositions that do not contain serotype 6C, but instead contain serotype 6A or serotypes 6A and 6B. In other embodiments, the immunogenic composition contains conjugates of pneumococcal serotypes 6A, 6B and 6C. In specific embodiments of the above methods, the S. pneumoniae serotypes comprise a set of serotypes selected from the group consisting of la) 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, PA, 12F, 15C,

17F and 20A;

lb) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, PA, 12F,

15C, 17F and 20A;

1c) 3, 7F, 19A, 22F, 33F, 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, PA,

12F, 15C, 17F and 20A; And

I-d) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, PA, 12F, 15C, 17F and 20A.

In specific embodiments of the above set of serotypes (I-a through I-d), serotype 20 or 20B may be replaced by serotype 20A.

In other embodiments of the above methods, the S. pneumoniae serotypes include a set of serotypes selected from the group consisting of

P-a) 15A, 16F, 23A, 23B, 24F, 31 and 35B;

Π-b) 6A, 15A, 16F, 23A, 23B, 24F, 31 and 35B;

P-c) 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31 and 35B;

II-d) 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B;

P-e) 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9H, 10A, PA, 12F, 15C, 17F and 20A;

ΙΙ-f) 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, PA, 12F, 15C, 17F and 20A;

ΙΙ-g) 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9H, 10A, PA, 12F, 15C, 17F and 20A;

And

ΙΙ-h) 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, PA, 12F, 15C, 17F and 20A.

In specific embodiments of the above set of serotypes (II-e-II-h), serotype 20 or 20B may be replaced by serotype 20A.

In further embodiments of the above methods, the S. pneumoniae serotypes comprise the set of serotypes specified in any of embodiments Ia), II-a) or II-e), and further comprise serotypes 6A, 6B and 6C.

It has also been shown that pneumococcal conjugate vaccine containing serotype 10A may provide some cross-protection against serotype 39 (see WO 2017/085586). Therefore, in some embodiments of the above methods, the invention also provides for the use of multivalent immunogenic compositions that do not contain serotype 10A, but instead contain serotype 39. In other embodiments, the immunogenic composition contains pneumococcal conjugates of serotypes 10A and 39. In specific embodiments of the above methods S. pneumoniae serotypes contain a set of serotypes selected from the group consisting of

Ш-а) 3, 7F, 19А, 22F, 33F, 15А, 16F, 23А, 23В, 24F, 31, 35В, 8, 9N, 39, PA, 12F,

15C, 17F and 20A;

Ш-b) 3, 7F, 19А, 22F, 33F, 6А, 15А, 16F, 23А, 23В, 24F, 31, 35В, 8, 9N, 39, PA, 12F,

15C, 17F and 20A;

Ш-с) 3, 7F, 19А, 22F, 33F, 6А, 6В, 15А, 16F, 23А, 23В, 24F, 31, 35В, 8, 9N, 39, PA,

12F, 15C, 17F and 20A;

ΙΙΙ-d) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 39, PA, 12F, 15C, 17F and 20A;

Ш-е) 15А, 16F, 23А, 23В, 24F, 31, 35В, 8, 9Н, 39, PA, 12F, 15С, 17F and 20А;

ΙΙΙ-f) 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9H, 39, PA, 12F, 15C, 17F and 20A;

ΙΠ-g) 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9H, 39, PA, 12F, 15C, 17F and 20A;

and

ΙΙΙ-h) 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 39, PA, 12F, 15C, 17F and 20A.

In specific embodiments of the above set of serotypes (II-a-III-h), serotype 20 or 20B may be replaced by serotype 20A.

In further embodiments of the above methods, the S. pneumoniae serotypes

- 33 043489 contain a set of serotypes specified in any of embodiments III-a)-III-b), and additionally contain serotype 10A.

It has also been shown that immunogenic conjugates containing the S. pneumoniae serotype 15B capsular polysaccharide covalently linked to a carrier protein may provide some cross-protection against serotype 15C and/or serotype 15A (see WO 2015/110942). Therefore, in some embodiments of the above methods, the invention also relates to the use of multivalent immunogenic compositions that do not contain serotype 15C (or de-Oacetylated 15B), but instead contain serotype 15B (i.e., a serotype 15B polysaccharide that is not O- acetylated). In other embodiments, the immunogenic composition comprises pneumococcal conjugates of serotypes 15B and 15C (or de-O-acetylated 15B). In specific embodiments of the above methods, the S. pneumoniae serotypes comprise a set of serotypes selected from the group consisting of

IV-a) 3, 7F, 19A, 22F, 33F, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, PA, 12F,

15V, 17F and 20A;

IV-b) 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, PA, 12F, 15B, 17F and 20A;

IV-c) 3, 7F, 19A, 22F, 33F, 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A,

PA, 12F, 15B, 17F and 20A;

IV-d) 3, 7F, 19A, 22F, 33F, 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, PA,

12F, 15B, 17F and 20A;

IV-e) 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9H, 10A, PA, 12F, 15B, 17F and 20A;

IV-f) 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, PA, 12F, 15B, 17F and 20A;

IV-g) 6A, 6B, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9H, 10A, PA, 12F, 15B, 17F and 20A; And

IV-h) 6C, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, PA, 12F, 15B, 17F and 20A.

In specific embodiments of the above set of serotypes (IV-a -IV-h), serotype 20 or 20B may be replaced by serotype 20A.

In further embodiments of the above methods, the S. pneumoniae serotypes comprise the set of serotypes specified in any of embodiments IV-a)-VI-h), and further comprise serotype 15C (and/or de-O-acetylated 15B).

The compositions of the invention can be used in methods of providing additional protection against S. pneumoniae in patients who have previously received a multivalent pneumococcal vaccine. When used in this manner, the compositions of the invention may provide protection against specific serotypes of S. pneumoniae against which the patient has not previously been vaccinated, or may provide additional protection against serotypes of S. pneumoniae against which the patient has been previously vaccinated, or may provide protection against serotypes of S. pneumoniae against which the patient has not previously been vaccinated, and against S. pneumoniae serotypes against which the patient has been previously vaccinated.

Thus, the invention relates to a method for inducing an immune response, vaccination or inducing a protective immune response against S. pneumoniae in a patient, comprising administering a multivalent immunogenic composition to the patient, wherein the composition contains several S. pneumoniae polysaccharide-protein conjugates, wherein the polysaccharide-protein conjugates include a capsular a polysaccharide of the S. pneumoniae serotype conjugated to a carrier protein, and the patient has previously been vaccinated against S. pneumoniae. In embodiments of this aspect of the invention, the multivalent immunogenic composition can be any multivalent immunogenic composition disclosed herein. In specific embodiments of the methods of the invention, the multivalent immunogenic composition is administered to a patient who has previously been treated with a multivalent pneumococcal vaccine. A multivalent immunogenic vaccine can be any vaccine that is indicated for the prevention of pneumococcal disease caused by more than one serotype of S. pneumoniae.

In specific embodiments of the above method, the patient has previously been treated with a multivalent pneumococcal vaccine that is indicated for the prevention of pneumococcal infection caused by S. pneumoniae serotypes selected from the group consisting of

- 34 043489

a) 4, 6B, 9B, 14, 18C, 19F and 23F;

b) 4, 6B, 9B, 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, 9B, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F and 33F;

e) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 22F, 33F, 2, 8, 9N, 10A, PA, 12F,

15B, 17F and 20; And

f) 4, 6B, 9B, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F, 33F, 8, 10A, IA, 12F and 15B.

In specific embodiments of the above method, the polyvalent pneumococcal vaccine contains capsular polysaccharides of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 22F, 33F, 2, 8, 9N, 10A, 11A, 12F, 15B, 17F and 20A. In specific embodiments of the above method, the polyvalent pneumococcal vaccine contains capsular polysaccharides of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 22F, 33F, 2, 8, 9N, 10A, 11A, 12F, 15B, 17F and 20. In specific embodiments of the above method, the polyvalent pneumococcal vaccine contains capsular polysaccharides of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 22F, 33F, 2, 8, 9N, 10A, 11A, 12F, 15B, 17F and 20B.

In specific embodiments of the above method, the multivalent pneumococcal vaccine contains several polysaccharide-protein conjugates containing a S. pneumoniae serotype polysaccharide conjugated to a carrier protein. In other embodiments, the multivalent pneumococcal vaccine contains multiple S. pneumoniae capsular polysaccharides that are not conjugated to a carrier protein.

In additional embodiments of the above method, the patient has previously been treated with PREVNAR® 13 (pneumococcal 13-valent conjugate vaccine [diphtheria toxin protein CRM197], Pfizer, Inc., Philadelphia, PA, USA).

In other embodiments of the above method, the patient has previously been treated with PNEUMOVAX® 23 (pneumococcal multivalent vaccine, Merck & Co., Inc., Kenilworth, NJ, USA).

In other embodiments of the above method, the patient has previously been treated with SYNFLORIX™ (pneumococcal polysaccharide-conjugate vaccine (adsorbed), GlaxoSmithKline Biologicals s.a., Rixensart, Belgium).

In embodiments of the above method, the multivalent immunogenic composition of the invention is administered to a patient at any time after the patient has received the multivalent pneumococcal vaccine, in accordance with a treatment regimen prescribed by a healthcare professional, such as a physician. In specific embodiments, the multivalent immunogenic composition of the invention is administered to a patient 1 month to 5 years after the patient has received the multivalent pneumococcal vaccine, alternatively 1 month to 1 year, 1 month to 2 years, 1 month to 3 years, from 1 month to 4 years, from 1 to 6 months, from 2 to 6 months, from 2 months to 1 year, from 1 year to 5 years, from 6 months to 5 years, from 6 months to 4 years, from 6 months to 3 years, 6 months to 2 years, 6 months to 1 year, 1 year to 4 years, 1 year to 3 years, or 1 year to 2 years after the patient has received the multivalent pneumococcal vaccine. In other embodiments, the multivalent immunogenic composition is administered to the patient after about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, approximately 11 months, approximately 1 year, approximately 1.25 years, approximately 1.5 years, approximately 1.75 years, approximately 2 years, approximately 2.25 years, approximately 2.5 years, approximately 2.75 years, approximately 3 years, approximately 3.25 years, approximately 3.5 years, approximately 3.75 years, approximately 4 years, approximately 4.25 years, approximately 4.5 years, approximately 4.75 years or approximately 5 years after the patient received the polyvalent pneumococcal vaccine.

In further embodiments, the invention provides a method of (1) inducing an immune response in a human patient;

(2) inducing a protective immune response in a human patient;

(3) vaccinating the human patient against S. pneumoniae infection; or (4) reducing the likelihood of S. pneumoniae infection in a human patient, the method comprising administering a multivalent immunogenic composition of the invention and administering a multivalent pneumococcal vaccine to the patient in any order. A multivalent pneumococcal vaccine can be any vaccine indicated for the prevention of pneumococcal disease caused by more than one serotype of S. pneumoniae.

In specific embodiments of the above method, the patient is treated with a multivalent immunogenic composition of the invention and a multivalent pneumococcal vaccine that is indicated for the prevention of pneumococcal infection caused by S. pneumoniae serotypes selected from the group consisting of

- 35 043489

a) 4, 6B, 9B, 14, 18C, 19F and 23F;

b) 4, 6B, 9B, 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, 9B, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F and 33F;

e) 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 22F, 33F, 2, 8, 9N, 10A, PA, 12F,

15B, 17F and 20; And

f) 4, 6B, 9B, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F, 33F, 8, 10A, PA, 12F and 15B.

In specific embodiments of the above method, the polyvalent pneumococcal vaccine contains capsular polysaccharides of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 22F, 33F, 2, 8, 9N, 10A, 11A, 12F, 15B, 17F and 20A.

In specific embodiments of the above method, the multivalent pneumococcal vaccine contains several polysaccharide-protein conjugates containing a S. pneumoniae serotype polysaccharide conjugated to a carrier protein. In other embodiments, the multivalent pneumococcal vaccine contains multiple S. pneumoniae capsular polysaccharides that are not conjugated to a carrier protein.

In additional embodiments of the above method, the patient is treated with the multivalent immunogenic composition of the invention and PREVNAR® 13 (pneumococcal 13-valent conjugate vaccine (Diphtheria CRM197 Protein), Pfizer, Inc., Philadelphia, PA, USA), in any order. In one embodiment, the patient is first administered PREVNAR® 13, and then is administered a multivalent immunogenic composition according to the invention. In alternative embodiments, the patient is first administered a multivalent immunogenic composition of the invention and then PREVNAR® 13 is administered.

In additional embodiments of the above method, the patient is treated with the multivalent immunogenic composition of the invention and PNEUMOVAX® 23 (pneumococcal multivalent vaccine, Merck & Co., Inc., Kenilworth, NJ, USA) in any order. In one embodiment, the patient is first administered PREVNAR® 23, and then is administered a multivalent immunogenic composition according to the invention. In alternative embodiments, the patient is first administered a multivalent immunogenic composition of the invention and then PNEUMOVAX®23 is administered.

In other additional embodiments of the above method, the patient is treated with the multivalent immunogenic composition of the invention and SYNFLORIX™ (pneumococcal polysaccharide conjugate vaccine (adsorbed), GlaxoSmithKline Biologicals s.a., Rixensart, Belgium), in any order. In one embodiment, the patient is first administered SYNFLORIX™, and then is administered a multivalent immunogenic composition of the invention. In an alternative embodiment, the patient is first administered a multivalent immunogenic composition of the invention and then administered SYNFLORIX™.

In some embodiments of the above method, the multivalent immunogenic composition and the multivalent pneumococcal vaccine are administered simultaneously. As used herein, simultaneous administration is not limited to dosing two compositions simultaneously and includes sequential (one after the other) administration in any order. In some embodiments, the multivalent immunogenic composition and the multivalent pneumococcal vaccine are administered intramuscularly or subcutaneously to separate anatomical sites, such as two different arms.

In some embodiments of the above method, the time interval between administration of the multivalent immunogenic composition of the invention and the multivalent pneumococcal vaccine is from 4 weeks to about 1 year. In alternative embodiments, the time interval is from about 1 month to about 5 years.

In one embodiment, the patient is administered first a multivalent pneumococcal vaccine, and then a multivalent immunogenic composition according to the invention. In alternative embodiments, the patient is first administered a multivalent immunogenic composition of the invention and then is administered a multivalent pneumococcal vaccine.

Also provided is a method of inducing an immune response, vaccinating, or inducing a protective immune response against S. pneumoniae in a patient, comprising (1) administering a multivalent immunogenic composition to a patient comprising multiple S. pneumoniae polysaccharide-protein conjugates, each of the polysaccharide-protein conjugates comprising a capsular polysaccharide S. pneumoniae serotype conjugated to carrier protein;

(2) waiting for a predetermined period of time to expire; and (3) administering a multivalent pneumococcal vaccine to the patient.

In this method, the multivalent immunogenic composition may contain any combination of S. pneumoniae polysaccharide-protein conjugates disclosed herein, wherein the multivalent pneumococcal vaccine may be any vaccine indicated for the prevention of diseases caused by more than one serotype of S. pneumoniae.

- 36 043489

The invention also relates to a method of inducing an immune response, vaccination or inducing a protective immune response against S. pneumoniae in a patient, comprising (1) administering a polyvalent pneumococcal vaccine to the patient;

(2) waiting for a predetermined period of time to expire; and (3) administering a multivalent immunogenic composition to a patient, the composition comprising multiple S. pneumoniae polysaccharide-protein conjugates, wherein each of the polysaccharide-protein conjugates comprises a capsular polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein.

In this method, the multivalent immunogenic composition may comprise any combination of S. pneumoniae polysaccharide-protein conjugates disclosed herein, wherein the multivalent pneumococcal vaccine may be any vaccine indicated for the prevention of disease caused by more than one serotype of S. pneumoniae.

In some embodiments of the methods described above, the multivalent pneumococcal vaccine comprises multiple S. pneumoniae polysaccharide-protein conjugates, wherein the polysaccharide-protein conjugates comprise a capsular polysaccharide of the S. pneumoniae serotype conjugated to a carrier protein. In alternative embodiments, the multivalent pneumococcal vaccine contains S. pneumoniae capsular polysaccharides that are not conjugated to a carrier protein.

In any embodiments of the methods of the invention (ie, any of the methods disclosed herein), the method may further comprise administering to the patient one or more additional doses of the multivalent immunogenic composition of the invention. In such methods, the patient may have already received a multivalent pneumococcal vaccine prior to receiving the first dose of the multivalent immunogenic composition of the present invention, or may not have been vaccinated against S. pneumoniae prior to receiving the multivalent immunogenic composition of the invention. Thus, in one embodiment, a patient who has received a multivalent pneumococcal vaccine indicated for the prevention of pneumococcal disease caused by S. pneumoniae is administered two or more doses of a multivalent immunogenic composition of the invention. In alternative embodiments, a patient who has not previously been treated with any vaccine indicated for the prevention of pneumococcal disease is administered two or more doses of a multivalent immunogenic composition of the invention.

In embodiments of the method described above, two or more doses are the same multivalent immunogenic composition of the invention. In alternative embodiments, the two or more doses are different multivalent immunogenic compositions of the invention.

In specific embodiments of any of these methods, the patient is administered two, three or four doses of the multivalent immunogenic composition of the invention. In specific embodiments, the patient is immunocompromised (eg, immunosuppressed following a stem cell transplant), and the number of doses is three.

In some embodiments, the time interval between administration of each dose of the multivalent immunogenic composition of the invention ranges from about 4 weeks to about 1 year. In an alternative embodiment, the time interval between administration of each dose of the multivalent immunogenic composition of the invention ranges from about 1 month to about 5 years.

In embodiments of any of the methods of the invention, the patient being treated with the composition(s) of the invention is a human. In certain embodiments, the human patient is an infant (about 12 to 24 months) or a young child (about 2 to 5 years). The compositions of the present invention are also suitable for administration to older children, adolescents and adults (e.g., 18 to 45 years of age, 18 to 50 years of age, 18 to 55 years of age, 18 to 60 years of age, or from 18 to 65 years old). In other embodiments of any of the methods of the invention, the patient's age ranges from 2 to 18 years. In other embodiments of any of the methods of the invention, the patient is 18 years of age or older.

In other embodiments of the methods of the invention, the human patient is an elderly patient. In some embodiments of any of the methods of the invention, the patient is 50 years of age or older. In some embodiments of any of the methods of the invention, the patient is 55 years of age or older. In some embodiments of any of the methods of the invention, the patient is 60 years of age or older. In other embodiments of any of the methods of the invention, the patient is 65 years old. In additional embodiments of any of the methods of the invention, the patient is 70 years of age or older.

In some embodiments of any of the methods of the invention, the patient to be treated with the immunogenic composition of the invention is immunocompromised.

In some embodiments of any of the methods of the invention, the multivalent immunogenic composition is administered concomitantly with an influenza vaccine. In some embodiments, the influenza vaccine is an older adult influenza vaccine, a high dose influenza vaccine intended for older adults, such as people 65 years of age and older.

The invention relates to a method of inducing a protective immune response in a patient against a pneumococcal infection, comprising the step of administering to the patient an immunologically effective amount of any of the multivalent immunogenic pneumococcal polysaccharide-protein conjugate compositions disclosed herein. The optimal amount of components for a particular vaccine (ie, multivalent immunogenic composition) can be determined through standard studies involving observation of appropriate immune responses in subjects. For example, in another embodiment, the dose for human vaccination is determined by extrapolating data obtained from animal experiments to humans. In another embodiment, the dose is determined empirically.

The methods of the invention can be used to prevent and/or reduce the severity of the major clinical manifestations of syndromes caused by microbes, such as S. pneumonia, including both invasive infections (meningitis, pneumonia and bacteremia) and non-invasive infections (acute otitis media and sinusitis).

Administration of the compositions of the invention may include one or more of the following: intramuscular, intraperitoneal, intradermal or subcutaneous injection; or introduction through the mucous membrane into the oral/digestive or respiratory system or genitourinary tract. In one embodiment, intranasal administration is used to treat pneumonia or otitis media (since nasopharyngeal carriage of pneumococci can be more effectively prevented, resulting in attenuation of the infection at a very early stage). In specific embodiments, the compositions of the invention are administered to a patient intramuscularly or subcutaneously.

All publications referred to herein are incorporated by reference to describe and disclose methods and materials that can be used in connection with the present invention.

While various embodiments of the invention have been described with reference to the accompanying drawings, it is to be understood that the invention is not limited to these precise embodiments and that various changes and modifications can be made by one skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims. inventions.

The following examples illustrate but do not limit the invention.

Example 1. Preparation of capsular polysaccharides of S. Pneumoniae.

Methods for culturing pneumococci are well known in the art. See, for example, Chase, 1967, Methods of Immunology and Immunochemistry, 1:52. Methods for producing pneumococcal capsular polysaccharides are also well known in the art. See, for example, European patent no. EP 0497524 B1. The process described below generally corresponds to the method described in European patent No. EP 0497524 B1 and is generally applicable to all pneumococcal serotypes.

Isolates of pneumococcal strains serotypes 6C, 23B, and 31 were obtained from the Centers for Disease Control and Prevention (Atlanta, GA). Strains of serotypes 3, 8, 10A, 11A, 12F, 15B, 22F and 33F were obtained from the University of Pennsylvania (Dr. Robert Austrian). Strains serotypes 17F and 19A were obtained from the FDA Office of Biologics (Dr. John Robbins). Serotype 7F was obtained from the State University of New York, State of New York Medical Center (Dr. Gerald Schiffman). The pneumococcal serotype isolates listed above were obtained from the American Type Culture Collection (Manassas, VA). If necessary, subtypes were differentiated based on the Quellung reaction using specific antiserum. See, for example, US Pat. No. 5,847,112. Additionally, clonal isolation of the resulting isolates was performed by plating sequentially in two stages on agar plates consisting of an animal-free medium containing soy peptone, yeast extract and glucose without hemin. For serotype 7F, the agar plates used also contained hemin. Clonal isolates of each serotype were further propagated in liquid culture using animal-free media containing soy peptone, yeast extract, HEPES, sodium chloride, sodium bicarbonate, potassium phosphate, glucose, and glycerol to prepare preliminary master cell banks.

The production of each serotype of pneumococcal polysaccharide consisted of cell expansion and batch fermentation followed by chemical inactivation before subsequent purification. A bank of thawed cells of each serotype from a tube was propagated using a shake flask or culture bottle containing pre-sterilized animal-free growth medium containing soy peptone or soy peptone ultrafiltrate, yeast extract or yeast extract ultrafiltrate, HEPES, sodium chloride, sodium bicarbonate, phosphate potassium and glucose. The expansion cell culture was grown in a sealed shake flask or bottle to minimize gas exchange under controlled temperature and agitation conditions. For serotypes 3, 7F, 8, 9N, 10A, 11A, 12F, 15B, 17F, 19A, 20, 22F, and 33F, thawed cell banks from tubes were propagated using a fermenter containing the same media. Temperature, pH, pressure, and agitation were controlled during cell proliferation of these serotypes. Air flow in surface

- 38 043489 layer was also controlled, since bubbling was not used. After reaching the desired culture density, measured by optical density at 600 nm, part of the cell expansion culture was transferred to a production fermenter containing pre-sterilized growth medium without animal components containing soy peptone or soy peptone ultrafiltrate, yeast extract or yeast extract ultrafiltrate, chloride sodium, potassium phosphate and glucose. Temperature, pH, pressure and stirring were controlled. The air flow in the surface layer was also controlled since bubbling was not used.

Batch fermentation was stopped by adding a chemical inactivating agent, phenol, when virtually all the glucose had been used. Pure phenol was added to a final concentration of 0.8-1.2% to inactivate the cells and release the capsular polysaccharide from the cell wall. Primary inactivation was carried out for a certain time in a fermenter, in which temperature and stirring were also controlled. After the initial inactivation, the batch was transferred to another vessel, where it was kept for an additional specified time at controlled temperature and stirring to ensure complete inactivation. Inactivation was confirmed either by plate colony counting methods or by checking the phenol concentration and the specified time. The inactivated broth was then purified.

Example 2: Purification of pneumococcal polysaccharides.

The purification process of pneumococcal polysaccharides consisted of several processes: centrifugation, deep filtration, concentration/diafiltration and sedimentation. All procedures were performed at room temperature unless otherwise stated.

The inactivated S. pneumoniae culture broth from the fermenter was flocculated with a cationic polymer (such as BPA-1000, TRETOLITE® (Baker Hughes Inc., Houston, TX), Spectrum 8160, poly(ethyleneimine), and Millipore pDADMAC). Cationic polymers bind to admixtures of proteins, nucleic acids and cellular debris. After the flocculation step and maturation period, the flocculated solids were removed by centrifugation and several deep filtration steps. The clarified broth was concentrated and diafiltered using a 100 to 500 kDa MWCO (molecular weight cutoff) filter. Diafiltration was carried out using Tris, MgCl ₂ buffer and sodium phosphate buffer. Diafiltration removed residual nucleic acid and protein.

Removal of other impurities was carried out by reprecipitation of the polysaccharide in sodium acetate and phenol with denatured alcohol and/or isopropanol. During the phenol precipitation step, sodium acetate in sodium phosphate buffer and phenol (liquefied phenols or solid phenols) were loaded into the diafiltered retentate. Then alcohol fractionation of the polysaccharide was performed in two stages. In the first stage, a low percentage of alcohol was added to the preparation to precipitate cellular debris and other unwanted impurities, while the crude polysaccharide remained in solution. Impurities were removed by centrifugation followed by a deep filtration step. The polysaccharide was then removed from solution by adding more isopropanol or denatured alcohol to the batch. The precipitated polysaccharide precipitate was recovered by centrifugation, triturated and dried into powder and stored frozen at -70°C.

Example 3. Analysis of the structural identity of some pneumococcal serotypes by NMR.

Samples for NMR analysis were prepared by dissolving the polysaccharide powder in deuterium oxide (D2O) containing 0.01% dimethyl sulfoxide (DMSO) and 0.01% sodium salt of 2,2-dimethyl-2silapentane-5-sulfonate-d ₆ . (DSS-d ₆ ) at a concentration of 5 mg of powder per ml of solution. DMSO was the internal standard used for quantitative analysis, and DSS-d ₆ was used to set the chemical shift scale to 0 ppm. A proton 1D NMR data set was acquired at 50°C and the portion of the spectrum containing the anomeric resonances was then selectively recorded as x,y coordinates in an ASCII file for analysis using a Microsoft Excel workbook. The Y coordinates (i.e., spectral profile) were then compared with the spectral profiles of capsular bacterial polysaccharides in a reference database. Reference profiles were obtained in a similar manner on selected preparations of each serotype, which were then designated as the reference batch. Pairwise comparisons were performed between the y-values of the sample and the reference spectra to obtain the correlation coefficient (p _x , _y ) as a measure of the similarity between the spectra. A value >0.95 for any of the reference spectra was taken as a positive identification of the polysaccharide structure.

In fig. 1-4 show 600 MHz one-dimensional 1H NMR spectra of capsular polysaccharides of S. pneumonia serotypes 6C, 15A, de-O-acetylated 15B and 35B, respectively. The 1H NMR identity regions used to identify S. pneumonia serotypes 6C, 15A, de-O-acetylated 15B, and 35B are presented in FIG. 5-8 respectively.

Structure of capsular polysaccharides of de-O-acetylated 15B and 15C S. pneumonia serotypes.

Immunogenicity studies were performed by administering PCV21 to New Zealand White rabbits (see Example 43 below). In these studies, the de-O-acetylated polysaccharide of serotype 15B was used instead of serotype 15C in the polyvalent composition. To confirm that de-O-39 043489 acetylated polysaccharide 15B is equivalent to polysaccharide serotype 15C,

NMR studies.

Structural differences between capsular polysaccharide serotypes appear as differences in chemical shift in the NMR spectrum. The anomeric region (approximately 4.4 to 6.0 ppm) is sensitive to each structural feature in the polysaccharide repeat unit. Differences in stereochemistry, monosaccharide composition, O-acetylation, and gliosidic linkage influence the chemical shifts of the anomeric signals, leaving this region of the spectrum unique to each serotype. For the de-Oacetylated polysaccharide serotype 15B, the complete 1H NMR spectrum was identical to the 1H NMR spectrum of the serotype 15C polysaccharide, indicating that the repeat unit of both polysaccharides consists of the same monosaccharide substituents and gliosidic linkage sites (see Figs. 9B-9C and 1OV-1OS). It is also clearly shown that the de-O-acetylation step removes the O-acetate group present in polysaccharide 15B and that essentially no O-acetate groups are observed (see FIGS. 10A-10F).

Example 4. Preparation of a serotype 3 conjugate by conjugation in DMSO for the study of a polyvalent vaccine.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The homogenization pressure and the number of passes through the homogenizer were controlled and maintained at 380 bar/5 passes.

The reduced size polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.25 mol of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target polysaccharide activation level (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 12 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 2 mg Ps/ml with a sucrose concentration of 10% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 1.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.3 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 1 h at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1.6 h at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

- 40 043489

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 1

Characteristics of conjugate 3, obtained by conjugation in DMSO, for the study of a monovalent vaccine

Mn/Mw of oxidized Ps Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 186/253 kDa 862/1468 kDa 1.16 8.2 <1% <1%

Example 5. Preparation of a serotype 6C conjugate by conjugation in DMSO for the study of a monovalent vaccine.

The polysaccharide was dissolved, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in DMSO. The polysaccharide and CRM197 dissolved solutions were then combined and conjugated as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Oxidation of polysaccharide.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The filtered dissolved polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.10 mol sodium metaperiodate per mole polysaccharide repeat unit to achieve the target polysaccharide activation level (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained by expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2- micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 2.2 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.4 were achieved. The mass ratio was chosen to control the ratio of polysaccharide and CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was performed for 15 h at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 3 h at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, using a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate portion was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60 °C.

- 41 043489

table 2

Characteristics of the 6C conjugate obtained by conjugation in DMSO for the study of a monovalent vaccine

Mn/Mw of oxidized Ps Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 144/229 kDa 2038/4182 kDa 1.20 9.2 3.1% 5.8%

Example 6. Preparation of a serotype 6C conjugate by conjugation in DMSO for the study of a polyvalent vaccine.

The polysaccharide was dissolved, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in DMSO. The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage such as pH, temperature, concentration and time were controlled to obtain conjugates with the required characteristics.

Oxidation of polysaccharides.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45-micron filter. The filtered dissolved polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate. The amount of sodium metaperiodate added was 0.10 mol of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by difiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharides with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 1.9 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.4 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol polysaccharide repeat unit) was added and conjugation was performed for 15 hours at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 3 h at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

- 42 043489

Table 3

Characteristics of the 6C conjugate obtained by conjugation in DMSO for the study of a monovalent vaccine

Mn/Mw oxidized PS Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 144/229 kDa 2104/5006 kDa 1.11 8.4 4.9% 2.2%

Preparation of serotype 6A conjugate for the study of monovalent conjugate obtained by conjugation in DMSO.

The polysaccharide was dissolved and chemically activated. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in DMSO. The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The homogenization pressure and the number of passes through the homogenizer were controlled and maintained at 200 bar/5 passes. The reduced size polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.10 mol sodium metaperiodate per mole polysaccharide repeat unit to achieve the target polysaccharide activation level (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 1.5 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.4 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 15 h at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 3 h at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

- 43 043489

Table 3A

Characteristics of conjugate 6A, obtained by conjugation in DMSO, for the study of a monovalent vaccine

Mn/Mw oxidized PS Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 238/278 kDa 2565/6310 kDa 1.021 9.2 3% 5%

Example 7. Preparation of a serotype 7F conjugate by conjugation in DMSO for the study of a polyvalent vaccine.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The homogenization pressure and the number of passes through the homogenizer were controlled and maintained at 150 bar/7 passes.

The reduced size polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.24 moles of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 4 hours at 4°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 2.6 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 4 hours at 22°C.

Reduction with sodium borohydride

Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 3 h at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

- 44 043489

Table 4

Characteristics of the 7F conjugate obtained by conjugation in DMSO for the study of a polyvalent vaccine

Mn/Mw of oxidized Ps Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 76/118 kDa 1817/4026 kDa 1.55 6.7 <1% 3.2%

Example 8. Preparation of serotype 8 conjugate by conjugation in DMSO for polyvalent vaccine research.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The homogenization pressure and the number of passes through the homogenizer were controlled and maintained at 600 bar/5 passes.

The reduced size polysaccharide was concentrated and diafiltered against water through a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.18 mol of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target polysaccharide activation level (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 4 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 5 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 4.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 2 h at 22°C. Sodium cyanoborohydride was not added to the conjugation reaction because it was noted that addition of sodium cyanoborohydride during the conjugation reaction could result in irreversible precipitation of serotype 8. Omitting sodium cyanoborohydride from the reaction avoided precipitation without significantly affecting the performance of the conjugate.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 h at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through 300 kDa NMWCO

- 45 043489 ultrafiltration membrane with tangential flow.

A batch of retentate was filtered through a 0.2 micron filter (pre-filtered through a 0.5 micron filter), then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C.

Table 5

Characteristics of the serotype 8 conjugate obtained by conjugation in DMSO for the study of a polyvalent vaccine

Mn/Mw oxidized PS Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 110/128 kDa 1479/2196 kDa 1D7 10.3 3.7% <1%

Example 9. Preparation of a serotype 9N conjugate by conjugation in DMSO for the study of a polyvalent vaccine.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The homogenization pressure and the number of passes through the homogenizer were controlled at 250 bar/5 passes.

The reduced size polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.16 mol of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target polysaccharide activation level (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 4 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 3.25 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 1 h at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, 30 kDa NMWCO through a tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through 300 kDa NMWCO

- 46 043489 ultrafiltration membrane with tangential flow.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 6

Characteristics of the serotype 9N conjugate obtained by conjugation in DMSO for the study of a polyvalent vaccine

Mn/Mw of oxidized Ps Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 195/226 kDa 1407/3134 kDa 1.33 10.1 1.9% <1%

Example 10. Preparation of a serotype 10A conjugate by conjugation in DMSO for the study of a monovalent vaccine.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. Homogenization pressure and number of passes through the homogenizer were controlled at 200 bar/5 passes then 600 bar/5 passes to achieve the target molecular weight.

The reduced size polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.15 mol of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target polysaccharide activation level (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 5.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 2.0 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 4 hours at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 h at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine at 150 mM

- 47 043489 sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 7

Characteristics of the serotype 10A conjugate obtained by conjugation in DMSO for the study of a monovalent vaccine

Mn/Mw Mn/Mw Ps:Pr Lysine consumption Free Free oxidized PS conjugate (mol/mol CRM197) Ps/general PS protein/total protein 76/111 kDa 5137/7061 kDa 1.13 10.1 <1% 15%

Example 11. Preparation of a serotype 10A conjugate by conjugation in DMSO for the study of a polyvalent vaccine.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The homogenization pressure and the number of passes through the homogenizer were controlled and maintained at 600 bar/5 passes.

The reduced size polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.16 mol of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target polysaccharide activation level (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 4.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.75 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 4 hours at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 h at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine at 150 mM

-48043489 sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 8

Characteristics of the serotype 10A conjugate obtained by conjugation in DMSO for the study of a polyvalent vaccine

Mn/Mw of oxidized Ps Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 94/130 kDa 1540/3000 kDa 1.54 7.7 8.5% <1%

Example 12. Preparation of serotype 11A conjugate by conjugation in DMSO for polyvalent vaccine research.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The size of the dissolved polysaccharide was reduced by acid hydrolysis by adding acetic acid to 200 mM, incubating at 92°C for 75 min, then neutralizing by adding cold potassium phosphate buffer, pH 7, to 400 mM.

The reduced size polysaccharide was concentrated and diafiltered against water through a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.13 mol of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 5 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 3.5 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 4 hours at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 h at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine at 150 mM

- 49 043489 sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 9

Characteristics of a serotype IA conjugate obtained by conjugation in DMSO for the study of a polyvalent vaccine

Mn/Mw of oxidized Ps Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 63/96 kDa 1584/2804 kDa 0.89 7.4 2.2% 2.8%

Example 13. Preparation of a serotype 12F conjugate by conjugation in DMSO for the study of a polyvalent vaccine.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The size of the dissolved polysaccharide was reduced by acid hydrolysis by adding acetic acid to 200 mM, incubating at 90°C for 45 min, then neutralizing by adding cold potassium phosphate buffer, pH 7, to 400 mM.

The reduced size polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.26 moles of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 5 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg PS/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 3.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 4 hours at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 3 h at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine at 150 mM

-50043489 sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 10

Characteristics of the serotype 12F conjugate obtained by conjugation in DMSO for the study of a polyvalent vaccine

Mn/Mw oxidized PS Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 54/73 kDa 1766/3119 kDa 1.20 9.6 1.3% 1.5%

Example 14.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The homogenization pressure and the number of passes through the homogenizer were controlled and maintained at 200 bar/5 passes.

The reduced size polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 1.75 moles of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target polysaccharide activation level (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 20 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 6.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 2.0 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 2.5 h at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 h at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through 300 kDa NMWCO

- 51 043489 ultrafiltration membrane with tangential flow.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 11

Characteristics of the serotype 15A conjugate obtained by conjugation in DMSO for the study of a polyvalent vaccine

Mn/Mw oxidized PS Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 158/200 kDa 6949/9235 kDa 1.05 9.7 10% 5.0%

Example 15. Preparation of serotype 15A conjugate by conjugation in an aqueous medium to study 15A/B/C cross-protection.

The polysaccharide was dissolved, reduced in size, chemically activated, and buffer exchanged by ultrafiltration. Purified CRM197 was then conjugated to the activated polysaccharide using nickel chloride in an aqueous reaction mixture, and the resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The homogenization pressure and the number of passes through the homogenizer were controlled and maintained at 200 bar/5 passes to achieve the target molecular weight. The reduced size polysaccharide was then concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.45 mol of sodium metaperiodate per mole of polysaccharide repeat unit. The oxidation reaction lasted 20 hours at 22°C. The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C. Further activation was achieved by heating the ultrafiltered product to 22°C and adjusting the pH to 5. Activation was performed by adding a 100 mM sodium metaperiodate solution with a concentration of 2.0 mol sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target level of polysaccharide activation (moles of aldehyde per mole repeating unit of a polysaccharide). The oxidation reaction lasted 20 hours at 22°C. The second activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

A solution of oxidized polysaccharide was mixed with water and 1.5 M potassium phosphate, pH 6.0. The pH of the buffer was chosen to improve the stability of the activated polysaccharide during the conjugation reaction. Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was filtered through a 0.2 micron filter and combined with a buffered polysaccharide solution at a polysaccharide to CRM197 weight ratio of 0.6. The mass ratio was chosen to control the ratio of polysaccharide and CRM197 in the resulting conjugate. The polysaccharide and phosphate concentrations were 9.75 g/L and 100 mM, respectively. The polysaccharide concentration was chosen to control the size of the resulting conjugate. Nickel chloride was added to a concentration of approximately 2 mM using a 100 mM nickel chloride solution. Sodium cyanoborohydride (2 moles per mole of polysaccharide repeat unit) was added. The conjugation reaction was carried out for 148 h at 10°C to maximize polysaccharide and protein consumption.

Reduction with sodium borohydride.

After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.0 g/L, cooled to 2-8°C and filtered through a 1.2 μm filter. The batch was diafiltered against 100 mM potassium phosphate, pH 7.0, at 2-8°C through a 100 kDa NMWCO tangential flow ultrafiltration membrane. The batch recovered from the retentate was then diluted to a concentration of approximately 2.0 g polysaccharide/L, and the pH was adjusted by adding 1.2 M sodium bicarbonate, pH 9.4. Sodium borohydride (1 mol per mole of polysaccharide repeat unit) was added. Then 1.5 M potassium phosphate, pH 6.0, was added.

- 52 043489

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane. Polysorbate 20 was added to the retentate batch to a concentration of 0.05% (w/v), then the batch was filtered through a 0.2 micron filter (pre-filtered through a 0.5 micron filter).

The batch was adjusted to a polysaccharide concentration of 1.0 g/L by adding 10 mM L-histidine buffer with 150 mM sodium chloride, pH 7.0, in the presence of 0.03% (w/v) polysorbate 20. The batch was aliquoted and frozen at <-60°C.

Table 12

Characteristics of the serotype 15A conjugate obtained by conjugation in an aqueous medium for the study of 15A/B/C cross-protection

Mn/Mw oxidized Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol Free Ps/general Free protein/total PS CRM197) PS protein 118/154 kDa 465/695 kDa 0.99 4.5 9.6% <1%

Example 16. Preparation of serotype 3 conjugate by conjugation in DMSO for polyvalent vaccine research.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The homogenization pressure and the number of passes through the homogenizer were controlled and maintained at 200 bar/5 passes.

The reduced size polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 1.75 moles of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target polysaccharide activation level (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 20 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO, which were prewarmed to 34°C. Sodium chloride was added to the polysaccharide solution to a concentration of 50 mM. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 5.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 2.0 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 2 h at 34°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 h at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 13

Characteristics of the serotype 15A conjugate obtained by conjugation in DMSO for the study of a polyvalent vaccine

Mn/Mw oxidized PS Mn/Mw of the conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 214/231 kDa 1628/3518 kDa 1.57 7.3 17% 7.8%

Example 17. Preparation of serotype 15B conjugate by conjugation in DMSO for 15A/B/C cross-protection studies.

The polysaccharide was dissolved, reduced in size, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and dissolved in DMSO. The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The homogenization pressure and the number of passes through the homogenizer were controlled and maintained at 300 bar/5 passes.

The reduced size polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.20 mol sodium metaperiodate per mole polysaccharide repeat unit to achieve the target polysaccharide activation level (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 4 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 3.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 2.0 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 5 h at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 14

Characterization of the serotype 15B conjugate obtained by conjugation in DMSO for the study of 15A/B/C cross-protection

Mn/Mw of oxidized Ps Mn/Mw of the conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 203/255 kDa 2329/3881 kDa 1.67 7.9 3.6% 4.4%

Example 18. Preparation of serotype 15C conjugate by conjugation in DMSO for monovalent vaccine studies and 15A/B/C cross-protection studies.

A polysaccharide derived from Streptococcus pneumoniae serotype 15B was dissolved, adjusted to target molecular weight, mildly hydrolyzed in an alkaline environment to release O-acetyl groups, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in the size of polysaccharides, hydrolysis in an alkaline environment and oxidation.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The homogenization pressure and the number of passes through the homogenizer were controlled and maintained at 300 bar/5 passes.

The reduced size polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was heated to 60°C and sodium bicarbonate buffer, pH 9, was added to a final concentration of 50 mM. The batch was incubated with stirring for 13 hours at 60°C to release the O-acetyl groups. Potassium phosphate buffer, pH 6, was added to a final concentration of 136 mM to neutralize the pH and the solution was cooled to ambient temperature. The solution was then concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.20 mol sodium metaperiodate per mole polysaccharide repeat unit to achieve the target polysaccharide activation level (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 3.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 2.0 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to

CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 4 hours at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 h at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 15

Characteristics of serotype 15C conjugate obtained by conjugation in DMSO for monovalent vaccine studies and 15A/B/C cross-protection studies ABOUT- acetyl/Ps Mn/Mw of oxidized Ps Mn/Mw conjugate Ps: Rg Lysine consumption (mol/mol CRM197) Free Ps/general Ps Free protein/total protein Not found 182/246 kDa 1962/4019 kDa 1.3 1 4.8 3.8% 13%

Example 19. Preparation of a serotype 15C conjugate by conjugation in DMSO for the study of a polyvalent vaccine.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Size reduction, hydrolysis under alkaline conditions and oxidation of polysaccharides.

Purified pneumococcal capsular Ps serotype 15B powder was dissolved in water and filtered through a 0.45-micron filter. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The homogenization pressure and the number of passes through the homogenizer were controlled and maintained at 300 bar/5 passes.

The reduced size polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was heated to 60°C and sodium bicarbonate buffer, pH 9, was added to a final concentration of 50 mM. The batch was incubated with stirring for 13 hours at 60°C to release the O-acetyl group. Potassium phosphate buffer, pH 6, was added to a final concentration of 136 mM to neutralize the pH, and the solution was cooled to ambient temperature. The solution was then concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.2 mol sodium metaperiodate per mole polysaccharide repeat unit to achieve the target polysaccharide activation level (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

- 56 043489

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a kDa NMWCO tangential flow ultrafiltration membrane and filtered through

0.2 micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 1.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.3 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 1 h at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 h at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 16

Characteristics of the serotype 15C conjugate obtained by conjugation in DMSO for the study of a polyvalent vaccine

ABOUT- acetyl/Ps Mn/Mw of oxidized Ps Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general Ps Free protein/total protein Not found 182/246 kDa 1540/3230 kDa 1.34 6.6 9.2% 5.7%

Example 20. Preparation of a serotype 16F conjugate by conjugation in an aqueous medium for the study of a monovalent vaccine.

The polysaccharide was dissolved, reduced in size, chemically activated, and buffer exchanged by ultrafiltration. Purified CRM197 was then conjugated to the activated polysaccharide using nickel chloride in an aqueous reaction mixture, and the resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The homogenization pressure and the number of passes through the homogenizer were controlled at 200 bar/5 passes then 500 bar/5 passes to achieve the target molecular weight. The reduced size polysaccharide was then concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.15 mol sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target polysaccharide activation level (moles of aldehyde per mole

- 57 043489 polysaccharide repeating unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

A solution of oxidized polysaccharide was mixed with water and 1.5 M potassium phosphate, pH 7.0. The pH value of the buffer chosen was to improve the stability of the activated polysaccharide during the conjugation reaction. Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was filtered through a 0.2 micron filter and combined with a buffered polysaccharide solution at a polysaccharide to CRM197 weight ratio of 0.7. The mass ratio was chosen to control the ratio of polysaccharide and CRM197 in the resulting conjugate. The polysaccharide and phosphate concentrations were 7.5 g/L and 100 mM, respectively. The polysaccharide concentration was chosen to control the size of the resulting conjugate. The solution was then filtered through a 0.2 micron filter. Nickel chloride was added to a concentration of approximately 2 mM using a 100 mM nickel chloride solution. Sodium cyanoborohydride (2 moles per mole of polysaccharide repeat unit) was added. The conjugation reaction was carried out for 122 h at 22°C to maximize polysaccharide and protein consumption.

Reduction with sodium borohydride.

After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.0 g/L, cooled to 2-8°C and filtered through a 1.2 μm filter. The batch was diafiltered against 100 mM potassium phosphate, pH 7.0, at 2-8°C through a 100 kDa NMWCO tangential flow ultrafiltration membrane. The batch recovered from the retentate was then diluted to a concentration of approximately 2.0 g polysaccharide/L, and the pH was adjusted by adding 1.2 M sodium bicarbonate, pH 9.4. Sodium borohydride (1 mol per mole of polysaccharide repeat unit) was added. Then 1.5 M potassium phosphate, pH 6.0, was added.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane. Polysorbate 20 was added to the retentate batch to a concentration of 0.05% (w/v), then the batch was filtered through a 0.2 micron filter (pre-filtered through a 0.5 micron filter).

The batch was adjusted to a polysaccharide concentration of 1.0 g/L by adding 10 mM L-histidine buffer with 150 mM sodium chloride, pH 7.0, in the presence of 0.03% (w/v) polysorbate 20. The batch was aliquoted and frozen at <-60°C.

Table 17

Characteristics of the serotype 16F conjugate obtained by conjugation in an aqueous medium for the study of a monovalent vaccine

Mn/Mw of oxidized Ps Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 90/139 kDa 1860/5539 kDa 1.10 3.3 7.0% <1%

Example 21. Preparation of serotype 16F conjugate by conjugation in DMSO for polyvalent vaccine research.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The homogenization pressure and the number of passes through the homogenizer were controlled and maintained at 1000 bar/5 passes.

The reduced size polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.15 moles of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed to achieve a polysaccharide concentration of 2.0 g Ps/L g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 2 h at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 h at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 18

Characteristics of the serotype 16F conjugate obtained by conjugation in DMSO for the study of a polyvalent vaccine

Mn/Mw of oxidized Ps Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 91/177 kDa 2075/3966 kDa 1.29 11.0 <1% <1%

Example 22. Preparation of serotype 17F conjugate by conjugation in DMSO for monovalent vaccine research.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The homogenization pressure and the number of passes through the homogenizer were controlled and maintained at 200 bar/5 passes.

The reduced size polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.11 moles of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 2.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 2 h at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 h at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C

Table 19

Characteristics of the serotype 17F conjugate obtained by conjugation in DMSO for the study of a monovalent vaccine

Mn/Mw oxidized Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol Free Ps/general Free protein/total PS CRM197) PS protein 179/216 kDa 2630/4632 kDa 1.20 8.0 1.9% 4.5%

Example 23. Preparation of serotype 17F conjugate by conjugation in DMSO for polyvalent vaccine research.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The homogenization pressure and the number of passes through the homogenizer were controlled and maintained at 200 bar/5 passes.

The reduced size polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.11 moles of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 2.1 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.3 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 2 h at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 h at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 20

Characteristics of the serotype 17F conjugate obtained by conjugation in DMSO for the study of a polyvalent vaccine

Mn/Mw oxidized PS Mn/Mw of the conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general Ps Free protein/total protein 150/212 kDa 2650/4110 kDa 1.10 7.7 2.8% 1.5%

Example 24. Preparation of a serotype 19A conjugate by conjugation in DMSO for the study of a polyvalent vaccine.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The homogenization pressure and the number of passes through the homogenizer were controlled and maintained at 380 bar/5 passes.

The reduced size polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.26 moles of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 20 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed to achieve a polysaccharide concentration of 3.8 g Ps/L and a polysaccharide to CRM197 mass ratio of 1.33. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol polysaccharide repeat unit) was added and conjugation was carried out for 1.5 h at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 3 h at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 21

Characteristics of the serotype 19A conjugate obtained by conjugation in DMSO for the study of a polyvalent vaccine

Mn/Mw of oxidized Ps Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 96/186 kDa 1714/3585 kDa 1.22 9.1 7.0% 1.4%

Example 25. Preparation of a serotype 20A conjugate by conjugation in DMSO for a monovalent vaccine study.

Polysaccharide previously identified as serotype 20A (Calix et al., Biochemical, Genetic, and Serological Characterization of Two Capsule Subtypes among Streptococcus pneumoniae Serotype 20 Strains, J. Biol. Chem., 287(33):27885-27894 (2012)) , dissolved, adjusted to target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The homogenization pressure and the number of passes through the homogenizer were controlled and maintained at 200 bar/5 passes.

The reduced size polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and the pH was adjusted to 5 using sodium acetate

- 62 043489 buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.11 mol of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 2.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 6 h at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 h at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 22

Characteristics of the serotype 20A conjugate obtained by conjugation in DMSO for the study of a monovalent vaccine

Mn/Mw of oxidized Ps Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 165/215 kDa 5159/7778 kDa 1.17 6.1 <1% 6.5%

Example 26. Preparation of a serotype 20A conjugate by conjugation in DMSO for the study of a polyvalent vaccine.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The homogenization pressure and the number of passes through the homogenizer were controlled and maintained at 220 bar/5 passes.

The reduced size polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and the pH was adjusted to 5 using sodium acetate

- 63 043489 buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.16 mol of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target polysaccharide activation level (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 1.4 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 4 hours at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 23

Characteristics of the serotype 20A conjugate obtained by conjugation in DMSO for the study of a polyvalent vaccine

Mn/Mw of oxidized Ps Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 178/215 kDa 2478/3863 kDa 1.10 8.3 1.0% 1.0%

Example 27. Preparation of serotype 22F conjugate by conjugation in DMSO for polyvalent vaccine research.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The homogenization pressure and the number of passes through the homogenizer were controlled and maintained at 400 bar/5 passes.

The reduced size polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and the pH was adjusted to 5 using sodium acetate

- 64 043489 buffer to minimize reduction in polysaccharide size due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.12 mol sodium metaperiodate per mole polysaccharide repeat unit to achieve the target polysaccharide activation level (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 1.8 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.3 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 1 h at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 h at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 24

Characteristics of the serotype 22F conjugate obtained by conjugation in DMSO for the study of a polyvalent vaccine

Mn/Mw of oxidized Ps Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 170/196 kDa 1800/3970 kDa 1.13 7.0 <1% 1.1%

Example 28. Preparation of a serotype 23A conjugate by conjugation in DMSO for the study of a monovalent vaccine.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The size of the dissolved polysaccharide was reduced by acid hydrolysis by adding acetic acid to 200 mM, incubating at 90°C for 1.5 h, then neutralizing by adding cold potassium phosphate buffer, pH 7, to 400 mM.

The reduced size polysaccharide was concentrated and diafiltered against water through a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and the pH was adjusted to 5 using sodium acetate

- 65 043489 buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.20 mol sodium metaperiodate per mole polysaccharide repeat unit to achieve the target polysaccharide activation level (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 5 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Sodium chloride was added to the polysaccharide solution to a concentration of 50 mM. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 3.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 2 h at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 3 hours at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 25

Characteristics of the serotype 23A conjugate obtained by conjugation in DMSO for the study of a monovalent vaccine

Mn/Mw oxidized PS Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 70/97 kDa 2183/3837 kDa 1.20 11.8 <1% <1%

Example 29. Preparation of a serotype 23A conjugate by conjugation in DMSO for the study of a polyvalent vaccine.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The size of the dissolved polysaccharide was reduced by acid hydrolysis by adding acetic acid to 200 mM, incubating at 90°C for 1.5 h, then neutralizing by adding cold potassium phosphate buffer, pH 7, to 400 mM.

The reduced size polysaccharide was concentrated and diafiltered against water through a 5 kDa NMWCO tangential flow ultrafiltration membrane.

- 66 043489

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.20 mol sodium metaperiodate per mole polysaccharide repeat unit to achieve the target polysaccharide activation level (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 5 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 3.5 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 2 h at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 26

Characteristics of the serotype 23A conjugate obtained by conjugation in DMSO for the study of a polyvalent vaccine

Mn/Mw oxidized PS Mn/Mw of the conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 116/175 kDa 2156/4933 kDa 1.12 6.3 3.3% 2.1%

Example 30. Preparation of a serotype 23B conjugate by conjugation in DMSO for the study of a monovalent vaccine.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The homogenization pressure and the number of passes through the homogenizer were controlled and maintained at 400 bar/5 passes.

The reduced size polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

- 67 043489

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.10 mol sodium metaperiodate per mole polysaccharide repeat unit to achieve the target polysaccharide activation level (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Sodium chloride was added to the polysaccharide solution to a final concentration of 50 mM. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 5.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 2 h at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 27

Characteristics of the serotype 23B conjugate obtained by conjugation in DMSO for the study of a monovalent vaccine

Mn/Mw of oxidized Ps Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 155/179 kDa 1322/3299 kDa 1.28 6.2 13% 5.1%

Example 31. Preparation of a serotype 23B conjugate by conjugation in DMSO for the study of a polyvalent vaccine.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The homogenization pressure and the number of passes through the homogenizer were controlled and maintained at 400 bar/5 passes.

The reduced size polysaccharide was concentrated and diafiltered against water

- 68 043489 via 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.13 mol of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 5.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 4 hours at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 h at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 28

Characteristics of the serotype 23B conjugate obtained by conjugation in DMSO for the study of a polyvalent vaccine

Mn/Mw of oxidized Ps Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 172/197 kDa 1076/2514 kDa 1.26 7.4 12% 3.2%

Example 32. Preparation of serotype 24F conjugate by conjugation in DMSO for monovalent vaccine research.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The size of the dissolved polysaccharide was reduced by acid hydrolysis by adding acetic acid to 200 mM, incubating at 92°C for 50 min, then neutralizing by adding cold potassium phosphate buffer, pH 7, to 400 mM.

The reduced size polysaccharide was concentrated and diafiltered against water

- 69 043489 via 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.18 mol of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target polysaccharide activation level (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 5 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 2 mg Ps/ml with a sucrose concentration of 10% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Sodium chloride was added to the polysaccharide solution to a final concentration of 50 mM. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 1.5 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 2 h at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 h at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 29

Characteristics of the serotype 24F conjugate obtained by conjugation in DMSO for the study of a monovalent vaccine

Mn/Mw of oxidized Ps Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 56/100 kDa 2233/4875 kDa 0.94 7.3 4.4% 1.4%

Example 33. Preparation of serotype 31 conjugate by conjugation in DMSO for monovalent vaccine research.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The size of the dissolved polysaccharide was reduced by acid hydrolysis by adding acetic acid to 200 mM, incubating at 90°C for 30 min, then neutralizing by adding cold potassium phosphate buffer, pH 7, to 400 mM.

- 70 043489

The reduced size polysaccharide was concentrated and diafiltered against water through a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.16 mol of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target polysaccharide activation level (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 5 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately.

Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 4.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 1 h at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 30

Characteristics of the serotype 31 conjugate obtained by conjugation in DMSO for the study of a monovalent vaccine

Mn/Mw of oxidized Ps Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 96/119 kDa 1818/2999 kDa 1.15 9.5 <1% <1%

Example 34. Preparation of serotype 3 conjugate by conjugation in DMSO for polyvalent vaccine research.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The homogenization pressure and the number of passes through the homogenizer were controlled by maintaining 71 043489 at 400 bar/5 passes.

The reduced size polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.12 mol sodium metaperiodate per mole polysaccharide repeat unit to achieve the target polysaccharide activation level (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 3.5 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 1 hour at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 31

Characteristics of the serotype 31 conjugate obtained by conjugation in DMSO for the study of a polyvalent vaccine

Mn/Mw of oxidized Ps Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 149/186 kDa 2354/5971 kDa 1.26 8.9 <1% 1.8%

Example 35. Preparation of serotype 33F conjugate by conjugation in DMSO for polyvalent vaccine research.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was homogenized to reduce the molecular weight of Ps. The homogenization pressure and the number of passes through the homogenizer were controlled by maintaining 72043489 live at 350 bar/4 passes.

The reduced size polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.12 mol sodium metaperiodate per mole polysaccharide repeat unit to achieve the target polysaccharide activation level (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 1.8 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.75 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 4 hours at 22°C.

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 h at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 32

Characteristics of the serotype 33F conjugate obtained by conjugation in DMSO for the study of a polyvalent vaccine

Mn/Mw of oxidized Ps Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 186/220 kDa 1630/2470 kDa 1.28 8.4 1.3% 5.4%

Example 36. Preparation of a serotype 35B conjugate by conjugation in DMSO for the study of a monovalent vaccine.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

-73 043489

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation of the polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.05 mol sodium metaperiodate per mole polysaccharide repeat unit to achieve the target polysaccharide activation level (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 10 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.3 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 2.5 h at 22°C. During incubation in the conjugation reaction, sodium borohydride was added twice (0.025 mol per polysaccharide repeating unit).

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1.5 h at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 33

Characteristics of the serotype 35B conjugate obtained by conjugation in DMSO for the study of a monovalent vaccine

Mn/Mw of oxidized Ps Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 48/82 kDa 5494/7385 kDa 1.26 4.5 1.8% eleven%

Example 37. Preparation of a serotype 35B conjugate by conjugation in DMSO for the study of a polyvalent vaccine.

The polysaccharide was dissolved, adjusted to the target molecular weight, chemically activated, and buffer exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were lyophilized separately and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide and CRM197 solutions were then combined and conjugation was performed as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to obtain conjugates with the desired characteristics.

Reduction in size and oxidation of polysaccharides.

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 μm filter. The dissolved polysaccharide was concentrated and diafiltered against water through a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then heated to 22°C and adjusted to pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Activation

- 74 043489 polysaccharide was initiated by adding 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.05 mol sodium metaperiodate per mole polysaccharide repeat unit to achieve the target polysaccharide activation level (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction lasted 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water through a 5 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2-8°C.

Conjugation of polysaccharide with CRM197.

Purified CRM197, obtained from expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.2, through a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 - micron filter.

Activated polysaccharides were prepared for lyophilization at a concentration of 6 mg Ps/ml with a sucrose concentration of 5% w/v. CRM197 was prepared for lyophilization at a concentration of 6 mg Pr/ml with a sucrose concentration of 1% w/v.

The prepared solutions of Ps and CRM197 were lyophilized separately. Lyophilized Ps and CRM197 materials were redissolved separately in equal volumes of DMSO. Solutions of polysaccharide and CRM197 were mixed until a polysaccharide concentration of 5.25 g Ps/L and a mass ratio of polysaccharide to CRM197 of 3.5 were achieved. The mass ratio was chosen to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added and conjugation was carried out for 3 h at 22°C. During incubation in the conjugation reaction, sodium borohydride was added twice (0.0375 mol per polysaccharide repeating unit).

Reduction with sodium borohydride.

Sodium borohydride (2 mol per mol polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22°C. The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7, through a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and storage of the product.

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20 at 4°C through a 300 kDa NMWCO tangential flow ultrafiltration membrane.

A batch of retentate was filtered through a 0.2 micron filter, then diluted with 10 mM histidine in 150 mM sodium chloride, pH 7.0, in the presence of 0.015% (w/v) polysorbate 20, aliquoted and frozen at <-60°C .

Table 34

Characteristics of the serotype 35B conjugate obtained by conjugation in DMSO for the study of a polyvalent vaccine

Mn/Mw of oxidized Ps Mn/Mw conjugate Ps:Pr Lysine consumption (mol/mol CRM197) Free Ps/general PS Free protein/total protein 48/82 kDa 1287/2585 kDa 2.1 6.9 7.8% 4.6%

Some aqueous conjugation methods have not been described for all serotypes used in Examples 38-51. For aqueous conjugation methods that are not disclosed in this application, similar methods can be found in WO 2018/156491.

Example 38. Composition of pneumococcal conjugate vaccines.

Individual pneumococcal polysaccharide-protein conjugates prepared using various chemistries as described in the above examples were used to prepare 1-, 7-, 8-, 14-, 15-, 16-, 21-, and 31-valent pneumococcal conjugate vaccine formulations , designated PCV1, PCV7, PCV8, PCV14, PCV15, PCV16, PCV21 and PCV31, respectively.

The PCV1 vaccine drug product consisted of serotype 3 conjugated by a reductive amination mechanism in either a protic (aqueous) solvent or an aprotic (DMSO) solvent, and was formulated with 20 mM L-histidine, pH 5.8, 150 mM NaCl and 0 .1% (w/v) PS-20 with a target final vaccine concentration of 84 μg/mL (w/v) pneumococcal polysaccharide (PnPs).

The PCV7 vaccine drug product consisted of 3, 8, 9N, 10A, 11A, 15A, and 19A conjugated by a reductive amination mechanism in either a protic (aqueous) solvent or an aprotic (e.g., DMSO) solvent and was formulated with 20 mM L-histidine,

- 75 043489 pH 5.8, 150 mM NaCl and 0.1% (w/v) PS-20. Each polysaccharide-protein conjugate in PCV7 was used at a concentration of 12 μg/ml (wt/vol) pneumococcal polysaccharide (PnPs) to produce a target final vaccine concentration of 84 μg/ml PnPs.

The PCV8 vaccine drug product consisted of serotypes 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B, conjugated by reductive amination or in an aprotic solvent (eg, DMSO), and was formulated with 20 mM L-histidine, pH 5.8, 150 mM NaCl, and 0.2% (w/v) PS-20. Each polysaccharide-protein conjugate in PCV8 was used at a concentration of 4 μg/ml (w/v) pneumococcal polysaccharide (PnPs) to produce a target final vaccine concentration of 32 μg/ml PnPs.

The PCV14 vaccine drug product consisted of serotypes 3, 7F, 8, 9N, 10A, 11A, 12F, 15A, 16F, 17F, 19A, 20A, 22F and 33F, conjugated by a reductive amination mechanism in either a protic (aqueous) solvent or an aprotic (eg, DMSO) solvent and was formulated with 20 mM L-histidine, pH 5.8, 150 mM NaCl, and 0.1% (w/v) PS-20. Each polysaccharide-protein conjugate in PCV14 was used at a concentration of 6 μg/ml (w/v) pneumococcal polysaccharide (PnPs) to produce a target final vaccine concentration of 84 μg/ml PnPs.

The PCV15 vaccine drug consisted of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated in a protic (aqueous) solvent and was formulated with 20 mM L-histidine, pH 5.8, 150 mM NaCl, and 0.2% (w/v) PS-20. Each polysaccharide-protein conjugate was used at a concentration of 4 μg/ml (w/v) pneumococcal polysaccharide (PnPs), with the exception of 6B, which was used at a concentration of 8 μg/ml, resulting in a target final vaccine concentration of 64 μg/ml PnPs.

The PCV16 vaccine drug consisted of serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 17F, 20A, 23A, 23B, 24F, 31, and 35B, conjugated by a reductive amination mechanism in an aprotic solvent (e.g. DMSO) and was formulated with 20 mM L-histidine, pH 5.8, 150 mM NaCl, and 0.2% (w/v) PS-20. Each polysaccharide-protein conjugate was used at a concentration of 4 or 8 μg/ml (w/v) pneumococcal polysaccharide (PnPs) to obtain a target final vaccine concentration of 64 μg/ml or 128 μg/ml PnPs.

The PCV21 vaccine drug consisted of serotypes 3, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 20A, 22F, 23A, 23B, 24F, 31, 33F and 35B conjugated by a reductive amination mechanism in an aprotic solvent (eg, DMSO) and was formulated with 20 mM L-histidine, pH 5.8, 150 mM NaCl, and various concentrations of PS-20 as determined in each example. Each polysaccharide-protein conjugate was used at a concentration of 4 μg/ml (wt/vol) or 8 μg/ml (wt/vol) pneumococcal polysaccharide (PnPs) to obtain a target final vaccine concentration of 84 or 168 μg/ml PnPs . In some examples, PCV21 contained 250 μg [A1]/ml as aluminum phosphate.

The PCV31 vaccine drug consisted of serotypes 1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9B, 10A, 11A, 12F, 14, 15A, 15C, 16F, 17F, 18C, 19A, 19F . 20A, 22F, 23A, 23B, 23F, 24F, 31, 33F, and 35B conjugated by a reductive amination mechanism in either a protic (aqueous) solvent or an aprotic solvent (e.g., DMSO) and was formulated with 20 mM L-histidine , pH 5.8 150 mM NaCl and 0.2% PS-20. Each polysaccharide-protein conjugate was used at a concentration of 4 μg/ml (w/v) pneumococcal polysaccharide (PnPs), with the exception of 6B, which was used at a concentration of 8 μg/ml PnPs, resulting in a target final vaccine concentration of 128 μg/ml PnPs. In some examples, PCV31 contained 250 μg [A1]/ml as aluminum phosphate.

Additional monovalent PCV drug formulations were prepared using a conjugate of pneumococcal polysaccharide serotypes 6A, 6B, 6C, 10A, 15A, 15B, 15C, 16F, 17F, 20A, 23A, 23B, 24F, 31, or 35B with protein. Additional details are described in the specific examples for these compositions.

To prepare the PCV drug formulation, the required volumes of starting conjugates needed to obtain the specified final concentration (wt/vol) of pneumococcal polysaccharide (also called PnPs) were calculated based on the batch size and the concentration of the starting polysaccharide.

The composition preparation process consisted of preparing an initial mixture of conjugates with 1X-4X final concentration of mixtures of PnPs in 10-80 mM histidine, 0.0-0.8% (w/v) PS-20 and 150 mM sodium chloride, pH 5 ,8.

Histidine pH 5.8, PS-20 (if used), and 150 mM sodium chloride solution were prepared and added to the preparation vessel. Individual pneumococcal polysaccharide-protein conjugates, stored frozen, were thawed at 2-8°C and then added to the preparation vessel. While adding the polysaccharide-protein conjugate to the buffer for preparing the composition (mixture of conjugates), the contents of the vessel were stirred until a homogeneous mixture was obtained using a magnetic rod or magnetic wheel. After all additives were added and the solution was mixed, the conjugate mixture was passed through sterilizing filters and collected in a vessel with or without aluminum phosphate adjuvant. In some cases, sterilizing filters were alternated with 150 mM sodium chloride to bring the batch to the target concentration.

The compositions were poured into plastic syringes, glass syringes or vials.

Example 39: Stability assessment of pneumococcal conjugate vaccines.

PCV16 (128 μg/ml PnPs) or two formulations of the pneumococcal conjugate vaccine PCV21 (PnPs 84 or 168 μg/ml) were filled into syringes or vials. Polysaccharide-protein conjugates were prepared by a reductive amination mechanism in an aprotic solvent (e.g., DMSO) and formulated with 20 mM L-histidine, pH 5.8, 150 mM NaCl, and 0.2% (w/v) PS- 20. These drugs were maintained at 25 or 37°C for up to four weeks and at 4°C for up to twelve weeks. In some cases, syringes were placed on a platform with horizontal rotation and shaken for up to 18 hours after storage for 1 week at 4, 25, or 37°C. This simulated the shipping and handling stress that can accompany drug production and distribution. HPSEC UV/MALS/RI methods were used to evaluate drug stability of PCV16 or PCV21: high performance size exclusion chromatography (HPSEC) with two Shodex columns (803 and 806) connected together in series, and UV multi-angle light scattering (MALS) and refractive index detection (RI) to measure the concentration and molar mass of PCV drug formulations during storage. The concentration was calculated for each time interval from the peak width and then integrated over all intervals to obtain the final concentration value. Light scattering is proportional to the product of molecular weight and analyte concentration. The molar mass or molecular weight in each interval was calculated from the detector signals. The weight average (Mw) or number average (Mn) molecular weight was calculated over all intervals for the peak and recorded as the average molecular weight.

Storage may damage the protein and/or carbohydrates in the conjugate vaccine. Carbohydrate antigens that contain phosphodiester linkages in the backbone or other unstable elements in the polysaccharide repeat unit may undergo depolymerization through hydrolysis during long-term storage and exposure to temperature. In addition, during storage and exposure to physical stress, the carrier protein or carbohydrate used in the conjugate vaccine may aggregate.

As shown in FIG. 11, PCV16 and PCV21 drugs were stable for 4 weeks at 25°C and 37°C and up to 12 weeks at 4°C regardless of polysaccharide concentration. Moreover, when subjected to horizontal stirring after thermal stress, the drugs PCV16 and PCV21 were found to be stable. There was no loss of antigen during these thermal and physical stresses due to nonspecific adsorption on the walls of containers or vessels. In addition, no aggregation of the drug product that could affect its performance on the column (eg, irreversible binding to the column or failure to enter/exit the separation columns) was observed when assessing the total vaccine dose (Figure 12).

HPSEC UV/MALs/RI was used to measure the molecular weight of the polysaccharide-protein conjugate. If the polysaccharide-protein conjugates in the drug product degrade or depolymerize, a decrease in molecular weight will be apparent, and if aggregation of the drug product occurs, an increase in molecular weight will occur. This measurement provides additional characterization of the quality of the vaccine medicinal product or intermediate product. As shown in FIG. 13, drug-containing drug formulations PCV16 and PCV21, each prepared by a reductive amination scheme in an aprotic solvent (e.g., DMSO), were resistant to depolymerization or chemical carbohydrate degradation and resistant to protein aggregation within the drug product. This indicates that the drugs PCV16 and PCV21, which use polysaccharide-protein conjugates produced by a reductive amination pathway in an aprotic solvent such as DMSO, are stable at elevated temperature and exercise. The compositions demonstrate high stability in both quantitative and qualitative terms. The formulation does not lose total dose due to nonspecific adsorption to the side of containers or other surfaces, and the formulation does not degrade or aggregate, as evidenced by the stable molecular weight of the vaccine drug product (weight average molecular weight (Mw) and number average molecular weight (Mn)) .

Example 40. Effect of conjugation chemistry on the stability of pneumococcal conjugate vaccine.

To prepare 15- and 16-valent pneumococcal conjugate vaccines, designated PCV15 and PCV16, at a concentration of 64 μg/ml, separate pneumococcal polysaccharide protein conjugates were used, prepared using different chemical media, as described in the examples above. The PCV15 vaccine product contained serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated by reductive amination in a protic solvent (all aqueous) and was prepared in 20 mM L-histidine, pH 5.8, 150 mM NaCl and 0.2% w/v. PS-20 (see example 38). The drug product PCV16 contained serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 17F, 20A, 23A, 23B, 24F, 31 and 35B, conjugated by the mechanism of reductive amination in an aprotic solvent ( e.g. all DMSO) and was prepared at 20 mM

L-histidine, pH 5.8, 150 mM NaCl and 0.2% (w/v) PS-20 (see example 38). Each polysaccharide-protein conjugate was used at a concentration of 4 μg/ml (w/v) pneumococcal polysaccharide (PnPs) with the exception of 6B in PCV15, which was used at a concentration of 8 μg/ml, resulting in a final concentration of 64 μg/ml PnPs in each vaccine .

Vaccine-filled containers were placed at 4°C or 37°C for up to 1 week. Intrinsic protein fluorescence spectroscopy (IPFS) was used to evaluate the stability of the pneumococcal polysaccharide-conjugated carrier protein, CRM197, in PCVI5 or PCV16 formulations when stored for up to 1 week at 4°C and 37°C. Fluorescence emission spectra (Em 290-400 nm) of undiluted samples were collected using a Jasco FP-6500 spectrofluorimeter at room temperature in a quartz cuvette with a path length of 1 cm. An excitation wavelength of 280 nm was used with a scan speed of 100 nm/min and an excitation bandwidth and radiation 3 nm.

As shown in FIG. 14, a drug formulation containing drugs prepared by a reductive amination mechanism in an aprotic solvent (e.g., all in DMSO) showed superior physical and chemical stability compared to a vaccine that used drugs prepared in a protic solvent during conjugation by the mechanism of reductive amination (all aqueous conjugates). The fluorescence intensity of the drug product PCV15 at a concentration of 64 μg/ml PnPs, prepared by the mechanism of reductive amination in a protic solvent (all aqueous) during the conjugation process, decreased in just 16 hours at 37°C, and the maximum emission of the vaccine shifted from 332 nm to 338 nm. The PCV16 vaccine drug product at 64 μg/mL PnPs, which used drugs prepared by a reductive amination mechanism in an aprotic solvent (e.g., all in DMSO), showed no change in fluorescence intensity or 338 nm emission maximum. It should be understood that the resulting signal intensity and maximum emission obtained from IPFS measurements are due to the overall stability of the individual drug substances in the multivalent complex drug product. It has been shown that a vaccine drug product that uses drugs formulated in an aprotic solvent remains stable, while a vaccine drug product that uses drugs formulated in a protic solvent is unstable.

Without being limited to any particular theory, this stability could be due to conjugation in DMSO leading to unfolding and denaturation of the carrier protein. Conjugation to a denatured carrier protein would block the conformation in the denatured state after conjugation. If this is the case, then the study in question also indicates that the final conformation remains stable in the stress experiments described herein. Therefore, it is preferable to use a conjugate vaccine, mainly prepared in aprotic solvents, to ensure an adequate and stable vaccine drug product.

If the vaccine medicinal product contains a mixture of drugs, glycoconjugates or polysaccharide-protein conjugates obtained through conjugation in various solvents by the mechanism of reductive amination (aqueous or DMSO), then the average maximum or intensity of radiation measured for the composition of the medicinal product will be weighted due to the contribution of the emission of each aqueous polysaccharide-protein conjugate at 332 nm and the emission of each non-aqueous polysaccharide-protein conjugate at 338 nm. A change would be expected to be observed in either intensity or elevated temperature emission maxima obtained for such a mixture in a multivalent drug product that uses polysaccharide-protein conjugates, not all of which were produced by reductive amination in an aprotic solvent. Therefore, it is preferable to use a vaccine conjugate mainly prepared in aprotic solvents to ensure an adequate and stable vaccine drug product.

Example 41. Generalized immunogenicity studies in rabbits of monovalent drugs of selected serotypes.

Adult New Zealand White rabbits (NZWR, n=3/group) were immunized intramuscularly (IM) with 0.25 ml or 0.5 ml (15C only) of the respective monovalent conjugate vaccine on day 0 and day 14 (alternating sides). Monovalent pneumococcal vaccines prepared in 20 mM L-histidine, pH 5.8, 150 mM NaCl and 0.2% (w/v) PS-20 using the preparation method described in Example 38 were dosed as follows:

(1) 1 µg PnPs (6C, 10A, 15A, 16F, 17F, 20A, 23A, 23B, 24F, 31 or 35B, each conjugated to CRM197) with 62.5 µg aluminum phosphate adjuvant (APA) per every immunization; or (2) 2 μg PnPs (15C-CRM197 with 125 μg APA per immunization.

Sera were collected at baseline (pre-immunization) and on days 14 (post-dose 1,

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PD1) and 28 (after dose 2, PD2). NZWR were monitored at least daily by trained animal care staff for signs of illness or distress. The vaccine formulations were considered safe and well tolerated by the NZWR as no vaccine-related adverse events were noted. All animal experiments were performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The NZWR experimental protocol was approved by the Institutional Animal Care and Use Committees at Merck &amp; Co., Inc. (Kenilworth, NJ) and Covance (Denver, PA).

Sera obtained from NZWR were tested in ELISA assays to assess IgG immunogenicity using a coating concentration of 1-2 mg/ml of the respective PnPs. Functional antibodies were determined using opsonophagocytosis assays (OPAs) based on previously described protocols available online from the Bacterial Respiratory Pathogens Reference Laboratory at the University of Alabama at Birmingham using Opsotiter® 3 software (UAB Research Foundation, Caro-Aguilar et al., Immunogenicity differences of a 15-valent pneumococcal polysaccharide conjugate vaccine (PCV15) based on vaccine dose, route of immunization and mouse strain, Vaccine, 35(6):865-72 (2017); Burton et al., Development and validation of a fourfold multiplexed opsonization assay (MOPA4) for pneumococcal antibodies, Clin. Vaccine Immunol., 13(9):1004-9 (2006)).

All monovalent pneumococcal conjugate vaccines were found to be immunogenic in rabbits (Fig. 15) and generate a functional antibody that kills the corresponding bacterial strain (Fig. 16).

Example 42. Assessment of cross-protection of serotypes 15A, 15B, 15C.

Rabbits were immunized with 15A-CRM197/APA, 15B-CRM197/APA, or 15C-CRM197/APA to assess cross-reactivity between each serotype.

Adult New Zealand White rabbits (NZWR, n=3/group) were immunized intramuscularly (IM) with 0.5 ml of the corresponding monovalent conjugate vaccine on day 0 and day 14 (alternating sides). A monovalent pneumococcal conjugate vaccine prepared in 20 mM L-histidine, pH 5.8, 150 mM NaCl and 0.2% (wt/vol) PS-20 in the preparation described in Example 38 was administered at a dose of 2 μg PnPs (15A, 15B, or 15C, each conjugated to CRM197) with 125 μg of APA per immunization. Sera were collected at baseline (pre-immunization) and on days 14 (post-dose 1, PD1) and 28 (post-dose 2, PD2). NZWR were monitored at least daily by trained animal care staff for signs of illness or distress. The vaccine formulations were found to be safe and well tolerated by the NZWR as there were no vaccine-related adverse events noted. All animal experiments were performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The NZWR experimental protocol was approved by the Institutional Animal Care and Use Committees at both Merck and Co., Inc. and Covance (Denver, PA).

Sera obtained from NZWR were tested in ELISA assays to assess IgG immunogenicity using a coating concentration of 1-2 mg/ml of the respective PnPs. Functional antibody was determined by OPA based on previously described protocols available online from the Bacterial Respiratory Pathogen Reference Laboratory at the University of Alabama at Birmingham using Opsotiter® 3 software (UAB Research Foundation (Caro-Aguilar et al., 2017; Burton et al. ., 2006).

All three monovalent serogroup 15 pneumococcal conjugate vaccines were found to be immunogenic in rabbits (Fig. 17) and generated functional antibodies that killed the corresponding bacterial strain (Fig. 18). In addition, rabbits immunized with monovalent pneumococcal serogroup 15 conjugate vaccines had equivalent IgG and OPA PD2 titers to the homologous and heterologous polysaccharide and bacterial strain, respectively. Rabbits immunized with 15A-CRM197/APA, 15B-CRM197/APA or 15C-CRM197/APA were cross-reactive to each pneumococcal polysaccharide (15A, 15B, 15C) (Fig. 17). Analysis of log-transformed post-dose 2 (PD2) IgG data by one-way ANOVA with Tukey's multiple comparison test (P-value 0.354) showed no significant differences in serogroup 15 IgG titers. Additionally, rabbits immunized with 15A-CRM197/APA , 15B-CRM197/APA or 15C-CRM197/APA, were cross-reactive to each strain of S. pneumoniae bacteria (15A, 15B, 15C), as all hyperimmune rabbit sera had functional antibodies to each strain assessed and killed the bacteria (Fig. 18). Similarly, analysis of log-transformed OPA data obtained after dose 2 (PD2) by one-way ANOVA with Tukey's multiple comparison test (P-value=0.054) showed no significant differences in OPA serogroup 15 titers.

Example 43: Immunogenicity of PCV21 in New Zealand White Rabbits.

Adult New Zealand White rabbits (NZWR, n=5/group) were immunized intramuscularly with 0.1-0.5 ml pneumococcal conjugate vaccine on days 0 and 14 (alternating sides). Pneumococcus

- 79 043489 PCV21 vaccine, prepared in 20 mM L-histidine, pH 5.8, 150 mM NaCl, and 0.2% (w/v) PS-20 by the preparation method described in Example 38, was administered in doses 4, 2, 1, 0.4, 0.08 or 0.016 μg PnPs (3, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 20A, 22F, 23A , 23B, 24F, 31, 33F, or 35B, each conjugated to CRM197) for immunization. Sera were collected at baseline (pre-immunization) and on days 14 (post-dose 1, PD1) and 28 (post-dose 2, PD2). NZWR were monitored at least daily by trained animal care staff for signs of illness or distress. The vaccine formulations were found to be safe and well tolerated by the NZWR as there were no vaccine-related adverse events noted. All animal experiments were performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The NZWR experimental protocol was approved by the Institutional Animal Care and Use Committees at both Merck and Co., Inc. and Covance (Denver, PA).

Sera obtained from NZWR were assessed for IgG immunogenicity using a multiplex electrochemiluminescence (ECL) assay. This assay was developed to work with rabbit serum based on the human serum assay described by Marchese et al. (Optimization and validation of a multiplex, electrochemiluminescence-based detection assay for the quantitation of immunoglobulin G serotype-specific antipneumococcal antibodies in human serum, Clin. Vaccine Immunol., 16(3):387-96 (2009)) using a method developed by MesoScale Discovery (a division of MesoScale Diagnostics, LLC, Gaithersburg, MD), which uses a SULFO-TAG™ tag that emits light in response to electrochemical stimulation. SULFO-TAG™-labeled anti-rabbit IgG was used as a secondary antibody to test NZWR serum samples. Functional antibodies were determined using a multiplex opsonophagocytic reaction assay (MOPA) based on previously described protocols available online from the Bacterial Respiratory Pathogen Reference Laboratory at the University of Alabama at Birmingham using Opsotiter® 3 software (UAB Research Foundation, Caro-Aguilar et al, 2017, supra; Burton et al., 2006, supra).

PCV21 pneumococcal conjugate vaccines were found to be immunogenic in rabbits (FIGS. 19A, 19B, 20A and 20B) and generated functional antibodies that killed vaccine-type bacterial strains (FIGS. 21A and 21B) at all doses tested.

Comparable PD1 immunogenicity was observed for PCV21 at doses of 4, 2, 1, and 0.4 μg on PnPs, with the exception of 24F, which had higher immunogenicity at the 2 μg dose compared to the 4 or 1 μg doses, and 15A, 15B, and 15C. which had higher immunogenicity at the 0.4 μg dose compared to the 2 μg dose (FIGS. 19A and 19B). Thus, at higher doses of PnPs (0.4-4 μg/PnPs), the vaccine dose response was negligible. Lower immunogenicity was observed at vaccine doses of 0.08 and 0.016 μg PnPs, as the abundance of many serotypes was significantly lower than in the 2 μg vaccine dose.

Comparable PD2 immunogenicity was observed for PCV21 at doses of 4, 2, 1 and 0.4 μg on PnPs (Fig. 20A and 20B).

Overall, comparable MOPA PD2 titers were observed for PCV21 at doses of 4, 2, 1, and 0.4 μg on PnPs, with a downward trend observed in MOPA titers for the 0.08 and 0.016 μg PnPs vaccine doses, although many did not reach statistical significance (data not shown). PCV21 at 2 μg on PnPs was selected as a representative data set for MOPA. Specifically, rabbits immunized with PCV21 at a dose of 2 μg had significantly higher MOPA PD1 titers for all serotypes compared to rabbit sera before immunization, with the exception of serotype 3 (Fig. 21A). It should be noted that preimmunization rabbit sera from this study had higher background titers for serotypes 16F, 31, and 35B. Rabbits immunized with PCV21 at a dose of 2 μg had significantly higher MOPA PD2 titers for all serotypes compared to rabbit sera before immunization (Fig. 21B). Log-transformed data were analyzed by one-way ANOVA using Dunnett's test to determine significance.

Example 44. Materials and methods.

Testing of free polysaccharides.

Free polysaccharide (polysaccharide that is not conjugated to CRM197) in the conjugate sample was measured by first precipitating the free protein and conjugates with deoxycholate (DOC) and hydrochloric acid. The precipitates were then filtered and the filtrates analyzed for free polysaccharide concentration using HPSEC/UV/MALS/RI. Free polysaccharide was calculated as a percentage of total polysaccharide measured by HPSEC/UV/MALS/RI.

Free protein testing.

Free polysaccharide, polysaccharide-CRM197 conjugate, and free CRM197 in conjugate samples were separated by micellar electrokinetic chromatography (MEKC) capillary electrophoresis. Briefly, samples were mixed with MEKC running buffer containing 25 mM borate, 100 mM SDS, pH 9.3, and separated in a pre-prepared uncoated fused silica capillary. Separation was monitored at 200 nm and free CRM197 was quantified using the CRM197 standard curve. Free protein results are presented as a percentage of the total protein content determined by the procedure

HPSEC/UV/MALS/RI.

Analysis of molecular weight and concentration of conjugates using HPSEC/UV/MALS/RI assay

Conjugate samples were injected and separated using high performance size exclusion chromatography (HPSEC). Detection was performed sequentially using an ultraviolet (UV) detector, multi-angle light scattering (MALS) and refractive index (RI) detectors. Protein concentration was calculated from UV280 using the extinction coefficient. Polysaccharide concentration was deconvoluted from the RI signal (provided by both protein and polysaccharide) using dn/dc coefficients, which represent the change in the refractive index of a solution with a change in solute concentration, expressed in ml/g. The average molecular weight of the samples was calculated using Astra software (Wyatt Technology Corporation, SantaBarbara, CA) using the measured concentration information and light scattering across the sample peak. There are several forms of expressing average molecular weights for polydisperse molecules. For example, number average molecular weight Mn, weight average molecular weight Mw and z-average molecular weight Mz (Molecules, 2015, 20, 10313-10341). Unless otherwise stated, molecular weights are weight average molecular weights.

Determination of lysine consumption in a conjugated protein as a measure of the number of covalent bonds between the polysaccharide and the carrier protein.

The Waters AccQ-Tag Amino Acid Assay (AAA) was used to measure the degree of conjugation in the conjugate samples. Samples were hydrolyzed using vapor phase acid hydrolysis on an Eldex workstation to separate carrier proteins into their constituent amino acids. Free amino acids were derivatized using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). The derivatized samples were then analyzed by UPLC with UV detection on a C18 column. Average protein concentration was obtained using representative amino acids other than lysine. Lysine consumption during conjugation (i.e., lysine loss) was determined by the difference between the average measured amount of lysine in the conjugate and the expected amount of lysine in the starting protein.

Example 45. Effect of the conjugation process on the stability of pneumococcal conjugate vaccine.

Individual pneumococcal polysaccharide-protein conjugates, prepared using different solvents (aqueous or DMSO) by the reductive amination mechanism as described in the examples above, were used to prepare 1-, 7-, 14-, and 21-valent pneumococcal conjugate vaccine drug products designated as PCV1, PCV7, PCV14 and PCV21, at a concentration of 84 μg/ml.

Containers filled with vaccine medicinal product were placed at 4 or 37°C for up to 7 days. Intrinsic protein fluorescence spectroscopy (IPFS) was used to evaluate the stability of pneumococcal polysaccharide-conjugated CRM197 carrier protein in PCV1, PCV7, PCV14, or PCV21 formulations when stored at 4°C and 37°C. Fluorescence emission spectra (Em 290-400 nm) of undiluted samples were obtained using a Jasco FP-6500 spectrofluorimeter at room temperature in a quartz cuvette with a path length of 1 cm. An excitation wavelength of 280 nm was used with a scan speed of 100 nm/min and an excitation bandwidth and radiation 3 nm.

As shown in FIG. 22A-22C, drug product formulations in which all polysaccharide-protein conjugates were produced by a reductive amination mechanism in an aprotic solvent (e.g., DMSO) had superior physical and chemical stability compared to a vaccine in which all polysaccharide-protein conjugates were obtained by conjugation in aqueous solvent (protic) according to the mechanism of reductive amination. In just 16 hours at 37°C, the fluorescence intensity of the drug products PCV1, PCV7 and PCV14, obtained using all polysaccharide-protein conjugates prepared by the mechanism of reductive amination in an aqueous solvent at a concentration of 84 μg/ml PnPs, was reduced, and the maximum vaccine emission was shifted from 332 nm towards 338 nm. The 84 μg/mL PnPs vaccine formulations PCV1, PCV7, and PCV14, which use drugs produced by a reductive amination mechanism in an aprotic solvent (e.g., DMSO), showed no change in fluorescence intensity or emission maximum at 338 nm. Additionally, the drug PCV21 was studied and formulated with 20 mM L-histidine, pH 5.8, 150 mM NaCl, and 0.1% (w/v) PS-20 as described in Example 38 , and contained polysaccharide-protein conjugates obtained by the mechanism of reductive amination in an aprotic solvent (for example, DMSO). The study showed excellent physical and chemical stability after storage at 37°C. PCV21 showed no change in fluorescence intensity or maximum emission at 338 nm (Fig. 22D).

It should be understood that the resulting signal intensity and maximum emission obtained from the IPFS measurement (Example 40) are due to the overall stability of the individual polysaccharide-protein

- 81 043489 conjugates in a polyvalent complex medicinal product. It has been shown that a vaccine drug product in which all polysaccharide-protein conjugates are formulated in an aprotic solvent is stable, while a vaccine drug product in which all drugs are formulated in a protic solvent is unstable under stress conditions. If a vaccine medicinal product contains a mixture of polysaccharide-protein conjugates resulting from a conjugation process in both a protic and an aprotic solvent, then the average maximum or emission intensity measured for the medicinal product will be weighted by the contribution of the emission of each protic polysaccharide. protein conjugate at 332 nm and emission of each aprotic polysaccharide-protein conjugate at 338 nm. In such a mixture, a weighted change would be expected to occur in intensity or emission maxima at elevated temperature due to the presence of polysaccharide-protein conjugates produced by the proton conjugation process.

As shown in FIG. 23, Nanoparticle trajectory analysis (NTA) was used to evaluate the stability of the PCV21 drug product prepared at 0.04 mg/mL PnPs. This method collects video of directly tracked populations of nanoparticles as they move through Brownian motion to extrapolate particle size and concentration. The 635 nm Class 1 laser focuses an 80 µm red laser beam through a liquid sample, illuminating the particles as rapidly diffusing points of light. The CCD camera records video at 30 frames per second, tracking the movement of each individual illuminated particle over time. The system software identifies the center of each individual particle from the video and tracks the distance traveled independently to determine the root mean square displacement. This tracking is performed simultaneously for each particle in the sample population in each frame until the raw data collected from the entire video has been analyzed. By simultaneously measuring the root mean square displacement of each individual tracked particle, its diffusion coefficient (Dt) and spherical equivalent hydrodynamic radius (rh) are determined using the Stokes-Einstein equation. The software then presents this accumulated data as a distribution of particle size and concentration. Raw data is collected not only on particle size and concentration, but also on the intensity or brightness of an individual particle. All of these data are approximated and plotted separately as particle intensity versus particle size and particle concentration versus particle size, and then as three-dimensional contour plots comparing particle size, concentration, and intensity across all particle populations.

A conjugate vaccine consisting of a carrier protein and a polysaccharide antigen can, together or separately, be susceptible to aggregation in the particle size range of 10–1000 nm, making NTA a suitable stability-indicating method for assessing and quantifying aggregation phenomena. After storing the drug product at 4°C and 37°C for 1 week, NTA results showed no significant drug product accumulation in formulations containing PS-20 at concentrations of 0.025, 0.05, 0.1, 0.15 and 0. 2% w/v PS20 (as shown in Fig. 23). However, after mixing the formulations for 6 hours at 4°C, the formulation that did not contain polysorbate 20 was ineffective in reducing agitation-induced drug product aggregation, as evidenced by increased D90 particle size, expanded particle size distribution, and increased particle intensity. according to NTA results. Therefore, it is preferable to maintain polysorbate 20 at a level of 0.025 to 0.2% or higher (w/v) to stabilize the drug product during normal manufacturing, storage, delivery and processing.

As described in Example 39, to measure the molecular weight of a 21-valent (PCV21) polysaccharide-protein conjugate drug product formulated with 20 mM L-histidine, pH 5.8, 150 mM NaCl, and 0 to 0.2% ( wt./vol.) PS-20 according to the preparation method described in example 38, HPSEC UV/MALS/RI was used. The compositions were placed at 4 or 37°C for up to one week. In some cases, syringes were placed on a platform with horizontal rotation and shaken for up to 6 hours after storage for 1 week at 4 or 37°C. This simulated the shipping and handling stress that can accompany the production and delivery of a drug product. In the event of degradation or depolymerization of polysaccharide-protein conjugates in drug products, there would be an apparent decrease in molecular weight, and in the event of aggregation of the composition of the drug product, an increase in molecular weight would occur. This measurement provides additional characterization of the quality of the vaccine drug product or polysaccharide-protein conjugates as a function of PS-20 concentration. As shown in FIG. 24, PCV21 drug products, in which all drug substances are prepared by a reductive amination mechanism in an aprotic solvent (eg, DMSO), were resistant to depolymerization or chemical degradation of carbohydrate and resistant to protein aggregation in the drug product vehicle caused by heat treatment, at 0 .05 to 0.15% PS-20. Taken together, this indicates that the PCV21 drug is stable at elevated temperature and exercise when formulated using PS-20 at levels of 0.025 to 0.2% or higher.

Example 46. PCV21 protects mice from infection.

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Young female Swiss Webster mice (6-8 weeks old, n=10 per group) were immunized intraperitoneally (IP) with 0.1-0.5 ml 21-valent pneumococcal conjugate vaccine (PCV21) on days 0, 14 and 28. Pneumococcal vaccine PCV21 were administered in doses of 4, 2, 0.4, 0.08 or 0.016 μg PnPs (3, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 20A, 22F, 23A , 23B, 24F, 31, 33F and 35B, each conjugated to CRM197) for immunization. Mice were monitored at least daily by trained animal care personnel for signs of disease or distress. The vaccines were considered safe and well tolerated in mice as no vaccine-related side effects were noted. On day 52, mice were challenged intratracheally with Streptococcus pneumoniae serotype 24F. Exponential phase cultures of S. pneumoniae were centrifuged, washed, and suspended in sterile PBS. Before infection, mice were anesthetized with isoflurane. 105 CFU of S. pneumoniae in 0.1 ml PBS were placed down the throats of mice suspended vertically by their incisors. Aspiration of bacteria was induced by gently extending the tongue outward and closing the nostrils. Mice were weighed daily and sacrificed if weight loss exceeded 20% of baseline. Blood was collected at 24, 48, and 72 h to assess bacteremia. Mice were monitored at least twice daily by trained animal care personnel for signs of disease or distress. All animal experiments were performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The mouse experimental protocol was approved by the Institutional Animal Care and Use Committee of Merck &amp; Co., Inc.

Mouse sera were assessed for IgG immunogenicity using a multiplex electrochemiluminescence (ECL) assay. This assay was developed for the use of mouse serum based on the assay for human serum described by Marchese et al., Clin. Vaccine Immunol. (2009), 16(3):387-96, using a method developed by MesoScale Discovery (a division of MesoScale Diagnostics, LLC, Gaithersburg, MD), which uses a SULFO-TAG™ tag that emits light upon electrochemical stimulation. SULFO-TAG™ labeled anti-mouse IgG was used as a secondary antibody to test mouse serum samples. Functional antibody was determined using a multiplex opsonophagocytic reaction assay (MOPA) based on previously described protocols at www.vaccine.uab.edu and Opsotiter® 3 software owned and licensed by the University of Alabama (UAB) Research Foundation (see Caro-Aguilar I. et al., Vaccine (2017), 35(6):865-72; and Burton R.L., Nahm M.H., Clin. Vaccine Immunol. (2006), 13(9):1004-9).

Immunization with PCV21 generated antibody titers in Swiss Webster mice for all serotypes in the vaccine (data not shown). PCV21 was also immunogenic in Balb/c and CD1 mice (data not shown). Swiss Webster mice immunized with PCV21 were also protected from challenge with S. pneumoniae serotype 24F (Fig. 25). The Mantel-Cox log-rank test indicated that all PCV21 immunized groups were significantly protected from infection compared with the naïve group (P<0.05). Similarly, in groups of mice immunized with PCV21, there was virtually no bacteremia, which was significantly less compared to the group of naïve animals (data not shown).

Example 47 PCV immunogenicity and functional antibody in rabbits.

Adult New Zealand White rabbits (NZWR, n=5 per group) were immunized intramuscularly (IM) with 0.1 ml PCV on day 0 and day 14 (alternating sides). PCV was administered at doses of 0.4 μg of PnPs per serotype per immunization with 25 μg of [Al] in the form of aluminum phosphate adjuvant (APA). For PCV31, the serotype 6B dose concentration was doubled to 0.8 μg of PnPs per immunization. PCV21 (as described in Example 38) includes purified polysaccharides of serotypes 3, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 20A, 22F, 23A, 23B, 24F, 31, 33F and 35B, each conjugated to CRM197. Each polysaccharide-protein conjugate was prepared without or with adjuvant at a concentration of 250 μg/ml/ml in the form of aluminum phosphate or APA to evaluate the effect of the adjuvant (PCV21/APA). Lower valence PCV formulations were also prepared (as described in Example 38), which included 8 new STs (PCV8: 6C, 15A, 16F, 23A, 23B, 24F, 31 and 35B, each conjugated to CRM197, without adjuvants ), 16 ST is not contained in any licensed PCV (PCV16: 6C, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 17F, 20, 23A, 23B, 24F, 31 and 35B, each conjugated to CRM197 , without adjuvants) or PCV31 (1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15C, 16F, 17F, 18C, 19A, 19F (PCV31/APA) . Each vaccine used polysaccharide-protein conjugates formulated at 4 μg/ml (w/v) pneumococcal polysaccharide (PnPs), with the exception of 6B, which was adjusted to 8 μg/ml (w/v) pneumococcal polysaccharide (PnPs). polysaccharides (PnPs) in PCV31 or PCV31/APA. The final concentration of pneumococcal polysaccharide (PnPs) in each vaccine was 128 μg/ml PnPs in PCV31, 84 μg/ml PnPs in PCV21, 64 μg/ml in PCV16, and 32 μg/ml in PCV8.

Sera were collected at baseline (pre-immunization) and on days 14 (PD1) and 28 (PD2). NZWR were supervised at least daily by trained abdominal care staff

- 83 043489 for signs of illness or distress. The vaccine formulations were considered safe and well tolerated by the NZWR as no vaccine-related adverse events were noted. All animal experiments were performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The NZWR experimental protocol was approved by the Institutional Animal Care and Use Committees at Merck &amp; Co., Inc and Covance (Denver, PA).

Rabbit sera were assessed for IgG immunogenicity using a multiplex electrochemiluminescence (ECL) assay. This assay was developed for the use of rabbit serum based on the human serum assay described by Marchese et al., Clin. Vaccine Immunol. (2009), 16(3):387-96, using a method developed by MesoScale Discovery (a division of MesoScale Diagnostics, LLC, Gaithersburg, MD), which uses a SULFO-TAG™ tag that emits light upon electrochemical stimulation. SULFO-TAG™ labeled anti-rabbit IgG was used as a secondary antibody to test NZWR serum samples. Functional antibody was determined using a multiplex opsonophagocytic reaction assay (MOPA) based on previously described protocols at www.vaccine.uab.edu and Opsotiter® 3 software owned and licensed by the University of Alabama (UAB) Research Foundation (see Caro-Aguilar I. et al., Vaccine (2017), 35(6):865-72; and Burton R.L., Nahm M.H., Clin. Vaccine Immunol. (2006), 13(9):1004-9).

Rabbit sera were tested as a pool before immunization and PD1 and PD2 separately in multiplex electrochemiluminescence assays to determine antibody titers. PCVs generated antibody titers in rabbits for all serotypes following vaccine immunization (data not shown). The serotype 15B polysaccharide was not included in PCV16, PCV21, or PCV31, but serotype 15B antibody titers were observed following immunization with both PCV16, PCV21, and PCV31.

PCV21 without adjuvant had comparable or significantly higher immunogenicity (serotypes 3, 7F, 10A, 19A, 23A and 23B) compared to PCV21 with ARA (Figs. 26A, 26B and 28), indicating no additional immunogenicity benefit in rabbits with ARA included in the vaccine.

Carrier-induced epitopic suppression refers to interference with an antibody response to an antigen (such as a capsular polysaccharide) that is bound to the same carrier protein (such as CRM197). The interference is thought to arise from competition for a limited number of host-specific primed helper T cells. As a result, a decreased response to capsular polysaccharide may occur. Pfizer found a decrease in the immunogenicity of common serotypes in vaccines as the valency of the vaccine increased from 7-valent to 13-valent [Comparison of IgG antibodies GMC Prevnar (7 valent) with Prevnar13 (Table 9, p. 29 of the PCV13 monograph). Thus, the purpose of the experiment was to study the immunogenicity of PCV with a lower valency compared to PCV21 (Fig. 27A-27C). PCV8 (8 new serotypes) showed comparable immunogenicity to PCV21 for 8 common serotypes (Fig. 29). PCV16 (PCV21 minus the 5 overlapping serotypes from PCV15) showed comparable immunogenicity to PCV21 for 15 of the 16 common serotypes (Fig. 29). Serotype 31 showed higher immunogenicity against PCV16 (Fig. 29). Overall, no significant trends in PCV21-induced carrier suppression were observed in NZWR. PCV31/APA showed comparable immunogenicity to PCV21/APA for 12 of 21 common serotypes. Ten serotypes (6C, 7F, 9N, 12F, 15A, 15B (not in PCV21), 15C, 17F, 19A and 23A) were 2.9- to 11-fold more immunogenic in PCV31 (FIG. 30). Overall, no significant trends in PCV31-induced carrier suppression were observed in NZWR.

PCVs have been found to be immunogenic in rabbits and generate functional antibodies that kill vaccine-type bacterial strains. Rabbit sera were tested in multiplex opsonophagocytic reaction assays (MOPA) to determine functional antibody titers. PCV21 had comparable or higher titers of OPA PDA compared to PCV21/APA, PCV8 and PCV16 (Fig. 31). Log-transformed data were analyzed by one-way ANOVA using Dunnett's test to determine significance. PCV21 had significantly higher OPA titers for serotype 16F compared with PCV8 and for serotypes 12F, 23A, and 19A compared with PCV21/APA. PCV31 had significantly higher OPA titers for serotype 23A compared to PCV21.

Example 48 Immunogenicity of PCV21 in adult rhesus monkeys.

PCV21 was also evaluated in immunogenicity models in adult rhesus monkeys. Rhesus macaques were immunized intramuscularly with PCV21 on days 0, 28 and 56. PCV21 was administered in doses of 1 μg PnPs in a volume of 0.25 ml (3, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F , 17F, 19A, 20A, 22F, 23A, 23B, 24F, 31 33F and 35B, each conjugated to CRM197) per immunization. Sera were collected at baseline (pre-immunization, day 0) and on days 14 (PD1), 28, 42 (PD2), 56, 70 (PD3), and 84.

Rhesus serum was assessed for IgG immunogenicity using a multiplex electrochemiluminescence (ECL) assay. This assay was developed for the use of rhesus serum based on the human serum assay described by Marchese et al. and Skinner et al. (Marchese R.D. et al., Clin. Vaccine Immunol. (2009), 16(3):387-96; and Skinner, J.M. et al., Vaccine (2011), 29(48):8870-8876) with

- 84 043489 using a method developed by MesoScale Discovery (a division of MesoScale Diagnostics, LLC, Gaithersburg, MD), which uses a SULFO-TAG™ tag that emits light upon electrochemical stimulation. SULFO-TAG™ labeled anti-human IgG was used as a secondary antibody to test rhesus serum samples. Functional antibody was determined using a multiplex opsonophagocytic reaction assay (MOPA) based on previously described protocols at www.vaccine.uab.edu and Opsotiter® 3 software owned and licensed by the University of Alabama (UAB) Research Foundation (see Caro-Aguilar I. et al., Vaccine (2017), 35(6):865-72; and Burton R.L., Nahm M.H., Clin. Vaccine Immunol. (2006), 13(9):1004-9).

PCV21 was found to be immunogenic in adult monkeys and generated functional antibodies that killed vaccine-type bacterial strains at all time points tested (FIGS. 32 and 33). It is also noteworthy that PCV21, which contains the polysaccharide conjugates 15A-CRM197 and 15C-CRM197, also provides cross-reactivity with 15B, as evidenced by the ECL results. PCV21 was immunogenic in monkeys after one dose of vaccine (Fig. 32). Most serotypes reached their maximum titer at PD1, with the exception of ST 6C, 12F and 24F, which were effective after additional immunizations. Adult rhesus monkeys showed higher titers of pre-existing antibodies compared with previous PCV studies in infant rhesus monkeys. Pre-existing antibody titers were most evident when serum samples were tested by MOPA, making it difficult to determine the opsonophagocytic titer. All study time points were MOPA tested as pooled or individual samples, but for ease of viewing, only pre-immunization, PD1 and PD3 data are shown (FIG. 33) as OPA titers between PD1 and PD3 were not significantly different.

PCV21 immunized rhesus macaque sera were assessed for cross-protection against other S. pneumoniae bacteria (Fig. 34). Sera from macaques immunized with PCV21 had cross-protection against serotypes 6A, 6B, and 23F, but not against 19F. Cross-protection against 6A and 6B is likely due to immunization with the polysaccharide conjugate 6C-CRM197 in the polyvalent PCV21. Similarly, immunization with polysaccharide conjugates 23A-CRM197 and 23B-CRM197 in polyvalent PCV resulted in cross-protection against serotype 23F. Immunization with PCV21 containing the 19A-CRM197 polysaccharide conjugate did not provide cross-protection against 19F.

Example 49 Immunogenicity of PCV21 in adult rhesus monkeys. Assessing adjuvant and transporter suppression.

Another study in monkeys involved evaluating PCV21 with and without APA and comparing overall serogenic immunogenicity to assess host suppression. Adult rhesus monkeys (n=5 per group) were immunized intramuscularly with half human dose vaccines on day 0 of the study. PCV21 was administered at a dose of 1 μg of PnPs in a volume of 0.25 ml (3, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 20, 22F, 23A, 23B, 24F, 31 33F and 35B, each conjugated to CRM197) per immunization. One arm included PCV21 adjuvanted with 62.5 μg [Al] in the form of aluminum phosphate. The other group included PCV15 administered at a dose of 1 μg of PnPs in a volume of 0.25 ml (1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F, each of which is conjugated with CRM197, and the dose of 6B was 2 μg) per immunization and with an adjuvant of 62.5 μg [Al] in the form of aluminum phosphate. The other group included Prevnar13® (PCV13), administered at a dose of 1.1 μg PnPs in a volume of 0.25 ml (1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F and 23F, each of which is conjugated to CRM197, and the dose of 6B was 2.2 μg) per immunization. Sera were collected at baseline (pre-immunization, day 0) and on day 28.

PCV21 was found to be immunogenic in adult monkeys and generate functional antibodies that kill vaccine-type bacterial strains. PCV21 without adjuvant showed comparable or significantly higher immunogenicity (serotypes 15A and 15B) compared to PCV21 with ARA (Fig. 35). PCV21 did not include serotype 15B.

PCV21 immunized monkeys were compared with monkeys immunized with PCV15 and Prevnar13. PCV21 and PCV15 have 5 common serotypes (3, 7F, 19A, 22F, 33F); in monkeys immunized with PCV21 or PCV15, there was no difference in the immunogenicity of the 5 serotypes (Fig. 36). PCV21 and Prevnar13 have 3 common serotypes (3, 7F, 19A); there was no difference in the immunogenicity of serotypes 7F and 19A. Prevnar13 immunized monkeys had lower antibody titers to serotype 3 compared to PCV21 (Fig. 36). This finding is consistent with clinical data for PCV15 in humans, suggesting that serotype 3 is more immunogenic in the Merck PCV15 vaccine. Together, these results suggest that carrier suppression is not observed in adult rhesus macaques immunized with PCV21 compared with that of Prevnar13 or PCV15.

Example 50: Cross-reactivity of serotypes 6A, 6B and 6C - monovalent test.

The monovalent drug product was prepared using pneumococcal polysaccharide conjugate 6A-CRM197 or pneumococcal polysaccharide conjugate 6B-CRM197, which was formulated with 20 mM histidine, pH 5.8, 150 mM sodium chloride and 0.1% w/v. polysorbate 20

- 85 043489 (PS-20) with a target concentration of total polysaccharide of 4.0 μg/ml of any serotype. Conjugates were prepared by conjugating CRM197 protein separately to each type of pneumococcal polysaccharide (PnPs) (-6A or -6B). The required volume of starting conjugates needed to obtain the target concentration of individual serotypes was calculated based on the batch size and the concentration of the individual starting polysaccharides. The individual conjugates were added to a solution of histidine, sodium chloride and PS-20 to produce a 2X conjugate mixture. The preparation vessel containing the 2X conjugate mixture was mixed using a magnetic stirrer and sterile filtration was performed into another vessel. The sterile filtered mixture was then diluted 2X with saline to achieve the required target concentrations of total polysaccharide and vehicle. The formulations were then placed in vials and stored at 2-8°C.

Rabbits were immunized with 6A-CRM197 or 6B-CRM197 to assess cross-reactivity in serogroup 6. Adult New Zealand White rabbits (NZWR, n=3/group) were immunized intramuscularly (IM) with 0.25 ml of the respective monovalent conjugate vaccine on day 0 and day 14 ( with alternating sides). A monovalent pneumococcal conjugate vaccine formulated with 20 mM L-histidine, pH 5.8, 150 mM NaCl, and 0.1% (w/v) PS-20 using the preparation method described in Example 38 was administered into dose of 1 μg of PnPs (6A or 6B, each conjugated to CRM197). Sera were collected at baseline (pre-immunization) and on days 14 (post-dose 1, PD1) and 28 (post-dose 2, PD2). NZWR were monitored at least daily by trained animal care staff for signs of illness or distress. The vaccine formulations were considered safe and well tolerated by the NZWR as no vaccine-related adverse events were noted. All animal experiments were performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The NZWR experimental protocol was approved by the Institutional Animal Care and Use Committees at Merck &amp; Co., Inc and Covance (Denver, PA).

NZWR sera were tested in ELISA assays to assess IgG immunogenicity using an appropriate coating concentration of 2 μg/ml PnPs. Functional antibody was determined using opsonophagocytosis assays (OPAs) based on previously described protocols at www.vaccine.uab.edu and Opsotiter® 3 software owned and licensed by the UAB Research Foundation (see Caro-Aguilar I. et al., Vaccine ( 2017), 35(6):865-72; and Burton R.L., Nam M.H., Clin. Vaccine Immunol. (2006), 13(9):1004-9).

Both 6A-CRM197 and 6B-CRM197 were found to be immunogenic in rabbits (FIG. 37) and generate a functional antibody that kills the corresponding bacterial strain (FIG. 38). In addition, rabbits immunized with monovalent pneumococcal serogroup 6 conjugate vaccines had equivalent PD2 IgG titers and ORA results for homologous and heterologous polysaccharide and bacterial strain, respectively. All rabbits immunized with 6A-CRM197 or 6B-CRM197 were cross-reactive to each pneumococcal polysaccharide (PnPs 6A, PnPs 6B and PnPs 6C) (Fig. 37). Using log-transformed post-dose 2 (PD2) IgG data analyzed by one-way ANOVA, no significant differences in IgG titers were observed in serogroup 6. Additionally, rabbits immunized with 6A-CRM197 or 6B-CRM197 were cross-reactive to each bacterial strain of S. pneumoniae (6A, 6B and 6C), since all hyperimmune rabbit sera had functional antibody to each strain evaluated and killed the bacteria (Fig. 38). Similarly, OPA data obtained after dose 2 (PD2), analyzed logarithmically with one-way ANOVA, showed no significant differences in OPA titers in serogroup 6.

Example 51. Cross-reactivity of serotypes 20A and 20B - monovalent study.

The monovalent drug product was prepared using pneumococcal polysaccharide conjugate 20A-CRM197 formulated with 20 mM histidine, pH 5.8, 150 mM sodium chloride and 0.2% w/v. polysorbate-20 (PS-20) with a concentration of 4.0 μg/ml. The formulation was prepared with 250.0 μg [A1]/ml as aluminum phosphate as an adjuvant (20A-CRM197/APA). The conjugate was prepared by conjugating the CRM197 protein separately to pneumococcal polysaccharides (PnPs) type 20. The required volume of input conjugate needed to obtain the target concentration of an individual serotype was calculated based on the batch volume and the concentration of the individual parent polysaccharide. A single conjugate was added to a solution of histidine, sodium chloride and PS-20 to obtain a 4X conjugate mixture at 16.0 μg/ml. The formulation vessel containing the conjugate mixture was mixed using a magnetic stirrer and sterile filtration was performed into another vessel. The sterile filtered 4X mixture was then added to another vessel containing aluminum phosphate adjuvant until the required target concentrations of polysaccharide, vehicle and adjuvant were achieved. The formulations were then placed in glass vials and stored at 2-8°C.

Rabbits were immunized with 20A-CRM197/APA or PCV21 to assess cross-reactivity with serogroup 20. Adult New Zealand White rabbits (NZWR, n=3/group) were immunized intramuscularly (IM) with pneumococcal conjugate vaccine on day 0 and day 14 (alternating sides).

- 86 043489

Monovalent pneumococcal conjugate vaccine, prepared as described above, was administered at a dose of 1 μg PnPs in 0.25 ml (ST20A conjugated to CRM197 and 62.5 μg [Al] in the form of aluminum phosphate was used as an adjuvant), multivalent pneumococcal conjugate PCV21 vaccine (84 μg/ml PnPs, formulated with 20 mM L-histidine, pH 5.8, 150 mM NaCl and 0.2% (w/v) PS-20 according to the preparation described in Example 38, administered at a dose of 0.4 μg of PnPs in 0.1 ml (serotypes 3, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 20, 22F, 23A, 23B, 24F , 31, 33F and 35B, each conjugated to CRM197. Sera were collected at baseline (pre-immunization) and on days 14 (post-dose 1, PD1) and 28 (post-dose 2, PD2). by at least daily observation by trained animal care personnel for signs of illness or distress.Vaccine formulations were considered safe and well tolerated by the NZWR as no vaccine-related adverse events were noted. All animal experiments were performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The NZWR experimental protocol was approved by the Institutional Animal Care and Use Committees at Merck &amp; Co., Inc and Covance (Denver, PA).

NZWR sera were tested in opsonophagocytosis assays (OPAs) to evaluate functional antibodies based on previously described protocols at www.vaccine.uab.edu and Opsotiter® 3 software owned and licensed by the UAB Research Foundation (see Caro-Aguilar I. et al. , Vaccine (2017), 35(6):865-72; and Burton R.L., Nahm M.H., Clin. Vaccine Immunol. (2006), 13(9):1004-9).

Rabbits immunized with both monovalent 20A-CRM197/APA and polyvalent PCV21 vaccines generated functional antibodies that killed both S. pneumoniae serotypes 20A and 20B (Fig. 39). This suggests that serotypes 20A and 20B share structural similarities, allowing cross-reactivity within the serogroup.

Claims (4)

1. A polyvalent immunogenic composition containing conjugates of S. pneumonia polysaccharides with a carrier protein, where each of the conjugates contains a polysaccharide of a specific S. pneumoniae serotype conjugated to a carrier protein, where S. pneumoniae serotypes consist of 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, de-O-acetylated 15B, 17F and 20, and the carrier protein is CRM197. 2. The polyvalent immunogenic composition according to claim 1, wherein the de-O-acetylated polysaccharide 15B has an O-acetyl content per repeat unit of less than 5%. 3. A polyvalent immunogenic composition containing conjugates of S. pneumonia polysaccharides with a carrier protein, where each of the conjugates contains a polysaccharide of a specific S. pneumoniae serotype conjugated to a carrier protein, where S. pneumoniae serotypes consist of 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15C, 17F and 20, and the carrier protein is CRM197. 4. A polyvalent immunogenic composition containing conjugates of S. pneumonia polysaccharides with a carrier protein, where each of the conjugates contains a polysaccharide of a specific S. pneumoniae serotype conjugated to a carrier protein, where S. pneumoniae serotypes consist of 3, 7F, 19A, 22F, 33F, 6A, 15A, 16F, 23A, 23B, 24F, 31, 35B, 8, 9N, 10A, 11A, 12F, 15B, 17F and 20, and the carrier protein is CRM197.